Life on Plastics: Deep-Sea Foraminiferal Colonization Patterns and Reproductive Morphology

Abstract

Plastic debris has become a persistent feature of deep-sea ecosystems, yet its role as a habitat for calcifying organisms remains poorly understood. Foraminifera colonization has been observed in significant numbers on plastic surfaces, suggesting that these materials serve as novel and significant deep-sea colonization sites for these abundant calcifying organisms. This study uses deep-sea experimental plastic substrates to examine the colonization and reproductive morphology of the benthic foraminifera Lobatula wuellerstorfi across three locations: Station M (4000 m), Oregon OOI (575 m), and Southern Hydrate Ridge (774 m). A total of 482 individuals were analyzed for morphometric traits, including proloculus diameter, to investigate reproductive morphotypes. The Oregon samples displayed a clear bimodal proloculus size distribution, consistent with alternating reproductive strategies, while Station M populations exhibited a broader, less defined bimodal distribution skewed toward megalospheric forms. A weak but significant increase in proloculus diameter over deployment duration was observed at Station M, suggesting a possible influence of experiment duration and/or substrate maturity and environmental conditions. These findings demonstrate that plastics can serve as persistent colonization sites for deep-sea foraminifera, offering a unique experimental platform to investigate benthic population dynamics, ecological plasticity, and potential geochemical implications, as well as the broader impacts of foraminifera on deep-sea biodiversity and biogeochemical cycling.

1. Introduction

It is estimated that between 4.8 to 12.7 million metric tons of plastic enter the ocean each year, with untold quantities settling to the deep-sea floor [1]. Plastic accumulation represents the latest major anthropogenic disturbance, altering deep-sea habitats and affecting benthic communities, including foraminifera [2,3,4]. Plastics provide novel substrates which may significantly alter patterns of colonization and succession in benthic faunal communities, influencing both biodiversity and species distributions, i.e., [2,3,4]. The long-term breakdown of plastics in these environments is also a poorly understood process, raising concerns about the effects of plastic degradation and the potential impacts of the release of associated additives/contaminants on the global ocean carbon cycle [5,6]. Plastics are primarily polymeric materials that are often considered chemically inert; however, they commonly contain hundreds of additives and trapped contaminants. These additives, including plasticizers, stabilizers, UV protective coatings, and flame retardants, can leach into the surrounding environment, acting as endocrine disruptors, toxins, or nutrients for microbial communities. Deep-sea macroplastics remain understudied as habitats, especially for calcifying taxa like foraminifera. Recent studies have demonstrated that plastics can serve as early-stage colonization substrates for meiofauna and foraminifera, and evidence suggests that plastic incorporation into foraminiferal structures may already be leaving a stratigraphic signal [7,8,9]. Despite widespread concern about microplastic toxicity, the biological colonization of deep-sea macroplastics, especially by calcifying organisms, remains extremely understudied [8]. This research helps fill that gap, offering new insights into habitat use, substrate affinity, and morphological expression on artificial surfaces.

Epibenthic foraminifera such as Lobatula wuellerstorfi are well-established bioindicators, sensitive to environmental shifts and bottom-water geochemistry, i.e., [10,11]. Foraminifera are increasingly recognized as sensitive bioindicators of environmental disturbances, including anthropogenic pollution [see review in [8]]. Their consistent abundance in deep-sea ecosystems provides a reliable baseline for assessing community changes caused by environmental disturbances and ecological change. Deployments of Seafloor Epibenthic Attachment Cubes (SEA³s) have effectively captured colonization dynamics of benthic foraminifera, demonstrating their utility in deep-sea investigations, i.e., [3,4]. Notably, plastic-derived compounds have been detected in foraminiferal tests [7,9] and cytoplasm [12], suggesting biological interactions with plastic substrates beyond simple settlement. Previous studies have documented the colonization of over 1000 individual Lobatula wuellerstorfi on deep-sea plastics, revealing that these epibenthic foraminifera—commonly associated with well oxygenated environments—can thrive in low-oxygen habitats [2]. Subsequent work expanded SEA³s into the abyssal settings at Station M (4000 m water depth) at for durations [3]. At Station M, naturally occurring elevated substrates, such as Hyalonema glass sponge stalks, also host significant populations of Lobatula wuellerstorfi morphotypes, providing a natural analog for comparisons with colonization on artificial substrates [13,14]. Comparisons between plastic substrates and adjacent sediments demonstrated that foraminiferal communities on plastics were compositionally distinct [3]. These differences provide valuable insights into potential source populations, recruitment strategies, and the influence of substrate type on benthic colonization.

This study presents an overview of opportunistically deployed SEA³ experimentation across different environmental settings—including abyssal plains (Station M) [3,4], methane seep environments (Hydrate Ridge) [2], and productive upwelling ecosystems (OOI Moorings; Figure 1)—enabling comparative analyses of colonization patterns and morphometric variability in Lobatula wuellerstorfi populations colonizing SEA³ plastics (Figure 2). Morphological characterization using MicroCT 2D x-rays facilitated visualization of internal structures unable to be viewed without mechanical breakage and documented differences in morphologies—potentially associated with reproduction—of the populations examined herein.

