Abstract: Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species. Description Editor’s summary DNA methylation installs a methyl group to cytosine, placing an epigenetic mark that regulates gene expression. Comparative epigenomics combines epigenetic signatures with phylogenetic relationships to understand species characteristics. Haghani et al. evaluated methylation levels in highly conserved DNA sequences, profiling ~15,000 samples across 348 mammalian species (see the Perspective by de Mendoza). Phylogenetic trees suggest that the divergence of DNA methylation profiles closely mirrors genetic evolution. Species with longer maximum life spans have developed tidier methylation patterns within the genome, characterized by unique peaks and troughs of methylation. Methylation patterns associated with maximum life spans generally differ from those connected to age or interventions that affect mortality risk in mice. These data provide a rich resource of information for fields including evolutionary biology and longevity research. —Di Jiang Phyloepigenetic trees derived from DNA methylation profiles mirror mammalian evolution and are related to traits including maximum life span. INTRODUCTION Comparative epigenomics is an emerging field that combines epigenetic signatures with phylogenetic relationships to elucidate species characteristics such as maximum life span. For this study, we generated cytosine DNA methylation (DNAm) profiles (n = 15,456) from 348 mammalian species using a methylation array platform that targets highly conserved cytosines. RATIONALE Nature has evolved mammalian species of greatly differing life spans. To resolve the relationship of DNAm with maximum life span and phylogeny, we performed a large-scale cross-species unsupervised analysis. Comparative studies in many species enables the identification of epigenetic correlates of maximum life span and other traits. RESULTS We first tested whether DNAm levels in highly conserved cytosines captured phylogenetic relationships among species. We constructed phyloepigenetic trees that paralleled the traditional phylogeny. To avoid potential confounding by different tissue types, we generated tissue-specific phyloepigenetic trees. The high phyloepigenetic-phylogenetic congruence is due to differences in methylation levels and is not confounded by sequence conservation. We then interrogated the extent to which DNA methylation associates with specific biological traits. We used an unsupervised weighted correlation network analysis (WGCNA) to identify clusters of highly correlated CpGs (comethylation modules). WGCNA identified 55 distinct comethylation modules, of which 30 were significantly associated with traits including maximum life span, adult weight, age, sex, human mortality risk, or perturbations that modulate murine life span. Both the epigenome-wide association analysis (EWAS) and eigengene-based analysis identified methylation signatures of maximum life span, and most of these were independent of aging, presumably set at birth, and could be stable predictors of life span at any point in life. Several CpGs that are more highly methylated in long-lived species are located near HOXL subclass homeoboxes and other genes that play a role in morphogenesis and development. Some of these life span–related CpGs are located next to genes that are also implicated in our analysis of upstream regulators (e.g., ASCL1 and SMAD6). CpGs with methylation levels that are inversely related to life span are enriched in transcriptional start site (TSS1) and promoter flanking (PromF4, PromF5) associated chromatin states. Genes located in chromatin state TSS1 are constitutively active and enriched for nucleic acid metabolic processes. This suggests that long-living species evolved mechanisms that maintain low methylation levels in these chromatin states that would favor higher expression levels of genes essential for an organism’s survival. The upstream regulator analysis of the EWAS of life span identified the pluripotency transcription factors OCT4, SOX2, and NANOG. Other factors, such as POLII, CTCF, RAD21, YY1, and TAF1, showed the strongest enrichment for negatively life span–related CpGs. CONCLUSION The phyloepigenetic trees indicate that divergence of DNA methylation profiles closely parallels that of genetics through evolution. Our results demonstrate that DNA methylation is subjected to evolutionary pressures and selection. The publicly available data from our Mammalian Methylation Consortium are a rich source of information for different fields such as evolutionary biology, developmental biology, and aging. DNAm network relates to mammalian phylogeny and traits. (A) Phyloepigenetic tree from the DNAm data generated from blood samples. (B) Unsupervised WGCNA networks identified 55 comethylation modules. (C) EWAS of log-transformed maximum life span. Each dot corresponds to the methylation levels of a highly conserved CpG. Shown is the log (base 10)–transformed P value (y axis) versus the human genome coordinate Hg19 (x axis). (D) Comethylation module correlated with maximum life span of mammals. Eigengene (first principal component of scaled CpGs in the midnightblue module) versus log (base e) transformed maximum life span. Each dot corresponds to a different species.

