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Novel alkenone-producing strains of genus Isochrysis (Haptophyta)isolated from Canadian saline lakes show temperature sensitivity ofalkenones and alkenoatesAraie, 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
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Download date:29. Apr. 2022
Accepted Manuscript
Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolatedfrom Canadian saline lakes show temperature sensitivity of alkenones and al-kenoates
Hiroya Araie, Hideto Nakamura, Jaime L. Toney, Heather A. Haig, JulienPlancq, Takashi Shiratori, Peter R. Leavitt, Osamu Seki, Ken-ichiro Ishida, KenSawada, Iwane Suzuki, Yoshihiro Shiraiwa
PII: S0146-6380(18)30082-2DOI: https://doi.org/10.1016/j.orggeochem.2018.04.008Reference: OG 3712
To appear in: Organic Geochemistry
Received Date: 29 March 2018Revised Date: 12 April 2018Accepted 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, OrganicGeochemistry (2018), doi: https://doi.org/10.1016/j.orggeochem.2018.04.008
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1
Novel alkenone-producing strains of genus Isochrysis (Haptophyta)
isolated from Canadian sal ine lakes show temperature sensi t ivi ty of
alkenones and alkenoates
H iroya Araie a,bϯ, H ideto Nakamura
c,d,eϯ, Jaime L. Toney f, Heather A.
Haig g, Jul ien Plancq
f, Takashi Shirator i
a, Peter R. Leavi t t
g,h, Osamu
Seki d,i
, Ken-ichiro Ishida a, Ken Sawada
c,d,
Iwane Suzuki
a,b, Yoshihiro
Shiraiwa a,b*
a Faculty of L i fe and Environmental Sciences, Universi ty 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 Universi ty, Sapporo, 060-0810, Japan
d CREST, Japan Science and Technology Agency (JST), Sapporo, 060-
0810, Japan
e Department of Geosciences, Osaka City Universi ty, Osaka, 558-8585,
Japan (present address of HN)
f School of Geographical and Earth Sciences, Universi ty of Glasgow,
Glasgow, G12 8QQ, UK
g L imnology Laboratory, Department of Biology, Universi ty of Regina,
Regina, Saskatchewan, S4S 0A2, Canada
2
h Inst i tute of Environmental Change and Society, Faculty of Science,
Universi ty of Regina, Regina, Saskatchewan, S4S 0A2, Canada
i Inst i tute of Low Temperature Science, Hokkaido Universi ty, Sapporo,
060-0814, Japan
ϯ Equal contr ibut ion
*Corresponding author. Tel .: +81-29-853-4668. E-mai l address:
emilhux@biol .tsukuba.ac.jp (Y. Shiraiwa).
Abbr eviat ions: LCAs, long chain alkenones; LSU, Rubisco large
subunit ; Rubisco, r ibulose bisphosphate carboxylase/oxygenase; SST,
sea sur face temperature; SSU, Rubisco small subunit ; ,
, RIA38
and A37
/A38,
alkenoate indices; ,
, ,
Et, Me and
Et,
alkenone unsaturat ion indices.
3
ABSTRACT
Alkenone-producing species have been recent ly found in diverse
lacustr ine environments, albei t wi th taxonomic informat ion der ived
indirect ly from environmental genomic techniques. In this study, we
isolated alkenone-producing algal species from Canadian sal ine lakes
and establ ished unialgal cul tures of individual strains to ident i fy their
taxonomical and molecular biological character ist ics. Water and
sediments col lected from the lakes were fi rst enr iched in ar t i ficial
seawater medium over a range of salini t ies (5–40 ppt) to cul t ivate taxa
in vi t ro. Unialgal cul tures of seven haptophyte strains were isolated
and categor ized in the Isochrysis clade using SSU and LSU rRNA gene
analysis. The alkenone distr ibut ions within isolated strains were
determined to be novel compared with other previously reported
alkenone-producing haptophytes. While al l st rains produced the typical
C37
and C38
range of isomers, one strain isolated from Canadian sal t
lakes also produced novel C41
and C42
alkenones that are temperature
sensi t ive. In addit ion, we showed that all alkenone unsaturat ion
indices (e.g., and
) are temperature-dependent in cul ture
exper iments, and that alkenoate indices (e.g., ,
, RIA38
and A37
/A38)
provide al ternat ive opt ions for temperature cal ibrat ion based on these
new lacustr ine algal strains. Important ly, these indices show
temperature dependence in cul ture exper iments at temperatures below
10 °C, where t radi t ional alkenone proxies were not as sensi t ive. We
4
hypothesize that this sui te of cal ibrat ions may be used for
reconstruct ions of past water temperature in a broad range of lakes in
the Canadian prair ies.
Keywords: Alkenoates, Alkenones, Alkenone unsaturat ion index,
Canadian sal t lakes, Chemotaxonomy, Haptophytes, I sochrysis, Long-
chain alkyl ketones, Paleothermometer,
5
1. I nt r oduct ion
Long-chain alkenones (LCAs) were or iginally reported in mar ine
sediments ca. 35 years ago (Boon et al ., 1978; Brassel l et al ., 1980; de
Leeuw et al ., 1980; Volkman et al ., 1980a, b; Mar lowe et al ., 1984) and
in Quaternary lacustr ine sediments a few years later (Cranwel l , 1985;
Volkman et al ., 1988). Typical ly, these LCAs exhibi t chain lengths from
C35
to C40
and contain 2–4 trans double bonds, with C37
–C39
LCAs
appear ing as the most common chain lengths in previous studies. The
mar ine coccol i thophore, Emil iania huxleyi , was the fi rst haptophyte
ident i fied to have produced LCAs (Volkman et al ., 1980a, b). These
microalgae have been widely studied, in part because haptophytes
change the proport ion of alkenones having a di fferent number of
double bonds depending on growth temperature. Consequent ly, the
rat io of LCAs with di fferent unsaturat ion levels is now used to
calculate indices, such as and
, that can be used as
paleotemperature proxies to reconstruct past sea sur face temperature
(SST) (Brassel l et al ., 1986; Prahl and Wakeham, 1987; Brassel l , 1993).
Only five species within the order Isochrysidales, phylum
Haptophyta are reported to produce LCAs and analogous compounds
such as alkyl alkenoates (Medl in et al ., 2008). Al though LCAs are now
found frequent ly in sal ine 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), i t appears that these compounds are st i l l
6
constrained to the phylum Haptophyta. At present, taxa known to
produce alkenones fi t into three taxonomical groups as defined by
Theroux et al . (2010). Group I I I includes E. huxleyi and Gephyrocapsa
oceanica (Family Noëlaerhabdaceae), which are character ist ic of
mar ine environments. Group I I includes Isochrysis galbana,
Tisochrysis lutea (Bendif et al ., 2013), Ruttnera lamel losa (revised from
Chrysot i la lamel losa; 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 sal ine lakes.
Group I is composed of haptophytes from which no l iving algal strains
have been isolated, but for which putat ive haptophyte strains have
been ident i fied by using environmental genomics of Rubisco smal l
subunit (SSU) rRNA from environmental samples col lected 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 potent ial for
their LCAs as indices of past cont inental cl imates (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 speci fic LCA-
producing strains have not been isolated from the source lakes, and
l i t t le is known of the environmental preferences of producer
populat ions. Analysis of environmental SSU rRNA suggests that
7
several alkenone-producing haptophyte species closely related to
Isochrysis and/or Ruttnera (family I sochrysidaceae) l ive in sal ine lakes
and brackish waters where the LCAs are associated with organic
matter suspended in water and sediments (Coolen, 2004, Coolen et al.,
2009; Theroux et al., 2010; Toney et al ., 2012, Randlet t et al ., 2014). The
most recent invest igat ion of nor thern Alaskan lakes reveals that those
LCAs are character ized by abundant C37:4
homologues and a ser ies of
C37:3
alkenone isomers. Fur thermore, Longo et al . (2016) used
suspended par t iculate matter in Tool ik Lake to determine an in si tu
-temperature cal ibrat ion for that freshwater si te. However, despite
these important advances, the absence of strain-specific informat ion on
environmental preferences makes i t difficult to determine whether
si te-, habitat - and species-speci fic calibrat ions may be required for
haptophytes from non-mar ine set t ings. Whi le alkenone-der ived, -
temperature cal ibrat ions have been developed for several genet ical ly
dist inct strains of haptophytes (Versteegh et al ., 2001; Rontani et al .,
2004; Sun et al ., 2007; Ono et al ., 2012; Nakamura et al ., 2014, 2016;
Zheng et al ., 2016), isolates from addit ional environments are st i l l
needed to determine the appl icabi l i ty of a universal calibrat ion.
