Nova Hedwigia, Beiheft 148, p. 101–112 C Stuttgart, February 2019

Potential use of chrysophyte cyst morphometrics as a tool for reconstructing ancient lake environments

Peter A. Siver

Department of Botany, Connecticut College, New London, CT 06320, USA; [email protected]

With 8 fi gures

Abstract: Chrysophycean algae produce a siliceous resting stage, the cyst, within the confi nes of the cell as a response to environmental or population stimuli. Modern cysts range in diameter from approximately 3 to 35 μm, the external morphology is species-specifi c, and the diff erent morphotypes can serve as valuable bioindicators in paleolimnological investigations. In this study, massive numbers of fossil cysts were obtained from an extensive core representing an Arctic maar lake in northern Canada that existed during the globally-warm middle Eocene. The exquisite preservation of microfossils makes the locality an especially valuable site for understanding impacts of warming on Arctic water bodies. Since the vast majority of fossil cysts in the core could not be linked to modern analogs, cyst morphology could not be easily used to infer past conditions. Instead, a method was devised to extract the cysts from the core and then measure morphometric characters on hundreds of specimens using a FlowCam. The goal was to characterize the size distributions of cysts in each stratum, and use the information to aid in reconstructing lake history. Over 25,000 cysts ranging in diameter from 3 to 32 μm were measured from 33 strata, classifi ed into frequency distributions, and used to trace changes over the history of the ancient lake. The cyst size categories were found to be highly correlated with other biological proxies, and relationships between mean cyst diameter and overall cyst diversity were established. Findings indicate that cyst size classes are potentially valuable proxies for reconstructing historical conditions of aquatic habitats. Key words: cysts, Chrysophyceae, Eocene, FlowCam, Synurophyceae. Introduction The Chrysophyceae and Synurophyceae, commonly referred to as golden-brown algae, are diverse, cosmopolitan, and ecologically signifi cant groups of heterokont organisms that are especially important in freshwater ecosystems (Andersen, 2004; Kristiansen, 2005; Adl et al., 2012; Siver, 2015a). Species are mostly microscopic and may be planktonic or attached, autotrophic or heterotrophic, naked or with a cell covering, motile or non-motile, and the group embraces a wide diversity of vegetative forms (Kristiansen, 2005; Nicholls & Wujek, 2015; Siver, 2015a). The Synurophyceae include golden-brown organisms characterized by distinctive siliceous scales thatuncorrected_proof produce a highly organized covering around the cell (Siver, 2015a). Even though the Chrysophyceae and Synurophyceae are closely related, the precise phylogenetic relationship between the two groups of organisms requires further investigation (Andersen et al., 1999; Škaloud et al., 2013; Siver et al., 2015). Some molecular phylogenies conclude that the Synurophyceae forms a distinct class separate from the Chrysophyceae (Andersen, 2007; Yang et al., 2012), yet other works suggest the synurophytes are a monophyletic clade nested within the Chrysophyceae (del Campo & Massana, 2011; Škaloud et al., 2013). The Chrysophyceae (Nicholls & Wujek, 2015) and Synurophyceae (Siver, 2015a) are dis- tri buted worldwide from the tropics to high latitudes with highest diversities often associated with habitats that are slightly acidic, poorly buff ered, dilute, humic-stained and with low to moderate nutrient levels. Despite this generalization, both groups of organisms exist in virtually any water body and numerous species have optima at diff erent positions along environmental gradients making them excellent bioindicators (Siver & Smol, 1993; Stevenson & Smol, 2015).

© 2019 J. Cramer in der Gebr. Borntraeger Verlagsbuchhandlung, Stuttgart, Germany www.borntraeger-cramer.de DOI 10.1127/nova-suppl/2019/115 1438-9134/2019/115 $ 2.75

