Regional restructuring in planktic foraminifera communities through Pliocene-early Pleistocene climate variability

Domain: earth_science
Published: 2025-05-30
Editorial outcome: accept

Abstract
Recent studies highlight asymmetrical range shifts within plankton due to spatial variability in climate change, impacting marine ecosystem functioning and biogeochemical cycling. The Pliocene—early Pleistocene interval, characterized by significant climatic fluctuations, provides a framework to study regional responses of marine organisms, such as planktic foraminifera. Using bipartite network analysis of the Triton database, we investigate biogeographic shifts in macroperforate planktic foraminifera ecogroups, tracking taxonomic diversity and distribution. Here we show high turnover between symbiont-bearing warm-water and high-latitude dwellers, isolated to the North Atlantic, and an expansion of cold-water sub-thermocline taxa across basins, particularly in the South Pacific. Enhanced water column stratification and nutrient export to mesopelagic depths, associated with the intensification of the Northern Hemisphere Glaciation, likely drove shifts in species diversity and ecogroup latitudinal gradients toward modern patterns. This localized community restructuring emphasizes the importance of regional to hemispheric heterogeneity in understanding biodiversity responses to future climate change.

Introduction
Marine ecosystem services directly impact billions of people. It is essential to understand the response of these ecosystems to ongoing anthropogenic climate changes1–4. Given recently documented latitudinal shifts in species ranges2,3,5, understanding how latitudinal and functional diversity respond to major climatic changes is particularly important. Due to the complexity and short timescale of modern records, exploring pre-historical climate events and the corresponding adaptive response of marine life offers invaluable insights for future ecological trajectories. Here, the deep-sea sediment record, which represents the largest and most complete archive of ancient ecosystem responses to climate change, can help better characterize how biological systems respond to changes in the Earth system.

During the Pliocene—early Pleistocene interval (3.9-1.8 million years ago (Ma)), the planet underwent major climate shifts that shaped both atmospheric and ocean circulation as we know them today e.g., refs.6–8. These changes began with the rise of the Isthmus of Panama, marking the constriction and closure of the Central American Seaway9,10 (~5–3.6 Ma). It is important to note that the Central American Seaway closure proceeded in stages: the earlier cessation of deep-water connections, the reduction in surface flow between the Caribbean and Pacific (~5–3 Ma), and the later complete closure that prevented faunal migrations between the two ocean basins7,8 (~3 Ma or later). Our study primarily considers the consequences of the earlier closure, which triggered significant reorganization of global ocean circulation, salinity, and heat transport, ultimately contributing to the intensification of the Northern Hemisphere Glaciation6,7,10. While the timing of the Central American Seaway closure remains debated6,8,10,11, we follow the general consensus that significant restriction occurred by ~3.6 Ma9.

The Pliocene (5.33–2.58 Ma) began with relative cooling, followed by warming during the mid-Piacenzian Warm Period (mPWP) (~3.3–3 Ma). The mPWP is linked to atmospheric CO2 concentrations similar to the present (380–420 ppm) with global temperatures ~2–3°C higher than pre-industrial levels12–14. This interval provides critical insights into future global climate and biodiversity dynamics, serving as a widely regarded analog to climate change projected for the end of this century12,13,15,16.

The mPWP was followed by the intensification of the Northern Hemisphere Glaciation (~2.7 Ma), marking the establishment of glacial-interglacial cycles that define the modern climate system6,17,18. This transition was marked by global mean cooling of 2–4°C associated with amplified latitudinal temperature gradients, and increased ocean stratification, seasonality, and regional variability in circulation19,20. These oceanographic changes substantially affected pelagic habitats, compressing thermal niches, shifting species distributions, and altering trophic structure9,20. The Pliocene—early Pleistocene thus offers a compelling test case to enhance our understanding of marine life responses to anthropogenically-relevant climate change.

Macroperforate planktic foraminifera (single-celled calcareous zooplankton) are a particularly informative fossil group for the study of past climate events because they (1) are an exemplary organism with the most complete species-level fossil record known to science3,21–25; (2) are key contributors to the biological carbonate pump1,26,27 and nutrient cycling24,28; (3) generally exhibit thermal niche stability across past climate change events5,24,29–31; and (4) are linked to biodiversity of important pelagic groups, including sharks, krill, and squid, amongst others3,32,33. Additionally, their distinctive carbon and oxygen isotope signatures offer invaluable insights into their vertical position within the water column21, allowing for the assignment of extant and extinct species into discrete ecological niche groups (ecogroups) (Fig. 1; Supplementary Table 1-2). In the context of a warming ocean, the thermal niche stability of planktic foraminifera is a significant concern, due to their tendency to track rather than adapt to temperature shifts, likely affecting their future distribution and the trophic dynamics of associated organisms1,4,5,34. Asymmetrical range shifts in modern plankton communities, partially driven by the spatial heterogeneity of climate change, significantly impact the structure and functioning of pelagic ecosystems34–36. Understanding the drivers and consequences of these shifts requires high-resolution temporal and spatial data that can link past biodiversity patterns to environmental changes.

Although comprehensive studies of planktic foraminiferal diversity over various temporal scales, ranging from thousands34,37,38 to millions3,39 of years, exist; to date, few have investigated regionally resolved patterns across intermediate timescales using hundreds of sites. The recently collated Triton dataset, which provides an extensive record of global fossil occurrences of planktic foraminifera22, enables high-resolution analyses of species diversity and functional dynamics of planktic foraminifera. This dataset offers new opportunities to investigate shifts in vertical ocean temperature structure and the corresponding responses of plankton populations to paleoclimate change at regional scales3,22, with relevance to ongoing and future climate crises.

Here, we use the Triton database to assess spatial and ecological patterns of macroperforate planktic foraminifera across the Pliocene—early Pleistocene interval (3.9 Ma–1.8 Ma), focusing on biogeographic shifts in ecological groups and standing species diversity. Our basin-scale analyses cover the Atlantic (n=21,547 species occurrences, 127 sediment cores), Pacific (n=18,834 species occurrences, 156 sediment cores), and Indian (n=10,459 species occurrences, 87 sediment cores) oceans22, with temporal resolution of 150 kyr (thousand years). By integrating basin-scale analyses with ecological niche metrics such as the Ecogroup Specialization Index (ESI), we reveal distinct patterns of ecological restructuring and hemispheric heterogeneity that are not evident in global averages lacking spatial context e.g., ref. 3. We show that the Pliocene—early Pleistocene period was a time of significant reorganization of planktic foraminiferal communities, characterized by variability in the response of ecogroups and species diversity across different ocean basins. Specifically, we find high turnover between symbiont-bearing tropical taxa and high-latitude dwellers isolated to the North Atlantic, alongside a trans-basin expansion of cold-water sub-thermocline taxa, most prominently in the South Pacific. Enhanced water column stratification and mesopelagic nutrient export associated with the intensification of Northern Hemisphere Glaciation possibly drove these shifts. This approach enhances our understanding of how regional and temporal variations of environmental drivers shaped marine biodiversity during this pivotal interval.

Results
Bipartite network analysis and data validation
Network studies, particularly bipartite network analyses, have been increasingly applied in paleoecology to explore the complex relationships between species, environments, and ecological interactions across deep time3,39. Bipartite networks have been used to reconstruct trophic interactions40,41, examine latitudinal biodiversity patterns, and assess species-environment dependencies during key climatic and geological events3,39. These methods provide a framework for understanding how species co-occurrences and ecological roles vary spatially and temporally, offering insights into species turnover, extinction events, and ecosystem restructuring in response to past environmental changes3,39,42.

Here we implemented bipartite network analyses following the methods outlined in Woodhouse and Swain et al.3, which consist of two classes of nodes: ecogroups as one class and 5° palaeolatitudinal bands as the other. Links in these networks represent the biogeographical co-occurrences between ecogroups and palaeolatitudinal bands, providing a holistic and innovative approach to capture their interconnected nature. In the bipartite network analysis, the width of the links between an ecogroup and a palaeolatitudinal band denotes the number of occurrences of that ecogroup at a given palaeolatitudinal band within that temporal bin (see Methods).