To date, exist ing -temperature cal ibrat ions exhibi t simi lar
relat ionships (slopes) to environmental temperature, suggest ing a
simi lar dependence of unsaturat ion on temperature (Theroux et al .,
2010; Bendif et al ., 2013; Nakamura et al ., 2016). However, the y-
8
intercepts of -temperature cal ibrat ions vary among indicator rat ios
and may reflect the influence of other physiological , taxonomic or
environmental parameters. In addit ion, most LCA-based
paleotemperature reconstruct ions have been per formed in
environments where diverse haptophyte species with dist inct ive LCA
composit ion are expected to co-occur, including Chesapeake Bay
(Schwab and Sachs, 2011), the Black Sea (Coolen et al ., 2009), the
Nordic Sea (Bendle et al ., 2005) and the Balt ic Sea (Schulz et al ., 2000),
making i t di fficul t to determine whether reconstruct ions ar ise from
temperature-related shi fts in saturat ion level or environmentally
control led changes in species composit ion. Consequent ly, fur ther
detai led studies are needed on the thermal ecology of producing
organisms and on how LCA composit ions may change through t ime due
to di fferent control pathways (temperature, nutr ient status, etc.) to
ful ly understand the controls on unsaturat ion dependence on
temperature.
In this study, we isolated and ident i fied seven alkenone-
producing algae from Canadian sal ine lakes to bet ter establ ish the
relat ionship between environmental condit ions and LCA product ion .
Our new strains were grouped at unique phylogenet ic posi t ions within
the genus Isochrysis, including the fi rst report of alkenone-producing I .
galbana from inland lakes. The LCA composit ions of some strains were
di fferent from those of mar ine I . galbana, which was reported by
9
Theroux et al . (2013). Al though the unsaturat ion dependence of the
LCAs on temperature for the newly isolated Isochrysis strains was
simi lar to those from unident i fied alkenone-producing sedimentary
isolates in a previous study (Toney et al ., 2012), the y-intercept of new
LCA rat ios was di fferent , suggest ing another control on the y-intercept
of the cal ibrat ion. The addit ion of the mult iple, phylogenet ically
dist inct I sochrysis strains enabled us to discuss the relat ionship
between phylogeny and LCA composi t ion, including that between the
degree of compound unsaturat ion and environmental temperature for
species within the genus Isochrysis.
2. M at er ial s and met hods
2.1. Locat ion of Canadian lakes for isolat ing alkenone-producing
microalgae
We selected ten Canadian saline lakes where LCAs were already
detected by Toney et al . (2011) and environmental data were avai lable
from an ear l ier comprehensive survey (Pham et al . 2009). Lakes
Antelope, Snakehole, Success, Fishing, Humboldt , Waldsea, Deadmoose,
Charron, Rabbit , and Redberry are sal ine lakes located in
Saskatchewan, Canada (Supplementary Fig. S1, Table 1). In the
present study, temperature, pH and sal ini ty profi les were measured at
1 m depth intervals using a YSI Pro Plus meter (YSI Inc., Yel low
Spr ings, Ohio, USA).
10
2.2. I solat ion and establ ishment of alkenone-producing microalgal
strains as unialgal cul ture
Samples of lake water, lake sediments, shore sand and plankton
were col lected with a 4 L Van-Dorn water sampler, Ekman grab, and
plankton net (mesh pore size 5 μm), respect ively, in September 2014.
The samples were t ransported to the laboratory of the Universi ty of
Regina, Regina, Canada, for the isolat ion of microalgae. The sample
types (water, sediment, and plankton) were individual ly combined with
a fresh cul ture medium for microalgae using ei ther AF6 modified
(prepared according to NIES-Culture Col lect ion Media L ist , NIES,
Japan; or iginal ly from Watanabe et al ., 2000) or an ar t i ficial seawater
(Mar ine Art SF-1; Osaka Yakken, Osaka, Japan) enr iched with a
modified Erd-Schreiber 's medium containing 10 nM disodium seleni te
(MA-ESM as descr ibed in Danbara and Shiraiwa, 1999).
MA-ESM media was prepared with a range of sal ini t ies to match
the sal ini ty of each lake by adjust ing the amount of Mar ine Art SF-1
powder to achieve sal ini t ies varying from 5 ppt to 40 ppt . Then the
medium was di luted using techniques for isolat ing single species of
microalgae, as descr ibed by Allen and Stanier (1968). The algal
suspension di luted with the fresh medium was dispensed into wells of
a t ransparent , plast ic, 96 wel l microplate. The sal ini ty of the cul ture
medium was set at 40, 30 or 10 ppt , as shown in Table 2. For the
11
cult ivat ion of microalgae, the plates were maintained in a plant growth
chamber under i l luminat ion by 20 W fluorescent lamp with an
intensi ty range of 28 to 43 μmol photons m-2 s
-1 (range: 400–700 nm)
with a 12 h l ight /12 h dark regime. The temperature in the chamber
was kept constant at 10 °C dur ing cul ture.
2.3. Culture of the establ ished strains as unialgal cul ture for test ing
temperature effect
Algal strains isolated from Canadian lakes were individual ly
establ ished as unialgal cul tures by using the di lut ion method, as
descr ibed above. Those strains were grown in 50 mL plast ic flasks
containing the MA-ESM medium with var ious ranges of sal ini ty from
10 to 40 ppt (Table 2). Al l cul tures were maintained in the algal growth
chamber where l ight intensi ty and temperature were control led. The
cul tures were cont inuously i l luminated by 20 W fluorescent lamps at
the intensi ty of 100 μmol photons m-2s
-1. For test ing temperature effect ,
three representat ive strains were used. The temperature was
separately set at 5, 10, 15, 20 and 25 °C. Cel ls were grown unt i l the late
l inear or the ear ly stat ionary growth phases when they were harvested.
Monitor ing was achieved using the opt ical cel l densi ty of the cel l
suspension. The cul ture per iods were different among cul ture vessels
depending on growth rate in each cul ture and ranged from 16 days to
23 days. The harvested cel ls were used for the l ipid analysis of LCAs
12
and alkenoates.
2.4. DNA extract ion, polymerase chain react ion (PCR) and sequencing
After algal cel ls were harvested by centr i fugat ion, DNA was
extracted from the cel ls using a DNeasy plant mini k i t (Qiagen, H i lden,
Germany). The D1–D2 region of the large subunit (LSU) rRNA gene
was ampl i fied by the PCR react ion using the haptophyte specific pr imer
set Hapto_4 (5'-ATGGCGAATGAAGCGGGC-3') and Euk_34r (5'-
GCATCGCCAGTTCTGCTTACC-3') (L iu et al ., 2009). The
ampl i ficat ions consisted of 30 cycles of denatur ing at 98 °C for 10 s,
anneal ing at 55 °C for 15 s, and extension at 72 °C for 1 min by using
Pr imeSTAR GXL DNA polymerase (Takara Bio, Ohtsu, Japan). The
SSU rRNA gene of isolates was ampl ified by the PCR react ion using
pr imer set 18F (5'-AACCTGGTTGATCCTGCCAG-3') and 18R (5'-
CYGCAGGTTCACCTACGGAA-3') (Yabuki et al ., 2010). The
ampl i ficat ions consisted of 30 cycles of denatur ing at 98 °C for 10 s,
anneal ing at 55 °C for 15 s, and extension at 72 °C for 2 min by using
Pr imeSTAR GXL DNA polymerase (Takara Bio). These ampl i fied DNA
fragments were sub-cloned into E. col i st rain JM109 and then
sequenced using a 3130 Genet ic Analyzer (Appl ied Biosystems,
Waltham, MA, USA) with a BigDye Terminator v3.1 cycle sequencing
ki t (Applied Biosystems).