eschweizerbart_xxx 102 Peter A. Siver

As a result, these organisms have been eff ectively used to reconstruct acidity (e.g., Cumming et al., 1992; 1994; Facher & Schmidt, 1996; Siver et al., 1999), dissolved salts (Siver, 1993), nutrients (Siver & Marsicano, 1996; Kamenik et al., 2001), eff ects of deforestation (Lott et al., 1994), temperature (Siver & Hamer, 1992), and climate change (Kamenik & Schmidt, 2005; Arseneau et al., 2016). All golden-brown algae form cysts, hollow siliceous structures produced in response to environmental (Cronberg, 1986; Sandgren, 1981) or population (Sandgren, 1988) cues. Cysts range in diameter from ca. 3-35 μm, and vary immensely in surface design from smooth to highly ornamented (Duff et al., 1995; Pla, 2001; Wilkinson et al., 2001). Since each living cell can form a cyst, a trace of its existence can be deposited in the sediment record. Cysts are the most common type of microfossil representing chrysophytes and synurophytes in the geologic record, for which the oldest specimens are Late Triassic (ca. 230 million years before present; Ma) in age (Zhang et al., 2016). Hundreds of cysts have been described from an extensive diversity of modern aquatic environments (e.g., Duff et al., 1995; Pla, 2001), and cyst morphotypes have been used to successfully reconstruct conditions over the last several hundred years (Kamenik & Schmidt, 2005). A stunning array of exquisitely preserved cysts has been documented from a middle Eocene freshwater lake situated near the Arctic Circle in northern Canada known as the Giraff e Pipe locality (Siver & Wolfe, 2005; 2009). The idea has been put forward to use the numerous cysts to help reconstruct past conditions of the Giraff e lake since this water body existed under warm greenhouse conditions during the middle Eocene (Zachos et al., 2008). However, assigning specifi c conditions to these ancient fossil cysts has proven problematic because many fossil morphotypes lack modern analogs. The purpose of this study is to explore the possibility of using cyst diameter, instead of morphology, as a proxy of past environmental conditions for the Giraff e Pipe lake. To achieve this goal, three steps needed to be carried out. First, a technique was devised to extract cysts from the Giraff e mudstones. Second, a method to measure morphometric data on hundreds to thousands of cysts was perfected using a FlowCam. Third, changes in cyst size classes were traced over the sediments representing the ancient water body and correlated with the remains of other groups of organisms to further understand the utility of this metric in reconstructing lake history. Study site The Giraff e locality (65º N, 110º W) is a kimberlite diatreme crater that formed following a phreatomagmatic volcanic eruption (Heaman et al., 2004), subsequently infi lled with a sequence of Eocene lacustrine and then paludal sediments, and later became entombed with Neogene glacial deposits. The kimberlite pipe intruded through the Slave Province in the Northwest Territories of Canada approximately 48 Ma based on Rb-Sr model ages from kimberlitic phlogopite (Creaser et al., 2004; Siver & Wolfe, 2009). A 165-meter long drilled core was obtained from the crater by BHP Billitonuncorrected_proof Inc. during diamond exploration and later archived at the Canadian Geological Survey, Calgary, Canada. The core contains 113 m of stratifi ed organic sediment of middle Eocene age, including 68 m of lacustrine mudstone overlain with 45 m of terrestrial sediments. The lacustrine phase includes two air-fall tephra beds near the transition from aquatic to terrestrial remains (Siver & Wolfe, 2009). The Rb-Sr model age estimate is corroborated by the presence of pollen throughout the lacustrine and paludal sections that are diagnostic of the middle Eocene, including Platycarya swasticoides and Pistillipollenites mcgregorii (Rouse, 1977; Hamblin et al., 2003). Diameter-corrected and isothermal-plateau fi ssion tracking age estimates of the tephra beds center on 38 Ma (Doria et al., 2011), constraining the lacustrine sequence to late middle Eocene. The protracted regional tectonic and thermal stability of the Slave Craton, into which the Giraff e Pipe was intruded, has resulted in near-zero diagenetic alteration of the fossil content, as testifi ed by exquisite preservation, even at the sub-cellular level (Wolfe et al., 2006).

