Hoshino and Inagaki ﻿Progress in Earth and Planetary Science (2024) 11:19
https://doi.org/10.1186/s40645-024-00621-2

RESEARCH ARTICLE

Progress in Earth andPlanetary Science

Open AccessDistribution of eukaryotic environmental
DNA in global subseafloor sediments
Tatsuhiko Hoshino1,2* and Fumio Inagaki2,3

Abstract
The analysis of eukaryotic environmental DNA (eDNA) in sediment has the potential for understanding past ecosystems, even for taxa lacking skeletons or preserved only as a part of necromass. Despite the paleoenvironmental
and ecological importance of eukaryotic eDNA in marine sediment, the duration of remaining eDNA and the species
of eDNA present in marine sediment has not been well investigated. Here, we analyzed eDNA extracted from 299
sediment samples down to 678 m below the seafloor at 40 geologically distinct sites. The results showed that eukaryotic eDNA was amplified from more than 80% of the sediments with a depositional age of less than 100,000 years.
The eDNA was well conserved in anoxic sediments than in oxic sediments, with PCR success rates of 48% and 18%,
respectively. The eukaryotic communities include non-benthic organisms such as marine plankton, including diatoms,
dinoflagellates, and coccolithophores. A freshwater diatom genus Aulacoseira was detected in the Baltic Sea sediments from the last glacial lacustrine environment. These results provide new insights into the global-scale distribution of the past eukaryotic eDNA preserved in marine sediment.
Keywords Environmental DNA, Eukaryote, Marine sediment, Past environment
1 Introduction
Environmental DNA remaining in the environment,
including sediment, over time scales of hundreds or
thousands of years is referred to as “ancient eDNA
(aeDNA)” (Taberlet et al. 2018), “fossil DNA” or “paleomes” (Inagaki et al. 2005; Coolen et al. 2013). Paleoenvironmental studies utilizing those eDNA have been
mainly progressed for localized settings; however, thespatiotemporal distribution of eukaryotic eDNA in deep
marine sediments that capture a global historical record
has not been thoroughly investigated. Knowledge gained
from the comprehensive analysis of eukaryotic eDNA
*Correspondence:
Tatsuhiko Hoshino
hoshinot@jamstec.go.jp
1
Japan Agency for Marine‑Earth Science and Technology (JAMSTEC),
Kochi Institute for Core Sample Research, Kochi, Japan
2
JAMSTEC, Advanced Institute for Marine Ecosystem Change (WPIAIMEC), Yokohama, Japan3
Department of Earth Sciences, Graduate School of Science, Tohoku
University, Sendai, Japanin deep marine sediments with a more diverse range of
sedimentological, geochemical, and paleontological characteristics would provide a valuable benchmark for establishing sedimentary eukaryotic eDNA as a genetic proxyfor past ecosystems.
The relatively well-preserved eDNA has been extracted
and sequenced, successfully, including Holocene andPleistocene plant and animal DNA from Siberian permafrost (Willerslev et al. 2003) and the diatomaceous
DNA from the Holocene sediment of the Antarctic
Lake (Coolen et al. 2004). DNA of fish and other aquaticorganisms has also been recovered from surface sediments outside of permafrost and polar regions, which
are cold and thus favorable for DNA preservation, being
studied as “sedimentary ancient DNA (sedaDNA)” forunderstanding past ecosystems (Turner et al. 2015;
Kuwae et al. 2020; Ogata et al. 2021). In addition, it has
been often pointed out that the adsorption of DNA to the
inorganic matter (e.g., sediment particles) might facilitate the long-term preservation of DNA under anaerobic© The Author(s) 2024, corrected publication 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes
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permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

