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The coastal waters of Cuba are home to a small, endangered population of West Indian manatee, which would benefit from a comprehensive characterization of the population's genetic variation. We conducted the first genetic assessment of Cuban manatees to determine the extent of the population's genetic structure and characterize the neutral genetic diversity among regions within the archipelago. We genotyped 49 manatees at 18 microsatellite loci, a subset of 27 samples on 1703 single nucleotide polymorphisms (SNPs), and sequenced 59 manatees at the mitochondrial control region. The Cuba manatee population had low nuclear (microsatellites H

Millette et al. (Ecology Letters, 2020, 23:55-67) reported no consistent worldwide anthropogenic effects on animal genetic diversity using repurposed mitochondrial sequences. We describe limitations to this study, some of which are common to other macrogenetic studies, that may lead to misinterpretations and unintended consequences for conservation.

Florida manatees depend on freshwater environments as a source of drinking water and as warm-water refuges. These freshwater environments are in direct contact with human activities where glyphosate-based herbicides are being used. Glyphosate is the most used herbicide worldwide and it is intensively used in Florida as a sugarcane ripener and to control invasive aquatic plants. The objective of the present study was to determine the concentration of glyphosate and its breakdown product, aminomethylphosphonic acid (AMPA), in Florida manatee plasma and assess their exposure to manatees seeking a warm-water refuge in Crystal River (west central Florida), and in South Florida. We analyzed glyphosate’s and AMPA’s concentrations in Florida manatee plasma (n = 105) collected during 2009–2019 using HPLC-MS/MS. We sampled eight Florida water bodies between 2019 and 2020, three times a year: before, during and after the sugarcane harvest using grab samples and molecular imprinted passive Polar Organic Chemical Integrative Samplers (MIP-POCIS). Glyphosate was present in 55.8% of the sampled Florida manatees’ plasma. The concentration of glyphosate has significantly increased in Florida manatee samples from 2009 until 2019. Glyphosate and AMPA were ubiquitous in water bodies. The concentration of glyphosate and AMPA was higher in South Florida than in Crystal River, particularly before and during the sugarcane harvest when Florida manatees depend on warm water refuges. Based on our results, Florida manatees were chronically exposed to glyphosate and AMPA, during and beyond the glyphosate applications to sugarcane, possibly associated with multiple uses of glyphosate-based herbicides for other crops or to control aquatic weeds. This chronic exposure in Florida water bodies may have consequences for Florida manatees’ immune and renal systems which may further be compounded by other environmental exposures such as red tide or cold stress.

The rapidly emerging field of macrogenetics focuses on analysing publicly accessible genetic datasets from thousands of species to explore large-scale patterns and predictors of intraspecific genetic variation. Facilitated by advances in evolutionary biology, technology, data infrastructure, statistics and open science, macrogenetics addresses core evolutionary hypotheses (such as disentangling environmental and life-history effects on genetic variation) with a global focus. Yet, there are important, often overlooked, limitations to this approach and best practices need to be considered and adopted if macrogenetics is to continue its exciting trajectory and reach its full potential in fields such as biodiversity monitoring and conservation. Here, we review the history of this rapidly growing field, highlight knowledge gaps and future directions, and provide guidelines for further research.

The northern shrimp (Pandalus borealisKrøyer) population in the Gulf of Maine collapsed during an extreme heatwave that occurred across the Northwest Atlantic Ocean in 2012. Northern shrimp is a boreal species, and reaches its southern limit in the Gulf of Maine. Here we investigate proximate causes for the population collapse using data from fishery-independent surveys, environmental monitoring, and the commercial fishery. We first examined spatial data to confirm that the decline in population estimates was not due to a major displacement of the population, and then tested hypotheses related to fishing mortality and shifts in predation pressure. Fishing mortality may have contributed but could not explain the magnitude of the decline or the disappearance of pre-exploitable size individuals. Stomach contents analysis and biomass trends revealed no new fish predators of shrimp. However, longfin squid (Doryteuthis pealeiiLesueur) was unique among all species in showing time-series biomass peaks during spring, summer and fall of 2012, and spatial overlap with northern shrimp was unusually high in 2012. Longfin squid is a voracious and opportunistic predator that consumes crustaceans as well as fish. We hypothesize that the warmer temperatures of 2012 not only led to expansion of longfin squid distribution in Gulf of Maine, but had differential effects on migration phenology that further increased spatial overlap with northern shrimp. The weight of our evidence suggests that longfin squid predation was likely a significant factor in the collapse of northern shrimp in the Gulf of Maine.

