==== Front Sci Rep Sci Rep Scientific Reports 2045-2322 Nature Publishing Group UK London 77722 10.1038/s41598-020-77722-7 Article Human proximity suppresses fish recruitment by altering mangrove-associated odour cues Brooker Rohan M. rohan.m.brooker@gmail.com 12 Seyfferth Angelia L. 3 Hunter Alesia 3 Sneed Jennifer M. 4 Dixson Danielle L. 2 Hay Mark E. 5 1 grid.1021.20000 0001 0526 7079Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Queenscliff, VIC 3225 Australia 2 grid.33489.350000 0001 0454 4791School of Marine Science and Policy, University of Delaware, Lewes, DE 19958 USA 3 grid.33489.350000 0001 0454 4791Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716 USA 4 grid.452909.30000 0001 0479 0204Smithsonian Marine Station at Fort Pierce, Fort Pierce, FL 34949 USA 5 grid.213917.f0000 0001 2097 4943School of Biological Sciences and Aquatic Chemical Ecology Center, Georgia Institute of Technology, Atlanta, GA 30332-0230 USA 3 12 2020 3 12 2020 2020 10 2109113 5 2020 13 11 2020 © The Author(s) 2020Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Human-driven threats to coastal marine communities could potentially affect chemically mediated behaviours that have evolved to facilitate crucial ecological processes. Chemical cues and their importance remain inadequately understood in marine systems, but cues from coastal vegetation can provide sensory information guiding aquatic animals to key resources or habitats. In the tropics, mangroves are a ubiquitous component of healthy coastal ecosystems, associated with a range of habitats from river mouths to coral reefs. Because mangrove leaf litter is a predictable cue to coastal habitats, chemical information from mangrove leaves could provide a source of settlement cues for coastal fishes, drawing larvae towards shallow benthic habitats or inducing settlement. In choice assays, juvenile fishes from the Caribbean (Belize) and Indo-Pacific (Fiji) were attracted to cues from mangroves leaves and were more attracted to cues from mangroves distant from human settlement. In the field, experimental reefs supplemented with mangrove leaves grown away from humans attracted more fish recruits from a greater diversity of species than reefs supplemented with leaves grown near humans. Together, this suggests that human use of coastal areas alters natural chemical cues, negatively affecting the behavioural responses of larval fishes and potentially suppressing recruitment. Overall, our findings highlight the critical links that exist between marine and terrestrial habitats, and the importance of considering these in the broader conservation and management of coastal ecosystems. Subject terms Behavioural ecologyEnvironmental impacthttp://dx.doi.org/10.13039/100000002National Institutes of Health2U19TW007401-10Hay Mark E. issue-copyright-statement© The Author(s) 2020 ==== Body Introduction Connectivity between emergent coastal vegetation and subtidal marine ecosystems can play a crucial environmental role, mediating productivity, community composition, and ecosystem functioning1–3. However, many coastal habitats, both above and below water, are rapidly changing due to anthropogenic pressure; becoming degraded, fragmented, and less biodiverse4–6, with the effects of these impacts often unclear7,8. Thus, identifying the ecological links between associated ecosystems is essential for effective conservation and resource management9,10. While human-driven changes to coastal landscapes are often clearly apparent, for instance the conversion of wild areas to agriculture or urban environments, the consequences of these actions for adjacent marine systems is generally less obvious. However, even small changes to land use can alter coastal marine environments; for instance, pollutants and nutrients in runoff that alter marine chemistry can affect the behaviour of marine animals11–13. Because ecological processes that can enhance or decrease coastal resilience are often behaviourally driven and mediated by chemosensory cues14,15, subtle changes to the chemosensory environment could appreciably affect ecosystem function and dynamics. A diverse array of aquatic taxa has evolved acute chemosensory systems, with waterborne chemicals providing a rich source of environmental information15. These chemical cues can mediate a range of important behavioural processes, informing foraging patterns16, navigation17, predator–prey dynamics18, habitat selection14, and interspecies interactions19. For species with an initial planktonic life stage, such as most fishes and marine invertebrates, chemoreception can play an especially important role, helping to orient larvae and juveniles towards key habitats and facilitating recruitment processes20. Because chemical signals influence critical ecological processes, it is crucial that we know cue sources and consequences. Information cues often have a marine origin, such as the odour of corals, seaweeds, or conspecifics14,21. However, due to the intimate relationship between coastal plant communities and marine habitats, some marine species respond to cues from emergent, or even non-aquatic coastal vegetation22,23. How such cues may be affected by coastal development is inadequately understood. Because coastal plant communities are being lost, or altered, at alarming rates24–26, and produce chemical cues that are critical for coastal marine species and communities, understanding the sources of these cues and processes affected by them is both timely and crucial. Along tropical and subtropical coastlines, mangrove forests comprise one of the most ubiquitous plant communities. These salt-tolerant plants represent globally important ecosystems, providing habitat for communities of