Environmental DNA reveals temporal variation in mesophotic reefs of the Humboldt upwelling ecosystems of central Chile: Toward a baseline for biodiversity monitoring of unexplored marine habitats.

Temperate mesophotic reef ecosystems (TMREs) are among the least known marine habitats. Information on their diversity and ecology is geographically and temporally scarce, especially in highly productive large upwelling ecosystems. Lack of information remains an obstacle to understanding the importance of TMREs as habitats, biodiversity reservoirs and their connections with better-studied shallow reefs. Here, we use environmental DNA (eDNA) from water samples to characterize the community composition of TMREs on the central Chilean coast, generating the first baseline for monitoring the biodiversity of these habitats. We analyzed samples from two depths (30 and 60 m) over four seasons (spring, summer, autumn, and winter) and at two locations approximately 16 km apart. We used a panel of three metabarcodes, two that target all eukaryotes (18S rRNA and mitochondrial COI) and one specifically targeting fishes (16S rRNA). All panels combined encompassed eDNA assigned to 42 phyla, 90 classes, 237 orders, and 402 families. The highest family richness was found for the phyla Arthropoda, Bacillariophyta, and Chordata. Overall, family richness was similar between depths but decreased during summer, a pattern consistent at both locations. Our results indicate that the structure (composition) of the mesophotic communities varied predominantly with seasons. We analyzed further the better-resolved fish assemblage and compared eDNA with other visual methods at the same locations and depths. We recovered eDNA from 19 genera of fish, six of these have also been observed on towed underwater videos, while 13 were unique to eDNA. We discuss the potential drivers of seasonal differences in community composition and richness. Our results suggest that eDNA can provide valuable insights for monitoring TMRE communities but highlight the necessity of completing reference DNA databases available for this region.

Spatio-temporal patterns of the crustacean demersal fishery discard from the south Humboldt Current System, based on scientific observer program (2014-2019).

This study summarises six years of spatio-temporal patterns of the discarded demersal community fauna recorded by onboard scientific observer program for both artisanal and industrial crustacean fisheries between 2014 and 2019, from mesophotic to aphotic depths (96 to 650 m) along the southern Humboldt Current System (28-38°S). In this period, one cold and two warm climatic events were observed during the austral summer 2014, 2015-2016 (ENSO Godzilla), and 2016-2017 (coastal ENSO), respectively. Satellite information showed that Chlorophyll-a concentration varied seasonally and latitudinally, associated with upwelling centres, while equatorial wind stress decreased southward of 36°S. Discards were composed of 108 species, dominated by finfish and molluscs. The Chilean hake Merluccius gayi was dominant and ubiquitous (occurrence, 95% of 9104 hauls), being the most vulnerable species of the bycatch. Three assemblages were identified: assemblage 1 (~200 m deep), dominated by flounders Hippoglossina macrops and lemon crabs Platymera gaudichaudii, assemblage 2 (~260 m deep), dominated by squat lobsters Pleuroncodes monodon and Cervimunida johni and assemblage 3 (~320 m depth), dominated by grenadiers Coelorinchus aconcagua and cardinalfish Epigonus crassicaudus. These assemblages were segregated by depth, and varied by year, and geographic zone. The latter represented changes in the width of the continental shelf, increasing southward of 36°S. Alpha-diversity indexes (richness, Shannon, Simpson, and Pielou) also varied with depth and latitude, with higher diversity in deeper continental waters (>300 m), between 2018-2019. Finally, at a spatial scale of tens of kilometres, and a monthly basis, interannual variations of biodiversity occurred in the demersal community. Surface sea temperature, chlorophyll-a, or wind stress did not correlate with discarded demersal fauna diversity of the crustacean fishery operating along central Chile.

Long-term exposure to artificial light at night in the wild decreases survival and growth of a coral reef fish.

