# Radiolarian and Phaeodarian high-rank assemblage change through time in the Scotia Sea: A paleo-genomics approach (IODP Exp. 382)

Vikki Lowe, Tristan Biard, G Cortese, I Hernández-Almeida, K-A Lawler, L

Armbrecht

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Vikki Lowe, Tristan Biard, G Cortese, I Hernández-Almeida, K-A Lawler, et al., Radiolarian and Phaeodarian high-rank assemblage change through time in the Scotia Sea: A paleo-genomics approach (IODP Exp. 382). Marine Micropaleontology, 2025, Part of special issue The invisible complement: using sedimentary ancient DNA (sedaDNA) to improve marine ecosystem reconstructions, 200, pp.102500. 10.1016/j.marmicro.2025.102500. hal-05372823

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Research paper

# Radiolarian and Phaeodarian high-rank assemblage change through time in the Scotia Sea: A paleo-genomics approach (IODP Exp. 382)☆

V. Lowe<sup>a,b,c,*</sup>, T. Biard<sup>d</sup>, G. Cortese<sup>e</sup>, I. Hernández-Almeida<sup>f</sup>, K.-A. Lawler<sup>g</sup>, L. Armbrecht<sup>a</sup>

## ARTICLE INFO

Keywords:
* sedaDNA
* Radiolaria
* Phaeodaria
* Changepoint
* Southern Ocean

## ABSTRACT

Radiolaria and Phaeodaria are single-celled eukaryotic zooplankton that inhabit diverse marine environments. While the sedimentary fossil record reveals their ancient origins and morphological diversity, recent advances in metagenomics and sedimentary ancient DNA (sedaDNA) techniques have highlighted the significant contributions of rarely preserved taxa (e.g., those that are only lightly silicified or soft-bodied) to marine biodiversity and the carbon cycle. Using a paleo-genomic approach, we produced a sedaDNA-based time series of the Radiolarian and Phaeodarian community from the Scotia Sea covering the last 500 kyr. We compared the use of two reference databases, one Radiolarian- and Phaeodarian-specific (RPD) and one more comprehensive eukaryote database (PR²), to assess the validity of a small, taxa-specific database. We determined that the two databases produced comparable results, showing that the overall assemblage changes at the glacial-interglacial scale were similar. At the Class level, Radiolarian and Phaeodarian assemblages appear to have experienced compositional changes during termination events and warm Interglacial periods. This study produced the first Radiolarian- and Phaeodarian-specific reference database. We showed that the Class-level changes that occurred over the last 500 kyr were associated with glacial-interglacial (G-I) cycles, and no permanent shifts away from the natural variation associated with the G-I cycles were found.

## 1. Introduction

Radiolaria and Phaeodaria are single-celled eukaryotic zooplankton that inhabit the water column from surface to sea floor and from tropics to polar regions (Boltovskoy and Correa, 2019). They are ancient eukaryotes that has been present in the world's oceans since the Cambrian (e.g., Braun et al., 2007; Zhang and Feng, 2019). While their evolutionary history is poorly understood, their fossil record reveals a wide range of skeletal structures in the past, ranging from single spicules to complex multi-shelled species and colonial groups (Biard, 2022). Historically, four major groups – Acantharea, Spumellaria, Nassellaria, and Phaeodaria – were classified by Haeckel (1887) as Radiolarians based on skeletal morphology. This classification persisted through much of the 20th century. However, advances in molecular phylogenetics have since

revealed that the radiolarian group is not monophyletic (Polet et al., 2004; Adl et al., 2019). In particular, Phaeodaria has been reclassified within the Phylum Cercozoa, distinct from the Radiolarian group, which now contains Acantharea, Taxopodida and Polycystinea (Spumellaria, Nassellaria, and Orodaria) (Polet et al., 2004). In this study, we will examine Phaeodaria and Radiolaria (the two groups will hereafter be referred to as the RP group), as their siliceous skeletons are found in sediments alongside one another, albeit rarely, and both are usually reported together (See Box 1).

Within the RP group, most biomineralise either opaline silica (Polycystinea, Phaeodaria and Taxopodida) or strontium sulphate (Acantharea; Decelle and Not, 2015) to produce their skeletons. Polycystinea are the most well-known group of Radiolaria. The robust skeletons and good preservation potential of polycystines mean that they are common

<footnote><sup>a</sup> Institute of Marine and Antarctic Studies, University of Tasmania, Hobart, Australia</footnote>

<footnote><sup>b</sup> University of Bordeaux, CNRS, Bordeaux INP, EPOC, UMOR 5805, F-33600 Pessac, France</footnote>

<footnote><sup>c</sup> EPHE/PSL Research University, Paris, France</footnote>

<footnote><sup>d</sup> LOG, Laboratoire d'Océanologie et de Géosciences, Université du Littoral Côte d'Opale, Université de Lille, CNRS, IRD, UMR8187, Wimereux, France</footnote>