terrestrial, estuarine, and marine organisms27, including the juvenile stages of aquatic species that migrate elsewhere as adults, such as to nearby coral reefs28. In addition to facilitating biodiversity, mangroves sequester carbon, provide coastal protection, build land, accumulate and assimilate pollutants, and stabilize water conditions29. Several aspects of mangrove ecology suggest they could provide recruitment cues for coastal fishes, including fish species associated with fringing and lagoonal coral reefs. The worldwide distribution of tropical mangroves largely overlaps that of reef building scleractinian corals30, with mangroves growing in oligotrophic areas with limited freshwater input often occurring alongside fringing coral reefs and associated habitats28,31–33. In addition, numerous ‘coral reef fishes’ are in fact, multi-habitat species, with the juveniles and adults of many also associated with mangroves34. Finally, while mangroves hold leaves year-round, they continuously drop some leaves with rates of litter fall often peaking in warmer, or wetter months35,36, overlapping with periods of high recruitment by reef fishes37,38. Thus, standing plants could produce cues to mark coastal systems, degraded leaves that sink could provide similar cues, and leaves drifting from shore could provide a trail of cues leading back to the shallow, structurally complex benthic environments essential for post-settlement survival of coastal fishes. If this is the case, variations in the chemical composition of mangroves or their decomposing litter could alter the sensory information subsequently released. Globally, mangrove ecosystems are at high risk, reducing in area at rates equal to, or greater than, coral reefs and rainforests25,39. Documented mangrove losses over the last quarter of the twentieth century were consistently between 35–86%, with stands becoming smaller, more fragmented, and less biodiverse due to the combined effects of agriculture, aquaculture, tourism, urban development and overexploitation25,40,41. While recent analysis suggests that rates of loss have substantially slowed in the twenty-first century42,43, the future of mangrove ecosystems remains uncertain. Pollution is also a major cause of mangrove deterioration, with many of the chemicals present in sewage, runoff, and other direct inputs (e.g. nitrates, phosphates, and heavy metals) reducing mangrove growth and condition44,45. As mangroves naturally accumulate compounds from their environments, many pollutants are incorporated into the plant’s tissues46. These then have the potential to be subsequently remobilized when those tissues degrade, with greater bioavailability than those held in sediments47. The presence of pollutants will also alter the microbial community associated with the water column, sediment, and plant material44,48, which could further impact litter decomposition and associated chemical cues. Given that environmental chemicals can influence the behaviour of marine larvae and that anthropogenic impacts to mangroves are likely altering marine chemistry, it is critical to determine if and how mangroves mediate juvenile recruitment, and if this role is compromised due to anthropogenic activities. To this end we examined, (1) if juvenile fishes are attracted to the odour of mangrove leaf litter, (2) how this varied among different leaf types, (3) if juvenile fishes distinguished between litter collected near versus remote from human settlements, and (4) if these behavioural responses reflected patterns of recruitment under field conditions. Results Do chemical cues from mangroves attract reef fishes? To assess whether different mangrove odours influenced the behaviour of juvenile reef fishes, and whether response patterns were generalizable between species or geographic locations, we conducted a series of paired-choice experiments in a two-channel choice flume. These experiments were conducted in both Fiji (South Pacific Ocean) and Belize (Western Atlantic Ocean), with two common reef-associated fish species used per location; Chromis viridis and Dascyllus reticulatus in Fiji and Thalassoma bifasciatum and Stegastes partitus in Belize. All four fishes exhibited similar responses to the odour of mangrove leaves (Fig. 1a–d). All four species preferred the odour of young leaves over blank seawater, no species distinguished between the odour of young green and old yellow leaves still on the tree, and all four preferred the odour of senescent, submerged leaves to young leaves collected from the tree.Figure 1 Results of paired-choice trials in Belize and Fiji testing the responses of two fish species in each region to the odour of mangroves leaves at different stages of growth and decay. Belizean species were (a) Thalassoma bifasciatum, (b) Stegastes partitus. Fijian species were (c) Chromis viridis, and (d) Dascyllus reticulatus. Three comparisons were conducted; either (i) young leaves vs. blank sea water, (ii) young leaves vs. old leaves from the plants, or (iii) young leaves vs. decaying leaves from beneath the plants. Boxplots show median values (horizontal lines), interquartile range (boxes), and minimum and maximum values (whiskers). p values are displayed below each comparison, calculated using either a paired-sample t-test or paired-sample Wilcoxon test. n = number of fish per comparison. When presented with senescent mangrove leaves from a site with human development versus senescent leaves from a site with limited human development both Belizean species preferred the odour of those from the undeveloped location (Fig. 2a, b). This preference was also seen towards water collected from each site. Interestingly, when leaves from both sites were treated to remove the