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Palaeontologia Electronica palaeo-electronica.org

Methods for isolation and quantification of teeth and elasmobranch dermal denticles (ichthyoliths) from marine

Elizabeth C. Sibert, Katie L. Cramer, Philip A. Hastings, and Richard D. Norris

ABSTRACT

Ichthyoliths—microfossil fish teeth and dermal scales (denticles)—are found in nearly all marine sediments. Their small size and relative rarity compared to other microfossil groups means that they have been largely ignored by the paleontol- ogy and communities, except as carriers of certain isotopic sys- tems. Yet, when properly concentrated, ichthyoliths are sufficiently abundant to reveal patterns of fish abundance and diversity at unprecedented temporal and spatial resolu- tion, in contrast to the typical millions of years-long gaps in the body record. In addition, ichthyoliths are highly resistant to dissolution, making it possible to reconstruct whole fish communities over highly precise and virtually continuous times- cales. Here we present methods to isolate and utilize ichthyoliths preserved in the sed- imentary record to track fish community structure and ecosystem productivity through geological and historical time periods. These include techniques for isolation and con- centration of these from a wide range of sediments, including deep- and coral carbonates, clays, shales, and -rich sediments. We also present a novel protocol for ichthyolith staining using Alizarin Red S to easily visualize and dis- tinguish small teeth from debris in the sample. Finally, we discuss several metrics for quantification of ichthyolith community structure and abundance, and their applications to reconstruction of ancient marine food webs and environments.

Elizabeth C. Sibert. Society of Fellows, Harvard University, 78 Mount Auburn Street, Cambridge, Massachusetts 02138, USA; Scripps Institution of Oceanography, University of California, San Diego, 9500 Gilman Drive MC 0208, La Jolla, California 92093, USA. [email protected] Katie L. Cramer. Scripps Institution of Oceanography, University of California, San Diego, 9500 Gilman Drive MC 0208, La Jolla, California 92093, USA. [email protected] Philip A. Hastings. Scripps Institution of Oceanography, University of California, San Diego, 9500 Gilman Drive MC 0208, La Jolla, California 92093, USA. [email protected] Richard D. Norris. Scripps Institution of Oceanography, University of California, San Diego, 9500 Gilman Drive MC 0244, La Jolla, California 92093, USA. [email protected]

Keywords: fish teeth; denticles; ichthyoliths; coral reefs; historical ;

Submission: 4 May 2016 Acceptance: 13 March 2017

Sibert, Elizabeth C., Cramer, Katie L., Hastings, Philip A., and Norris, Richard D. 2017. Methods for isolation and quantification of microfossil fish teeth and elasmobranch dermal denticles (ichthyoliths) from marine sediments. Palaeontologia Electronica 20.1.2T: 1-14 palaeo-electronica.org/content/2017/1800-quantifying-ichthyoliths

Copyright: April 2017 Palaeontological Association SIBERT: QUANTIFYING ICHTHYOLITHS

INTRODUCTION and therefore the abundance and composition of ichthyoliths may be a reasonable for ecosys- Despite being relatively common in marine tem structure and function. Moreover, there are sediments, ichthyoliths, the isolated microfossil typically excellent chronologies and relatively con- teeth, dermal denticles, and fragments of tinuous rates in many deep-sea sed- and bony (Figure 1), have been over- imentary sequences (e.g., Hilgen, 1991; looked by much of the scientific community, over- Westerhold et al., 2008; Hilgen et al., 2010). Thus, shadowed by more abundant and better studied it is possible to capture unusually detailed histories , nannofossils, and other microfossils, of , as compared to the typical temporal for research into biological responses to ancient and spatial fragmentation of the terrestrially climate and environmental change (Cifelli, 1969; exposed body-fossil record. In recent, shallow Frerichs, 1971; Smit, 1982; Hallock and Schlager, marine sediments, ichthyoliths can reveal changes 1986; Kelly et al., 1998; Hull et al., 2011). While in both diversity and abundance of fishes and understanding the response of these unicellular sharks in coastal systems—making it possible to to climate and biotic events provides reconstruct fish community responses to overfish- insight into the sensitivity of marine ecosystems to ing, reef environmental decline and anthropogenic global change, unicellular and are climate change (Jackson et al., 2001). only the of a complex marine ecosystem, While ichthyoliths have a rich his- which support a diverse array of consumers, tory of study (Maisey, 1984; Turner, 2004), younger including marine vertebrates. ichthyoliths (Late and Cenozoic) have Fishes are one of the most diverse and eco- largely been ignored by the commu- logically successful vertebrate clades (Nelson, nity, excepting large shark teeth (Cappetta and 2006; Friedman and Sallan, 2012; Near et al., Schultze, 2012), as the majority of stem diversity 2013) and are a hallmark of nearly all marine eco- for living clades was established by the Mesozoic. systems. The presence and abundance of fish bio- Cretaceous and Cenozoic ichthyoliths have been mass is an indicator of how efficiently an used as carriers of several isotopic proxies, includ- ecosystem is functioning, in terms of transferring ing neodymium as a -mass tracer (Martin and energy from the base of the food web to the upper Haley, 2000; Scher and Martin, 2004), and stron- tiers (Sprules and Munawar, 1986; Iverson, 1990), tium, which can be used both as a

FIGURE 1. An assortment of large (>106 μm fraction) denticles (elasmobranch scales; left) and fish teeth (right) from DSDP Site 596, a red core in the South Pacific. These ichthyoliths are approximately 52 million years old. Image was taken on the Hull Lab Imaging System, Yale University. Scale bar is 500 μm.