13
2.5. Sequence al ignments and phylogeny
For molecular phylogenet ic analysis, we newly created two
datasets of haptophytes; one for the D1–D2 region of LSU rRNA gene
sequences, whi le the other was the whole region of SSU rRNA gene
sequences. The datasets were automat ical ly al igned with mafft -l insi
(Katoh and Standley, 2013), and then edited manual ly with SeaView
(Galt ier et al ., 1996). Ambiguously al igned regions were manual ly
deleted from the al ignments. Final ly, we prepared a LSU rRNA gene
al ignment with 58 operat ional taxonomic uni ts (OTUs) and 995
posi t ions, and a SSU rRNA gene al ignment with 100 OTUs and 1713
posi t ions. The maximum l ikel ihood (ML) t ree was constructed using
IQ-TREE (Nguyen et al ., 2015) under the best -fi t model (TIM2+G4 for
LSU rRNA and TNe+G4 for SSU rRNA gene) determined by IQ-TREE.
Non-parametr ic bootstrap analysis with 100 repl icates was conducted
under the best-fi t models. The Bayesian analysis was per formed on
each alignment using MrBayes v. 3.2.2 (Ronquist et al ., 2012) with the
GTR +Γ model. Two separated Metropol is-coupled Markov chain Monte
Car lo, each with one cold and three heated chains (defaul t chain
temperature = 0.1), were run for 5 106 generat ions. The lnL values
and t rees were sampled at every 100 generat ion intervals. The
convergence was assessed based on the average standard deviat ion of
spl i t frequencies, and the fi rst 2 106 generat ions of each run were
discarded as “burn-in.” Bayesian poster ior probabi l i ty (BPP) and
14
branch lengths were calculated from the remaining of the t rees.
2.6. L ipid extract ion and fract ionat ion by organic solvents
The total l ipid content of cel ls were extracted and separated into
di fferent compound classes for analysis using methods of Sawada et al .
(1996) and Nakamura et al . (2014). Br iefly the l ipids were successively
extracted from whole algal cells with methanol (MeOH),
dichloromethane (DCM):MeOH (1:1, v:v) and DCM. The combined
extracts were vigorously shaken after the addit ion of dist i l led water,
and subsequent ly centr i fuged to separate into two layers: l ipid and
water-soluble fract ions. The result ing organic solvent layer was passed
through an anhydrous Na2SO
4 column to remove water. The l ipid
extract was dr ied in a rotary evaporator and subsequent ly re-dissolved
with n-hexane. The l ipid-containing hexane extracts were separated
using a si l ica gel column into three fract ions with n-hexane, n-
hexane:ethyl acetate (9:1, v:v) and ethyl acetate:MeOH (1:1, v:v) to
yield hydrocarbons, LCAs, and polar l ipids (e.g., sterols and fat ty acids),
respect ively. After adding an internal standard (n-hexatr iacontane),
those three fract ions were analyzed by GC and GC–MS to quant i fy the
var ious compounds they contained.
The LCA fract ion was fur ther cleaned up by saponificat ion to
reduce contaminat ion from non-LCA compounds. Here a por t ion of the
alkenone fract ion was saponified by heat ing at 70 °C for 3 h in 1 N
15
KOH MeOH:H2O (95:5, v:v). After saponificat ion, resul tant products
were extracted three t imes with 1 ml each of n-hexane and
subsequent ly brought to the analysis of components using both GC and
GC–MS.
LCAs were also extracted from the lake sediments col lected at
the Canadian si tes. Br iefly, freeze-dr ied sediments were homogenized
and extracted with DCM:MeOH (9:1, v:v) using a Dionex model
ASE350 accelerated solvent extractor. Fol lowing evaporat ion of the
solvent, the total l ipid extracts were separated into neutral and acid
fract ions by elut ion through a LC-NH2 SPE column using
DCM:isopropyl alcohol (1:1, v:v) followed by ether with 4% acet ic acid
(v:v) as eluents, respect ively. The neutral fract ions were fur ther
separated into four fract ions of increasing polar i ty by chromatography
over a si l ica gel column packed with 35–70 μm part icles using hexane,
DCM, ethyl acetate:hexane (1:3, v:v) and MeOH as eluents. The second
fract ions (DCM fract ion) containing LCAs were saponified using the
same procedure as descr ibed above.
2.7. GC and GC–MS analyses
GC was conducted using a Shimadzu GC-2025 instrument
equipped with FID for quant i ficat ion of alkenone fract ions. Two
methods were appl ied with di fferent columns and respect ive
temperature programs: Agi lent VF-200ms column (60 m × 0.25 mm ×
16
0.10 µm), temperature program was 50 °C (1 min) to 255 °C at
20 °C/min, to 300 °C at 3 °C/min, and subsequent ly to 320 °C at
10 °C/min (held 10 min); Agi lent CPSil5CB column (50 m × 0.32 mm ×
0.12 µm), temperature program was 90 °C (2 min) to 255 °C at
40 °C/min, to 300 °C at 1 °C/min, and subsequent ly to 320 °C at
10 °C/min (held 10 min).
The use of the VF-200ms column was demonstrated by Longo et
al . (2013) to signi ficant ly improve the separat ion for long-chain LCAs.
However, C37:4
alkenone and C36:2
FAEE alkenoate co-eluted under these
condit ions, therefore the CPSil5CB column was used to separate both
compounds clearly, quant i fy the rat io of C37:4
to C37:2
compounds, and
calculate the abundances of C37:4
and C36:2
FAEE (Nakamura et al ., 2014).
The LCAs and alkenoates were ident i fied by Agi lent 6890N GC
instrument coupled to an Agi lent 5975 iner t XL MSD quadruple mass
spectrometer (electron ionizat ion: 70 eV; emission current : 350 μA; m/z
50–650). The VF-200ms column and the ident ical temperature program
as for GC analysis were used for GC–MS analysis. Helium was the
carr ier gas in both GC and GC–MS.
The LCAs and alkenoates consist of di -, t r i - and tetra-
unsaturated homologues. General ly, di -unsaturated LCAs possess
double bonds at Δ14
and Δ21
posi t ions (Δ14,21
), t r i -unsaturated LCAs have
a third double bond at the Δ7 posi t ion (Δ
7,14,21) and the tetra-unsaturated
alkenone has a four th double bond at Δ28
(Δ7,14,21,28
) (Di l lon et al ., 2016;
17
Longo et al ., 2016; Zheng et al ., 2017). By using a GC column lined with
a VF-200ms column, isomers of t r i -unsaturated LCAs and alkenoates
exhibi t doublet -l ike peaks consist ing of a left -hand peak and a r ight -
hand peak correspond to Δ7,14,21
(Δ7 isomer; isomer a) at Δ
14,21,28 (Δ
28 isomer;
isomer b), respect ively.
2.8. Temperature indices
The alkenone unsaturat ion indices ( ,
, ,
Et, Me
and Et), alkenoate unsaturat ion indices (
and ), methyl and
ethyl alkenoate rat io (A37
/A38), and the isomer ic rat io of alkenoates
(RIA38
) were al l calculated based on biochemical profiles of Canadian
algal samples raised under diverse temperature regimes. Refer to
Nakamura et al . (2016) for or iginal references for these indices. Final ly,
RIA38
was determined as the isomer ic rat io of alkenoates (C36:3
FAEE)
according to the defini t ion of RIK indices proposed by Longo et al .
(2016), calculated by the fol lowing equat ion; RIA38
= C36:3a
FAEE /
(C36:3a
FAEE + C36:3b
FAEE), where C36:3a
FAEE and C36:3b
FAEE are isomers.
3. Resul t s
3.1. Haptophyte strains isolated from Canadian lakes
Haptophyte strains were isolated successfully from nearshore
sand or sediments of three central Canadian lakes, speci fical ly lakes
Snakehole, Success and Deadmoose (Tables 1 and 2A). Al l basins were
18
sal ine lakes, and two exhibi ted a clear ly defined thermocl ine
accompanied by changes in pH and sal ini ty (Supplementary Fig. S2).
In contrast , algal isolat ion was unsuccessful for samples obtained from
lake water and plankton net samples, even though marker genes were
ident i fied in some l iving algal samples and whole-water environmental
samples of lakes Snakehole, Success and Waldsea (Table 1).
Strains isolated from Lake Snakehole were named as Sh 1 and 2
(Fig. 1A and B), two from Lake Success were named as Sc 1 and 2 (Fig.