eschweizerbart_xxx Reconstructing lake environments using cyst size 103

Methods

Chips of mudstone (0.1–0.5 g) from the Giraff e core were oxidized using 30% H2O2 under low heat for one to four hours, rinsed four times with distilled water, and the resulting slurries stored in glass vials. For most samples this oxidation procedure resulted in separation of cysts from the mudstone matrix as observed with light microscopy. Sonication for 1 min could further separate the microfossils, but in most cases this was not needed. Other more resistant chips were subjected to a strong acid treatment involving a mixture of potassium dichromate and sulfuric acid after initial oxidation with H2O2. Signifi cant numbers of cysts were fully extracted from all strata examined in this study. Aliquots of each oxidized sample were air dried onto pieces of heavy duty aluminum foil. The aluminum foil samples were trimmed, attached to aluminum stubs with Apiezon® wax, coated with a mixture of gold and palladium for 2 min with a Polaron Model E sputter coater, and examined with a Leo 982 fi eld emission scanning electron microscope (FE-SEM) or an FEI Nova FE-SEM. Observations of the cysts in each sample were made with the SEM, including estimates of the <10 μm and >10 μm percentages, and the smallest and largest specimens. The SEM observations were used to ensure the full complement of cyst sizes was captured with the FlowCam (see below). Once cysts were extracted from core samples and studied with the SEM, a FlowCam VS cytometer (Fluid Imaging Technologies) was used to image and measure the cyst sizes in each sample (Fig. 1). The FlowCam VS is a continuous imaging fl ow cytometer that uses a high speed camera to capture images of passing particles, and simultaneously records morphometric data using Visual Spreadsheet software. The following procedure was used to capture cyst data. First, each slurry was diluted with distilled water to a fi nal turbidity between 8-10 NTUs (Nephelometric Turbidity Units) using a Micro TPI Turbidimeter (HF Scientifi c, Inc). Samples were initially studied with scanning electron microscopy. Based on the SEM observations, cysts below 3 μm or over 32 μm in diameter were not recorded. Therefore, each sample was prefi ltered using a nitex net with a 38 μm mesh size to remove large particles that could clog the fl ow cell, and the FlowCam was set to record data only on particles larger than 2 μm. These settings assured that the full range of cyst sizes would be captured with the FlowCam. Next, 0.5 ml of each sample was pumped through a FC50 (50 μm) fl ow cell, where particles were magnifi ed with a 20× lens, imaged with a high speed camera, and measured with the Visual Spreadsheet software. A digital photo mask was used to inspect eachuncorrected_proof cyst to be sure the perimeter was complete and intact. Visual Spreadsheet software was then used to select and classify the cysts. Four runs were made on each sample and all cyst data combined into one spreadsheet. This procedure yielded over 300 cysts for all but three samples. For samples with fewer cysts, additional runs were made until a minimum of 300 cysts was imaged.

Fig. 1. Flow diagram depicting the major steps used to collect and evaluate morphometric data on fossil cysts from the Giraff e locality.

eschweizerbart_xxx 104 Peter A. Siver

The equivalent spherical diameter (ESD) metric estimated from the Visual Spreadsheet software was used as an estimate of cyst diameter. ESD is the mean value of 36 feret measurements, taken at 5 degree intervals. A feret measurement is the perpendicular distance between parallel tangents touching opposite sides of the particle (i.e., cyst). Ideally, for a perfect sphere all feret lengths would be identical. Diff erences in the 36 feret measurements were minimal for the vast majority of cysts, refl ecting the fact that most cysts were close to spherical. The largest diff erences were between 1–1.5 μm, observed for a small percentage of oval cysts. ESD measurements for the cysts were exported to SigmaPlot ver. 13 where they were distributed into 1 μm size bins (e.g. diameter), and used to generate frequency plots. Bins ranged from 2 to 30 μm (28 bins). Since only four cysts had an ESD between 30–32 μm, these were combined with the 29–30 μm bin category. The bin data were then imported into Primer-E software (ver. 6.1) and used to estimate cyst size diversity and to run non-metric multidimensional scaling (MDS) ordination analyses. The DIVERSE routine in Primer-E was used to estimate the Shannon-Wiener diversity index for each sample based on cyst diameter. MDS was used to ordinate and display diff erences between samples based on a rank order resemblance matrix of Bray-Curtis measurements using the cyst data. Two MDS analyses were performed and used to trace changes over the length of the core. The fi rst MDS was run using remains of over 100 microfossil taxa, including diatoms, chrysophytes, synurophytes, heliozoans, euglyphids and sponges, previously enumerated from each stratum included in the study. In this case, cysts were lumped into a single category regardless of size. The second MDS was based solely on the percentages of cysts in each of the 28 bin classes. Samples that are closely aligned on the MDS plots represent those with similar complements of microfossils, whereas the more dissimilar two samples are with respect to their fossil remains, the farther apart they will be on the plots. Records of cyst diameter for modern freshwater chrysophytes were made from a review of over 75 literature publications. A total of 1409 records were recovered from the review, most made with scanning electron microscopy, and used to estimate the mean diameter and evaluate the frequency distribution of cyst sizes for modern species. For many of the 1409 cyst records, only the minimum and maximum diameters were given. For these cases, the midpoint between the minimum and maximum values was used to represent the mean diameter. Samples from the Giraff e core are identifi ed with a three-part number (Siver, 2015b). The fi rst number represents the core box. The larger the number, the deeper the section is within the core. Box 11 represents the top and end of the lacustrine phase within the sequence. Each box contains three 1.5 m core lengths, identifi ed as channels 1, 2 and 3. The second number represents the channel. The third number is the measurement in cm down from the top of a core length. For example, sample 13-1-130 represents a sample taken from 130 cm down along the core length positioned in channel 1 from box 13. This study includes 33 samples from the lacustrine phase representing depths in the core from 70 to 112.1 meters. Results uncorrected_proof Modern versus fossil cyst diameter The range in cyst diameter and the overall frequency distribution for modern cysts (n = 1409) was remarkably similar to those for fossil cysts uncovered from the Giraff e locality (Figs 2A, B). Except for several records of cysts < 1μm in diameter, and one record of a cyst 40.5 μm in diameter, the range for modern specimens was 2 to 30 μm, close to the range of 3 to 32 μm for Giraff e fossils. The frequency distribution for modern cysts had slightly higher numbers of cysts between 5 and 10 μm, whereas there were greater numbers of smaller fossil cysts between 4 and 5 μm (Figs 2A, B). As a result, the mean and median measures for modern cysts were 8.6 μm and 8.2 μm, respectively, slightly higher than the estimates of 7.8 μm and 7.5 μm for fossil cysts. All of the modern records of cyst sizes taken from literature sources and used in this study reported the minimum and maximum diameters. The range in diameter for each cyst morphotype was estimated for each of the 1409 records. The range in cyst diameter per morphotype was