Recent taxonomic revisions have reclassified Cibicidoides wuellerstorfi (Schwager, 1866) as Lobatula wuellerstorfi (originally referred to as Anomalina wuellerstorfi in Schwager, 1866). In this study, this updated nomenclature will be followed. The more traditional test morphology of L. wuellerstorfi—characterized by a flattened, biconvex test and consistently elevated and pore-free sutures—is well represented in the Oregon OOI and Southern Hydrate Ridge (SHR) specimens, as illustrated in Figure 3. Similar morphological variability has been reported across environmental gradients in the Denmark Strait, where chamber expansion, whorl number, and aperture shape in C. wuellerstorfi varied systematically with food availability, hydrodynamics, and carbonate chemistry, consistent with ecophenotypic responses [15]. Specimens collected from Station M, which exhibit consistent morphological differences from classical descriptions of L. wuellerstorfi [16] but were genetically identified within that clade [3], are herein referred to as Lobatula wuellerstorfi morphotype A. The morphological distinction of Station M specimens, despite clear genetic identification as L. wuellerstorfi, highlights the species’ capacity for substantial ecophenotypic plasticity. This variation likely reflects environmentally driven morphologic responses rather than taxonomic divergence. While previously designated as Cibicidoides wuellerstorfi var. lobatulus (in [3]), the reclassification of this species into the genus Lobatula and the existence of Lobatula lobatula (Walker & Jacob, 1798) necessitate the use of a morphotype designation. For clarity and consistency, L. wuellerstorfi morphotype A will be used throughout this manuscript when discussing Station M specimens, as described in [3]. Lobatula wuellerstorfi morphotype A displays a plano-convex test with trochospiral coiling, less pronounced chamber sutures, and moderate to large unevenly distributed pores on both the spiral and umbilical sides (Figure 4). The aperture is interiomarginal with a prominent imperforate lip, often extending toward the spiral side. Compared to typical L. wuellerstorfi, these tests are less flattened, and chamber sutures are not raised or consistently imperforate [4]. Some individuals exhibited slightly concave sides consistent with qualitative impressions of substrate conformity, though this observation was not systematically assessed [see Figure 4 and examples in 2, 3, 4]. This morphology matches that of C. lobatulus described in [13,14], although genetically these specimens align with L. wuellerstorfi.

Some foraminifera exhibit generational alternation in reproductive mode. Sexual reproduction occurs through gametogenesis, where gametes combine to form zygotes that develop into diploid agamont adults. These produce microspheric individuals with small proloculi (the proloculus is the first chamber formed and in L. wuellerstorfi is the spherical feature visible in the center in X-ray view; see Figure 5). When mature, these in turn reproduce asexually via multiple fission, producing megalospheric offspring with large proloculi [17]. This generational switching may be facultative or obligatory, but remains poorly understood, especially for deep-sea taxa where direct observation is limited. Because proloculus size is thought to reflect reproductive mode and remains fixed after chamber formation, it has been widely used as a proxy for assessing life history and reproductive dynamics. SEA³s offer a promising platform for reconstructing recruitment and dispersal patterns based on such morphologic traits.

However, recent studies advise caution, as proloculus size may also vary with environmental parameters, such as salinity [18], and fitness, rather than external conditions [19]. Final test size has also been shown to correlate with initial chamber volume, implying that terminal morphology may result passively from early growth [20]. Together, these findings emphasize the importance of developmental context when using proloculus size to infer reproductive strategies in natural populations.

By understanding how foraminifera interact with plastic substrates at both the morphological and the ecological level, this work provides insights into the resilience and adaptation of benthic microfauna in a changing ocean, highlighting potential pathways through which anthropogenic materials may influence deep-sea biodiversity and carbon cycling processes. This study evaluates the morphometric variation of Lobatula wuellerstorfi (and morphotype A) populations on plastic substrates deployed at multiple deep-sea locations in the Pacific and over varying durations.

2. Materials and Methods

Morphometric variables, station information, and deployment details are summarized in

Table 1. Summary of deployment locations, recovery details, and analyzed Lobatula wuellerstorfi individuals from plastic and natural substrates. Oregon deployments include experimental plastic substrates recovered from the Coastal Endurance Oregon Offshore Surface Mooring (CE04OSSM) [21] and Southern Hydrate Ridge (RS01SUM1) [22]. Station M deployments include experimental SEA³ substrates from previous studies [3,4] and natural Hyalonema sponge stalks [13,14]. “Days Deployed” refers to the total time substrates remained on the seafloor. Natural substrates, noted as NS, were collected from 14. The number of foraminifera analyzed represents individuals used in the morphometric analysis. Note that the OOI and SHR data are combined when referred to as Oregon Margin.

Deployment Location	Collection Details	Water Depth (m)	Days Deployed	Number of Foraminifera Analyzed	Date of Recovery	Latitude (N)	Longitude (W)
Oregon OOI	CE04OSSM	575	184	17	27 September 2022	44°22′52.0	124°57′21.6
Oregon OOI	CE04OSSM	575	264	153	21 October 2019	44°22′40.8″	124°56′45.6″
Oregon OOI	CE04OSSM	575	325	31	1 June 2021	44°22.721″	124°56.705′
Southern Hydrate Ridge (SHR)	RS01SUM1	774	347	26	15 August 2021	44°34′08.8″	125°08′53.2″
Station M	3	4000	365	34	13 November 2017	34°50′	123°00′
Station M	4	4000	703	35	17 October 2019	34°50′	123°00′
Station M		4000	1095	98	17 October 2022	34°50′	123°00′
Station M	Beaulieu SE (2001b)-Stalks 3, 4, and 6 from DV 2828 and 2831	4000	NS	86	17 and 20 September 1994

and detailed in Supplementary Table S1. It is important to note that the number of foraminifera collected from the SEA³s was not standardized by the surface area of the substrates or the number of SEA³ units processed during each deployment. Instead, this dataset serves as a means to assess significant populations for morphological analysis, rather than as a quantitative measure of colonization densities and patterns.