associated microbial film the one labrid tested, T. bifasciatum, ceased to distinguish between odours; however, whether this is due to differences in the microbiome or is related to the treatment itself is not clear Similarly, the one Fijian species tested, D. reticulatus, preferred water from undeveloped locations in two separate comparisons; Suva (developed) versus Nukulau Island (undeveloped), and Korovou (developed) versus Namuka (undeveloped). This preference was extended to the odour of senescent leaves from Namuka versus those from Korovou, but not for leaves from Nukulau versus those from Suva (Fig. 2c).Figure 2 Results of paired-choice trials in Belize and Fiji testing the responses of fish species in each region to the odour of mangroves leaves or water collected from sites with human development vs limited human development. Belizean species were (a) Thalassoma bifasciatum, (b) Stegastes partitus. The sole Fijian species was Dascyllus reticulatus (c). For both Belizean species three comparisons were conducted; either (1) decaying leaves from an undeveloped site (Twin Cays) vs. decaying leaves from a developed site (South Water Cay), (2) decaying leaves from each site treated with NaOCl, and (3) water from each site. For D. reticulatus, Comparison A tested responses to either decaying leaves or water from an undeveloped site (Nukulau Island) or developed site (Suva). Comparison B tested responses to either decaying leaves or water from a second undeveloped site (Namuka) or developed site (Korovou). Boxplots and statistical procedures are as in Fig. 1. Do mangrove chemical cues influence settlement site selection? To test whether the odour of decaying mangrove leaves from sites near human development vs those from sites with limited human development influenced natural patterns of fish settlement we conducted an experiment in Belize using constructed patch reefs that contained either NaOCl-treated or unmodified leaves from the developed or undeveloped location along with a control containing no leaves. NaOCl treatment reduced the microbial load on the leaves. Nine fish species settled onto the experimental patch reef sets: Canthigaster rostrata, Gymnothorax moringa, Halichoeres bivittatus, Pomacanthus arcuatus, a Scarus sp., Stegastes adustus, Stegastes leucostictus, S. partitus, and T. bifasciatum. However, T. bifasciatum was by far the most common settler accounting for 88.6% of fish recorded (101 fish out of 114). Treatment had a significant effect on settlement (Fig. 3), with both total settlement and the diversity of settlers significantly higher on patch reefs containing unmodified undeveloped leaves than those containing unmodified leaves from the developed location. Total settlement was also significantly higher on both the control reefs and those containing treated leaves from the undeveloped site than to reefs containing the unmodified leaves from developed sites. There was no difference in either variable between the treated leaves from the undeveloped and developed site. However, as in the paired choice experiment results above, it is possible the treatment itself had an effect.Figure 3 Results of a patch reef experiment testing whether the odour of mangrove leaves collected near human development (from South Water Cay) vs those from near limited human development (Twin Cays) influenced natural patterns of fish settlement in Belize. Patch reef treatments were: empty control (EC), leaves from the undeveloped site (LU), treated leaves from the undeveloped site (TLU), leaves from the developed site (LD), and treated leaves from the developed site (TLD). Values displayed are for total settlement or species number residualised with respect to even distribution within the replicate (n = 18 per treatment). Letters indicate significant differences as determined by post hoc multiple comparisons (p < 0.05; see Supplementary Tables S2, S3). Boxplots are as in Fig. 1. Discussion Mangrove-associated chemical cues influenced the behaviour of multiple species of settlement-stage fishes in both the Caribbean and tropical Pacific with our findings suggesting that these cues are diminished or reversed by mangrove proximity to humans. This was true for mangroves growing adjacent to a large city (Suva, Fiji with a population of ~ 170,000), but also for those growing on South Water Cay, Belize (an island of ~ 0.06 km2 comprised primarily of tourist resorts). This suggests that even sparse human populations on isolated islands may suppress the effectiveness of chemical cues that fishes use to find and recruit to appropriate coastal habitats. Thus, humans not only remove adult fish and brood-stock by fishing but may also be indirectly suppressing the ability of local fish populations to recover via interference with the chemical cues that organisms use to identify suitable habitat choices during recruitment. For coastal fishes, an ability to identify and orientate towards mangroves would be invaluable as mangrove roots, detritus, and associated benthic communities create structural complexity and habitat diversity where this may otherwise be limited, offering shelter from predators, increasing the abundance of food resources, and reducing competition. As the distribution of mangrove stands often overlaps that of corals and other sessile invertebrates within oligotrophic environments, the innate ability to recognize mangrove cues would likely prove valuable for fishes that associate with a range of shallow habitats, but especially those species that recruit to mangroves as juveniles and move to reefs as they mature28,34,49. Our choice experiments demonstrated that fish species associated with both reefs and rubble were attracted to mangrove odours, likely