Multiple and contrasting pressures determine intraspecific phytochemical variation in a tropical shrub.

Intraspecific phytochemical variation across a landscape can cascade up trophic levels, potentially mediating the composition of entire insect communities. Surprisingly, we have little understanding of the processes that regulate and maintain phytochemical variation within species, likely because these processes are complex and operate simultaneously both temporally and spatially. To assess how phytochemistry varies within species, we tested the degree to which resource availability, contrasting soil type, and herbivory generate intraspecific chemical variation in growth and defense of the tropical shrub, Piper imperiale (Piperaceae). We quantified changes in both growth (e.g., nutritional protein, above- and below-ground biomass) and defense (e.g., imide chemicals) of individual plants using a well-replicated fully factorial shade-house experiment in Costa Rica. We found that plants grown in high light, nutrient- and richer old alluvial soil had increased biomass. High light was also important for increasing foliar protein. Thus, investment into growth was determined by resource availability and soil composition. Surprisingly, we found that chemical defenses decreased in response to herbivory. We also found that changes in plant protein were more plastic compared to plant defense, indicating that constitutive defenses may be relatively fixed, and thus an adaptation to chronic herbivory that is common in tropical forests. We demonstrate that intraspecific phytochemical variation of P. imperiale is shaped by resource availability from light and soil type. Because environmental heterogeneity occurs over small spatial scales (tens of meters), herbivores may be faced with a complex phytochemical landscape that may regulate how much damage any individual plant sustains.

A biological condition gradient for Caribbean coral reefs: Part II. Numeric rules using sessile benthic organisms.

The Biological Condition Gradient (BCG) is a conceptual model used to describe incremental changes in biological condition along a gradient of increasing anthropogenic stress. As coral reefs collapse globally, scientists and managers are focused on how to sustain the crucial structure and functions, and the benefits that healthy coral reef ecosystems provide for many economies and societies. We developed a numeric (quantitative) BGC model for the coral reefs of Puerto Rico and the US Virgin Islands to transparently facilitate ecologically meaningful management decisions regarding these fragile resources. Here, reef conditions range from natural, undisturbed conditions to severely altered or degraded conditions. Numeric decision rules were developed by an expert panel for scleractinian corals and other benthic assemblages using multiple attributes to apply in shallow-water tropical fore reefs with depths <30 m. The numeric model employed decision rules based on metrics (e.g., % live coral cover, coral species richness, pollution-sensitive coral species, unproductive and sediment substrates, % cover by Orbicella spp.) used to assess coral reef condition. Model confirmation showed the numeric BCG model predicted the panel's median site ratings for 84% of the sites used to calibrate the model and 89% of independent validation sites. The numeric BCG model is suitable for adaptive management applications and supports bioassessment and criteria development. It is a robust assessment tool that could be used to establish ecosystem condition that would aid resource managers in evaluating and communicating current or changing conditions, protect water and habitat quality in areas of high biological integrity, or develop restoration goals with stakeholders and other public beneficiaries.

A biological condition gradient for coral reefs in the US Caribbean Territories: Part I. Coral narrative rules.

As coral reef condition and sustainability continue to decline worldwide, losses of critical habitat and their ecosystem services have generated an urgency to understand and communicate reef response to management actions, environmental contamination, and natural disasters. Increasingly, coral reef protection and restoration programs emphasize the need for robust assessment tools for protecting high-quality waters and establishing conservation goals. Of equal importance is the need to communicate assessment results to stakeholders, beneficiaries, and the public so that environmental consequences of decisions are understood. The Biological Condition (BCG) model provides a structure to evaluate the condition of a coral reef in increments of change along a gradient of human disturbance. Communication of incremental change, regardless of direction, is important for decision makers and the public to better understand what is gained or lost depending on what actions are taken. We developed a narrative (qualitative) Biological Condition Gradient (BCG) from the consensus of a diverse expert panel to provide a framework for coral reefs in US Caribbean Territories. The model uses narrative descriptions of biological attributes for benthic organisms to evaluate reefs relative to undisturbed or minimally disturbed conditions. Using expert elicitation, narrative decision rules were proposed and deliberated to discriminate among six levels of change along a gradient of increasing anthropogenic stress. Narrative rules for each of the BCG levels are presented to facilitate the evaluation of benthic communities in coral reefs and provide specific narrative features to detect changes in coral reef condition and biological integrity. The BCG model can be used in the absence of numeric, or quantitative metrics, to evaluate actions that may encroach on coral reef ecosystems, manage endangered species habitat, and develop and implement management plans for marine protected areas, watersheds, and coastal zones. The narrative BCG model is a defensible model and communication tool that translates scientific results so the nontechnical person can understand and support both regulatory and non-regulatory water quality and natural resource programs.

