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\begin{document}

\title{Group 2i Isochrysidales thrive in marine and lacustrine systems
with ice cover}



\author[1]{Karen Wang}%
\author[1]{Yongsong Huang}%
\author[1]{Tyler Kartzinel}%
\author[1]{Markus Majaneva}%
\author[1]{Nora Richter}%
\author[1]{Sian Liao}%
\author[1]{Camilla S Andresen}%
\author[1]{Flor Vermassen}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 18, 2024}


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Karen J Wang\textsuperscript{1,2,*}, Yongsong
Huang\textsuperscript{1,2,*}, Tyler Kartzinel\textsuperscript{2,3},
Markus Majaneva\textsuperscript{4}, Nora Richter\textsuperscript{1,2,5},
Sian Liao\textsuperscript{2,6}, Camilla S Andresen\textsuperscript{7},
Flor Vermassen\textsuperscript{7}

\begin{enumerate}
\tightlist
\item
  Department of Earth, Environmental and Planetary Sciences, Brown
  University, Providence, RI, 02912, USA
\item
  Institute at Brown for Environment and Society, Brown University,
  Providence, RI, 02912, USA
\item
  Department of Ecology and Evolutionary Biology, Brown University,
  Providence, RI, 02912, USA
\item
  Norwegian Institute for Nature Research (NINA), NO-7485, Trondheim,
  Norway
\item
  Department of Marine Microbiology \& Biogeochemistry, NIOZ Royal
  Netherlands Institute for Sea Research, 1790 AB Den Burg, the
  Netherlands
\item
  Department of Chemistry, Brown University, Providence, RI, 02912, USA
\item
  Department of Glaciology and Climate,~Geological Survey of Denmark and
  Greenland
\end{enumerate}

\par\null

\textbf{Abstract}

Global warming is causing rapid changes to the cryosphere. Predicting
the future trajectory of the cryosphere requires quantitative
reconstruction of its past variations. A recently identified
sea-ice-associated haptophyte, known as Group 2i Isochrysidales, has
given rise to a new quantitative sea-ice proxy with its characteristic
alkenone distributions. However, apart from the occurrence of Group 2i
Isochrysidales in regions with sea ice, and the empirical relationship
between C\textsubscript{37:4} alkenone abundance and sea-ice
concentration, little is known about the ecology of these haptophyte
species. Here, we systematically mapped the spatial and temporal
occurrence of known Group 2i Isochrysidales based on environmental DNA
and alkenones in both marine and lacustrine environments. Our results
indicate Group 2i is widely distributed in icy marine and lacustrine
environments in both Northern and Southern Hemisphere, but absent in
ice-free environments. Temporally, Group 2i is part of the sea-ice algae
bloom during the cold seasons, in contrast to other Isochrysidales that
bloom in open waters during warm seasons. Our results indicate that ice
is a prerequisite for the occurrence of the psychrophilic Group 2i
haptophytes in marine and lacustrine ecosystems and further affirms its
value for past ice reconstructions.

\textbf{Main}

The cryosphere, including sea ice, lake ice, and glaciers, is a crucial
element of the Earth's climate system. It regulates the Earth's heat
budget with its high albedo and energy transfer between the Earth's
surface and atmosphere. Pervasive changes in the cryosphere have already
occurred under a warming climate \textsuperscript{1}. Current Arctic sea
ice cover has decreased to its lowest level since at least 1850 CE, with
a continuous loss of \textasciitilde{} 12.3 \% per decade since 1978
when satellite observation began\textsuperscript{1}. Loss of lake ice
has also been observed over the recent decades, and the decrease in ice
duration and thickness is projected to intensify at an unprecedented
pace\textsuperscript{2}. As sea ice continues to melt, the Arctic Ocean
becomes increasingly accessible for resource exploration, shipping
routes, and military activities, all of which are rapidly and
drastically reshaping the global economical and geopolitical frameworks.
We urgently need to accurately and quantitatively project the courses of
Arctic sea ice change in the coming decades under various scenarios of
anthropogenic greenhouse gas emissions. Quantitative reconstruction of
past cryosphere changes beyond instrumental records, especially during
warmer periods in the geological past, is essential for calibrating
future projections using climate models.

