DNA-metabarcoding supports trophic flexibility and reveals new prey species for the Galapagos sea lion.

Tropical ecosystems are challenging for pinnipeds due to fluctuating food availability. According to previous research, the Galapagos sea lion (GSL, Zalophus wollebaeki) adopts trophic flexibility to face such conditions. However, this hypothesis comes from studies using traditional methods (hard-parts analysis of scat and isotopic analysis from tissue). We studied the diet of five rookeries in the southeastern Galapagos bioregion (which harbors the highest GSL density), via DNA-metabarcoding of scat samples. The DNA-metabarcoding approach may identify consumed prey with a higher taxonomic resolution than isotopic analysis, while not depending on hard-parts remaining through digestion. Our study included five different rookeries to look for evidence of trophic flexibility at the bioregional level. We detected 98 prey OTUs (124 scats), mostly assigned to bony-fish taxa; we identified novel prey items, including a shark, rays, and several deep-sea fish. Our data supported the trophic flexibility of GSL throughout the studied bioregion since different individuals from the same rookery consumed prey coming from different habitats and trophic levels. Significant diet differentiations were found among rookeries, particularly between Punta Pitt and Santa Fe. Punta Pitt rookery, with a more pronounced bathymetry and lower productivity, was distinguished by a high trophic level and consumption of a high proportion of deep-sea prey; meanwhile, Santa Fe, located in more productive, shallow waters over the shelf, consumed a high proportion of epipelagic planktivorous fish. Geographic location and heterogeneous bathymetry of El Malecon, Española, and Floreana rookeries would allow the animals therein to access both, epipelagic prey over the shelf, and deep-sea prey out of the shelf; this would lead to a higher prey richness and diet variability there. These findings provide evidence of GSL adopting a trophic flexibility to tune their diets to different ecological contexts. This strategy would be crucial for this endangered species to overcome the challenges faced in a habitat with fluctuating foraging conditions.

Seroconversion in Galapagos Sea Lions (Zalophus wollebaeki) Confirms the Presence of Canine Distemper Virus in Rookeries of San Cristóbal Island.

BACKGROUND: The emblematic Galapagos sea lion (GSL-Zalophus wollebaeki) has faced an important population decline over the last four decades. There are multiple environmental and biological factors that might be implied in this decrease. Recently, evidence of various zoonotic infectious diseases that can be potential threats has been reported. Considering that in some islands of the archipelago the risk of transmission of infectious diseases may be promoted by the increasing population of domestic dogs, epidemiological vigilance and search of new pathogens are essential. The canine distemper virus (CDV), one of the viral pathogens that generate the most concern for the agencies responsible for the management and conservation of the Galapagos pinnipeds, was detected in the GSL in 2010. However, there is scarce information about its impact on GSL health and about its epidemiology. METHODS: In this study, 110 GSL serum samples were collected during the summer of 2016 and 2017. All samples were exposed to VERO dog SLAM cells expressing the canine SLAM receptor. RESULTS: Our results showed a significative increase (p = 0.04) in the frequency of neutralizing antibodies to CDV in the 2017 (53.1%) samples compared to the 2016 samples (19.6%). CONCLUSIONS: Our work confirmed the continuous and increasing circulation of the CDV in the GSL and highlights the importance of monitoring emerging diseases that can be transmitted from domestic to wildlife species. Vigilance of CDV is essential to understand the role of this virus in GSL mortality and to take informed decisions for wildlife conservation.

Time-calibrated phylogeny and full mitogenome sequence of the Galapagos sea lion (Zalophus wollebaeki) from scat DNA.

