Function and stability of mesophotic coral reefs.

The function and stability of mesophotic coral ecosystems (MCEs) have been extensively studied in recent years. These deep reefs are characterized by local physical processes, particularly the steep gradient in irradiance with increasing depth, and their impact on trophic resources. Mesophotic reefs exhibit distinct zonation patterns that segregate shallow reef biodiversity from ecologically unique deeper communities of endemic species. While mesophotic reefs are hypothesized as relatively stable refuges from anthropogenic stressors and a potential seed bank for degraded shallow reefs, these are site-specific features, if they occur at all. Mesophotic reefs are now known to be susceptible to many of the same stressors that are degrading shallow reefs, suggesting that they require their own specific conservation and management strategies.

Cophylogeny and convergence shape holobiont evolution in sponge-microbe symbioses.

Symbiotic microbial communities of sponges serve critical functions that have shaped the evolution of reef ecosystems since their origins. Symbiont abundance varies tremendously among sponges, with many species classified as either low microbial abundance (LMA) or high microbial abundance (HMA), but the evolutionary dynamics of these symbiotic states remain unknown. This study examines the LMA/HMA dichotomy across an exhaustive sampling of Caribbean sponge biodiversity and predicts that the LMA symbiotic state is the ancestral state among sponges. Conversely, HMA symbioses, consisting of more specialized microorganisms, have evolved multiple times by recruiting similar assemblages, mostly since the rise of scleractinian-dominated reefs. Additionally, HMA symbioses show stronger signals of phylosymbiosis and cophylogeny, consistent with stronger co-evolutionary interaction in these complex holobionts. These results indicate that HMA holobionts are characterized by increased endemism, metabolic dependence and chemical defences. The selective forces driving these patterns may include the concurrent increase in dissolved organic matter in reef ecosystems or the diversification of spongivorous fishes.

Microbiome diversity and metabolic capacity determines the trophic ecology of the holobiont in Caribbean sponges.

Sponges are increasingly recognized as an ecologically important taxon on coral reefs, representing significant biomass and biodiversity where sponges have replaced scleractinian corals. Most sponge species can be divided into two symbiotic states based on symbiont community structure and abundance (i.e., the microbiome), and are characterized as high microbial abundance (HMA) or low microbial abundance (LMA) sponges. Across the Caribbean, sponge species of the HMA or LMA symbiotic states differ in metabolic capacity, as well as their trophic ecology. A metagenetic analysis of symbiont 16 S rRNA and metagenomes showed that HMA sponge microbiomes are more functionally diverse than LMA microbiomes, offer greater metabolic functional capacity and redundancy, and encode for the biosynthesis of secondary metabolites. Stable isotope analyses showed that HMA and LMA sponges primarily consume dissolved organic matter (DOM) derived from external autotrophic sources, or live particulate organic matter (POM) in the form of bacterioplankton, respectively, resulting in a low degree of resource competition between these symbiont states. As many coral reefs have undergone phase shifts from coral- to macroalgal-dominated reefs, the role of DOM, and the potential for future declines in POM due to decreased picoplankton productivity, may result in an increased abundance of chemically defended HMA sponges on tropical coral reefs.

Incident light and morphology determine coral productivity along a shallow to mesophotic depth gradient.

While the effects of irradiance on coral productivity are well known, corals along a shallow to mesophotic depth gradient (10-100 m) experience incident irradiances determined by the optical properties of the water column, coral morphology, and reef topography.Modeling of productivity (i.e., carbon fixation) using empirical data shows that hemispherical colonies photosynthetically fix significantly greater amounts of carbon across all depths, and throughout the day, compared with plating and branching morphologies. In addition, topography (i.e., substrate angle) further influences the rate of productivity of corals but does not change the hierarchy of coral morphologies relative to productivity.The differences in primary productivity for different coral morphologies are not, however, entirely consistent with the known ecological distributions of these coral morphotypes in the mesophotic zone as plating corals often become the dominant morphotype with increasing depth.Other colony-specific features such as skeletal scattering of light, Symbiodiniaceae species, package effect, or tissue thickness contribute to the variability in the ecological distributions of morphotypes over the depth gradient and are captured in the metric known as the minimum quantum requirements.Coral morphology is a strong proximate cause for the observed differences in productivity, with secondary effects of reef topography on incident irradiances, and subsequently the community structure of mesophotic corals.