2.1. SEA³ Deployment and Recovery

Plastic colonization experiments were conducted using Seafloor Epibenthic Attachment Cubes (SEA³s), which are 10 cm cubes constructed from welded galvanized steel and coated in Performix Brand Plasti Dip^® (Blaine, MN, USA) to minimize corrosion and surface abrasion. Each frame was wrapped in commercially available TENAX^® Hardware Cloth (Baltimore, MD, USA), composed primarily of high-density polyethylene (HDPE), with ~2.4 mm thick plastic strands arranged in a 1.27 cm × 1.27 cm square grid. These materials were selected for their durability and mechanical stability in marine environments and are commonly used in aquaculture and erosion control applications. SEA³s were deployed at abyssal depths at Station M (4000 m) and on the continental margin off the Oregon on Ocean Observatories Initiative (OOI) Moorings (575 m) and Southern Hydrate Ridge (SHR) infrastructure, with deployment durations ranging from 184 to 1095 days (Table 1). Recoveries were performed using remotely operated vehicles (ROVs) at Station M and SHR (Figure 2A–C), or via acoustic release from the OOI Moorings (Figure 2D). Colonization of the plastic substrates by Lobatula wuellerstorfi is documented in Figure 2E,F, including individuals tightly adhered to the mesh and in some cases exhibiting test morphologies that conformed to the plastic surface.

Previous deployments using the same SEA³ design (2–4) have demonstrated consistent colonization by foraminifera across various polymer types, including HDPE, polyethylene, polypropylene, and plastic welding rods made of PET, ABS, PVC, and fiberglass composites. No significant differences in settlement numbers or species composition were observed across polymer types or between elevated and sedimented positions. Colonization has never been observed directly on the Plasti Dip^® coating in any previous deployments; foraminifera consistently settled only on the mesh material.

In [4], similar colonization densities were also observed on a variety of experimental substrates made from plastic welding rods, including silicon polymer and PET (polyethylene terephthalate), ABS (acrylonitrile-butadiene-styrene), PVC (polyvinyl chloride), polypropylene (PP), and fiberglass poles. While no conclusions were drawn about non-foraminiferal taxa, these results suggest that foraminiferal colonization is not strongly constrained by the polymer composition of available substrates.

Deployment and recovery of SEA³s were subject to the logistical constraints of larger oceanographic expeditions, including maintenance of OOI and deep-sea research cruises led by the Monterey Bay Aquarium Research Institute (MBARI, Monterey Bay, CA, USA). As a result, the deployment strategy was necessarily opportunistic, with SEA³s deployed during available seafloor operations and recovered as ship time, weather, and ROV access permitted. This led to variability in the number of SEA³s deployed and recovered at each site, as well as unequal deployment durations between regions. While this approach limited the ability to maintain a fully balanced experimental design, it provided a unique opportunity to collect large-scale morphological data across a range of timeframes and environmental settings. The resulting dataset contains unequal numbers of foraminifera analyzed (Figure 6) and is best interpreted as a morphometric survey of colonizing populations of L. wuellerstorfi, rather than a direct measure of colonization rate or recruitment efficiency.

At Station M (4000 m water depth), naturally occurring elevated substrates were collected to investigate foraminiferal colonization patterns. These substrates consisted of Hyalonema glass sponge stalks previously described in [13], specifically Stalks 3, 4, and 6 collected on September 17 and 20, 1994 during dives DV2828 and DV2831. These sponge stalks provide persistent, elevated hard surfaces that function similarly to SEA³ experimental platforms by raising organisms above the sediment–water interface. Previous studies demonstrated that glass sponge stalks at Station M support substantial populations of the Lobatula wuellerstorfi morphotype A, making them valuable natural analogs for evaluating colonization behavior on artificial substrates. In this study, natural substrates are included alongside experimental plastics and plotted with the label Natural Substrates (Figure 6 and Figure 7). Experimental plastic substrates at Station M included materials recovered after approximately 365 days [3], 703 days [4], and 1095 days.

Off the Oregon Margin (575 m depth), foraminiferal colonization was assessed from experimental SEA³s attached to mooring infrastructure at the Coastal Endurance Oregon Offshore Surface Mooring, Newport, OR, USA [21]. At Southern Hydrate Ridge (774 m depth), SEA³s were deployed and recovered around methane seep environments at the Oregon Margin Southern Hydrate Summit 1 Seafloor site [22], characterized by active venting of methane-rich fluids and dense chemosynthetic biological communities. Whenever possible, these populations are discussed separately, but often OOI and SHR data are combined when referred to as Oregon Margin (see grouping in Figure 8).

Given that the number of SEA³s deployed and recovered varied between locations and timepoints, the results presented herein focus on a subset of Lobatula wuellerstorfi individuals recovered and analyzed with 2D X-ray imaging with MicroCT (totals reported in Table 1), rather than normalizing colonization rates by substrate area or the number of platforms deployed. As many specimens as could be obtained and were intact were included in the analysis presented here. It should be noted that some deployments had more substrate examined than others based on recovery and transportation (i.e., roughness in shipping), resulting in different numbers of foraminifera per deployment analyzed herein, but these numbers do not likely reflect total colonization differences between experimental deployment duration.