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sedimentary conditions (Corinaldesi et al. 2011; Pedersen
et al. 2015).
Microfossils of plankton (diatoms, coccolithophorids,
foraminifera, etc.) in sediment have been widely used forgeological dating and paleoenvironmental reconstruction. Nevertheless, restoration of past ecosystems basedon non-fossilized organisms has not been thoroughly
explored, and technical difficulties remain due to theirdecomposable nature. For example, analysis of eukaryotic eDNA in Black Sea sediments revealed that the same
virus–host relationship had continued to occur in phytoplanktonic populations for centuries (Coolen 2011). In
addition, those marine planktonic populations over about
11,400 years have responded to the gradual increase insalinity after the latest marine reconstruction (Coolen
et al. 2013). In the northeastern Arabian Sea, the depth
and age profile of sedimentary eDNA and paleoceanographic proxies synchronously demonstrated that the
protist community has responded to the variability of theoxygen minimum zone over the last 43,000 years (Moreet al. 2018). Recently, metagenomic analysis of eukaryotic eDNA in sediments from the Scotia Sea has shownchanges in diatom species composition corresponding
to glacial–interglacial cycles over the past few hundred
thousand years (Armbrecht et al. 2022). However, thesestudies have focused on one specific site in each study,
and sufficient information on the distribution of eDNA

and its persistence in marine sediments of different geological settings has not yet been obtained.
In this study, we evaluated the presence of eukaryotic
eDNA and its taxonomic composition by PCR amplification of 18S rRNA genes in 299 eDNA assemblages
from global subseafloor sediments, including those from
depths down to 678 m below the seafloor (mbsf ) anddates to over 10 million years before present. The consistent methodology used in this study for DNA extraction,library preparation, and sequencing allowed for unbiased
comparisons and analysis across all samples. This broad
analysis of eDNA recovered from marine sediments
around the world has confirmed that eukaryotic eDNAof past organisms is long preserved globally in oxic andanoxic sediment after burial from the surface world andis potentially useful for understanding ancient ecosystems that include non-fossilized organisms.

2 Methods
2.1 Sediment samples and DNA

The present study used 299 subseafloor sediment samples collected from depths of up to 678 mbsf at 40 sites
over the past 20 years (Fig. 1, Additional file 1: Table S1).
The all DNA samples used in this study were alreadyextracted in an earlier study and stored at − 80 °C(Hoshino and Inagaki 2019; Wörmer et al. 2019; Hoshino
et al. 2020) except for sediments of KR0805 from which

Fig. 1 A site location map. Red and dark blue squares indicate margin and open ocean sites, respectively. Sediment samples were collected
at different depths from the surface to 678 mbsf during 14 scientific cruises from 40 sites. A total of 299 sediment samples were used. Leg
201: Ocean Drilling Program Leg 201 Peru Deep Biosphere, Exp. 301: Integrated Ocean Drilling Program (IODP) Expedition 301 Juan de Fuca
Hydrogeology, Exp. 307: IODP Expedition 307 Modern Carbonate Mounds: Porcupine Drilling, Exp. 308: IODP Expedition 308 Gulf of Mexico
Hydrogeology, Exp. 315 & 316: IODP Expeditions 315 and 316 NanTroSEIZE, Exp. 346: IODP Expedition 346 Asian Monsoon, Exp. 347: IODP Expedition
347 Baltic Sea Paleoenvironment, Exp. 353: International Ocean Discovery Program (IODP) Expedition 353 Indian Monsoon Rainfall, Exp. 354: IODP
Expedition 354 Bengal Fan, CK06-06: D/V Chikyu Shakedown Cruise off Shimokita Peninsula, KN223: R/V Knorr Cruise 223 in North Atlantic, KR0805:
R/V Kairei KR08-05 Cruise. This figure is adapted from Hoshino and Inagaki 2019

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DNA was extracted in this study. To briefly describe the
DNA extraction method, sediment samples were frozen
immediately after the core sample delivery onboard andstored at − 80 °C until DNA extraction. To avoid external contamination as much as possible, all DNA extractions were performed from 5 g of the frozen sediments
in a clean room of the Kochi Institute for Core Sample
Research, JAMSTEC, using a Dneasy PowerMax Soil
Kit (QIAGEN). The sedimentation age of each sediment
sample was described in the previous study (Hoshino
et al. 2020, Additional file 1: Table S1). Sedimentologicalcharacteristics by smear slide observations in the previous study were also used as part of the data analysis in
this study. In brief, the sediments were smeared on slide
glasses. These smear slides were then examined using atransmitted-light, petrographic microscope. The relative proportions of biogenic, mineralogic, and authigenic
components were determined.