Abstract: Introduction Domoic acid (DA) is a glutaminergic excitatory neurotoxin that causes the morbidity and mortality of California sea lions (Zalophus californianus; CSL) and other marine mammals due to a suite of effects mostly on the nervous and cardiac systems. Between 1998 and 2019, 11,737 live-stranded CSL were admitted to The Marine Mammal Center (TMMC; Sausalito, CA, USA), over 2,000 of which were intoxicated by DA. A plethora of clinical research has been performed over the past 20 years to characterize the range of toxic effects of DA exposure on CSLs, generating the largest dataset on the effects of natural exposure to this toxin in wildlife. Materials and methods In this study, we review published methods for diagnosing DA intoxication, clinical presentation, and treatment of DA-intoxicated CSL and present a practical, reproducible scoring system called the neuroscore (NS) to help assess whether a DA-affected CSL is fit for release to the wild following rehabilitation. Logistic regression models were used to assess the relationships between outcome (released vs. euthanized or died) and multiple variables to predict the outcome for a subset of 92 stranded CSLs. Results The largest proportion of DA-intoxicated CSLs was adult females (58.6%). The proportions of acute and chronic cases were 63.5 and 36.5% respectively, with 44% of affected CSL released and 56% either dying naturally or euthanized. The average time in rehabilitation was 15.9 days (range 0–169) for all outcomes. The best-performing model (85% accuracy; area under the curve = 0.90) assessing the relationship between outcome and predictor variables consisted of four variables: final NS, change in NS over time, whether the animal began eating in rehabilitation, and the state of nutrition on admission. Discussion Our results provide longitudinal information on the symptomatology of CSL intoxicated by domoic acid and suggest that a behavioral scoring system is a useful tool to assess the fitness for the release of DA-intoxicated CSL.

Abstract: (1) Background: When a human or animal is recovering from general anesthesia, their medical team uses several behavioral and physiological parameters to assess their emergence from the unconscious state to complete wakefulness. However, the return of auditory and acoustic behaviors indicative of the complete return of consciousness in humans can be difficult to assess in a completely aquatic non-human mammal. Dolphins produce sound using the nasal system while using both passive auditory and active biological sonar (echolocation) to navigate and interrogate their environment. The sounds generated by dolphins, such as whistles and clicks, however, can be difficult to hear when the animal is submerged. (2) Methods: We implemented a system to audibly and visually (i.e., using spectrograms) monitor the underwater acoustic behavior of dolphins recovering from anesthesia. (3) Results: Eleven of the twelve recorded dolphins began echolocating within 92 min (Mean = 00:43:41 HH:MM:SS) following spontaneous respirations. In all cases, the dolphins echolocated prior to whistling (Mean = 04:57:47). The return of echolocation was significantly correlated to the return of the righting reflex (Mean = 1:13:44), a commonly used behavioral indicator of dolphin emergence. (4) Conclusions: We suggest that acoustic monitoring for the onset of click production may be a useful supplement to the established medical and behavioral biomarkers of restoring consciousness following anesthesia in bottlenose dolphins.