<footnote><sup>e</sup> Surface Geosciences Department, GNS Sciences, Lower Hutt, New Zealand</footnote>

<footnote><sup>f</sup> PAGES IPO, University of Bern, Switzerland</footnote>

<footnote><sup>g</sup> Research School of Earth Sciences, Australian National University, Canberra, Australia</footnote>

<footnote>* This article is part of a Special issue entitled: 'Marine sedaDNA' published in Marine Micropaleontology.</footnote>

<footnote>* Corresponding author at: Institute of Marine and Antarctic Studies, University of Tasmania, Hobart, Australia.
E-mail address: info@vikkilowe.com (V. Lowe).</footnote>
in marine sediments, particularly those from the Southern Ocean, and so are utilised as proxies for past environmental conditions, for example, oceanic surface and sub-surface sea temperature (CLIMAP Project Members, 1976; Cortese and Prebble, 2015; Hernández-Almeida et al., 2020; Civel-Mazens et al., 2023) and water mass structures (Iizuka et al., 2024; Lowe et al., 2024). The other three groups are preserved at much lower rates than Polycystinea, either due to their lightly biomineralised skeletons that are highly subject to dissolution (e.g., Phaeodaria), or because they do not possess biomineralised body parts at all (e.g., ‘naked’ Collodaria; Ishitani et al., 2012; Biard, 2022).

The RP group contributes significantly to marine biodiversity (de Vargas et al., 2015) and many species have symbiotic relationships with other organisms, ranging from prokaryotes to eukaryotes such as dinoflagellates (Anderson, 2014). They are an essential part of the marine food web, frequently found in the gut content of many organisms, from microzooplankton to adult fish (Biard, 2022). Recent studies on the modern RP group indicate that they are important contributors to the export of organic matter in deep waters, where they often dominate the mesozooplankton community (Biard et al., 2016). RP group surface-dwellers alone (predominantly Collodaria and Phaeodaria) contribute an estimated 5.2 % of the total biotic carbon reservoir (Biard et al., 2016), and up to 60 % of the organic flux from the surface to 3000 m depth (Lampitt et al., 2009). As it has been recently demonstrated that RP group deep-dwellers comprise a high proportion of the eukaryote communities in bottom waters (Armbrecht et al., 2023), those estimates (Lampitt et al., 2009; Biard et al., 2016) may likely rise significantly if deeper-dwelling species were considered.

While we have known that the RP group is biodiverse and important for the silica and carbon cycles, recent advancements in metagenomics research have increased our appreciation of their contributions to the marine food-web and biogeochemical cycles (Biard et al., 2016; Armbrecht et al., 2023). Even in paleo-studies, Rhizaria, which includes the RP group (Fig. 1), have been reported to be the most dominant group of eukaryotes based on sedimentary ancient DNA (sedaDNA) found in marine sediments (Armbrecht et al., 2022; Armbrecht et al., 2023). As such, it seems timely to explore the dynamics of the broader RP community through time and over periods of environmental perturbation, including taxa with and without robust silica-skeletons. Understanding the glacial-interglacial (G-I) scale changes in the broader RP community, particularly for those taxa that do not preserve well, or at all, in sediment, will provide greater insight into their role and response to current and future climate scenarios.

To study this, we used a paleo-genomics approach to track changes in assemblage compositions over the past 500 kyr at several sites located in the Southern Ocean. We collated an RP-specific reference sequence

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Location of IODP Exp. 382 Sites U1534 (Falkland Plateau), U1536 (Dove Basin) and U1538 (Pirie Basin). Front locations are according to Park et al. (2019). Northern Boundary – NB; Subantarctic Front – SAF; Antarctic Polar Front – APF; Southern Antarctic Circumpolar Current Front – SACCF; Southern Boundary – SB. Map created in R using package "S maps" Maschette et al. (2019).</figcaption></figure>

database (RPD) of small subunit ribosomal RNA genes (hereafter, 18S), and used this to assign obtained sequence reads to specific species or clades for the extracted sedaDNA from the core samples. This produced a time series of the Radiolarian and Phaeodarian communities from three International Ocean Discovery Program (IODP) Expedition 382 Sites (U1534, U1536, and U1538; Weber et al., 2021a) in the Scotia Sea from the Subantarctic Zone to the Sea-Ice Zone, covering the last 500 kyr. We also used the comprehensive Protist Ribosomal reference database (PR², Guillou et al., 2013) to reproduce an RP group time series on the same samples, and compared the results against those of our smaller, RPD to validate the usefulness of a small, taxa-specific reference dataset. We specifically focused on the nuclear taxonomic marker gene 18S as it occurs in high copy numbers in RPD (Gutierrez-Rodriguez et al., 2019), and to allow comparisons to previous work on the Exp. 382 sedaDNA data investigating marine protist communities using this gene (Armbrecht et al., 2022). We explored the relationship between Radiolarian and Phaeodarian community composition and major environmental shifts in G-I cycles using cluster and changepoint analyses.