2.2. Imaging and Morphological Measurement

Recovered foraminifera were gently rinsed and transferred to microscope slides to allow a consistent orientation scheme when imaging. Each specimen was aligned with the spiral side down, the proloculus toward the center of the image, and the ultimate chamber directed to the left or right (depending on coiling direction; see Figure 5). This setup ensured consistent measurement angles across all individuals. Imaging was performed using a ZEISS Xradia Micro-CT instrument (Carl Zeiss AG, Oberkochen, Germany), capturing high-resolution radiographic images (2D X-ray images views) of the tests. Rather than fully reconstructing CT volumes, individual X-ray images were imported into the image processing software, ImageJ 1.54g, where scale bar-based calibration was applied and measurements were made. Figure 5 shows some typical foraminifera in X-ray cross-sectional view. Maximum test diameter and proloculus diameter were measured manually from 2D X-ray images in ImageJ to efficiently process a large number of specimens without the time-intensive reconstruction and segmentation required for full 3D volumetric analysis. Each specimen was assigned to a statistical group (SG) based on deployment location and duration to allow comparison across environmental settings and water depth (WD). The duration of seafloor exposure is recorded as days deployed (DD). To evaluate reproductive expression, the proloculus-to-test percentage (PT%) was calculated by dividing the proloculus diameter (PD)—the size of the initial chamber—by the maximum test diameter (MTD), which reflects the largest cross-sectional dimension of the test. Additional measurements included chamber-specific diameters: the ultimate diameter (UD), penultimate diameter (PnD), and antepenultimate diameter (APD), representing the last, second-to-last, and third-to-last formed chambers, respectively. Internal whorl diameter (IWD), the width across the inner coiling of the test, provides information about early test development, while the number of chambers (NC) represents the total count of chambers formed throughout growth. These variables collectively capture test architecture and growth trajectories across morphotypes and environmental contexts. These measurements were documented for all 480 specimens analyzed in this study (Supplementary Table S1 through manual measurements, as indicated in number 4 of Figure 5).

In addition to morphometric measurements, scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) was used to visualize the materials covering the foraminifera at the time of collection (Figure 2F and Figure S1A–D). The plastic materials were snipped around selected specimens, which were then attached to SEM stubs with silver conductive paint (p/n. 12686-15) from Electron Microscopy Sciences and (without being plated i.e., with gold) inserted into the imaging apparatus (Figure S1E,G). Imaging revealed the presence of fine-grained minerals (Figure S1F,H), likely adhered with protoplasm and interpreted as clay cysts.

2.3. Data Processing and Statistical Analysis

The total number of foraminifera included from each location and duration grouping is reported in Figure 6. Proloculus diameters were binned into 5 μm intervals to explore morphotype distribution patterns between Station M and the Oregon Margin—it should be noted that Oregon Margin refers to the combination of Oregon OOI and Southern Hydrate Ridge (SHR) specimens (Figure 7). To investigate temporal trends, data were grouped by deployment duration and location (Figure 8). Regression analysis and Pearson correlation were used to assess the relationship between morphologic variables for all 480 individuals in this study (

Table 2. Pearson correlation coefficients (R² values) between morphometric variables measured across Lobatula wuellerstorfi individuals from all samples analyzed in this study. Morphometric variables were measured for each specimen, including abbreviations used throughout the manuscript. Statistical group (SG) corresponds to deployment location and duration and water depth (WD). Days deployed (DD) indicates the length of time each sample remained on the seafloor. Maximum test diameter (MTD) is the largest cross-sectional diameter of the test. Proloculus diameter (PD) reflects the size of the initial chamber, and proloculus-to-test percentage (PT%) is calculated as the ratio of PD to MTD. Internal whorl diameter (IWD) captures early test development across the inner coil. Ultimate (UD), penultimate (PnD), and antepenultimate diameters (APD) refer to the diameters of the last, second-to-last, and third-to-last formed chambers, respectively. Number of chambers (NC) is the total chamber count in each test.

	SG	WD	DD	PT%	MTD	PD	UD	PnD	APD
SG
WD	0.63
DD	0.16	0.69
PT%	0.03	0	0.69
MTD	0.11	0	0.1	0.3
PD	0.02	0.02	0.13	0.2	0.15
UD	0.17	0.01	0.05	0.28	0.93	0.14
PnD	0.14	0.01	0.03	0.24	0.8	0.12	0.82
APD	0.14	0.01	0.05	0.29	0.94	0.14	0.91	0.82
IWD	0.07	0	0.1	0.08	0.28	0.83	0.28	0.24	0.27
NC	0.02	0.05	0.05	0.66	0.18	0.22	0.12	0.11	0.14

), as well as those only from Station M (

Table 3. Pearson correlation coefficients (R² values) for morphometric variables measured in Lobatula wuellerstorfi morphotype A individuals from Station M (4000 m depth). Variables include deployment duration, proloculus diameter, maximum test diameter, chamber diameters (ultimate, penultimate, and antepenultimate), internal whorl diameter, and number of chambers. Strong internal correlations were observed among chamber measurements, while proloculus diameter showed only weak correlation with deployment time. Morphometric variables and corresponding abbreviations for specimens included in the Station M subset analysis. Statistical group (SG) identifies each deployment event. Days deployed (DD) reflects the duration each SEA³ platform remained on the seafloor. Maximum test diameter (MTD) represents the largest diameter measured across the test. Proloculus diameter (PD) indicates the size of the initial chamber, and proloculus-to-test percentage (PT%) is the proportion of PD relative to MTD. Internal whorl diameter (IWD) captures the diameter across the coiled inner portion of the test. Ultimate (UD), penultimate (PnD), and antepenultimate diameters (APD) refer to the final three chambers in reverse sequence. Number of chambers (NC) denotes the total number of chambers present in each specimen.

