Oxygen isotopes in orangutan teeth reveal recent and ancient climate variation.

Studies of climate variation commonly rely on chemical and isotopic changes recorded in sequentially produced growth layers, such as in corals, shells, and tree rings, as well as in accretionary deposits-ice and sediment cores, and speleothems. Oxygen isotopic compositions (δ18O) of tooth enamel are a direct method of reconstructing environmental variation experienced by an individual animal. Here, we utilize long-forming orangutan dentitions (Pongo spp.) to probe recent and ancient rainfall trends on a weekly basis over ~3-11 years per individual. We first demonstrate the lack of any consistent isotopic enrichment effect during exclusive nursing, supporting the use of primate first molar teeth as environmental proxies. Comparisons of δ18O values (n=2016) in twelve molars from six modern Bornean and Sumatran orangutans reveal a high degree of overlap, with more consistent annual and bimodal rainfall patterns in the Sumatran individuals. Comparisons with fossil orangutan δ18O values (n=955 measurements from six molars) reveal similarities between modern and late Pleistocene fossil Sumatran individuals, but differences between modern and late Pleistocene/early Holocene Bornean orangutans. These suggest drier and more open environments with reduced monsoon intensity during this earlier period in northern Borneo, consistent with other Niah Caves studies and long-term speleothem δ18O records in the broader region. This approach can be extended to test hypotheses about the paleoenvironments that early humans encountered in southeast Asia.

When an animal drinks water, two naturally occurring variants of oxygen ­ known as oxygen-18 and oxygen-16 ­ are incorporated into its growing teeth. The ratio of these variants in water changes with temperature, rainfall and other environmental conditions and therefore can provide a record of the climate during an animal's life. Teeth tend to be well preserved as fossils, which makes it possible to gain insights into this climate record even millions of years after an animal's death. Orangutans are highly endangered great apes that today live in rainforests on the islands of Borneo and Sumatra. During a period of time known as the Pleistocene (around 2.6 million years to 12,000 years ago), these apes were more widely spread across Southeast Asia. Climate records from this area in the time before human-induced climate change are somewhat limited. Therefore, fossilized orangutan teeth offer a possible way to investigate past seasonal rainfall patterns and gain insight into the kind of environments early humans would have encountered. To address this question, Smith et al. measured oxygen-18 and oxygen-16 variants in thin slices of modern-day orangutan teeth using a specialized analytical system. This established that the teeth showed seasonal patterns consistent with recent rainfall trends, and that the ratio of these oxygen variants did not appear to be impacted by milk intake in young orangutans. These findings indicated that the oxygen variants could be a useful proxy for predicting prehistoric weather patterns from orangutan teeth. Further measurements of teeth from fossilized Sumatran orangutans showed broadly similar rainfall patterns to those of teeth from modern-day orangutans. On the other hand, fossilized teeth from Borneo suggested that the environment used to be drier, with less intense wet seasons. The approach developed by Smith et al. provides an opportunity for scientists to leverage new fossil discoveries as well as existing collections to investigate past environments. This could allow future research into how climate variation may have influenced the spread of early humans through the region, as well as the evolution of orangutans and other endangered animals.

Formation and Replacement of Bone and Tooth Mineralized Tissues in Green Iguanas (Iguana iguana) Revealed by In-Vivo Fluorescence Marking.

Hard tissue formation patterns and rates reveal details of animal physiology, life history, and environment, but are understudied in reptiles. Here, we use fluorescence labels delivered in vivo and laser confocal scanning microscopy to study tooth and bone formation in a managed group of green iguanas (Iguana iguana, Linné 1758) kept for 1.5 years under experimentally controlled conditions and undergoing several dietary switches. We constrain rates of tooth elongation, which we observe to be slow when enamel is initially deposited (c. 9 µm/day), but then increases exponentially in the dentin root, reaching c. 55 µm/day or more after crown completion. We further constrain the total timing of tooth formation to ∼40-60 days, and observe highly variable timings of tooth resorption onset and replacement. Fluorescent labels clearly indicate cohorts of teeth recruited within Zahnreihen replacement waves, with faster sequential tooth recruitment and greater wave sizes posteriorly, where each wave initiates. Fluorescence further reveals enamel maturation after initial deposition. Rates of hard tissue formation in long bones range from 0.4 to 3.4 µm/day, correlating with animal weight gain and cortical bone recording the entire history of the experiment. We suggest additional labeling experiments to study hard tissue formation patterns in other reptiles, and propose strategies for chemical analyses of hard tissues in order to extract temporal information about past environments, behaviors, and diets from reptilian fossils throughout the Phanerozoic.