Abstract: Marine Mammal ScienceEarly View NOTE Observations of tissue healing around an implanted “C” tag in a Pacific Coast Feeding Group gray whale (Eschrichtius robustus) P. Dawn Goley, Corresponding Author P. Dawn Goley [email protected] orcid.org/0000-0001-8021-1286 Marine Mammal Education and Research Program, Department of Biological Sciences, Cal Poly Humboldt, Arcata, California Correspondence P. Dawn Goley, Marine Mammal Education and Research Program, 1 Harpst Street, Department of Biological Sciences, Cal Poly Humboldt, Arcata, CA 95521. Email: [email protected] Contribution: Conceptualization, Data curation, Funding acquisition, ​Investigation, Project administration, Resources, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorJohn Calambokidis, John Calambokidis orcid.org/0000-0002-5028-7172 Cascadia Research Collective, Olympia, Washington Contribution: Conceptualization, Data curation, ​Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorPádraig Duignan, Pádraig Duignan The Marine Mammal Center, Sausalito, California Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Project administration, Supervision, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorFrances M. D. Gulland, Frances M. D. Gulland The Marine Mammal Center, Sausalito, California Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorBarbie Halaska, Barbie Halaska The Marine Mammal Center, Sausalito, California Contribution: Conceptualization, Data curation, ​Investigation, Methodology, Resources, Writing - review & editingSearch for more papers by this authorAllison Lui, Allison Lui Marine Mammal Education and Research Program, Department of Biological Sciences, Cal Poly Humboldt, Arcata, California Contribution: Data curation, ​Investigation, Visualization, Writing - review & editingSearch for more papers by this authorMargaret Martinez, Margaret Martinez Wildlife Health Center, School of Veterinary Medicine, Davis, California Contribution: Visualization, Writing - review & editingSearch for more papers by this authorBruce Mate, Bruce Mate Marine Mammal Institute, Department of Fisheries, Wildlife and Conservation, Oregon State University, Hatfield Marine Science Center, Newport, Oregon Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Project administration, Validation, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this author P. Dawn Goley, Corresponding Author P. Dawn Goley [email protected] orcid.org/0000-0001-8021-1286 Marine Mammal Education and Research Program, Department of Biological Sciences, Cal Poly Humboldt, Arcata, California Correspondence P. Dawn Goley, Marine Mammal Education and Research Program, 1 Harpst Street, Department of Biological Sciences, Cal Poly Humboldt, Arcata, CA 95521. Email: [email protected] Contribution: Conceptualization, Data curation, Funding acquisition, ​Investigation, Project administration, Resources, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorJohn Calambokidis, John Calambokidis orcid.org/0000-0002-5028-7172 Cascadia Research Collective, Olympia, Washington Contribution: Conceptualization, Data curation, ​Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorPádraig Duignan, Pádraig Duignan The Marine Mammal Center, Sausalito, California Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Project administration, Supervision, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorFrances M. D. Gulland, Frances M. D. Gulland The Marine Mammal Center, Sausalito, California Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorBarbie Halaska, Barbie Halaska The Marine Mammal Center, Sausalito, California Contribution: Conceptualization, Data curation, ​Investigation, Methodology, Resources, Writing - review & editingSearch for more papers by this authorAllison Lui, Allison Lui Marine Mammal Education and Research Program, Department of Biological Sciences, Cal Poly Humboldt, Arcata, California Contribution: Data curation, ​Investigation, Visualization, Writing - review & editingSearch for more papers by this authorMargaret Martinez, Margaret Martinez Wildlife Health Center, School of Veterinary Medicine, Davis, California Contribution: Visualization, Writing - review & editingSearch for more papers by this authorBruce Mate, Bruce Mate Marine Mammal Institute, Department of Fisheries, Wildlife and Conservation, Oregon State University, Hatfield Marine Science Center, Newport, Oregon Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Project administration, Validation, Visualization, Writing - original draft, Writing - review & editingSearch for more papers by this author First published: 31 August 2023 https://doi.org/10.1111/mms.13066Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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Abstract: Abstract A proper assessment of the foraging habits of the Guadalupe fur seal (GFS; Arctocephalus townsendi ) is a priority to better understand its recovery, in which the potential for intraspecific competition for prey and space resources is expected to lead to segregation. This study aimed to determine the foraging habits of different sex and age classes. A total of 146 GFS fur samples was collected at Guadalupe Island, Mexico (2014–2020) for stable isotopes (δ 13 C and δ 15 N) analysis. Isotopic areas were created (SIBER package in R). Significant isotopic differences were observed between classes. Male (3.6‰ 2 ) and female (3.0‰ 2 ) juveniles had the largest isotopic areas due to a greater foraging dispersion. Adult females showed the lowest mean δ 15 N value (16.1‰ ± 0.5‰) due to foraging trips that are mostly performed towards high latitudes. Except for pups, adult and subadult males presented the highest mean δ 15 N (17.4‰ ± 0.4‰) and δ 13 C values (−17.0‰ ± 0.8‰) due to a possible higher trophic level and coastal foraging habits, whereas pups presented the highest mean δ 15 N value (17.6‰ ± 0.3‰) because of lactation, which reflects their mothers δ 15 N signal plus their own enrichment. Our findings suggest a segregation explained by differences in life history, energy requirements, and a possible strategy to avoid competition.

Abstract: Abstract: A retrospective study was conducted to categorize and describe the causes of mortality in harbor seals (Phoca vitulina) along the British Columbia coast that presented to the Vancouver Aquarium Marine Mammal Rescue Centre (MMR) for rehabilitation from 2012 to 2020. Medical records for 1,279 predominantly perinatal live-stranded harbor seals recovered in this region were reviewed. Approximately 20.0% (256 individuals; 137 males, 118 females, 1 unknown) of these animals died while at MMR. Infectious disease was the most common cause of death, accounting for 60.5% of mortality across all age classes. This was followed by nonanthropogenic trauma (7.1%), metabolic illness (5.4%), nutritional deficiency (5.0%), parasitic illness (5.0%), congenital disorders (2.5%), and human-associated trauma (0.4%). Pups were the most common age class (87.4%) amongst mortalities and predominantly died of an infectious process (62.5%). Phocid herpesvirus-1 infection was identified in 18.9% of the mortalities, with the highest prevalence occurring in 2019 (30.8%). Fungal disease was detected in six seals: three cases of pulmonary mycosis due to Cryptococcus gattii and three cases consistent with mucormycosis. In six cases, mortality was attributed to congenital disorders. Two of these cases involved axial skeletal malformities that are not currently described in the literature. This is the first study to describe the causes of mortality in harbor seals undergoing rehabilitation in British Columbia.

