Author Correction: The dental proteome of Homo antecessor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The dental proteome of Homo antecessor.

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated1-5. For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge6-8. Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain)9,10 and Homo erectus from Dmanisi (Georgia)1, two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor-that is, similar to that of modern humans-may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Dating the Paleolithic: Trapped charge methods and amino acid geochronology.

Despite the vast array of different geochronological tools available, dating the Paleolithic remains one of the discipline's greatest challenges. This review focuses on two different dating approaches: trapped charge and amino acid geochronology. While differing in their fundamental principles, both exploit time-dependent changes in signals found within crystals to generate a chronology for the material dated and hence, the associated deposits. Within each method, there is a diverse range of signals that can be analyzed, each covering different time ranges, applicable to different materials and suitable for different paleoenvironmental and archaeological contexts. This multiplicity of signals can at first sight appear confusing, but it is a fundamental strength of the techniques, allowing internal checks for consistency and providing more information than simply a chronology. For each technique, we present an overview of the basis for the time-dependent signals and the types of material that can be analyzed, with examples of their archaeological application, as well as their future potential.

Alkylresorcinol detection and identification in archaeological pottery using ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry.

RATIONALE: Alkylresorcinols (AR) are cereal-specific biomarkers and have recently been found in archaeological pots. However, their low concentrations and high susceptibility to degradation make them difficult to detect using conventional gas chromatography mass spectrometry (GC/MS). Here we describe the development of a more sensitive liquid chromatography mass spectrometry (LC/MS) method to detect these compounds. METHOD: A method based on the use of ultra-high-performance liquid chromatography (UHPLC) coupled to an Orbitrap mass analyser was established and validated for the detection of low-concentration ARs in pottery. During the preliminary experiments, UHPLC-Q/Orbitrap MS (ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry) was demonstrated to be more sensitive, and a wide range of AR homologues in cereal extracts were detected, unlike UHPLC-QTOFMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry) and GC/MS. The developed method was utilised to profile AR homologue distribution in modern cereal samples and reanalyse AR-containing pots from the archaeological site of Must Farm. RESULTS: A highly sensitive LC/MS method with a limit of detection (LOD) of 0.02 µg/g and a limit of quantification (LOQ) of 0.06 µg/g was used to profile ARs in five modern cereal grains. The obtained LOD is 250 times lower than that obtained using the conventional GC/MS approach. AR 21:0 was the most abundant homologue in all four Triticum spp.-einkorn, emmer, Khorasan wheat and common wheat. Meanwhile, AR 25:0 was the predominant homologue in barley, potentially enabling differentiation between wheat and barley. The developed LC/MS-based method was successfully used to analyse ARs extracted from Must Farm potsherds and identified the cereal species most likely processed in the pots-emmer wheat. CONCLUSION: The described method offers an alternative and more sensitive approach for detecting and identifying ARs in ancient pottery. It has been successfully utilised to detect AR homologues in archaeological samples and discriminate which cereal species-wheat and barley-were processed in the pots.

Ionisation bias undermines the use of matrix-assisted laser desorption/ionisation for estimating peptide deamidation: Synthetic peptide studies demonstrate electrospray ionisation gives more reliable response ratios.

RATIONALE: Although mass spectrometry (MS) is routinely used to determine deamination in peptide mixtures, the effects of the choice of ionisation source have not yet been investigated. In particular, matrix-assisted laser desorption/ionisation (MALDI) has become a popular tool with which to measure levels of glutamine deamidation in ancient proteins. Here we use model synthetic peptides to rigorously compare MALDI and electrospray ionisation (ESI). METHODS: We used two synthetic peptides, with glutamine (Q) in one substituted for glutamic acid (E) in the other, to investigate the suitability of MALDI and ESI sources for the assessment of deamidation in peptides using MS. We also compared measurements of the same Q- and E-containing peptide mixtures using two different mass analysers (time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR)). RESULTS: When standard mixtures of the Q- and E-containing peptides were analysed using MALDI, under-representation of the E-containing peptide was observed. This observation was consistent between analyses carried out using either TOF or FT-ICR-MS. When the same mixtures were analysed using ESI FT-ICR-MS, no ionisation bias was observed. CONCLUSIONS: MALDI may not be a suitable ionisation method for the determination of deamidation in peptide mixtures. However, ESI was successfully used to determine the ratio in known mixtures of Q- and E-containing peptides. These preliminary observations warrant further investigation into ionisation bias when measuring deamidation in other peptide sequences.