A new quantitative sea-ice proxy based on characteristic alkenone
distributions produced by Group 2i Isochrysidales (an order of
haptophytes) was recently proposed by Wang et al. \textsuperscript{3}.
Phylogenetically based on 18S rRNA gene, Isochrysidales has been
classified into 3 groups, with Group 1 inhabiting freshwater and
oligohaline environments, Group 2 species in saline lakes and estuaries,
and Group 3 in open ocean settings\textsuperscript{4,5}. Group 2 can be
further separated into ice-associated Group 2i and warm-season blooming
Group 2w (e.g., \emph{Isochrysis galbana} , \emph{Ruttnera lamellosa}
)\textsuperscript{6,4,7,8,3,9}. Alkenones produced by Group 3
Isochrysidales have been widely used for paleo sea surface temperature
(SST) reconstructions since the 1980s \textsuperscript{10,11}. However,
alkenones from sea-ice-laden oceans often lead to abnormal SST
reconstructions and display high value of
\%C\textsubscript{37:4}(C\textsubscript{37:4}/( C\textsubscript{37:2}+
C\textsubscript{37:3}+ C\textsubscript{37:4})) compared to mid-to-low
latitude oceans, which was previously attributed to meltwater input and
decreased salinity\textsuperscript{12,13}. Recent culture experiments
show that \%C\textsubscript{37:4} of alkenones produced by Group 2i and
3 Isochrysidales is not affected by salinity\textsuperscript{14}. The
increased \%C\textsubscript{37:4} observed in high-latitude ocean is
rather an effect of input from Group 2i during increased sea-ice cover
which coincide with low surface salinity\textsuperscript{3}. Cells and
DNA sequences of Group 2i has been widely observed within sea
ice\textsuperscript{3}, and the correlation between occurrence of Group
2i and high \%C\textsubscript{37:4} has been used to reconstruct past
changes in sea ice in regions such as the Gulf of Alaska and the Fram
Strait\textsuperscript{15,16,3}. However, we have a limited
understanding of the ecology of Group 2i Isochrysidales in the natural
environments. For instance, it is unclear whether ice is a prerequisite
for the presence of Group 2i in the global ocean and lacustrine
environments. Further, there are currently no constraints on the growth
conditions (e.g., temperature or salinity) or seasonality of Group 2i
phytoplankton blooms in natural waters.

Here, we made the first global map of known occurrences of Group 2i and
its habitable temperature ranges based on environmental DNA data
sequenced from sediment samples collected in Baltic Sea, Chesapeake Bay,
and Greenland fjords and re-analyzed environmental DNA data from
published studies in global marine and lacustrine environments
(Supplementary Data). We also examined the seasonal distribution of
Group 2i during annual cycles to understand their role in bloom
successions. We propose potential growth strategies adopted by Group 2i
that enable its success in cold settings and discuss the implications
for paleoclimate reconstructions.

\textbf{Results}

\textbf{Spatial distribution of Group 2i Isochrysidales in marine and
lacustrine environments}

We compiled global environmental DNA data from ice, water, and sediment
samples in lacustrine and marine environments that targeted haptophytes,
and mapped the global distribution of Group 2 Isochrysidales detected in
different ocean environments (Fig.1). Based on previous study, Group 2i
is widely identified in seawater and ice samples in the Arctic Ocean,
where other Isochrysidales are rare \textsuperscript{3}. Here, we
investigated DNA recovered from seawater samples collected in the
Antarctic Region and report the first identification of Group 2i in the
Southern Ocean, from seawater samples collected during the austral
summer in the northern Antarctic Peninsula region
\textsuperscript{17}(Fig. S1). Group 2i DNA is found in water column
samples from ocean adjacent to the South Orkney Islands. The water
temperature at the time of sampling (January, 2018) range from -0.5 to 3
@\selectlanguage{english}C and salinity range from 33.1 to 34.4 psu. No other Group 2
Isochrysidales has been found in the Southern Ocean water samples
studies we examined (Supplementary Data).

We analyzed Isochrysidales DNA and alkenone profiles from sediment
samples in estuaries that encompass three temperature regimes: Greenland
fjords, the Baltic Sea, and Chesapeake Bay (Fig. S2). The Upernavik
Fjord and Kangerlussuaq Fjord are located on the west and east coast of
Greenland, respectively (Fig. S3). The modern annual SST of the two
sample sites ranges between -2 \textasciitilde{} 4@\selectlanguage{english}C (Fig. S2). Both
fjords are major outlets of the retreating Greenland Ice Sheet
\textsuperscript{18,19}. Isochrysidales 28S rRNA gene sequences were
recovered from downcore sediment samples from two core sites along
Upernavik Fjord. Phylogenetic analyses based on 28S rRNA gene sequences
show the Upernavik Fjord Isochrysidales sequences belong to Group 2i, as
it clustered with Group 2i sequences recovered from surface sediment
from Lake Fryxell\textsuperscript{3,5} (Fig. S4). Group 2i
Isochrysidales was also detected through the 18S rRNA gene from surface
sediment at the Kangerlussuaq Fjord. The DNA of other Isochrysidales is
not detected in Greenland fjords during this study. However, the
alkenone profile from Kangerlussuaq Fjord suggests \emph{E. huxleyi}
also exists at the site, and the alkenones are produced by both Group 2i
and \emph{E. huxleyi} , as \%C\textsubscript{37:4} (14\% in
Kangerlussuaq sample) is not as high as in typical Group 2i cultures
(\textasciitilde{}80\% for strain RCC5486 under 3@\selectlanguage{english}C
\textsuperscript{14}) and C\textsubscript{38Me}alkenones are relatively
high as well (Fig. S5).