Background: The Galapagos sea lion, Zalophus wollebaeki, is an endemic and endangered otariid, which is considered as a sentinel species of ecosystem dynamics in the Galapagos archipelago. Mitochondrial DNA is an important tool in phylogenetic and population genetic inference. In this work we use Illumina sequencing to complement the mitogenomic resources for Zalophus genus-the other two species employed Sanger sequencing-by a complete mitochondrial genome and a molecular clock of this species, which is not present in any case. Materials and Methods: We used DNA obtained from a fresh scat sample of a Galapagos sea lion and shotgun-sequenced it on the Illumina NextSeq platform. The obtained raw reads were processed using the GetOrganelle software to filter the mitochondrial Zalophus DNA reads (∼16% survive the filtration), assemble them, and set up a molecular clock. Results: From the obtained 3,511,116 raw reads, we were able to assemble a full mitogenome of a length of 16,676 bp, consisting of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNA), and two ribosomal RNAs (rRNA). A time-calibrated phylogeny confirmed the phylogenetic position of Z. wollebaeki in a clade with Z. californianus, and Z. japonicus, and sister to Z. californianus; as well as establishing the divergence time for Z. wollebaeki 0.65 million years ago. Our study illustrates the possibility of seamlessly sequencing full mitochondrial genomes from fresh scat samples of marine mammals.

Detection and quantification of microplastic pollution in the endangered Galapagos sea lion.

Marine debris pollution poses a significant global threat to biodiversity, with plastics being the primary debris type found in oceans due to their low-cost production and high demand worldwide. Microplastics (MPs, <5 mm in size) are highly bioavailable to a wide range of marine taxa, including marine mammals, through direct and indirect ingestion routes (i.e., trophic transfer). Recently, MP pollution has been detected on the Galapagos Marine Reserve, so in this study we developed a baseline framework for MP pollution in the Galapagos sea lion (GSL, Zalophus wollebaeki) through scat-based analysis. We collected 180 GSL scat samples from the southeast region following strict quality assurance/quality control protocols to detect, quantify and characterize physical-chemical properties of MPs through visual observations and µFT-IR spectroscopy. We recovered 81 MPs of varying sizes and colors in 37 % of samples (n = 66/180), consisting mostly of fibers (69 %, x¯ = 0.31 ± 0.57 particles scat-1). The number of particles per gram of sample wet weight ranged from 0.02 to 0.22 (x¯ = 0.04 ± 0.05 particles scat wet g-1). El Malecón and Punta Pitt rookeries at San Cristobal Island had the highest number of MPs (x¯ = 0.67 ± 0.51 and 0.43 ± 0.41 particles scat-1, respectively), and blue-colored particles were the most common in all samples. We identified eleven polymers in 46 particles, consisting mostly of polypropylene-polyethylene copolymer, polypropylene, cellulose, polyethylene, and polyvinyl chloride. The textile, fishing, and packaging industries are likely significant sources of microfibers into this insular ecosystem. Our results suggest that the GSL is exposed to MPs due to anthropogenic contamination that is subsequently transferred through trophic processes. These findings provide an important baseline framework and insights for future research on MP pollution in the region, as well as for management actions that will contribute to the long-term conservation of the GSL.

Correction: Culda et al. The Presence of Dirofilaria immitis in Domestic Dogs on San Cristobal Island, Galapagos. Pathogens 2022, 11, 1287.

There was an error in the original publication [...].

Genomic evidence for homoploid hybrid speciation in a marine mammal apex predator.

Hybridization is widespread and constitutes an important source of genetic variability and evolution. In animals, its role in generating novel and independent lineages (hybrid speciation) has been strongly debated, with only a few cases supported by genomic data. The South American fur seal (SAfs) Arctocephalus australis is a marine apex predator of Pacific and Atlantic waters, with a disjunct set of populations in Peru and Northern Chile [Peruvian fur seal (Pfs)] with controversial taxonomic status. We demonstrate, using complete genome and reduced representation sequencing, that the Pfs is a genetically distinct species with an admixed genome that originated from hybridization between the SAfs and the Galapagos fur seal (Arctocephalus galapagoensis) ~400,000 years ago. Our results strongly support the origin of Pfs by homoploid hybrid speciation over alternative introgression scenarios. This study highlights the role of hybridization in promoting species-level biodiversity in large vertebrates.

Dirofilaria immitis Identified in Galapagos Sea Lions (Zalophus wollebaeki): A Wildlife Health and Conservation Concern.

