Sampling multiple life stages significantly increases estimates of marine biodiversity.

Biodiversity assessments are critical for setting conservation priorities, understanding ecosystem function and establishing a baseline to monitor change. Surveys of marine biodiversity that rely almost entirely on sampling adult organisms underestimate diversity because they tend to be limited to habitat types and individuals that can be easily surveyed. Many marine animals have planktonic larvae that can be sampled from the water column at shallow depths. This life stage often is overlooked in surveys but can be used to relatively rapidly document diversity, especially for the many species that are rare or live cryptically as adults. Using DNA barcode data from samples of nemertean worms collected in three biogeographical regions-Northeastern Pacific, the Caribbean Sea and Eastern Tropical Pacific-we found that most species were collected as either benthic adults or planktonic larvae but seldom in both stages. Randomization tests show that this deficit of operational taxonomic units collected as both adults and larvae is extremely unlikely if larvae and adults were drawn from the same pool of species. This effect persists even in well-studied faunas. These results suggest that sampling planktonic larvae offers access to a different subset of species and thus significantly increases estimates of biodiversity compared to sampling adults alone. Spanish abstract is available in the electronic supplementary material.

Tropical dead zones and mass mortalities on coral reefs.

Degradation of coastal water quality in the form of low dissolved oxygen levels (hypoxia) can harm biodiversity, ecosystem function, and human wellbeing. Extreme hypoxic conditions along the coast, leading to what are often referred to as "dead zones," are known primarily from temperate regions. However, little is known about the potential threat of hypoxia in the tropics, even though the known risk factors, including eutrophication and elevated temperatures, are common. Here we document an unprecedented hypoxic event on the Caribbean coast of Panama and assess the risk of dead zones to coral reefs worldwide. The event caused coral bleaching and massive mortality of corals and other reef-associated organisms, but observed shifts in community structure combined with laboratory experiments revealed that not all coral species are equally sensitive to hypoxia. Analyses of global databases showed that coral reefs are associated with more than half of the known tropical dead zones worldwide, with >10% of all coral reefs at elevated risk for hypoxia based on local and global risk factors. Hypoxic events in the tropics and associated mortality events have likely been underreported, perhaps by an order of magnitude, because of the lack of local scientific capacity for their detection. Monitoring and management plans for coral reef resilience should incorporate the growing threat of coastal hypoxia and include support for increased detection and research capacity.

Early Activation of MAPK and Apoptosis in Nutritive Embryos of Calyptraeid Gastropods.

Investigation of alternative phenotypes, different morphologies produced by a single genome, has contributed novel insights into development and evolution. Yet, the mechanisms underlying developmental switch points between alternative phenotypes remain poorly understood. The calyptraeid snails Crepidula navicella and Calyptraea lichen produce two phenotypes: viable and nutritive embryos, where nutritive embryos arrest their development after gastrulation and are ingested by their viable siblings as a form of intracapsular nutrition. Here, we investigate the activity of mitogen-activated protein kinase (MAPK, ERK1/2) and apoptosis during early cleavage. MAPK and apoptosis, found in a previous transcriptomic study, are known to be involved in organization of other spiralian embryos and nutritive embryo development, respectively. In the model Crepidula fornicata, MAPK activation begins at the 16-cell stage. In contrast, we discovered in C. navicella and C. lichen that many embryos begin MAPK activation at the one-cell stage. A subset of embryos shows a similar pattern of MAPK activation to C. fornicata at later stages. In all stages where MAPK is detected, the activation pattern is highly variable, frequently occurring in all quadrants or in multiple tiers of cells. We also detected apoptosis in cleaving embryos, while C. fornicata and Crepidula lessoni, which do not produce nutritive embryos, show no signs of apoptosis during cleavage. Our results show that MAPK and apoptosis are expressed during early development in species with nutritive embryos, and raises the possibility that these processes may play a role and even interact with one another in producing the nutritive embryo phenotype.

Comparative Transcriptomics of Alternative Developmental Phenotypes in a Marine Gastropod.