Lessons from Star Carr on the vulnerability of organic archaeological remains to environmental change.

Examples of wetland deposits can be found across the globe and are known for preserving organic archaeological and environmental remains that are vitally important to our understanding of past human-environment interactions. The Mesolithic site of Star Carr (Yorkshire, United Kingdom) represents one of the most influential archives of human response to the changing climate at the end of the last glacial in Northern Europe. A hallmark of the site since its discovery in 1948 has been the exceptional preservation of its organic remains. Disturbingly, recent excavations have suggested that the geochemistry of the site is no longer conducive to such remarkable survival of organic archaeological and environmental materials. Microcosm (laboratory-based) burial experiments have been undertaken, alongside analysis of artifacts excavated from the site, to assess the effect of these geochemical changes on the remaining archaeological material. By applying a suite of macroscopic and molecular analyses, we demonstrate that the geochemical changes at Star Carr are contributing to the inexorable and rapid loss of valuable archaeological and paleoenvironmental information. Our findings have global implications for other wetland sites, particularly archaeological sites preserved in situ.

Collagen proteins exchange oxygen with demineralisation and gelatinisation reagents and also with atmospheric moisture.

RATIONALE: The oxygen (O) isotope composition of collagen proteins is a potential indicator of adult residential location, useful for provenancing in ecology, archaeology and forensics. In acidic solution, proteins can exchange O from carboxylic acid moieties with reagent O. This study investigated whether this exchange occurs during demineralisation and gelatinisation preparation of bone/ivory collagen. METHODS: EDTA and HCl demineralisation or gelatinisation reagents were made up in waters with different δ18 O values, and were used to extract collagen from four skeletal tissue samples. Aliquots of extracted collagen were exposed to two different atmospheric waters, at 120°C and ambient temperature, and subsequently dried in a vacuum oven at 40°C or by freeze drying. Sample δ18 O values were measured by HT-EA pyrolysis/IRMS using a zero-blank autosampler. RESULTS: Collagen samples exchanged O with both reagent waters and atmospheric water, which altered sample δ18 O values. Exchange with reagent waters occurred in all extraction methods, but was greater at lower pH. Damage to the collagen samples during extraction increased O exchange. The nature of exchange of O with atmospheric water depended on the temperature of exposure: kinetic fractionation of O was identified at 120°C but not at ambient temperature. Exchange was difficult to quantify due to the high variability of δ18 O values between experimental replicates. CONCLUSIONS: Studies of δ18 O values in collagen proteins should avoid extraction methods using acidic solutions.

Tuning hardness in calcite by incorporation of amino acids.

Structural biominerals are inorganic/organic composites that exhibit remarkable mechanical properties. However, the structure-property relationships of even the simplest building unit-mineral single crystals containing embedded macromolecules-remain poorly understood. Here, by means of a model biomineral made from calcite single crystals containing glycine (0-7 mol%) or aspartic acid (0-4 mol%), we elucidate the origin of the superior hardness of biogenic calcite. We analysed lattice distortions in these model crystals by using X-ray diffraction and molecular dynamics simulations, and by means of solid-state nuclear magnetic resonance show that the amino acids are incorporated as individual molecules. We also demonstrate that nanoindentation hardness increased with amino acid content, reaching values equivalent to their biogenic counterparts. A dislocation pinning model reveals that the enhanced hardness is determined by the force required to cut covalent bonds in the molecules.

A chronological framework for the British Quaternary based on Bithynia opercula.

Marine and ice-core records show that the Earth has experienced a succession of glacials and interglacials during the Quaternary (last ∼2.6 million years), although it is often difficult to correlate fragmentary terrestrial records with specific cycles. Aminostratigraphy is a method potentially able to link terrestrial sequences to the marine isotope stages (MIS) of the deep-sea record. We have used new methods of extraction and analysis of amino acids, preserved within the calcitic opercula of the freshwater gastropod Bithynia, to provide the most comprehensive data set for the British Pleistocene based on a single dating technique. A total of 470 opercula from 74 sites spanning the entire Quaternary are ranked in order of relative age based on the extent of protein degradation, using aspartic acid/asparagine (Asx), glutamic acid/glutamine (Glx), serine (Ser), alanine (Ala) and valine (Val). This new aminostratigraphy is consistent with the stratigraphical relationships of stratotypes, sites with independent geochronology, biostratigraphy and terrace stratigraphy. The method corroborates the existence of four interglacial stages between the Anglian (MIS 12) and the Holocene in the terrestrial succession. It establishes human occupation of Britain in most interglacial stages after MIS 15, but supports the notion of human absence during the Last Interglacial (MIS 5e). Suspicions that the treeless 'optimum of the Upton Warren interstadial' at Isleworth pre-dates MIS 3 are confirmed. This new aminostratigraphy provides a robust framework against which climatic, biostratigraphical and archaeological models can be tested.