## Box 1

### The RP Group

The RP group, as defined in this study, includes four groups of marine protists:

* **Acantharea:** Classified as Radiolarians, Acantharians produce strontium sulfate skeletons. Due to the undersaturation of strontium in seawater, these skeletons dissolve rapidly, leaving no fossil record (Biard, 2022; Decelle and Not, 2015).

* **Polycystinea:** A well-known group of Radiolarians that includes Spumellaria and Nassellaria. Polycystines possess siliceous skeletons that are commonly preserved in marine sediments and widely used as paleoceanographic proxies. Collodaria, previously considered a separate class of Radiolaria, are now classified as a subgroup within Nassellaria, based on molecular work (Adl et al., 2019).

* **Taxopodida:** Classified as Radiolarians, Taxopodids produce skeletons composed of siliceous spicules. They are considered “Polycystine-like” in their morphology, but molecular evidence indicates that they are phylogenetically distinct from Polycystines (Adl et al., 2019).

* **Phaeodaria:** Originally morphologically grouped with Radiolarians, molecular work has now classified Phaeodarians within the Phylum Cercozoa of the Infrakingdom Rhizaria. Fossil remains of their lightly silicified skeletons have been reported alongside Polycystines. However, Phaeodaria are only preserved under very favourable conditions due to their high susceptibility to dissolution (Polet et al., 2004; Adl et al., 2019).
## 2. Methods

### 2.1. Sampling location, sediment coring and age determination

This study is a re-analysis of sedaDNA data from sediment samples that were collected during IODP Exp. 382 'Iceberg Alley and Subantarctic Ice and Ocean Dynamics' on-board RV JOIDES Resolution in 2019 (Weber et al., 2021a). Specifically, samples were obtained from 3 sites: Sites U1534 (Falkland Plateau, 606 m water depth), U1536 (Dove Basin, Scotia Sea, 3220 m water depth), and U1538 (Pirie Basin, Scotia Sea, 3130 m water depth; Fig. 1). sedaDNA samples collected at Site U1534 were from Hole C, at Site U1536 from Hole B, and at Site U1538 from Holes C and D (Supplementary Table 1), and in the following we refer to site names only. Age control for Site U1534 was based on tuning of benthic foraminifera δ¹⁸O to the LR04 stack (Lisiecki and Raymo, 2005) and biostratigraphy (Peck et al., 2021; Peck and Alcock, 2022; Weber et al., 2022). Age control for Sites U1536 and U1538 was established using shipboard magneto- and biostratigraphy (Reilly et al., 2021; Weber et al., 2021a; Weber et al., 2022) and by correlating sedimentary dust proxy records (such as magnetic susceptibility and sedimentary Ca and Fe records) to ice-core dust proxy records over the last 800 ka (Weber et al., 2022) and to a benthic isotopic stack (Lisiecki and Raymo, 2005) before that. For detailed information on the sampling locations and age determinations, see Armbrecht et al. (2021).

### 2.2. Sampling of sedaDNA

A detailed description of sedaDNA sampling methods can be found in Weber et al. (2021b). In brief, advanced piston coring (APC) was used to acquire sediment cores, and clean sampling procedures were followed, including sterilising the core liners and using sterilised cutting and sampling tools and equipment (treated with 3 % sodium hypochlorite, 'bleach', and 80 % ethanol). The 10 m core was cut into 1.5 m sections, and ~ 3 mm of surface material was removed from the bottom of each core section. A cylindrical sample was taken from the core centre using a sterile (autoclaved) 10 mL cut-tip syringe, providing ~5 cm³ of sediment material. The syringe was placed in a sterile plastic bag (Whirl-Pak) and immediately frozen at -80 °C. The mudline (sediment/seawater interface) was transferred from the core liner into a sterile bucket, and 10 mL sample was retained in a sterile 15 mL centrifuge tube (Falcon) and frozen at -80 °C. Samples were collected at various depth intervals depending on the site to span the Holocene up to ~1 million years (Supplementary Table 1).

Two types of controls were taken on the ship, to test for airborne and drill fluid contamination. For the 'air controls', empty syringes (one per site) were held for a few seconds in the sampling area and then transferred into a sterile plastic bag and frozen at -80 °C. For the drill fluid controls, a sample of tracer-infused drill fluid (~10 mL) was collected in a sterile 15 mL centrifuge tube (inside a sterile plastic bag) and frozen at -80 °C. The frozen air and drill fluid controls, as well as extraction of blank controls (EBCs, empty tubes that were added during the extraction process in the lab) were processed and analysed, including sequencing, in the same way as the sedaDNA samples. Sediment samples (~3 cm³) for tracer testing (perfluoromethyldecalin, PFMD) were also collected at each sedaDNA sampling depth and analysed via gas chromatography (GC-µECD; Hewlett-Packard 6890). A detailed description of the control sampling and PFMD GC measurements is provided in Armbrecht et al. (2021) and Weber et al. (2021b).

