Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolated from Canadian saline lakes show temperature sensitivity of alkenones and alkenoates Araie, H., Nakamura, H., Toney, J. L., Haig, H. A., Plancq, J., Shiratori, T., Leavitt, P. R., Seki, O., Ishida, K. I., Sawada, K., Suzuki, I., & Shiraiwa, Y. (2018). Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolated from Canadian saline lakes show temperature sensitivity of alkenones and alkenoates. Organic Geochemistry, 121, 89-103. https://doi.org/10.1016/j.orggeochem.2018.04.008 Published in: Organic Geochemistry Document Version: Peer reviewed version Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights © 2018 Elsevier Ltd. 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Sep. 2021 Accepted Manuscript Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolated from Canadian saline lakes show temperature sensitivity of alkenones and al- kenoates Hiroya Araie, Hideto Nakamura, Jaime L. Toney, Heather A. Haig, Julien Plancq, Takashi Shiratori, Peter R. Leavitt, Osamu Seki, Ken-ichiro Ishida, Ken Sawada, Iwane Suzuki, Yoshihiro Shiraiwa PII: S0146-6380(18)30082-2 DOI: https://doi.org/10.1016/j.orggeochem.2018.04.008 Reference: OG 3712 To appear in: Organic Geochemistry Received Date: 29 March 2018 Revised Date: 12 April 2018 Accepted Date: 13 April 2018 Please cite this article as: Araie, H., Nakamura, H., Toney, J.L., Haig, H.A., Plancq, J., Shiratori, T., Leavitt, P.R., Seki, O., Ishida, K-i., Sawada, K., Suzuki, I., Shiraiwa, Y., Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolated from Canadian saline lakes show temperature sensitivity of alkenones and alkenoates, Organic Geochemistry (2018), doi: https://doi.org/10.1016/j.orggeochem.2018.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolated from Canadian saline lakes show temperature sensitivity of alkenones and alkenoates Hiroya Araie a,bϯ, Hideto Nakamura c,d,eϯ, Jaime L. Toney f, Heather A. Haig g, Julien Plancq f, Takashi Shiratori a, Peter R. Leavitt g,h, Osamu Seki d,i, K en-ichiro Ishida a, K en Sawada c,d, Iwane Suzuki a,b, Yoshihiro Shiraiwa a,b* a Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 305-8572, Japan b CREST, Japan Science and Technology Agency (JST), Tsukuba, 305- 8572, Japan c Department of Earth and Planetary Sciences, Faculty of Science, Hokkaido University, Sapporo, 060-0810, Japan d CREST, Japan Science and Technology Agency (JST), Sapporo, 060- 0810, Japan e Department of Geosciences, Osaka City University, Osaka, 558-8585, Japan (present address of HN) f School of Geographical and Earth Sciences, University of Glasgow, Glasgow, G12 8QQ, UK g Limnology Laboratory, Department of Biology, University of Regina, Regina, Saskatchewan, S4S 0A2, Canada 1 h Institute of Environmental Change and Society, Faculty of Science, University of Regina, Regina, Saskatchewan, S4S 0A2, Canada i Institute of Low Temperature Science, Hokkaido University, Sapporo, 060-0814, Japan ϯ Equal contribution *Corresponding author. Tel.: +81-29-853-4668. E-mail address: [email protected] (Y. Shiraiwa). Abbr eviat ions: LCAs, long chain alkenones; LSU, Rubisco large subunit; Rubisco, ribulose bisphosphate carboxylase/oxygenase; SST, sea surface temperature; SSU, Rubisco small subunit; , , RIA38 and A37/A38, alkenoate indices; , , , Et, Me and Et, alkenone unsaturation indices. 2 ABSTRACT Alkenone-producing species have been recently found in diverse lacustrine environments, albeit with taxonomic information derived indirectly from environmental genomic techniques. In this study, we isolated alkenone-producing algal species from Canadian saline lakes and established unialgal cultures of individual strains to identify their taxonomical and molecular biological character istics. Water and sediments collected from the lakes were first enriched in artificial seawater medium over a range of salinities (5–40 ppt) to cultivate taxa in vitro. Unialgal cultures of seven haptophyte strains were isolated and categorized in