To address potential sampling biases between hemispheres and basins, we performed resampling of ecogroup distribution data without replacement (n=1000) for each 150 kyr time bin, matching the smallest number of cores in any 150 kyr bin (Supplementary Fig. 9). This analysis revealed that both raw and subsampled datasets exhibit identical distributions, indicating that observed patterns are not influenced by sampling differences. Additionally, sampling coverage metrics and Pielou’s Evenness confirmed high data completeness and consistent representation of ecogroup distributions across regions (Supplementary Figs. 13–17).

After verifying robust sampling coverage and evenness, we calculated the Ecogroup Specialization Index to investigate biogeographic patterns across paleolatitudes at a 150 kyr resolution for the Atlantic, Pacific, and Indian oceans during the Pliocene—early Pleistocene (Fig. 2). The ESI quantifies the degree of evenness in the distribution of ecogroups within a paleolatitudinal band relative to other bands in each temporal bin. Lower ESI values (bluer colors in Fig. 2b, d, f) indicate higher evenness, where all functional groups are equally represented within a paleolatitudinal band. In contrast, higher ESI values (more yellow colors in Fig. 2b, d, f) correspond to dominance by a single group (for further methodological details, see the Methods section). To further validate our results on species specialization, we ran null models to evaluate deviations from random expectations of occurrences while accounting for spatiotemporal constraints (Supplementary Figs. 10–12; see Methods section for details).

We examined five ecogroups, previously classified by Aze et al.21 based on their stable isotopic geochemical fingerprint and biogeography: ecogroup 1, mixed layer tropical/subtropical species with symbionts; ecogroup 2, mixed layer tropical/subtropical species without symbionts; ecogroup 3, thermocline species; ecogroup 4, sub-thermocline species, and ecogroup 5, high-latitude species (Fig. 1). We constructed a table of extant species of planktic foraminifera and compared their ecogroup classifications from Aze et al.’s21 evaluation to modern plankton to wand sediment trap studies, finding a strong correlation between the two (see Supplementary Table 1).

The benefit of assessing community structure through the lens of functional groups, such as ecogroups, is their consistency over geological time, irrespective of the contemporaneous evolution and extinction of individual species within said groups3. Ecogroup data allows quantitative investigations of ecological niche space occupation in the past and its response to global climate change.

Woodhouse and Swain et al.3 analyzed the global record of planktic foraminiferal ESI, demonstrating that zones with low ESI had migrated equatorward over the past ~8 Myr. Their findings focused on a global compilation; however, recent studies of zooplankton dynamics suggest a high degree of regional heterogeneity to global change31. It is, therefore, important to examine metrics such as ESI and species diversity on a basin-scale to comprehensively understand how different ocean basins respond to climate shifts and how regional changes contribute to the global average.

Spatial patterns in Ecogroup Specialization Index
In our study, the lowest ESI zones, indicative of the highest ecogroup evenness, are predominantly located in mid-latitude regions compared to lower latitudes across all basins (Fig. 2b, d, f). These mid-latitude zones align with areas of higher species richness3,43 and species turnover areas31. This distribution pattern potentially results from steeper vertical temperature gradients at mid-latitudes, which foster stratification of vertically stacked niches, accommodating a large variety of equally represented ecogroups3,43. Following the mPWP, a shift in decreased ESI from mid-latitudes to lower latitudes is observed in the North Atlantic and southern Indian oceans (Fig. 2b, f). These collective shifts manifested an equatorward movement of global ecogroup evenness (or equitability) zones, as noted by Woodhouse & Swain et al.3. It is important to note that our study excludes high-latitude data due to a lack of continuous temporal and spatial coverage. A study of the last 20,000 years by Jonkers et al.31, a period with climate perturbations much stronger than those recorded during our study interval, identified key biogeographic changes at mid-latitudes, aligning with the latitudinal limits of our analysis.

Hemispheric variability in ecogroup distributions
To assess the hemispheric-scale variability in ESI and identify the specific ecogroups contributing to these fluctuations, we calculated the average ESI (Fig. 2a, c, e) and the proportional distribution of each ecogroup for each hemisphere in each ocean basin over time (Fig. 3; Supplementary Figs. 2–7). The most prominent decrease in ESI occurred in the North Atlantic between 3.1 and 1.8 Ma, driven by increased relative occurrences of high-latitude (ecogroup 5) and sub-thermocline (ecogroup 4) dwellers, coupled with decreased relative occurrences of symbiotic surface mixed layer dwellers (ecogroup 1) (Figs. 2a, 3; Supplementary Figs. 2–3, 6, 7). Our linear model highlights a negative correlation restricted to the North Atlantic, specifically between the warm-water taxa of ecogroup 1 and the high-latitude, cool-water taxa of ecogroup 5. The negative correlation emerged concurrently with the onset of the intensification of the Northern Hemisphere Glaciation at ~2.7 Ma, and ecogroup 5 played a significant role in driving the observed ecological changes during the Pliocene—early Pleistocene interval (Supplementary Fig. 8). The population of ecogroup 5 in our study is represented by only two species—Neogloboquadrina pachyderma and Turborotalita quinqueloba—in contrast to other ecogroups (See supplementary Table 1). Despite its lower diversity, ecogroup 5 plays a significant role in driving changes observed during the Pliocene—early Pleistocene interval. It is important to note that Neogloboquadrina atlantica, although known to be common in the North Atlantic during the study interval, was excluded from our study due to a low number of records in the Triton dataset, likely a result of historical misidentification and synonymization with other taxa. While its absence does not affect the broader interpretations presented here it highlight the importance of continued taxonomic refinement in future iterations of the database.

The ESI decline in the South Atlantic was less pronounced and began at ~3.5 Ma (Fig. 2a, b). In the North Pacific, a steeper ecogroup evenness gradient was observed between 3.9 and 3.2 Ma, followed by an increase in ESI after the mPWP (Fig. 2c, d). This variation in ecogroup evenness is primarily driven by a decrease in the relative abundance of thermocline dwellers (ecogroup 3) and an increase in high-latitude dwellers (ecogroup 5) during these periods (Fig. 3; Supplementary Figs. 2, 5b, 7b). Conversely, ESI remained relatively flat in the Southern Hemisphere of the Pacific, suggesting stable vertical temperature gradient conditions during this interval (Fig. 2c). As the climate began to cool again following the mPWP (Fig. 2g), there was an increase in the proportion of sub-thermocline dwellers (ecogroup 4) across all basins, with the highest rate of expansion in the Southern Hemisphere (Fig. 3; Supplementary Fig. 2, 6).

Temporal shifts in species and ecogroup diversity
To gain insights into standing diversity and functional dynamics across the Pliocene—early Pleistocene, we analyzed the corrected sample-in-bin metric44, using shareholder quorum subsampling (SQS), of both species and ecogroup diversity (Fig. 4; see Methods). The modern latitudinal diversity gradient of planktic foraminifera was established ~15 Ma, where diversity increases towards lower latitudes with a slight equatorial depression3,28,45,46. Studies of planktic foraminiferal biodiversity document a species diversity peak in the Pliocene43 followed by a decline towards modern levels3,37. Our data reveal that this trend occurred asynchronously, characterized by hemispheric heterogeneity (Fig. 4). The decline in species diversity initiated across all studied ocean basins in the Southern Hemisphere coincided with the mPWP. This was subsequently followed by a decline in planktic foraminiferal diversity across both hemispheres coinciding with the onset of the intensification of the Northern Hemisphere Glaciation (Fig. 4a).

The decoupling of ecogroup and species diversity across the Pliocene—early Pleistocene is observed in all ocean basins (Fig. 4). The most significant changes in ecogroup diversity occurred in both the North Atlantic and North Pacific, although these shifts occurred during different timeframes (Fig. 4b). In the North Atlantic, ecogroup diversity began increasing immediately after the mPWP. In the North Pacific, ecogroup diversity increased between 3.6 and 3.0 Ma (Fig. 4b). In the Indian Ocean, ecogroup diversity remained relatively stable in the Northern Hemisphere, while the Southern Hemisphere displayed a gradual increase between the closure of the Central American Seaway and the onset of the mPWP. Overall, these shifts in ecogroup diversity appear to occur over a broad interval spanning the closure of the Central American Seaway, the mPWP, and the intensification of the Northern Hemisphere Glaciation, rather than being tightly constrained to a specific event. We note, however, that the confidence intervals for ecogroup diversity often overlap, especially in the Atlantic and Pacific oceans (Supplementary Fig. 19), which limits the statistical support for some of these observed differences across time and space.