Fine-scaled climate variation in equatorial Africa revealed by modern and fossil primate teeth.

Variability in resource availability is hypothesized to be a significant driver of primate adaptation and evolution, but most paleoclimate proxies cannot recover environmental seasonality on the scale of an individual lifespan. Oxygen isotope compositions (δ18O values) sampled at high spatial resolution in the dentitions of modern African primates (n = 2,352 near weekly measurements from 26 teeth) track concurrent seasonal precipitation, regional climatic patterns, discrete meteorological events, and niche partitioning. We leverage these data to contextualize the first δ18O values of two 17 Ma Afropithecus turkanensis individuals from Kalodirr, Kenya, from which we infer variably bimodal wet seasons, supported by rainfall reconstructions in a global Earth system model. Afropithecus' δ18O fluctuations are intermediate in magnitude between those measured at high resolution in baboons (Papio spp.) living across a gradient of aridity and modern forest-dwelling chimpanzees (Pan troglodytes verus). This large-bodied Miocene ape consumed seasonally variable food and water sources enriched in 18O compared to contemporaneous terrestrial fauna (n = 66 fossil specimens). Reliance on fallback foods during documented dry seasons potentially contributed to novel dental features long considered adaptations to hard-object feeding. Developmentally informed microsampling recovers greater ecological complexity than conventional isotope sampling; the two Miocene apes (n = 248 near weekly measurements) evince as great a range of seasonal δ18O variation as more time-averaged bulk measurements from 101 eastern African Plio-Pleistocene hominins and 42 papionins spanning 4 million y. These results reveal unprecedented environmental histories in primate teeth and suggest a framework for evaluating climate change and primate paleoecology throughout the Cenozoic.

A simple CO2 equilibration method for measuring blood oxygen isotope compositions.

RATIONALE: Blood water oxygen isotope compositions can provide valuable insights into physiological processes and ecological patterns. While blood samples are commonly drawn for medical or scientific purposes, blood fractions are infrequently measured for oxygen isotopic compositions (δ18 O) because such measurements are time consuming and expensive. METHODS: We sampled blood from sheep, goats, and iguanas raised in field and animal laboratories into serum, EDTA, heparin, and uncoated plastic vials commonly used in medical and scientific research, then separated red blood cell (RBC) and plasma or serum blood fractions. These were injected into helium-flushed Exetainer tubes where they naturally outgassed endogenous CO2 (goat blood), or into He- and CO2 -flushed tubes (iguana blood). The CO2 gas was sampled on a GasBench II system, and δ18 O was measured by an isotope ratio mass spectrometer (IRMS). RESULTS: Repeated δ18 O measurements were stable over multiple days. The addition of desiccated blood solids to water standards had little impact on their δ18 O measurements, suggesting that organic molecular constituents within blood serum and plasma do not interfere with blood water δ18 O values. We observed slight but statistically significant δ18 O offsets between plasma, serum and RBC fractions. Mass-dependent body water turnover times for iguanas were derived from the data. CONCLUSIONS: We demonstrate that a simple blood-CO2 equilibration method using the GasBench can quickly, reliably and accurately characterize water δ18 O in the plasma, RBC, and whole blood fractions of mammalian and reptilian blood samples (precision ≤ 0.1‰). This method will expand the application of blood stable isotope analysis in physiological and medical research.

Teeth reveal juvenile diet, health and neurotoxicant exposure retrospectively: What biological rhythms and chemical records tell us.

Integrated developmental and elemental information in teeth provide a unique framework for documenting breastfeeding histories, physiological disruptions, and neurotoxicant exposure in humans and our primate relatives, including ancient hominins. Here we detail our method for detecting the consumption of mothers' milk and exploring health history through the use of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) mapping of sectioned nonhuman primate teeth. Calcium-normalized barium and lead concentrations in tooth enamel and dentine may reflect milk and formula consumption with minimal modification during subsequent tooth mineralization, particularly in dentine. However, skeletal resorption during severe illness, and bioavailable metals in nonmilk foods, can complicate interpretations of nursing behavior. We show that explorations of the patterning of multiple elements may aid in the distinction of these important etiologies. Targeted studies of skeletal chemistry, gastrointestinal maturation, and the dietary bioavailability of metals are needed to optimize these unique records of human health and behavior.