	SG	DD	PT%	MTD	PD	UD	PnD
SG
DD	0.14
PT%	0.25	0.05
MTD	0.53	0.19	0.52
PD	0.29	0.14	0	0.32
UD	0.49	0.17	0.49	0.94	0.3
PnD	0.34	0.14	0.39	0.74	0.23	0.75
APD	0.47	0.18	0.5	0.95	0.32	0.91	0.75
IWD	0.37	0.18	0.04	0.5	0.73	0.48	0.37
NC	0.19	0.03	0.71	0.5	0	0.44	0.36

). All statistical analyses and visualizations were performed using Rstudio 2024.12.1-563 and Excel.

3. Results

Colonization abundance data by site and deployment duration are summarized in Figure 6 and Table 1, providing essential context for the morphometric results that follow.

3.1. Morphotype Distribution Across Regions

A total of 480 Lobatula wuellerstorfi specimens were analyzed across seven deployments (Figure 6). Proloculus diameter suggests the presence of reproductive morphotypes (Figure 7) with different patterns between Station M and Oregon Margin samples. The Oregon population displayed a clearly bimodal pattern consistent with microspheric (~15–40 μm) and megalospheric (~55–85 μm) forms (orange bars in Figure 7). In contrast, the Station M population (L. wuellerstorfi morphotype A) exhibited a broader range and a less defined bimodal distribution skewed toward larger proloculus diameters, suggesting a dominant megalospheric signal (blue bars in Figure 7). While there is a bimodal peak in the data, the central portion is not as pronounced as it is for Oregon Margin samples, and it is very difficult to define sphericities based on proloculus diameters in Station M specimens.

3.2. Colonization Abundance by Deployment Duration

Total L. wuellerstorfi colonization varied across both region and deployment time (Figure 6). The number of foraminifera used in this study is not standardized per unit area or per SEA³ substrate, and the number of SEA³s sampled varied between deployments. Therefore, comparisons among deployments (especially those with fewer than 30 individuals) should be interpreted with caution. The highest number of foraminifera included in this study comes from Station M after 1095 days, while Oregon deployments generally contain fewer individuals overall.

3.3. Pyrgo

Pearson correlation analysis was conducted across all 480 individuals in the dataset to assess relationships among measured morphometric variables (Table 2). The significant relationships summarized here are those with R² > 0.75. The strongest relationship existed between maximum test diameter and antepenultimate diameter, with an R² of 0.94. Maximum test diameter was also highly correlated with ultimate diameter (R² = 0.93) and penultimate diameter (R² = 0.80). Individual chamber diameters are correlated with one another, with ultimate and antepenultimate at R² = 0.914, ultimate and penultimate at R² = 0.82, and penultimate and antepenultimate at R² = 0.82. These values suggest consistent covariation among test dimensions related to growth and chamber development. When all foraminifera are considered, proloculus diameter is highly correlated with internal whorl diameter (R² = 0.83), and proloculus-to-test percentage and days deployed have an R² = 0.69.

3.4. Temporal Trends in Reproductive Morphology at Station M

A subset analysis was performed on samples from Station M (208 individuals) to investigate whether these morphometric relationships were consistent at a single site (Table 3). The significant relationships summarized here are those with R² > 0.75. The strongest relationships existed between maximum test diameter and antepenultimate diameter (R² = 0.95) and maximum test diameter and ultimate diameter (R² = 0.94). Again, chamber measurements were highly correlated with ultimate and antepenultimate at R² = 0.91, penultimate and antepenultimate at R² = 0.75, and ultimate and penultimate at R² = 0.75. In only Station M foraminifera, proloculus diameter is highly correlated with internal whorl diameter, with an R² = 0.74, and proloculus-to-test percentage and days deployed have an R² = 0.05. Regression analysis was performed to evaluate the relationship between proloculus diameter and deployment duration for all specimens collected from Station M. The data included specimens from deployments of 365, 703, and 1095 days and individuals colonizing natural sponge stalk substrates (Figure 9). The regression revealed a weak but statistically significant positive correlation between proloculus diameter and days deployed (r = 0.293, p = 2.06 × 10⁻⁶). The fitted trendline indicates an approximate increase of 0.018 μm in proloculus diameter per additional day of deployment. Despite the low trend, the consistent presence of larger proloculus diameters at longer deployments suggests a possible association between substrate exposure time and morphologic expression.