2 PALAEO-ELECTRONICA.ORG proxy, and for rough dating of sediments (Ingram, that they can be translated to other marine and 1995; Gleason et al., 2002, 2004, 2008). The field lacustrine sediments as well. of ichthyolith was developed in the early 1970s (Doyle et al., 1974; Edgerton et al., METHODS FOR ICHTHYOLITH ISOLATION 1977). Composed of calcium-, ichthyo- AND CONCENTRATION liths are extremely dissolution resistant, and are We present methods for isolation of ichthyo- one of the last microfossil groups remaining in liths from a variety of types, summarized marine sediments exposed to corrosive bottom in a flow chart (Figure 2), and discuss the specifics water and can be used to date fossil-poor pelagic of each protocol within the text. It is usually imprac- red clays (Doyle and Riedel, 1979b, 1985; Doyle et tical to sort through disaggregated sediments for al., 1988). An updated ichthyolith biostratigraphy ichthyoliths due to their small size and rarity com- for the Eastern North Pacific was developed in pared to other microfossils such as benthic and 2006 (Johns et al., 2005, 2006). planktonic foraminifera and other coarse-grained However, the Cretaceous and Cenozoic ich- sediment clasts. Because metrics of ichthyolith thyolith record can reveal important information accumulation (abundance) and community struc- about the role of fishes in aquatic ecosystems and ture rely on the quantification of all ichthyoliths in a their response to global change events (Sibert et sample, as opposed to a randomly sampled sub- al., 2014, 2016; Sibert and Norris, 2015). As ichthy- set, it is necessary to concentrate the full ichthyo- oliths are found in nearly all sediment types, includ- lith assemblage from a raw sediment sample. ing those of the open , which are rarely Processing a sediment sample for ichthyoliths is a preserved on land, pelagic ichthyoliths represent a balance between efficient concentration (typically fossil record virtually untouched by traditional by disaggregation of sediment and washing paleoichthyology. Further, ichthyolith through a fine sieve), and minimization of potential records have the potential for identification to loss of teeth by dissolution, fragmentation or extant taxa, and can show changes in functional adherence onto surfaces such as paintbrushes, and taxonomic groups over prehistorical and his- splitters, vials, or other surfaces during processing torical time periods resulting from environmental and picking. While ichthyoliths are composed of and/or anthropogenic change (Cramer et al., calcium phosphate (bio-apatite), which is resistant 2017). For example, on modern coral reefs, the to dissolution, care must be taken to counteract abundance of coral-associated fishes is a reliable potential destruction and loss of ichthyoliths when indicator of coral abundance and growth, and using methods of acid preparation or bleach-medi- intensive algal grazing by herbivorous fishes facili- ated disaggregation of sediments. Once washed, tates coral dominance (Randall, 1961; Bellwood ichthyoliths are picked out of the remaining residue and Wainwright, 2002). Thus the ichthyolith record, using a high-power dissection microscope and in conjunction with other microfossil and geochemi- extremely fine paintbrush. cal records, can provide insight into ecosystem A challenge in working with ichthyoliths is their response and resilience to climatic, biotic, and small size: the vast majority of teeth in pelagic sed- even anthropogenic perturbations (Sibert et al., iments are only retained on a 38 μm screen, pass- 2014; Sibert and Norris, 2015; Cramer et al., ing through the typical 63 μm sieves used for most 2017). Lastly, understanding how this group of con- foraminifera work. Modern reef fish teeth are sumers has responded to global change events somewhat larger, retained on 63 µm screens, how- may also yield insights into the mechanisms ever they are some of the smallest-sized compo- behind Cenozoic marine vertebrate evolution and nents of reef sediments. As a practical matter, most the development of the vast diversity of fish clades pelagic fish teeth are conical or triangular, and will (Nelson, 2006; Near et al., 2012, 2013; Betancur-R slip through the larger 63μm sieve even if they are et al., 2013; Broughton et al., 2013). much longer than 63 μm. We have found that Here, we provide a detailed methodological upwards of 50–80% of the total ichthyolith assem- framework for the isolation, concentration, and blage in pelagic sediments is represented by the analysis of ichthyoliths as a paleoceanographic, 38–63 μm fraction in pelagic sediments. It is likely paleoecological, and paleontological resource. that using a sieve smaller than 38 µm would yield Although the methods presented here have been additional ichthyoliths, as the majority of teeth in developed and tested with deep-sea sediments our samples are in the 38–63 μm fraction, however and near-modern sediments, we believe the <38 μm fraction presents significant technical