Elevated atmospheric concentrations of CO2 increase endogenous immune function in a specialist herbivore.

Animals rely on a balance of endogenous and exogenous sources of immunity to mitigate parasite attack. Understanding how environmental context affects that balance is increasingly urgent under rapid environmental change. In herbivores, immunity is determined, in part, by phytochemistry which is plastic in response to environmental conditions. Monarch butterflies Danaus plexippus, consistently experience infection by a virulent parasite Ophryocystis elektroscirrha, and some medicinal milkweed (Asclepias) species, with high concentrations of toxic steroids (cardenolides), provide a potent source of exogenous immunity. We investigated plant-mediated influences of elevated CO2 (eCO2 ) on endogenous immune responses of monarch larvae to infection by O. elektroscirrha. Recently, transcriptomics have revealed that infection by O. elektroscirrha does not alter monarch immune gene regulation in larvae, corroborating that monarchs rely more on exogenous than endogenous immunity. However, monarchs feeding on medicinal milkweed grown under eCO2 lose tolerance to the parasite, associated with changes in phytochemistry. Whether changes in milkweed phytochemistry induced by eCO2 alter the balance between exogenous and endogenous sources of immunity remains unknown. We fed monarchs two species of milkweed; A. curassavica (medicinal) and A. incarnata (non-medicinal) grown under ambient CO2 (aCO2 ) or eCO2 . We then measured endogenous immune responses (phenoloxidase activity, haemocyte concentration and melanization strength), along with foliar chemistry, to assess mechanisms of monarch immunity under future atmospheric conditions. The melanization response of late-instar larvae was reduced on medicinal milkweed in comparison to non-medicinal milkweed. Moreover, the endogenous immune responses of early-instar larvae to infection by O. elektroscirrha were generally lower in larvae reared on foliage from aCO2 plants and higher in larvae reared on foliage from eCO2 plants. When grown under eCO2 , milkweed plants exhibited lower cardenolide concentrations, lower phytochemical diversity and lower nutritional quality (higher C:N ratios). Together, these results suggest that the loss of exogenous immunity from foliage under eCO2 results in increased endogenous immune function. Animal populations face multiple threats induced by anthropogenic environmental change. Our results suggest that shifts in the balance between exogenous and endogenous sources of immunity to parasite attack may represent an underappreciated consequence of environmental change.

Proximity to canopy mediates changes in the defensive chemistry and herbivore loads of an understory tropical shrub, Piper kelleyi.

Phytochemical traits are a key component of plant defense theory. Chemical ecology has been biased towards studying effects of individual metabolites even though effective plant defenses are comprised of diverse mixtures of metabolites. We tested the phytochemical landscape hypothesis, positing that trophic interactions are contingent upon their spatial location across a phytochemically diverse landscape. Specifically, intraspecific phytochemical changes associated with vertical strata in forests were hypothesised to affect herbivore communities of the neotropical shrub Piper kelleyi Tepe (Piperaceae). Using a field experiment, we found that phytochemical diversity increased with canopy height, and higher levels of phytochemical diversity located near the canopy were characterised by tradeoffs between photoactive and non-photoactive biosynthetic pathways. For understory plants closer to the ground, phytochemical diversity increased as direct light transmittance decreased, and these plants were characterised by up to 37% reductions in herbivory. Our results suggest that intraspecific phytochemical diversity structures herbivore communities across the landscape, affecting total herbivory.

Phytochemical diversity drives plant-insect community diversity.