Alternative phenotypes are discrete phenotypic differences that develop in response to both genetic and environmental cues. Nutritive embryos, which arrest their development to serve as nutrition for their viable siblings, are an example of an alternative developmental phenotype found in many animal groups. Females of the marine snail, Crepidula navicella, produce broods that consist mainly of nutritive embryos and a small number of viable embryos. In order to better understand the genetic mechanisms that lead to the development of alternative phenotypes in this species, we compared the transcriptomes of viable and nutritive embryos at the earliest stage that we were able to distinguish visually between the two. Using high-throughput Illumina sequencing, we assembled and annotated a de novo transcriptome and compared transcript levels in viable and nutritive embryos. Viable embryos express high levels of transcripts associated with known developmental events, while nutritive embryos express high levels of apoptosis-related transcripts. Gene Ontology term enrichment with GOSeq found that these are associated with the negative regulation of apoptotic processes. This enrichment, combined with morphological evidence, suggests that apoptosis is important in the formation of gastrula-like nutritive embryos. Apoptosis has been implicated in the development of alternative phenotypes in other animal groups, raising the possibility that this mechanism's role in alternative phenotypes is conserved in gastropod development. We suggest possible alternative mechanisms of nutritive embryo development. Most importantly, we contribute further evidence to the hypothesis that nutritive embryos are an alternative developmental phenotype.

Once Is Not Enough: Seasonal Patterns in Abundance of Hydromedusae Are Not Consistent in the Coastal Waters of the Bay of Panama.

Hydromedusae and other gelatinous zooplankton are poorly understood but important consumers in the plankton. Blooms of large-bodied medusae which may have detrimental ecological impacts have captured significant attention, yet the seasonal patterns of local abundance and diversity, and the factors that control them, are not well documented for the much more diverse tiny hydromedusae. There is virtually no published information on their phenology or ecology in tropical coastal systems. It has previously been suggested that increased productivity stimulated by upwelling promotes production of tiny hydromedusa and blooms as their abundance can be much higher during upwelling than during non-upwelling periods in the tropics. However, this conclusion was based on a single year of sampling in the Bay of Panama. To determine if a single year of sampling is sufficient to understand seasonal patterns in abundance and occurrence of blooms, we repeated this study 20 years later. Sampling 153 times between April 2022 and April 2023, we found similar genus-level diversity and overall counts of animals compared to the previous study, with medusae of Bougainvillia, Campalecium, Obelia, and Turritopsis accounting for 79% of the animals collected. However, we found that relative abundance was much higher during the non-upwelling season compared to the upwelling season, the opposite result compared to the previous study. These results highlight how strong baseline understanding of phenologies and the ecological conclusions drawn from them must be based on multiple years of data, even in regions with very strong seasonal changes in environmental conditions. In the case of the upwelling regions of the Tropical Eastern Pacific, further research is necessary to understand the generalizability of these patterns, and to determine if contrasting results are due to inter-annual differences in oceanographic drivers or changing environmental baselines.

Frequency of Temperature Fluctuations Subtly Impacts the Life Histories of a Tropical Snail.

AbstractMost organisms are faced with daily cyclic changes in a suite of environmental conditions, including temperature. In shallow marine waters, populations of the same species may experience either intertidal or subtidal conditions, such that some individuals experience extreme daily fluctuations as the tide ebbs and flows, while others only a few meters away experience less pronounced or less frequent fluctuations or almost constant thermal conditions. This study used a fully factorial combination of three thermal treatments and two diet treatments to test the hypotheses that (1) individuals experiencing fluctuating temperatures perform more poorly than those experiencing the same mean temperature under constant conditions and that (2) the negative impact of fluctuating temperatures is greater under food-limiting conditions. Five life-history components of the slipper snail Crepidula cf. marginalis were used as response variables. We found that temperature fluctuations impacted size at hatching and time to hatching, as well as growth rate, to some extent. Diet treatments impacted growth rates, clutch sizes, time to first brood, and time to hatching. There were no statistically significant interactions between the two factors. These results show that fluctuations between two temperatures that are typically experienced by these animals in the field can significantly affect fitness-related characters and, therefore, suggest the tidal height at which larvae settle can significantly impact individual fitness. This is one of the first studies to demonstrate that differences in the frequency of fluctuations, in the absence of differences in the magnitude or the mean, can have significant impacts on invertebrate life histories.