Methods
Data
We quantified the spatiotemporal shifts of macroperforate planktic foraminifera using a total of 50,840 species-by-locality-by-age records from the Triton dataset; specifically with 21,547 unique occurrences from the Atlantic (number of cores=127), 18,834 unique occurrences from the Pacific (number of cores=156), and 10,459 unique occurrences from the Indian oceans (Supplementary Fig. 1). Planktic foraminifera with microperforate and medioperforate wall textures were excluded from the analysis since they are currently characterized by a less refined phylogeny compared to macroperforate forms21,61. In addition, the relative lack of ecogroup assignments among microperforate species, only 43 out of 81 in the latest phylogeny, would significantly limit the completeness and comparability of the dataset. For our analysis, speciation and extinction data for all macroperforate planktic foraminiferal species were determined based on the frameworks provided by Aze et al.21 and Fenton & Woodhouse et al.22. We excluded any species occurrences falling outside their established stratigraphic ranges to minimize inaccuracies in the occurrence data likely resulting from misidentification or stratigraphic reworking. We included a total of 74 species of planktic foraminifera, comprising 36 extant species and 38 extinct species (see Supplementary Table 1).

Raw counts of each species within each ecogroup, binned in 150 kyr intervals, are provided in Supplementary Data 1. The studied interval covered the Pliocene—early Pleistocene, spanning from 3.9 to 1.8 Ma, and utilized foraminiferal data categorized into 150 kyr time bins, which were further divided into paleolatitudinal bands of 5°. In our study, the Atlantic Ocean dataset included 226 combined time-latitude bins, the Pacific Ocean included 207 bins, and the Indian Ocean included 162 bins.

To investigate whether species ecology played a role in their biogeographic network interactions across the Pliocene—early Pleistocene, we also assigned species to the five specified ecogroups from Aze et al.21. Ecogroups are defined as following: ecogroup 1, mixed layer tropical/subtropical species with symbionts; ecogroup 2, mixed layer tropical/subtropical species without symbionts; ecogroup 3, thermocline species; ecogroup 4, sub-thermocline species, and ecogroup 5, high-latitude species (Fig. 1; Supplementary Table 1, 2). Ecogroups are determined by examining the biogeographic distributions, phylogenetic relationships, and the stable oxygen (δ18O) and carbon (δ13C) isotopic signature of planktic foraminiferal species skeletons. These analyses allow us to assess the extent of bathymetric and ecological differentiation among both extant and extinct species enabling us to deduce species-specific ecological niches, influenced by varying physicochemical and biological processes, as well as depth-related variations within the water column21,48,62.

Calculating network-associated ecological metrics
In this study, we used the modified Triton dataset22, partitioning planktic foraminifera into 150 kyr time bins and 5° paleolatitude bands. We constructed bipartite networks using these bins where paleolatitude represented one node class and ecogroup the other node class. Bipartite networks are used to analyze interactions between two distinct sets of nodes and can provide insights into the ecological structure and relationships within datasets such as Triton. The links between these nodes represent the presence of a certain ecogroup within a specific paleolatitudinal band, where the width of these connections indicates the frequency of each ecogroup’s occurrence in the respective paleolatitudinal band6,58. To assess dataset completeness, we examined the sampling coverage (frequency of each unique ecogroup within a paleolatitudinal band during each time interval) using the iNEXT v3.0.1R63 package, which is based on Good’s U estimator64,65. Our analysis showed that all samples achieved sufficient and consistent coverage levels for our interpretations (See Supplementary Figs. 13, 14, 17).

The bipartite package66 v2.20 in R v4.3.2 calculated network-associated ecological metrics for each paleolatitudinal band node: (1) number of ecogroups at that paleolatitudinal band, and (2) the Ecogroup Specialization Index (ESI). The ESI calculates the coefficient of variation in the number of occurrences of ecogroups within a particular paleolatitudinal band, with its values normalized to a range between 0 and 1. Here, a value of 0 signifies low specialization, indicating generalized functional communities with an even distribution of ecogroups, whereas a value of 1 suggests high specialization, indicative of specialized functional communities dominated by one ecogroup. This index is based on the Species Specificity Index concept introduced by Julliard67 and Poisot68 (see cited papers for detailed explanation), tailored to quantify the uniformity or disparity in ecogroup distribution across a paleolatitudinal band for each time interval. Thus, a band characterized by an identical occurrence count for all ecogroups would exhibit an ESI of 0 (dark blue in Fig. 2), whereas a band dominated by a single ecogroup would display an ESI approaching 1 (yellow in Fig. 2). The ESI is weighted by the relative occurrence counts of different 397 ecogroups within a paleolatitudinal band. To address potential biases, such as the under representation of depauperate ecogroups (e.g., the polar group), we calculated ESI using relative frequencies rather than raw counts, ensuring proportional contributions. Additionally, we used null model analyses to interpret the ESI robustly and identify statistically significant patterns of specialization (See Supplementary Figs. 10–12 for a summary of these patterns across ecogroups).

To understand species specialization within a biogeographical framework and determine how observed patterns deviate from random expectations, we utilized the econullnetr v0.2.1 R package69. These null models simulate the distribution of various ecogroups across each latitudinal band while accounting for (1) the number of occurrences across all ecogroups within specific latitudinal bands, and (2) the temporal occurrences within ecogroups across all latitudes. We generated 500 sampling distributions by iterating the null model and calculated 95% confidence intervals for occurrence frequencies of each ecogroup within each latitudinal and time bin. If the empirical occurrence frequencies fall outside these confidence intervals, occurrence values are either significantly greater or less than expected under a random distribution. This method provides a statistical framework for detecting deviations and interpreting specialization trends calculated from the observed occurrence values (see Supplementary Figs. 10–12 for a summary of these patterns across ecogroups). As most ecogroups do not show significantly different distributions from the null expectations, the results suggest that shifts in community composition over time are primarily driven by temporal or environmental changes rather than fixed biogeographic or regional constraints.

Species and ecogroup diversity
To explore the standing diversity of species and functional dynamics of planktic foraminiferal ecogroups across the Pliocene—early Pleistocene Period, we conducted the analysis based on the corrected sample-in-bin metric, utilizing the shareholder quorum subsampling (SQS). The corrected sampled-in-bin metric is used to account for sampling biases when estimating the presence of taxa within a specific time bin44. It adjusts the observed data to better reflect true ecological or evolutionary signals by correcting for variations in sampling effort across different intervals. This approach is particularly useful in paleontological and ecological studies to avoid over- or under-representing taxa due to uneven sampling70. To mitigate the effects of uneven sampling intensity across different times and locations for species and ecogroups, the SQS quorum level was set at 0.8 and we ran 1000 iterations in 150 kyr time bins using the divDyn v0.8.2 R package71.

The SQS method is designed to normalize raw species richness to a consistent share of the total frequency distribution. Its key strength lies in its enhanced ability to include rare taxa in the analysis, providing a more comprehensive and accurate representation of biodiversity70. The standing diversity is characterized by the corrected sample-in-bin metric, which is an assessment of the richness of both species and ecogroups over time, thereby providing a more refined understanding of ecological and evolutionary trends during the studied interval.

Ecogroup trends and variability
To elucidate temporal trends in ecogroup distribution and identify specific ecogroups contributing to the variability of the ESI in the Atlantic, Pacific, and Indian Ocean basins, we calculated the relative abundance of each ecogroup within 15 kyr time bins. Initially, we performed resampling of ecogroup distribution data without replacement 1000 times for each 150 kyr time bin, matching the smallest number of cores observed in any 150 kyr bin. Subsequent analysis of both the original dataset and the subsampled datasets revealed congruent distributions, affirming that patterns and trends in the relative ecogroup distribution and ESI variability are robust and not artifacts of sampling (Supplementary Fig. 9).