The Galapagos sea lion (Zalophus wollebaeki), an endemic and endangered pinniped, faces an increasing threat due to infectious diseases related to domestic animals. Dirofilaria immitis, the parasite responsible for canine heartworm disease, is one such threat, as canine infections on the archipelago have been documented. We used a canine heartworm antigen test kit to analyze the blood from 25 juvenile Galapagos sea lions for D. immitis. Two (8%) sea lions tested positive for D. immitis antigen. Using morphologic and genetic assessments, we evaluated 20 filarial-like worms collected from within the heart of an adult male Galapagos sea lion during a previous routine postmortem examination. The intracardiac worms were morphologically consistent with adult D. immitis, and sequence analysis of targeted PCR amplicons confirmed their identity. This is the first report of D. immitis infection in Galapagos sea lions, which could become a major health problem for these pinnipeds. Further studies are necessary to confirm the level of threat from this parasite; however, widespread adoption of routine heartworm testing, prevention, and treatment in the canine population, and the control of mosquitos, could potentially reduce the disease impact on this endangered pinniped species.

The Presence of Dirofilaria immitis in Domestic Dogs on San Cristobal Island, Galapagos.

This study's concept was outlined around the principle of conservation medicine in a biodiversity hotspot from the Neotropical realm: the Galapagos Islands. The wildlife balance has been modified by multi-host parasites introduced with some domestic animals (dogs and cats). The endemic and endangered species, the Galapagos sea lion (GSL, Zalophus wollebaeki), has been exposed to pathogens of canine and feline origin that could become a significant conservation problem for this species. One of these potential cases is the filarial heartworm infection, Dirofilaria immitis, which has been reported on other pinnipeds, with fatalities and clinical symptoms. Therefore, this study evaluated the presence of the microfilaria of D. immitis in dogs from Puerto Baquerizo Moreno, San Cristobal Island, where the largest rookery of GSLs lives and where the proximity to domestic dogs is the most intimate compared to other rookeries of the archipelago. Between July and September 2021, 587 blood samples were collected from owned dogs of Puerto Baquerizo Moreno. Overall, 10 dogs (1.7%) were positive for the presence of the microfilaria of D. immitis with a confidence interval of 0.7%-2.8%. No other filarial species were identified. Significant differences in prevalence between different dog categories were observed only for the age (p = 0.001). This study represents the first report of D. immitis, the agent of canine heartworm disease, in dogs from San Cristobal Island. Hence, the presence of the microfilaria of D. immitis in the blood of dogs could increase the risk of infection to which the GSL is exposed in the region.

Using stable isotopes analysis to understand ontogenetic trophic variations of the scalloped hammerhead shark at the Galapagos Marine Reserve.

Changes in life-history requirements drive trophic variations, particularly in large marine predators. The life history of many shark species is still poorly known and understanding their dietary ontogeny is a challenging task, especially for highly migratory species. Stable isotope analysis has proven as a useful method for examining the foraging strategies of sharks and other marine predators. We assessed the foraging strategies and ontogenetic changes of scalloped hammerhead sharks, Sphyrna lewini, at Galapagos Marine Reserve (GMR), by analysing δ13C and δ15N signatures in different maturity stages. Our isotopic results suggest ontogenetic shifts in resource use between sub-adult and adult stages, but not between adult and juvenile stages. Carbon isotopic signatures found in the juvenile stage were enriched in contrast to sub-adults (~0.73‰) suggesting a combination of the maternal input and the use of coastal resources around the Galapagos Islands. Adult female sharks also showed enrichment in δ13C (~0.53‰) in comparison to sub-adult stages that suggest feeding in high primary productivity areas, such as the GMR. This study improves the understanding of the trophic ecology and ontogenetic changes of a highly migratory shark that moves across the protected and unprotected waters of the Eastern Tropical Pacific.

Population Genetics and Phylogeography of Galapagos Fur Seals.

Pinnipeds found across islands provide an ideal opportunity to examine the evolutionary process of population subdivision affected by several mechanisms. Here, we report the genetic consequences of the geographic distribution of rookeries in Galapagos fur seals (GFS: Arctocephalus galapagoensis) in creating population structure. We show that rookeries across four islands (nine rookeries) are genetically structured into the following major groups: 1) a western cluster of individuals from Fernandina; 2) a central group from north and east Isabela, Santiago, and Pinta; and possibly, 3) a third cluster in the northeast from Pinta. Furthermore, asymmetric levels of gene flow obtained from eight microsatellites found migration from west Isabela to Fernandina islands (number of migrants Nm = 1), with imperceptible Nm in any other direction. Our findings suggest that the marked structuring of populations recovered in GFS is likely related to an interplay between long-term site fidelity and long-distance migration in both male and female individuals, probably influenced by varying degrees of marine productivity.

