Remains of megatheres have been known since the 18th -century and were among the first megafaunal vertebrates to be studied. While several examples of preserved integument show a thick coverage of fur for smaller ground sloths living in cold climates such as Mylodon and Nothrotheriops, comparatively very little is known about megathere skin. Assuming a typical placental mammal metabolism, it was previously hypothesized that megatheres would have had little-to-no fur as they achieved giant body sizes. Here the “hairless model of integument” is tested using geochemical analyses to estimate body temperature to generate novel models of ground sloth metabolism, fur coverage, and paleoclimate with Niche Mapper software. The simulations assuming metabolic activity akin to those of modern xenarthrans suggest that sparse fur coverage would have resulted in cold stress across most latitudinal ranges inhabited by extinct ground sloths. Specifically, Eremotherium predominantly required dense 10 mm fur with implications for seasonal changes of coat depth in northernmost latitudes and sparse fur in the tropics; Megatherium required dense 30 mm fur year-round in its exclusive range of cooler, drier climates; Mylodon and Nothrotheriops required dense 10–50 mm fur to avoid thermal stress, matching the integument remains of both genera, and further implying the use of behavioral thermoregulation. Moreover, clumped isotope paleothermometry data from the preserved teeth of four genera of ground sloth yielded reconstructed body temperatures lower than those previously reported for large terrestrial mammals (23 ± 5–32 ± 3° C). This combination of low metabolisms and thick fur allowed ground sloths to inhabit various environments.

AbstractEnvironmental managers need a rapid and cost‐effective monitoring tool for tracking the spread of invasive species, particularly at the onset of introduction. The macroalgae Caulerpa prolifera is considered an invasive species outside its native range, colonizing large patches of seafloor, reducing native species, and altering ecosystem functioning. Here, we developed a droplet digital PCR assay for detection of C. prolifera from environmental DNA seawater samples using the internal transcribed spacer (ITS) region. While the assay itself was confirmed to be highly efficient, we discovered concentrations of C. prolifera eDNA were present below detectable levels in the water column surrounding an outbreak. To understand why, we conducted tank‐based experiments for two California invasive algae species, Caulerpa prolifera and Sargassum horneri. The steady‐state eDNA concentration (eDNA copies/ gram of biomass detected) of C. prolifera was found to be two orders of magnitude lower than S. horneri. A meta‐analysis of steady‐state concentrations reported in the literature showed a remarkable range from ~104–1011 (copies/g), revealing C. prolifera to have the lowest recorded steady‐state concentrations of eDNA of any known species. We attribute C. prolifera's low steady‐state eDNA concentration to its unique biology as a unicellular macroscopic algae which reduces the possible modes of eDNA release compared to similarly sized multicellular organisms. Critically our results demonstrate the potential limits of eDNA approaches, the influence of shedding rates in the reliability of species detections, and the vital importance of benchmarking and validating eDNA assays in both field and laboratory settings.

Shark teeth are abundant in the fossil record and integrate physiological information, ecological interactions, and paleo-oceanographic conditions in their chemistry. Fossil shark teeth are well suited for stable isotope analysis because their enameloid is resistant to diagenetic alteration due to its high chemical stability. Although often used in paleoecological studies of mammals, carbonate carbon isotope compositions (delta C-13(CO3)) in shark enameloid have remained enigmatic. Here, we investigate multiple stable isotope systems (delta C-13(org), delta C-13(CO3), delta O-18(CO3), delta O-18(PO4)) within modern shark teeth to determine relationships between the different systems and build an interpretative framework for future studies of both modern and fossil sharks. There is a weaker than expected correlation between delta O-18(PO4) and delta O-18(CO3) values in modern shark teeth (r(2) = 0.44), which contrasts with mammalian studies to date and suggests this metric is not an appropriate test for diagenetic alteration in fossil shark teeth. Organic carbon isotope composition (delta C-13(org)) measured from modern dental collagen ranges from 16.0 parts per thousand to 10.8 parts per thousand. The enameloid delta C-13(CO3) values we measured are much higher than collagen, ranging from 6.0 parts per thousand to 10.3 parts per thousand, and there is no direct relationship between delta C-13(org) and delta C-13(CO3) values in shark teeth. Instead, we found the fractionation (epsilon) between delta C-13(org) and delta C-13(CO3) values to correspond with delta O-18(CO3) values but not delta O-18(PO4) values. This could be due to the carbon source in shark enameloid being partitioned between dietary carbon and dissolved inorganic carbon (DIC) or physiological differences in the tooth formation process changing the fractionation of carbonate isotopes. We applied the fractionation factor from modern teeth to carbonate isotope compositions of fossil shark teeth to predict delta C-13(org) values. Although the carbon sources to shark enameloid carbonate needs further investigation, our results suggest that fossil shark teeth could provide insights into carbon cycling of ancient marine ecosystems.