eschweizerbart_xxx Reconstructing lake environments using cyst size 105

Fig. 2. Characteristics of fossil cysts from the Giraff e locality and modern cysts based on literature records. Histograms displaying size categories for fossil (A) and modern (B) cysts are given. The diff erence in cyst diameter for 1409 records of modern species taken from the literature, calculated as the diff erence between the minimum and maximum diameter, is given in (C).

uncorrected_proof

Fig. 3. The fi rst 75 cysts captured by the FlowCam for samples representing strata from 11-3-75 and 18-1-30. Histograms of cyst diameter representing all cysts measured for both samples are also given.

eschweizerbart_xxx 106 Peter A. Siver within 1 μm for 54% of the records, within 2 μm for 75% of the records, and within 3 μm for 85% of the records (Fig. 2C). The few literature records reporting diff erences larger than 5 μm for an individual cyst morphotype were largely based on light microscopic observations, and it is likely that these records represent cysts from more than one species. Frequency distributions for fossil cysts A total of 33 fossil strata taken from between 112.1 meters (box 23-3-116) and 70.0 meters (11- 3-75) in the Giraff e core were used in this investigation. The fossil cysts imaged and measured in each of the four runs from each sample were combined and used to estimate the cyst percentages in 1 μm wide bins from 2 to 30 μm. Images of the initial 75 cysts captured by the FlowCam, the total number included in four runs, and the frequency distributions for samples taken from the 11-3-75 and 18-1-30 strata are given in Fig. 3 as examples of the type of data obtained from each of the 33 samples. The complement of cysts and mean cyst diameter in sample 11-3-75, which represents the shallow and terminal stages of the water body,ody, are signifi cantly larger than the measures made from 18-1-30, which is believed to represent a deep phase in the history of the lake. Models relating cyst size categories to concentrations of diff erent microfossil groups, and cyst diversity The percentages of fossil cysts in the bins with 1 μm increments from 2 to 30 μm (n = 28) were initially used as independent variables in multiple regressions to predict percentages of other groups of microfossils. Analyses of four very diff erent types of organisms, including centric diatoms (valves), euglyphid testate amoebae (plates), sponges (spicules), and Mallomonas porifera (scales), are presented (Figs 4A–D). In this case, centric diatomsdia represent percentages of two to three co-occurring species of Aulacoseira. In each case, a highly signifi cant relationship (p < 0.01) was observed with r2 values of 0.85, 0.83, 0.79 and 0.71 for centrics, M. porifera, sponges and euglyphids, respectively. Signifi cant relationships were also found between cyst size categories and eunotioid diatoms, fragilarioid diatoms, heliozoans, and multiple other species of synurophytes. A signifi cant relationship (r2 = 0.39; p < 0.001) was found between the mean diameter of cysts and the diversity of cysts within a sample (Fig. 5). Samples with collections of cysts yielding a lower mean diameter also had lower Shannon-Wiener diver- sity. uncorrected_proof