1C and D), and three from Lake Deadmoose named as Dm 1, 2 and 3
(Fig. 1E–G). DNA sequences of SSU and LSU rRNA were ampl i fied
from seven isolated haptophytes and algal mixtures from
environmental samples such as water, plankton net and sediment by
using haptophyte-speci fic pr imers. The results of successful
ampl i ficat ion are as shown in Table 2 with the GenBank accession
numbers. DNA sequences obtained from environmental samples, the
prefix E was added to the strain number (e.g., Sh E1). The LSU rRNA
sequence data of the environment DNA sample from Lake Waldsea was
coded Ws E1.
The phylogenet ic analysis showed that DNA sequences of the
Canadian lake isolates and algal mixture from environmental samples
were composed of four groups: namely Group A including Sh E2, Sh 2,
Sh 1 and Sh E1, Group B including Sc E2, Sc 1, Sc 2, Group C including
Dm 2, Dm 3, Dm 1, Sc E1, Sc E3 and Group D including Ws E1. Group
19
A from Lake Snakehole occupied a unique phylogenet ic posi t ion in the
I . galbana clade. This phylogenet ic posi t ion was suggested by both
maximum l ikel ihood t ree analysis of SSU and LSU rRNA sequences of
the Canadian haptophyte strains (Figs. 2 and 3). Groups B and C were
included in the I . galbana clade. Ws E1 obtained from Lake Waldsea
suggested the existence of R. lamel losa (Fig. 3). Overal l , unialgal
strains isolated from lakes Success and Deadmoose showed a
swimming abi l i ty dr iven by flagel la under microscopic analysis;
whereas, that from Lake Snakehole did not (Supplementary Fig. S3).
This observat ion is also supported by determinat ions of the
phylogenet ic posi t ions of Groups A, B and C.
3.2. Growth character ist ics of the Canadian lake haptophyte strains
Strains Sh 1, Sc 2 and Dm 2 showed simi lar temperature-
dependent growth patterns when cul tured over the range 5–25 °C (Fig.
4). In all st rains, the logar i thmic growth phase ended within 100 h and
then proceeded to the l inear growth phase above 15 °C up to 25 °C. The
opt imum growth temperature was 25 °C for Sc 2 and 20 °C for Sh 1
and Dm 2. Below 20 °C, the growth pattern of those three strains was
simi lar. Algal growth also showed a clear lag phase slowly fol lowed by
the logar i thmic growth phase with a low rate below 10 °C.
The strain Sh 1 showed the greatest preference for cold waters,
with a high growth rate at 5 °C and suppressed growth at 25 °C (Fig. 4).
20
The strain Sc 2 showed an opposite t rend to Sh 1, with low growth at
low temperatures and increased growth rates at high temperatures.
The strain Dm 2 showed intermediate propert ies. In addit ion to growth
parameters, some st rains exhibi ted physiological or morphological
responses to changes in temperature. For example, the number of cells
swimming decreased below 10 °C in strains Sc 2 and Dm 2 (data not
shown), whereas the shape of some cel ls became more round than
oblong (Supplementary Fig. S3).
3.3. LCAs and alkenoates in newly establ ished Canadian lake
haptophyte strains
Al l haptophyte strains (Table 2A) were analyzed for LCAs using
a GC–FID approach (Supplementary Fig. S4). As there was minimal
var iat ion in GC–FID profi les for samples obtained from individual
lakes, only one strain from each lake was presented as representat ive
of the composit ion of LCAs and alkenoates including der ivat ives (Fig. 5,
Supplementary Table S1).
The distr ibut ions of LCAs and alkenoates of the Canadian lake
strains were character ized by the occurrence of major components such
as C37
methyl alkenones, C38 ethyl alkenones, and relat ively minor
components such as C39
and C40
alkenones. Overal l , these patterns were
simi lar to those already publ ished from other Isochrysidaceae strains
(Rontani et al., 2004; Sun et al ., 2007; Theroux et al ., 2013; Nakamura
21
et al ., 2014, 2016; Zheng et al ., 2016). Addit ional ly, C38
methyl and C39
ethyl LCAs were also detected as minor alkenone components.
Fur thermore, along with LCAs, C36
methyl alkenoates (C36
FAMEs) and
C36
ethyl alkenoates (C36
FAEEs) were also detected as signi ficant
components in the non-saponified alkenone fract ions (Fig. 5C).
I somers of t r i -unsaturated LCAs and alkenoates exhibi ted
doublet -l ike peaks consist ing of a left -hand peak and a r ight-hand peak
that may correspond to Δ7,14,21
(Δ7 isomer; isomer a) at Δ
14,21,28 (Δ
28 isomer;
isomer b), respect ively (Fig. 5). Among the previously reported t r i -
unsaturated isomers of LCAs, only minor amounts of the Δ28
isomer of
C38:3
alkenone (C38:3b
Et in Fig. 5C; Supplementary Table S1) were
ident i fied in Canadian lake samples. However, the double bond
posi t ions are st i l l considered tentat ive in our analysis, even though the
ident i ficat ion of isomer ic alkenones and alkenoates using DMDS
treatment in Di l lon et al . (2016) and Zheng et al . (2017) is a robust
protocol .
There were also lake-specific character ist ics in LCAs among
strains grown at 20 °C. Specifical ly, Sh 1 was character ized by the
product ion of extended LCAs that eluted after C40
LCAs in GC–FID
analysis (peaks 23–26 in Fig. 5). Previously, simi lar peaks have been
reported as C41
methyl and C42 ethyl LCAs with two and three double
bonds in samples isolated from Chinese inland sal ine lakes (Zhao et al.,
2014). Peaks 23–26 were assigned as C41
methyl - and C42
ethyl -
22
alkenones by compar ing elut ion patterns and mass spectra reported by
Zhao et al . (2014). C41:3
Me (peak 23) and C41:2
Me (peak 24) were
character ized by M+. at m/z 584 and 586, respect ively. Both compounds
exhibi ted [M –18]+ ion, indicat ing a methyl ketone. C
42:3Et (peak 25) and
C42:2
Et (peak 26) exhibi ted [M+.] (m/z 598 and 600, respect ively) and [M–
29]+ ion, indicat ing a ethyl ketone.
The sedimentary LCAs from lakes Snakehole, Success and
Deadmoose showed simi lar LCA profiles to the cul tured isolates with
some notable di fferences. For instance, only the Snakehole sedimentary
LCAs were character ized by the presence of C41:3
Et. Meanwhi le, the
sedimentary LCA profi les showed consistent ly fewer numbers of LCAs
compared to the cul ture isolates; lacking C36:3
FAME, C36:2
FAME,
C36:3b
FAEE, C38:3b
Et, C38:3
Me, C38:2
Me, C39:3
Et, C41:2
Me, C42:3
Et, and C42:2
Et.
The Snakehole sedimentary LCA profi les also lacked C36:4
FAME and
C36:4
FAEE that were present in the cul ture samples. The Success
sedimentary LCA profi le also lacked C39:2
Me, C40:3
Et, and C40:2
Et that
were present in the cul ture samples. The Deadmoose sedimentary LCA
profi les also lacked C40:3
Et, C40:2
Et that were present in the cul ture
samples. Of those “cul ture-only” LCAs, C38
methyl -, C39
ethyl-, and above
C40
alkenones were minor compounds in cul ture isolates, so their lack
in sedimentary LCA profiles might be due to smal l amount of these
compounds.
23
3.4. Growth temperature-dependent changes in alkenone and
alkenoate composit ions and the alkenone unsaturat ion index
The unsaturat ion indices of C37
–C42
LCAs were establ ished using
Canadian lake haptophyte strains of Sh 1, Sc 2 and Dm 2 (Table 3, Fig.
6, Supplementary Table S2). Important ly, Me and
Et were
avai lable only for Sh 1 strain, because only Sh 1 produces C41
and C42
LCAs. Overal l , al l alkenone unsaturat ion indices increased above 10 °C,
but were relat ively invar iant below that temperature. Such a low-
temperature plateau in alkenone unsaturat ion index values between 5
and 10 °C is seen in other isolates (Conte et al ., 1998; Versteegh et al .,
2001; Nakamura et al ., 2014).