What are the ecological causes and consequences of variation in phytochemical diversity within and between plant taxa? Despite decades of natural products discovery by organic chemists and research by chemical ecologists, our understanding of phytochemically mediated ecological processes in natural communities has been restricted to studies of either broad classes of compounds or a small number of well-characterized molecules. Until now, no studies have assessed the ecological causes or consequences of rigorously quantified phytochemical diversity across taxa in natural systems. Consequently, hypotheses that attempt to explain variation in phytochemical diversity among plants remain largely untested. We use spectral data from crude plant extracts to characterize phytochemical diversity in a suite of co-occurring plants in the tropical genus Piper (Piperaceae). In combination with 20 years of data focused on Piper-associated insects, we find that phytochemical diversity has a direct and positive effect on the diversity of herbivores but also reduces overall herbivore damage. Elevated chemical diversity is associated with more specialized assemblages of herbivores, and the cascading positive effect of phytochemistry on herbivore enemies is stronger as herbivore diet breadth narrows. These results are consistent with traditional hypotheses that predict positive associations between plant chemical diversity, insect herbivore diversity, and trophic specialization. It is clear from these results that high phytochemical diversity not only enhances the diversity of plant-associated insects but also contributes to the ecological predominance of specialized insect herbivores.

Similarity in volatile communities leads to increased herbivory and greater tropical forest diversity.

A longstanding paradigm in ecology is that there are positive associations between herbivore diversity, specialization, and plant species diversity, with a focus on taxonomic diversity. However, phytochemical diversity is also an informative metric, as insect herbivores interact with host plants not as taxonomic entities, but as sources of nutrients, primary metabolites, and mixtures of attractant and repellant chemicals. The present research examines herbivore responses to phytochemical diversity measured as volatile similarity in the tropical genus Piper. We quantified associations between naturally occurring volatile variation and herbivory by specialist and generalist insects. Intraspecific similarity of volatile compounds across individuals was associated with greater overall herbivory. A structural equation model supported the hypothesis that plot level volatile similarity caused greater herbivory by generalists, but not specialists, which led to increased understory plant richness. These results demonstrate that using volatiles as a functional diversity metric is informative for understanding tropical forest diversity and indicate that generalist herbivores contribute to the maintenance of diversity.

Intraspecific phytochemical variation shapes community and population structure for specialist caterpillars.

Chemically mediated plant-herbivore interactions contribute to the diversity of terrestrial communities and the diversification of plants and insects. While our understanding of the processes affecting community structure and evolutionary diversification has grown, few studies have investigated how trait variation shapes genetic and species diversity simultaneously in a tropical ecosystem. We investigated secondary metabolite variation among subpopulations of a single plant species, Piper kelleyi (Piperaceae), using high-performance liquid chromatography (HPLC), to understand associations between plant phytochemistry and host-specialized caterpillars in the genus Eois (Geometridae: Larentiinae) and associated parasitoid wasps and flies. In addition, we used a genotyping-by-sequencing approach to examine the genetic structure of one abundant caterpillar species, Eois encina, in relation to host phytochemical variation. We found substantive concentration differences among three major secondary metabolites, and these differences in chemistry predicted caterpillar and parasitoid community structure among host plant populations. Furthermore, E. encina populations located at high elevations were genetically different from other populations. They fed on plants containing high concentrations of prenylated benzoic acid. Thus, phytochemistry potentially shapes caterpillar and wasp community composition and geographic variation in species interactions, both of which can contribute to diversification of plants and insects.

Dearomative Indole Cycloaddition Reactions of Aza-Oxyallyl Cationic Intermediates: Modular Access to Pyrroloindolines.

A regioselective dearomative aza-(3 + 2) cycloaddition reaction of substituted indoles with α-halohydroxamates has been developed. This transformation provides rapid access to highly functionalized pyrroloindolines that are represented in large number of bioactive compounds. The natural product, physostigmine, has been concisely synthesized utilizing this method.

exo-Brevicomin biosynthesis in the fat body of the mountain pine beetle, Dendroctonus ponderosae.

exo-Brevicomin (exo-7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octane) is an important semiochemical for a number of beetle species, including the highly destructive mountain pine beetle, Dendroctonus ponderosae. It also has been found in other insects and even in the African elephant. Despite its significance, little is known about its biosynthesis. In order to fill this gap and to identify new molecular targets for potential pest management methods, we performed gas chromatography-mass spectrometry analyses of cell cultures and in vitro assays of various D. ponderosae tissues with exo-brevicomin intermediates, analogs, and inhibitors. We confirmed that exo-brevicomin was synthesized by "unfed" males after emerging from the brood tree. Furthermore, in contrast to the paradigm established for biosynthesis of monoterpenoid pheromone components in bark beetles, exo-brevicomin was produced in the fat body, and not in the anterior midgut. The first committed step involves decarboxylation or decarbonylation of ω-3-decenoic acid, which is derived from a longer-chain precursor via ß-oxidation, to (Z)-6-nonen-2-ol. This secondary alcohol is converted to the known precursor, (Z)-6-nonen-2-one, and further epoxidized by a cytochrome P450 to 6,7-epoxynonan-2-one. The keto-epoxide is stable at physiological pH, suggesting that its final cyclization to form exo-brevicomin is enzyme-catalyzed. exo-Brevicomin production is unusual in that tissue not derived from ectoderm apparently is involved.