To quantify the rates of change in the relative abundance of each ecogroup across hemispheres and basins, we calculated the slopes of these fitted linear models for ecogroup relative abundance over time. A positive slope indicates an increase in the number of ecogroup dwellers from 3.9 to 1.8 Ma, while a negative slope indicates a decrease. Time series showing a decreasing proportion of ecogroup occurrences over time are characterized by a negative slope, and vice versa (Supplementary Figs. 3–7).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I appreciate your careful and thorough revision. Figures are now much better and I like them very much.\nOnly a few things:\nLine 76 “linked to biodiversity hotspots of important marine groups, including sharks”\nBetter to say “pelagic group” and just “biodiversity (instead of biodiversity hotspot)”, as coastal benthic groups have very different biodiversity trend/hotspot and also pelagic groups don’t show very distinct “hotpots”.\nFrom line 88: “Although comprehensive studies of planktic foraminiferal 88 diversity over various\n89 scales, ranging from thousandse.g.,34,37-38 to millionse.g.,3,39 of years, exist; to date, none have\n90 investigated patterns on large spatial scales with hundreds of sites.”\n“none have (should be “has”?) investigated patterns on large spatial scales with hundreds of sites”\nThis is bit misleading as Swain et al Woodhouse et al and Fenton et al actually did such using Triton.\nYou may add a few more words to better specify (such as intermediate time scale or regionally resolved? I suspect somehow you missed to specify what you like to say here enough).\nThe paragraph from Line 100 on Triton:\nYou may consider to combine it into the previous paragraph, as it looks a bit redundant.\nOtherwise I think the manuscript is now well revised.\nSincerely yours,\nMoriaki Yasuhara", "reviewer_2": "Helen Coxall review round 2.\nThis is a much improved manuscript. My major comments have been addressed more or less completely – i.e. the authors have given attention to the details of the planktonic foraminifera assemblages and eco-groupings at the heart of this study, connecting us back to the data and observations.\nI am largely happy, however, the new list of species raises a couple of foram specific questions that I would like to see addressed before this should be accepted for publication.\n• Figure 1. A representative diagram of the planktic foraminifera ecogroups used in this\n695 study.\nGreat. A vast improvement (even if the Neogene species do not have such nice silhouettes!)\n• Figure 5. Conceptual representation of eco-group distributions during warmer (mPWP = the mid-Pliocene Warm Period\nThis is also a great addition and complement to Fig 1 bringing the paper together. Really helpful.\n• Supplementary Table 1. Species of planktic foraminifera with assigned eco-groups used in our study from Aze, compared to ecogroups assigned by modern studies.\nThank you for making this table. Even though the eco-group interpretations have not much changed since Aze et al., its important and useful for the modern planktonic foraminifera expert to keep these check lists of species alive, and keep the references up to date. It also highlights the level of taxonomic detail that is contained in the data base, which is important.\n-Please in the SI Table caption include a list of the eco-groups (again) to remind the reader what the numbers mean. Or perhaps since this is all about the ecor groups its worth while sorting them by eco group so we see the composition of each group.\n• Now I see the lists of species I have a question about the North Atlantic. Why is the species Neogloboquadrina atlantica not listed in the table or eco-groups? It is extremely abundant/dominant in the North Atlantic in the Pliocene going extinct in the early Pleistocene – it is also an important biostratigraphic marker in the North Atlantic/subpolar region. It seems to be the key high latitude North Atlantic species before N. pachyderma took over that niche. If it is not in the Triton database then it must have been overlooked and binned in e.g. N. dutertrei. But is this true? It is important comment on this.\nThe data base and paper focus on macroperforate planktic foraminifera (single-celled calcareous zooplankton), the reason given that “Planktic foraminifera with microperforate and medioperforate wall textures were excluded from the analysis since they are currently characterized by a less refined phylogeny compared to macroperforate forms.” That may be the case, although there are some species that become important at high latitudes eg. Globigerinita uvula/glutinata. Morad et al., 2019, hints of hidden diversity among some of these small tax such as these.\n-Can the authors at last comment on the extent to which the omission of microperforates might result in significant information missing in terms of diversity signals?\nMorard, R., Vollmar, N.M., Greco, M., Kucera, M., 2019. Unassigned diversity of planktonic foraminifera from environmental sequencing revealed as known but neglected species. PLOS ONE 14, e0213936.\nThis microperforate issue also becomes relevant for this section:\nLine 192-: “The negative correlation emerged concurrently with the onset of the intensification of the Northern Hemisphere Glaciation at ~2.7 Ma, and ecogroup 5 played a significant role in driving the observed ecological changes during the Pliocene - early Pleistocene interval (Supplementary Fig. 8). Curiously, the population of ecogroup 5 is represented by only two species — Neogloboquadrina pachyderma and Turborotalita quinqueloba — in contrast to other ecogroups (See supplementary table 1). Yet, despite its lower diversity, ecogroup 5 plays a significant role in driving changes observed during the Pliocene - early Pleistocene interval.”\nThis is where inclusion of microperforate taxa would make a difference to the diversity (+ 1 or 2 species). Also the inclusion of N. atlantica.\nImportant N. atlantica references\nWeitkamp, T.M., Razmjooei, M.J., Pearson, P.N., Coxall, H.K., 2025. Upper Oligocene to Pleistocene planktonic foraminifera stratigraphy at North Atlantic DSDP Site 407, Reykjanes Ridge: diversity trends and biozonation using modern Neogene taxonomic concepts. J. Micropalaeontol. 44, 1-78.\nBolton, C.T., Bailey, I., Friedrich, O., Tachikawa, K., de Garidel-Thoron, T., Vidal, L., Sonzogni, C., Marino, G., Rohling, E.J., Robinson, M.M., Ermini, M., Koch, M., Cooper, M.J., Wilson, P.A., 2018. North Atlantic Midlatitude Surface-Circulation Changes Through the Plio-Pleistocene Intensification of Northern Hemisphere Glaciation. Paleoceanography and Paleoclimatology 33, 1186-1205.\nDowsett, H., Robinson, M., Foley, K., Herbert, T., Hunter, S., Andersson, C., Spivey, W., 2023. The Relative Stability of Planktic Foraminifer Thermal Preferences over the Past 3 Million Years. Geosciences 13, 71.\nFlower, B.P., 1999. Data report: Planktonic foraminifers from the subpolar North Atlantic and Nordic Seas: Sites 980-987 and 907. Proceedings of the Ocean Drilling Program: Scientific Results 162, 19-34.\nWeaver, P.P.E., Clement, B.M., 1986. Synchroneity of Pliocene planktonic foraminiferal datums in the North Atlantic. Marine Micropaleontology 10, 295-307.", "reviewer_3": "Please see attached file."}, "author_response": "Point-by-point responses to the reviewer comments are provided\nbelow in italics and in green font. All line numbers in our responses\nrefer to the updated submission.\nReviewer #1:\nThis ms is very important study on 4D reconstruction of global plankton diversity in the\nintermediate time scale. While it’s known in longer (Cenozoic through, eg Woodhouse, Swain,\nFenton) and shorter time scales (Yasuhara et al 2020 PNAS), it’s not very well understood in this\ntime scale. It’s exciting results and I enthusiastically recommend publication in your journal after\nmoderate revision as explained below.\nThank you for your positive feedback and for recognizing the significance of our study on the\nintermediate time scale of global plankton diversity, Moriaki Yasuhara. We greatly appreciate\nyour recommendation for publication and will address the suggested revisions carefully to\nfurther improve the manuscript.\nMajor comments\n1. While the MS is generally well written, it’s bit rough here and there.\nYou may polish introduction more (in general, I tend to feel you can add a few more words there\nand there. Eg, justification to your statement that you say foram trend is similar to shark etc).\nAlso, some parts of discussion is little confusing (Pls see my specific comments below).\nThank you for your thoughtful comments. We have revised the introduction to improve its clarity\nand flow. While we did not explicitly add a statement about sharks to the paper, we would like to\nprovide additional context and references to clarify our reasoning.\nThe study by Worm et al. (2003, PNAS) discusses diversity hotspots for oceanic predators,\nstating:\n\"In the open ocean, many large predators such as tunas, sharks, billfishes, and sea turtles are of\ncurrent conservation concern because of their vulnerability to overfishing and ecosystem role.