Combined and independent effects of hypoxia and tributyltin on mRNA expression and physiology of the Eastern oyster (Crassostrea virginica).

Oyster reefs are vital to estuarine health, but they experience multiple stressors and globally declining populations. This study examined effects of hypoxia and tributyltin (TBT) on adult Eastern oysters (Crassostrea virginica) exposed either in the laboratory or the field following a natural hypoxic event. In the laboratory, oysters were exposed to either hypoxia followed by a recovery period, or to hypoxia combined with TBT. mRNA expression of HIF1-α and Tß-4 along with hemocyte counts, biomarkers of hypoxic stress and immune health, respectively, were measured. In field-deployed oysters, HIF1-α and Tß-4 expression increased, while no effect on hemocytes was observed. In contrast, after 6 and 8 days of laboratory-based hypoxia exposure, both Tß-4 expression and hemocyte counts declined. After 8 days of exposure to hypoxia + TBT, oysters substantially up-regulated HIF1-α and down-regulated Tß-4, although hemocyte counts were unaffected. Results suggest that hypoxic exposure induces immunosuppression which could increase vulnerability to pathogens.

Will coral reef sponges be winners in the Anthropocene?

Recent observations have shown that increases in climate change-related coral mortality cause changes in shallow coral reef community structure through phase shifts to alternative taxa. As a result, sponges have emerged as a potential candidate taxon to become a "winner," and therefore a numerically and functionally dominant member of many coral reef communities. But, in order for this to occur, there must be sufficient trophic resources to support larger populations of these active filter-feeding organisms. Globally, climate change is causing an increase in sea surface temperatures (SSTs) and a decrease in salinity, which can lead to an intensification in the stratification of shallow nearshore waters (0-200 m), that affects both the mixed layer depth (MLD) and the strength and duration of internal waves. Specifically, climate change-driven increases in SSTs for tropical waters are predicted to cause increased stratification, and more stabilized surface waters. This causes a shallowing of the MLD which prevents nutrients from reaching the euphotic zone, and is predicted to decrease net primary production (NPP) up to 20% by the end of the century. Lower NPP would subsequently affect multiple trophic levels, including shallow benthic filter-feeding communities, as the coupling between water column productivity and the benthos weakens. We argue here that sponge populations may actually be constrained, rather than promoted, by climate change due to decreases in their primary trophic resources, caused by bottom-up forcing, secondary to physical changes in the water column (i.e., stratification and changes in the MLD resulting in lower nutrients and NPP). As a result, we predict sponge-dominated tropical reefs will be rare, or short-lived, if they occur at all into the future in the Anthropocene.

Annual Thermal Stress Increases a Soft Coral's Susceptibility to Bleaching.

Like scleractinian corals, soft corals contain photosymbionts (Family Symbiodiniaceae) that provide energy for the host. Recent thermal events have resulted in soft coral bleaching in four of five years on Guam, where they dominated back-reef communities. Soft coral bleaching was examined in Sinularia maxima, S. polydactyla, and their hybrid S. maxima x polydactyla. Results from annual field surveys indicated that S. maxima and the hybrid were more susceptible to bleaching than S. polydactyla, and this was related to differences in their Symbiodiniaceae communities in 2016 and 2017. The photosymbionts of S. polydactyla were apparently more stress tolerant and maintained higher photosynthetic potential through three years of bleaching, in contrast to the other species that exhibited a decline in photosynthetic potential after the first year of bleaching. Nonetheless, by the 2017 bleaching event all soft coral populations exhibited significant bleaching-mediated declines and loss of photosynthetic efficiency suggesting a declining resiliency to annual thermal stress events. While S. polydactyla initially looked to succeed the other species as the dominant space occupying soft coral on Guam back-reefs, cumulative bleaching events ultimately turned this "winner" into a "loser", suggesting the trajectory for coral reefs is towards continued loss of structure and function.