### 2.3. sedaDNA extractions, metagenomic library preparations and raw sequencing data processing

A total of 80 sedaDNA extracts (Supplementary Table 1) were prepared using the "combined" protocol described in Armbrecht et al. (2020). This technique involves an overnight sediment incubation step in ethylenediaminetetraacetic acid (EDTA) to isolate and stabilise

extracellular DNA, followed by bead-beating to lyse cells and DNA binding in silica-solution to retain small DNA fragments (Armbrecht et al., 2020). Metagenomic shotgun double-barcoded libraries were prepared, using a first amplification with IS7/IS8 primers, and then a second amplification using IS4 and GAI Indexing Primers (Meyer and Kircher, 2010; Armbrecht et al., 2020). The libraries were sequenced at the Garvan Institute for Medical Research, Sydney, Australia (Illumina NovaSeq 2 × 100 bp) (Armbrecht et al., 2021). The sequencing data was processed and filtered, which involved the removal of sequences <25 bp (AdapterRemoval v. 2.1.7-foss-2016a, Schubert et al., 2016), removal of low-complexity (Komplexity software (Clarke et al., 2019), -cl-threshold 0.55) and duplicate reads ('dedupe' tool in BBMap v.37.36), and quality control after each step (FastQC v.0.11.4, Babraham Bioinformatics; MultiQC v.1.0.dev0; Ewels et al., 2016). Up until this step procedures resembled those described in detail in Armbrecht et al. (2021), as this is a re-analysis of the sequencing data from that study. Below, we outline the details of the new analysis of the filtered sequencing data, focussing on Radiolarians and Phaeodaria.

### 2.4. Alignments with PR² and a custom RPD database

Using MALT (version 0.4.0; semi-global alignment (Herbig et al., 2016), we ran the shotgun data against the curated Protist Ribosomal Database (PR², version 5.0; https://pr2-database.org/). We also ran the data against a custom-made database containing exclusively RP group sequences (Supplementary Material). This database contained 4532 sequences that were extracted from PR² (18S rRNA sequences) and 377 sequences downloaded from NCBI on 03/08/2023 (18S and 28S rRNA sequences; https://www.ncbi.nlm.nih.gov/nucleotide/).

As we expected a limited number of reads to map to RPD, we maximised the number of reads by avoiding normalising (rarefying) the aligned data and worked with relative abundances. The resulting .blastn files were converted to .rma6 files using the Blast2RMA tool in MEGAN (version 6.18.9), with the default settings except for a minimum support percent of zero ('off') and a minimum percent identity of 95 %.

Subtractive filtering (i.e., subtracting reads for species identified in EBCs, air, and drill fluid controls from samples) was conducted with MEGAN Community Edition version 6.21.12 (Huson et al., 2016). The filtered read counts per taxon, Class and site were exported for downstream analysis. All taxa determined in the controls post-alignments are listed in Supplementary Table 2.

### 2.5. Statistics

The total number of reads and their proportions were determined for Acantharea, Phaeodaria, Polycystinea, and Taxopodida for both the RSD and PR² alignments. We applied average agglomerative cluster analysis using Chord distance to the relative abundance data among samples using code provided by (Borcard et al., 2018). Changepoint analysis using Pruned Exact Linear Time (PELT) was applied to the relative abundances of assigned reads for each RP group individually and to the combined groups from each core to identify when significant changes in mean and variance occurred using R package "Rbeast" (Zhao et al., 2019). Pearson correlation analyses were performed on the relative abundances of each Class-level within the RP group to compare the results produced by the two datasets.

### 2.6. Data availability

The following databases were used in this study: PR² version 5.0 (https://pr2-database.org/), and the custom-made 'RPD' database, which is provided as supplementary material (RPD.fasta) with this paper. Detailed Supplementary Information on methods and analysis is provided with this submission. The demultiplexed raw sequencing data generated and analysed during this study have been deposited in the NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.
gov/sra) under Accession code/BioProject PRJNA861836 (http s://www.ncbi.nlm.nih.gov/bioproject/PRJNA861836/; BioSamples SAMN29928044 - SAMN29928123; Armbrecht et al., 2022), and includes metadata for each sediment and control sample. For further requests please contact the corresponding author.

## 2.7. Code availability

The bioinformatic pipeline used in this study was previously published in Armbrecht et al. (2020), and the R code used for analyses of the data is provided in Supplementary Material.