The Baltic Sea is an intra-continental shelf sea characterized by strong
salinity and temperature gradients (Fig. 2, Fig. S2). The south of the
Baltic Sea is connected with the North Sea, where salinity is
\textasciitilde{}35 psu, in contrast to the northern end of Bothnian Bay
and the Gulf of Finland where salinity is \textasciitilde{}2 psu. During
winter, Bothnian Bay is covered by consolidated ice. Moving towards the
North Sea along the transect, winter ice cover transitions to more
fractured ice and open water. All three groups of Isochrysidales were
detected in the Baltic Sea region \textsuperscript{20--22}. Skagerrak is
dominated by \emph{E. huxleyi} with small alkenone contributions from
other Groups. From Skagerrak toward the Gulf of Bothnian estuary, the
alkenone-producing community changed progressively (Fig. S5). The inner
part of the Baltic Sea, where sea ice is more prominent, is dominated by
Group 2i, in addition to Group 1 Isochrysidales in low salinity
(\textless{} 6 psu) waters\textsuperscript{21,22}.

Chesapeake Bay's annual SST ranges from 4 to 27 @\selectlanguage{english}C, and the seawater
remains unfrozen during winter (Fig. S2). Isochrysidales 18S rRNA gene
sequences were recovered from seven surface sediment samples in the
lower part of Chesapeake Bay through next-generation sequencing
(NGS).\emph{Emiliania huxleyi} was detected in one sample near the mouth
of the bay. The dominant amplicon sequence variants (ASVs) in all
samples were \emph{Isochrysis galbana} (a Group 2w Isochrysidales),
which agrees with the microscopic identification conducted in a previous
study\textsuperscript{23}. The unidentified ASVs recovered all fall into
Group 2w and have 99.2-99.7\% similarity to \emph{Isochrysis
galbana}(Fig. S6). Importantly, no Group 2i was identified in these
samples despite the recovery of abundant Group 2w and Group 3
Isochrysidales reads. The alkenone profile from surface sediment is
similar to the\emph{Isochrysis galbana} culture with a small input
from\emph{Emiliania huxleyi} (Fig. S5).

Our results show that although Group 2i can occur across a wide salinity
range from the typical open ocean (\textasciitilde{} 35 psu) to
oligohaline waters, as shown in the Baltic Sea and off shore Antarctic
Peninsula, they are strongly associated with low temperatures. The
absence of Group 2i in Chesapeake Bay demonstrates that they are limited
to regions with seasonal or perennial ice cover.

Group 2i is also widespread in icy lacustrine environments (Fig. 1).
However, it is generally absent in freshwater lakes where Group 1
Isochrysidales flourish \textsuperscript{24,25,5,26}, regardless of the
temperature. A combination of Group 2i and 2w is commonly found in
surface sediments from saline lakes with seasonal ice cover, throughout
a range of minimal monthly temperatures from -23 @\selectlanguage{english}C to 3 @\selectlanguage{english}C. In modern
samples from lakes with perennial ice cover (e.g., Lakes Fryxell and
Bonney in the Antarctic Dry Valley), Group 2i was the only
Isochrysidales found \textsuperscript{5,27,28}. In contrast, only Group
2w was found in temperate saline lakes in Spain, France, Tanzania, and
Brazil, where lake water remains unfrozen throughout the
year\textsuperscript{29--31}.

\textbf{Phenology of Group 2i Isochrysidales}

Within the same location where annual temperature varies from around
freezing to over 10 @\selectlanguage{english}C, Group 2i only occurs in winter and spring when
the water temperature is below \textasciitilde{}12@\selectlanguage{english}C (Fig. 3). Group 2i
was detected in water samples with 1) temperatures below 0 @\selectlanguage{english}C, 2)
temperature above 0@\selectlanguage{english}C collected under-ice or samples collected shortly
after ice melt in spring; but is absent in samples collected during the
summer. Group 2i was not detected in any samples collected during an
extensive survey of haptophytes conducted in the central North Pacific
from 0@ \selectlanguage{english}to 60@ \selectlanguage{english}N along the transect of 170@\selectlanguage{english}W during the summer of 2014
where seawater temperatures ranged between
9.8-29.3@\selectlanguage{english}C\textsuperscript{32}.

We examined Isochrysidales DNA data from ice and seawater samples
collected across seasonal cycles from two regions in the Baltic Sea,
Gulf of Finland and Oslofjorden (Fig. 2). Isochrysidales sequences were
found in the ice and water column during the cold seasons (Oct.-May) in
the two monitored sites at the northwest of the Gulf of
Finland---Krogarviken and Storfja\selectlanguage{english}rden by Enberg et
al.\textsuperscript{21}. Both of the sites were covered by sea ice from
January to April, and the total algal biomass was higher in the ice than
in the water column during the ice-covered season and slowly increased
as ice thickness increased (Fig. 4; Enberg et al., 2018). Isochrysidales
communities in the ice, under ice water (collected at the ice-water
interface), and water bottom were composed of Group 2i and Group 1
Isochrysidales. Group 2i was the predominant haptophyte in the ice
samples based on relative read abundance, where the brine salinity
ranged widely from approximately 4-45 psu. It made up 40-85\% of the
total haptophyte read in Krogarviken and 58-86\% in Storfja\selectlanguage{english}rden, and
accounted for up to 0.2-2.0\%, and 0.3-2.2\% of the total Eukaryotic
reads in the Krogarviken and Storfja\selectlanguage{english}rden sea ice from January to April
(Fig. 4). In contrast, zero or only a few (\textless{}5 reads) Group 1
were detected in each ice sample, the majority of Group 1 was detected
in low salinity (\textasciitilde{}4 psu) water samples. Group 2i's
relative read abundance to total eukaryotes and absolute read count were
lower in the water column than in the ice from February through early
April, and the abundance in the water decreased sharply as the ice
started to melt in mid-April. The Group 2i population in the water
column decreased as water temperatures rose and was absent in most of
the water samples collected during July-November at both sites (Fig. 4).