Allelopathy-mediated competition in microbial mats from Antarctic lakes.

Microbial mats are vertically stratified communities that host a complex consortium of microorganisms, dominated by cyanobacteria, which compete for available nutrients and environmental niches, within these extreme habitats. The Antarctic Dry Valleys near McMurdo Sound include a series of lakes within the drainage basin that are bisected by glacial traverses. These lakes are traditionally independent, but recent increases in glacial melting have allowed two lakes (Chad and Hoare) to become connected by a meltwater stream. Microbial mats were collected from these lakes, and cultured under identical conditions at the McMurdo Station laboratory. Replicate pairings of the microbial mats exhibited consistent patterns of growth inhibition indicative of competitive dominance. Natural products were extracted from the microbial mats, and a disk diffusion assay was utilized to show that allelochemical compounds mediate competitive interactions. Both microscopy and 16S rRNA sequencing show that these mats contain significant populations of cyanobacteria known to produce allelochemicals. Two compounds were isolated from these microbial mats that might be important in the chemical ecology of these psychrophiles. In other disk:mat pairings, including extract versus mat of origin, the allelochemicals exhibited no effect. Taken together, these results indicate that Antarctic lake microbial mats can compete via allelopathy.

Biochemical Mechanisms for Geographical Adaptations to Novel Toxin Exposures in Butterflyfish.

Some species of butterflyfish have had preyed upon corals for millions of years, yet the mechanism of butterflyfish specialized coral feeding strategy remains poorly understood. Certain butterflyfish have the ability to feed on allelochemically rich soft corals, e.g. Sinularia maxima. Cytochrome P450 (CYP) is the predominant enzyme system responsible for the detoxification of dietary allelochemicals. CYP2-like and CYP3A-like content have been associated with butterflyfish that preferentially consumes allelochemically rich soft corals. To investigate the role of butterflyfish CYP2 and CYP3A enzymes in dietary preference, we conducted oral feeding experiments using homogenates of S. maxima and a toxin isolated from the coral in four species of butterflyfish with different feeding strategies. After oral exposure to the S. maxima toxin 5-episinulaptolide (5ESL), which is not normally encountered in the Hawaiian butterflyfish diet, an endemic specialist, Chaetodon multicinctus experienced 100% mortality compared to a generalist, Chaetodon auriga, which had significantly more (3-6 fold higher) CYP3A-like basal content and catalytic activity. The specialist, Chaetodon unimaculatus, which preferentially feed on S. maxima in Guam, but not in Hawaii, had 100% survival, a significant induction of 8-12 fold CYP3A-like content, and an increased ability (2-fold) to metabolize 5ESL over other species. Computer modeling data of CYP3A4 with 5ESL were consistent with microsomal transformation of 5ESL to a C15-16 epoxide from livers of C. unimaculatus. Epoxide formation correlated with CYP3A-like content, catalytic activity, induction, and NADPH-dependent metabolism of 5ESL. These results suggest a potentially important role for the CYP3A family in butterflyfish-coral diet selection through allelochemical detoxification.

Hard coral (Porites lobata) extracts and homarine on cytochrome P450 expression in Hawaiian butterflyfishes with different feeding strategies.

Dietary specialists tend to be less susceptible to the effects of chemical defenses produced by their prey compared to generalist predators that feed upon a broader range of prey species. While many researchers have investigated the ability of insects to detoxify dietary allelochemicals, little research has been conducted in marine ecosystems. We investigated metabolic detoxification pathways in three species of butterflyfishes: the hard coral specialist feeder, Chaetodon multicinctus, and two generalist feeders, Chaetodon auriga and Chaetodon kleinii. Each species was fed tissue homogenate of the hard coral Porites lobata or the feeding deterrent compound homarine (found in the coral extract), and the expression and catalytic activity of cytochrome P450 (CYP) 3A-like and CYP2-like enzymes were examined after one-week of treatment. The P. lobata homogenate significantly induced content and catalytic activity of CYP2-like and CYP3A-like forms, by 2-3 fold and by 3-9 fold, respectively, in C. multicinctus. Homarine caused a significant decrease of CYP2-like and CYP3A-like proteins at the high dose in C. kleinii and 60-80% mortality in that species. Homarine also induced CYP3A-like content by 3-fold and catalytic activity by 2-fold in C. auriga, while causing non-monotonic increases in CYP2-like and CYP3A-like catalytic activity in C. multicinctus. Our results indicate that dietary exposure to coral homogenates and the feeding deterrent constituent within these homogenates caused species-specific modulation of detoxification enzymes consistent with the prey selection strategies of generalist and specialist butterflyfishes.