Use of the 2-chlorotrityl chloride resin for microwave-assisted solid phase peptide synthesis.

A fast and efficient microwave (MW)-assisted solid-phase peptide synthesis protocol using the 2-chlorotrityl chloride resin and the Fmoc/tBu methodology, has been developed. The established protocol combines the advantages of MW irradiation and the acid labile 2-chlorotrityl chloride resin. The effect of temperature during the MW irradiation, the degree of resin substitution during the coupling of the first amino acids and the rate of racemization for each amino acid were evaluated. The suggested solid phase methodology is applicable for orthogonal peptide synthesis and for the synthesis of cyclic peptides.

Biomineralisation by earthworms - an investigation into the stability and distribution of amorphous calcium carbonate.

BACKGROUND: Many biominerals form from amorphous calcium carbonate (ACC), but this phase is highly unstable when synthesised in its pure form inorganically. Several species of earthworm secrete calcium carbonate granules which contain highly stable ACC. We analysed the milky fluid from which granules form and solid granules for amino acid (by liquid chromatography) and functional group (by Fourier transform infrared (FTIR) spectroscopy) compositions. Granule elemental composition was determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and electron microprobe analysis (EMPA). Mass of ACC present in solid granules was quantified using FTIR and compared to granule elemental and amino acid compositions. Bulk analysis of granules was of powdered bulk material. Spatially resolved analysis was of thin sections of granules using synchrotron-based µ-FTIR and EMPA electron microprobe analysis. RESULTS: The milky fluid from which granules form is amino acid-rich (≤ 136 ± 3 nmol mg-1 (n = 3; ± std dev) per individual amino acid); the CaCO3 phase present is ACC. Even four years after production, granules contain ACC. No correlation exists between mass of ACC present and granule elemental composition. Granule amino acid concentrations correlate well with ACC content (r ≥ 0.7, p ≤ 0.05) consistent with a role for amino acids (or the proteins they make up) in ACC stabilisation. Intra-granule variation in ACC (RSD = 16%) and amino acid concentration (RSD = 22-35%) was high for granules produced by the same earthworm. Maps of ACC distribution produced using synchrotron-based µ-FTIR mapping of granule thin sections and the relative intensity of the ν2: ν4 peak ratio, cluster analysis and component regression using ACC and calcite standards showed similar spatial distributions of likely ACC-rich and calcite-rich areas. We could not identify organic peaks in the µ-FTIR spectra and thus could not determine whether ACC-rich domains also had relatively high amino acid concentrations. No correlation exists between ACC distribution and elemental concentrations determined by EMPA. CONCLUSIONS: ACC present in earthworm CaCO3 granules is highly stable. Our results suggest a role for amino acids (or proteins) in this stability. We see no evidence for stabilisation of ACC by incorporation of inorganic components. Graphical abstractSynchrotron-based µ-FTIR mapping was used to determine the spatial distribution of amorphous calcium carbonate in earthworm-produced CaCO3 granules.

New experimental evidence for in-chain amino acid racemization of serine in a model peptide.

The facile racemization of protein-bound amino acids plays an important role in the aging and pathologies of living tissues, and it can be exploited for protein geochronological studies in subfossil biominerals. However, the in-chain degradation pathways of amino acids are complex and difficult to elucidate. Serine has proven to be particularly elusive, and its ability to racemize as a peptide-bound residue (like asparagine and aspartic acid) has not been demonstrated. This study investigates the patterns of degradation of a model peptide (WNSVWAW) at elevated temperatures, quantifying the extent of racemization and peptide bond hydrolysis using reverse-phase high-performance liquid chromatography (RP-HPLC) and tracking the presence of degradation products by MALDI-MS. We provide direct evidence that, under these experimental conditions, both serine and asparagine are able to undergo racemization as internally bound residues, which shows their potential for initiating protein breakdown and provides an explanation for the presence of d-enantiomers in living mammalian tissues.

An aminostratigraphy for the British Quaternary based on Bithynia opercula.