Egge et al.\textsuperscript{20} collected water samples in outer
Oslofjorden (59.19@\selectlanguage{english}N, 10.69@\selectlanguage{english}E) at 1 m depth monthly and sequenced
haptophytes through NGS from September 2009 to June 2011. Group 1, 2i,
2w, and 3 were all detected at this site. \emph{Emiliania huxleyi} was
detected in all months except April and is one of the predominant
haptophyte species during the high-temperature months from summer to
early autumn (Fig. 5). Group 2w was detected in September 2009 and May
2011 when the surface temperature reached 16@\selectlanguage{english}C. Group 2i, in contrast,
was absent during the warm season and only detected from December
2010-April 2011 when the surface water temperature was around freezing
for a prolonged period and upper Oslofjorden was frozen extensively. The
relatively low sequence reads of Group 2i at this site suggest that
Group 2i might have been transported from the ice in Upper Oslofjorden.
Group 1 was detected in February and May 2010, and March-May 2011,
likely controlled by freshwater input and reduced surface salinity
during those periods. The sequences in Oslofjorden further demonstrate
the substantial difference between the key environmental controls on the
bloom phenology of the three groups.

\textbf{Discussions}

\emph{Growth strategies} : The Group 2i Isochrysidales global
distribution shows that the lineage is restricted to marine and
lacustrine habitats with seasonal or perennial ice cover. The time
series of water and ice samples from the Baltic Sea shows that Group 2i
is a part of the ice algae community and blooms before other
Isochrysidales in the early spring. Potential growth strategies that
contribute to its prosperity under cold climates in both hemispheres
include 1) the forming of resting stages; 2) mixotrophy; 3) a preference
for low growing temperatures and adaptability to a wide range of
salinities.

Forming of cysts was observed in Group 2 species, which allows the cells
to stay in sea ice or surface sediments for a long
time\textsuperscript{33}. In an enrichment experiment, Group 2i was
triggered out of its resting stage (during which the cells were not
active and alkenone production was paused) in the sediment by increasing
the light intensity from 100 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{2}s\textsuperscript{-1} to 200 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{2}s\textsuperscript{-134,35}. Group 2i likely forms
cysts in the sediment and sea ice during prolonged winters when light
availability is limited, and can be activated when light availability
increases. This was observed in the Canadian Arctic Archipelago, Baffin
Bay, and Greenland fjords, where the bathymetry of these regions
enhances vertical mixing from tidal energy which can re-suspend cysts
from the sediment and leads to activation of the
cysts\textsuperscript{3,36,37}.

Group 2i could potentially use mixotrophy to compensate for reduced
photosynthesis under light-limited conditions. Multiple Isochrysidales
species are capable of mixotrophy, including \emph{Emiliania huxleyi}
,\emph{Gephyrocapsa oceanica} , \emph{Isochrysis galbana} ,
and\emph{Tisochrysis lutea} \textsuperscript{38--41}. Increased biomass
production and nutrient conversion efficiency under mixotrophic
conditions has been reported in Group 2 species \emph{Isochrysis
galbana} and \emph{Tisochrysis lutea} \textsuperscript{39,40}.
Mixotrophy of Group 2i has been observed in the McMurdo Dry Valley Lake
located in Antarctica \textsuperscript{28}. Culture enrichment of
Isochrysidales isolated from this site could grow in either dark or
light conditions but achieved optimal growth under mixotrophic
conditions with organic carbon sources such as cereal grass, rice, and
yeast extract \textsuperscript{28}. Group 2i Isochrysidales takes up a
significant proportion (80\% based on DNA sequencing) of the mixotrophic
community in the McMurdo Dry Valley Lake and is an important primary
producer in this lake. Moreover, the gene copy abundance of Group 2i
measured through qPCR is not correlated with light availability in this
lake \textsuperscript{28,42}. The capability of mixotrophy could give
Group 2i an advantage during the early stages of the phytoplankton bloom
succession around ice melt where light availability was still low.
Mixotrophy could potentially compensate for photosynthesis for Group 2i
when photosynthetically active radiation was relatively
low\textsuperscript{27,28}.

Culture experiments also demonstrate Group 2i Isochrysidales' preference
for low growing temperatures and adaptability to a wide range of
salinities. Liao and Huang \textsuperscript{14} cultured the Group 2i
strain RCC5486 (isolated from Baffin Bay) at different temperatures from
0-12 @\selectlanguage{english}C at 31 ppt under light: dark cycle of 16:8 hrs and found that
RCC5486 growth rates were high at 0, 3, 6, 9 @\selectlanguage{english}C, but failed to grow at
10.5 and 12 @\selectlanguage{english}C. Liao et al. (2022) also tested different salinities (15,
21, 26, 31, and 38 ppt) under 3@\selectlanguage{english}C and did not observe a significant
difference in growth rate at different salinities. The preference of low
temperature and flexibility with salinity enable Group 2i to survive in
sea ice brine channels, melt ponds, and under-ice water, where the
temperature is low, and salinity fluctuates greatly when ice forms and
melts. The temperature preference explains why Group 2i is not detected
in sediments from Chesapeake Bay and temperate lakes and water columns
during summer, i.e. Lake George and the Baltic Sea\textsuperscript{35}.