# 3. Results

## 3.1. Comparison of reads aligned with PR² and RPD

The total number of reads assigned using the PR² reference library was 1339 compared to the 441 produced by the RPD. Phaeodaria and Polycystinea accounted for most of the differences between the two datasets, particularly at Site U1538 and, to a lesser extent, Polycystinea at Site U1534 (Table 1). There was a much higher proportion of Polycystinea reference sequences within the RPD compared to the PR². However, the proportion of the resulting assigned reads for each group was similar between the two datasets (Fig. 2).

No reads were assigned to the RP group using PR² for 5 samples at Site U1534 (0.7, 16, 333, 400 and 700 kyr). Using the RPD, no reads were assigned to the RP Group in 1 sample (16 kyr). For Site U1536, no reads were assigned to the RP Group for 1 sample using PR² (300 kyr) but using RPD, reads were assigned to all samples. For Site U1538, no reads were assigned to the RP Group for 3 samples (203, 215 and 392 kyr) using both PR² and the RPD.

The relative abundance of assigned reads for each taxonomic group downcore using the two databases followed similar trends and amplitude of changes through time (Fig. 3; Table 2). The strongest positive correlation (Pearson) was observed in the relative abundance of Polycystinea between the two datasets ($r$ (13) = 0.82, $p$ < 0.01). Phaeodaria relative abundance showed the next strongest correlation, followed by Acantharea (Table 2). Relative abundance of *Taxopodida* showed the weakest correlation between the two datasets ($r$ (30) = 0.11, $p$ > 0.05), more frequent assigned reads, and higher relative abundances for RPD (Fig. 3; Table 2).

## 3.2. Changepoint analysis

Four changepoints were identified for both datasets (Fig. 4). A changepoint was identified at Site U1534 in the Subantarctic Zone at ~250 kyr at Termination III (TIII, MIS 8 to 7). The change was driven predominantly by an increase in Acantharea and a decrease in Phaeodaria (RPD; Fig. 5). At Site U1536 in the SIZ a changepoint was identified during MIS 5 (Fig. 4). This was driven by an increase in Acantharea and Phaeodaria (PR² and RPD), a decrease in Polycystinea (PR²) and an increase in Taxopodida (RPD; Fig. 5). Two changepoints were identified at Site U1538 near the modern sea-ice margin. The first change occurred at ~200 kyr using both sets of alignments (Fig. 4) and was predominantly driven by an increase in Polycystinea (PR² and RPD) and

Taxopodida (RPD). The second change occurred at ~12 kyr, with a decrease in Acantharea and Polycystinea (PR² and RPD), and an increase in Phaeodaria (PR² and RPD) and Taxopodida (RPD; Fig. 5)

## 3.3. Cluster analysis

Cluster analysis for the Subantarctic Zone (U1534) indicated that for both datasets, there was an assemblage composition change after MIS 11 (411 kyr, Fig. 6a). After this time, there appears to have been less variability in assemblages, except for one sample (0.7 kyr), for which reads were assigned to Acantharea and Phaeodaria using RPD, while there were no reads assigned using PR².

In the modern sea ice zone (U1536), cluster analysis indicated that the 0.1 kyr assemblage was the more distinct assemblage using PR² assigned reads, while for RPD the 300 kyr assemblage was the most distinctive (Fig. 6c). There was little agreement between the cluster analyses using the two different datasets at this site.

At Site U1538, at the modern sea ice margin, cluster analysis of the assigned reads for both datasets indicated that the assemblages of the Holocene were the most distinct group. However, the exact samples in which distinct assemblages were determined varied. There was agreement that the assemblages of MIS 6 were similar and that MIS 5e (126 kyr) clustered with MIS 6 for both databases.

The cluster analysis indicates that, for all sites, the changes detected were shifts between two assemblage composition types, and largely clustered as glacial and interglacial samples. Samples representing times before and after the changepoints were present in the clusters, indicating that the compositional shifts were associated with the G-I cycles.

# 4. Discussion

## 4.1. Comparison of PR² and RPD datasets and outputs

The RPD contained all 4532 Radiolarian and Phaeodarian sequences from PR² plus an additional 377 sequences from NCBI, totalling 4909 sequences – a sizable database for a single taxonomic group. However, the fact that exclusively RP group taxa are contained in RPD could introduce reference bias, causing sequences to align with RPD instead of other groups if a larger, more taxonomically diverse database were used (e.g., if a sequence covers a more conserved section of the 18S rRNA gene). To address this, the IODP Exp. 382 shotgun data were also aligned to the more comprehensive PR² database, which includes 221,085 sequences from 10 supergroups of eukaryotic plankton. As expected, more reads could be assigned taxonomy after aligning using PR² (3 times higher). The sedaDNA-derived RPD assemblages of both the RPD and PR² datasets were relatively consistent, and there was good agreement in the relative abundances of Acantharea, Phaeodaria, and Polycystinea between the two databases for each sample, but not for Taxopodida (Table 1; Fig. 4).