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FIGURE 2. A flowchart showing the steps for sediment processing for efficient and effective ichthyolith isolation from a variety of sediment types. Sediment types are in boxes, while processing steps are shown in ovals. challenges for reflected-light microscope-based elasmobranch abundance or productivity. Com- work. bined with the rich abundance of microfossil plank- ton, or the diverse hard parts of reef-associated Carbonates , foraminifera and algae, a well-studied car- Acid-resistant calcium phosphate ichthyoliths bonate section can yield information about many can be extracted from marine carbonates by acetic components of an ecosystem through an interval of acid dissolution of the matrix. In interest, giving environmental and ecological con- deep-sea sediments, carbonate-hosted ichthyolith text to an ichthyolith record (Sibert et al., 2014; assemblages can be placed on the highly resolved Cramer et al., 2017). time scales derived from analysis of other micro- Deep-sea carbonate ooze and . Simply fossil groups, magnetic reversals, or astrochronol- picking ichthyoliths out of the coarse fraction of car- ogies (e.g., Sibert et al., 2014). In Holocene coral bonate sediments is time consuming, and often reef sediments, high-precision uranium-thorium leads to poor data quality, as the small teeth are dating of coral can provide extremely obscured by the high abundance of foraminifera well-resolved chronologies of fish communities and siliceous microfossils. To concentrate ichthyo- over prehistorical and historical time (Cramer et al., liths effectively and address these issues, samples 2017). These precise time scales provide esti- are dried to a constant weight and then dissolved mates of sedimentation rate and mass accumula- in 5–10% acetic acid. Acid is added to the samples tion rate, which can be used to estimate fish and in 100–200 ml increments and stirred every ~20–

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30 minutes, until no carbonate remains, usually to other records. Additionally, large teeth (>150 after ~2–5 hours. The reaction is considered com- μm) are relatively rare in pelagic sediments and, if plete when no bubbles are released when adding ignored or under-counted, will not greatly bias the acid or stirring. We find that between 30 and 80 ml total ichthyolith accumulation rate. Indeed, it is also of dilute acetic acid is needed per gram of dry sedi- possible to count only the fraction subjected to acid ment to completely dissolve all of the carbonate in treatment since the fine fractions retain the vast a sample, depending both on the concentration of majority of teeth in a given sample; however in this acid used and the percent-carbonate composition case, information on the maximum size of teeth, or of the sediments. We do not observe any etching the change in abundance of specific large teeth or other damage to ichthyoliths during this process, and denticles, which are almost exclusively >100 and indeed, due to their high abundance and μm will be missed, which can preclude significant exceptional preservation in red clays, which were biological findings (Sibert and Norris, 2015). There- exposed to millions of years of relatively corrosive fore, the exact method employed will depend on bottom water, it is unlikely that this limited exposure the goals of the study. Above all, it is important to to weak acetic acid damages the ichthyoliths. maintain consistency in processing method Indeed, time-series experiments comparing acetic throughout the entire record. acid dissolution to hydrochloric acid dissolution . Lithified also yields ichthy- show that while HCl causes significant degradation oliths in the acid-insoluble fraction, however, the to ichthyoliths within 30 minutes of exposure, ace- processing is slightly different from that used for tic acid does not harm the ichthyoliths even after deep-sea carbonate sediments. Limestones should prolonged soaking. However, to avoid any potential be broken up into ~1 cm pieces, to increase sur- destruction of ichthyoliths, acid exposure should be face area exposed to acid, while preserving the limited and dilute acetic acid should be used. Once microfossils – in our experience, shatterboxes and dissolved, the sample is washed over a 38 μm other crushing tools can damage the . Our screen, and the residue is transferred to filter paper approach comprises barely covering samples with in a funnel and dried in a 50°C oven. 10% acetic acid—we have found that 5% is ineffec- Although it is destructive of the tive for the majority of limestones—and changing fossils, dissolution of bulk carbonate samples for the acid bath every 24 hours. When changing the ichthyoliths as outlined above, is by far the most acid, the sample is washed over a stack of sieves effective method for ichthyolith work. It also yields with all pieces of limestone >150 μm returned to the highest data quality, as every transfer of the fresh acid, and all residues 38–150 μm are sample between containers leads to some loss of retained to pick through for ichthyoliths. The pro- ichthyoliths. Bulk dissolution followed by washing cess takes approximately 5–12 washes, depending also uses the least amount of water per sample. on the degree of of the and the However, if it is imperative to preserve certain car- size of the original limestone fragments (Sibert et bonate microfossils in a sample, such as larger al., 2014). benthic foraminifera for isotope studies, or foramin- Coral reef sediments. Modern reef sediments are ifera from a critical interval, we suggest a double- comprised almost entirely of carbonate grains from washing procedure: the first wash is carried out calcifying organisms including corals, mollusks, with de-ionized water only, to retain the coarse echinoderms, foraminifera, calcareous algae, fraction of carbonates >38 µm. All material below a , and crustaceans. To preserve these specific size threshold (e.g., 150 μm or 250 μm, other taxonomic groups, which are mostly >500 study-specific) is then dissolved to concentrate the μm, only the fraction <500 μm of these cores is smaller ichthyoliths. We have found that ichthyo- digested in acid and picked through for ichthyoliths, liths are selectively lost in sample splitters due to the majority of which are <250 μm. Due to the sand static adhesion and recommend against their use. to -sized carbonate grains in reef sediments, As the volume of coarse-grained carbonate sedi- 10% acetic acid is required. Two to four applica- ment is relatively small, it is feasible, although time tions of approximately 200 ml of acid are added to consuming, to pick out all of the ichthyoliths in the dry, size-fractioned sediments every 24 hours. >150 µm or >250 µm calcareous residues. In this When gentle stirring of acid and residues fails to case, it is most important that the processing cause further reaction and residues darken due to method be internally consistent for an entire sam- dominance of organic material following elimination ple set, and the potential biases recognized when of carbonates, samples are transferred to a 63 μm comparing absolute ichthyolith abundance values sieve and washed with DI water until the water