Isotopic replicate data for standards and samples.

The late Neogene megatooth shark, Otodus megalodon (Lamniformes: Otodontidae), is mostly known for its gigantic teeth and vertebrae. Re-examination of the rock matrix surrounding a previously described associated tooth set of O. megalodon from the upper Miocene of Japan resulted in the observation of numerous fragments of tessellated calcified cartilage and placoid scales. The morphology of each tessera and the arrangement of overall tessellated calcified cartilage are practically identical to those of extant chondrichthyans. Many placoid scales possess pronounced, rather broadly-spaced keels. A quantitative relationship between interkeel distances of keeled scales and reported cruising speeds across extant pelagic lamniforms and carcharhiniforms suggests that O. megalodon with a representative interkeel distance of ca. 100 & mu;m was not a fast swimmer. We propose that O. megalodon was generally a slow cruising shark with occasional burst swimming for prey capture, where much of its metabolic heat through regional endothermy was possibly used to facilitate the digestion of large pieces of ingested meat as well as absorbing and processing nutrients. If so, the relative importance of the functional roles of regional endothermy possibly shifted from maintaining high cruising speeds to visceral food processing through the evolution towards gigantism in otodontids.

The evolution of the extinct megatooth shark, Otodus megalodon, and its close phylogenetic relatives remains enigmatic. A central question persists regarding the thermophysiological origins of these large predatory sharks through geologic time, including whether O. megalodon was ectothermic or endothermic (including regional endothermy), and whether its thermophysiology could help to explain the iconic shark's gigantism and eventual demise during the Pliocene. To address these uncertainties, we present unique geochemical evidence for thermoregulation in O. megalodon from both clumped isotope paleothermometry and phosphate oxygen isotopes. Our results show that O. megalodon had an overall warmer body temperature compared with its ambient environment and other coexisting shark species, providing quantitative and experimental support for recent biophysical modeling studies that suggest endothermy was one of the key drivers for gigantism in O. megalodon and other lamniform sharks. The gigantic body size with high metabolic costs of having high body temperatures may have contributed to the vulnerability of Otodus species to extinction when compared to other sympatric sharks that survived the Pliocene epoch.

Carbonate clumped isotope geochemistry has primarily focused on mass spectrometric determination of m/z 47 CO2 for geothermometry, but theoretical calculations and recent experiments indicate paired analysis of the m/z 47 (13C18O16O) and m/z 48 (12C18O18O) isotopologues (referred to as Δ47 and Δ48) can be used to study non-equilibrium isotope fractionations and refine temperature estimates. We utilize 5,448 Δ47 and 3,400 Δ48 replicate measurements of carbonate samples and standards, and 183 Δ47 and 195 Δ48 replicate measurements of gas standards from 2015 to 2021 from a multi-year and multi-instrument data set to constrain Δ47 and Δ48 values for 27 samples and standards, including Devils Hole cave calcite, and study equilibrium Δ47-Δ48, Δ47-temperature, and Δ48-temperature relationships. We compare results to previously published findings and calculate equilibrium regressions based on data from multiple laboratories. We report acid digestion fractionation factors, Δ*63-47 and Δ*64-48, and account for their dependence on the initial clumped isotope values of the mineral.