\emph{Habitats, bloom successions and implications for paleoclimatology}
: The seasonal succession of Group 2i in the Gulf of Finland suggests
that it does not solely live in the meltwater, in the under-ice water,
or was merely ''trapped into'' the sea ice. Instead, Group 2i was
concentrated into the sea ice during ice formation via certain selective
mechanisms, and can thrive in the brine channels under fluctuating
salinity \textsuperscript{21}. In contrast to\emph{Emiliania huxleyi} ,
which blooms during warmer temperatures, Group 2i was absent in water
samples collected during the summer months in the Gulf of Finland and
Skagerrak. Group 2i is not a part of the summer phytoplankton bloom;
instead, they are part of the ice algae assemblage and potentially
seeded the under-ice bloom through brine drainage in the spring. Group
2i likely permeates all three phases of the ice algal bloom from
pre-bloom, bloom, to post-bloom, and is most prominent in the first two
phases before the ice starts to break up, as Group 2i was the most
abundant lineage among haptophyte in the sea ice during the entire ice
season in Gulf of Finland and active Group 2i cells were isolated from
an ice core in Baffin bay during pre-bloom and bloom
development\textsuperscript{21,37}. The exact timing of Group 2i growth
and its role in the bloom succession likely differs depending on
available sunlight, ice properties, temperature, and nutrient
conditions.

Similar Isochrysidales bloom successions exist in lacustrine
environments with seasonal ice cover. In a water column time series from
Lake George during the spring, two non-overlapping Isochrysidales blooms
were observed \textsuperscript{35}. The early bloom that consisted of
Group 2i peaked shortly after ice melt when the surface water was around
7 @\selectlanguage{english}C, and the population quickly declined as water temperature continued
to rise \textsuperscript{35}. In contrast, the later bloom consisted of
Group 2w, and their DNA read abundance peaked around 15 @\selectlanguage{english}C. The two
separate blooms are also evident in different alkenone profiles, which
are dominated by C\textsubscript{37:4} and
C\textsubscript{37:3,}respectively \textsuperscript{34}.

A holistic picture of Group 2i's ecology greatly extends alkenones as
paleoclimate proxies: 1) it reaffirms the underlying basis for using
\%C\textsubscript{37:4} as a sea-ice proxy in high latitude oceans for
both Northern Hemisphere and Southern Hemisphere; 2) In temperate
regions with seasonal ice where both 2i and 2w are present in saline
lakes, interpretation of \%C\textsubscript{37:4} is less straight
forward, as Group 2w is also capable of producing variable amounts of
C\textsubscript{37:4}. However, C\textsubscript{39:4}Me alkenone has
been found exclusively in 2i Isochrysidales and is not present is 2w
species \textsuperscript{9,14}. Thus, the relatively abundance of
C\textsubscript{39:4}Me alkenone (i.e., \%C\textsubscript{39:4} ) may be
better suited for reconstructing past lake ice coverages. 3) In settings
where only Group 2i or 2w is present, alkenones may allow reconstruction
of cold or warm season temperatures, respectively. For example, in warm
settings with no seasonal ice cover that only have Group 2w, such as
Spanish lakes and Chesapeake Bay. In such settings, alkenone
unsaturation ratios should primarily reflect warm season temperatures.

\textbf{Methods and materials}

\textbf{Surface sediments from the Chesapeake Bay and Greenland Fjords}

Sediment samples from the Baltic Sea, Chesapeake Bay, USA, and two
Greenland Fjords were collected for DNA and alkenone analyses (Table
S1). Surface sediment samples were collected across the Baltic Sea from
Skagerrak to the Bothnian Sea during expeditions M86/1 (R/V Meteor;
November 2011), P435 (R/V Poseidon; June 2012), 6EZ1215 (R/V Elisabeth
Mann Borgese; July 2012), EMB046 (R/V Elisabeth Mann Borgese; May 2013)
through multi- corer \textsuperscript{43}. Surface sediment samples were
collected in the Chesapeake Bay from July-September 2017 and kept frozen
prior to DNA extraction. Two sediment cores (POR13\_05 and POR13\_08)
from Upernavik Fjord were collected in August 2013 onboard R/V Porsild
with Rumohr corer \textsuperscript{18}. One surface sediment sample from
Kangerlussuaq Fjord was collected during JR106 (James Clark Ross; 2004).

\textbf{DNA sequencing}

Surface sediment samples from the Baltic Sea are sequenced through
Sanger with haptophyte-specific primers targeting 18S rRNA as described
in \textsuperscript{22}. In short, DNA was extracted and purified from
0.5 g of dried sediments. One round of PCR was performed with primer
pair Prym-429F and Prym-887R \textsuperscript{44}. The PCR products were
run on gel electrophoresis, and gel bands with targeted length were
purified and cloned. The clone products were sequenced on an ABI 3730XL
capillary sequencer (Applied Biosystems, Foster City, CA). Operational
Taxonomic Units (OTUs) were clustered by UCLUST. Representative
sequences of each OTU were deposited in GenBank under accession numbers
MH472563-MH472566.