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leaving the sieve runs clear. Coral reef sediments ica is insoluble in acetic acid and thus increases often have considerable organic matter and debris, the volume of the acid-insoluble coarse-fraction which causes excessive clumping of ichthyoliths containing ichthyoliths. Additionally, many and highly abundant but acid-insoluble siliceous grains have a significant visual similarity to tiny ich- spicules. To remove excess organic matter, thyoliths at first glance, making visual identification the washed samples can be treated with 25 ml and picking a challenge. We have found two meth- dilute chlorine bleach poured directly onto the ods to be effective in isolation of teeth in siliceous sieve following an initial wash, left for approxi- sediments—the use of Alizarin Red S, a calcium- mately 1 minute, then rinsed with DI water and specific stain to color ichthyoliths and make them lightly agitated until water runs clear below the visible against a backdrop of translucent silica. For sieve. The samples are then transferred to filter particularly challenging samples, heavy liquid sep- paper in a funnel and dried at 50°C. In contrast to aration may remove most of the low density sili- pelagic sediments, where the majority of ichthyo- ceous sediment relative to ichthyoliths, however, liths are within the 38–63 μm fraction, the vast we do not recommend this as a first line of ichthyo- majority of ichthyoliths preserved in reef sediments lith concentration methods. are >63 μm, so the 63 μm sieve size is used to Alizarin Red S. Visual differentiation of fish teeth facilitate washing the larger initial sample volumes from other small triangular sediment grains can necessary in these high sedimentation rate sys- often be confounded at small size fractions (<63 tems (Cramer et al., 2017). μm). However, ichthyoliths can be preferentially stained by Alizarin Red S (1,2-dihydroxyanthraqui- Pelagic Clays none, C14H8O4), a calcium-specific dye commonly Pelagic clays yield, by far, the greatest abun- used in clearing-and-staining fish (Taylor, 1967; dance of ichthyoliths per gram sediment: the slow Song and Parenti, 1995). Alizarin Red S gives ich- sedimentation rate below the carbonate compen- thyoliths a pink tinge, while leaving the silica grains sation depth, and small means that ich- untouched (Figure 3). Alizarin is a pH sensitive thyoliths are highly concentrated, and are typically dye, which turns a deep purple in basic solution, well-preserved in these sediments. However, the and when in contact with calcium, will adhere to it, slow sedimentation rate and lack of other biostrati- leaving a pink or red color. Alizarin Red S is not a graphically well-calibrated microfossils such as cal- panacea: it will also dye calcium carbonate grains, careous nannofossils, mean that clays often have and thus is used most effectively after the - poor age constraints, and there may be very little ate fraction has been removed from a sample via paleoenvironmental context from traditional prox- acid dissolution (see Figure 2). However we have ies. found that even in samples with carbonate, ichthy- To isolate ichthyoliths from pelagic clay, the oliths are preferentially stained a more intense samples are dried completely to enable the calcu- color than foraminifera. We modified a clearing- lation of ichthyolith accumulation rates. We have and-staining protocol for fishes, based on both a found that many pelagic clay samples fail to published protocol (step 9, Song and Parenti, achieve stable dry weights for many weeks, per- 1995), and the protocol used by Scripps Marine haps because of water bound in clays. However, Vertebrate Collection, to use a 1% potassium once the samples are completely dried, they are hydroxide (KOH) solution with enough Alizarin to simply disaggregated in de-ionized water, washed turn the solution a deep purple (a surprisingly small over a 38μm sieve, transferred to filter paper in a amount). The KOH/Alizarin Red S solution is funnel, dried in a 50°C oven, and examined under added to the post-acid, washed and dried residue. the microscope. These residues, in the best cir- The volume of Alizarin + KOH solution needed is cumstance, may contain only fish teeth and dermal dependent on the amount of residue: generally, denticles, however, in some cases may also con- just a few drops of the solution, enough to cover tain micro- nodules, siliceous microfos- the sample residue in its container, is more than sils, terrigenous sediment clasts, zeolites, or sufficient to produce the desired effect. This is left clumps of Fe-oxides. for 24 to 48 hours, and then washed over a 38 μm screen, transferred to filter paper in a funnel, and Silica-dominated Sediments dried overnight in a 50°C oven before picking. This Siliceous sediments, whether from quartz technique is extremely effective, staining >95% of or biogenic , create a distinctive challenge in the ichthyoliths in a sample a pink color (Figure 3). the isolation and quantification of ichthyoliths. Sil- The intensity of the color is dependent on both the

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FIGURE 3. Paleocene-aged ichthyoliths from ODP Site 1262, stained with Alizarin Red S. The scale bar is 500 μm, with teeth >106 μm in the upper row and teeth <106 μm in the lower. Note that in the coloring effect is present in all teeth, however, the degree of staining varies.