\nHere we use scientific-observer records from pelagic longline fisheries in the Atlantic and Pacific\nOceans to show that oceanic predators concentrate in distinct diversity hotspots. Predator\ndiversity consistently peaks at intermediate latitudes (20–30° N and S), where tropical and\ntemperate species ranges overlap. Individual hotspots are found close to prominent habitat\nfeatures such as reefs, shelf breaks, or seamounts and often coincide with zooplankton and coral\nreef hotspots.\" This study includes planktic foraminifera as part of the zooplankton associated\nwith these hotspots.\nSimilarly, Titterson et al. (2010, Nature) provide a visual representation in their Figure 1, showing\noverlapping patterns of species richness for sharks, squids, and foraminifera, highlighting the\nconsistency in diversity trends across these groups.\nWe have also refined the discussion to address the specific points raised and to improve its overall\nclarity. We hope these changes better address your comments and clarify our reasoning.\n2. The discussion is rather complicated (I am not criticizing about it per se. Good to know such\ndetails that we didn’t know before). If possible, it’s good to try to add a summary figure (cartoon,\nconceptual diagram, etc).\nThank you for your suggestion. We have created a conceptual figure to summarize the discussion;\nplease see Figure 5. We hope this addition provides clarity and enhances the overall\nunderstanding of the key points.\n3. Some S-Figs (on ecogroup relative abundances) show more distinct/remarkable trends than eg\nfig 3. You may consider to bring some to the main text fig (perhaps by combining some).\nAs suggested, we have moved SM Figure 2 into the main text, where it is now Figure 3, and\nmoved the previous Figure 3 to the supplementary material. We hope this improves the clarity\nand impact of the presentation.\nSpecific comments:\n1. Line 50,\nIt’s good to note that the closure of CAS has two meanings.\nPaleoceanographers tend to think it as closure of deep water connection or major water\nexchange.\nPaleontologists (coastal and terrestrial) thin it as complete closure that prevent coastal benthos to\nmove between Caribbean and Pacific and allow terrestrial mammals to move between S and N\nAmerica. The timing of the former is much earlier (may see eg Daniela Schmidt paper,\ndiscussion between Aaron O’dea and Carlos Jaramillo).\nSo, CAS closure that affect planktic foram may be the former (and so may be earlier than the\ncoastal benthic extinction of the Caribbean Sea eg studies by Aaron O’Dea and Jeremy Jackson)?\nThank you for your insightful comment. The planktic foraminiferal distribution should not be\naffected by the closure of deep water flow, and the isolation of the two ocean basins at mixed-layer\ndepth is clearly demonstrated by the divergent values of planktonic isotopes starting around 4 Ma\n(see Figure 3D in O’Dea et al., 2016). We have revised the text as suggested (Lines 47ff; Line 232-\n236).\n2. Good to explain how new from Woodhouse & Swain bit more. One is as I mentioned above\nyou focused on the intermediate time scale that remained elusive. You may like to cite Yasuhara\net al 2020 PNAS for short time scale study.\nThank you for your comment. We have clarified how our study differs from Woodhouse & Swain\nand other. Additionally, we have cited Yasuhara et al. (2020, PNAS) to provide context on\nshorter timescale studies (see lines 88-99.)\n3. Line 117-118. A bit confusing sentence. I read as the LDG was stable for the last 15 but\nchanged from 8 Ma? It sounds these two conflict? You may rephrase.\nThank you for pointing out the potential confusion in this sentence. To improve clarity and avoid\nconflicting interpretations, we have removed the discussion of the last 15 Ma.\n4. Line 123-124, ESI lowest at mid latitudes. This mean you have no high latitude data, right?\nThis bit sounds ESI is high in low and high latitudes (=misleading).\nYou may revise like “ESI lower in higher latitudes”?\nThank you for your thoughtful comment. The Triton database does include records from high\nlatitudes, and in our study, we analyzed data from the North Atlantic up to 65°N, covering the\nsubpolar region. However, high-latitude data availability is often limited to specific intervals, such\nas the mid-Piacenzian Warm Period, due to patchy and inconsistent sampling across the Pliocene–\nearly Pleistocene. Given the high-resolution nature of our study, which required consistent\ntemporal and spatial coverage, these regions frequently failed to meet the criteria for sample\ncompleteness when analyzed in 150-kyr by 5° latitude bins. We have revised the text to clarify this\nand to emphasize that the lowest ESI zones, indicative of the highest ecogroup equitability, are\npredominantly located in mid-latitude regions compared to lower latitudes across all basins (see\nlines 170-171; 175-177).\n5. Line 128-129, this shift to the (sub)tropics is not super clear in fig 2C andf G.\nI see what you mean though, Any way to better visualize this trend?\nTo address your suggestion and enhance the visualization, we have added a black line to the\nfigure to better highlight the pattern. See Figure 2b,2f.\n6. Line 133, FIg. 2B-D. You mean 2B, D, and F?\nWe updated the figure numbering.\n7. How about the artefact from differences in latitudinal range covered by S and N hemispheres.\nIt could affect S and N hemisphere averaged ESIs (fig 2B, D, F)?\nFor sampling bias (between high and low latitudes and also N and S hemispheres),\nyou may like to have a look at discussion&summary in Yasuhara et al 2024 (hope this is useful):\nYasuhara et al 2024 Tropical paleobiology discovers biodiversity in a warmer past. PNAS\nThank you for your suggestion. We have included a discussion of the statistical tests performed to\naddress the limitations of unequal sample sizes between hemispheres and basins. Specifically, we\nincorporated resampling analyses and sampling coverage metrics to ensure the robustness of our\nfindings (see lines 133-139; Supplementary Figs. 13-17). These analyses confirm that observed\npatterns are not artifacts of sampling bias.\n8. Line 166. New paragraph starting from “this”. Meaning of “this” is not very clear.\nWe edited the sentence for further clarity.\n9. Line 168-169. N Atlantic ecogroup diversity decline after the closure of CAS.\nI don’t see it in fig 4 green curve. Instead it look decreasing in the mPWP?\nWe have reviewed the text and corrected it to align with the pattern observed in the Northern\nHemisphere of the Atlantic in Fig. 4. We appreciate your careful observation.\n10. Line 182-183, stability before 3.6 Ma. To show it, it’s good to show longer data like past 10\nor 15 myr? Otherwise, it’s bit difficult to clearly understand or see for readers. You have the\ndata.\nThank you for your suggestion. Although we have data extending beyond 3.6 Ma, the analysis of\nthe last 15 Ma falls outside the scope of this study. To address your concern, we have revised the\ntext (see lines 234-239). For broader context, the study by Woodhouse and Swain et al. (2023)\ndemonstrates similar patterns over longer timescales, albeit at a coarser resolution and at an\naveraged global scale, supporting the overall picture presented in our analysis.\n11. Line 186-187. It’s good to specify ecogroups 1-5 that you mean in fig 3.\n(for high-latitude (cool-water) and subthermocline (deep-water) ecological communities)\nWe have revised the text as suggested.\n12. Line 188. you refer fig 2C but it;’s on the Atlantic, and here you are talking about Pacific?\nYou mean E?\nAlso here you say “high-latitude (cool-water) and subthermocline (deep-water) ecological\n187 communities (Fig. 3, Supplementary Figs. 2, 6B, 7B) expanded their niches from mid to\nlow188 latitudes (Fig. 2C),”\nBut Fig 2 shows only ESI and doesn’t show such community information.\nIt may be something wrong here.\nThank you for your detailed feedback. You are correct that Fig. 2 shows only ESI and does not\ndisplay community information, and that the reference should be to Fig. 2E, not Fig. 2C. We have\nrevised the text to clarify that we are referring to changes in the proportional abundances of the\necogroups rather than niche expansion.\n13. Line 205–. the formation of cryosphere. Good to specify the timing you are discussing here\n(when it was happened?). Bit confusing what you are discussing here.\nWe have revised the text as suggested by the Reviewer 1. See line 271.\n14. Line 230-235, discussion on ecogroups. This is very important discovery!! I love this!!\nThank you for your enthusiastic feedback! We’re thrilled that you find the discovery significant\nand appreciate your support for our work.\n15. Line 234 “higher trophic levels are expected to migrate towards the poles”\nYou may like to cite Yasuhara and Deutsch 2022 that has very relevant conceptual fig:\nYasuhara, M., Deutsch, C. A., 2022. Paleobiology provides glimpses of future ocean: Fossil\nrecords from tropical oceans predict biodiversity loss in a warmer world. Science: 375 (6576),\n25–26.\nWe have added the citation for Yasuhara and Deutsch (2022) to support the statement regarding\nthe migration of higher trophic levels towards the poles. We appreciate you pointing us to this\nrelevant reference and conceptual figure.