Aminostratigraphies of Quaternary non-marine deposits in Europe have been previously based on the racemization of a single amino acid in aragonitic shells from land and freshwater molluscs. The value of analysing multiple amino acids from the opercula of the freshwater gastropod Bithynia, which are composed of calcite, has been demonstrated. The protocol used for the isolation of intra-crystalline proteins from shells has been applied to these calcitic opercula, which have been shown to more closely approximate a closed system for indigenous protein residues. Original amino acids are even preserved in bithyniid opercula from the Eocene, showing persistence of indigenous organics for over 30 million years. Geochronological data from opercula are superior to those from shells in two respects: first, in showing less natural variability, and second, in the far better preservation of the intra-crystalline proteins, possibly resulting from the greater stability of calcite. These features allow greater temporal resolution and an extension of the dating range beyond the early Middle Pleistocene. Here we provide full details of the analyses for 480 samples from 100 horizons (75 sites), ranging from Late Pliocene to modern. These show that the dating technique is applicable to the entire Quaternary. Data are provided from all the stratotypes from British stages to have yielded opercula, which are shown to be clearly separable using this revised method. Further checks on the data are provided by reference to other type-sites for different stages (including some not formally defined). Additional tests are provided by sites with independent geochronology, or which can be associated with a terrace stratigraphy or biostratigraphy. This new aminostratigraphy for the non-marine Quaternary deposits of southern Britain provides a framework for understanding the regional geological and archaeological record. Comparison with reference to sites yielding independent geochronology, in combination with other lines of evidence, allows tentative correlation with the marine oxygen isotope record.

Optimising a method for aragonite precipitation in simulated biogenic calcification media.

Resolving how factors such as temperature, pH, biomolecules and mineral growth rate influence the geochemistry and structure of biogenic CaCO3, is essential to the effective development of palaeoproxies. Here we optimise a method to precipitate the CaCO3 polymorph aragonite from seawater, under tightly controlled conditions that simulate the saturation state (Ω) of coral calcification fluids. We then use the method to explore the influence of aspartic acid (one of the most abundant amino acids in coral skeletons) on aragonite structure and morphology. Using ≥200 mg of aragonite seed (surface area 0.84 m2), to provide a surface for mineral growth, in a 330 mL seawater volume, generates reproducible estimates of precipitation rate over Ωaragonite = 6.9-19.2. However, unseeded precipitations are highly variable in duration and do not provide consistent estimates of precipitation rate. Low concentrations of aspartic acid (1-10 µM) promote aragonite formation, but high concentrations (≥ 1 mM) inhibit precipitation. The Raman spectra of aragonite precipitated in vitro can be separated from the signature of the starting seed by ensuring that at least 60% of the analysed aragonite is precipitated in vitro (equivalent to using a seed of 200 mg and precipitating 300 mg aragonite in vitro). Aspartic acid concentrations ≥ 1mM caused a significant increase in the full width half maxima of the Raman aragonite v1 peak, reflective of increased rotational disorder in the aragonite structure. Changes in the organic content of coral skeletons can drive variations in the FWHM of the Raman aragonite ν1 peak, and if not accounted for, may confuse the interpretation of calcification fluid saturation state from this parameter.

The earliest record of human activity in northern Europe.

The colonization of Eurasia by early humans is a key event after their spread out of Africa, but the nature, timing and ecological context of the earliest human occupation of northwest Europe is uncertain and has been the subject of intense debate. The southern Caucasus was occupied about 1.8 million years (Myr) ago, whereas human remains from Atapuerca-TD6, Spain (more than 780 kyr ago) and Ceprano, Italy (about 800 kyr ago) show that early Homo had dispersed to the Mediterranean hinterland before the Brunhes-Matuyama magnetic polarity reversal (780 kyr ago). Until now, the earliest uncontested artefacts from northern Europe were much younger, suggesting that humans were unable to colonize northern latitudes until about 500 kyr ago. Here we report flint artefacts from the Cromer Forest-bed Formation at Pakefield (52 degrees N), Suffolk, UK, from an interglacial sequence yielding a diverse range of plant and animal fossils. Event and lithostratigraphy, palaeomagnetism, amino acid geochronology and biostratigraphy indicate that the artefacts date to the early part of the Brunhes Chron (about 700 kyr ago) and thus represent the earliest unequivocal evidence for human presence north of the Alps.

Amino acid racemization dating of marine shells: A mound of possibilities.