Crystal misorientation correlates with hardness in tooth enamels.

The multi-scale hierarchical structure of tooth enamel enables it to withstand a lifetime of damage without catastrophic failure. While many previous studies have investigated structure-function relationships in enamel, the effects of crystal misorientation on mechanical performance have not been assessed. To address this issue, in the present study, we review previously published polarization-dependent imaging contrast (PIC) maps of mouse and human enamel, and parrotfish enameloid, in which crystal orientations were measured and displayed in every 60-nm-pixel. By combining those previous results with the PIC maps of sheep enamel presented here we discovered that, in all enamel(oid)s, adjacent crystals are slightly misoriented, with misorientation angles in the 0°-30° range, and mean 2°-8°. Within this limited range, misorientation is positively correlated with literature hardness values, demonstrating an important structure-property relation, not previously identified. At greater misorientation angles 8°30°, this correlation is expected to reverse direction, but data from different non-enamel systems, with more diverse crystal misorientations, are required to determine if and where this occurs. STATEMENT OF SIGNIFICANCE: We identify a structure-function relationship in tooth enamels from different species: crystal misorientation correlates with hardness, contributing to the remarkable mechanical properties of enamel in diverse animals.

Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns.

Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

Determinants of blood water δ 18O variation in a population of experimental sheep: implications for paleoclimate reconstruction.

Mammalian body, blood and hard tissue oxygen isotope compositions (δ 18O values) reflect environmental water and food sources, climate, and physiological processes. For this reason, fossil and archaeological hard tissues, which originally formed in equilibrium with body chemistry, are a valuable record of past climate, landscape paleoecology, and animal physiology and behavior. However, the environmental and physiological determinants of blood oxygen isotope composition have not been determined experimentally from large herbivores. This class of fauna is abundant in Cenozoic terrestrial fossil assemblages, and the isotopic composition of large herbivore teeth has been central to a number of climate and ecological reconstructions. Furthermore, existing models predict blood water, or nearly equivalently body water, δ 18O values based on environmental water sources. These have been evaluated on gross timescales, but have not been employed to track seasonal variation. Here we report how water, food, and physiology determine blood water δ 18O values in experimental sheep (Ovis aries) subjected to controlled water switches. We find that blood water δ 18O values rapidly reach steady state with environmental drinking water and reflect transient events including weaning, seasons, and snowstorms. Behavioral and physiological variation within a single genetically homogenous population of herbivores results in significant inter-animal variation in blood water δ 18O values at single collection times (1 s.d. = 0.1-1.4 ‰, range = 3.5 ‰) and reveals a range of water flux rates (t1/2 = 2.2-2.9 days) within the population. We find that extant models can predict average observed sheep blood δ 18O values with striking fidelity, but predict a pattern of seasonal variation exactly opposite of that observed in our population for which water input variation was controlled and the effect of physiology was more directly observed. We introduce to these models an evaporative loss term that is a function of environmental temperatures. The inclusion of this function produces model predictions that mimic the observed seasonal fluctuations and match observations to within 1.0 ‰. These results increase the applicability of available physiological models for paleoseasonality reconstructions from stable isotope measurements in fossil or archaeological enamel, the composition of which is determined in equilibrium with blood values. However, significant blood δ 18O variation in this experimentally controlled population should promote caution when interpreting isotopic variation in the archaeological and paleontological record.

Wintertime stress, nursing, and lead exposure in Neanderthal children.

Scholars endeavor to understand the relationship between human evolution and climate change. This is particularly germane for Neanderthals, who survived extreme Eurasian environmental variation and glaciations, mysteriously going extinct during a cool interglacial stage. Here, we integrate weekly records of climate, tooth growth, and metal exposure in two Neanderthals and one modern human from southeastern France. The Neanderthals inhabited cooler and more seasonal periods than the modern human, evincing childhood developmental stress during wintertime. In one instance, this stress may have included skeletal mobilization of elemental stores and weight loss; this individual was born in the spring and appears to have weaned 2.5 years later. Both Neanderthals were exposed to lead at least twice during the deep winter and/or early spring. This multidisciplinary approach elucidates direct relationships between ancient environments and hominin paleobiology.