To investigate a Florida manatee (Trichechus manatus latirostris) mortality event following a red tide bloom in Southwest Florida, an RNA sequencing experiment was conducted. Gene expression changes in white blood cells were assessed in manatees rescued from a red tide affected area (n = 4) and a control group (n = 7) using RNA sequencing. The genes with the largest fold changes were compared between the two groups to identify molecular pathways related to cellular and disease processes. In total, 591 genes (false discovery rate

Invasive species provide powerful in situ experimental systems for studying evolution in response to selective pressures in novel habitats. While research has shown that phenotypic evolution can occur rapidly in nature, few examples exist of genomewide adaptation on short "ecological" timescales. Burmese pythons (Python molurus bivittatus) have become a successful and impactful invasive species in Florida over the last 30 years despite major freeze events that caused high python mortality. We sampled Florida Burmese pythons before and after a major freeze event in 2010 and found evidence for directional selection in genomic regions enriched for genes associated with thermosensation, behaviour and physiology. Several of these genes are linked to regenerative organ growth, an adaptive response that modulates organ size and function with feeding and fasting in pythons. Independent histological and functional genomic data sets provide additional layers of support for a contemporary shift in invasive Burmese python physiology. In the Florida population, a shift towards maintaining an active digestive system may be driven by the fitness benefits of maintaining higher metabolic rates and body temperature during freeze events. Our results suggest that a synergistic interaction between ecological and climatic selection pressures has driven adaptation in Florida Burmese pythons, demonstrating the often-overlooked potential of rapid adaptation to influence the success of invasive species.

Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee-specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity.

The Burmese python (Python bivittatus) is now established as a breeding population throughout south Florida, USA. However, the extent of the invasion, and the ecological impacts of this novel apex predator on animal communities are incompletely known, in large part because Burmese pythons (hereafter "pythons") are extremely cryptic and there has been no efficient way to detect them. Pythons are recently confirmed nest predators of long-legged wading bird breeding colonies (orders Ciconiiformes and Pelecaniformes). Pythons can consume large quantities of prey and may not be recognized as predators by wading birds, therefore they could be a particular threat to colonies. To quantify python occupancy rates at tree islands where wading birds breed, we utilized environmental DNA (eDNA) analysis-a genetic tool which detects shed DNA in water samples and provides high detection probabilities. We fitted multi-scale Bayesian occupancy models to test the prediction that pythons occupy islands with wading bird colonies at higher rates compared to representative control islands containing no breeding birds. Our results suggest that pythons are widely distributed across the central Everglades in proximity to active wading bird colonies. In support of our prediction that pythons are attracted to colonies, site-level python eDNA occupancy rates were higher at wading bird colonies (ψ = 0.88, 95% credible interval [0.59-1.00]) than at the control islands (ψ = 0.42 [0.16-0.80]) in April through June (n = 15 colony-control pairs). We found our water temperature proxy (time of day) to be informative of detection probability, in accordance with other studies demonstrating an effect of temperature on eDNA degradation in occupied samples. Individual sample concentrations ranged from 0.26 to 38.29 copies/μL and we generally detected higher concentrations of python eDNA in colony sites. Continued monitoring of wading bird colonies is warranted to determine the effect pythons are having on populations and investigate putative management activities.

Objective We use Tempus blood RNA tubes (Applied Biosystems) during health assessments of American moose (Alces alces spp.) as a minimally invasive means to obtain RNA. Here we describe a novel protocol to additionally isolate high-quality DNA from the supernatant remaining after the RNA isolation methodology. Metrics used to qualify DNA quality included measuring the concentration, obtaining a DNA integrity number from a genomic DNA ScreenTape assay (Agilent), and running the isolated DNA on an agarose gel. Results Of the 23 samples analyzed, the average DNA concentration was 121 ng/µl (range 4–337 ng/µl) and a genomic DNA ScreenTape assay of seven samples indicated high DNA integrity values for 6 of the 7 samples (range 9.1–9.4 out of 10). Of the DNA sent for genotyping by sequencing, all proved to be of sufficient integrity to yield high-quality next-generation sequence results. We recommend this simple procedure to maximize the yield of both RNA and DNA from blood samples.