The surface sediment samples from the Chesapeake Bay and Kangerlussuaq
Fjord were sequenced through Next Generation Sequencing (NGS) with
haptophyte-specific primers targeting 18S rRNA following methods
described in Wang et al. (2021), and stored under accession number
PRJNA835469 in GenBank. In short, DNA was extracted from the samples,
followed by a two-step PCR. The first round of PCR used an adapted
version of the primer pair 528Flong and PRYM01+7 (Egge et al., 2013)
followed by a second round of PCR which gives each sample a unique index
sequence. The cleaned and normalized amplicons were then sequenced on an
Illumina Miseq with V2 600-Cycle Kit (San Diego, CA). Sequences were
verified using FastQC v0.11.8 and clustered into Amplicon Sequence
Variants (ASVs). Taxonomy of ASVs were assigned using the SILVA
reference database.

Water and ice sample collection and sequencing in the Gulf of Finland is
described in Enberg et al\textsuperscript{21}. Relative read abundance
(RRA) to total haptophyte of Eukaryotes for the \emph{i} th OTU is
defined as:\(\text{RRA}_{i}=\frac{1}{S}\sum_{k=1}^{S}{\frac{n_{i,k}}{\sum_{i=1}^{T}n_{i,k}}\times 100\%}\), where S is the number of samples,
\emph{T} is the total number of haptophyte or Eukaryotes OTU,
\(n_{i,k}\) is the number of sequences of OTU \emph{i} in sample
\emph{k} \textsuperscript{46}. The presence/absence of Isochrysidales
sequences was defined as if any ASV or OTU were identified as
Isochrysidales in the sample.

The downcore sediment samples from Upernavik Fjord were sequenced on an
Illumina MiSeq 500-cycle paired-end run with haptophyte-specific primers
(HAP\_LSU\_F and LHapto20R\_bis) targeting D1-D2 region of 28S rRNA
following methods described in Richter et al. (2019). Isochrysidales
sequences were selected through BLAST and aligned to the 28S rRNA
reference sequences in Richter et al. (2019) by PyNast.
Maximum-likelihood phylogenetic trees for 18S and 28S rRNA were built by
RAxML under GTRCAT approximation with 1000 bootstrap on CIPRES to
determine the sub-groups of the Isochrysidales
sequences\textsuperscript{47,48}.

We also reanalyzed environmental sequences recovered in seawater samples
from Southern Ocean near Antarctica Peninsula (Supplementary Data),
following methods described in Wang et al.\textsuperscript{3}. Sequences
classified as Isochrysidales are included in phylogeny tree shown in
Fig. S1.

\textbf{Alkenones analyses}

Freeze-dried sediment samples were extracted by an accelerated solvent
extraction system with DCM: methanol mixture (9:1, v/v). The total lipid
extraction was further separated into three fractions of increasing
polarity by silica gel columns eluting with hexane, DCM, and methanol
subsequently. The mid-polar fractions containing alkenones were analyzed
on a gas chromatography--flame ionization detection Agilent 7890N Series
instrument with mid-polarity column RTX-200 (105 m \selectlanguage{ngerman}× 250 \selectlanguage{greek}μ\selectlanguage{english}m \selectlanguage{ngerman}× 0.25 \selectlanguage{greek}μ\selectlanguage{english}m)
with the oven program described in Zheng et al. (2017).

\textbf{Acknowledgement}

This work is supported by NSF grants 1762431, 2234875 and IBES seed
grant to Y.H. The authors thank the R/V Porsild team, the VILLUM FONDEN
project, and BOSCORF for providing sediment samples~from Greenland. We
thank~Anthony Rodi,~Cindy Johnson, and Daniel M.~Dauer for sample
collection in the Chesapeake Bay.

\textbf{Author contributions}

Y.H. initiated the research idea. K.J.W. and Y.H. discussed with T.R.K.
and codesigned the study. C.S.A. and F.V. collected and processed
sediment samples.~ K.J.W., M.M., and N.R. analyzed the DNA and the
phylogenetic data. S.L. and K.J.W. conducted the alkenone analyses.
K.J.W. and Y.H. cowrote the manuscript with contributions from
coauthors.

\textbf{Data availability}

Representative sequences from each OTU recovered from Baltic Sea
sediment using Sanger sequencing are stored in NCBI GenBank under
accession number MH472563-MH472566. Raw sequencing data from sediment
samples in Chesapeake Bay and Kangerlussuaq Fjord are stored in Sequence
Read Archive (SRA) database of NCBI under accession number PRJNA835469.
List of published datasets re-investigated in this study are included in
supplementary materials. Processed data outcomes are available upon
request.

\textbf{Competing interests}

The authors declare no competing interests.