Taxopodida are little-known, and their siliceous skeletons consist only of spicules (Suzuki and Not, 2015). Currently, only one species is described, and the group has undergone the least morpho-molecular work compared to the other RP groups (Biard, 2022). The discrepancy between the relative abundances of Taxopodida between the RPD and PR² results could be due to the low occurrence of assigned reads (RPD –

<caption>**Table 1**</caption>

Total number of assigned reads per group at each site using the PR² and RPD reference databases.

<table><thead><tr><th>Database</th><th>Site</th><th>Acantharea</th><th>Phaeodaria</th><th>Polycystinea</th><th>Taxopodida</th><th>Total</th></tr></thead><tbody><tr><td rowspan="4">PR<sup>2</sup></td><td>U1534</td><td>44</td><td>26</td><td>259</td><td>0</td><td>329</td></tr><tr><td>U1536</td><td>14</td><td>40</td><td>113</td><td>4</td><td>171</td></tr><tr><td>U1538</td><td>93</td><td>230</td><td>512</td><td>4</td><td>839</td></tr><tr><td>U1534</td><td>30</td><td>26</td><td>106</td><td>2</td><td>164</td></tr><tr><td rowspan="2">RPD</td><td>U1536</td><td>17</td><td>35</td><td>93</td><td>9</td><td>154</td></tr><tr><td>U1538</td><td>40</td><td>42</td><td>181</td><td>20</td><td>283</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>Fig. 2. Total reads produced by a) RPD and b) PR² for each Rhizarian group, and the relative proportion of the total group reads.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 3. Comparison of the relative abundances for a) Acantharea, b) Phaeodaria, c) Polycystinea, and d) Taxopodida, resulting from running the data against both databases. Black – PR²; Grey – RPD.</figcaption></figure>

5.2 %; PR².- 0.6 %, Fig. 2). Such low occurrences may account for the low correlation between the RPD and PR² Taxopodida relative abundances (Fig. 3, Table 2).

Polycystinea skeletons are well-preserved in sediments (de Weaver et al., 2001), while the soft-tissue-only and strontium-shelled RP are

rarely preserved (Decelle et al., 2012). Good preservation of Polycystinea was also observed in this study (a higher number of reads were assigned to Polycystinea than to the other groups in both datasets). This suggests that the group's DNA preserves well in the sediment. Possibly, the surrounding siliceous skeletons offer protection from predation and
<caption>**Table 2**</caption>

Pearson's correlations between the relative abundances of the two databases (PR² and RPD) assigned reads for each taxonomic group at the three IODP Exp. 382 Sites.

<table><thead><tr><th>Group</th><th>U1534</th><th>U1536</th><th>U1538</th></tr></thead><tbody><tr><td>Acantharea</td><td>0.08</td><td>0.26</td><td>0.41</td></tr><tr><td>Phaeodaria</td><td>0.67</td><td>0.26</td><td>0.5</td></tr><tr><td>Polycystinea</td><td>0.47</td><td>0.82</td><td>0.77</td></tr><tr><td>Taxopodida</td><td>-</td><td>0.21</td><td>0.11</td></tr></tbody></table>

digestion, and provide greater weight for accelerated sinking through the water column to the sea floor. The high binding affinity of DNA to the silica, in this case skeletal material, may also play a role in favouring Polycystinea DNA preservation, as suggested by Armbrecht et al. (2020, 2023). Further, some Rhizarian groups such as Foraminifera and the Polycystinea group Collodaria, are known to have high copy numbers (Decelle et al., 2014; Biard, 2022). High copy numbers may lead to an over-representation of a group's abundance within a meta-barcoding survey, a possible contributor toward high Polycystinea abundance within this study.

Phaeodaria are rarely preserved in sediments due to their hollow skeleton being susceptible to silica dissolution (Erez et al., 1982). They are most commonly (but infrequently) found in recent sediment deposits from the Late Quaternary, but their delicate and porous skeletons mean they rarely survive lithification (Dumitrica and Hollis, 2004). However, in the modern oceans, they are frequently found in plankton tows, including in the Southern Ocean close to Antarctica (Morley and Stepien, 1984, 1985). The presence of Phaeodarian DNA in 78 % of our sediments, at abundances of between 10 and 40 % as far back as 450 kyr, is somewhat remarkable. While there may be little to no physical fossil evidence of this group within the core material, the abundance of their DNA in the sediments confirms their presence, potentially in much higher abundances than the fossil record indicates (Fig. 3). While this group has often been overlooked due to poor preservation in sediments, the results of this study indicate that *sedaDNA* is useful for generating more information on Phaeodaria.