due to the close relationship between these habitats and mangroves at the study sites. Fishes were not simply responding to novel chemical cues; all four species distinguished between leaves at different stages of growth and decay, while two of the three tested distinguished between those collected near versus far from humans. Thus, the attraction for mangrove odour and the ability to distinguish nuances of this odour seems to have been selected for among different species from at least two families occupying different oceans. Recruiting reef species may use numerous odours20, as well as other sensory modes (e.g. vision and hearing50) to select recruitment sites; decaying plant material may act as a sensory ‘booster’ in tandem with other sensory cues, increasing the likelihood that larvae will identify appropriate habitats. The similar behavioural responses observed between locations suggests that human activities have a generalized effect on recruiting fishes in that they can initiate an avoidance behaviour. In Fiji, our developed sampling locations were close to the capital city of Suva, within several miles of a fishing port, an industrial area, and dense urban development. In contrast, activities at the developed Belizean site of South Water Cay consisted primarily of tourist resorts and associated boat traffic. Our low-impact sites were more similar between locations, with little human activity occurring near collection sites. The similar behavioural responses observed highlight the need to identify the underlying causes of these negative chemical cues and determine the concentrations at which their presence has an adverse effect on fish behaviour. Differences in the composition of the chemical cues produced as leaf litter breaks down could involve a number of pathways. For instance, chemicals may accumulate from the environment that are subsequently released as leaves decay or cues may differ due to environmental effects on leaf microbiomes. While the chemical composition of the leaves used in our behavioural experiments was not tested, there is some evidence that this can vary between sites occupied and unoccupied by humans. For instance, both Cu and Zn were 4–5 times higher in leaves subsequently obtained from the developed site, South Water Cay, compared to the undeveloped site, Twin Cays, Belize (Supplementary Table S1). Because the presence of toxic metals can directly impact the health of aquatic environments51, excess amounts of these or other pollutants in mangrove leaves from the developed site could have repelled fishes directly, or indirectly via effects on associated microbes. Treating leaves with NaOCl to reduce the microbial load on the leaves had a variable effect on both behaviour and settlement in Belize. While it is possible that the treatment itself had some effect, in behavioural assays the labrid, Thalassoma bifasciatum ceased to differentiate between leaves from each site while the pomacentrid, Stegastes partitus, continued to prefer leaves from the undeveloped site. The variable responses between species suggests that, while broad trends are consistent, subtle differences in how species or families perceive and respond to these cues exist. In the field, both total settlement and settler species richness did not differ between NaOCl-treated leaves from sites with versus without humans but did differ between these sites for untreated leaves. The increased settlement on artificial patch reefs in Belize that contained NaOCl-treated leaves from the developed site compared to untreated leaves from the developed site suggests that microbes associated with the developed site may be deterring fish recruitment, although the treatment itself may have also had an effect. Little is known about the relationship between microbial communities and fish recruitment, however, it is well established that bacterial biofilms and the chemicals they produce act as settlement cues for many invertebrate larvae (see52), and in some cases both juvenile fishes and coral larvae respond similarly to chemical cues14. Analysis of microbiomes from decaying leaves from each site in Belize found 20 OTUs in significantly higher relative abundances on leaves from the developed site (p < 0.001, Supplementary Fig. S1). These include sulfate-reducing taxa as well as copiotrophic taxa that are often associated with shifts in marine microbial communities exposed to anthropogenic nutrient enrichment53. That mangrove-associated chemicals provide recruitment cues for fishes and that these cues are modified by even modest-scale human occupation of nearby sites highlight the importance of incorporating the impacts of terrestrial landscapes into marine spatial planning and management, and indicate the value of implementing management strategies that limit the amount or type of chemicals entering the marine environment via terrestrial sources. Additional work of value would include determining the importance of these cues relative to other biological and hydrodynamic processes as well as the chemicals to which the juvenile fishes are responding. In addition, future work should examine the role of mangrove odours and related chemical cues on the settlement and behaviour of mangrove specialist species, in particular those of ecological or economic importance. However, that we found behavioural responses to mangrove leaves in species not tightly associated with mangroves highlights that mangroves provide critical cues and habitats for a range of juvenile fishes and that their continuing losses affect not only mangrove systems, but also adjacent communities such as coral reefs. Methods Study sites and species Portions of this study were conducted within the Western