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\textbf{Main Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} \textbf{Global distribution of known Group 2w and 2i
Isochrysidales based on environmental DNA.} a) The contour shows maximum
monthly lake ice thickness in each region averaged over 2005-2015,
modeled in ERA5-Land \textsuperscript{1}. b) Zoomed in map of Chesapeake
Bay surface sediment samples examined in this study.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Sample sites in the Baltic Sea.} The contour shows
averaged cold season (Feb-Apr) sea ice concentration during 2005-2015
calculated from ERA5 monthly averaged data\textsuperscript{2}. The
filled circles indicate sediment samples, and the numbers show
Isochrysidales groups detected by environmental DNA and biomarkers from
sediment samples, the asterisks show predominant alkenone-producers. The
stars indicate locations where timeseries samples were collected. Note
that the Group 2 Isochrysidales found in the Baltic Sea samples were
exclusively 2i. In contrast, the Group 2 Isochrysidales found in the
Chesapeake Bay samples were exclusively 2w species (Fig.1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} \textbf{Temperature range of three groups of
Isochrysidales.} Scatter plot of temperature of ice, water, and surface
sediment samples where Isochrysidales was detected in environmental DNA.
Blue markers represent detection of Group 2i, red and yellow markers
represent absence of Group 2i where there were positive detections of
Group 2w and 3, respectively. Temperature of ice and water samples
are\emph{in situ} temperature measured during sample collection. Surface
sediment temperature are modern coldest month temperature calculated
from ERA5 monthly averaged data\textsuperscript{2} over 2005-2015.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} \textbf{Isochrysidales detected from ice and water
samples collected during Oct. 2012-Jul. 2013 at two sites from the Gulf
of Finland.} a-f) The bar plots show Group 1 and 2i Isochrysidales
relative read abundance to total Eukaryotes. The black lines
show\emph{in situ} total algal biomass measured during the sample
collection. g-h) Ice depth and under-ice water temperature measured at
sample collection. The gray dashed lines indicate the time window during
which ice existed. Next generation sequencing data from: Enberg et
al.\textsuperscript{3}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} \textbf{Haptophytes successions in Oslofjorden,
Norway during Sept. 2009-June 2011.} Barplot of Group 2i and \emph{E.
huxleyi} relative read abundance to total haptophytes from ice and water
samples collected, note the y-axis is in log-scale, and the bars
representing the two lineages are overlaid instead of stacked. The black
line represents \emph{in situ} water temperatures at 1m depth when
samples were collected. Next-generation sequencing data from: Egge et
al.\textsuperscript{4}

\textbf{Reference}

1. Mu\selectlanguage{ngerman}ñoz-Sabater, J. \emph{et al.} ERA5-Land: a state-of-the-art global
reanalysis dataset for land applications. \emph{Earth Syst. Sci.
Data}\textbf{13} , 4349--4383 (2021).

2. Hersbach, H. \emph{et al.} The ERA5 global reanalysis. \emph{Q.J.R.
Meteorol. Soc.} \textbf{146} , 1999--2049 (2020).

3. Enberg, S., Majaneva, M., Autio, R., Blomster, J. \& Rintala, J.
Phases of microalgal succession in sea ice and the water column in the
Baltic Sea from autumn to spring. \emph{Marine Ecology Progress
Series}\textbf{599} , 19--34 (2018).

4. Egge, E. S., Eikrem, W. \& Edvardsen, B. Deep-branching Novel
Lineages and High Diversity of Haptophytes in the Skagerrak (Norway)
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\textbf{Supplementary Material}

Karen J Wang\textsuperscript{1,2}, Yongsong Huang\textsuperscript{1,2},
Tyler Kartzinel\textsuperscript{2,3}, Markus
Majaneva\textsuperscript{4}, Nora Richter\textsuperscript{1,2,5}, Sian
Liao\textsuperscript{2,6},~Camilla S Andresen\textsuperscript{7},~Flor
Vermassen\textsuperscript{7}

\begin{enumerate}
\tightlist
\item
  Department of Earth, Environmental and Planetary Sciences, Brown
  University, Providence, RI, 02912, USA
\item
  Institute at Brown for Environment and Society, Brown University,
  Providence, RI, 02912, USA
\item
  Department of Ecology and Evolutionary Biology, Brown University,
  Providence, RI, 02912, USA
\item
  Norwegian Institute for Nature Research (NINA), NO-7485, Trondheim,
  Norway
\item
  Department of Marine Microbiology \& Biogeochemistry, NIOZ Royal
  Netherlands Institute for Sea Research, 1790 AB Den Burg, the
  Netherlands
\item
  Department of Chemistry, Brown University, Providence, RI, 02912, USA
\item
  Department of Glaciology and Climate,~Geological Survey of Denmark and
  Greenland
\end{enumerate}