Simi lar to findings with the LCAs, alkenoate unsaturat ion
indices based on both methyl and ethyl alkenoates ( and
,
respect ively) increased l inear ly with incubat ion temperature, whereas,
RIA38
and A37
/A38
decreased with growth temperature from 10 °C to
25 °C (Table 3, Fig. 7, Supplementary Table S2). Compar ison of l inear
and second-order polynomial regressions of the alkenone and alkenoate
indices (10 °C to 25 °C) revealed that the second-order polynomial
regressions gave a sl ight ly bet ter fi t due to a sl ight curvature of
thermal relat ionships, for example, r2 = 0.99 vs r
2 = 0.93, respect ively
( of Sh 1).
4. Discussion
24
4.1. I solat ion and establ ishment of new strains of alkenone-producing
haptophytes from Canadian lakes
This study succeeded in the isolat ion and establ ishment of seven
new strains of alkenone-producing haptophytes from Canadian sal ine
lakes. Study basins (lakes Snakehole, Success and Deadmoose) are
located immediately nor th of the North Amer ican freshwater and
sal ine lakes where alkenone-producing microalgae were fi rst ident i fied
based on SSU rRNA analysis (Toney et al . 2010, 2012). Interest ingly,
both Lakes Success and Deadmoose exhibi ted strong ver t ical
strat i ficat ion of both temperature and sal inity, whereas shallow, but
sal ine, Lake Snakehole seemed to be wel l -mixed due to vigorous wind
at the t ime of sampl ing (Supplementary Fig. S2). These di fferences are
consistent with the recent environmental survey of 106 lakes in this
region that shows that sal ini ty is the pr imary control on alkenone
presence and concentrat ion, with a secondary influence of strat i ficat ion
(Plancq et al ., 2018).
Alkenones were most commonly present in deep lakes that
ranged in sal ini ty from 2.4 g/L to 44.4 g/L. Al though there is no direct
exper imental evidence on the relat ionship between such environmental
character ist ics and the presence of alkenone-producers, the fact that
isolat ion of haptophytes was only successful at si tes with hypersal ine
deep waters suggest that chemical character ist ics may be important at
some phase of the haptophyte l i fe cycle. In par t icular, high sal ini ty is
25
often associated with strong anoxia in deep waters (strat i fied) or
sediments (al l lakes), consistent with the prior observat ion that LCAs
are associated in lakes with anoxic bot tom waters (e.g., Toney et al .
2010; Plancq et al ., 2018).
The presence of flagel la and swimming abi l ity in isolates from
deep, meromict ic lakes Success and Deadmoose, but not isolates from
shal lower Lake Snakehole may suggest that access to anoxic water
depends in par t on the mot i l i ty of the haptophytes present
(Supplementary Fig. S3). On the other hand, non-mot i le strains might
have a tolerance for dry condit ions, since i t was isolated from shore
sand. However, as the isolat ion process employed in this study was just
per formed once in late September 2014 when the ambient temperature
was very low, i t is possible that cel l growth rates were depressed and
inadequate to establ ish high populat ions of alkenone-producing
microalgae at some si tes. Fur ther , as there should be pronounced
seasonal var iat ion in haptophyte abundance, we speculate that i t may
be necessary to col lect samples throughout a year from diverse habitats
in each lake to ful ly character ize the presence of alkenone-producing
taxa. Thus, the presence of alkenones in si tes where viable populat ions
were not isolated most l ikely reflects di fferences in the t iming of
haptophyte growth and alkenone deposit ion in sediments.
The phylogenet ic analysis showed that the Canadian lake-
isolates can be grouped into three groups, namely Group A including
26
the Lake Snakehole strains closely related to I . l i toral is and I . nuda,
Group B including the Lake Success strains, and Group C including the
Lake Deadmoose strains. The LSU rRNA sequences from Lake
Snakehole occupy a unique phylogenet ic posi t ion in the I . galbana
clade (Fig. 3). Simi lar relat ionships were confi rmed by the analysis of
SSU rRNA sequencing data (Fig. 2). Here, Lake Snakehole strains are
posi t ioned near to Dicrater ia sp. ALGO HAP49 al though the Dicrater ia
sp. was renamed to Isochrysis nuda after Bendif et al . (2013). The
microscopic observat ions also supported the phylogenet ical analyses
and showed that the Lake Snakehole strains are di fferent species from
I . galbana; whereas, the strains from Lake Success and Deadmoose are
I . galbana.
4.2 Character ist ics of LCA and alkenoate composit ions
4.2.1. Tetra-unsaturated LCAs and alkenoates
Product ion of abundant tetra-unsaturated LCAs is considered a
common feature of non-calci fying, LCA-producing haptophyte algae
that are classi fied into Groups I and I I (Theroux et al ., 2010). This
pat tern appears to also hold for alkenone distr ibut ions obtained from
ei ther unialgal isolates or environmental samples from a diverse range
of lake water sal ini ty, ranging from highly sal ine brackish and sal ine
inland waters to relat ively di lute ol igotrophic freshwater systems
(Theroux et al ., 2010; Longo et al ., 2016) (Fig. 2). Among the
27
I sochrysidaceae, both genera Ruttnera and Isochrysis are known to
produce relat ively high amounts of tetra-unsaturated LCAs, whereas
Tisochrysis produces only di- and t r i -unsaturated LCAs (Nakamura et
al ., 2016). Accordingly, the newly isolated Canadian haptophytes
strains, such as Sh 1, Sc 2 and Dm 2, were character ized by product ion
of tetra-unsaturated LCAs, especial ly at lower temperatures. We infer
that these results suggest that Sh 1, Sc 2 and Dm 2 are closely related
to genus Isochrysis and note that this hypothesis is also consistent
with phylogenet ic t rees based on SSU and LSU rRNA sequences (Fig. 2,
Fig. 3).
4.2.2. Double bond posi t ion of t r i -unsaturated LCA and alkenoate
isomers
A smal l amount of the t r i -unsaturated isomer of alkenone C38:3b
Et
was detected from newly isolated Isochrysis strains Sh 1, Sc 2 and Dm
2, whi le incomplete chromatographic separat ion with closely elut ing
major peak of C38:3a
Et precludes accurate quant i ficat ion of C38:3b
Et (Fig. 5,
Supplementary Table S1). Patterns of presence/absence of t r i -
unsaturated LCAs and alkenoates are considered to serve as
chemotaxonomic character ist ics for taxonomic assessments of alkenone
producers. For example, in general , the LCA profi les of the Group I
haptophytes are di fferent from those of the Group I I and I I I
haptophytes, including the product ion of t r i -unsaturated alkenone
28
isomers throughout both ethyl and methyl LCAs of the whole range of
chain-lengths (Longo et al ., 2016). Zheng et al . (2017) fur ther observed
R. lamel losa LG strain (Group I I ) and E. huxleyi Van 556 (Group I I I ) do
not produce t r i -unsaturated LCAs, other than a relat ively minor
amount of C38:3b
Et. In addit ion, confi rmat ion of simi lar pat terns (i .e., the
sole occurrence of C38:3b
Et isomer among known tr i -unsaturated LCA
isomers) in three genet ical ly different strains of I sochrysis reinforces
the idea that this pat tern is a shared feature for representat ives of
both Isochrysis and Ruttnera genera within the Family I sochrysidaceae
(Fig. 3).
Relat ive (%) abundance of C36:3
FAEE isomers di ffered between
strains closely related to both I . l i toral is and I . nuda (Sh 1) and those
more closely related to I . galbana (Sc 2, Dm 2). In par t icular, Sh 1
exhibi ted a dominant α-type isomer of C36:3
FAEE, whi le the β-type
isomer was recorded in both Sc 2 and Dm 2 strains (Supplementary
Table S1). Interest ingly, t r i -unsaturated isomers were detected only in
C36:3
FAEE, but not in C36:3
FAME. This result is in accordance with the
sole occurrence of C38:3
Et isomers in LCAs. These data provide insights
on the synthesis of LCAs, and alkenoates in par t icular, as unsaturat ion
may be speci fic to a speci fic chain-length or methyl and ethyl group in
the unsaturat ion at the posi t ion of Δ28
in the Group I I haptophytes.
Overal l , the isolates from lakes Snakehole, Success and
Deadmoose were from Group I I haptophytes. However, while the LCA
29
profi le of Sh 1 and Dm 2 showed the C38:3b
Et isomer, we detected no
C38:3b
Et isomer from the sediment of Lake Snakehole. These results
suggest that the presence/absence of t r i -unsaturated isomers of
alkenoates (Zheng et al ., 2016), as wel l as their relat ive abundance and
response to environmental temperatures, might serve as a
chemotaxonomic feature of alkenone-producing haptophyte algae.