Amorphous precursors to minerals have been observed in laboratory materials and in nature, including across diverse phyla. These metastable phases allow for the incorporation of cations at higher concentrations than classical crystallization pathways, thus, their chemistry and behavior have implications in an array of disciplines. Currently, little is known about the isotopic composition of the anion in amorphous carbonates and how isotopic values evolve during transformation into a mineral. Here, we examined the evolution of isotopic values in amorphous carbonates and mineral transformation products to identify the potential origins of disequilibrium isotopic compositions in carbonate minerals that form from an amorphous precursor. We measured dual carbonate clumped isotopes (13C18O16O -d47; 12C18O18O -d48), bulk stable isotope ratios (613C, 618O), and chemical and structural data throughout the transformation of amorphous calcium magnesium carbonate (ACMC) into high Mg-calcite (HMC) over 1 year, with crystallization occurring in solutions from 10 to 60 degrees C. The d47, d48, and 618O values evolved significantly during transformation, indicating dissolution of ACMC and reprecipitation of HMC. After crystallization, the d47 and d48 values achieved a disequilibrium steady state, while 618O values continued to evolve. For the fully crystallized HMC samples, the low temperature samples formed at 10 degrees C had the greatest extent of oxygen isotope disequilibrium (measured value - equilibrium value = -39%o); conversely; the greatest clumped isotope disequilibrium was observed in high temperature samples formed at 40 and 60 degrees C (0.068%o for d47, 0.072%o for d48). These results are consistent with a new potential mechanism of disequilibrium clumped isotope values in carbonate minerals. Specifically, the dissolution of ACMC during transformation causes disequilibrium oxygen and clumped isotope values in the dissolved inorganic carbon (DIC) pool. The extent of isotopic disequilibrium in DIC during transformation is temperature dependent, and is recorded by the forming mineral. Isotopic results may also reflect mixing effects, as scanning electron microscopy (SEM) showed ACMC and HMC existing simultaneously during transformation, indicating that ACMC likely progressively dissolves and reprecipitates as the DIC pool isotopically evolves. This may result in heterogeneous isotopic values in HMC. In total, these data suggest a highly dynamic localized environment could exist in biomineralizing organisms and abiotic systems that utilize amorphous precursors to form carbonate minerals, potentially resulting in isotopic values that are not representative of formation temperature.

The Cedars is an area in Northern California with a chain of highly alkaline springs resulting from CO-charged meteorological water interacting with a peridotite body. Serpentinization resulting from this interaction at depth leads to the sequestration of various carbonate minerals into veins accompanied by a release of Ca and OH enriched water to the surface, creating an environment which promotes rapid precipitation of CaCO at surface springs. This environment enables us to apply the recently developed Δ-Δ dual clumped isotope analysis to probe kinetic isotope effects (KIEs) and timescales of CO transformation in a region with the potential for geological CO sequestration. We analyzed CaCO recovered from various localities and identified significant kinetic fractionations associated with CO absorption in a majority of samples, characterized by enrichment in Δ values and depletion in Δ values relative to equilibrium. Surface floes exhibited the largest KIEs (ΔΔ­: 0.163‰, ΔΔ­: -0.761‰). Surface floe samples begin to precipitate out of solution within the first hour of CO absorption, and the dissolved inorganic carbon (DIC) pool requires a residence time of >100 hours to achieve isotopic equilibria. The Δ/Δ slope of samples from the Cedars (-3.223±0.519; 1 SE) is within the range of published theoretical values designed to constrain CO hydrolysis-related kinetic fractionation (-1.724 to -8.330). The Δ/δO slope (-0.009±0.001) and Δ/δC slope (0.009±0.001) are roughly consistent with literature values reported from a peridotite in Oman of -0.006±0.002 and -0.005±0.002, respectively. The consistency of slopes in the multi-isotope space suggests the Δ-Δ dual carbonate clumped isotope framework can be applied to study CO-absorption processes in applied systems, including sites of interest for geological sequestration.