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure S1.} Phylogenetic tree of Isochrysidales 18S rRNA
detected in the northern Antarctic Peninsula region\textsuperscript{1}.
Bootstrap values that are \textgreater{}50\% are shown. Liu et
al.\textsuperscript{1} collected surface water samples and water column
samples in the northern Antarctic Peninsula region. Only three samples
collected from water column at depth of 150m, 200m, 340m at 60.85@\selectlanguage{english}S,
45.64@\selectlanguage{english}W recovered Isochrysidales DNA, all three ASVs falls into Group
2i.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure S2.} Monthly SST of sediment sample sites in Chesapeake
Bay, Baltic Sea, and Greenland fjords. The climate data is from
ERA5\textsuperscript{2} monthly averaged data averaged over 2005-2015.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure S3.} Maps of Group 2i detected in Greenland fjords
surface and downcore sediment samples (Table 1). a) locations of
Upernavik Fjord and Kangerlussuaq Fjord with bathymetry adapted from
IBCAO Version 3\textsuperscript{3}. b, c) Maps of Upernavik Fjord and
Kangerlussuaq Fjord (Image U.S. Geological Survey, Image IBCAO, Google
Earth).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Figure S4.} Phylogenetic tree of Isochrysidales 28S rRNA.
Bootstrap values that are \textgreater{}50\% are shown. Sequences found
from Upernavik Fjord are clustered with Group 2i detected from Fryxell
Lake.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

\textbf{Figure S5.} Representative GC-FID chromatograms of alkenones in
surface sediment from Kangerlussuaq Fjord (GC13), Chesapeake Bay (RB12),
central Baltic Sea (Landsort Deep), western Baltic Sea (Skagerrak)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

\textbf{Figure S6.} Phylogenetic tree of Isochrysidales 18S rRNA
detected in Chesapeake Bay. View of the same phylogenetic tree with
expanded Group 2i is shown in Fig. S1. Bootstrap values that are
\textgreater{}50\% are shown. Sequences from the Chesapeake Bay are
clustered with cultured \emph{Emiliania huxleyi} and \emph{Isochrysis
galbana} .\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{Sample ID} & \textbf{Region} & \textbf{Lat.} & \textbf{Lon.} &
\textbf{Depth} & \textbf{Sequencing method} & \textbf{Group 1} &
\textbf{Group 2w} & \textbf{Group 2i} & \textbf{Group 3}\tabularnewline
\midrule
\endhead
1CB & Chesapeake Bay, USA & 36.985 & -76.166 & Surface & 528Flong and
PRYM01+7; NGS & N & Y & N & Y\tabularnewline
RR14 & & 37.774 & -76.619 & Surface & & N & Y & N & N\tabularnewline
RB3 & & 37.011 & -76.252 & Surface & & N & Y & N & N\tabularnewline
RB9 & & 37.425 & -76.109 & Surface & & N & Y & N & N\tabularnewline
RB12 & & 37.549 & -76.205 & Surface & & N & Y & N & N\tabularnewline
RB16 & & 37.653 & -76.209 & Surface & & N & Y & N & N\tabularnewline
RB22 & & 37.783 & -76.203 & Surface & & N & Y & N & N\tabularnewline
Skagerrak & Baltic Sea & 58.429 & 9.477 & Surface & Prym-429F and
Prym-887R; Sanger & N & N & Y & N\tabularnewline
Arkona Basin & & 54.852 & 13.433 & Surface & & Y & N & Y &
N\tabularnewline
Eastern Gotland Basin & & 57.083 & 19.985 & Surface & & Y & N & Y &
N\tabularnewline
Landsort Deep 1 & & 57.267 & 19.796 & Surface & & Y & N & Y &
N\tabularnewline
Landsort Deep 2 & & 58.641 & 18.265 & Surface & & Y & N & Y &
N\tabularnewline
Bothnian Sea 1 & & 61.085 & 19.579 & Surface & & N & N & Y &
N\tabularnewline
Bothnian Sea 2 & & 62.845 & 18.889 & Surface & & Y & N & Y &
N\tabularnewline
Bothnian Bay 1 & & 63.833 & 21.583 & Surface & & Y & N & Y &
N\tabularnewline
Bothnian Bay 2 & & 64.204 & 22.029 & Surface & & Y & N & Y &
N\tabularnewline
JR106 GC13 & Kangerlussuaq Fjord & 68.26 & -31.89 & Surface & 528Flong
and PRYM01+7; NGS & N & N & Y & N\tabularnewline
POR13\_05 5-6 cm & Upernavik Fjord & 72.945 & -55.62 & Core, 5-6 cm &
HAP LSU F, LHapto20R bis; NGS & N & N & Y & N\tabularnewline
POR13\_05 45-46 cm & & 72.945 & -55.62 & Core, 45-46 cm & & N & N & Y &
N\tabularnewline
POR13\_08 48-49 cm & & 72.869 & -54.558 & Core, 48-49 cm & & N & N & Y &
N\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 1.} Sediment samples examined in this study and the
presence/absence of Isochrysidales (Y=yes, N=no).

\textbf{Reference}

1. Liu, C., Zhang, X. \& Wang, X. DNA metabarcoding data reveals harmful
algal-bloom species undescribed previously at the northern Antarctic
Peninsula region. \emph{Polar Biol} \textbf{45} , 1495--1512 (2022).

2. Hersbach, H. \emph{et al.} The ERA5 global reanalysis. \emph{Q.J.R.
Meteorol. Soc.} \textbf{146} , 1999--2049 (2020).

3. Jakobsson, M. \emph{et al.} The International Bathymetric Chart of
the Arctic Ocean (IBCAO) Version 3.0: IBCAO VERSION
3.0.\emph{Geophysical Research Letters} \textbf{39} , n/a-n/a (2012).

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