concentration of dye, and the length of time in solu- since both are non-toxic and have low viscosity at tion. Time step experiments have shown that the room temperature. Both liquids have the disadvan- Alizarin protocol does not cause physical degrada- tage of being relatively expensive (~$1000/liter) tion of the teeth, even after weeks in the solution, and can be destroyed by contamination with cal- though the color gets progressively darker. How- cium. Therefore, the use of heavy liquids on sam- ever, as the Alizarin staining protocol requires toxic ples containing calcium carbonate grains should be chemicals (KOH) and a second wash, which can avoided. We use a heavy liquid density of about increase the amount of teeth lost to processing, it 2.3–2.4 g/cm3 to capture most of the is generally best saved for particularly challenging or 2.85 g/cm3 to separate ichthyoliths from quartz residues, where silica consistently confounds silt. counts of small ichthyoliths and used consistently In our practice, heavy liquid of suitable density within a single record. is poured into a 25–50 mL tube containing the pre- Heavy liquid separation. Heavy liquids have been pared sample residues and mixed until the sample historically used to isolate calcium phosphate is completely wetted; sufficient heavy liquid should from acid-prepared limestone, and the be added to the tube so that the silica can float. methods have been described extensively else- The tube is capped and centrifuged for five minutes where (Leiggi and May, 2005). This procedure can at 1000–1500 rpm to concentrate the ichthyoliths in effectively separate ichthyoliths from biogenic silica the bottom of the tube. The light fraction is scooped and quartz silt, however heavy liquids are expen- or poured off the top of the liquid. Both the light and sive and often toxic, making them a last resort for heavy fractions are rinsed in de-ionized water over ichthyolith isolation. We have used both sodium a 38 μm screen, retaining the rinse solution, then metatungstate hydrate (Na6W12O39 xH2O) and LST transferred to filter paper in a funnel and dried in at solution (heteropolytungstate) as heavy liquids

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50°C oven. The dilute heavy liquid is recovered any concentration is potentially damaging to the and cleaned by passing it through a 0.4 µm filter in organic components in ichthyoliths and should be a vacuum filtration system, and then placed in an limited if possible. oven to evaporate the rinse water and restore its Isolating modern ichthyoliths. Similar to remov- density. ing organic material from sediments, flesh can be removed from jaws or skin patches of modern Organic Rich Sediments specimens to isolate taxonomically known fish While the majority of deep-sea sediments are teeth and shark scales. In this case, the jaw (for carbonate or silica-dominated, there are distinct fish teeth) or a patch of skin (for shark denticles) is horizons, such as the Mediterranean sapropels dissected from a modern specimen and placed in (Cramp and O'Sullivan, 1999), which are organic- dilute (5–10%) bleach until all flesh is dissolved, rich, and ichthyolith concentration using other usually 1–4 hours. Since bleach will attack the methods is hampered. In addition, modern coral organic compounds in teeth and bone as well as reef sediments, though carbonate dominated, may the softer tissues, we recommend removing the still have considerable amounts of organic matter. ichthyoliths from the bleach and washing the newly Organic matter can lead to sediment clumping and isolated modern ichthyoliths as soon as is practi- adds extra challenges to sample processing. To cal. These isolated modern ichthyoliths are then address this, samples are first disaggregated and washed over a 38 μm screen and dried in at 50°C dissolved in weak acid, following the carbonate oven. deep-sea sediments or coral reef sediments proto- Comments about Ichthyolith-specific Washing cols (see Figure 2), and washed over a 38 μm and Picking Techniques sieve. However, in the case of high organic matter content, there may be numerous organic-rich Traditional uses of ichthyoliths, for biostratig- clumps of sediment remaining. Once the sample raphy or as carriers of isotopes, do not require that has been thoroughly washed, a rinse while on the all teeth be retained and accounted for in a sample. sieve with dilute (5–10%) bleach solution promotes However, to assess the ichthyolith accumulation disaggregation and dissolution of the remaining rate, ichthyolith community structure, and the role organic matter. However, it is important to note that of fishes within an ecosystem through time, all of bleach and acetic acid produce chlorine gas when the ichthyoliths within a certain size range must be mixed, so caution is advised to ensure that the quantified. The methods presented here aim to sample is sufficiently rinsed from acid before any improve the fidelity of isolation and concentration bleach is used. of ichthyoliths, to make this robust quantification In the case where disaggregation of organic- both possible and repeatable. Due to their small rich sediments does not occur with the addition of size and unusual shape, care must be taken when de-ionized water or acetic acid, an additional short handling the concentrated ichthyolith residue to

soak in bleach, hydrogen peroxide (H2O2), avoid losing any teeth. As most teeth are triangular, BoraxTM, CalgonTM, or OxiCleanTM are a potential they tend to stick point-down into the sieve when alternatives, although prolonged exposure to any washing. Running water up through the back of the of these chemicals can damage the ichthyoliths. sieve, a technique often used when separating bio- Most commercial grades of bleach contain perfume logical samples, will help to dislodge any teeth that and colorants besides pure sodium hypochlorate, are stuck point-down. and various formulations produce different results. Earlier ichthyolith work mounted resi- For instance, in work with Turonian black shales dues in optical medium and viewed them using from Ocean Drilling Program Site 1259, we transmitted light (Doyle et al., 1977; achieved the best disaggregation using pure com- Doyle and Riedel, 1979a, 1979b, 1985). Transmit- mercial bleach, rather than making a dilute mixture ted light imaging is particularly useful for observing (Bice and Norris, 2005). In our experience, com- the details of the interior of the pulp cavity and the mercial grades of bleach vary in their content of structure of the enamel cap and may have value sodium hypochlorate from 5.25% to 6%. Dilution for identification of teeth to taxonomic group (Doyle lowers the pH of bleach solutions, potentially and Riedel, 1979b; Johns, 1993). Strewn slides increasing the etching of microfossils with sus- made by embedding the entire sample residue in tained contact and may reduce the effectiveness of Canada Balsam or Norland optical medium can the solution for breaking down organic-rich sedi- also be used to count the abundance of extremely ments. However, prolonged exposure to bleach at small teeth, which can be re-located by use of a