Artificial light at night (ALAN) is an increasing anthropogenic pollutant, closely associated with human population density, and now well recognized in both terrestrial and aquatic environments. However, we have a relatively poor understanding of the effects of ALAN in the marine realm. Here, we carried out a field experiment in the coral reef lagoon of Moorea, French Polynesia, to investigate the effects of long-term exposure (18-23 months) to chronic light pollution at night on the survival and growth of wild juvenile orange-fin anemonefish, Amphiprion chrysopterus. Long-term exposure to environmentally relevant underwater illuminance (mean: 4.3 lux), reduced survival (mean: 36%) and growth (mean: 44%) of juvenile anemonefish compared to that of juveniles exposed to natural moonlight underwater (mean: 0.03 lux). Our study carried out in an ecologically realistic situation in which the direct effects of artificial lighting on juvenile anemonefish are combined with the indirect consequences of artificial lighting on other species, such as their competitors, predators, and prey, revealed the negative impacts of ALAN on life-history traits. Not only are there immediate impacts of ALAN on mortality, but the decreased growth of surviving individuals may also have considerable fitness consequences later in life. Future studies examining the mechanisms behind these findings are vital to understand how organisms can cope and survive in nature under this globally increasing pollutant.

Evolutionary origin of the Atlantic Cabo Verde nibbler (Girella stuebeli), a member of a primarily Pacific Ocean family of antitropical herbivorous reef fishes.

Nibblers (family Girellidae) are reef fishes that are mostly distributed in the Indo-Pacific, with one exception: Girella stuebeli, which is found in the Cabo Verde Archipelago, in the Atlantic Ocean. We capitalized on this unusual distribution to study the evolutionary history of the girellids, and determine the relationship between G. stuebeli and the remaining nibbler taxa. Based on thousands of genomic markers (RAD sequences), we identified the closest relatives of G. stuebeli as being a clade of three species endemic to the northwestern Pacific, restricted to the Sea of Japan and vicinity. This clade diverged from G. stuebeli approximately 2.2 Mya. Two alternative potential routes of migration may explain this affinity: a western route, from the Tropical Eastern Pacific and the Tropical Western Atlantic, and an eastern route via the Indian Ocean and Southern Africa. The geological history and oceanography of the regions combined with molecular data presented here, suggest that the eastern route of invasion (via the Indian Ocean and Southern Africa) is a more likely scenario.

Hormonal and behavioural effects of motorboat noise on wild coral reef fish.

Anthropogenic noise is an emergent ecological pollutant in both terrestrial and aquatic habitats. Human population growth, urbanisation, resource extraction, transport and motorised recreation lead to elevated noise that affects animal behaviour and physiology, impacting individual fitness. Currently, we have a poor mechanistic understanding of the effects of anthropogenic noise, but a likely candidate is the neuroendocrine system that integrates information about environmental stressors to produce regulatory hormones; glucocorticoids (GCs) and androgens enable rapid individual phenotypic adjustments that can increase survival. Here, we carried out two field-based experiments to investigate the effects of short-term (30 min) and longer-term (48 h) motorboat-noise playback on the behaviour, GCs (cortisol) and androgens of site-attached free-living orange-fin anemonefish (Amphiprion chrysopterus). In the short-term, anemonefish exposed to motorboat-noise playback showed both behavioural and hormonal responses: hiding and aggression increased, and distance moved out of the anemone decreased in both sexes; there were no effects on cortisol levels, but male androgen levels (11-ketotestosterone and testosterone) increased. Some behaviours showed carry-over effects from motorboat noise after it had ceased, and there was no evidence for a short-term change in response to subsequent motorboat-noise playback. Similarly, there was no evidence that longer-term exposure led to changes in response: motorboat noise had an equivalent effect on anemonefish behaviour and hormones after 48 h as on first exposure. Longer-term noise exposure led to higher levels of cortisol in both sexes and higher testosterone levels in males, and stress-responses to an additional environmental challenge in both sexes were impaired. Circulating androgen levels correlated with aggression, while cortisol levels correlated with hiding, demonstrating in a wild population that androgen/glucocorticoid pathways are plausible proximate mechanisms driving behavioural responses to anthropogenic noise. Combining functional and mechanistic studies are crucial for a full understanding of this global pollutant.

Near-future ocean warming and acidification alter foraging behaviour, locomotion, and metabolic rate in a keystone marine mollusc.