Compound Extreme Events Induce Rapid Mortality in a Tropical Sea Urchin.

AbstractThe frequency, magnitude, and duration of marine heatwaves and deoxygenation events are increasing globally. Recent research suggests that their co-occurrence is more common than previously thought and that their combination can have rapid, dire biological impacts. We used the sea urchin Echinometra lucunter to determine whether mortality occurs faster when deoxygenation events are combined with extreme heating (compound events), compared to deoxygenation events alone. We also tested whether prior exposure to local heatwave conditions accentuates the impacts of compound events. Animals were first exposed for five days to either ambient temperature (28 °C) or a warmer temperature that met the minimum criteria for a local heatwave (30.5 °C). Animals were then exposed to hypoxia, defined as oxygen levels 35% below their average critical oxygen limit, combined with ambient or extreme field temperatures (28 °C, 32 °C). Subsets of animals were removed from the hypoxic treatments every 3 hours for 24 hours to determine how long they could survive. Prior exposure to heatwave conditions did not help or hinder survival under hypoxic conditions, and animals exposed to hypoxia under ambient temperatures experienced little mortality. However, when hypoxia was coupled with extreme temperatures (32 °C), 55% of the animals died within 24 hours. On the reefs at our Panama study site, we found that extreme hypoxic conditions only ever occurred during marine heatwave events, with four compound events occurring in 2018. These results show that short durations (∼1 day) of compound events can be catastrophic and that increases in their duration will severely threaten sea urchin populations.

Spanish AbstractLa frecuencia, magnitud y duración de las olas de calor marinas y los eventos de desoxigenación están aumentando a nivel mundial. Investigaciones recientes sugieren que su coocurrencia es más común de lo que se pensaba anteriormente y que su combinación puede tener impactos biológicos rápidos y nefastos. Usamos el erizo de mar Echinometra lucunter para determinar si la mortalidad de estos ocurre más rápido cuando los eventos de desoxigenación se combinan con un calentamiento extremo (eventos compuestos), en comparación con los eventos de desoxigenación solos. También probamos si la exposición previa a las condiciones locales de olas de calor acentúa los impactos de los eventos compuestos. Primero se expusieron a los animales durante cinco días a temperatura ambiente (28 °C) o una temperatura más cálida que cumpliera con los criterios mínimos para una ola de calor local (30.5 °C). Luego, los animales se expusieron a hipoxia, definida como niveles de oxígeno un 35% por debajo de su límite de oxígeno crítico promedio, combinado con temperaturas ambientales o de campo extremas (28 °C, 32 °C). Se retiraron los subconjuntos de animales de los tratamientos hipóxicos cada 3 horas durante 24 horas para determinar los tiempos de supervivencia. La exposición previa a condiciones de olas de calor no ayudó ni obstaculizó la supervivencia en condiciones hipóxicas, y los animales expuestos a hipoxia en temperatura ambiente experimentaron poca mortalidad. Sin embargo, cuando la hipoxia se combinó con temperaturas extremas (32 °C), el 55% de los animales murió en 24 horas. Las condiciones compuestas en nuestro sitio de estudio en Panamá fueron poco frecuentes y cortas (3 horas). Pero cuando ocurrieron, fueron durante olas de calor marinas, con un total de cuatro eventos compuestos observados en los arrecifes locales durante las olas de calor en 2018. Estos resultados muestran que las duraciones cortas (∼1 día) de eventos compuestos pueden ser catastróficas y aumentar debido al calentamiento, en su duración amenazarán severamente las poblaciones de erizos de mar.

Integrative Approaches to Understanding Organismal Responses to Aquatic Deoxygenation.