\n16. North Atlantic is something special, right? As far as I remember, future cooling is projected\nin mid-high latitudes? I think I discussed a bit in Yasuhara et al 2020 PNAS.\nWe have added the suggested citation (Yasuhara et al., 2020 PNAS) to emphasize the unique\ncharacteristics of the North Atlantic, particularly its sensitivity to climate-driven changes and\nthe strong influence of sea surface temperature on planktic species distribution.\n17. Fig 3. shading for iNHG, nPWP, and cCAS is in light colors. Good to make sure they are\nclear enough to be distinguiished.\nAs suggested, we improved the figure quality.\n18. Fig 4. may consider to change NH (green) and SH (blue) colors to be more distinct each\nother?\nAs suggested, we changed the colors. See Figure 4.\nSincerely yours,\nMoriaki Yasuhara (this is signed review)\nReviewer #2:\nThis is the 3rd in a series of studies (Woodhouse et al., 2023; Swain et al., 2024) utilizing the\nTriton data base of geological planktonic foraminifera records to explore spatial and temporal\nvariation in responses of pelagic biodiversity to climate change during major events in the\nCenozoic.\nSwain, A., Woodhouse, A., Fagan, W.F., Fraass, A.J., Lowery, C.M., 2024. Biogeographic\nresponse of marine plankton to Cenozoic environmental changes. Nature 629, 616-623.\nWoodhouse, A., Swain, A., Fagan, W.F., Fraass, A.J., Lowery, C.M., 2023. Late Cenozoic\ncooling restructured global marine plankton communities. Nature 614, 713-718.\nTriton is an exceptional resource, that was always going to have great potential to illuminate\npelagic ecosystem responses to during significant ocean-climate episodes, leading to high impact\npapers (Nature and Science). Although the previous 2 papers coming out of Triton analysis, and\nnow this one, follow a similar formula, i.e. bipartite analysis, and presentation of diversity\nmetrics, the focus on different major climatic states or transitions for each manuscript, is\nsufficiently novel and the results sufficiently interesting and relevant to concerns about marine\necosystem changes, to be paradigm shifting in each case, and thus, potentially, worthy of high\nprofile publication.\nThis current ms probes the major climate transitions of the Pliocene-Pleistocene, including the\nPliocene warm period and the Pliocene-early Pleistocene onset of Northern hemisphere\nglaciation, which, as stated, “provides critical information about global climate and biodiversity\ndynamics that are widely regarded as the best analog to climate change projected for the end of\nthis century”.\nThank you for recognizing the importance of our study and for your detailed and thoughtful\nfeedback.\nThe manuscript is well written and well-organized and the results are extremely interesting.\nHowever, I have a major issue that to me stalls this manuscript going forward.\nThe connection to actual planktonic foraminifera observations, a reality check linking the black-\nbox of a large database to the original foraminifera observations, including subsequent\ntransformations to ecogroups, is missing. That is, the practical aspects of what is actually in the\nassemblage referred to, which species and species groups respond, and the robustness of their\ninterpretation/classification into ecogroups. We are merely referred to previous interpretations of\nspecies ecology from a paper by Aze et al. 2011. Even the opening figure (Fig. 1) leaves me with\ndoubt that the authors are in touch with the actual organisms in question because the icons used\nto represent the various ecogroups are a series of species that went extinct in the early Cenozoic.\nMaybe this is an oversight but combined with the minimal reference to species throughout the\npaper or methods or SI section (N. pachyderma and Pulleniatina are mentioned once) it does not\nadd confidence. This to me is really important, since many of the species that existed during the\ntime period of study, in contrast to the studies of earlier time periods, exist today and have an\necology that is known not only from stable isotope inferences (which is the case of extinct taxa)\nbut from real plankton net studies.\nFor this reason I suggest rejection of the manuscript in this stage. The authors could come back\nand say this is an oversight, easily resolved, or if they cannot present convincing background\nmethodology to show that the ecogroups are valid then reanalysis would be needed. This\nbecomes especially relevant because the Pliocene -Pleistocene species involved have living\ncounterparts whose ecology is known from direct plankton net and culture studies, some\nknowledge on which might have evolved since the Aze et al. 2011 study.\nThus any revised manuscript needs at least in the supplementary information tables showing the\nspecies that make up the eco groups, modern updates, and a digest of the results /discussion, that\nconnect the broad ecogroup diversity evolution back to tangible variations at the species level in\nthe text, that is useful not only for big picture conclusions/predictions about patterns of change,\nbut also to the fossil/plankton-collector observer level, who have work remaining to fill gaps in\ndocumenting and understanding especially higher latitude changes.\nAnother angle to emphasize in any revised manuscript would be the latitudinal limits of the study\ndue to the data base content: it is limited to mid latitudes/temperate latitudes (no data from polar\nor subpolar latitudes).\nWe have addressed your concerns as follows:\nConnection to Observations and Ecogroup Validity:\nWe acknowledge the importance of validating the ecogroup classifications against modern\nstudies and observations. To address this, we have included a supplementary table listing the\nspecies that comprise each ecogroup, updated with modern ecological studies of extant planktic\nforaminifera (see supplementary Table 1). Specifically, we compared the ecogroups used in our\nstudy, based on Aze et al. (2011), with modern studies employing geochemical and plankton net\ndata, and updated them where appropriate. The only discrepancy identified in our study was the\nclassification of Turborotalita humilis. While we initially placed it within ecogroup 2, modern\nstudies confirm that it belongs to ecogroup 1. We have updated this information and included T.\nhumilis in our analysis as part of ecogroup 1. This adjustment did not impact any results, and the\ncurrent manuscript now contains the updated figures.\nAdditionally, we have included spatio-temporal raw occurrences of species across various\necogroups in the supplementary materials. See extended data files. We hope this addition\nimproves transparency and connects our ecogroups back to species-level observations.\nCorrection of Figure 1: Thank you for noting the issue with the icons used in Figure 1. We have\ncorrected this oversight and replaced the faulty forms with representations of extant species\ncorresponding to the studied time interval. We believe this revision aligns the figure with the scope\nof the study and addresses your concerns.\nLatitudinal Limits and Subpolar Regions: The Triton database does include records from high\nlatitudes, and in our study, we analyzed data from the North Atlantic up to 65°N, covering the\nsubpolar region. However, in high latitudes across different basins, data availability is often\nlimited to specific intervals, such as the mid-Piacenzian Warm Period or other short timeframes.\nGiven the high-resolution nature of our study, which required consistent temporal and spatial\ncoverage, high-latitude regions frequently failed to meet the criteria for sample completeness when\nanalyzed in 150 kyr by 5° latitude bins. These regions often showed patchy, inconsistent data\nacross the Pliocene–early Pleistocene. Therefore, to maintain the robustness and resolution of our\nanalyses, we excluded these regions.\nWe hope these revisions address your concerns and further strengthen the manuscript. Thank\nyou again for your time and effort in reviewing our work.\nOTHER COMMENTS\nLines 58-60: “The intensification of the Northern Hemisphere Glaciation impacted ocean\ntemperatures and circulation and began a climate system dominated by glacial and interglacial\ncycles that continues to the present day”. The important aspects for planktonic forams will be\nocean temperature, stratification, seasonality and food. Please connect these factors more directly\nto the climate transition in question upfront in the introductions. What happened to ocean\ncirculation, how much cooling was there across latitudes in the different basins, how did this\nimpact planktonic foram habitat distributions? These are background palaeoceanographic factors\nthat can be summarized.\nThank you for your thoughtful feedback. Considering that this is the introduction and needs to\nremain succinct, we have incorporated key aspects of ocean circulation, cooling across latitudes,\nand their impacts on marine organisms into this section to provide essential context (see lines 63-\n70). Additional details addressing your questions are included in the discussion to elaborate on\nthese palaeoceanographic factors in the appropriate depth (lines 270ff). We hope this approach\nbalances clarity and conciseness.