of Mexico, and in all 25 sediment samples from Expedition 347 in the Baltic Sea. However, PCR amplifications
were observed in only 3 out of 39 sediment samples(7%) from Expeditions 315 and 316 in the Nankai Troughforearc basin off the Kii Peninsula. No amplification wasobserved from all ten sediment samples cored during
the D/V Chikyu Shakedown Expedition CK06-06 off the
Shimokita Peninsula and collected in KR0805 at Mariana
Trench. The deepest sediment sample from which amplicons were obtained was from Expedition 308 in the Gulf
of Mexico, at Site U1324 392.2 mbsf, with a depositionalage of ~ 94,000 years, while the oldest sediment withamplification was from Expedition 353 in the Bay of Bengal, at Site U1443, 310.7 mbsf, with an age of ~ 65 Ma.
The subseafloor sedimentary biosphere is generally
anoxic in eutrophic marginal areas and oxic in oligotrophic open ocean gyres. It was estimated that oxygen
reaches the basement rock in up to 37% of global marine
sediments (D’Hondt et al. 2015). In the oxic oligotrophic
ocean, amplification products were obtained from only
12 of 66 sediment samples (18.2%), whereas 111 of 233
samples (47.6%) were obtained from anoxic sediment
samples (Fig. 2B). The higher PCR success rate in anaerobic sediments suggests a higher input of eDNA at thetime of sedimentation and/or a more stable condition for
DNA than in aerobic sediments.
PCR success rates were 52%, 44%, and 16% on averagefor 0–10 mbsf, 10–100 mbsf, and depths below 100 m,
respectively. This trend suggests that PCR products weremore readily obtained from shallower and younger sediments than deeper and older sediments, most likely due
to the fragmentation and decomposition of the eukaryotic eDNA during burial processes (Fig. 2C). The PCR
success rate for sediments deposited after 100,000 years
ago including all the Exp.347 sediments (Andrén et al.
2015) was approximately 80%, which is higher than forthe older sediment samples (Fig. 2D). The PCR success rate declined sharply as the sediment age exceeded
100,000 years: 27% from 100,000 to 1 million years, 11%from 1 to 10 million years, and 7.4% from > 10 million
years. This trend strongly indicates that increase in depth
and burial time has a critical impact on eukaryotic eDNA
preservation in marine sediment.

2.2 PCR amplification and library preparation

By using the primer set Euk_1391f-EukBr (Stoeck et al.
2010), a fragment of the eukaryotic 18S rRNA gene wasamplified by PCR. The PCR was performed using MigtyAmp DNA polymerase (Takara Bio) for 40 cycles of(98 °C for 10 s, 58 °C for 15 s, and 68 °C for 15 s) after
initial denaturation at 98 °C for 2 min. The PCR products
obtained were purified by agarose gel electrophoresis,
followed by index PCR for sequencing. After measuring
the concentration of the products from the index PCR,
equal amounts of the PCR products from each sample
were mixed to prepare a sequence library. Sequencing
was performed by MiSeq using the MiSeq Reagent Kit
v3 (600 cycles; Illumina), according to the manufacturer’s
instructions. The resulting sequences were quality filtered, merged, and clustered into ZOTUs (referred to as
sequence variants: ASVs in this study) using Usearch (64bit version; www.​drive5.​com/​usear​ch/) and phylogenetically identified by Mothur (Schloss et al. 2009) using the
SILVA 138 SSU database (Quast et al. 2012; https://​www.​arb-​silva.​de/). Analysis of the obtained data was performed using vegan, and plotting was performed using
the ggplot2 package in R.

3 Results
3.1 Amplification of 18S rRNA gene

3.2 Sequencing and eukaryotic community composition

Amplification of the V9 region of the eukaryotic 18SrRNA gene from 299 sediment samples was attempted,
and 124 samples yielded visible bands on agarose gelsafter PCR. The success rate of PCR amplification varied
from site to site (Fig. 2A): For example, in sediment samples obtained from depths shallower than 100 mbsf, successful PCR amplifications were observed in 21 out of 23(91%) sediment samples from Expedition 308 in the Gulf

After quality filtering, a total of 10.1 million 18S rRNA
gene sequences were obtained. The average numberof sequences per sample was 5.73 × ­104. The obtained
sequences were clustered into amplicon sequence variants (ASVs), resulting in 20,658 ASVs, of which 12,918
ASVs were classified as eukaryotes. For further analysis, only the sequences of those eukaryotic ASVs wereused, and the number of sequences in each sample