Environmentally-induced changes in fitness are mediated by direct effects on physiology and behaviour, which are tightly linked. We investigated how predicted ocean warming (OW) and acidification (OA) affect key ecological behaviours (locomotion speed and foraging success) and metabolic rate of a keystone marine mollusc, the sea hare Stylocheilus striatus, a specialist grazer of the toxic cyanobacterium Lyngbya majuscula. We acclimated sea hares to OW and/or OA across three developmental stages (metamorphic, juvenile, and adult) or as adults only, and compare these to sea hares maintained under current-day conditions. Generally, locomotion speed and time to locate food were reduced ~1.5- to 2-fold when the stressors (OW or OA) were experienced in isolation, but reduced ~3-fold when combined. Decision-making was also severely altered, with correct foraging choice nearly 40% lower under combined stressors. Metabolic rate appeared to acclimate to the stressors in isolation, but was significantly elevated under combined stressors. Overall, sea hares that developed under OW and/or OA exhibited a less severe impact, indicating beneficial phenotypic plasticity. Reduced foraging success coupled with increased metabolic demands may impact fitness in this species and highlight potentially large ecological consequences under unabated OW and OA, namely in regulating toxic cyanobacteria blooms on coral reefs.

Anemone bleaching increases the metabolic demands of symbiont anemonefish.

Increased ocean temperatures are causing mass bleaching of anemones and corals in the tropics worldwide. While such heat-induced loss of algal symbionts (zooxanthellae) directly affects anemones and corals physiologically, this damage may also cascade on to other animal symbionts. Metabolic rate is an integrative physiological trait shown to relate to various aspects of organismal performance, behaviour and locomotor capacity, and also shows plasticity during exposure to acute and chronic stressors. As climate warming is expected to affect the physiology, behaviour and life history of animals, including ectotherms such as fish, we measured if residing in bleached versus unbleached sea anemones (Heteractis magnifica) affected the standard (i.e. baseline) metabolic rate and behaviour (activity) of juvenile orange-fin anemonefish (Amphiprion chrysopterus). Metabolic rate was estimated from rates of oxygen uptake [Formula: see text], and the standard metabolic rate [Formula: see text] of anemonefish from bleached anemones was significantly higher by 8.2% compared with that of fish residing in unbleached anemones, possibly due to increased stress levels. Activity levels did not differ between fish from bleached and unbleached anemones. As [Formula: see text] reflects the minimum cost of living, the increased metabolic demands may contribute to the negative impacts of bleaching on important anemonefish life history and fitness traits observed previously (e.g. reduced spawning frequency and lower fecundity).

Ephemeral and Localized Outbreaks of the Coral Predator Acanthaster cf. solaris in the Southwestern Lagoon of New Caledonia.

Mehdi Adjeroud, Mohsen Kayal, Christophe Peignon, Matthieu Juncker, Suzanne C. Mills, Ricardo Beldade, and Pascal Dumas (2018) Outbreaks of the coral predator Acanthaster spp., the crown-of-thorns seastar (COTS), cause major coral declines across the Indo-Pacific. However, the processes surrounding the initiation and propagation of COTS outbreaks are still unclear. We observed COTS outbreak abundances on several mid-shelf and inner-barrier reefs in the southern section of the New Caledonian lagoon, a multi-location initiation event that we expected to precede a broader region-wide disturbance. However, reef monitoring over 3 years revealed the highly localized and ephemeral character of these outbreaks. Outbreaks that were observed at four reef locations at the beginning of the survey simply faded away, without any specific management actions such as culling efforts. We also found no distinct reef biotope on which COTS outbreaks originated, although mid-shelf and inner-shelf barrier reefs seem to be favoured. New Caledonia has been exempted from to widespread regional COTS outbreaks, which is surprising given its proximity to the Australian Great Barrier Reef - a reef complex particularly vulnerable to COTS - and Vanuatu - where large-scale outbreaks were recorded during the same period. The availability of coral prey is probably not a limiting factor for the propagation of COTS outbreaks on New Caledonian reefs, given the high abundance and diversity of coral assemblages. Our findings reveal that localized and ephemeral COTS outbreaks can be naturally contained and do not necessarily result in widespread disturbances.

Cascading effects of thermally-induced anemone bleaching on associated anemonefish hormonal stress response and reproduction.