Fig. 4. Results of multiple regres- sion analyses relating cyst size categories to percentages of (A) sponge megascleres, (B) scales of Mallomonas porifera, (C) testate amoeba plates and, (D) centric diatoms. Each relation ship was highly signifi cant (p < 0.001).

eschweizerbart_xxx Reconstructing lake environments using cyst size 107

Fig. 5. The relationship between the mean cyst diameter and  overall cyst diversity of samples analyzed from the Giraff e core. Note that as the mean diameter of cysts in a sample  increases so does the overall diversity of cyst size. 

 Mapping changes in cyst size    The mean cyst diameter was found to signifi cantly shift 5  

6KDQQRQ:LHQHU'LYHUVLW\ S  over the 42 meter-long segment of the core examined in  this study (Fig. 6). Mean cyst diameter was high in the  lower sections of the core (boxes 21–23; a on Fig. 6), with 0HDQ&\VW'LDPHWHU —P values above 8, then declined to a low of 5.6 μm at 93 m (boxes 17–20; b on Fig. 6). Over the next fi ve meters the mean size increased rapidly reaching values above 9 μm at 90.2 meters (end of box 17), became relatively stable between 78–88 meters (boxes 13–16; d on Fig. 6) with values fl uctuating around 7.5 μm, and terminated with higher mean values at the end of the lacustrine phase (box 11, e on Fig. 6). General changes in complements of the microorganisms within each core section are given below.

Fig. 6. Changes in the mean diameter of cysts over a 42-meter length of the Giraff e core. The letters correspond to the following core sections: a) Boxes 21-23, dominated with euglyphid plates and valves of the benthic diatom, Fragilaria; b) Boxes 17-20, increasing concentrations of the planktonic diatom, Aulacoseira; c) End of Box 17, abrupt decline in Aulacoseira and increase in euglyphid plates; d) Boxes 13-16, increases in acidic diatoms, synurophytes and heliozoans; e) post a nearby volcanic event and; f) Box 11, end of theuncorrected_proof lake phase before transitioning to a terrestrial environment. See text for more details.

The MDS ordination plot based solely on cyst size categories showed a directional trend over the lacustrine phase examined in the study (Fig. 7) that was similar to the MDS based on all microfossils found in the core (Fig. 8). In both analyses large shifts were observed between boxes 20 and 21, and between 12 and 13, the latter being more prominent on the cyst-based ordination. The large shift between boxes 16 and 17 based on all organisms was less obvious on the cyst-based plot. In both cases, the terminal phases of the water body (box 11) became more similar to the early stages of the aquatic phase (box 23).

eschweizerbart_xxx 108 Peter A. Siver

Fig. 7. An MDS ordination plot of sam- ples representing boxes 11 through 23 of the Giraff e core. The plot is based on 28 size categories of chrysophyte cysts. The closer samples are to each other the more similar they are with respect to their complement of cyst sizes.

Fig. 8. An MDS ordination plot of samples representing boxes 11 through 23 of the Giraff e core. The plot is based on percentages of all microfossil organisms found in each sample. The more similar the organisms are between samples, the closer the samples are to each other on the plot.

A few general observations regarding the groups of organisms most dominant along the MDS trajectory based on all microfossils (Fig. 8), and shown on Fig. 6, are noteworthy. First, samples from boxes 21–23 contained large concentrations of euglyphids, species from the diatom genus Fragilaria, and numerous scales of an organism closely related to the modern synurophyte, Mallomonas insignis (a on Fig. 6). Microfossils in samples from boxes 17–20 correspond to overwhelminglyuncorrected_proof high concentrations of the planktonic diatom genus Aulacoseira, and several species of the synurophyte Synura, coupled with a disappearance in euglyphids (b on Fig. 6). A large shift in the complement of microfossils was also recorded between box 17 and boxes 13–16. First, the concentration of Aulacoseira began to decline, coupled with a resurgence of euglyphid plates (c on Fig. 6). This was followed with a continued decline of Aulacoseira, being replaced with numerous specimens of the acidic diatom genera Eunotia, Actinella and Oxyneis, numerous species of synurophytes, and higher concentrations of heliozoans (d on Fig. 6). The largest deviation in the MDS plot based on cyst sizes (Fig. 7), and observed to a lesser extent in the MDS based on all organisms (Fig. 8), occurred in samples taken immediately after a 20 cm- thick tephra layer was deposited in the water body as a result of a nearby volcanic eruption. This event corresponds to an increase in the mean cyst diameter (e on Fig. 6). The end of the aquatic phase is represented by box 11 (f on Fig. 6), where the overall composition of microfossils was similar to those in box 22.