4.2.3. Unique occurrence of LCAs >C40
in the Sh 1 strain
The carbon chain length of LCAs in ocean sediments commonly
ranges from C37
to C39
, whereas isolates from lake sediments somet imes
contain C40
LCAs as minor components. The occurrence of LCAs longer
than C40
are except ional ly rare as C41
and C42 LCAs have only been
reported from two recent hypersal ine lakes in the ar id nor thwestern
China (Zhao et al ., 2014) and from the Cretaceous mar ine sediment
(Cenomanian black shale, ca. 95 Ma) from western North At lant ic
(Deep Sea Dr i l l ing Project (DSDP) Site 534; Farr imond et al ., 1986).
In this context , the Sh 1 strain establ ished in this study is the
fi rst cul tured strain that is character ized as a producer of C41 and C
42
LCAs (Fig. 5, Supplementary Table S1). The molecular phylogenet ic
t ree of LSU and SSU rRNA gene sequences indicate that the Sh 1
strain is most closely related to both I . l i toral is and I . nuda (Figs. 2 and
3). Fur ther invest igat ion on the alkenone profi les of I . l i toralis and I .
nuda wi l l be necessary to clar ify whether such chemotaxonomic
30
character ist ic of >C40
LCAs is speci fic to the Snakehole Lake strain, or
whether i t is also representat ive of I . l i toral is and I . nuda. The present
resul ts also suggests that the Sh 1 strain might be a candidate taxon
responsible for the product ion of C41
and C42
LCAs in the hypersaline
lakes in the nor thwestern China reported by Zhao et al . (2014).
4.3. Changes in alkenone and alkenoate composit ions with growth
temperature
Simi lar to previous cul ture studies, our resul ts show that the
ent i re sui te of alkenone homologues, as well as alkenoates, is engaged
in the adjustment of alkenone unsaturat ion and great ly affected by
growth temperature of alkenone-producing species (e.g., Prahl et al .,
1988; Conte et al ., 1998). However, the consistency in the alkenone and
alkenoate unsaturat ion dependence on temperature in the lacustr ine
isolates is unusual relat ive to studies from mar ine isolates, in which a
number of other factors have been ci ted as potent ial ly inter fer ing with
the temperature dependence in cul tures (e.g., Epstein et al . 1998, 2001;
Conte et al ., 1995, 1998; Popp et al ., 1998; Laws et al ., 2001). For
example, previous reports suggested that alkenone unsaturat ion
degree in some strains of E. huxleyi may also be affected by the
di fference in physiological condit ions/status of cel ls. For example, the
alkenone unsaturat ion degree increases (i .e. decreases) more in the
stat ionary growth phase in compar ison with the exponent ial growth
31
phase (Conte et al ., 1995, 1998).
In this study, isolate cul tures were al l grown at the same t ime,
under the same condit ions, with an exper imental design that control led
for temperature as the only var iable (i .e., not nutr ients, l ight , water
chemistry, etc.). I t is worth not ing that simi lar haptophyte species in
other lacustr ine environments (e.g., Lake George, USA) have a simi lar
temperature dependence, based on slopes (Fig. 8) with the isolate
cul tures (i .e. I . galbana CCMP715 and Dm 2). However, in this instance,
the y-intercepts observed here di ffer from that of cul ture exper iments,
which suggests that simi lar biot ic and abiot ic condit ions l ikely
influence the y-intercept of this correlat ion as the mar ine findings, but
not the temperature dependency (Toney et al . 2010, 2012).
The effects of other biot ic and abiot ic controls on the y-intercept
would benefi t from fur ther research. Previous studies (Toney et al .
2010) show that an in si tu temperature cal ibrat ion could be der ived for
a given lake despite sampl ing over mult iple years, seasons and water
depths and that the temperature cal ibrat ion was valid from 2 °C in
more sal ine bottom waters to 25 °C in sur face water. I t is possible that
the lacustr ine algal taxa are bet ter adapted to a var iable environment,
whereas mar ine taxa are used to a relat ively constant environment. On
an annual basis, lakes undergo more extreme var iat ions than oceans,
including pH (several uni ts), photon flux (near-sur face i r radiance to
zero at depth), and oxygen (over-saturat ion to microaerobic condit ions
32
over 10–20 m); whereas such var iables are more stable in both t ime
and space in many mar ine systems. Whi le these di fferences in observed
environmental condit ions may hold clues to the di fference between
reported mar ine haptophyte cul tures versus lacustr ine haptophyte
cul tures, our data suggest that the temperature dependency of the
alkenone and alkenoate unsaturat ion is consistent in both cul ture and
environmental set t ings for the lake haptophytes.
4.4. Compar ison of cal ibrat ions with other species and the
lacustr ine alkenone profiles: Impl icat ions for environmental
reconstruct ion
The -temperature cal ibrat ions obtained for the three newly
isolated Canadian haptophyte strains (Sh 1, Sc 2, Dm 2) can be
compared with those known previously for other strains of
I sochrysidales (Fig. 8). E. huxleyi 55a represents a typical planktonic
mar ine producer and serves as a global marine SST cal ibrat ion (Prahl
and Wakeham, 1987). Cal ibrat ions of our three Canadian haptophyte
cul tures most closely resemble to that of I . galbana CCMP715 (Theroux
et al ., 2013) rather than R. lamel losa (Nakamura et al ., 2014) and T.
lutea (Nakamura et al ., 2016). Whi le simi lar cal ibrat ions were observed
at the genus and species-level , var iat ions within the Isochrysis clade
become obvious with the compar ison of multiple strains enabled by
addit ion of our new cul tures. Consequent ly, future work on I . l i toral is
33
and I . nuda is cr i t ical to reinforce the potential species-specific
character ist ics of strains in the Isochrysis clade (i .e. higher
temperature sensi t ivi ty of Sh 1 strain and product ion of chain lengths
>C40
).
Appl icat ion of Sh 1 and Sc 2 calibrat ions to the index
inferred from the sediments of lakes Snakehole and Success gives
modern temperature values of 11.2 °C and 10.3 °C, respect ively (Fig. 8).
This reconstructed temperature corresponds to a water column
temperature above the thermocl ine in Lake Success in autumn
(Supplementary Fig. S2). However, al l values of strain Dm 2 (> –
0.35) were higher than that of environmental samples obtained from
Lake Deadmoose (–0.41 as shown by a horizontal l ine), which suggests
that those values are within range of expected environmental
temperatures (Fig. 8). To understand these resul ts, we speculate that
other strains which possess a high abi l i ty to produce C37:4
LCAs may
have contr ibuted to the C37:4
alkenone pool in sediments. Al ternately, we
suggest that al though the slope of the cal ibrat ion is defined by
temperature dependency, other lake propert ies, such as nutr ients,
sal ini ty, etc., may regulate the exact y-intercept. These findings suggest
that an in si tu cal ibrat ion (e.g., Toney et al ., 2010; 2012) may st i l l be
the best approach to capture natural var iat ions in LCA distr ibu t ions
and temperature sensi t ivi ty among alkenone producers.
34
5. Conclusions
Seven algal strains were successful ly isolated from three sal t
lakes in Saskatchewan, Canada, and used to establ ish unialgal
cul tures. Those strains were classi fied into the genus Isochrysis clade
of haptophytes according to SSU and LSU rRNA sequence analysis.
One of strains, Sh 1 from Lake Snakehole, was closely related to both I .
l i toral is and I . nuda according to i ts phylogenet ic posi t ion and
morphological character ist ics (i .e., mucoid polysacchar ide sheath). The
Sh 1 strain exhibi ted a unique alkenone composit ion with presence of
C41
and C42
LCAs. This is the fi rst finding of C41
and C42
LCAs in l iving
haptophytes and the ident i ficat ion of alkenone producer from
Canadian saline lakes.