For both datasets, the number of assignments at the species level was low (<5 % of total assignments) and so did not produce enough reads to perform cluster or changepoint analyses. Thus, in this study, we have

focused on the composition of the RP group at the Class level, and assessed the contribution of the groups to the assemblage composition.

## 4.2. Comparison of Changepoint (CP) and cluster analysis

In this study, the CP analysis provided information on changes within the RP group (Class level), and the cluster analysis helped assess if those changes were permanent shifts in assemblage composition, or related to cyclic G-I climate conditions (i.e., did similar assemblages before the CP occur again at any stage after the CP?). We identified four changes in the assemblages that were common to both datasets. Each change was associated with a warm period (MIS 7, 5 and 1). However, the cluster analysis confirmed that these changes were likely related to G-I cycles, and that there was a return to glacial assemblages as conditions cooled again.

The results of this study indicate that changepoint analysis was able to detect rapid and distinct changes. In this case, changes were detected at termination events when there was rapid warming after a glacial peak. However, CP analysis could not detect any significant changes in assemblages over the more gradual shifts associated with the slow cooling toward glaciations. Cluster analysis assisted in interpreting these more gradual shifts, revealing that lower-level clusters (those with higher similarity) often comprised assemblages from the same glacial period, although not exclusively.

## 4.3. Temporal changes in RP group assemblages of the Scotia Sea

The changes in the RP group assemblage compositions at the Class level from the three core sites over the last 500 kyr were centred around interglacial periods and termination events. The temporal resolution of the study was low (~25 ky on average), and with greater resolution, it may be possible to identify other changepoints, particularly associated with the more gradual glaciations. However, even at this low resolution, it is clear that rapid assemblage changes occur concurrently with warming events.

During MIS 7, the initial impact of warming on the RP group assemblages occurred at the northernmost site (U1534) in the modern Subantarctic Zone (increase in Acantharea (both datasets) and Polycystinea (PR²), decrease in Phaeodaria (RPD)). A brief cool period was

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Timing of changepoints identified for each site (U1534, U1536 and U1538). Changepoints are based on relative abundances of the PR² and RPD assigned reads, showing their association with marine isotope stages (MIS) and EPICA Dome C (EDC) temperature anomalies for the last 500 kyr. Changepoints occur at Termination 1 and 3, and during warm interglacial events, MIS 7 and 5.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Changepoint analysis results for sites a) U1534 b) U1538 and c) U1536, including the variability trend of each Rhizarian group, the overall probability of a changepoint and the calculated error for each Rhizarian groups changepoint. The dashed line indicates identified changepoint. The left side panel are the changepoint results for the PR² alignments, and the right-side panel are the changepoint results for the RPD alignments.</figcaption></figure>

followed by a second MIS 7 warming event at ~225 kyr, and this led to a change in the RP group assemblages further south (U1538) at the modern sea-ice margin (increase in Acantharea, Phaeodaria and Polycystinea, both datasets). The warmest of the interglacials, MIS 5e, saw a change in assemblages at the southernmost site (U1536) within the modern sea-ice zone (Increase in Acantharea and Phaeodaria (both datasets), decrease in Polycystinea (PR²)). During MIS 1, a change

occurred at the modern sea-ice margin (U1538; Increase in Phaeodaria and decrease in Acantharea and Polycystinea, both datasets). Interestingly, Acantharea was the only group involved in all detected changepoints, and future work may target understanding their association with and sensitivity to environmental perturbations.

There was no indication of any change in the RP group contributions to assemblages outside of the variation seen across G-I cycles. This aligns
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Cluster analysis performed for PR² and RPD datasets at Sites a) U1534, b) U1536, and c) U1538. The red 'x' denotes samples with zero assigned reads. Each change is represented by either the red or blue boxes; each box denotes samples between which a significant change in assemblage composition was determined based on changepoint analysis (red denotes the first changepoint at that site, blue the second changepoint at that site). Each sample name denotes the Marine Isotope Stage, followed by G-Glacial, I-Interglacial or T-Transition, and then age (kyr). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

well with the findings of Pérez et al. (2021), who showed that the
oceanography of the region experienced a major change in circulation
patterns around 600 to 400 kyr and has remained in a similar state since
then. Late Pleistocene interglacials have been demonstrated to have
individual signatures in the Southern Ocean (Cortese et al., 2007), with
some of them resulting in distinct large-scale oceanographic reorgan-
isations in the Southern Ocean. Water mass shifts associated with these
reorganisations have been hypothesised for very high latitude sites (e.g.,
in the Ross Sea, Marschaleck et al., *In Prep*). This observation might
explain the occurrence of assemblage shifts during interglacials MIS 7, 5
and 1 at our study location.