Proteomic profiling of healthy and diseased hybrid soft corals Sinularia maxima × S. polydactyla.

Emerging diseases of marine invertebrates have been implicated as one of the major causes of the continuing decline in coral reefs worldwide. To date, most of the focus on marine diseases has been aimed at hard (scleractinian) corals, which are the main reef builders worldwide. However, soft (alcyonacean) corals are also essential components of tropical reefs, representing food, habitat and the 'glue' that consolidates reefs, and they are subject to the same stressors as hard corals. Sinularia maxima and S. polydactyla are the dominant soft corals on the shallow reefs of Guam, where they hybridize. In addition to both parent species, the hybrid soft coral population in Guam is particularly affected by Sinularia tissue loss disease. Using label-free shotgun proteomics, we identified differences in protein expression between healthy and diseased colonies of the hybrid S. maxima × S. polydactyla. This study provided qualitative and quantitative data on specific proteins that were differentially expressed under the stress of disease. In particular, metabolic proteins were down-regulated, whereas proteins related to stress and to symbiont photosynthesis were up-regulated in the diseased soft corals. These results indicate that soft corals are responding to pathogenesis at the level of the proteome, and that this label-free approach can be used to identify and quantify protein biomarkers of sub-lethal stress in studies of marine disease.

Allelopathy in the tropical alga Lobophora variegata (Phaeophyceae): mechanistic basis for a phase shift on mesophotic coral reefs?

Macroalgal phase shifts on Caribbean reefs have been reported with increasing frequency, and recent reports of these changes on mesophotic coral reefs have raised questions regarding the mechanistic processes behind algal population expansions to deeper depths. The brown alga Lobophora variegata is a dominant species on many shallow and deep coral reefs of the Caribbean and Pacific, and it increased in percent cover (>50%) up to 61 m on Bahamian reefs following the invasion of the lionfish Pterois volitans. We examined the physiological and ecological constraints contributing to the spread of Lobophora on Bahamian reefs across a mesophotic depth gradient from 30 to 61 m, pre- and post-lionfish invasion. Results indicate that there were no physiological limitations to the depth distribution of Lobophora within this range prior to the lionfish invasion. Herbivory by acanthurids and scarids in algal recruitment plots at mesophotic depths was higher prior to the lionfish invasion, and Lobophora chemical defenses were ineffective against an omnivorous fish species. In contrast, Lobophora exhibited significant allelopathic activity against the coral Montastraea cavernosa and the sponge Agelas clathrodes in laboratory assays. These data indicate that when lionfish predation on herbivorous fish released Lobophora from grazing pressure at depth, Lobophora expanded its benthic cover to a depth of 61 m, where it replaced the dominant coral and sponge species. Our results suggest that this chemically defended alga may out-compete these species in situ, and that mesophotic reefs may be further impacted in the near future as Lobophora continues to expand to its compensation point.

Ecology of Caribbean sponges: are top-down or bottom-up processes more important?

Benthic-pelagic coupling and the role of bottom-up versus top-down processes are recognized as having a major impact on the structure of marine communities. While the roles of bottom-up processes are better appreciated they are still viewed as principally affecting the outcome of top-down processes. Sponges on coral reefs are important members of the benthic community and provide a critically important functional linkage between water-column productivity and the benthos. As active suspension feeders sponges utilize the abundant autotrophic and heterotrophic picoplankton in the water column. As a result sponges across the Caribbean basin exhibit a consistent and significant pattern of greater biomass, tube extension rate, and species numbers with increasing depth. Likewise, the abundance of their food supply also increases along a depth gradient. Using experimental manipulations it has recently been reported that predation is the primary determinant of sponge community structure. Here we provide data showing that the size and growth of the sponge Callyspongia vaginalis are significantly affected by food availability. Sponges increased in size and tube extension rate with increasing depth down to 46 m, while simultaneously exposed to the full range of potential spongivores at all depths. Additionally, we point out important flaws in the experimental design used to demonstrate the role of predation and suggest that a resolution of this important question will require well-controlled, multi-factorial experiments to examine the independent and interactive effects of predation and food abundance on the ecology of sponges.