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Fig. 2 PCR amplification of 18S rRNA genes from eDNA extracted from 299 marine sediment samples. A Success or failure of PCR amplificationof 18S rRNA gene in every sample. Red and gray circles indicate PCR success and failure, respectively. Only the plots for which sediment ages
are available are shown in the lower panel. The red dashed squares indicate successful amplification from all Expedition 347 sediment samples
known to be younger than 105 years. B The summary of PCR success rates in the oxic and anoxic sediments. C The summary of PCR success rates
in the three different sediment depth classes, 0–10, 10–100, and 100–677.9 mbsf. D The PCR success rates in the four different sediment age classes,
0–0.1, 0.1–1, 1–10, and > 10 million years ago

used for phylogenetic and beta-diversity analyses
was randomly rarefied to 5,000 sequences. Phylogenetic classification was performed using the ARB silva
v138 database as a reference. However, many of thesequences could not be classified even at the phylum
level, which was either due to the lack of referencesin the database or to short sequence length (approximately 130 bases; see “Eukaryotic_unclassified” inFig. 3). Excluding those unclassified sequences, Phragmoplastophyta was the most predominantly detectedeukaryotic clade, followed by Ascomycota, Dinoflagellata, Vertebrata, and Basidiomycota. In the sediments
in Baltic Sea collected during Expedition 347, Diatomea was the most predominant eukaryotic clade at Site
M0059, whereas sequences affiliated with the phylum
Dinoflagellata were more predominantly detected at
Sites M0063 and M0065 than at any other sites. Threesites of Expedition 353, U1443, U1444, and U1450 in
the Bay of Bengal were also distinguished by the predominance of Phragmoplastophyta and Vertebrata.
The beta-diversity of eukaryotic community compositions within the recoverable pool of eDNA indicatedthat the communities differed significantly between
marginal and open ocean sediments (PERMANOVA,
p = 0.001, Fig. 4). The samples from the Baltic Sea(Expedition 347) constituted an independent cluster, representing different eukaryotic communities
between the shallower and deeper sediment horizons.
The samples from the Bay of Bengal (Expeditions 353
and 354) derived from greater than 50 m-deep beneaththe open ocean were clustered in the upper right-handcorner (Fig. 4). These global-scale data set suggeststhat the distribution of eukaryotic eDNA is spatiotemporally vast in the marine sedimentary environment

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Fig. 3 Phylum-level taxonomic composition of eukaryotic communities in subseafloor sediment. The upper red line chart shows sediment depth
on a logarithmic (log10) scale. The colored bar below the bar chart indicates expeditions. Classification is based on the SILVA 138 SSU database(https://​www.​arb-​silva.​de/)Fig. 4 NMDS ordination plots for the sedimentary eukaryotic
communities. The Jaccard index was derived from the rarefied
amplicon sequence variants composition. Triangles and squares
indicate marginal and open ocean sediments, respectively

and depends on the geological and paleoenvironmental history (Orsi and Inagaki 2023).
3.3 Correlation of eukaryotic eDNA with microfossils

Microfossils of marine plankton are generally preservedin sediment (e.g., diatom spicules). Therefore, their stratigraphy (occurrence and disappearance) has historically
been employed as an index fossil record for dating andpaleoenvironmental reconstructions. We determined
whether there is a correlation between the occurrenceof microfossils in those sediments and the results of
eDNA analysis. Smear slide analysis results from theprevious studies were used to determine the occurrenceof three typical microfossils, Diatomea, Prymnesiophyceae, and Retaria, in each of the sediment samples. We
then examined whether there were differences in therelative abundance of eDNA of organisms corresponding to the microfossils in the sediments with and without the microfossils (Fig. 5). Among these, foraminiferal
microfossils were observed on smear slides in several
sediment samples; however, they were not detected by
18S rRNA gene sequencing probably because the primer
set does not amplify those sequences. For Diatomea,
the median relative abundance of sequences in samples
where smear slides did not contain Diatomea microfossils was 4.95 × ­10–3 and that of samples with microfossils was 1.25 × ­10–2, showing a significant difference in
the Welch two-sample t test (p = 0.0159, Fig. 5A). On theother hand, for the nannoplankton Prymnesiophyceae,
there was no significant difference (p = 0.198, Fig. 5B),
with 7.90 × ­10–4 and 1.66 × ­10–4 for the fossil-undetected
and -detected smear slides, respectively.
3.4 Correlation between paleoclimate and eukaryotic
eDNA data