eschweizerbart_xxx Reconstructing lake environments using cyst size 109

Discussion The fact that the direction of the trend line observed in the MDS plot based only on cyst sizes is so strikingly similar to the MDS based on all microfossils suggests that the two groups of organisms are independently capturing similar changes that took place in the ancient aquatic ecosystem. This concept is further supported by the signifi cant relationships observed between concentrations of diff erent sizes of cysts and specifi c types of aquatic organisms, including sponges, euglyphids, diatoms, and synurophyte species. In this respect, a few preliminary conclusions can be made regarding changes that took place in the Giraff e water body over time. First, during time periods represented by samples from boxes 21–23, that contained large concentrations of euglyphids, Fragilaria, and remains of M. insignis, the water body was a shallow and alkaline pond or marsh. Microfossils in samples from boxes 17–20, including large concentrations of Aulacoseira spp. and Synura spp, coupled with a steep decline in euglyphids, suggest a deepening of the water body, and a dominance of planktonic communities. The large shift on the MDS plot that occurred between box 17 and boxes 13–16, highlighted by the disappearance of Aulacoseira, the rise in acidic diatom and synurophyte taxa, and higher concentrations of heliozoans, likely corresponds to a shallowing and acidifi cation of the water body. The largest deviation observed in the MDS plot based on cyst sizes, also observed to a lesser extent in the MDS based on all organisms, was immediately after the occurrence of a nearby volcanic eruption. The complement of organisms found in samples deposited after the volcanic event changed signifi cantly, becoming more like those in box 22. The major shifts identifi ed on the MDS plot using all organism remains are largely and independently tracked using cyst size classes. In conclusion, separating cysts by size classes, instead of lumping them into a single category, should improve and strengthen reconstruction eff orts using microfossil proxies. Another intriguing observation made during this study was the signifi cant and positive relationship found between mean cyst diameter and cyst size diversity. Samples with smaller cysts had signifi cantly lower Shannon-Wiener diversity scores than those containing larger cysts. In the case of the Giraff e locality, many of the samples with smaller cysts and lower size diversity measures correspond to time periods when the crater was believed to be fi lled with water and representing a deep maar lake with a reduced littoral zone. This idea is based on the fact these samples contain high concentrations of planktonic diatoms and synurophytes, coupled with low levels of sponge spicules and testate amoeba remains. Under this scenario, cyst-forming organisms were likely restricted to the plankton community. In contrast, strata containing higher diversities of cyst sizes, including many more large-diameter cysts, are largely ones with higher concentrations of sponges, testate amoebae, and periphytic diatoms, suggesting shallower aquatic habitats. With further observations it may be possible to also correlate cyst sizes and diversity measures with other characteristics, such as pH, nutrient levels and dissolved salt concentrations (Siver, 2015a; Nicholls & Wujek, 2015). The fact that only slight diff erences in the mean sizes of modern and fossil cysts were observed is remarkableuncorrected_proof given that the modern records represent numerous types of water bodies from around the world, while the fossil cysts come from only one locality. However, the reality is that although Giraff e is a single fossil locality, it represents aquatic conditions that existed for hundreds of thousands of years during which time signifi cant changes were evident. As such, Giraff e actually represents many diff erent types of aquatic habitats ranging from shallow marsh and bog-like conditions to a deep lake. The larger percentage of smaller fossil cysts from Giraff e could be due, in part, to biases resulting from diff erent numbers of cysts being extracted per sample. For this preliminary study, the same volume of slurry was analyzed with the FlowCam for each sample. This means that more cyst specimens were included from samples with greater concentrations of cysts. Alternatively, in future studies, it may be worthwhile to compare this method to one using the same number of cysts per sample, for example, only the fi rst 300 specimens measured. Preliminary observations of the Giraff e cyst dataset indicate that only slight diff erences in cyst metrics would result between these two procedures. In future work