Organisms can behaviorally, physiologically, and morphologically adjust to environmental variation via integrative hormonal mechanisms, ultimately allowing animals to cope with environmental change. The stress response to environmental and social changes commonly promotes survival at the expense of reproduction. However, despite climate change impacts on population declines and diversity loss, few studies have attributed hormonal stress responses, or their regulatory effects, to climate change in the wild. Here, we report hormonal and fitness responses of individual wild fish to a recent large-scale sea warming event that caused widespread bleaching on coral reefs. This 14-month monitoring study shows a strong correlation between anemone bleaching (zooxanthellae loss), anemonefish stress response, and reproductive hormones that decreased fecundity by 73%. These findings suggest that hormone stress responses play a crucial role in changes to population demography following climate change and plasticity in hormonal responsiveness may be a key mechanism enabling individual acclimation to climate change.Elevated temperatures can cause anemones to bleach, with unknown effects on their associated symbiotic fish. Here, Beldade and colleagues show that climate-induced bleaching alters anemonefish hormonal stress response, resulting in decreased reproductive hormones and severely impacted reproduction.

Motorboat noise disrupts co-operative interspecific interactions.

Human-made noise is contributing increasingly to ocean soundscapes. Its physical, physiological and behavioural effects on marine organisms are potentially widespread, but our understanding remains largely limited to intraspecific impacts. Here, we examine how motorboats affect an interspecific cleaning mutualism critical for coral reef fish health, abundance and diversity. We conducted in situ observations of cleaning interactions between bluestreak cleaner wrasses (Labroides dimidiatus) and their fish clients before, during and after repeated, standardised approaches with motorboats. Cleaners inspected clients for longer and were significantly less cooperative during exposure to boat noise, and while motorboat disturbance appeared to have little effect on client behaviour, as evidenced by consistency of visit rates, clientele composition, and use of cleaning incitation signals, clients did not retaliate as expected (i.e., by chasing) in response to increased cheating by cleaners. Our results are consistent with the idea of cognitive impairments due to distraction by both parties. Alternatively, cleaners might be taking advantage of distracted clients to reduce their service quality. To more fully understand the importance of these findings for conservation and management, further studies should elucidate whether the efficacy of ectoparasite removal by cleaners is affected and explore the potential for habituation to boat noise in busy areas.

Spatial patterns of self-recruitment of a coral reef fish in relation to island-scale retention mechanisms.

Oceanographic features influence the transport and delivery of marine larvae, and physical retention mechanisms, such as eddies, can enhance self-recruitment (i.e. the return of larvae to their natal population). Knowledge of exact locations of hatching (origin) and settlement (arrival) of larvae of reef animals provides a means to compare observed patterns of self-recruitment 'connectivity' with those expected from water circulation patterns. Using parentage inference based on multiple sampling years in Moorea, French Polynesia, we describe spatial and temporal variation in self-recruitment of the anemonefish Amphiprion chrysopterus, evaluate the consistency of net dispersal distances of self-recruits against the null expectation of passive particle dispersal and test the hypothesis that larvae originating in certain reef habitats (lagoons and passes) would be retained and thus more likely to self-recruit than those originating on the outer (fore) reef. Estimates of known self-recruitment were consistent across the sampling years (~25-27% of sampled recruits). For most (88%) of these self-recruits, the net distance between hatching and settlement locations was within the maximum dispersal distance expected for a neutrally buoyant passive particle based on the longest duration of the larval dispersive phase and the average direction and speed of current flow around Moorea. Furthermore, a parent of a given body size on the outer (fore) reef of Moorea was less likely to produce self-recruits than those in passes. Our findings show that even a simple dispersal model based on net average flow and direction of alongshore currents can provide insight into landscape-scale retention patterns of reef fishes.

Lime Juice and Vinegar Injections as a Cheap and Natural Alternative to Control COTS Outbreaks.

Outbreaks of the corallivorous crown-of-thorns seastar Acanthaster planci (COTS) represent one of the greatest disturbances to coral reef ecosystems in the Indo-Pacific, affecting not only coral reefs but also the coastal communities which rely on their resources. While injection approaches are increasingly used in an attempt to control COTS densities, most of them display severe drawbacks including logistical challenges, high residual environmental impacts or low cost-effectiveness. We tested a new alternative control method based upon acidic injections of cheap, 100% natural products. We investigated the lethal doses, intra- and inter-specific disease transmission and immune responses of COTS when injected with fresh lime juice (extracted from local Citrus arantifolia) and white spirit vinegar. High COTS mortality was achieved with small volumes: 10-20 ml per seastar induced death in 89%/97% of injected specimens after an average 34.3 h/29.8 h for lime juice and vinegar respectively. Highest efficiency was reached for both solutions with double shots of (2 × 10 ml) in two different areas on the body: 100% mortality occurred within 12-24 h, which is similar or faster compared with other current injection methods. Multiple immune measures suggested that death was very likely caused by pH stress from the acidic solutions rather than a bacterial infection. Contagion to either conspecifics or a variety of other reef species was not observed, even at COTS densities 15 times higher than the highest naturally reported. 10 to 20 l lime juice/vinegar could kill up to a thousand COTS at a cost of less than 0.05 USD per specimen; no permits or special handling procedures are required. We conclude that injections of lime juice and vinegar offer great advantages when compared to current best practises and constitute a cheap and natural option for all reefs affected by COTS.