Antiherbivore prenylated benzoic acid derivatives from Piper kelleyi.

The known prenylated benzoic acid derivative 3-geranyl-4-hydroxy-5-(3″,3″-dimethylallyl)benzoic acid (1) and two new chromane natural products were isolated from the methanolic extract of the leaves of Piper kelleyi Tepe (Piperaceae), a midcanopy tropical shrub that grows in lower montane rain forests in Ecuador and Peru. Structure determination using 1D and 2D NMR analysis led to the structure of the chromene 2 and to the reassignment of the structure of cumanensic acid as 4, an isomeric chromene previously isolated from Piper gaudichaudianum. The structure and relative configuration of new chromane 3 was determined using 1D and 2D NMR spectroscopic analysis and was found to be racemic by ECD spectropolarimetry. The biological activity of 1-3 was evaluated against a lab colony of the generalist caterpillar Spodoptera exigua (Noctuidae), and low concentrations of 2 and 3 were found to significantly reduce fitness. Further consideration of the biosynthetic relationship of the three compounds led to the proposal that 1 is converted to 2 via an oxidative process, whereas 3 is produced through hetero-[4+2] dimerization of a quinone methide derived from the chromene 2.

An assessment of chemical contaminants in sediments from the St. Thomas East End Reserves, St. Thomas, USVI.

The St. Thomas East End Reserves or STEER is located on the southeastern end of the island of St. Thomas, USVI. The STEER contains extensive mangroves and seagrass beds, along with coral reefs, lagoons, and cays. Within the watershed, however, are a large active landfill, numerous marinas, resorts, various commercial activities, an EPA Superfund Site, and residential areas, all of which have the potential to contribute pollutants to the STEER. As part of a project to develop an integrated assessment for the STEER, 185 chemical contaminants were analyzed in sediments from 24 sites. Higher levels of chemical contaminants were found in Mangrove Lagoon and Benner Bay in the western portion of the study area. The concentrations of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT), zinc, copper, lead, and mercury were above a NOAA Effects Range-Low (ERL) sediment quality guideline at one or more sites, indicating impacts may be present in more sensitive species or life stages. Copper at one site in Benner Bay was above a NOAA Effects Range-Median (ERM) guideline indicating effects on benthic organisms were likely. The antifoulant boat hull ingredient tributyltin (TBT) was found at the third highest concentration in the history of NOAA's National Status and Trends (NS&T) Program, which monitors the nation's coastal and estuarine waters for chemical contaminants and bioeffects. The results from this project will provide resource managers with key information needed to make effective decisions affecting coral reef ecosystem health and gauge the efficacy of restoration activities.

Bacterial dealkylation of benzalkonium chlorides in wastewater produces benzyldimethylamine, a potent N-nitrosodimethylamine precursor.

N-nitrosodimethylamine (NDMA) is a carcinogenic disinfection byproduct that forms during chloramine disinfection of municipal wastewater effluents which are increasingly used to augment drinking water supplies due to growing water scarcity. Knowledge of wastewater NDMA precursors is limited and the known pool of NDMA precursors has not closed the mass balance between precursor loading, precursor NDMA yield, and formed NDMA. Benzalkonium chlorides (BACs) are the most prevalent quaternary ammonium surfactants and have antimicrobial properties. The extensive utilization of BACs in household, commercial and industrial products has resulted in their detection in wastewater at elevated concentrations. We report the formation of a potent NDMA precursor, benzyldimethylamine (BDMA) from the biodegradation of BACs during activated sludge treatment. BDMA formation and NDMA formation potential (FP) were functions of BAC and mixed liquor suspended solids concentration at circumneutral pH, and the microbial community source. Sustained exposure to microorganisms reduced NDMA FP through successive dealkylation of BDMA to less potent precursors. BAC alkyl chain length (C8 - C16) had little impact on NDMA FP and BDMA formation because chain cleavage occurred at the C-N bond. Wastewater effluents collected from three facilities contained BDMA from 15 to 106 ng/L, accounting for an estimated 4 to 38 % of the NDMA precursor pool.