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England Finder, similar to the study of calcareous are found) and the number of years represented by nannoplankton. However, there are a number of a sample. The number of years in a sample can be disadvantages of embedding teeth in a mounting computed from U/Th-derived sediment accumula- medium, including the formation of bubbles in the tion rates (Cramer et al., 2017). This produces an pulp cavity, the difficulty in achieving standard ori- ichthyolith abundance accumulation rate (AAR): entations given the very small size of many teeth, and the three-dimensional aspect of large teeth in Ichthyoliths contrast with the narrow depth of field in transmit- AAR =  gram year ted light microscopy. An alternative approach is to pick ichthyoliths with a fine paint brush and mount equals ichthyoliths years them with water soluble glue on cardboard micro-   grams paleontology slides. This method retains the most sample sample options for quantifying ichthyoliths. It also ensures This calculation of IAR or AAR normalizes for that teeth are not overlooked in original count anal- sedimentation rate and changes in lithology. There- yses. Once picked, these assemblage slides are a fore, we can compare the flux of ichthyoliths to the resource, which can be worked with directly or eas- sea floor between sites with very different back- ily be used for many other imaging techniques, ground sedimentation rates, such as between open including transmitted light microscopy, scanning ocean gyre sites and those from the high-produc- electron microscopy, and even microCT or nanoCT tivity equatorial . We can also correct for scanning. This also leaves the ichthyoliths accessi- variations in sedimentation rate time in a single site ble for geochemical analyses. that result from changes in fish production, sedi- ment delivery, or carbonate dissolution. However, BIOLOGICAL ICHTHYOLITH METRICS the calculation of accumulation rate is highly sensi- Fish Production: Ichthyolith Accumulation tive to the accuracy of the time scale used to esti- Rates mate sedimentation rate. Bulk density is also a component of accumulation rate, but contributes Once isolated and quantified, ichthyoliths pro- relatively little to variation in accumulation rate in vide a unique view of fish production and commu- most pelagic sediments (see supplement to Sibert nity dynamics through time. However, changes in et al., 2014). An exception is where there are major sedimentation rate as well as sediment composi- changes in lithology, such as from carbonates to tion and density can have a profound effect on the claystone or calcareous ooze to limestone; in these absolute abundance of ichthyoliths in a sample, cases accurate measurement of bulk density and which bias any estimations of fish production or sedimentation rate can be important in the calcula- flux. To correct for this, we calculate an ichthyolith tion of accumulation. accumulation rate (IAR; eq. 1), yielding a metric of Sample size. The size fractions quantified can be ichthyolith flux of ichthyoliths falling to a fixed area study-specific, to balance between statistical confi- of seafloor over a fixed time interval. Thus, dence in the data (enough ichthyoliths available), changes in IAR can be interpreted as increases or time committed by the researcher, and preserva- decreases in total ichthyolith production, a proxy tion of other microfossils. We have found through for overall fish production (Sibert et al., 2014, 2016; our work that for pelagic marine carbonates, where Sibert and Norris, 2015). IAR in pelagic sediments sedimentation rate is 1–2 cm/kyr, quantification of is calculated as: all ichthyoliths >38 μm in a 10–20cc sample is nec- Ichthyolith Accumulation Rate = Abundance * Dry Bulk essary for sufficiently robust abundances of >30– Density * Sedimentation Rates 100 teeth/sample. The same sample volume in can yield hundreds to thousands of Ichthyoliths ichthyoliths grams cm teeth, and statistically significant samples of sev- =  eral hundred teeth may be found in the >106 μm cm2 kyr gram cm3 kyr fraction. In contrast, in coastal sediments and reef carbonates, the high degree of dilution of ichthyo- In the case of sediments from reef matrix liths by other grains and higher sedimentation rates cores, which have large fragments of subfossil can require much larger sample volumes to obtain coral or mollusk shell (>2 mm), IAR is calculated by statistically representative ichthyolith samples. For normalizing by the weight of sediments in the size example, in our work in modern Caribbean reef fraction <2 mm (where the vast majority of teeth sediments, we routinely sample volumes of 400 cc