Exploring individual- to population-level impacts of disease on coral reef sponges: using spatial analysis to assess the fate, dynamics, and transmission of Aplysina Red Band Syndrome (ARBS).

BACKGROUND: Marine diseases are of increasing concern for coral reef ecosystems, but often their causes, dynamics and impacts are unknown. The current study investigated the epidemiology of Aplysina Red Band Syndrome (ARBS), a disease affecting the Caribbean sponge Aplysina cauliformis, at both the individual and population levels. The fates of marked healthy and ARBS-infected sponges were examined over the course of a year. Population-level impacts and transmission mechanisms of ARBS were investigated by monitoring two populations of A. cauliformis over a three year period using digital photography and diver-collected data, and analyzing these data with GIS techniques of spatial analysis. In this study, three commonly used spatial statistics (Ripley's K, Getis-Ord General G, and Moran's Index) were compared to each other and with direct measurements of individual interactions using join-counts, to determine the ideal method for investigating disease dynamics and transmission mechanisms in this system. During the study period, Hurricane Irene directly impacted these populations, providing an opportunity to assess potential storm effects on A. cauliformis and ARBS. RESULTS: Infection with ARBS caused increased loss of healthy sponge tissue over time and a higher likelihood of individual mortality. Hurricane Irene had a dramatic effect on A. cauliformis populations by greatly reducing sponge biomass on the reef, especially among diseased individuals. Spatial analysis showed that direct contact between A. cauliformis individuals was the likely transmission mechanism for ARBS within a population, evidenced by a significantly higher number of contact-joins between diseased sponges compared to random. Of the spatial statistics compared, the Moran's Index best represented true connections between diseased sponges in the survey area. This study showed that spatial analysis can be a powerful tool for investigating disease dynamics and transmission in a coral reef ecosystem.

Chlorinated didemnins from the tunicate Trididemnum solidum.

Chemical investigation of the tunicate Trididemnum solidum resulted in the isolation of two new chlorinated compounds belonging to the didemnin class, along with two known compounds didemnin A and didemnin B. The structural determination of the compounds was based on extensive NMR and mass spectroscopic analysis. The isolated compounds 1-4 were tested for their anti-inflammatory activity using in vitro assays for inhibition of inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) activity. The anti-cell proliferative activity of the above compounds was also evaluated against four solid tumor cell lines.

Genetic structure in the coral, Montastraea cavernosa: assessing genetic differentiation among and within Mesophotic reefs.

Mesophotic coral reefs (30-150 m) have recently received increased attention as a potential source of larvae (e.g., the refugia hypothesis) to repopulate a select subset of the shallow water (<30 m) coral fauna. To test the refugia hypothesis we used highly polymorphic Amplified Fragment Length Polymorphism (AFLP) markers as a means to assess small-scale genetic heterogeneity between geographic locations and across depth clines in the Caribbean coral, Montastraea cavernosa. Zooxanthellae-free DNA extracts of coral samples (N = 105) were analyzed from four depths, shallow (3-10 m), medium (15-25 m), deep (30-50 m) and very deep (60-90 m) from Little Cayman Island (LCI), Lee Stocking Island (LSI), Bahamas and San Salvador (SS), Bahamas which range in distance from 170 to 1,600 km apart. Using AMOVA analysis there were significant differences in ΦST values in pair wise comparisons between LCI and LSI. Among depths at LCI, there was significant genetic differentiation between shallow and medium versus deep and very deep depths in contrast there were no significant differences in ΦST values among depths at LSI. The assignment program AFLPOP, however, correctly assigned 95.7% of the LCI and LSI samples to the depths from which they were collected, differentiating among populations as little as 10 to 20 m in depth from one another. Discriminant function analysis of the data showed significant differentiation among samples when categorized by collection site as well as collection depth. FST outlier analyses identified 2 loci under positive selection and 3 under balancing selection at LCI. At LSI 2 loci were identified, both showing balancing selection. This data shows that adult populations of M. cavernosa separated by depths of tens of meters exhibits significant genetic structure, indicative of low population connectivity among and within sites and are not supplying successful recruits to adjacent coral reefs less than 30 m in depth.