Evaluating the Possibility of Transfusion Medicine, Through Crossmatching in Juvenile Galapagos Sea Lions (Zalophus wollebaeki).

The Galapagos sea lion (Zalophus wollebaeki) is an endemic pinniped to the Galapagos archipelago, and like most wild mammals, is at risk for anemia due to trauma, infectious disease, and poor nutrition. This study evaluated the health status of 26 juvenile Galapagos sea lions on the island of San Cristobal prior to evaluating 100 crossmatch combinations. On evaluation, all but one sea lion had no major systemic abnormalities. Of the 100 crossmatches performed, 23% had minor reactions. The most significant reaction was weak macroscopic agglutination found in 4% of samples. The small percentage of agglutination reactions suggests a small proportion of naturally occurring alloantibodies in this species and may be consistent with a low risk of acute immune-mediated hemolytic transfusion reaction.

Correction: A matter of taste: Spatial and ontogenetic variations on the trophic ecology of the tiger shark at the Galapagos Marine Reserve.

[This corrects the article DOI: 10.1371/journal.pone.0222754.].

Fish diversity patterns along coastal habitats of the southeastern Galapagos archipelago and their relationship with environmental variables.

Coastal habitats are essential for ecological processes and provide important ecosystem services. The Galapagos archipelago has a wide diversity of ichthyofauna which preservation guarantees the functioning of the marine ecosystem. In this study, we used ecological and taxonomic indices as well as multivariate analysis to identify spatiotemporal changes in fish community structure in coastal habitats of San Cristóbal Island in the southeastern Galapagos archipelago. We analyzed how the patterns of variability were related to the abiotic conditions (substrate, sea temperature and depth) of each habitat. Nine sites affected by anthropogenic influence (fishing and tourism) representing different habitats/substrates were sampled. Underwater surveys were conducted during the warm and cold seasons in 2010 and 2011 at transects that varied in depth according to site. Artificial habitat, followed by coral and rocky habitats, had the highest diversity, evenness, and taxonomic distinctness, while mangrove habitats had the lowest values. This was related to the habitat complexity and possible anthropogenic influences. While the diversity patterns were more strongly related to the type of substrate, followed by the combination of substrate and depth, and the sea temperature had less influence. These findings were related to the ecological traits of the fish communities and their mobility between habitats. Temporal changes in fish community diversity and composition were not detected at all sites, suggesting that these species have high fidelity to their habitats and a high environmental tolerance that allows them to persist in their habitats despite strong changes in sea temperature on the Galapagos archipelago.

A stable foraging polymorphism buffers Galápagos sea lions against environmental change.

Understanding the ability of animals to cope with a changing environment is critical in a world affected by anthropogenic disturbance.1 Individual foraging strategies may influence the coping ability of entire populations, as these strategies can be adapted to contrasting conditions, allowing populations with foraging polymorphisms to be more resilient toward environmental change.2,3 However, environmentally dependent fitness consequences of individual foraging strategies and their effects on population dynamics have not been conclusively documented.4,5 Here, we use biologging data from endangered Galápagos sea lion females (Zalophus wollebaeki) to show that benthically foraging individuals dig after sand-dwelling prey species while pelagic foragers hunt in more open waters. These specialized foraging behaviors result in distinct and temporally stable patterns of vibrissae abrasion. Using vibrissae length as a visual marker for the benthic versus pelagic foraging strategies, we furthermore uncovered an environment-dependent fitness trade-off between benthic and pelagic foragers, suggesting that the foraging polymorphism could help to buffer the population against the negative effects of climate change. However, demographic projections suggest that this buffering effect is unlikely to be sufficient to reverse the ongoing population decline of the past four decades.6 Our study shows how crucial a deeper understanding of behavioral polymorphisms can be for predicting how populations cope within a rapidly changing world. VIDEO ABSTRACT.