AbstractOxygen bioavailability is declining in aquatic systems worldwide as a result of climate change and other anthropogenic stressors. For aquatic organisms, the consequences are poorly known but are likely to reflect both direct effects of declining oxygen bioavailability and interactions between oxygen and other stressors, including two-warming and acidification-that have received substantial attention in recent decades and that typically accompany oxygen changes. Drawing on the collected papers in this symposium volume ("An Oxygen Perspective on Climate Change"), we outline the causes and consequences of declining oxygen bioavailability. First, we discuss the scope of natural and predicted anthropogenic changes in aquatic oxygen levels. Although modern organisms are the result of long evolutionary histories during which they were exposed to natural oxygen regimes, anthropogenic change is now exposing them to more extreme conditions and novel combinations of low oxygen with other stressors. Second, we identify behavioral and physiological mechanisms that underlie the interactive effects of oxygen with other stressors, and we assess the range of potential organismal responses to oxygen limitation that occur across levels of biological organization and over multiple timescales. We argue that metabolism and energetics provide a powerful and unifying framework for understanding organism-oxygen interactions. Third, we conclude by outlining a set of approaches for maximizing the effectiveness of future work, including focusing on long-term experiments using biologically realistic variation in experimental factors and taking truly cross-disciplinary and integrative approaches to understanding and predicting future effects.

Predator control of marine communities increases with temperature across 115 degrees of latitude.

Early naturalists suggested that predation intensity increases toward the tropics, affecting fundamental ecological and evolutionary processes by latitude, but empirical support is still limited. Several studies have measured consumption rates across latitude at large scales, with variable results. Moreover, how predation affects prey community composition at such geographic scales remains unknown. Using standardized experiments that spanned 115° of latitude, at 36 nearshore sites along both coasts of the Americas, we found that marine predators have both higher consumption rates and consistently stronger impacts on biomass and species composition of marine invertebrate communities in warmer tropical waters, likely owing to fish predators. Our results provide robust support for a temperature-dependent gradient in interaction strength and have potential implications for how marine ecosystems will respond to ocean warming.

Temperature-Induced Changes in Hatching Size of a Tropical Snail Occur During Oogenesis and Can Persist for Several Weeks.

AbstractIt is accepted that temperature affects offspring size in ectotherms. However, the processes that result in temperature-induced changes are not well understood. We sought to determine when temperature changes during development induce changes in hatching size and how long hatchlings reflect the previous thermal experiences of their mother. Juveniles of the common tropical slipper snail Crepidula cf. marginalis were collected at Playa Venado, Panama; were raised in the laboratory at either 24 °C or 28 °C, temperatures experienced in nature; and were reciprocally moved between the two temperatures. In the first experiment, the animals were moved immediately after oviposition to determine whether temperatures experienced during oogenesis or embryogenesis contribute to differences in hatching size. The second experiment transplanted animals between the same two temperatures after the first brood hatched. The subsequent three broods were measured to determine how long the legacy of the first temperature persists. We found that (i) the temperature the mother experienced during oogenesis significantly affects hatching size, whereas the temperature experienced during embryogenesis does not; and (ii) hatching size is impacted for at least two broods after a change in temperature (≥17 days). These results show that hatching size is a legacy of temperatures experienced prior to oviposition and that this legacy does not persist for more than two brooding cycles. It remains unclear whether this rapid response to environmental temperature is adaptive or the result of a physiological constraint on oogenesis. Understanding the process whereby temperature influences offspring size will provide insight into the potential for organisms to respond to temperature changes and, ultimately, climate change.

Ancestral form and function of larval feeding structures are retained during the development of non-planktotrophic gastropods.

Mode of development (MOD) is a key feature that influences the rate and direction of evolution of marine invertebrates. Although many groups include species with different MODs, the evolutionary loss of feeding larvae is thought to be irreversible, as the complex structures used for larval feeding and swimming are lost, reduced, or modified in many species lacking feeding larvae. This view is largely based on observations of echinoderms. Phylogenetic analysis suggests that feeding larvae have been re-gained in at least one species of calyptraeid gastropod. Further, its sister species has retained the velum, the structure used for larval feeding and swimming. Here, we document velar morphology and function in calyptraeids with 4 different MODs. Embryos of Crepidula navicella, Crepidula atrasolea, Bostrycapulus aculeatus, Bostrycapulus odites, Bostrycapulus urraca, Crepipatella dilatata, Crepipatella occulta, Crucibulum quiriquinae and Crepidula coquimbensis all hatch as crawling juveniles, yet only Crepidula coquimbensis does not make a well-formed velum during intracapsular development. The velar dimensions of 6 species with non-planktotrophic development were similar to those of planktotrophic species, while the body sizes were significantly larger. All of the species studied were able to capture and ingest particles from suspension, but several non-planktotrophic species may ingest captured particles only occasionally. Video footage suggests that some species with adelphophagic direct development capture but frequently fail to ingest particles compared to species with the other MODs. Together these lines of evidence show that, among calyptraeids at least, species that lack planktotrophic larvae often retain the structures and functions necessary to successfully capture and ingest particles, reducing the barriers to the re-evolution of planktotrophy.