Shell middens are one of the most important and widespread indicators for human exploitation of marine resources and occupation of coastal environments. Establishing an accurate and reliable chronology for these deposits has fundamental implications for understanding the patterns of human evolution and dispersal. This paper explores the potential application of a new methodology of amino acid racemization (AAR) dating of shell middens and describes a simple protocol to test the suitability of different molluscan species. This protocol provides a preliminary test for the presence of an intracrystalline fraction of proteins (by bleaching experiments and subsequent heating at high temperature), checking the closed system behaviour of this fraction during diagenesis. Only species which pass both tests can be considered suitable for further studies to obtain reliable age information. This amino acid geochronological technique is also applied to midden deposits at two latitudinal extremes: Northern Scotland and the Southern Red Sea. Results obtained in this study indicate that the application of this new method of AAR dating of shells has the potential to aid the geochronological investigation of shell mounds in different areas of the world.

Assessment of different screening methods for selecting palaeontological bone samples for peptide sequencing.

Ancient proteomics is being applied to samples dating further and further back in time, with many palaeontological specimens providing protein sequence data for phylogenetic analysis as well as protein degradation studies. However, fossils are a precious material and proteomic analysis is destructive and costly. In this paper we consider three different techniques (ATR-FTIR, MALDI-ToF MS and chiral AA analysis) to screen fossil material for potential protein preservation, aiming to maximise the proteomic information recovered and saving costly time consuming analyses which may produce low quality results. It was found that splitting factor and C/P indices from ATR-FTIR were not a reliable indicator of protein survival as they are confounded by secondary mineralisation of the fossil material. Both MALDI-ToF MS and chiral AA analysis results were able to successfully identify samples with surviving proteins, and it is suggested that one or both of these analyses be used for screening palaeontological specimens. SIGNIFICANCE: This study has shown both chiral amino acid analysis and MALDI-ToF MS are reliable screening methods for predicting protein survival in fossils. Both these methods are quick, cheap, minimally destructive (1 mg and 15 mg respectively) and can provide crucial additional information about the endogeneity of the surviving proteins. It is hoped that the use of these screening methods will encourage the examination of a wide range of palaeontological specimens for potential proteomic analysis. This in turn will give us a better understanding of protein survival far back in time and under different environmental conditions.

Estimating the dwarfing rate of an extinct Sicilian elephant.

Evolution on islands, together with the often extreme phenotypic changes associated with it, has attracted much interest from evolutionary biologists. However, measuring the rate of change of phenotypic traits of extinct animals can be challenging, in part due to the incompleteness of the fossil record. Here, we use combined molecular and fossil evidence to define the minimum and maximum rate of dwarfing in an extinct Mediterranean dwarf elephant from Puntali Cave (Sicily).1 Despite the challenges associated with recovering ancient DNA from warm climates,2 we successfully retrieved a mitogenome from a sample with an estimated age between 175,500 and 50,000 years. Our results suggest that this specific Sicilian elephant lineage evolved from one of the largest terrestrial mammals that ever lived3 to an island species weighing less than 20% of its original mass with an estimated mass reduction between 0.74 and 200.95 kg and height reduction between 0.15 and 41.49 mm per generation. We show that combining ancient DNA with paleontological and geochronological evidence can constrain the timing of phenotypic changes with greater accuracy than could be achieved using any source of evidence in isolation.

The role of aspartic acid in reducing coral calcification under ocean acidification conditions.

Biomolecules play key roles in regulating the precipitation of CaCO3 biominerals but their response to ocean acidification is poorly understood. We analysed the skeletal intracrystalline amino acids of massive, tropical Porites spp. corals cultured over different seawater pCO2. We find that concentrations of total amino acids, aspartic acid/asparagine (Asx), glutamic acid/glutamine and alanine are positively correlated with seawater pCO2 and inversely correlated with seawater pH. Almost all variance in calcification rates between corals can be explained by changes in the skeletal total amino acid, Asx, serine and alanine concentrations combined with the calcification media pH (a likely indicator of the dissolved inorganic carbon available to support calcification). We show that aspartic acid inhibits aragonite precipitation from seawater in vitro, at the pH, saturation state and approximate aspartic acid concentrations inferred to occur at the coral calcification site. Reducing seawater saturation state and increasing [aspartic acid], as occurs in some corals at high pCO2, both serve to increase the degree of inhibition, indicating that biomolecules may contribute to reduced coral calcification rates under ocean acidification.