To test whether eukaryotic eDNA in marine sedimentcan be used as a proxy for paleoenvironmental changes,
we focused on diatom eDNA in sediment samples fromthe Baltic Sea collected during Expedition 347. Expedition 347 explored a sedimentary sequence from the last

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Fig. 5 Correlation between the presence or absence of microfossils in sediment samples (using the results of smear slide analysis in Ref. 3)
and the relative proportion of environmental DNA. The panels A and B indicate the results for Diatomea and Prymnesiophyceae, respectively

glacial and interglacial periods, and thus the obtained
sediment samples are distinguishable between freshwater and seawater depositional environments according
to the depth, age, and sedimentological characteristics(Andrén et al. 2015).
Of the top 20 ASVs classified in Diatomea, 14 ASVs
were identified at the genus level. These were marine
genera Chaetoceros, Skeletonema, Thalassiosira,Eucampia, Attheya, and Cylindrotheca, and freshwater genera Aulacoseira, Stephanodiscus. We focusedon the frequency of occurrence of ASVs in freshwaterand marine sediments, focusing on Aulacoseira, which
has a high abundance. Aulacoseira was detected in only
seven sediments, six of which were from the Baltic Sea,out of a total of 114 sediments used for ASV analysis(Fig. 6). Among the Baltic Sea sites, Aulacoseira was
detected at Site M0063, located in Landsort Deep, andSite M0065, located in the Borhholm Basin, while it
was not detected at Sites M0059 and M0060, locatedin the Little Belt and southeast of Anholt at the bay
entrance. At Site M0063, Aulacoseria were frequently
detected in deep sediments, 5.6%, 2.0%, and 1.0% of
5,000 sequences at 64.2 m, 70.1 m, and 87.8 m, respectively. Four sediment samples greater than 64.2 m at
Site M0063 are identified as freshwater deposits in the
Glacio-lacustrine with the detection of Aulocoseira.
On the other hand, the 17.5 m sediment with only
0.02% (1/5000sequences) of Aulascoseira detected is a

Holocene marine sediment, so the detected sequence
probably is distal origin. At Site M0065, 1.6% Aulacoseira was detected in the sediments from 10.3 m in thelacustrine depositional environment, while they were
not detected in the upper marine sediments.
In contrast, no Aulacoseira were detected in the 29.2
and 33.9 m sediments of Site M0063, which are classified
as Ancylus Lake sediments, or in the 36.4 m deep sediment of M0065, which is a glacial lacustrine depositional
environment. The absence of Aulacoseira may be due
to environmental factors other than salinity, but further
research is needed to understand the diatom community
at the time. These data indicate that Aulacoseira eDNA
can serve as a proxy for a freshwater ecosystem in theBaltic Sea during the last glacial period.

4 Discussion
In this study, we showed the distribution of eukaryotic
eDNA in marine sediments by analyzing eukaryotic
eDNA in sediments from a wide range of sites around theworld. With greater sediment depth or age, lower PCR
success was observed, as seen at Sites U1301, C0002,
U1450, U1371, and 1226 (Fig. 2). The lower PCR successrate could be due to the gradual degradation of eukaryotic eDNA over time. Deeper and older subseafloor sediments are typically unfavorable habitats for active growthand/or long-term survival of most eukaryotes due toenergy and space limitations, with some exceptions,Hoshino and Inagaki ﻿Progress in Earth and Planetary Science (2024) 11:19

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Fig. 6 Relative abundance of top 20 ASVs belonging to phylum Diatomea in sediments of the Baltic Sea, IODP Expedition 347. Genus Aulacoseira,enclosed by green dashed line, is known as freshwater diatom

including spore-forming organisms. This is one of thereasons why the depth-dependent decrease in amplifiable 18S rRNA genes was consistently observed at allsites investigated in this study. A previous study of Moa
bones (flightless birds endemic to New Zealand) suggested that the half-life for 242-base mitochondrial DNA