The alkenone and alkenoate distr ibut ions that character ize the
newly isolated haptophyte strains are in good agreement with their
taxonomic posi t ion as Group I I haptophytes and relat ive other known
species. For example, the product ion of tetra-unsaturated alkenone and
alkenoates are common features of the family I sochrysidaceae (except
for T. lutea), whi le Sh 1 strain (closely related to both I . l i toral is and I .
nuda) has a unique alkenone/alkenoate distr ibut ion (i .e. >C40
LCAs,
C36:3
FAEE composit ions) compared to the Sc 2 and Dm 2 strains, which
are closely related to I . galbana. Compar ison of the -growth
temperature cal ibrat ions among var ious species also showed that Sh 1,
Sc 2 and Dm 2 strains isolated from Lakes Snakehole, Success and
35
Deadmoose were simi lar to those from I . galbana and R. lamel losa of
the Isochrysis clade. Overal l , i t is hoped that the findings of this study
wi l l promote fur ther study on the reconstruct ion of paleotemperature
using the alkenone paleothermometer in inland lakes, a task which is
substant ial ly less developed in compar ison to mar ine alkenone-
paleothermometer studies. Future studies should also include a more
comprehensive lake sampl ing program, to bet ter character ize
var iabi l i ty in the t iming and spat ial extent of alkenone-producing
haptophyte populat ions.
Acknowledgment s
This study was par t ly supported by a fund of Core Research for
Evolut ional Science and Technology in Japan Science and Technology
Agency (CREST/JST), Japan, to YS (FY2010–2016) and ERC funding
for the ALKENoNE project [Project ID 63776] to JT (FY2015–2020). We
thank Ms. M. Atsumi for her technical contr ibut ion to the isolat ion and
establ ishment of newly isolated haptophyte algal strains. We also
thank Ms. K. Makino for her technical contr ibut ion to extract DNA
from algal isolates and environmental samples from these Canadian
lakes. Field work and prel iminary sample preparat ion was funded by
an NSERC Canada and Canada Research Chair award to PRL. We are
grateful for the construct ive comments of two anonymous reviewers,
and also for valuable suggest ions that helped to improve this
36
manuscr ipt by Editor-in-Chief Dr. John Volkman and Associate Editor
Dr. Phi l ip Meyers.
Associate Editor–Phi l ip M eyer s
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47
48
Table capt ions:
Table 1. L ist of Canadian lakes where the isolat ions of microalgae and
LSU rRNA were per formed from lake water and sediments with
corresponding environmental data for the lakes.
Footnote: aY and N indicate yes (posi t ive) and no (negat ive),
respect ively.
bThe results of alkenone analysis are also l isted according to
l i terature (Toney et al . 2010).
Table 2. L ist of seven algal strains establ ished as unialgal cul tures (A)
and six environmental samples of which haptophyte marker genes
were successful ly ampl i fied (B) accompanied with the names of
lakes, mater ials used for isolat ion, sal ini ty, cul ture medium used for
algal isolat ion, and the GenBank accession numbers of SSU and
LSU rRNAs registered in GenBank obtained by ampl ificat ion.
Footnote: aSal ini ty of MA-ESM medium used for establ ishing
unialgal strains and cul t ivat ion of algal mixture from environmental
samples, respect ively.
Table 3. Correlat ion equat ions between the LCAs and alkenoate
unsaturat ion indices and the growth temperature.
49
Footnote: aThese data were obtained from laboratory cul tured
alkenone-producing haptophyte strains Sh 1, Sc 2 and Dm 2 which
had been isolated from three Canadian sal t lakes of Snakehole,
Success and Deadmoose, respect ively.
bThe correlat ion equat ions were obtained by calculat ion of the l inear
and second-order polynomial regressions with correlat ion coefficient
(R2). For graphs, see Figs. 6 and 7 on the alkenone and alkenoate
unsaturat ion indices including related parameters, respect ively.
Figur e legends:
Fig. 1. L ight micrographs of seven algal strains establ ished as unialgal
cul tures after the isolat ion of algae from three Canadian sal ine
lakes. A and B, two strains isolated from Lake Snakehole (Sh),
named as Sh 1 and Sh 2; C and D, two strains isolated from Lake
Success (Sc), named as Sc 1 and Sc 2; E–G, three strains isolated
from Lake Deadmoose (Dm), named as Dm 1, Dm 2 and Dm 3. Scale
bar in each photo is 50 μm.
Fig. 2. Maximum l ikel ihood t ree of the three Canadian lakes
haptophyte strains constructed by the analysis of SSU rRNA gene
sequences. Six samples analyzed are prepared from strains Sh 1, Sh
2, Sc 1, Sc 2 and Dm 2, Dm 3 which were isolated from Lake
50
Snakehole, Success and Deadmoose, respect ively. The values of
bootstrap percentage (BP) and Bayesian poster ior probabi l i ty (BPP)
are expressed as BP/BPP on each note by select ing values with only
BP >P50% and BPP>P0.5.
Fig. 3. Maximum l ikel ihood t ree of the three Canadian lakes
haptophyte strains constructed by the analysis of D1–D2 region of
LSU rRNA gene sequences. For the values of BP/, see Fig. 2.
Symbols: ●, samples prepared from the seven isolated haptophyte
strains of Sh 1 and Sh 2, Sc 1 and Sc 2, Dm 1, Dm 2 and Dm 3
isolated from Lakes Snakehole (Sh), Success (Sc) and Deadmoose
(Dm), respect ively; ▲, six environmental samples expressed as Sh
E1 and Sh E2, Sc E1, Sc E2 and Sc E3, and Ws E1 extracted from
the algal mixture cul t ivated from environmental samples of three
Canadian high sal t lakes of Snakehole (Sh), Success (Sc) and
Waldsea (Ws), respect ively.
Fig. 4. Growth curves of three haptophyte strains establ ished as
unialgal cul tures of Sh 1 (isolated from Lake Snakehole), Sc 2
(isolated from Lake Success) and Dm 2 (isolated from Lake
Deadmoose) grown under di fferent temperatures. Compar ison of
growth curves among var ious temperatures at 5 (×), 10 (◆), 15 (■),
51
20 (▲) and 25 °C (●) in the strains Sh 1 (A), Sc 2 (B) and Dm 2 (C).
Compar ison of growth curves among the strains of Sh 1 (○), Sc 2 (△
) and Dm 2 (□) grown at 5 (D), 10 (E), 15 (F), 20 (G) and 25 °C (H).
The y-axes of graphs are logar i thmic scales. Error bars denote the
standard deviat ion of t r ipl icate cul tures.
Fig. 5. Par t ial GC-FID chromatograms of LCAs and alkenoates
extracted from the lake sediments (rows A and B) and the isolated
haptophyte strains of Sh 1, Sc 2 and Dm 2 (rows C and D) from the
three Canadian saline lakes of Lake Snakehole, Success and
Deadmoose, respect ively. Samples B and D were prepared by
removing alkyl alkenoates after saponificat ion of alkenone fract ions
A and C, respect ively. Peak assignments: 1. C36:4
FAME, 2. C36:3
FAME,
3: C36:2
FAME, 4. C36:4
FAEE, 5. C36:3a
FAEE, 6. C36:3b
FAEE, 7. C36:2
FAEE,
8. C37:4
Me, 9. C37:3
Me, 10. C37:2
Me, 11. C38:4
Et, 12. C38:3a
Et, 13. C38:3b
Et, 14.
C38:2
Et, 15. C38:3
Me, 16. C38:2
Me, 17. C39:3
Et, 18. C39:2
Et, 19. C39:3
Me, 20.
C39:2
Me, 21. C40:3
Et, 22. C40:2
Et, 23. C41:3
Me, 24. C41:2
Me, 25. C42:3
Et, 26.
C42:2
Et. * : unident i fied peaks.
Fig. 6. Relat ionship between growth temperature and the alkenone
unsaturat ion indices in the three haptophyte strains isolated from
Canadian saline lakes, namely Sh 1, Sc 2 and Dm 2 isolated from
52
lakes Snakehole, Success, and Deadmoose, respect ively. Correlat ion
equat ions (y) are shown in each figure with correlat ion coefficients
(R2). The defini t ions of var ious alkenone and alkenoate unsaturat ion
indices are shown in the panel.
Fig. 7. Relat ionship between growth temperature and the alkenoate
unsaturat ion indices and
, the rat io of isomer ic alkenoates
(RIA38
) and the rat io of methyl to ethyl alkenoates (A37
/A38
) in the
three establ ished haptophyte strains isolated from Canadian sal ine
lakes, namely Sh 1, Sc 2 and Dm 2. Correlat ion equat ions (y) are
shown in each figure with correlat ion coefficients (R2). For the
defini t ions of the unsaturat ion indices and the other parameters, see
the inset (in the r ight bot tom) of Fig. 6.