Discovery of octopamine and tyramine in nectar and their effects on bumblebee behavior.

Nectar chemistry can influence the behavior of pollinators in ways that affect pollen transfer, yet basic questions about how nectar chemical diversity impacts plant-pollinator relationships remain unexplored. For example, plants' capacity to produce neurotransmitters and endocrine disruptors may offer a means to manipulate pollinator behavior. We surveyed 15 plant species and discovered that two insect neurotransmitters, octopamine and tyramine, were widely distributed in floral nectar. We detected the highest concentration of these chemicals in Citrus, alongside the well-studied alkaloid caffeine. We explored the separate and interactive effects of these chemicals on insect pollinators in a series of behavioral experiments on bumblebees (Bombus impatiens). We found that octopamine and tyramine interacted with caffeine to alter key aspects of bee behavior relevant to plant fitness (sucrose responsiveness, long-term memory, and floral preferences). These results provide evidence for a means by which synergistic or antagonistic nectar chemistry might influence pollinators.

The chemical ecology of tropical forest diversity: Environmental variation, chemical similarity, herbivory, and richness.

Species richness in tropical forests is correlated with other dimensions of diversity, including the diversity of plant-herbivore interactions and the phytochemical diversity that influences those interactions. Understanding the complexity of plant chemistry and the importance of phytochemical diversity for plant-insect interactions and overall forest richness has been enhanced significantly by the application of metabolomics to natural systems. The present work used proton nuclear magnetic resonance spectroscopy (1 H-NMR) profiling of crude leaf extracts to study phytochemical similarity and diversity among Piper plants growing naturally in the Atlantic Rainforest of Brazil. Spectral profile similarity and chemical diversity were quantified to examine the relationship between metrics of phytochemical diversity, specialist and generalist herbivory, and understory plant richness. Herbivory increased with understory species richness, while generalist herbivory increased and specialist herbivory decreased with the diversity of Piper leaf material available. Specialist herbivory increased when conspecific host plants were more spectroscopically dissimilar. Spectral similarity was lower among individuals of common species, and they were also more spectrally diverse, indicating phytochemical diversity is beneficial to plants. Canopy openness and soil nutrients also influenced chemistry and herbivory. The complex relationships uncovered in this study add information to our growing understanding of the importance of phytochemical diversity for plant-insect interactions and tropical plant species richness.

Generation and reactivity of aza-oxyallyl cationic intermediates: aza-[4 + 3] cycloaddition reactions for heterocycle synthesis.

Aza-[4 + 3] cycloadditions of putative aza-oxyallyl cationic intermediates and cyclic dienes are reported. The intermediate is generated by the dehydrohalogenation of α-haloamides. The reaction is general to a variety of α-haloamides and is diastereoselective. Computational and experimental data suggest that an N-alkoxy substituent stabilizes the aza-oxyallyl cationic intermediate.

Phytochemistry reflects different evolutionary history in traditional classes versus specialized structural motifs.

Foundational hypotheses addressing plant-insect codiversification and plant defense theory typically assume a macroevolutionary pattern whereby closely related plants have similar chemical profiles. However, numerous studies have documented variation in the degree of phytochemical trait lability, raising the possibility that phytochemical evolution is more nuanced than initially assumed. We utilize proton nuclear magnetic resonance (1H NMR) data, chemical classification, and double digest restriction-site associated DNA sequencing (ddRADseq) to resolve evolutionary relationships and characterize the evolution of secondary chemistry in the Neotropical plant clade Radula (Piper; Piperaceae). Sequencing data substantially improved phylogenetic resolution relative to past studies, and spectroscopic characterization revealed the presence of 35 metabolite classes. Metabolite classes displayed phylogenetic signal, whereas the crude 1H NMR spectra featured little evidence of phylogenetic signal in multivariate tests of chemical resonances. Evolutionary correlations were detected in two pairs of compound classes (flavonoids with chalcones; p-alkenyl phenols with kavalactones), where the gain or loss of a class was dependent on the other's state. Overall, the evolution of secondary chemistry in Radula is characterized by strong phylogenetic signal of traditional compound classes and weak phylogenetic signal of specialized chemical motifs, consistent with both classic evolutionary hypotheses and recent examinations of phytochemical evolution in young lineages.