Seagrass beds are disappearing at a record pace despite their known value to our oceans and coastal communities. Simultaneously, our coastlines are under the constant pressure of climate change which is impacting their chemical, physical and biological characteristics. It is thus pertinent to evaluate and record habitat use so we can understand how these different environments contribute to local biodiversity. This study evaluates the assemblages of fish found at five Zostera beds in Southern California using environmental DNA (eDNA) metabarcoding. eDNA is a powerful biodiversity monitoring tool that offers key advantages to conventional monitoring. Results from our eDNA study found 78 species of fish that inhabit these five beds around Southern California representing embayment, open coastal mainland and open coastal island settings. While each bed had the same average number of species found throughout the year, the composition of these fish assemblages was strongly site dependent. There were 35 fish that were found at both open coast and embayment seagrass beds, while embayment seagrass sites had 20 unique fish and open coast sites had 23 unique fish. These results demonstrate that seagrass fish assemblages are heterogenous based on their geographic positioning and that marine managers must take this into account for holistic conservation and restoration efforts.

AbstractThe macroalgaeCaulerpa proliferais considered an invasive species in many environments and can colonize large patches of seafloor, reduce native species, and alter ecosystem functioning. Environmental managers need a rapid and cost-effective monitoring tool for tracking the spread of this invasive species. We developed a digital PCR assay for detection ofC. proliferafrom environmental DNA seawater samples. We demonstrate, in both field and laboratory experiments, that the invasive algaeC. proliferais undetectable in practical applications of eDNA due to its minimal shedding. To test why, we conducted tank-based shedding experiments for two California invasive algae species,C. proliferaandSargassum horneri. Copy numbers ofC. proliferaeDNA detected in the experimental tanks were found to be two orders of magnitude lower thanS. horneri. A meta-analysis of steady state eDNA produced by aquatic organisms reported in the literature showC. proliferato have the lowest recorded steady state concentrations of eDNA in the water column. We attributeC. proliferalow eDNA shedding to its unique biology as a unicellular, multinucleate, macroscopic siphonous algae which reduces the possible modes of eDNA release compared to multicellular organisms. Our results highlight the value of benchmarking and validating eDNA surveys in both field and laboratory settings and potential limits of eDNA approaches for some applications. These results also emphasize the importance of organismal physiology in eDNA shedding rates, variations in mechanisms of eDNA shedding between organisms, and characterizing shedding rates for accurate interpretation of eDNA results.