Synchrotron imaging and Markov Chain Monte Carlo reveal tooth mineralization patterns.

The progressive character of tooth formation records aspects of mammalian life history, diet, seasonal behavior and climate. Tooth mineralization occurs in two stages: secretion and maturation, which overlap to some degree. Despite decades of study, the spatial and temporal pattern of elemental incorporation during enamel mineralization remains poorly characterized. Here we use synchrotron X-ray microtomography and Markov Chain Monte Carlo sampling to estimate mineralization patterns from an ontogenetic series of sheep molars (n = 45 M1s, 18 M2s). We adopt a Bayesian approach that posits a general pattern of maturation estimated from individual- and population-level mineral density variation over time. This approach converts static images of mineral density into a dynamic model of mineralization, and demonstrates that enamel secretion and maturation waves advance at nonlinear rates with distinct geometries. While enamel secretion is ordered, maturation geometry varies within a population and appears to be driven by diffusive processes. Our model yields concrete expectations for the integration of physiological and environmental signals, which is of particular significance for paleoseasonality research. This study also provides an avenue for characterizing mineralization patterns in other taxa. Our synchrotron imaging data and model are available for application to multiple disciplines, including health, material science, and paleontological research.

Matrix assisted ionization: new aromatic and nonaromatic matrix compounds producing multiply charged lipid, peptide, and protein ions in the positive and negative mode observed directly from surfaces.

Matrix assisted inlet ionization (MAII) is a method in which a matrix:analyte mixture produces mass spectra nearly identical to electrospray ionization without the application of a voltage or the use of a laser as is required in laserspray ionization (LSI), a subset of MAII. In MAII, the sample is introduced by, for example, tapping particles of dried matrix:analyte into the inlet of the mass spectrometer and, therefore, permits the study of conditions pertinent to the formation of multiply charged ions without the need of absorption at a laser wavelength. Crucial for the production of highly charged ions are desolvation conditions to remove matrix molecules from charged matrix:analyte clusters. Important factors affecting desolvation include heat, vacuum, collisions with gases and surfaces, and even radio frequency fields. Other parameters affecting multiply charged ion production is sample preparation, including pH and solvent composition. Here, findings from over 100 compounds found to produce multiply charged analyte ions using MAII with the inlet tube set at 450 °C are presented. Of the compounds tested, many have -OH or -NH(2) functionality, but several have neither (e.g., anthracene), nor aromaticity or conjugation. Binary matrices are shown to be applicable for LSI and solvent-free sample preparation can be applied to solubility restricted compounds, and matrix compounds too volatile to allow drying from common solvents. Our findings suggest that the physical properties of the matrix such as its morphology after evaporation of the solvent, its propensity to evaporate/sublime, and its acidity are more important than its structure and functional groups.

Evolution of the complementary sex-determination gene of honey bees: balancing selection and trans-species polymorphisms.

The mechanism of sex determination varies substantively among evolutionary lineages. One important mode of genetic sex determination is haplodiploidy, which is used by approximately 20% of all animal species, including >200,000 species of the entire insect order Hymenoptera. In the honey bee Apis mellifera, a hymenopteran model organism, females are heterozygous at the csd (complementary sex determination) locus, whereas males are hemizygous (from unfertilized eggs). Fertilized homozygotes develop into sterile males that are eaten before maturity. Because homozygotes have zero fitness and because common alleles are more likely than rare ones to form homozygotes, csd should be subject to strong overdominant selection and negative frequency-dependent selection. Under these selective forces, together known as balancing selection, csd is expected to exhibit a high degree of intraspecific polymorphism, with long-lived alleles that may be even older than the species. Here we sequence the csd genes as well as randomly selected neutral genomic regions from individuals of three closely related species, A. mellifera, Apis cerana, and Apis dorsata. The polymorphic level is approximately seven times higher in csd than in the neutral regions. Gene genealogies reveal trans-species polymorphisms at csd but not at any neutral regions. Consistent with the prediction of rare-allele advantage, nonsynonymous mutations are found to be positively selected in csd only in early stages after their appearances. Surprisingly, three different hypervariable repetitive regions in csd are present in the three species, suggesting variable mechanisms underlying allelic specificities. Our results provide a definitive demonstration of balancing selection acting at the honey bee csd gene, offer insights into the molecular determinants of csd allelic specificities, and help avoid homozygosity in bee breeding.