Oxygen-mediated plasticity confers hypoxia tolerance in a corallivorous polychaete.

There is mounting evidence that the deoxygenation of coastal marine ecosystems has been underestimated, particularly in the tropics. These physical conditions appear to have far-reaching consequences for marine communities and have been associated with mass mortalities. Yet little is known about hypoxia in tropical habitats or about the effects it has on reef-associated benthic organisms. We explored patterns of dissolved oxygen (DO) throughout Almirante Bay, Panama and found a hypoxic gradient, with areas closest to the mainland having the largest diel variation in DO, as well as more frequent persistent hypoxia. We then designed a laboratory experiment replicating the most extreme in situ DO regime found on shallow patch reefs (3 m) to assess the response of the corallivorous fireworm, Hermodice carnaculata to hypoxia. Worms were exposed to hypoxic conditions (8 hr ~ 1 mg/L or 3.2 kPa) 16 times over an 8-week period, and at 4 and 8 weeks, their oxygen consumption (respiration rates) was measured upon reoxygenation, along with regrowth of severed gills. Exposure to low DO resulted in worms regenerating significantly larger gills compared to worms under normoxia. This response to low DO was coupled with an ability to maintain elevated oxygen consumption/respiration rates after low DO exposure. In contrast, worms from the normoxic treatment had significantly depressed respiration rates after being exposed to low DO (week 8). This indicates that oxygen-mediated plasticity in both gill morphology and physiology may confer tolerance to increasingly frequent and severe hypoxia in one important coral predator associated with reef decline.

World Travelers: DNA Barcoding Unmasks the Origin of Cloning Asteroid Larvae from the Caribbean.

AbstractThe identity of wild cloning sea star larvae has been a mystery since they were first documented in the Caribbean. The most commonly collected cloning species was thought to belong to the Oreasteridae, on the basis of similarity with sequences from Oreaster reticulatus and Oreaster clavatus. This larval form has recently been linked to a rare benthic juvenile. As part of two larger DNA barcoding projects, we collected cloning asteroid larvae from the Caribbean coast of Panama and compared them to a large reference database of tropical echinoderms. Morphological and DNA barcode data from the cytochrome c oxidase subunit I gene demonstrated that Panamanian larvae belonged to the same operational taxonomic unit as those recovered in previous studies of cloning larvae from the Caribbean. Much to our surprise, sequences from these larvae clearly identified them as belonging to Valvaster striatus, a species typically considered to be endemic to the Indo-West Pacific. A lineage of Mithrodia clavigera that occurs in both the Caribbean and the Indo-West Pacific also has cloning larvae, suggesting that this unusual life history has allowed larvae to pass around the Cape of Good Hope and the Benguela upwelling region, which is a barrier to dispersal for most tropical marine invertebrates.

Documenting decapod biodiversity in the Caribbean from DNA barcodes generated during field training in taxonomy.

DNA barcoding is a useful tool to identify the components of mixed or bulk samples, as well as to determine individuals that lack morphologically diagnostic features. However, the reference database of DNA barcode sequences is particularly sparsely populated for marine invertebrates and for tropical taxa. We used samples collected as part of two field courses, focused on graduate training in taxonomy and systematics, to generate DNA sequences of the barcode fragments of cytochrome c oxidase subunit I (COI) and mitochondrial ribosomal 16S genes for 447 individuals, representing at least 129 morphospecies of decapod crustaceans. COI sequences for 36% (51/140) of the species and 16S sequences for 26% (37/140) of the species were new to GenBank. Automatic Barcode Gap Discovery identified 140 operational taxonomic units (OTUs) which largely coincided with the morphospecies delimitations. Barcode identifications (i.e. matches to identified sequences) were especially useful for OTUs within Synalpheus, a group that is notoriously difficult to identify and rife with cryptic species, a number of which we could not identify to species, based on morphology. Non-concordance between morphospecies and barcode OTUs also occurred in a few cases of suspected cryptic species. As mitochondrial pseudogenes are particularly common in decapods, we investigate the potential for this dataset to include pseudogenes and discuss the utility of these sequences as species identifiers (i.e. barcodes). These results demonstrate that material collected and identified during training activities can provide useful incidental barcode reference samples for under-studied taxa.