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(about 200 g dry weight) to recover a range of 2 to those of species with robust dentitions, and miti- 250 teeth (mean = 74 teeth) and 0–5 denticles per gate against relatively lightly calcified pharyngeal sample. teeth and the teeth of some midwater species While the abundance of fish may be an indica- where an elongate pulp cavity can run almost the tor of primary or export productivity of an ecosys- full length of the teeth (Fink, 1981). Indeed, long- tem, this is not the only signal recorded in the term trends in changes in ichthyolith abundance, ichthyolith record. The overall efficiency of a particularly those associated with shifts in the size marine food web is determined by how many tro- structure of the assemblage, may reflect an evolu- phic steps are needed to transfer the carbon fixed tionary shift in fish community composition, (e.g., by primary producers up to higher-order consum- those documented in Sibert and Norris, 2015 and ers such as fish. In a large -domi- Sibert et al., 2016), rather than a change in overall nated system, such as a modern zone, a primary productivity or food web dynamics. modest will yield abundant fish Fish Community Structure: Ichthyolith with only 1–2 trophic steps. In contrast, a system Assemblage Metrics dominated by small phytoplankton (such as cyano- bacteria in the open ocean) can require 5–7 trophic While taxonomic identifications of most steps to produce a single fish (Moloney and Field, ancient ichthyoliths are presently elusive, a consid- 1991; Moloney et al., 1991). While both of these erable amount of information about marine verte- ecosystems may have similar levels of primary pro- brate community composition can be obtained by duction, the former will produce several orders of considering the composition of whole ichthyolith magnitude more fish biomass than the latter (Iver- assemblages, which preserve snapshots of the son, 1990) and thus should have a significantly entire community, rather than occurrences of a sin- higher ichthyolith accumulation rate. Indeed, a sub- gle species or morphotype. Since ichthyoliths are stantial portion of observed IAR patterns could be abundant in most sediment samples, we can eval- accounted for not by changing net primary produc- uate how the relative abundances of different tion, but instead by small shifts in the relative abun- marine vertebrate groups have changed through dance of certain size classes of phytoplankton. time. This food web imprint can also be exacerbated by Vertebrate community structure. Due to similar- changes in the efficiency of energy transfer ity in their chemical compositions, both elasmo- between trophic levels due to increases or branch dermal scales (denticles) and ray-finned decreases in metabolic rates of the organisms. fish teeth are preserved in the ichthyolith record. Accumulation rates may be also be affected by While the majority of denticles preserved in the ich- changes in habitat: for example, in coral reef sedi- thyolith record have been chipped, or preserve ments, abundances of teeth from coral-associated only the enamel crown of the scale, they are dis- taxa are tightly coupled with reef accretion rates tinctive from teeth and readily recognizable as den- (Cramer et al., 2017). ticles (Figure 1). The relative and absolute Ichthyolith accumulation is also influenced by abundances of teeth and denticles through time the production of ichthyoliths by individuals. Spe- can be used to study the response of different tro- cies which put considerable effort into growing their phic level organisms to global change (Sibert and teeth and have low turnover, or resorb teeth rather Norris, 2015; Sibert et al., 2016). than shedding them (Bemis et al., 2005), could pro- Ichthyolith functional group and taxonomic duce fewer ichthyoliths than a species which pro- composition. Individual ichthyolith size is also duces numerous, but oftentimes less sturdy teeth informative of evolutionary patterns. While fish which are regularly shed. The majority of the ich- tooth size is not necessarily correlated directly with thyolith accumulation rate signal is driven by the body size (e.g., deep-sea viperfish of the family smallest teeth, which likely are derived from a com- Stomiidae have fanglike teeth that are nearly the bination of small species, juvenile fish, and the length of their head), it is an indicator of diet. For pharyngeal tooth battery. At present we are unsure example, long, pointed teeth are more likely to be about the relative contribution of teeth from these used for handling larger or more active prey. The different sources, but we suspect that most teeth size structure of an ichthyolith assemblage, quanti- preserved in sediments are biased toward those fied either through changes in relative abundance with dissolution-resistant enamel caps. The excel- of different size fractions or by measuring the lent preservation of enamel relative to dentine is length of individual teeth (Sibert and Norris, 2015), likely to bias the tooth record toward oral teeth, and can reveal evolutionary or ecological trends. For

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FIGURE 4. Examples of select taxonomically identifiable fossil ichthyoliths and modern counterparts. All modern ich- thyoliths were isolated from specimens in the Scripps Marine Vertebrate Collection. The fossil Myctophidae and Triaki- dae specimens are from ODP Site 1262, and are 62 million years old. The Scaridae modern teeth are from Smithsonian National Museum of Natural History’s Fish Collection and subfossil teeth are from coral reef sediment cores taken off of the of Bocas del Toro, Panama, and are approximately 1200 years old. example, following the Cretaceous-Paleogene , and we believe that identification of extinction, the maximum size of the largest teeth in either taxonomic affinity or ecological group will an assemblage in open ocean sediments tripled also become possible for pelagic fishes as from pre-extinction values, suggesting that there research progresses. was a radiation of fishes preferring larger prey fol- Ichthyolith . While ichthyoliths are lowing the (Sibert and Norris, generally resistant to the dissolution effects that 2015). damage other microfossil groups, there are several While some ichthyoliths are taxonomically taphonomic processes that can affect the preser- identifiable (Figure 4), the majority remain unidenti- vation of ichthyoliths. It is rare to find highly frag- fied to taxonomic group at present. Teeth from mented samples, and ichthyoliths rarely display modern Caribbean reef sediments have a greater pitting or other signs of partial dissolution. How- variety of tooth morphotypes than those from ever, as many large teeth have an extensive, hol- pelagic sediments and can be divided into diet cat- low pulp cavity, these teeth are prone to splitting egories such as predators (raptorial or canine due to mechanical forces, either during preserva- teeth), herbivores (incisiform teeth), and duropha- tion or laboratory sediment processing. As the gous invertivores (molariform teeth), producing a large teeth most likely to break are relatively rare in record of fish trophic structure through time (Cra- a sample, it is often straightforward to piece a sin- mer et al., 2017). Utilizing a fish tooth reference gle large tooth back together following a fracture. collection for modern Caribbean reef fish (www.ich- We have also occasionally observed iron and man- thyolith.ucsd.edu), it is also possible to identify sev- ganese oxides growing in the pulp cavity of teeth eral distinctive tooth types to family level. Pelagic which can also cause splintering. These same ichthyoliths also have discrete morphological char- oxides can also grow around teeth, hiding them acters, such as the shape and structure of the pulp from observation. Finally, some parts of ichthyoliths cavity, which have been studied in depth for bio- are more durable than others. For example, the