Hoshino and Inagaki ﻿Progress in Earth and Planetary Science (2024) 11:19

Page 8 of 10(mtDNA) is estimated to be 521 years, and that the average strand length of mtDNA is 88 bp after 10,000 years at
5 °C, a temperature close to that of many shallow marine
sediments (Allentoft et al. 2012). Given this estimate andthe fact that the size of the PCR amplicons obtained inthis study was about 140–170 bp, it was somewhat unexpected that the PCR success rate was as high as approximately 80% for sediments younger than 100,000 years.
DNA degradation rate is influenced by physical, chemical, and biological factors of the surrounding environment; for example, minerals can stabilize eDNA in soil
where a substantial amount of eDNA can accumulate
over time (Kirkpatrick et al. 2016; Morrissey et al. 2015).
Similarly, the absorption of eukaryotic eDNA to mineral particles or skeletal remains in marine sediments
may also help to stabilize those eDNA from spontaneous decomposition. And thus, subseafloor sediment may
therefore be an environment in which eDNA is relatively
stable against degradation. Fungi and some other eukaryotes, including diatoms and dinoflagellates, are known
to form spores in their life cycle that permit long-term
survival (Ribeiro et al. 2011), and the intracellular DNA
is likely more protected within such dehydrated periplasmic structures. Alternatively, it has been reported
that marine phytoplankton including diatoms, dinoflagellates, and haptophytes have other resting stages thanspores, either with minimal activity or as dormant cells(Ellegaard and Ribeiro 2018). If these organisms are present, they might be preferentially detected as their DNA
is more easily extracted than that of spores.
Our research has shown that fragmented DNA
remaining in subseafloor sediments can also be amplified by PCR and sequenced to identify species. It should
be noted, however, that the community composition
obtained by PCR amplification may be biased becauseof the trace amount of target DNAs in the sediments. A
comparative study of sedimentary 16S rRNA gene amplicon libraries with shotgun metagenomic data suggested
that a shotgun metagenomic approach may help obtain
more quantitative data on the buried eukaryotic community in sediment (Ziesemer et al. 2016). In fact, metagenomic analysis without DNA amplification has been
applied to shallow sediments with ages up to 6000 years
from the Arabian Sea, demonstrating that the abundance of the chlorophyll biosynthesis gene of diatoms
can be used as a proxy for past photosynthetic plankton abundance (Giosan et al. 2018). However, eDNA ismore fragmented in older sediments, thus hampering
metagenomic analysis. In recent years, DNA enrichmentof specific taxa by hybridization capture has been applied
to eDNA analysis (Armbrecht et al. 2021), which may be
useful for metagenomic analysis of such samples withextensive DNA fragmentation.

Correlations between the presence/absence of Diatomeae and Prymnesiophyceae microfossils and the relative abundance of eDNA were found to be correlated forthe former, but not for the latter. These algal skeletal fossils and eDNA represent differences in the mechanismsof persistence in sediments. The coccolithophyte skeleton is composed of carbonate, which does not persist at
depths greater than the carbonate compensation depth,
whereas the diatom skeleton is composed of silicate,
which is more stable. Therefore, it is possible that eDNA
would persist even if the carbonate skeletons were dissolved. On the other hand, in the present study, Prymnesiophyceae eDNA was rarely detected (around 0–0.01%)
in sediments where nanofossil predominated in smear
analysis (Additional file 1: Table), e.g., sediments from
Site 3 of KN224 in Fig. 3. The relationship between theabundance of microfossils and eDNA in sediments andthe differences in their respective physicochemical andbiochemical degradation processes remains largely open
to research.
While diatom eDNA was detected in the Baltic Sea sediments, including those from glacial sediments, dinoflagellate DNA was also abundant in other inner bays (Fig. 3,
sites M0063 and M0065). The Baltic Sea is a region ofhigh primary production, resulting in a greater frequencyof these primary producers. In addition, the ratio of dinoflagellates to diatoms is reported to be high in the innerBaltic Sea and low near the outlet at present (Klais et al.
2011). This may be consistent with the high abundanceof diatom environmental DNA detected in the sedimentsof M0059 (located in the bays of Kiel and Mecklenburg),
which is the outlet to the Baltic Sea. However, as the proportion of diatoms is known to vary with environmental
change, it is debatable whether diatoms were the dominant species at the time of deposition at the M0059 site.
In this study, a high relative abundance of eukaryotic eDNA belonging to the phylum Phragmoplastophyta, most of which consist of terrestrialorganisms, was observed in samples from multiple
sites and depths/ages (Fig. 3). It may be of particularinterest to see the predominance of Phragmoplastophyta-related sequences dominated at sites U1443 andU1444 in the Bay of Bengal. The Bengal Fan is the largest estuarine fan on Earth and its massive terrigenous
influx likely leads to the wide dispersal of Phyragmoplastophyta from land to sea. Nonetheless, one could
argue that the eukaryotic eDNA of terrestrial plants
is difficult to distinguish from the modern DNA contaminants introduced into amplified DNA products
during sampling and laboratory analysis. Our negative control experiments where DNA extraction blanks
were applied to the same PCR procedure used for the
sediment samples resulted in no-visible amplification