The high-precision analysis of the abundance of the dominant m/z 47 CO2 isotopologue derived from acid digestion of carbonate minerals (13C18O16O; denoted by Δ47) forms the basis for carbonate clumped isotope thermometry. Since the first measurements were published 16 years ago, considerable effort has gone into characterizing the relationship between Δ47 and carbonate precipitation temperature, and in identifying carbonates that do not achieve isotopic equilibrium. Mass spectrometry is now capable of the paired measurement of the primary m/z 47 and m/z 48-isotopologues (Δ47 and Δ48; 12C18O2 is denoted by Δ48), which has the potential to place additional constraints on kinetic isotope effects in carbonate minerals and trace distinct reaction pathways. Here, we explored factors that contribute to calcite mineral equilibrium and disequilibrium in Δ47 and Δ48 using a combination of experiments and theoretical calculations with three types of models. We precipitated calcite at pH 8.3 with carbonic anhydrase (CA) to approach quasi-isotopic equilibrium in the dissolved inorganic carbon pool and report values for Δ47, Δ48, and oxygen isotopes (δ18O) for calcite grown over a temperature range from 5 to 25 oC and compare our findings to predictions from an ion-by-ion model that support equilibrium precipitation. We also compare results to the Devils Hole slow-growing cave calcite, and other published temperature calibration data. We report the following combined equilibrium calibration relationships: Δ48 CDES 90 = (0.429 ± 0.010) Δ47 CDES 90 - (0.006 ± 0.006); r2 = 0.98; Δ47 I-CDES = (0.037 ± 0.001) × 106T-2 + (0.178 ± 0.009); r2 = 0.99; Δ48 CDES 90 = (0.015 ± 0.0005) × 106T-2 + (0.078 ± 0.006); r2 = 0.98. We used paired measurements of Δ47 and Δ48 to constrain kinetic isotope effects in calcite precipitated at pH ranging from 8.3-11 and temperatures from 5 to 25 oC, with and without CA present, and observe kinetic enrichments in Δ47, negative (hyperstochastic) values for Δ48, and depleted values of δ18O, compared to equilibrium values. Experimentally constrained kinetic trajectories, when compared with an ion-by-ion model and IsoDIC theoretical predictions, are consistent with CO2 hydration/hydroxylation. Mixing drives elevated Δ47 and Δ48 values and was assessed using mixing experiments with endmembers of varying isotopic compositions and compared to a Δ47 and Δ48 mixing model that constrains nonlinear mixing trajectories for calcite. While mixing may induce artifacts in two-component mixtures when endmember bulk compositions differ by more than 7 ‰, or if endmember Δ47 and Δ48 differ by more than 0.03 ‰, this should be detectable and potentially correctible using paired clumped isotope measurements and is unlikely to be important for some materials.

Trophic position is a fundamental characteristic of animals, yet it is unknown in many extinct species. In this study, we ground-truth the 15N/14N ratio of enameloid-bound organic matter (δ15NEB) as a trophic level proxy by comparison to dentin collagen δ15N and apply this method to the fossil record to reconstruct the trophic level of the megatooth sharks (genus Otodus). These sharks evolved in the Cenozoic, culminating in Otodus megalodon, a shark with a maximum body size of more than 15 m, which went extinct 3.5 million years ago. Very high δ15NEB values (22.9 ± 4.4‰) of O. megalodon from the Miocene and Pliocene show that it occupied a higher trophic level than is known for any marine species, extinct or extant. δ15NEB also indicates a dietary shift in sharks of the megatooth lineage as they evolved toward the gigantic O. megalodon, with the highest trophic level apparently reached earlier than peak size.

It is thought that the active physiological regulation of the chemistry of a parent fluid is an important process in the biomineralization of scleractinian corals. Biological regulation of calcification fluid pH (pHCF) and other carbonate chemistry parameters ([CO32−]CF, DICCF, and ΩCF) may be challenged by CO2 driven acidification and temperature. Here, we examine the combined influence of changing temperature and CO2 on calcifying fluid regulation in four common Caribbean coral species—Porites astreoides, Pseudodiploria strigosa, Undaria tenuifolia, and Siderastrea siderea. We utilize skeletal boron geochemistry (B/Ca and δ11B) to probe the pHCF, [CO32−]CF, and DICCF regulation in these corals, and δ13C to track changes in the sources of carbon for calcification. Temperature was found to not influence pHCF regulation across all pCO2 treatments in these corals, in contrast to recent studies on Indo-Pacific pocilloporid corals. We find that [DIC]CF is significantly lower at higher temperatures in all the corals, and that the higher temperature was associated with depletion of host energy reserves, suggesting [DIC]CF reductions may result from reduced input of respired CO2 to the DIC pool for calcification. In addition, δ13C data suggest that under high temperature and CO2 conditions, algal symbiont photosynthesis continues to influence the calcification pool and is associated with low [DIC]CF in P. strigosa and P. astreoides. In P. astreoides this effect is also associated with an increase in chlorophyll a concentration in coral tissues at higher temperatures. These observations collectively support the assertion that physicochemical control over coral calcifying fluid chemistry is coupled to host and symbiont physiological responses to environmental change, and reveals interspecific differences in the extent and nature of this coupling.