Protistan plankton communities in the Galápagos Archipelago respond to changes in deep water masses resulting from the 2015/16 El Niño.

The Galápagos Archipelago lies within the Eastern Equatorial Pacific Ocean at the convergence of major ocean currents that are subject to changes in circulation. The nutrient-rich Equatorial Undercurrent upwells from the west onto the Galápagos platform, stimulating primary production, but this source of deep water weakens during El Niño events. Based on measurements from repeat cruises, the 2015/16 El Niño was associated with declines in phytoplankton biomass at most sites throughout the archipelago and reduced utilization of nitrate, particularly in large-sized phytoplankton in the western region. Protistan assemblages were identified by sequencing the V4 region of the 18S rRNA gene. Dinoflagellates, chlorophytes and diatoms dominated most sites. Shifts in dinoflagellate communities were most apparent between the years; parasitic dinoflagellates, Syndiniales, were highly detected during the El Niño (2015) while the dinoflagellate genus, Gyrodinium, increased at many sites during the neutral period (2016). Variations in protistan communities were most strongly correlated with changes in subthermocline water density. These findings indicate that marine protistan communities in this region are regimented by deep water mass sources and thus could be profoundly affected by altered ocean circulation.

Whole blood fatty acid concentrations in the San Cristóbal Galápagos tortoise (Chelonoidis chathamensis).

To continue releasing San Cristóbal Galápagos tortoises housed in managed-care facilities at the Giant Tortoise Breeding Center of Galápagos National Park (Galapaguera de Cerro Colorado) to the Otoy Ecological Farm, health assessments and physical examinations were conducted. As a part of these wellness examinations, blood was drawn from 11 tortoises to analyze fatty acid concentrations. Fatty acid levels can provide insight into the nutritional profiles, immune status, and reproductive health of vertebrates. To the co-author's knowledge, there is no current information about fatty acids in this species. It was hypothesized that there would be inherent differences based on the different geographic ranges, diets, sex, and age of turtles. It was noted that the ω-6/ω-3 ratio was higher for the breeding center than for the ecological farm and that overall polyunsaturated fatty acids (PUFAs) did not have any significant differences. The ω-6/ω-3 findings can contribute to a global picture of these fatty acids across taxa, as reptiles are underrepresented in this area of research. Additional results are a resourceful starting point for future investigations into how fatty acids are affected in Galápagos tortoises.

International fisheries threaten globally endangered sharks in the Eastern Tropical Pacific Ocean: the case of the Fu Yuan Yu Leng 999 reefer vessel seized within the Galápagos Marine Reserve.

Shark fishing, driven by the fin trade, is the primary cause of global shark population declines. Here, we present a case study that exemplifies how industrial fisheries are likely depleting shark populations in the Eastern Tropical Pacific Ocean. In August 2017, the vessel Fu Yuan Yu Leng 999, of Chinese flag, was detained while crossing through the Galápagos Marine Reserve without authorization. This vessel contained 7639 sharks, representing one of the largest seizures recorded to date. Based on a sample of 929 individuals (12%), we found 12 shark species: 9 considered as Vulnerable or higher risk by the IUCN and 8 listed in CITES. Four species showed a higher proportion of immature than mature individuals, whereas size-distribution hints that at least some of the fishing ships associated with the operation may have been using purse-seine gear fishing equipment, which, for some species, goes against international conventions. Our data expose the magnitude of the threat that fishing industries and illegal trade represent to sharks in the Eastern Tropical Pacific Ocean.

Hormone-mediated foraging strategies in an uncertain environment: Insights into the at-sea behavior of a marine predator.