Fig. 8. Compar ison of the cul ture-based -temperature cal ibrat ions
among the newly obtained strains Sh 1, Sc 2 and Dm 2 (classified
into Isochrysidaceae, Haptophyta) and those of the other species
reported in l i teratures. For reference, -values in environmental
samples extracted from sediments of Lakes Snakehole, Success and
Deadmoose are indicated by colored hor izontal l ines at the values of
–0.38, –0.41 and –0.30, respect ively. Symbols: Sh 1 (▲ wi th a red
l ine), strain Sh 1 isolated from Lake Snakehole; Sc 2 (□ wi th a
53
dashed line), strain Sc 2 isolated from Lake Success; Dm 2 (○ wi th a
dot ted l ine), strain Dm 2 isolated Lake Deadmoose; a, E. huxleyi 55a
(Noëlaerhabdaceae), representat ive of typical planktonic mar ine
species (Prahl and Wakeham, 1987); b, T. lutea CCMP 463
(Nakamura et al ., 2016); c, T. lutea NIES-2590 (Nakamura et al .
2016); d, I . galbana CCMP 715 (Nakamura et al ., 2014). e, R.
lamel losa CCMP 1307 (Nakamura et al ., 2014); L ake Geor ge, data
from in si tu cal ibrat ion of Lake George (Toney et al ., 2012).
54
55
56
57
58
59
Lake Name Isolationa
LSU rRNA amplificationa
Salinity (g L-1
) pH
Alkenones (μg g-1
sed) Alkenoates (μg g-1
sed) % Alkenones % Alkenoates %C37:4 UK
37 UK'
37 UK
38 C37/C38
Antelope N N 7.87 9.40 66.39 6.46 91.13 8.87 45.67 -0.66 0.17 -0.09 5.57
Snakehole Y Y 50.21 8.96 8.06 0.23 97.25 2.75 27.73 -0.38 0.13 0.07 1.97
Success Y Y 34.97 8.62 5.59 0.21 96.42 3.58 19.94 -0.30 0.28 -0.06 1.09
Fishing N N 1.31 9.17 4.90 0.90 84.49 15.51 27.10 -0.60 0.23 -0.42 1.32
Humboldt N N 0.84 9.13 2.34 0.59 79.77 20.23 41.64 -0.53 0.06 -0.14 7.05
Waldsea N Y 12.82 8.45 180.87 14.44 92.61 7.39 38.26 -0.68 0.17 -0.18 1.35
Deadmoose Y N 12.27 8.87 31.60 7.61 80.58 19.42 33.00 -0.41 0.27 -0.18 1.73
Charron N N 4.9 10.55 150.05 6.83 95.65 4.35 46.64 -0.58 0.19 0.04 3.53
Rabbit N N 4.23 9.21 8.95 0.98 90.17 9.83 50.28 -0.64 0.19 -0.01 4.59
Redberry N N 8.61 9.09 357.70 9.00 97.54 2.46 49.18 -0.67 0.18 -0.26 3.20
Table 1.
Alkenone parameters in the sedimentsb
Table 1
SSU rRNA LSU rRNA
Sh 1 Snakehole Shore sand 40 MA-ESM LC322312 LC322318
Sh 2 Snakehole Shore sand 40 MA-ESM LC322313 LC322319
Sc 1 Success Sediment 30 MA-ESM LC322314 LC322320
Sc 2 Success Sediment 30 MA-ESM LC322315 LC322321
Dm 1 Deadmoose Sediment 40 MA-ESM - LC322322
Dm 2 Deadmoose Sediment 40 MA-ESM LC322316 LC322323
Dm 3 Deadmoose Sediment 40 MA-ESM LC322317 LC322324
SSU rRNA LSU rRNA
Sh E1 Snakehole Surface water 40 MA-ESM - LC322325
Sh E2 Snakehole Shore sand 40 MA-ESM - LC322326
Sc E1 Success Sediment 40 MA-ESM - LC322327
Sc E2 Success Sediment 40 MA-ESM - LC322328
Sc E3 Success Sediment 40 MA-ESM - LC322329
Ws E1 Waldsea PlanktonNet 10 MA-ESM - LC322330
Table 2.
Salinity (ppt)/ Mediuma
B Sample name of
amplified DNA
Sample lake Salinity / MediumaSample used for
amplification
A Isolated
Strain Name
Isolated lake Sample used
for isolation
GenBank accession number
GenBank accession number
Table 2
Sh 1 R2
Sc 2 R2
Dm 2 R2
Alkenone indices
UK
37 10–25; polynom. -0.001T2 + 0.085T - 1.21 1.00 0.001T
2 + 0.0046T - 0.43 1.00 0.0006T
2 + 0.0086T - 0.48 0.97
10–25; linear 0.049T - 0.93 0.99 0.039T - 0.70 0.99 0.028T - 0.63 0.96
UK'
37 10–25; polynom. 0.0009T2 - 0.016T + 0.17 0.99 0.0017T
2 - 0.043T + 0.35 0.99 0.0004T
2 - 0.0068T + 0.11 0.93
10–25; linear 0.014T - 0.073 0.92 0.017T - 0.11 0.82 0.0066T - 0.0005 0.87
UK''
37 10–25; polynom. -0.0017T2 + 0.094T - 1.30 1.00 -0.0007T
2 + 0.046T - 0.76 1.00 0.0002T
2 + 0.0145T - 0.57 0.96
10–25; linear 0.033T - 0.82 0.95 0.022T - 0.57 0.98 0.021T - 0.61 0.96
UK
38Et 10–25; polynom. -0.001T2 + 0.082T - 0.86 1.00 0.0021T
2 - 0.022T - 0.21 1.00 0.0021T
2 - 0.038T + 0.06 0.94
10–25; linear 0.048T - 0.59 0.99 0.051T - 0.78 0.96 0.0342T - 0.50 0.87
UK
41Me 10–25; polynom. 0.0011T2 - 0.026T + 0.25 0.91
10–25; linear 0.012T - 0.041 0.79
UK
42Et 10–25; polynom. 0.0002T2 + 0.014T - 0.047 0.98
10–25; linear 0.023T - 0.11 0.98
Alkenoate indices
UA
37 10–25; polynom. -0.0029T2 + 0.15T - 1.85 0.99 -0.0003T
2 + 0.091T - 1.33 1.00 0.0006T
2 + 0.053T - 1.23 0.99
10–25; linear 0.053T - 1.07 0.93 0.081T - 1.25 1.00 0.075T - 1.41 0.99
UA
38 10–25; polynom. -0.0024T2 + 0.14T - 1.32 1.00 -0.0012T
2 + 0.099T - 0.85 1.00 0.0006T
2 + 0.030T - 0.36 0.98
10–25; linear 0.053T - 0.67 0.96 0.057T - 0.52 0.99 0.052T - 0.53 0.98
RIA38 10–25; polynom. 0.0013T2 - 0.064T + 0.90 0.98 -0.0002T
2 - 0.0026T + 0.83 0.82 -0.0003T
2 + 0.0004T + 0.83 0.92
10–25; linear -0.019T + 0.54 0.90 -0.0096T + 0.88 0.81 -0.011T + 0.92 0.91
A37/A38 10–25; polynom. 4.3×10-5
T2 + 0.0081T + 0.22 0.98 0.0006T
2 - 0.036T + 0.75 0.99 -0.0003T
2 - 0.0054T + 0.52 0.98
10–25; linear -0.0066T + 0.21 0.98 -0.016T + 0.59 0.97 -0.017T + 0.61 0.97
Unsaturation
indicesa
Temperature range
and type of fit
Regressions vs. temperature (T )b
Table 3.
Table 3
60
Highlights
Novel LCA-producing strains from Canadian lakes established as unialgal
cultures.
Seven strains were categorized in the Isochrysis clade using genomic analysis.
One strain produces C41 and C42 alkenones that are sensitive to temperature
changes.
New alkenoate-based indices from isolated strains for temperature reconstructions.
Photo by: Rick Bohn - CGREC
Max Depth = 60m
Graphical Abstract