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on agarose gel electrophoresis. However, it would bevery hard to eliminate this possibility at any stage of theanalyses completely. For example, it is physically impossible to achieve no contamination from the seawaterthat is used as a drilling fluid during the drilling process(Lever et al. 2006). In addition, onboard sampling was
not be conducted in a clean environment at that time,
although sediment samples of DNA analysis were takenfrom the near center of the core. Therefore, crossvalidation with other data such as lipid biomarkers
and geochemical proxies is necessary for interpreting
eukaryotic eDNA data, as discussed in earlier studies(Armbrecht et al. 2019 and papers cited therein). Also,
focusing only on organisms that are typically not present in the laboratory environment could minimize thecomplications of data interpretation (More et al. 2018;
Lejzerowicz et al. 2013). For example, the applicationof PCR targeting phytoplankton, fish and seaweeds,
rather than universal primer sets as used in this study,
is promising for obtaining data that will be useful forunderstanding past ecosystems.

Author contributions
Both authors designed the study. T.H. performed PCR, sequencing, and data
analysis. Both authors wrote and approved the final version of the manuscript.
TH and FI helped in conceptualization. TH contributed to methodology. TH
and FI assisted in investigation. TH helped in writing—original draft.

5 Conclusions
This study demonstrated that eukaryotic eDNA haslong been globally preserved in subseafloor sediment for as long as 100,000 years. Comparisons withpaleoenvironmental and microfossil records support
the notion that eukaryotic eDNA in the deep sedimentary biosphere includes taxa from the past surface biosphere and thus is a useful proxy for gaining a deeper
understanding of paleoenvironments and ecosystem’s
coevolution at the DNA level.
Abbreviations
eDNA	Environmental DNA
ASVs	Amplicon sequence variants
mbsf	Meters below sea floor

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s40645-​024-​00621-2.
Additional file 1: Metadata of the sediment samples.
Acknowledgements
The samples and/or data used in this study were provided by the Ocean Drilling Program (ODP), the Integrated Ocean Drilling Program, and the International Ocean Discovery Program (IODP). The authors thank all the crew, drillingteam members, and shipboard scientists and technical staff for core sampling
during ODP Leg 201; IODP Expeditions 301, 307, 308, 315, 316, 329, 346, 347,
353, and 354; D/V Chikyu shakedown cruise CK06-06; R/V Kairei Cruise KR08-05;
and R/V Knorr Cruise KN223. We thank R. R. Adhikari, Y. Morono, T. Nunoura,
G.-I. Uramoto, and N. Xiao for their assistance in sediment core sampling as a
shipboard scientist during the IODP expedition. We are also grateful for thetechnical assistance provided by S. Hashimoto and T. Terada at the Kochi Institute for Core Sample Research, JAMSTEC. This work was supported by World
Premier International Research Center Initiative (WPI), MEXT, Japan.

Funding
Japan Society for the Promotion of Science Grant-in-Aid for Science Research
22H00429 (FI, TH) and 21K19876 and 22H01347 (TH).
Availability of data and materials
The DNA sequence datasets supporting the conclusions of this article aredeposited in DDBJ/EMBL/GenBank under the accession number PRJDB14385.

Declarations
Competing interests
Authors declare that they have no competing interests.
Received: 5 December 2023 Accepted: 22 March 2024
Published: 7 April 2024

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