Atlantic (Belize) and portions within the South Pacific (Fiji). In Fiji, work was conducted in January–February 2015, while most work in Belize occurred in June 2015 with some additional sampling (for leaf chemical analysis, see Supplementary Materials) in March 2016. In Belize, laboratory and field-based work was conducted at the Smithsonian's Research Station at Carrie Bow Cay (16° 48′ 9.8316″ N, 88° 4′ 54.8148″ W) using fishes from Carrie Bow Cay and plant material collected from neighbouring islands, while in Fiji, laboratory-based experiments were conducted using fishes and plant material collected from reefs and islands offshore from Labasa, Vanua Levu (16° 23′ 08.5″ S, 179° 19′ 52.5″ E) and surrounding the capital city of Suva, Viti Levu (18° 9′ 1.8432″ S, 178° 27′ 13.392″ E). Laboratory-based behavioural work focused on two common fish species associated with fringing reefs at each location; the bluehead wrasse, Thalassoma bifasciatum, and bicolor damselfish, Stegastes partitus, in Belize, and the blue-green chromis, Chromis viridis, and two stripe damselfish, Dascyllus reticulatus, in Fiji. In each location, these species were selected due to the high number of recruits present during the study period. These species are common in reef and non-reef habitats near mangrove forests and associate with microhabitats such as corals and other reef invertebrates34. Do chemical cues from mangroves attract reef fishes? To assess the effects of odours from mangroves on reef fishes, we conducted choice assays using a two-channel flume (13 cm length × 4 cm width)54 in which individual fish were presented with two parallel water flows, each containing different chemical cues. In the flume, each water mass remained separated on either side of the main chamber without producing turbulence or eddies. Water was gravity fed into the flume at equivalent volumes (100 ml min−1) from both sources, with dye tests conducted to confirm that the two water sources continued to exhibit parallel water flow. Recently settled fish (1.5–2 cm total length) were collected by hand from reefs not associated with mangroves using nets and clove oil and held in aerated tanks until experimentation. Trials occurred within 6–24 h of collection with all fish used observed actively swimming and interacting with their environment prior to each trial. All replicates used unique individual fish and none were used in multiple assays. For each trial, a fish was placed into the centre of the flume at the downstream end. Following a two-minute habituation period, the position of the fish (left or right side of chamber) was recorded at five-second intervals for a period of two-minutes. The source of water to each side of the chamber was then reversed and the chamber was allowed to flush for a one-minute period. Then, a second two-minute habituation and two-minute test period were conducted. This controlled for any side preference fish may be exhibiting within the flume. Due to logistical constraints, the tester was aware of the cues being tested. For each test, significant differences in time spent in each water source were determined using either paired-sample t-tests, or Wilcoxon signed-rank tests if data did not met the assumption of normality. All analyses of paired-choice data were conducted using R55. Fish that remained on one side during the habituation and test periods were considered to not be exhibiting normal exploratory behaviour and so were excluded from the subsequent analysis (Supplementary Table S5)56. To assess whether fishes were attracted to odours from mangroves, we soaked 15 leaves (~ 10 cm L from tip to base × 6 cm width at widest point of blade) in 20 L of untreated seawater for a period of 2 h; and tested fish behavioural responses to this water versus the same water but without the leaves. In Belize, all water was taken from the Carrie Bow Cay seawater system (the intake for this water is located approximately 25 m seaward from the island); in Fiji, all baseline water was from collections made at least 1 km from any reef or land. For this, and all subsequent tests, treatment water was used within 4 h of production to limit deterioration of odour molecules. In both Belize and Fiji, Rhizophora spp. mangroves were used to produce leaf chemical cues. Mangroves from this circumtropical genus were dominant at all sites, forming large coastal and offshore stands. In Belize, the genus is represented by Rhizophora mangle57, while in Fiji, it is represented by R. stylosa, R. samoensis, and a hybrid of both, R. x selala58. In Fiji, plants were only distinguished to genus due to their overlapping distributions and morphological and genetic similarity. After determining that mangrove cues were attractive to all four species of reef fish tested, we also evaluated whether fishes were differentially attracted to (1) young (green) versus old (yellow) leaves collected while still on the plants, and (2) young leaves from the plants versus submerged, decaying leaves that had fallen from the plants and were collected as leaf litter at a depth of ~ 50 cm below standing plants. Latex gloves were worn during collection to reduce altering leaf microbial communities. In Belize, all fishes for these trials were collected from the Carrie Bow Cay reef, with leaves collected from the neighbouring Twin Cays (16° 49′ 43.2″ N, 88° 06′ 14.1″ W). In Fiji, fishes were either collected off shore from Labasa (C. viridis, young versus submerged leaves) or from fringing reef to the east of Suva (18° 08′ 45.7″ S, 178° 22′ 45.5″ E) (C. viridis, young versus older leaves and young leaves versus blank seawater; D. reticulatus, all trials). Leaves were either collected from offshore stands near Labasa or stands