Larvae of Caribbean Echinoids Have Small Warming Tolerances for Chronic Stress in Panama.

In species with complex life cycles, early developmental stages are often less thermally tolerant than adults, suggesting that they are key to predicting organismal response to environmental warming. Here we document the optimal and lethal temperatures of larval sea urchins, and we use those to calculate the warming tolerance and the thermal safety margin of early larval stages of seven tropical species. Larvae of Echinometra viridis, Echinometra lucunter, Lytechinus williamsi, Eucidaris tribuloides, Tripneustes ventricosus, Clypeaster rosaceus, and Clypeaster subdepressus were reared at 26, 28, 30, 32, and 34 °C for 6 days. The temperatures at which statistically significant reductions in larval performance are evident are generally the same temperatures at which statistically significant reductions in larval survival were detected, showing that the optimal temperature is very close to the lethal temperature. The two Echinometra species had significantly higher thermal tolerance than the other species, with some surviving culture temperatures of 34 °C and showing minimal impacts on growth and survival at 32 °C. In the other species, larval growth and survival were depressed at and above 30 or 32 °C. Overall, these larvae have lower warming tolerances (1 to 5 °C) and smaller thermal safety margins (-3 to 3 °C) than adults. Survival differences among treatments were evident by the first sampling on day 2, and survival at the highest temperatures increased when embryos were exposed to warming after spending the first 24 hours at ambient temperature. This suggests that the first days of development are more sensitive to thermal stress than are later larval stages.

Combined effects of temperature, salinity, and diet simulating upwelling and nonupwelling seasons alter life-history characteristics of a tropical invertebrate.

Upwelling is known to affect the ecology and life history of temperate nearshore organisms, and these effects are thought to be mediated by changes in temperature and food supply. However, little information is available for tropical systems. To understand how changes in the intensity of upwelling might impact marine invertebrates, we tested how factorial combinations of temperature, salinity, and phytoplankton availability affected growth and reproduction of a common intertidal snail, Crepidula cf. marginalis. We used temperatures typical of nonupwelling (29°C), moderate (26°C) and severe (23°C) upwelling, salinities typical of nonupwelling (30 ppt) and upwelling (34 ppt) and a good diet (Isochrysis) and a better diet (Isochrysis and Tetraselmis) as a proxy for increased productivity during upwelling. Overall, temperature and diet had consistent effects on body size, with better food and lower temperatures promoting larger size, as well as promoting shorter time to first reproduction. Diet had the largest effects on clutch size, with clutch size increasing with better diet. Temperature had the largest effect on offspring size and the frequency of discarded broods; offspring size decreased with increasing temperature and the frequency of discarded broods also decreased with increasing temperatures. We found no significant 3rd order interactions and few significant strong 2nd order interactions, which have often been found in similar experimental studies using stressful treatments. For this tropical slipper limpet, the effect of higher food and cooler temperatures during upwelling appears to be positive, promoting higher growth rates, larger clutch sizes, and larger offspring size suggesting that both factors likely play an important role underlying reproductive responses to upwelling. Climatic changes, like El Niño, which suppress upwelling in the Bay of Panama, appear likely to negatively impact this species.

Reference DNA barcodes and other mitochondrial markers for identifying Caribbean Octocorals.