The Florida manatee (Trichechus manatus latirostris) has limited diversity in the immunoglobulin heavy chain. We therefore investigated the antigen receptor loci of the other arm of the adaptive immune system: the T cell receptor. Manatees are the first species from Afrotheria, a basal eutherian superorder, to have an in-depth characterization of all T cell receptor loci. By annotating the genome and expressed transcripts, we found that each chain has distinct features that correlates to their individual functions. The genomic organization also plays a role in modulating sequence conservation between species. There were extensive V subgroup synteny blocks in the TRA and TRB loci between T. m. latirostris and human. Increased genomic locus complexity correlated to increased locus synteny. We also identified evidence for a VHD pseudogene for the first time in a eutherian mammal. These findings emphasize the value of including species within this basal eutherian radiation in comparative studies.

Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees ( Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α; and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida, US. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher ( P0.05) in manatees from the east coast of Florida than they were from those from the west coast. We found IL-10 and β-actin to be consistent between sites and identified β-actin as a good candidate for use as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty – indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis, and forensic and clinical diagnostics. This article is protected by copyright. All rights reserved.

To address the ongoing debate over the impact of invasive species on native terrestrial wildlife, we conducted a large-scale experiment to test the hypothesis that invasive Burmese pythons ( Python molurus bivittatus ) were a cause of the precipitous decline of mammals in Everglades National Park (ENP). Evidence linking pythons to mammal declines has been indirect and there are reasons to question whether pythons, or any predator, could have caused the precipitous declines seen across a range of mammalian functional groups. Experimentally manipulating marsh rabbits, we found that pythons accounted for 77% of rabbit mortalities within 11 months of their translocation to ENP and that python predation appeared to preclude the persistence of rabbit populations in ENP. On control sites, outside of the park, no rabbits were killed by pythons and 71% of attributable marsh rabbit mortalities were classified as mammal predations. Burmese pythons pose a serious threat to the faunal communities and ecological functioning of the Greater Everglades Ecosystem, which will probably spread as python populations expand their range.

Marine mammals from different mammalian orders share several phenotypic traits adapted to the aquatic environment and therefore represent a classic example of convergent evolution. To investigate convergent evolution at the genomic level, we sequenced and performed de novo assembly of the genomes of three species of marine mammals (the killer whale, walrus and manatee) from three mammalian orders that share independently evolved phenotypic adaptations to a marine existence. Our comparative genomic analyses found that convergent amino acid substitutions were widespread throughout the genome and that a subset of these substitutions were in genes evolving under positive selection and putatively associated with a marine phenotype. However, we found higher levels of convergent amino acid substitutions in a control set of terrestrial sister taxa to the marine mammals. Our results suggest that, whereas convergent molecular evolution is relatively common, adaptive molecular convergence linked to phenotypic convergence is comparatively rare.

In many North American rivers, populations of multiple species of non-native cyprinid fishes are present, including black carp (Mylpharyngodon piceus), grass carp (Ctenopharyngodon idella), bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio), and goldfish (Carassius auratus). All six of these species are found in the Mississippi River basin and tracking their invasion has proven difficult, particularly where abundance is low. Knowledge of the location of the invasion front is valuable to natural resource managers because future ecological and economic damages can be most effectively prevented when populations are low. To test the accuracy of environmental DNA (eDNA) as an early indicator of species occurrence and relative abundance, we applied eDNA technology to the six non-native cyprinid species putatively present in a 2.6 river mile stretch of the Chicago (IL, USA) canal system that was subsequently treated with piscicide. The proportion of water samples yielding positive detections increased with relative abundance of the six species, as indicated by the number of carcasses recovered after poisoning. New markers for black carp, grass carp, and a common carp/goldfish are reported and details of the marker testing to ensure specificity are provided.

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.