\nLine 78-79: ‘Modern’ is mentioned. Here an important comparison should also be the study\n(Strack et al., 2022-ref 31) about community shifts during one interglacial cycle, i.e. end of the\nlast ice age, which as far as I am aware, is the closest (large scale warming even) of the past and\nan important yard stick for this study, especially considering a similar suite or species (or their\nclose relatives) existed. I’d like to see more about this also in the discussion.\nAnd it is important to make comparisons to studies of the modern and the Strack et al., 2020\ndeglacial-transition study: How do the methodologies compare? Are the resulting data\ncomparable? Please elaborate.\nThank you for your insightful comment and for highlighting the importance of Strack et al. (2022)\nin understanding community shifts during the last deglacial transition. We recognize the\nsignificance of their work and have cited it where appropriate in the manuscript to acknowledge\nits relevance. However, we believe that detailed comparisons to the last ice age and its transitions\nare beyond the scope of this study. Our focus is on broader temporal trends and major climate\ntransitions during the Pliocene–Pleistocene, which provide critical insights into biodiversity\ndynamics over longer timescales.\nWhile comparisons to the deglacial-transition study would be valuable, there are many other\nimportant time intervals or other studies that could be used for comparison and exploring all of\nthese would dilute the focus of our current analysis.\nLine 106: Good to have this line explaining how one interprets ESI values, which is a tricky\nconcept to get your head around – you could prove this further by relating this directly to the figure,\ni.e. the red – blue colour scaling system on Figure 2. Its tricky to appreciate the ESI concepts and\nthis supporting guide to what e.g. a strong blue colour, or strong red colour means xxx, and the\nmeans inverse yy, would be really helpful. Maybe also add it to the Fig caption for clarity (will be\nappreciated). Also the axis limits and divisions on the time axis. Can you use something more\nlogical, than 0.3 my tick marks, i.e. integers (major ticks) and 0.2 or 0.5 myr (minor ticks) seems\nmore user friendly to me.\nThank you for your helpful feedback. We have updated Figure 2 and its caption to clarify how ESI\nvalues relate to the yellow-blue color scaling and what the colors represent. Additionally, we\nrevised the time axis to use more logical scaling. See Figure 2.\nLine 108-11\nGiven my comment about figure 1 (see above), please bring us back to something tangible\nregarding the species that go into these eco groups by including a table in the supplementary\ninformation (modified from Aze et al) presenting lists of the species belonging to each Eco group\nutilized here, with appropriate icons in Fig. 1. This will restore my faith in the authors having\nknowledge of the species whose diversity data they are analyzing and connect readers/uses back\nto micropalaeontological observations, which is really important. It feels like we have lost touch\nin the abyss of the database.\nThank you for your detailed comment. In response, we have updated Figure 1 to include\nappropriate icons representing the species relevant to the ecogroups studied. Additionally, as\nsuggested, we have included a supplementary table listing the species belonging to each ecogroup\nfrom Aze et al. (2011) and from modern studies (see Supplementary Table 1). We hope these\nadditions provide the tangible connection to species-level data and micropalaeontological\nobservations you requested and restore confidence in the robustness of our analysis.\nResults section\n-This section would benefit greatly from some anchoring sentences to frame the method here in\nthe ms body text - so that the reader can directly connect with the results. At the moment the body\ntext and analysis feels far way from the Triton data base and what it actually comprises. For\nexample, I suggest at least mention how many sites are included in the Triton data base for the\ntime internal of interest, and the latitudinal range (and refer directly to the Supp info figure here),\nalso mention directly the types of materials/lithology – e.g. fossil census data based on pelagic\ncarbonate rich pelagite core samples.\nAlso, Hhw did the Pliocene-early Pleistocene pelagic ecosystem changes compare with the older\nsystem responses? The Pliocene warming compared to previous Cenozoic warmings?\nThank you for your thoughtful suggestions. We have included the number of sites analyzed (370\ncores) in Supplementary Figure 1 as well as in the Methods section (see lines 331-333).\nAdditionally, the latitudinal range for each basin can be observed in Figure 2 and in\nSupplementary figures 1, 10-12, 17. Regarding lithology, due to the large number of cores\nanalyzed across 3 different basins (370 in total), the materials vary, making it impractical to\ndiscuss them in detail within the manuscript and beyond the scope of this study. However, anyone\ncan access the core IDs in our Triton_df_Oceans.RData file (available at\nhttps://github.com/ekatlarina/forams-ecogroups) and explore additional lithological details\nthrough the corresponding core repositories.\nDiscussion:\no In terms of the oceanographic explanation for the asynchronous patterns of biodiversity change\nduring the Pliocene and Pliocene-Pleistocene transition, i.e. heterogeneous between ocean basins.\nThe regions pointed to likely transitioned through different ocean overturning states/strengths\nthrough this period, for which there is a body of literature. AMOC is mentioned. Changes in\nsurface currnents is also mentioned but less about the overturning states and their possible impact\non stratificaiton, which would be important drivers of the surface current strengthening and\nstratification. This could be brought out more strongly, even if the data in the end point to relative\nstability in diversity through the peak warmth. There is even modelling evidence for major a\npossible PMOC in the Pliocene warm peak:\nBurls, N.J., Fedorov, A.V., Sigman, D.M., Jaccard, S.L., Tiedemann, R., Haug, G.H., 2017. Active\nPacific meridional overturning circulation (PMOC) during the warm Pliocene. Science Advances\n3, e1700156.\nThank you for your constructive feedback. We have incorporated a discussion on overturning\nstates and their impact on stratification and surface currents, referencing Burls et al. (2017) to\nhighlight the potential role of the Pacific Meridional Overturning Circulation (PMOC) during the\nmPWP. These revisions aim to provide a stronger oceanographic context to our findings (see lines\n252ff).\no Point out that the analysis is limited by the limited latitudinal extent of the records in the data\nbase. You do not have anything at polar latitudes, or even subpolar latitudes, the database content\nis limited to mid latitudes/temperate latitudes, according to ocean climate zone definitions.\nThank you for your thoughtful comment. The Triton database does include records from high\nlatitudes, and in our study, we analyzed data from the North Atlantic up to 65°N, covering the\nsubpolar region. However, in high latitudes across different basins, data availability is often\nlimited to specific intervals, such as the mid-Piacenzian Warm Period or other short timeframes.\nGiven the high-resolution nature of our study, which required consistent temporal and spatial\ncoverage, high-latitude regions frequently failed to meet the criteria for sample completeness when\nanalyzed in 150 kyr by 5° latitude bins. These regions often showed patchy, inconsistent data\nacross the Pliocene–early Pleistocene. Therefore, to maintain the robustness and resolution of our\nanalyses, we excluded these regions. See lines 178-180.\nFIGURES\nFigure 1. (see my comments above) I note that this figure has just been copied more or less from\nWoodhouse and Swain et al., 2024, showing silhouettes of planktonic forams representing the\ndifferent ecogroups that absolutely did not exist (had gone extinct) during the time-interval under\nconsideration (e.g. Morozovelloides/ hantkeninids). It’s a bit concerning -since it suggests a\ndisconnection between actual knowledge of Plio-Pleistocene planktonic foram assemblages and a\nblind analysis of data. This figure needs to be properly customized to include representative\nmorphologies for all the eco groups that are actually characteristic of the Plio-Pliestocene,\nunderpinned by isotopic/and or plankton net ecology data.\nThank you for noting the issue with the icons used in Figure 1. We have corrected this oversight\nand replaced the faulty forms with representatives of extant species corresponding to the studied\ntime interval. We believe this revision aligns the figure with the scope of the study and addresses\nyour concerns.\nFigure 2: Environmental proxies for the Pliocene-early Pleistocene 671 Climate and Ecogroup\nSpecialization Index.\n-I have to say, compared to the previous papers using the bipartite analysis, the more resticted\ntime interval focus (i.e. of the bitartie analysis space-time map) is here at the edge of comfortable\nvisual resolution. i.e. in the previous longer time studies these figures were visually impressive\nand informative, but here, with the 150 kyr-long time binds are stretched out to their limits so\nthat the figures are not pleasent on the eye and require some standing back and squinting to see a\npattern. If there is the possibilty to change the axis limits to 1.6- 4 Myrs, this could help, laterally\nsqueeze everything. Or can you resample at 100 kyr bins? This could help with the visuals.