Hormones are extensively known to be physiological mediators of energy mobilization and allow animals to adjust behavioral performance in response to their environment, especially within a foraging context.Few studies, however, have narrowed focus toward the consistency of hormonal patterns and their impact on individual foraging behavior. Describing these relationships can further our understanding of how individuals cope with heterogeneous environments and exploit different ecological niches.To address this, we measured between- and within-individual variation of basal cortisol (CORT), thyroid hormone T3, and testosterone (TEST) levels in wild adult female Galápagos sea lions (Zalophus wollebaeki) and analyzed how these hormones may be associated with foraging strategies. In this marine predator, females exhibit one of three spatially and temporally distinct foraging patterns (i.e., "benthic," "pelagic," and "night" divers) within diverse habitat types.Night divers differentiated from other strategies by having lower T3 levels. Considering metabolic costs, night divers may represent an energetically conservative strategy with shorter dive durations, depths, and descent rates to exploit prey which migrate up the water column based on vertical diel patterns.Intriguingly, CORT and TEST levels were highest in benthic divers, a strategy characterized by congregating around limited, shallow seafloors to specialize on confined yet reliable prey. This pattern may reflect hormone-mediated behavioral responses to specific risks in these habitats, such as high competition with conspecifics, prey predictability, or greater risks of predation.Overall, our study highlights the collective effects of hormonal and ecological variation on marine foraging. In doing so, we provide insights into how mechanistic constraints and environmental pressures may facilitate individual specialization in adaptive behavior in wild populations.

Influence of hunting strategy on foraging efficiency in Galapagos sea lions.

The endangered Galapagos sea lion (GSL, Zalophus wollebaeki) exhibits a range of foraging strategies utilising various dive types including benthic, epipelagic and mesopelagic dives. In the present study, potential prey captures (PPC), prey energy consumption and energy expenditure in lactating adult female GSLs (n = 9) were examined to determine their foraging efficiency relative to the foraging strategy used. Individuals displayed four dive types: (a) epipelagic (<100 m; EP); or (b) mesopelagic (>100 m; MP) with a characteristic V-shape or U-shape diving profile; and (c) shallow benthic (<100 m; SB) or (d) deep benthic (>100 m; DB) with square or flat-bottom dive profiles. These dive types varied in the number of PPC, assumed prey types, and the energy expended. Prey items and their energetic value were assumed from previous GSL diet studies in combination with common habitat and depth ranges of the prey. In comparison to pelagic dives occurring at similar depths, when diving benthically, GSLs had both higher prey energy consumption and foraging energy expenditure whereas PPC rate was lower. Foraging efficiency varied across dive types, with benthic dives being more profitable than pelagic dives. Three foraging trip strategies were identified and varied relative to prey energy consumed, energy expended, and dive behaviour. Foraging efficiency did not significantly vary among the foraging trip strategies suggesting that, while individuals may diverge into different foraging habitats, they are optimal within them. These findings indicate that these three strategies will have different sensitivities to habitat-specific fluctuations due to environmental change.

Declines and recovery in endangered Galapagos pinnipeds during the El Niño event.

Currently, the Galapagos sea lion (GSL, Zalophus wollebaeki) and Galapagos fur seal (GFS, Arctocephalus galapagoensis) are among the most important endemic species for conservation in the Galapagos Archipelago. Both are classified as "Endangered" since their populations have undergone drastic declines over the last several decades. In this study we estimated the abundance of both otariids, and their population trends based using counts conducted between 2014 and 2018 in all their rookeries, and we analyzed the influence of environmental variability on pup production. The GSL population size in 2018 in the archipelago was estimated to be between 17,000 to 24,000 individuals and has increased at an average annual rate of 1% over the last five years after applying correction factors. The highest number of GSL counted in the archipelago was in 2014 followed by a population decline of 23.8% in 2015 that was associated with the El Niño event that occurred during that year. Following this event, the population increased mainly in the northern, central and southeastern rookeries. The GSL pup abundance showed a decreasing trend with the increase in intensity of the El Niño. The GFS population in 2018 was counted in 3,093 individuals and has increased at an annual rate of 3% from 2014 to 2018. A high number of GFS counted in 2014 was followed by a population decrease of 38% in 2015, mainly in the western rookeries. There was interannual population fluctuations and different growth trends among regions of the archipelago. GSL and GFS pup abundance has a strong decreasing tendency with the increase in the subthermocline temperature (ST) and the El Niño 1 + 2 index. Our results provide evidence that both species are highly vulnerable to periodic oceanographic-atmospheric events in the Galapagos Archipelago which impact prey abundance and the flow of energy in the unique Galapagos ecosystem.