In addition to temporal differences, the spatial distribution of the four identified changepoints indicated that changepoint occurred concurrently in more than one location. It is possible that each of the four times of change saw the RP groups at the different sites being affected by environmental changes and oceanographic conditions to varying levels. Site U1538, located at the modern sea-ice margin, was the only site to experience two changepoints in RP assemblages during the last 450 kyr. This may be due to the north and south displacement of the sea-ice margin and seasonal sea ice cover across glacials and interglacials (Howe et al., 2002; Xiao et al., 2016) which would impact the conditions present during the study period, and thus strongly influence the high-rank assemblage present at a given time.

The study period covers a time interval during which no major RP
group extinctions at the species level are known to have occurred, which
would have greatly altered the assemblage composition. Abundances of
*Stylattractus universus*, the last known biostratigraphic Polycystinea

species to go extinct, had significantly declined in the Southern Ocean by
500 kyr (Lazarus, 1990, 1992). The last appearance of *S. universus* was
~430 kyr, although this is not well constrained due to this species'
infrequent and sporadic appearances in sediments around this time
(McKay et al., 2019). This bioevent was evident in the fossil record at the
study sites. However, the abundances were very low (Weber et al.,
2021b), and so the disappearance of this taxon from the radiolarian
community would likely not have caused a systematic assemblage shift
in the region. A larger shift in assemblages due to an extinction event
would have occurred prior to the study period, with the extinction of
*Antarctissa cylindrica*, which occurred at ~0.65 Ma (McKay et al., 2019).
No other major extinction events that could have significantly altered
assemblage composition are known to have occurred since then.

This study suggests that the RP group assemblages have remained relatively stable over the last 500 kyr and have not shown variations outside of what was related to the G-I cycles. Today, the RP group is a significant part of the marine zooplankton community (Biard et al., 2016; Armbrecht et al., 2022), likely due to population and evolutionary plasticity that allows them to adapt and survive through environmental changes, and has allowed them to be part of the marine food web since the Cambrian. This adaptability may be crucial as ocean conditions shift due to global warming, potentially enabling the RP group to continue as significant contributors to the marine zooplankton community.

**5. Conclusions**

The RP-specific reference sequence database produced similar results
to those of the larger, more comprehensive PR² when using the relative abundances of the Radiolarian and Phaeodarian groups to understand community changes over the last 500 kyr. The consistency of the results produced by the two datasets not only strengthens confidence in our findings, but also validates the use of smaller, taxa-specific reference datasets such as the RPD. The results of the Class-level changepoint analyses for the two databases were in close agreement, and indicate that RP group assemblages have been relatively stable in the Scotia Sea over the past 500 kyr. Their variability over that time has been primarily related to G-I cycles and the fluctuations in oceanographic conditions related to those cycles. The apparent stability suggests that the RP groups are robust and have evolved life strategies that are well-adjusted to the variability in environmental conditions over the study period.

### CRediT authorship contribution statement

V. Lowe: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. T. Biard: Writing – review & editing, Methodology, Data curation, Conceptualization. G. Cortese: Writing – review & editing, Conceptualization. I. Hernández-Almeida: Writing – review & editing, Conceptualization. K.-A. Lawler: Writing – review & editing, Conceptualization. L. Armbrecht: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Funding sources

Funding for this research was provided by the Australian and New Zealand International Ocean Discovery Program Consortium (ANZIC) Post-Cruise Analytical Funding (PC_LArmbrecht_0120) and ANZIC IODP Legacy Analytical Funding (AILAF2321_LArmbrecht). Additionally, funding was provided to VL by the Australian Centre for Excellence in Antarctic Science. GC contribution was supported by the New Zealand Ministry of Business, Innovation and Employment through the Antarctic Science Platform (ANTA1801) and the Global Change through Time Programme (GCT SSIF, contract C05X1702).

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Linda Armbrecht reports financial support was provided by Australia and New Zealand International Ocean Discovery Program Consortium. Vikki Lowe reports financial support was provided by Australian Centre of Excellence in Antarctic Science. Giuseppe Cortese reports financial support was provided by New Zealand Ministry of Business Innovation and Employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This research used samples provided by the International Ocean Discovery Program (IODP). Funding for this research was provided by the Australian and New Zealand International Ocean Discovery Program Consortium (ANZIC) Post-Cruise Analytical Funding (PC_LArmbrecht_0120) and ANZIC IODP Legacy Analytical Funding (AILAF2321_LArmbrecht). LA was further supported by an Australian Research Council (ARC) Discovery Early Career Researcher (DECRA) Fellowship (DE210100929). VL was supported by the Australian Centre of Excellence in Antarctic Science (ACEAS) through a Visiting Fellowship to the Institute for Marine and Antarctic Studies, University of Tasmania, 2023. We thank the IODP Exp. 382 crew and technical team for assistance during sampling.

We would like to thank the reviewers of this paper for their thoughtful and constructive comments, which helped improve the clarity and quality of this manuscript.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.marmicro.2025.102500.

### Data availability

I have attached the data at the attach file step

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