Constraints on the evolution of atmospheric CO2 levels throughout Earth's history are foundational to our understanding of past variations in climate. Despite considerable effort, records vary in their temporal and spatial coverage and estimates of past CO2 levels do not always converge, and therefore new records and proxies are valuable. Here we reconstruct atmospheric CO2 values across major climate transitions over the past 16 million years using the boron isotopic composition (δ11B) of planktic foraminifera from 89 samples obtained from two sites in the West Pacific Warm Pool, Ocean Drilling Program (ODP) Sites 806 and 807, measured using high-precision multi-collector inductively coupled plasma mass spectrometry. We compare our results to published data from ODP Site 872, also in the Western Equatorial Pacific, that goes back to 22 million years ago. These sites are in a region that today is near equilibrium with the atmosphere and are thought to have been in equilibrium with the atmosphere for the interval studied. We show that δ11B data from this region are consistent with other boron-based studies. The data show evidence for elevated pCO2 during the Middle Miocene and Early to Middle Pliocene, and reductions in pCO2 of ∼200 ppm during the Middle Miocene Climate Transition, ∼250 ppm during Pliocene Glacial Intensification and ∼50 ppm during the Mid-Pleistocene Climate Transition. During the Mid-Pleistocene Transition there is a minimum pCO2 at marine isotopic stage (MIS) 30. Our results are consistent with a coupling between pCO2, temperature and ice sheet expansion from the Miocene to the late Quaternary.

Diet is a crucial trait of an animal's lifestyle and ecology. The trophic level of an organism indicates its functional position within an ecosystem and holds significance for its ecology and evolution. Here, we demonstrate the use of zinc isotopes (δ66Zn) to geochemically assess the trophic level in diverse extant and extinct sharks, including the Neogene megatooth shark (Otodus megalodon) and the great white shark (Carcharodon carcharias). We reveal that dietary δ66Zn signatures are preserved in fossil shark tooth enameloid over deep geologic time and are robust recorders of each species' trophic level. We observe significant δ66Zn differences among the Otodus and Carcharodon populations implying dietary shifts throughout the Neogene in both genera. Notably, Early Pliocene sympatric C. carcharias and O. megalodon appear to have occupied a similar mean trophic level, a finding that may hold clues to the extinction of the gigantic Neogene megatooth shark.

Corals are globally important calcifiers that exhibit complex responses to anthropogenic warming and acidification. Although coral calcification is supported by high seawater pH, photosynthesis by the algal symbionts of zooxanthellate corals can be promoted by elevated pCO2. To investigate the mechanisms underlying corals’ complex responses to global change, three species of tropical zooxanthellate corals (Stylophora pistillata, Pocillopora damicornis, and Seriatopora hystrix) and one species of asymbiotic cold-water coral (Desmophyllum pertusum, syn. Lophelia pertusa) were cultured under a range of ocean acidification and warming scenarios. Under control temperatures, all tropical species exhibited increased calcification rates in response to increasing pCO2. However, the tropical species’ response to increasing pCO2 flattened when they lost symbionts (i.e., bleached) under the high-temperature treatments—suggesting that the loss of symbionts neutralized the benefit of increased pCO2 on calcification rate. Notably, the cold-water species that lacks symbionts exhibited a negative calcification response to increasing pCO2, although this negative response was partially ameliorated under elevated temperature. All four species elevated their calcifying fluid pH relative to seawater pH under all pCO2 treatments, and the magnitude of this offset (Δ[H+]) increased with increasing pCO2. Furthermore, calcifying fluid pH decreased along with symbiont abundance under thermal stress for the one species in which calcifying fluid pH was measured under both temperature treatments. This observation suggests a mechanistic link between photosymbiont loss (‘bleaching’) and impairment of zooxanthellate corals’ ability to elevate calcifying fluid pH in support of calcification under heat stress. This study supports the assertion that thermally induced loss of photosymbionts impairs tropical zooxanthellate corals’ ability to cope with CO2-induced ocean acidification.