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crowns of denticles are much more likely to be pre- methodological framework for isolation and quanti- served than the subcutaneous base, and the more fication of ichthyoliths from most heavily enameled tooth tips preserve more often as types ranging from coral reefs to the open ocean, well. Despite these potential taphonomic biases, however, these methods can also be applied to the ichthyolith record is generally well-preserved. lacustrine or other aquatic deposits. We have fur- Future applications of ichthyoliths. While we ther presented a novel protocol for staining ichthy- have addressed several applications of the ichthy- oliths pink for easier and more accurate visual olith record here, there are numerous other poten- identification using Alizarin Red S. The applications tial applications. For example, taxonomically of the ichthyolith record include more traditional identifiable pelagic ichthyoliths can provide signifi- biostratigraphy and geochemistry, alongside fish cantly better fossil calibration ages for molecular production, evolution, and ancient food web recon- clock estimates of divergence in open ocean fish struction. Taxonomic or ecological identification of lineages, which have a poor body fossil record. ichthyoliths will further reveal patterns in fish evolu- Comparison of ichthyolith records with other bio- tion, shed light on the development and rise to logical groups present in the same core (e.g., ich- dominance of the most diverse group of verte- thyoliths and coral community composition in the brates on the planet, and reveal the full magnitude Caribbean, or fish and foraminifera in the open of change in fish communities resulting from past ocean) can reveal trophic or community dynamics and present human activities. through time. IAR or community composition met- rics can also be compared to geochemical proxies, ACKNOWLEDGMENTS to assess the effects of local or global change on We thank J. Williams, K. McComas, D. fish population or community ecology. Establishing Pitassy, H.J. Walker, M. Alvarez, F. Rodriguez, M.P. the natural abundance, structure, and variability of Concepcion, and A. Castillo for help with develop- fish communities in coastal, reef, or even lake set- ing the modern Caribbean reef fish tooth reference tings, on historic or pre-historic timescales can pro- collection; A. Sanderson and D. Chen for help with vide a baseline for separating anthropogenic isolating and identifying teeth from coral reef cores; pressures and climate impacts on economically B. Oller, C. Carpenter, S. Buckley, and M. Siltanen significant fish stocks. Finally, archaeological mid- for help processing reef sediments; and J. Lyakov dens may have considerable amounts of ichthyo- for help with tooth dissolution experiments. E.C.S. liths, which could offer insight into how ancient was supported on an NSF Graduate Research Fel- humans interacted with marine resources. How- lowship. K.L.C. was supported by Smithsonian ever, archaeological practice commonly studies Institution MarineGEO Postdoctoral Fellowship and only the >5 mm fraction in screen washings, which UC San Diego Frontiers of Innovation Scholars will fail to recover virtually all teeth (e.g., Powell, Postdoctoral Fellowship. No permits were required 2003; Rainsford et al., 2014). Hence, future for the described study, which complied with all rel- archaeological use of ichthyoliths will require pre- evant regulations. serving at least a known proportion of the fine material ordinarily discarded during screen wash- REFERENCES ing. Bellwood, D.R. and Wainwright, P.C. 2002. The history CONCLUSIONS and of fishes on coral reefs, p. 5-32. In Sale, P.F. (ed.), Coral Reef Fishes: Dynamics and Ichthyoliths represent an important and under- Diversity in a Complex Ecosystem. Academic Press, studied microfossil group that preserves the record San Diego. of fishes and sharks at unprecedented temporal Bemis, W.E., Giuliano, A., and McGuire, B. 2005. Struc- resolution. Quantification of the relative and abso- ture, attachment, replacement and growth of teeth in lute abundance of ichthyoliths through time can bluefish, Pomatomus saltatrix (Linnaeus, 1766), a reveal changing patterns in fish production, food teleost with deeply socketed teeth. Zoology, 108:317- web stability, and ecosystem structure through 327. Earth’s history (including the Anthropocene) and Betancur-R., Broughton, R.E., Wiley, E.O., Carpenter, K., López, J.A., Li, C., Holcroft, N.I., Arcila, D., San- across global change events throughout much of ciangco, M., and Cureton, J. 2013. The tree of the Phanerozoic. Accurate quantification of these and a new classification of bony fishes. PLoS Cur- trends in ichthyolith accumulation and assemblage rents, 5:1-33. structure relies on quantification of all ichthyoliths Bice, K.L. and Norris, R.D. 2005. Data report: Stable iso- in each discrete sample. We have presented a tope ratios of foraminifers from ODP Leg 207, Sites

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