Secondary metabolites in a neotropical shrub: spatiotemporal allocation and role in fruit defense and dispersal.

Deciphering the ecological roles of plant secondary metabolites requires integrative studies that assess both the allocation patterns of compounds and their bioactivity in ecological interactions. Secondary metabolites have been primarily studied in leaves, but many are unique to fruits and can have numerous potential roles in interactions with both mutualists (seed dispersers) and antagonists (pathogens and predators). We described 10 alkenylphenol compounds from the plant species Piper sancti-felicis (Piperaceae), quantified their patterns of intraplant allocation across tissues and fruit development, and examined their ecological role in fruit interactions. We found that unripe and ripe fruit pulp had the highest concentrations and diversity of alkenylphenols, followed by flowers; leaves and seeds had only a few compounds at detectable concentrations. We observed a nonlinear pattern of alkenylphenol allocation across fruit development, increasing as flowers developed into unripe pulp then decreasing as pulp ripened. This pattern is consistent with the hypothesis that alkenylphenols function to defend fruits from pre-dispersal antagonists and are allocated based on the contribution of the tissue to the plant's fitness, but could also be explained by non-adaptive constraints. To assess the impacts of alkenylphenols in interactions with antagonists and mutualists, we performed fungal bioassays, field observations, and vertebrate feeding experiments. In fungal bioassays, we found that alkenylphenols had a negative effect on the growth of most fungal taxa. In field observations, nocturnal dispersers (bats) removed the majority of infructescences, and diurnal dispersers (birds) removed a larger proportion of unripe infructescences. In feeding experiments, bats exhibited an aversion to alkenylphenols, but birds did not. This observed behavior in bats, combined with our results showing a decrease in alkenylphenols during ripening, suggests that alkenylphenols in fruits represent a trade-off (defending against pathogens but reducing disperser preference). These results provide insight into the ecological significance of a little studied class of secondary metabolites in seed dispersal and fruit defense. More generally, documenting intraplant spatiotemporal allocation patterns in angiosperms and examining mechanisms behind these patterns with ecological experiments is likely to further our understanding of the evolutionary ecology of plant chemical traits.

Development of a reef fish biological condition gradient model with quantitative decision rules for the protection and restoration of coral reef ecosystems.

Coral reef ecosystems are declining due to multiple interacting stressors. A bioassessment framework focused on stressor-response associations was developed to help organize and communicate complex ecological information to support coral reef conservation. This study applied the Biological Condition Gradient (BCG), initially developed for freshwater ecosystems, to fish assemblages of U.S. Caribbean coral reef ecosystems. The reef fish BCG describes how biological conditions changed incrementally along a gradient of increasing anthropogenic stress. Coupled with physical and chemical water quality data, the BGC forms a scientifically defensible basis to prioritize, protect and restore water bodies containing coral reefs. Through an iterative process, scientists from across the U.S. Caribbean used fishery-independent survey data and expert knowledge to develop quantitative decision rules to describe six levels of coral reef ecosystem condition. The resultant reef fish BCG provides an effective tool for identifying healthy and degraded coral reef ecosystems and has potential for global application.

Incidence of marine debris and its relationships with benthic features in Gray's Reef National Marine Sanctuary, Southeast USA.

Gray's Reef National Marine Sanctuary (GRNMS) is an increasingly popular site for recreational fishing and diving in the South Atlantic Bight (SAB). As a result, there has been heightened concern about potential accumulation of marine debris and its consequent effects on sanctuary resources. Field surveys were conducted at GRNMS in 2004 and 2005 to provide a spatially comprehensive characterization of benthic communities and to quantify the distribution and abundance of marine debris in relation to bottom features. The spatial distribution of debris was concentrated in the center of the sanctuary and was most frequently associated with ledges rather than other bottom types. On ledges, the presence and abundance of debris was significantly related to observed boating activity and physiographic features including ledge height, ledge area, and percent cover of benthic organisms. The results from this study will aid managers in optimizing cleanup efforts and long-term monitoring of debris accumulation patterns at GRNMS and other hard bottom areas in the SAB.