3.5. Proloculus Diameters of All Foraminifera

The proloculus diameter distributions of Lobatula wuellerstorfi were analyzed by deployment duration and site to assess reproductive morphotype expression through time (Figure 8). At the Oregon Margin (OOI and SHR specimens), all deployment intervals exhibited clear bimodal distributions (although less so at 184 days, which may be the result of the low number of individuals assessed), consistent with the presence of both microspheric and megalospheric forms. The 184-day deployment displayed low overall abundance but distinct peaks near ~20–30 µm and ~70–80 µm. The 264-day deployment showed the highest sample size and a pronounced bimodal pattern, with dominant peaks at ~20 µm and ~75 µm. Oregon Margin deployments included those on the OOI for 184, 264, and 325 days at 575 m water depth. Southern Hydrate Ridge specimens were from 347-day deployments at 774 m water depth.

In contrast, specimens from Station M demonstrated broader and less distinct bimodal (365 and 703) or unimodal (1095 and Natural Substrates) distributions. At 365 and 703 days, distributions were skewed toward larger proloculus diameters, with weak separation between potential modes. The 1095-day deployment and natural substrate specimens both exhibited unimodal or overlapping bimodal patterns centered around 65–80 µm, with minimal representation of smaller microspheric forms. The natural substrate samples, in particular, showed a strongly peaked unimodal distribution with a mode around 75 µm. These patterns suggest differences in morphotype representation between regions and with increasing deployment duration, particularly with regard to the apparent decline of microspheric forms at Station M.

3.6. Cyst Coverings

Nearly all L. wuellerstorfi morphotype A specimens from Station M were completely to partially encased in fine-grained mineral coatings or “clay cysts,” which may serve protective or functional roles related to feeding, substrate interaction, or microenvironmental conditions. Using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS), dried tests covered in cysts and attached to plastics were imaged, and it was determined the cyst material was likely a combination of clay particles the foraminifera were able to extract from the water column as they floated past (Figure S1E–H). While most specimens observed in this study appeared to be coated in organic cysts, coated vs. non-coated counts were not recorded.

4. Discussion

The interpretations presented here align with prior observations in deep-sea foraminiferal ecology and morphometrics. Specimens from Station M display morphological traits consistent with descriptions of Cibicides lobatulus [3,13,14]. These subtle but documentable morphological deviations in L. wuellerstorfi morphotype A from Station M—most notably the departure from the typical bimodal distribution of proloculus diameters—reinforce the view that foraminiferal morphology, even within a genetically uniform species, may be highly sensitive to environmental cues.

4.1. Morphology of Foraminifera Colonizing Plastics

The strong correlations observed among test and chamber diameters across all specimens underscore a high degree of internal consistency in foraminiferal growth patterns. Maximum test diameter correlated with chamber diameters, indicating predictable allometric growth trajectory––a pattern observed in other species by [20]. These relationships were robust both across the full dataset and within the Station M subset, indicating that test scaling relationships are preserved across morphological divergence (i.e., morphotype A) and different environmental settings.

The correlation between proloculus diameter and internal whorl diameter (R² = 0.83 in the full dataset; R² = 0.74 at Station M) supports the idea that early growth patterns reflect broader test architecture. The consistent association between proloculus size and final test diameter observed in this study aligns with previous volumetric analyses demonstrating that terminal size is largely a passive outcome of embryonic chamber volume, rather than being due to active modulation of growth rate over time [20]. If this is the case, and proloculus size is influenced by non-physiological factors, including reproductive strategy [19], then final test size is not likely to be as significantly influenced by environmental factors like temperature or oxygen. In fact, foraminiferal size and surface-area-to-volume ratios have been shown to not reliably track oxygen, instead being influenced by resource availability and reproductive strategy [20]. It is possible that environmental conditions at Station M—such as depth, current velocity, or substrate type—may influence morphologic expression (hence proloculus size), although other studies have shown that salinity and temperature have influences on proloculus size [18,23], highlighting the complexity of environmental influences and emphasizing the need for site-specific calibrations.

The disconnect between morphology and genetic identity in these populations underscores the complexity of species delineation in deep-sea foraminifera. However, the observed morphological variability—likely reflecting ecophenotypic responses to local environmental gradients—also highlights the adaptive capacity of L. wuellerstorfi and its potential as a sensitive proxy for past environmental conditions. Test conformity to the shape of the plastic substrates (as seen in [24]) and similar morphologic shifts driven by current strength, food availability, and substrate interaction have been observed in L. wuellerstorfi populations across the Denmark Strait, further supporting the interpretation of these differences as ecophenotypic rather than taxonomic (reported as C. wuellerstorfi in [15]). The observations of proloculus diameter and internal whorl diameter may prove useful in identifying reproductive morphotypes, particularly when only partial specimens are recovered. However, proloculus diameter (R² = 0.14) and proloculus-to-test percentage (R² = 0.05) showed little correlation with days deployed at Station M or Oregon Margin proloculus diameter (R² = 0.13) and proloculus-to-test percentage (R² = 0.20), indicating that overall test structure is not strongly influenced by substrate exposure time alone. Maximum test diameter shows no correlation with days deployed at Oregon Margin (R² = 0.10) and Station M (R² = 0.19), suggesting no significant differences in test size between shorter and longer deployments.