Abstract: Abstract Domoic acid producing blooms of the diatom genus Pseudo-nitzschia are pervasive in coastal environments globally. Domoic acid, a neurotoxin, accumulates via trophic transfer into marine food webs and are often associated with mass marine mammal mortality and stranding events. In Southern California, California sea lions ( Zalophus californiaus ) are an indicator species for food web impacts of domoic acid because they are abundant secondary consumers, sensitive to domoic acid intoxication, and are actively monitored by stranding networks. However, domoic acid exposure may occur a distance from where a sea lion ultimately strands. This spatiotemporal variation complicates coupling domoic acid observations in water to strandings. Therefore, we sought to quantify whether monitoring data from four pier sites across the region, covering nearly 700 km of coastline from 2015-2019, could be used to predict adult and subadult sea lion strandings along the 68 km Orange County coastline surveyed by the Pacific Marine Mammal Center. We found that increased sea lion strandings were often observed just prior to an increase in particulate domoic acid at the piers, confirming that clusters of subadult and adult sea lion strandings with clinical signs of domoic acid intoxication a serve as indicators of bloom events. In addition, domoic acid concentrations at Stearns Wharf, nearly 200 km from stranding locations, best predicted increased total sea lion strandings, and strandings of sea lions with domoic acid intoxication symptoms. Particulate domoic acid concentrations greater than 0.05 μg/L at Stearns Wharf led to a detectable increase in stranding probability in Orange County, and concentrations over 0.25 μg/L resulted in a nearly 1.6-fold increase in stranding probabilities for a given week.

Abstract: Defining baseline mortality and trends in wildlife populations is imperative to understand natural and anthropogenic threats to overall population health and improve conservation measures for species, particularly in geographically confined habitats. The Guadalupe fur seal Arctocephalus townsendi (GFS) is a threatened pinniped that ranges throughout the west coast of Mexico with sporadic dispersion to higher latitudes. Their breeding habitat is restricted to Guadalupe Island, Mexico, which is vulnerable to periodic and cyclic warming of the Northeast Pacific Ocean. The impacts of environmental change on GFS health and reproductive success at Guadalupe Island are poorly defined and the aim of this study was to establish baseline pup (GFS n ) mortality rates and primary causes of death during the 2013-2016 breeding seasons at Guadalupe Island. Interannual mortality rates and causes of death were compared by year, breeding seasons and by geographic location. The highest mortality rate in GFS n was in 2015 (14.7%), followed by 2014 (8.2%), 2016 (6.7%) and 2013 (5.6%). The presumptive causes of mortality of GFS n were consistent with other published long term otariids health surveys and included: emaciation (49%), trauma (24%), infectious disease (8%), drowning (4%) stillbirth/perinatal mortality (4%) and undetermined (11%). However, in 2015 and coinciding with northeast Pacific marine heatwave in 2014-2016, emaciation accounted for 54% of GFS n mortality in contrast to 9% in 2013. For GFS n , terrestrial habitat may influence mortality rates and causes of mortality but like other marine predators, marine habitat features, such as an increase in sea surface temperature are associated with changes in maternal care, nutritional status and pups survival. Monitoring mortality rate and causes in GFS n at Guadalupe Island is crucial to establish baseline health trends, document potential impacts on species demographics and recruitment during marine heatwaves and potential consequences in population recovery.

Abstract: We report the first case series of osteofluorosis in free ranging marine mammals and documentation of baseline bone fluoride concentrations in this population; however, the exact source is not known. A stranded subadult male California sea lion, one subadult male carcass, and one partial carcass, presumably adult male, demonstrated osseous (bony) proliferations on various appendicular and axial bones. The most severe lesions were from the index case with symmetrical segmental circumferential periosteal new bone proliferation on the diaphysis of both femurs and humeri. This case, and the second case, also had significant segmental periosteal new bone proliferation on the mandibles as well as increased tooth wear with dull enamel. Total fluoride concentrations in various bones of the cases ranged from 3,700 to 9,700 ppm dw, with a mean of 5,431 ppm dw, approximately twice the concentration in adult cattle with osteofluorosis. Ribs from four control California sea lions of various ages and both sexes had an average total fluoride of 1,575 ppm dw, which is three times as high as the bone fluoride concentration of normal adult cattle, but four times lower than the mean rib fluoride concentrations in the cases. This is the first report of osteofluorosis in free ranging marine mammals and documentation of baseline bone fluoride concentrations in this population. The exact source of the excessive fluoride levels is not known as male California sea lions have a large range and varied diet.