DNA barcoding is a useful tool for documenting the diversity of metazoans. The most commonly used barcode markers, 16S and COI, are not considered suitable for species identification within some "basal" phyla of metazoans. Nevertheless metabarcoding studies of bulk mixed samples commonly use these markers and may obtain sequences for "basal" phyla. We sequenced mitochondrial DNA fragments of cytochrome oxidase c subunit I (COI), 16S ribosomal RNA (16S), NADH dehydrogenase subunits 2 (16S-ND2), 6 (ND6-ND3) and 4L (ND4L-MSH) for 27 species of Caribbean octocorals to create a reference barcode dataset and to compare the utility of COI and 16S to other markers more typically used for octocorals. The most common genera (Erythropodium, Ellisella, Briareum, Plexaurella, Muriceopsis and Pterogorgia) were effectively distinguished by small differences (5 or more substitutions or indels) in COI and 16S sequences. Gorgonia and Antillogorgia were effectively distinguished from each other by unique haplotypes, but the small genetic differences make distance approaches ineffective for these taxa. Plexaura, Pseudoplexaura and Eunicea were indistinguishable from each other but were generally effectively distinguished from other genera, further supporting the idea that these genera have undergone a rapid endemic radiation in the Caribbean.

Correction: Widespread local chronic stressors in Caribbean coastal habitats.

[This corrects the article DOI: 10.1371/journal.pone.0188564.].

Molecular phylogenetic and embryological evidence that feeding larvae have been reacquired in a marine gastropod.

Evolutionary transitions between different modes of development in marine invertebrates are thought to be biased toward the loss of feeding larvae. Because the morphology of feeding larvae is complex and nonfeeding larvae or encapsulated embryos with benthic development often have simplified morphologies, it is presumed to be easier to lose a larval stage than to reacquire it. Some authors have gone so far as to suggest that feeding larvae, morphologically similar to the ancestral feeding larvae, cannot be reacquired. However, the larval structures of some groups, most notably gastropods, are often retained in the encapsulated embryos of species that hatch as benthic juveniles. Therefore the re-evolution of feeding larvae using the same structures may be possible in these groups. Here we present the first well-substantiated case for the recent re-evolution of feeding larvae within a clade of direct-developers. DNA sequence data show that Crepipatella fecunda, a species of calyptraeid gastropod with planktotrophic development, is nested within a clade of species with direct development, and that Crepipatella dilatata, a species with direct development, appears to be paraphyletic with respect to C. fecunda. Observation of the embryos of C. dilatata shows that the features necessary for larval feeding and swimming are retained in the encapsulated veligers, suggesting that heterochronic shifts in hatching time and changes in nurse-egg allotment could have resulted in the re-evolution of feeding larvae in this species.

Reproductive cycles in tropical intertidal gastropods are timed around tidal amplitude cycles.

Reproduction in iteroparous marine organisms is often timed with abiotic cycles and may follow lunar, tidal amplitude, or daily cycles. Among intertidal marine invertebrates, decapods are well known to time larval release to coincide with large amplitude nighttime tides, which minimizes the risk of predation. Such bimonthly cycles have been reported for few other intertidal invertebrates. We documented the reproduction of 6 gastropod species from Panama to determine whether they demonstrate reproductive cycles, whether these cycles follow a 2-week cycle, and whether cycles are timed so that larval release occurs during large amplitude tides. Two of the species (Crepidula cf. marginalis and Nerita scabricosta) showed nonuniform reproduction, but without clear peaks in timing relative to tidal or lunar cycles. The other 4 species show clear peaks in reproduction occurring every 2 weeks. In 3 of these species (Cerithideopsis carlifornica var. valida, Littoraria variegata, and Natica chemnitzi), hatching occurred within 4 days of the maximum amplitude tides. Siphonaria palmata exhibit strong cycles, but reproduction occurred during the neap tides. Strong differences in the intensity of reproduction of Cerithideopsis carlifornica, and in particular, Littoraria variegata, between the larger and smaller spring tides of a lunar month indicate that these species time reproduction with the tidal amplitude cycle rather than the lunar cycle. For those species that reproduce during both the wet and dry seasons, we found that reproductive timing did not differ between seasons despite strong differences in temperature and precipitation. Overall, we found that most (4/6) species have strong reproductive cycles synchronized with the tidal amplitude cycle and that seasonal differences in abiotic factors do not alter these cycles.