Regression analysis between proloculus diameter and deployment duration at Station M revealed a weak but statistically significant positive relationship (Figure 9), indicating that longer deployments subtly influence proloculus size. While longer deployments may capture changes in reproductive morphotype distributions, this relationship likely reflects a complex interplay of biofilm development, substrate conditions, and environmental stabilization. The contrasting proloculus diameter distributions between Oregon Margin and Station M deployments suggest site-specific differences in reproductive mode expression and morphologic development (Figure 7). Oregon Margin samples exhibit strong bimodality regardless of deployment duration, reflecting the persistent expression of both microspheric and megalospheric forms (Figure 8). This distribution is consistent with classical models of dimorphic reproduction in foraminifera, where alternating generations cycle between sexual and asexual modes, i.e., [17], and may reflect the variable productivity and environmental conditions characteristic of the Oregon continental slope. In contrast, distributions from Station M (Figure 8) show a reduction in microspheric forms and a skewed dominance of megalospheric individuals. While some bimodality is visible in the 365- and 703-day deployments, this pattern becomes increasingly unimodal in the 1095-day and natural substrate samples. The narrow peak in the natural substrate samples centered around larger proloculus diameters suggests a more homogeneous population and possible selection for asexual reproduction in this stable abyssal environment. Similar patterns of morphotype dominance in resource-limited settings have been noted in other deep-sea foraminiferal studies [3].

The observed shift toward unimodal or skewed distributions with increasing deployment time at Station M also implies that prolonged substrate exposure—whether on plastic or biogenic surfaces—may influence morphotype expression. Factors contributing to these morphotype expressions are not able to be determined from this study alone. Potential influences include biofilm development, microbial succession, and longer-term settlement filtering, which have all been proposed as influential in plastic colonization dynamics [7,12,25]. Reproduction within the colonizing population may result in generationally dependent foraminiferal morphologies. For instance, if it were assumed that the first generation of colonizing foraminifera are recruited from the surrounding water, it may be likely that they were produced by sexual reproduction and released as gametes into the water column, ultimately producing agamont adults, which typically have a smaller proloculus. These agamont adults may then reproduce through multiple fission, producing several megalospheric offspring which will have a larger proloculus. At present, it is not possible to determine whether initial recruitment results from sexual reproduction, nor can it be ruled out that additional recruitment occurs continuously throughout the duration of deployment. Currently, no studies have been able to constrain key factors such as the timing of initial colonization, retention, and reproduction, or subsequent recruitment of individual foraminifera, over the course of an experiment.

Laboratory and field studies have demonstrated that benthic foraminiferal responses to microplastic and metal stressors are often taxon-specific and regionally variable, i.e., [26], consistent with the differences observed here between Oregon Margin and Station M samples. Experiments in seagrass found that foraminifera preferentially colonized natural Halophila stipulacea leaves over plastic mimics, primarily attributed to higher microbial abundance and diversity on the natural substrate, suggesting that foraminiferal settlement is not random, but is influenced by substrate-associated microbial communities [25]. Similarly, biofilm development on SEA³s may mediate settlement and morphotype expression. Additionally, the presence of naturally occurring elevated substrates at Station M may support local source populations of Lobatula wuellerstorfi morphotype A, which could facilitate more rapid or consistent colonization of artificial substrates. In contrast, such substrates are less abundant at other locations, potentially limiting nearby source populations. While speculative, this possibility warrants consideration in future studies.

Foraminifera are increasingly recognized as sensitive bioindicators of anthropogenic pollution, including plastic contamination, due to their morphological variability and responsiveness to environmental change [8]. Benthic foraminifera can incorporate plastic-derived compounds inside their tests and cytoplasm, inducing physiological stress and potentially altering biomineralization processes [12]. Additionally, agglutinated foraminifera from the Gulf of Mexico have been shown to incorporate microplastics directly into their test structures [8]. These findings suggest that foraminifera may serve not only as ecological indicators of plastic pollution, but also as stratigraphic recorders of anthropogenic influence in marine sediments.

4.2. Cysts in L. wuellerstorfi from Station M

Cyst formation is a common phenomenon among benthic foraminifera from various depths and habitats (e.g., [27,28,29,30,31]). At Station M, specimens of L. wuellerstorfi morphotype A and Pyrgo spp. were frequently enclosed in loose, sediment-laden coatings that could be gently removed with a fine brush and are. Similar to coatings previously reported, i.e., [3,4,27,28,29,30,31] and have been suggested to aid in feeding (e.g., rhizopodial strand formation; [27]) or chamber formation [29,30]. Observations from specimens collected in this study included extended structures radiating from the central cyst (typically containing the foraminifera––this can be seen in Figure S1, where cyst materials were wiped away with a paint brush from the foraminifera shown in A, revealing the foraminifera test under the cyst in B). These structures usually did not measure more than 1 mm between the test and the end of the cyst (Figure S1A,C,D). Similar structures have been described in Cibicides refulgens from Antarctic scallops, and are thought to facilitate protoplasmic streaming and food acquisition [31].

While cyst formation is common, the function of these structures remains speculative. They may create microenvironments distinct from the surrounding habitat, potentially playing roles in reproduction, feeding, chamber formation, and/or protection [27,28,29,30,31]. The prevalence of cysts at Station M suggests they confer adaptive advantages in elevated habitats at 4000 m depth in the Pacific. The composition of the structures indicates that foraminifera are incorporating clay particles, likely acquired from the water column as they pass by, implying that there is some selective benefit to investing energy in constructing these cysts. In some studies, cyst formation has been linked to low pH or corrosive conditions [29], although other evidence suggests this is to not the sole driver, particularly in high-productivity regions like Station M.