More coral, more fish? Contrasting snapshots from a remote Pacific atoll.

Coral reefs are in decline across the globe as a result of overexploitation, pollution, disease and, more recently, climate change. The impacts of changes in coral cover on associated fish communities can be difficult to predict because of the uneven dependence of reef fish species on corals for food, shelter or the three-dimensional structure they provide. We compared live coral cover, reef fish community metrics, and their associations in two surveys of the lagoon of the remote atoll of Mataiva (French Polynesia) carried out 31 years apart. In contrast to the general pattern of decreasing coral cover reported for many parts of the Indo-Pacific region, live coral cover increased 6-7 fold at Mataiva between 1981 and 2012, and fish density nearly doubled. The stable overall reef fish species richness belied a significant shift in community structure. There was little overlap in community composition across years, and fish assemblages in 2012 were more homogeneous in composition than they were in 1981. Changes in species abundance were not clearly related to species-specific reliance on corals. The strong positive relationships between live coral cover and fish diversity and abundance noted in 1981, when coral cover rarely exceeded 10%, were no longer present in 2012, when coral cover rarely fell below this value. The most parsimonious explanation for these contrasting relationships is that, over the combined range of coral cover observed in the 1981 and 2012 snapshots, there is a rapidly asymptotic relationship between coral and fish. Our results, and other data from the south and west Pacific, suggest that fish diversity and abundance might accumulate rapidly up to a threshold of approximately 10% live coral cover. Such a relationship would have implications for our expectations of resistance and recovery of reef fish communities facing an increasingly severe regime of coral reef disturbances.

Full-sibs in cohorts of newly settled coral reef fishes.

Reef fishes exhibit a bipartite life cycle where a benthic adult stage is preceded by a pelagic dispersal phase during which larvae are presumed to be mixed and transported by oceanic currents. Genetic analyses based on twelve microsatellite loci of 181 three-spot dascyllus (Dascyllus trimaculatus) that settled concurrently on a small reef in French Polynesia revealed 11 groups of siblings (1 full sibs and 10 half-sibs). This is the first evidence that fish siblings can journey together throughout their entire planktonic dispersal phase (nearly a month long for three-spot dascyllus). Our findings have critical implications for the dynamics and genetic structure of fish populations, as well as for the design of marine protected areas and management of fisheries.

Evidence for cohesive dispersal in the sea.

As with many marine species, the vast majority of coral-reef fishes have a bipartite life cycle consisting of a dispersive larval stage and a benthic adult stage. While the potentially far-reaching demographic and ecological consequences of marine dispersal are widely appreciated, little is known of the structure of the larval pool and of the dispersive process itself. Utilizing Palindrome Sequence Analysis of otolith micro-chemistry (PaSA;) we show that larvae of Neopomacentrus miryae (Pomacentridae) appear to remain in cohesive cohorts throughout their entire pelagic larval duration (PLD; ~28 days). Genetically, we found cohort members to be maternally (mtDNA) unrelated. While physical forcing cannot be negated as contributing to initial cohort formation, the small scale of the observed spatial structure suggests that some behavioral modification may be involved from a very early age. This study contributes to our ongoing re-evaluation of the processes that structure marine populations and communities and the spatial scales at which they operate.

Isolation and characterization of 13 polymorphic microsatellites for the black murex, Hexaplex nigritus.

Thirteen polymorphic microsatellite markers have been isolated and characterized for the black murex (Hexaplex nigritus). These loci are moderately to highly variable with seven to 37 alleles within 113 individuals from four populations in the Northern Gulf of California. Expected heterozygosities ranged from 0.43 to 0.98. High variability indicates that these markers will be useful for studying population structure and connectivity in this species.