inshore from the Suva fringing reef. We also assessed whether attraction to odours from senescent, submerged mangrove leaves differed depending on whether these leaves were collected beneath mangroves near human settlements or beneath mangroves remote from human settlements. Sites near versus remote from human settlements were determined based on proximity to anthropogenic development. In Fiji, developed sites were mangrove stands near central Suva city (18° 9′ 22.432″ S, 178° 26′ 47.468″ E) and the village of Korovou (18° 7′ 12.55″ S, 178° 25′ 51.269″ E). Undeveloped sites were: Nukulau Island (18° 10′ 27.632″ S, 178° 31′ 5.646″ E) and Namuka (18° 08′ 06.2 "S, 178° 21′ 22.8″ E). In Belize, the largely uninhabited Twin Cays was the undeveloped site while the neighbouring, and more populated, South Water Cay was the developed site (16° 49′ 43.2″ N, 88° 06′ 14.1″ W). These islands were approximately 2 km apart at their shortest distance. In addition to testing responses to leaves, we also tested the responses of fishes towards water collected at each of the leaf collection sites. Water was collected in 15 L containers and used within 4 h of collection. Water from each site did not differ in clarity or colour based on visual inspection. Fishes for comparison one (Suva vs Nukulau) were collected from the fringing reef near Makuluva Island (18° 11′ 17.9″ S, 178° 30′ 57.3″ E), while fishes for comparison two (Korovou vs Namuka) were collected from fringing reef to the east of Suva as above. Do mangrove chemical cues influence settlement site selection? At Carrie Bow Cay, three groups of five patch reefs (i.e., 15 in all) were built in a large, sandy area 40 m from shore and 100 m from the nearest reef, with each group of five patch reefs comprising one replicate block of treatments. For each block, the five patch reefs were arranged in a circle with each patch reef 1.5 m from its adjacent patch reefs. Each block was separated from the other two blocks by ≥ 15 m. Each patch reef was approximately 40 cm in diameter and consisted of equal parts coral rubble and live Acropora prolifera coral surrounding a stimulus emission device (SED)59. SEDs were rectangular plastic containers (L = 15 cm, W = 10, H = 10) with opaque mesh sides and top that allowed dispersal of the odour of the mangrove leaf litter inside. Materials used to make the reefs obscured the SEDs from view. The five treatments included; a control containing an empty SED (empty control = EC), unmodified mangrove leaves from the undeveloped site (leaves unmodified = LU), unmodified mangrove leaves from the developed site (LD), mangrove leaves from the undeveloped site treated to reduce the microbial biofilm (TLU), and mangrove leaves from the developed site treated to reduce the microbial biofilm (TLD). Treatment to remove the original leaf-associated microbiome consisted of spraying leaves with the bactericidal agent sodium hypochlorite (NaOCl) diluted to 0.005% with deionized water until saturated and holding these in a sterile container for 10-min. Leaves were then flushed with seawater until no chemical odour could be detected. The order and position of treatments within each patch reef block was randomised between each trial. This experiment was repeated for six consecutive nights during June 2015 yielding n = 18 per treatment (i.e. three replicates per treatment per night for six nights). Patch reefs were built each afternoon prior to sunset. The following morning at 0630 h, all fish recruits on each patch reef were identified and counted. All fish were removed from each patch reef and released onto nearby reef areas. To identify difference in settlement site selection while accounting for spatial and temporal variation in recruitment, total settlement and the total number of settler species to each patch reef was converted to a replicate residual for each block of treatments. This was done by dividing total settlement or species for all patch reefs in a block by five to give the expected numbers of settlers or species per reef if distributed evenly. Residual settlement and species for each patch reef equalled the actual minus expected settlement and species. As settlement data did not meet parametric assumptions, a Kruskal–Wallis rank sum test was used to determine whether residual settlement varied between treatments, with Dunn’s tests of multiple comparisons used for post hoc analysis. For species data, a one-way ANOVA was used with post hoc analysis conducted using Tukey’s HSD tests. All analyses of patch reef data were conducted using R55. Potential drivers of the behavioural patterns observed Following the behavioural components of this study, we collected leaves from each site and analysed these for differences in elemental composition and in the community composition of their microbiomes. Because leaves for chemical analysis were not collected synchronous with our behavioural assays and because leaves for both components were from only one developed and one undeveloped site (Twin Cay and South Water Cay in Belize), we view these as preliminary data indicating possible drivers of the behavioural patterns we observed. Results from these efforts are presented in the Supplementary Materials. Ethics approval All work using animals was approved by the Georgia Institute of Technology and University of Delaware ethics committees and followed relevant guidelines and regulations. Supplementary information Supplementary Informations. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information is available for this paper at 10.1038/s41598-020-77722-7. Acknowledgements We thank the Governments of Fiji and Belize for permission to conduct the study. Thanks to the University of the South Pacific, Smithsonian Institution, R. Poulin, Z. Foltz, M. Ashur, L. Johnston, K. Critchell, and V. Paul for logistical assistance. This is contribution 1048 of the Caribbean Coral Reef Ecosystems Program (CCRE), Smithsonian Institution. Author contributions R.M.B, D.L.D, and M.E.H conceived the study with input from all authors. R.M.B, A.L.S, A.H and J.M.S collected and analyzed the data. R.M.B wrote the manuscript with input from all authors. Funding Funding was provided by the National Institutes of Health (Grant No. 2U19TW007401-10). Data availability All data and code used to conduct the analysis in this manuscript is available at: 10.5281/zenodo.4284108. Competing interests The authors declare no competing interests. ==== Refs References 1. Graham NAJ Seabirds enhance coral reef productivity and functioning in the absence of invasive rats Nature 2018 559 250 253 10.1038/s41586-018-0202-3 29995864 2. Young HS McCauley DJ Dunbar RB Dirzo R Plants cause ecosystem nutrient depletion via the interruption of bird-derived spatial subsidies Proc. Natl. Acad. Sci. 2010 107 2072 2077 10.1073/pnas.0914169107 20133852 3. Hocking MD Reynolds JD Impacts of salmon on riparian plant diversity Science 2011 331 1609 1612 10.1126/science.1201079 21442794 4. Butchart SHM Global biodiversity: Indicators of recent declines Science 2010 328 1164 1168 10.1126/science.1187512 20430971 5. Haddad NM Habitat fragmentation and its lasting impact on Earth’s ecosystems Sci. Adv. 2015 10.1126/sciadv.1500052 26601154 6. Foley JA Global consequences of land use Science 2005 309 570 574 10.1126/science.1111772 16040698 7. Halpern BS A global map of human impact on marine ecosystems Science 2008 319 948 952 10.1126/science.1149345 18276889 8. Syvitski JPM Vörösmarty CJ Kettner AJ Green P Impact of humans on the flux of terrestrial sediment to the global coastal ocean Science 2005 308 376 380 10.1126/science.1109454 15831750 9. Polis GA Anderson WB Holt RD Toward an integration of landscape and food web ecology: The dynamics of spatially subsidized food webs Annu. Rev. Ecol. Syst. 1997 28 289 316 10.1146/annurev.ecolsys.28.1.289 10. Álvarez-Romero JG Integrated land-sea conservation planning: The missing links Annu. Rev. Ecol. Evol. Syst. 2011 42 381 409 10.1146/annurev-ecolsys-102209-144702 11. Hamilton TJ Kline DI Tresguerres M Shoaling behaviour is differentially altered by ethanol and dopamine D1 receptor modulators in tropical marine forage fish Can. J. Fish. Aquat. Sci. 2018 75 999 1004 10.1139/cjfas-2018-0084 12. Fabbri E Franzellitti S Human pharmaceuticals in the marine environment: Focus on exposure and biological effects in animal species Environ. Toxicol. Chem. 2016 35 799 812 10.1002/etc.3131 26111460 13. Scott GR Sloman KA The effects of environmental pollutants on complex fish behaviour: Integrating behavioural and physiological indicators of toxicity Aquat. Toxicol. 2004 68 369 392 10.1016/j.aquatox.2004.03.016 15177953 14. Dixson DL Abrego D Hay ME Chemically mediated behavior of recruiting corals and fishes: A tipping point that may limit reef recovery Science 2014 345 892 897 10.1126/science.1255057 25146281 15. Hay ME Marine chemical ecology: Chemical signals and cues structure marine populations, communities, and ecosystems Ann. Rev. Mar. Sci. 2009 1 193 212 10.1146/annurev.marine.010908.163708 21141035 16. DeBose JL Lema SC Nevitt GA Dimethylsulfoniopropionate as a foraging cue for reef fishes Science 2008 319 1356 1356 10.1126/science.1151109 18323445 17. Devine BM Munday PL Jones GP Homing ability of adult cardinalfish is affected by elevated carbon dioxide Oecologia 2012 168 269 276 10.1007/s00442-011-2081-2 21800059 18. Brooker RM Munday PL Chivers DP Jones GP You are what you eat: Diet-induced chemical crypsis in a coral-feeding reef fish Proc. R. Soc. B Biol. Sci. 2015 282 20141887 10.1098/rspb.2014.1887 19. Dixson DL Hay ME Corals chemically cue mutualistic fishes to remove competing seaweeds Science 2012 338 804 807 10.1126/science.1225748 23139333 20. Brooker RM Dixson DL Schulte BA Goodwin TE Ferkin MH Assessing the role of olfactory cues in the early life history of coral reef fish: Current methods and future directions Chemical Signals in Vertebrates 13 2016 Berlin Springer 17 31 21. Lecchini D Shima J Banaigs B Galzin R Larval sensory abilities and mechanisms of habitat selection of a coral reef fish during settlement Oecologia 2005 143 326 334 10.1007/s00442-004-1805-y 15647903 22. Dixson DL Coral reef fish smell leaves to find island homes Proc. R. Soc. B Biol. Sci. 2008 275 2831 2839 10.1098/rspb.2008.0876 23. Brooker RM Olfactory discrimination between chemical cues from coastal vegetation in two palaemonid shrimps, Palaemon vulgaris Say, 1818 and P. affinis H Milne Edwards, 1837 (Decapoda: Caridea: Palaemonidae) J. Crustac. Biol. 2019 39 312 315 24. Waycott M Accelerating loss of seagrasses across the globe threatens coastal ecosystems Proc. Natl. Acad. Sci. 2009 106 12377 12381 10.1073/pnas.0905620106 19587236 25. Alongi DM Present state and future of the world’s mangrove forests Environ. Conserv. 2002 29 331 349 10.1017/S0376892902000231 26. Spencer T Global coastal wetland change under sea-level rise and related stresses: The DIVA wetland change model Glob. Planet. Change 2016 139 15 30 10.1016/j.gloplacha.2015.12.018 27. Kathiresan K Bingham BL Biology of mangroves and mangrove ecosystems Adv. Mar. Biol. 2020 40 81 251 10.1016/S0065-2881(01)40003-4 28. Mumby PJ Mangroves enhance the biomass of coral reef fish communities in the Caribbean Nature 2004 427 533 10.1038/nature02286 14765193 29. Lee SY Ecological role and services of tropical mangrove ecosystems: a reassessment Glob. Ecol. Biogeogr. 