4.3. Implications for Deep-Sea Ecology and Future Research

SEA³s deployments provide a rare opportunity to study foraminifera colonization and morphological development in a controlled time-series framework not achievable with natural substrates alone. Elevated, standardized plastic substrates allow for direct observation of settlement, growth, and morphologic variability in situ. Prior deployments have shown that foraminiferal communities colonizing elevated substrates differ significantly from those found in surrounding sediments [4,24] and that L. wuellerstorfi populations can reach densities exceeding 1000 individuals, even in oxygen-limited environments [2,3,4]. These studies underscore the utility of this experimental design in capturing benthic taxa that are otherwise absent from (or rare in) soft-bottom habitats. The findings presented herein underscore the complex interplay among reproductive biology, environmental setting, and anthropogenic influence in shaping deep-sea foraminiferal assemblages. The SEA³ structures do not alter environmental conditions such as oxygen or food availability, but they provide an elevated, hard substrate that is typically absent from abyssal sediments.

Plastic colonization by calcifying taxa like foraminifera has implications for both substrate ecology and biogeochemical cycling. First, as plastics degrade, they release carbon-containing compounds; however, recent estimates suggest that the contribution of this carbon to atmospheric CO₂ is likely negligible [32]. More significant may be the role of plastics as novel substrates for colonization. Foraminifera, in particular, are major contributors to marine carbonate production through the formation of calcium carbonate (CaCO₃) shells. Planktonic species alone are estimated to contribute over 1.2 billion tons of CaCO₃ per year, representing more than 21% of the ocean’s carbonate production [33], with an additional 5% attributed to benthic species. The increasing presence of plastic substrates in the ocean may promote benthic foraminiferal settlement and calcification in areas where suitable natural substrates are lacking, potentially acting as localized carbon sinks. However, the broader ecological and geochemical consequences of plastic–foraminifera interactions remain poorly understood. Plastics serve as physical substrates and have the potential to leach additives and contaminants trapped within polymer matrices, which likely influences microbial colonization and overall ecological function. These additives and contaminants can include endocrine disruptors, toxins, and nutrients essential for bacterial growth. Laboratory studies have shown that plastic-associated stress can reduce metabolic activity and alter stable isotope incorporation in benthic foraminifera, pointing to potential impacts on biomineralization and carbonate chemistry [26]. These stressors include physiological effects such as oxidative damage, reduced metabolic activity, and impaired calcification, which may result from exposure to plastic-associated compounds or the physical presence of microplastics within the test or cytoplasm [12,26]. Thus, while plastics may facilitate colonization, they may also impair the physiological processes that underlie calcification. Understanding the balance of these effects is critical for refining present-day carbonate chemistry and anticipating how plastic pollution may influence future ocean chemistry and conditions. Continued research integrating field monitoring, genetic analysis, explicit characterization of polymer composition and additive profiles, and geochemical proxy development will be critical to deciphering these dynamics.

5. Conclusions

This study demonstrates that Lobatula wuellerstorfi readily colonize plastic substrates across a range of deep-sea environments, from the productive Oregon Margin to the abyssal plains at Station M. Colonization occurred within 184 days, with reproductive morphotypes present across all sites. The Oregon samples retained the expected bimodal distribution of microspheric and megalospheric individuals, while Station M populations—consisting of L. wuellerstorfi morphotype A—exhibited a skewed, often unimodal pattern heavily favoring megalospheric forms. While some variation in reproductive strategy is expected, the consistent shift toward megalospheric expression at Station M suggests that this pattern is not random, but is likely driven by ecological factors such as substrate maturity, colonization timing, or local energy constraints.

Despite genetic consistency with L. wuellerstorfi, specimens from Station M were morphologically distinct, both externally and in internal features such as proloculus diameter. These findings underscore the limitations of morphology-based taxonomy in deep-sea settings and suggest the potential for cryptic variation within genetic foraminiferal clades. The strong correlations between morphometric variables across all individuals also reinforce the internal consistency of test development, even under varied environmental conditions.

One notable feature of Station M populations was the widespread occurrence of sediment-laden cysts encasing individual foraminifera. These structures, common on elevated substrates, may represent adaptive responses to habitat conditions. Their presence points to an underexplored component of deep-sea foraminifera and highlights the importance of substrate context in shaping morphological traits. Although often linked to corrosive environments, the formation of these cysts at Station M—an oxygen-rich site—suggests that additional drivers such as substrate elevation, biofilm composition, or reproductive state may also be involved.

Together, these results emphasize the ecological and experimental value of plastics in deep-sea research. While anthropogenic in origin, plastic debris enables standardized and replicable studies of colonization, morphologic variation, and population structure. SEA³s, in particular, provide rare access to epibenthic foraminifera in environments where they are sparse or absent from sediment records and facilitate controlled comparisons across sites and time. The integration of morphological, ecological, and geochemical tools—such as stable isotopes—will be essential for disentangling environmental versus genetic drivers of morphologic variability and for improving the utility of foraminifera in paleoceanographic reconstructions.

Plastics in the deep sea act not only as pollutants, but also as novel substrates and experimental tools. The incorporation of plastic into benthic foraminiferal test structures [7,8], combined with their colonization behavior [this study, 2, 3, 4] and potential influences on physiological responses [12,26], may reflect a broader ecological shift in progress. Understanding how deep-sea microfauna interact with these substrates will be essential for forecasting the biological consequences of global plastic contamination—and may reveal new proxies for tracking environmental change and human impact in the deep ocean.