Marine proteomics: a critical assessment of an emerging technology.

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Antibacterial secondary metabolites from the cave sponge Xestospongia sp.

Chemical investigation of the cave sponge Xestospongia sp. resulted in the isolation of three new polyacetylenic long chain compounds along with two known metabolites. The structures of the new metabolites were established by NMR and MS analyses. The antibacterial activity of the new metabolites was also evaluated.

Spatial variability in secondary metabolites of the indo-pacific sponge Stylissa massa.

Chemical diversity represents a measure of selective pressures acting on genotypic variability. In order to understand patterns of chemical ecology and biodiversity in the environment, it is necessary to enhance our knowledge of chemical diversity within and among species. Many sponges produce variable levels of secondary metabolites in response to diverse biotic and abiotic environmental factors. This study evaluated intra-specific variability in secondary metabolites in the common Indo-Pacific sponge Stylissa massa over various geographic scales, from local to ocean basin. Several major metabolites were quantified in extracts from sponges collected in American Samoa, Pohnpei, Saipan, and at several sites and depths in Guam. Concentrations of several of these metabolites varied geographically across the Pacific basin, with American Samoa and Pohnpei exhibiting the greatest differences, and Guam and Saipan more similar to each other. There were also significant differences in concentrations among different sites and depths within Guam. The crude extract of S. massa exhibited feeding deterrence against the omnivorous pufferfish Canthigaster solandri at natural concentrations, however, none of the isolated compounds was deterrent at the maximum natural concentrations observed, nor were mixtures of these compounds, thus emphasizing the need for bioassay-guided isolation to characterize specific chemical defenses. Antibacterial activity against a panel of ecologically relevant pathogens was minimal. Depth transplants, predator exclusion, and UV protection experiments were performed, but although temporal variability in compound concentrations was observed, there was no evidence that secondary metabolite concentration in S. massa was induced by any of these factors. Although the reasons behind the variability observed in the chemical constituents of S. massa are still in question, all sponges are not created equal from a chemical standpoint, and these studies provide further insights into patterns of chemical diversity within S. massa.

Unique actinomycetes from marine caves and coral reef sediments provide novel PKS and NRPS biosynthetic gene clusters.

In the ever-expanding search for novel bioactive molecules and enzymes, marine actinomycetes have proven to be a productive source. While open reef sediment and sponge-associated actinomycetes have been extensively examined, their marine cave counterparts remain unevaluated. Anchialine cave systems in the Bahamas offered an ideal setting to evaluate the occurrence and variation within sediment-associated actinomycete communities. While in close geographical proximity to open reef environments, these systems provide a specialized environmental niche devoid of light and direct exposure to nutrient input. In the present study, selective isolation techniques and molecular methods were used to test the hypothesis that variable distribution of actinomycetes and secondary metabolite gene clusters occur between open reef and marine cave systems. The results indicated that differences exist within the culturable sediment-associated actinomycete communities between marine caves and open reef systems, with members of the genus Streptomyces dominating cultures from open reef sediments and a more diverse suite of actinomycetes isolated from marine cave sediment samples. Within the cave isolates, members of the proposed genus Solwaraspora were the most represented. Based on PKS- and NRPS-gene-targeted PCR amplification and sequencing, geographic variation in the occurrence of these biosynthetic pathways was also observed. These findings indicate that marine cave systems are a lucrative source in the search for novel secondary metabolite producers with biotechnological applications and that environmental and geographic factors likely affect the occurrence of these biosynthetic pathways.