2014 23 726 743 10.1111/geb.12155 30. Giri C Status and distribution of mangrove forests of the world using earth observation satellite data Glob. Ecol. Biogeogr. 2011 20 154 159 10.1111/j.1466-8238.2010.00584.x 31. Nagelkerken I How important are mangroves and seagrass beds for coral-reef fish? The nursery hypothesis tested on an island scale Mar. Ecol. Prog. Ser. 2002 244 299 305 10.3354/meps244299 32. Bengtsson Z Corals of the genus Porites are a locally abundant component of the epibiont community on mangrove prop roots at Calabash Caye, Turneffe Atoll, Belize Carib. Nat. 2019 67 1 16 33. Camp EF Edmondson J Doheny A Rumney J Grima AJ Huete A Suggett DJ Mangrove lagoons of the Great Barrier Reef support coral populations persisting under extreme environmental conditions Mar. Ecol. Prog. Ser. 2019 625 1 14 10.3354/meps13073 34. Sambrook K Hoey AS Andréfouët S Cumming GS Duce S Bonin MC Beyond the reef: The widespread use of non-reef habitats by coral reef fishes Fish. Fish. 2019 20 903 920 10.1111/faf.12383 35. Clough B Tan DT Phuong DX Buu DC Canopy leaf area index and litter fall in stands of the mangrove Rhizophora apiculata of different age in the Mekong Delta, Vietnam Aquat. Bot. 2000 66 311 320 10.1016/S0304-3770(99)00081-9 36. Mackey AP Smail G Spatial and temporal variation in litter fall of Avicennia marina (Forssk.) Vierh. in the Brisbane River, Queensland, Australia Aquat. Bot. 1995 52 133 142 10.1016/0304-3770(95)00490-Q 37. D’Alessandro E Sponaugle S Lee T Patterns and processes of larval fish supply to the coral reefs of the upper Florida Keys Mar. Ecol. Prog. Ser. 2007 331 85 100 10.3354/meps331085 38. Leis JM McCormick MI Sale PF The biology, behavior and ecology of the pelagic, larval stage of coral reef fishes Coral Reef Fishes: Dynamics and Diversity in a Complex Ecosystem 2002 San Diego Academic Press 171 199 39. Valiela I Bowen JL York JK Mangrove forests: one of the world’s threatened major tropical environments Bioscience 2001 51 807 815 10.1641/0006-3568(2001)051[0807:MFOOTW]2.0.CO;2 40. Duke NC A world without mangroves? Science 2007 317 41 42 10.1126/science.317.5834.41b 17615322 41. Richards DR Friess DA Rates and drivers of mangrove deforestation in SoutheastAsia, 2000–2012 Proc. Natl. Acad. Sci. 2016 113 344 349 10.1073/pnas.1510272113 26712025 42. Friess DA Rogers K Lovelock CE Krauss KW Hamilton SE Yip Lee S Lucas R Primavera J Rajkaran A Shi S The state of the world's mangrove forests: Past, present, and future Ann. Rev. Environ. Res. 2019 44 89 115 10.1146/annurev-environ-101718-033302 43. Friess DA Yando ES Abuchahla GMO Adams JB Cannicci S Canty SWJ Cavanaugh KC Connolly RM Cormier N Dahdouh-Guebas F Diele K Feller IC Fratini S Jennerjahn TC Yip Lee S Ogurcak DE Ouyang X Rogers K Rowntree JK Sharma S Sloey TM Mangroves give cause for conservation optimism, for now Curr. Biol. 2020 30 R153 R154 10.1016/j.cub.2019.12.054 32097637 44. Yim MW Tam NFY Effects of wastewater-borne heavy metals on mangrove plants and soil microbial activities Mar. Pollut. Bull. 1999 39 179 186 10.1016/S0025-326X(99)00067-3 45. Mandura AS A mangrove stand under sewage pollution stress: Red Sea Mangroves Salt Marshes 1997 1 255 262 10.1023/A:1009927605517 46. MacFarlane GR Koller CE Blomberg SP Accumulation and partitioning of heavy metals in mangroves: A synthesis of field-based studies Chemosphere 2007 69 1454 1464 10.1016/j.chemosphere.2007.04.059 17560628 47. Almahasheer H Serrano O Duarte CM Irigoien X remobilization of heavy metals by mangrove leaves Front. Mar. Sci. 2018 5 484 10.3389/fmars.2018.00484 48. Bayen S Occurrence, bioavailability and toxic effects of trace metals and organic contaminants in mangrove ecosystems: A review Environ. Int. 2012 48 84 101 10.1016/j.envint.2012.07.008 22885665 49. Rogers A Mumby PJ Mangroves reduce the vulnerability of coral reef fisheries to habitat degradation PLoS Biol. 2019 17 e3000510 10.1371/journal.pbio.3000510 31714938 50. Atema J Gerlach G Paris CB Mora C Sensory biology and navigation behavior of reef fish larvae Ecology of Fishes on Coral Reefs 2015 Cambridge Cambridge University Press 3 15 51. Laskowski R Hopkin SP Effect of Zn, Cu, Pb, and Cd on fitness in snails (Helix aspersa ) Ecotoxicol. Environ. Saf. 1996 34 59 69 10.1006/eesa.1996.0045 8793321 52. Hadfield MG Biofilms and marine invertebrate larvae: what bacteria produce thatlarvae use to choose settlement sites Ann. Rev. Mar. Sci. 2010 3 453 470 10.1146/annurev-marine-120709-142753 53. Nogales B Lanfranconi MP Piña-Villalonga JM Bosch R Anthropogenic perturbations in marine microbial communities FEMS Microbiol. Rev. 2011 35 275 298 10.1111/j.1574-6976.2010.00248.x 20738403 54. Atema J Kingsford MJ Larval reef fish could use odour for detection, retention and orientation to reefs Mar. Ecol. Prog. Ser. 2002 241 151 160 10.3354/meps241151 55. R Core Team R: A Language and Environment for Statistical Computing 2020 Vienna R Foundation for Statistical Computing 56. Jutfelt F Sundin J Raby GD Krång A-S Clark TD Two-current choice flumes for testing avoidance and preference in aquatic animals Methods Ecol. Evol. 2017 8 379 390 10.1111/2041-210X.12668 57. Murray MR Zisman SA Furley PA Munro DM Gibson J Ratter J Bridgewater S Minty CD Place CJ The mangroves of Belize: part 1. distribution, composition and classification For. Ecol. Manag. 2002 174 265 279 10.1016/S0378-1127(02)00036-1 58. Duke, N. C., Mackenzie, J., Wood, A. A revision of mangrove plants of the Solomon Islands, Vanuatu, Fiji, Tonga and Samoa. Centre for Tropical Water & Aquatic Ecosystem Research (TropWATER) Publication 12/13, James Cook University, Townsville (2012). 59. Vail AL McCormick MI Metamorphosing reef fishes avoid predator scent when choosing a home Biol. Lett. 2011 7 921 924 10.1098/rsbl.2011.0380 21653563