Allopatric divergence and speciation in coral reef fish: the three-spot dascyllus, Dascyllus trimaculatus, species complex.

Long pelagic larval phases and the absence of physical barriers impede rapid speciation and contrast the high diversity observed in marine ecosystems such as coral reefs. In this study, we used the three-spot dascyllus (Dascyllus trimaculatus) species complex to evaluate speciation modes at the spatial scale of the Indo-Pacific. The complex includes four recognized species and four main color morphs that differ in distribution. Previous studies of the group using mitochondrial DNA revealed a noncongruence between color morphs and genetic groupings; with two of the color morphs grouped together and one color morph separated into three clades. Using extensive geographic sampling of 563 individuals and a combination of mitochondrial DNA sequences and 13 nuclear microsatellites, we defined population/species boundaries and inferred different speciation modes. The complex is composed of seven genetically distinct entities, some of which are distinct morphologically. Despite extensive dispersal abilities and an apparent lack of barriers, observed genetic partitions are consistent with allopatric speciation. However, ecological pressure, assortative mating, and sexual selection, were likely important during periods of geographical isolation. This study therefore suggests that primarily historical factors later followed by ecological factors caused divergence and speciation in this group of coral reef fish.

Multiple paternity and competition in sympatric congeneric reef fishes, Embiotoca jacksoni and E. lateralis.

The black surfperch Embiotoca jacksoni and the striped surfperch E. lateralis (Embiotocidae, Perciformes) are livebearing temperate reef fishes that live sympatrically over a large portion of their distribution range, where they exhibit strong ecological competition. In order to assess whether mating strategies reflect competition, we investigated multiple paternity in these two species in an area of sympatry. We sampled 24 pregnant females (12 for each species) in Monterey Bay, California, used microsatellite analysis and assessed paternity with the COLONY software. While broods are relatively small (12 to 36 offspring), they were always sired by multiple fathers (2 to 9), with no correlation between the size of a brood and the number of fathers. The number of sires for each brood was not significantly different between the two species (approximately 3.5 sires per brood). We tested the deviation from stochasticity of fathered offspring for each father in one brood. Results showed a significant deviation for both E. jacksoni and E. lateralis. However, this deviation was not found to be significant between species. The striking similarity in the dynamics of multiple paternity in these species, when sampled in sympatry, may result from several alternative scenarios, including phylogenetic inertia, reproductive behaviour, and ecological competition.

Historical biogeography and speciation in the Creole wrasses (Labridae, Clepticus).

We tested whether vicariance or dispersal was the likely source of speciation in the genus Clepticus by evaluating the evolutionary timing of the effect of the mid-Atlantic barrier, which separates C. brasiliensis and C. africanus, and the Amazon barrier, which separates C. parrae and C brasiliensis. Genetic data from three mitochondrial genes and one nuclear gene were used. Mitochondrial genes separated Clepticus into three well supported clades corresponding to the three recognized allopatric morpho-species. All analyses provided consistent support for an initial separation (~9.68 to 1.86 mya; 4.84% sequence divergence) of the Caribbean and South Atlantic lineages, followed by a much more recent divergence (~ 0.60 to 0.12 mya; 0.3% sequence divergence) of the Brazilian and African sister morpho-species. Both these phylogenetic events occurred well after the formation of the two barriers that currently separate those three allopatric populations. The planktonic larval duration of these species (35-49 days) and coastal pelagic habits may have facilitated dispersal by this genus across those dispersal barriers after they formed.

Isolation and characterization of 13 polymorphic nuclear microsatellite primers for the widespread Indo-Pacific three-spot damselfish, Dascyllus trimaculatus, and closely related D. auripinnis.

A set of 13 simple sequence repeat markers was developed from D. trimaculatus genomic DNA, tested for D. auripinnis and characterized using 40 individuals per species. All the loci were polymorphic with a number of alleles ranging from three to 30. Observed heterozygosities varied from 0.23 to 0.89 for D. trimaculatus and from 0.11 to 0.85 for D. auripinnis. Early results show that these will be powerful markers for the study of ecological and evolutionary mechanism in this coral reef fish species complex.