eschweizerbart_xxx 110 Peter A. Siver on the Giraff e site, many additional samples will be included in order to reconstruct conditions more closely over this extensive core which will allow for a more in-depth comparison of the two methods. In conclusion, this study presents preliminary data that supports the use of chrysophyte cyst size as a new and valuable proxy for reconstructing historical conditions of aquatic habitats. The use of the FlowCam was instrumental in obtaining large volumes of data on cyst sizes used to develop the proxy. The procedure used in this study could easily be applied to any fossil locality where the microfossils can be separated from the mudstone matrix, as well as for more recent time periods (e.g., hundreds of years) represented by wet sediments. Acknowledgements This work was funded with support to PAS from the U.S. National Science Foundation (DEB- 0716606, DEB-1144098 and EAR-1725265), and by an NSF equipment grant (NSF#1126100) to Marie Cantino (University of Connecticut). The work was performed, in part, at the Biosciences Electron Microscopy Facility of the University of Connecticut. I would like to thank Anne Lott and Nels Christiansen (Connecticut College) for help with sample preparation, and James Romanow and Xuanhao Sun for assistance with the SEM facilities. Comments from two reviewers helped to strengthen the manuscript and are greatly appreciated. References Adl, S.M., Simpson, A.G.B., Lane, C.E., Lukes, J., Bass, D., Bowser, S.S., Brown, M.W., Burki, F., Dunthorn, M., Hampl, V., Heiss, A., Hoppenrath, M., Lara, E., Gall, I.L.E., Lynn, D.H., McManus, H., Mitchell, E.A.D., Mozley-Stanridge, S.E., Parfrey, L.W., Pawlowski, J., Rueckert, S., Shadwick, L., Schoch, C.L., Smirnov, S. & Spiegel, F.W., 2012: The revised classification of eukaryotes. – J. Eukaryot. Microbiol. 59: 429–493. Andersen, R.A., 2004: A historical review of heterokont phylogeny.phyloge – The Japanese Journal of Phycology 52: 153–162. Andersen, R.A., 2007: Molecular systematics of the Chrysophyceae and Synurophyceae. – In: Brodie, J. & Lewis, J. (ed.): Unravelling the Algae. – CRC Press, pp. 285–313. Andersen, R.A., van der Peer, Y., Potter, D., Sexton, J.P., Kawachi, M. & Lajeunesse, T., 1999: Phylogenetic analysis of the SSU rRNA from members of the Chrysophyceae. – Protist 150: 71–84. Arseneau, K.M.A., Driscoll, C.T., Cummings, C.M., Pope, G. & Cumming, B.F., 2016: Adirondack (NY, USA) reference lakes show a pronounced shift in chrysophyte speciesspe composition since ca. 1900. – J. Paleolimnol. 56: 349–364. Creaser, R., Grütter, H., Carlson, J. & Crawford, B., 2004: Macrocrystal phlogopite Rb-Sr dates for the Ekati property kimberlites, Slave Province, Canada: evidence for multiple intrusive episodes in the Paleocene and Eocene. – Lithos 76: 399–414. Cronberg, G., 1986: Chrysophycean cysts and scales in lake sediments: a review. – In: Kristiansen, J. & Andersen, R.A. (ed.): Chrysophytes: Aspects and Problems. – Cambridge Univ. Press, pp. 281–315. Cumming, B.F., uncorrected_proofSmol, J.P., Kingston, J.C., Charles, D.F., Birks, H.J.B., Camburn, K.E., Dixit, S.S., Uutala, A.J. & Selle, A.R., 1992: How much acidifi cation has occurred in Adirondack region lakes (New York, USA) since preindustrial times? – Canadian Journal of Fisheries and Aquatic Sciences 49: 128–141. Cumming, B.F., Davey, K.A., Smol, J.P. & Birks, H.J.B., 1994: When did acid-sensitive Adirondack lakes (New York, USA) begin to acidify and are they still acidifying? – Canadian Journal of Fisheries and Aquatic Sciences 51: 1550–1568. Del Campo, J. & Massana, R., 2011: Emerging diversity within chrysophytes, choanofl agellates and bicosoecids based on molecular surveys. – Protist 162: 435–448. Doria, G., Royer, D.L., Wolfe, A.P., Fox, A., Westgate, J.A. & Beerling, D.J., 2011: Declining atmospheric

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Manuscript received: 14.01.2017 Revisions required: 16.08.2017 Revised version received: 28.09.2017 Accepted for publication: 17.07.2018

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