the Isochrysis clade using SSU and LSU rRNA gene analysis. The alkenone distributions within isolated strains were determined to be novel compared with other previously reported alkenone-producing haptophytes. While all strains produced the typical C37 and C38 range of isomers, one strain isolated from Canadian salt lakes also produced novel C41 and C42 alkenones that are temperature sensitive. In addition, we showed that all alkenone unsaturation indices (e.g., and ) are temperature-dependent in culture experiments, and that alkenoate indices (e.g., , , RIA38 and A37/A38) provide alternative options for temperature calibration based on these new lacustrine algal strains. Importantly, these indices show temperature dependence in culture experiments at temperatures below 10 °C, where traditional alkenone proxies were not as sensitive. We 3 hypothesize that this suite of calibrations may be used for reconstructions of past water temperature in a broad range of lakes in the Canadian prairies. K eywords: Alkenoates, Alkenones, Alkenone unsaturation index, Canadian salt lakes, Chemotaxonomy, Haptophytes, Isochrysis, Long- chain alkyl ketones, Paleothermometer, 4 1. Introduction Long-chain alkenones (LCAs) were originally reported in marine sediments ca. 35 years ago (Boon et al., 1978; Brassell et al., 1980; de Leeuw et al., 1980; Volkman et al., 1980a, b; Marlowe et al., 1984) and in Quaternary lacustrine sediments a few years later (Cranwell, 1985; Volkman et al., 1988). Typically, these LCAs exhibit chain lengths from C35 to C40 and contain 2–4 trans double bonds, with C37–C39 LCAs appearing as the most common chain lengths in previous studies. The marine coccolithophore, Emiliania huxleyi, was the first haptophyte identified to have produced LCAs (Volkman et al., 1980a, b). These microalgae have been widely studied, in part because haptophytes change the proportion of alkenones having a different number of double bonds depending on growth temperature. Consequently, the ratio of LCAs with different unsaturation levels is now used to calculate indices, such as and , that can be used as paleotemperature proxies to reconstruct past sea surface temperature (SST) (Brassell et al., 1986; Prahl and Wakeham, 1987; Brassell, 1993). Only five species within the order Isochrysidales, phylum Haptophyta are reported to produce LCAs and analogous compounds such as alkyl alkenoates (Medlin et al., 2008). Although LCAs are now found frequently in saline and freshwater inland lakes (e.g., Pearson et al., 2008; Theroux et al., 2010; Toney et al., 2010, 2012; D’Andrea et al., 2011; Longo et al., 2013), it appears that these compounds are still 5 constrained to the phylum Haptophyta. At present, taxa known to produce alkenones fit into three taxonomical groups as defined by Theroux et al. (2010). Group III includes E. huxleyi and Gephyrocapsa oceanica (Family Noëlaerhabdaceae), which are characteristic of marine environments. Group II includes Isochrysis galbana, Tisochrysis lutea (Bendif et al., 2013), Ruttnera lamellosa (revised from Chrysotila lamellosa; Andersen et al., 2014), which belong to Family Isochrysidaceae and represent species found in a wide range of environments such as coastal regions, brackish waters and saline lakes. Group I is composed of haptophytes from which no living algal strains have been isolated, but for which putative haptophyte strains have been identified by using environmental genomics of Rubisco small subunit (SSU) rRNA from environmental samples collected in freshwater environments (D’Andrea et al., 2006; Theroux et al., 2010; Longo et al., 2013, 2016). Freshwater alkenone-producing haptophytes have gained the interest of the paleoclimate community because of the potential for their LCAs as indices of past continental climates (Zink et al., 2001; Chu et al., 2005, 2012; Hou et al., 2016). However, the use of these compounds has been constrained to date because specific LCA- producing strains have not been isolated from the source