\n-What are the white areas in the figure? Data gaps. Clarify.\nThank you for your detailed feedback. The white areas in the figure indeed represent data gaps or\nbins that did not meet our data completeness criteria and were therefore excluded from the\nanalysis. We clarified in the figure caption as suggested (see Figure 2). Regarding your suggestion\nabout time bins, we tested the analysis using different time bin sizes, including 100 kyr, but found\nthat 150 kyr time bins by 5° latitude provided the most complete and comprehensive coverage for\nour time frame. To address your concerns about visual resolution, we have improved the figure by\nadding space between panels, modifying the color scheme for better distinction, and including\nsolid black lines to emphasize key patterns. We hope these updates enhance the figure's clarity and\nvisual appeal.\nFig. 3. In the caption, explain ecogroup 3 for completeness.\n-the blue colours are not so easy to distinguish in as curves. Can you choose a set of more\n´distinctive colours for the ecogroup curves?\n-The axis tick mark choice is not look especially nice. Other options? The axis looks better in Fig\n4, so perhaps use the same axis limits and formatting for Fig. 3.\nThank you for your suggestions. We have updated the figure as recommended: the colors for the\necogroup curves have been adjusted to make them more distinctive, and the axis limits and\nformatting have been aligned with those used in Fig. 4 for consistency. Additionally, we have\nmoved this figure to the supplementary material as Fig. 2 and updated the caption to include an\nexplanation of all ecogroups, including ecogroup 3 for completeness.\nSM Figure 1. Please explain why there are no records above 60 degrees N in the North and nothing\nbelow 50 degrees S in the south. These high latitudes regions would be important for a holistic\nunderstanding of latitudinal pelagic sensitivities to global change but are missing. I know that there\nare deep sea drilling cores (and thus foram data) at least for the Atlantic and Indian Oceans with\nmove Antarctic and sub Arctic coverage. Is this excluded from Triton?\nThank you for your thoughtful comment. The Triton database does include records from high\nlatitudes, and in our study, we analyzed data from the North Atlantic up to 65°N, covering the\nsubpolar region. However, in high latitudes across different basins, data availability is often\nlimited to specific intervals, such as the mid-Piacenzian Warm Period or other short timeframes.\nGiven the high-resolution nature of our study, which required consistent temporal and spatial\ncoverage, high-latitude regions frequently failed to meet the criteria for sample completeness when\nanalyzed in 150 kyr by 5° latitude bins. These regions often showed patchy, inconsistent data\nacross the Pliocene–early Pleistocene.\nTherefore, to maintain the robustness and resolution of our analyses, we excluded these regions.\nHowever, we note that Woodhouse and Swain et al. (2023) provide complementary insights by\nanalyzing polar and subpolar regions of ESI for all basins combined at 250 kyr time bins. Their\nstudy highlights the expected pattern of high ESI, or lower evenness, in high latitudes, aligning\nwith ecological expectations. While our focus remains on high-resolution basin-specific analyses,\nthese findings further reinforce the patterns observed at higher latitudes.\nReviewer #2 (Remarks on code availability):\nThe code page exists where is says it exists. I have not looked further.\nReviewer #3:\nThe authors have analyzed taxonomic and functional diversity of a well-resolved plankton group\nacross an interval of Earth history relevant to present-day climate warming. I was interested to\nread this article from its premise alone, and I would be happy to see an article on this topic\nappear in Nature Communications. My comments are mostly minor (although see two larger\nmethodological comments/questions), and most relate to improving clarity, especially of the\nmethodological details, or softening the interpretive assertions to be less definitive. I apologize to\nthe authors for my long delay in returning this review and hope the detail of constructive\ncomments below helps offset the inconvenience.\nThank you for your thoughtful review and constructive comments. We are grateful for your positive\nfeedback on the relevance of our study and your detailed suggestions, which will help us improve\nthe clarity and robustness of the manuscript. We appreciate your time and effort in providing this\nvaluable input.\nAbstract and Introduction\n1. Line 21 and 42: I suggest rewording “recent observations” to something like “observations of\nliving species”. “Recent observations” could mean samples of living species taken in the last few\nyears or, geologically speaking, samples from the Recent.\nWe have edited the text to clarify that we are referring to recent studies of both past and modern\nplanktonic communities (lines 22, 37-38).\n2. Line 47: Do you mean “life system responses”? I wouldn’t consider plankton part of the\nEarth system. You could also be wordier but explicit, “life system responses to Earth system change”\nor similar.\nWe have revised the sentence to explicitly state \"how biological systems respond to changes in\nthe Earth system,\" as this more accurately conveys the intended meaning (line 44).\n3. Line 48: Define Ma at first use for non-geology/paleontology readers\nWe defined Ma as ‘millions of years old’ for non-geology readers (line 45).\n4. Line 50: This is referring to the rise of the Panama isthmus, correct? I would include reference\nto that by name, since I think it’s a more well-known geographic feature than the Central\nAmerican Seaway. Also consider additional references – the single citation for the given age date\nwas published several years ago now and there’s been heated debate about the exact timing of\nthe closure.\nThank you for your comment. We have updated the text and acknowledged the ongoing debate\nregarding the timing of the closure of the Central American Seaway (lines 48ff).\n5. Line 80: I understand the typical narrative structure of introductions is to include a sentence\nlike this (“despite some prior study, there is still a gap in understanding X”), but I suggest\nomitting the sentence here beginning “Despite ongoing efforts…”. There have arguably been\nmore comprehensive and high-resolution comparative studies of zooplankton responses to\npaleoclimate change than for any other group except bivalves. That doesn’t make the topic less\ndeserving of study! Simply merge some the language from the sentence with the next two\nsentences for flow in the paragraph. For example, something like,\n“The recently collated Triton dataset, which provides an extensive record of global fossil\noccurrences of planktic foraminifera, allows for comprehensive, high-resolution studies of\nzooplankton community responses to paleoclimate change across regional scales.”\nThank you for your suggestion. We have revised the text to merge the language for better flow and\nto avoid overstating gaps in prior research (See lines 88ff).\nResults\n6. The results section needs to include a several-sentence introduction to what bipartite\nnetwork analysis is and how it calculates ESI, including a definition of equitability that doesn’t\ninclude the word equitable. I know there is prior literature applying this method to similar questions, but\nthis paper needs to be fully understandable on its own for a reader who may be unfamiliar with\nthat previous work and with network theory. How would you explain this to an advanced\nundergraduate learning about it for the first time?\nThank you for your feedback. We have revised the text to clarify the concept of the Ecogroup\nSpecialization Index (ESI). Further clarification on how the Ecogroup Specialization Index (ESI)\nor Specificity Specialization Index is calculated can be found in Julliard67 and Poissot68. Instead\nof defining \"equitability\" in general, we provide an explanation of what equitable means within\nthe context of our study, which we hope increases clarity about this metric as suggested by the\nReviewer (Lines 142-149)\n7. The information currently in the Methods about how to interpret ESI (ranging from 0 to 1,\nwith 1 indicating specialization) needs to be repeated here as ESI results are presented. Readers\nneed this context to interpret Fig 2, for example.\nWe have revised the text as suggested by the Reviewer 3 (see lines 142-149).\n8. Line 126: I would say “might be” the result of thermocline differences rather than “likely” due\nto it. There are alternative hypotheses, and reality is usually a mix of processes at work. Without\ndata on the thermocline or an estimate of its effect relative to other processes, it feels too\nassertive to me to assume the pattern is due to thermocline differences alone.\nWe have revised the text as suggested by the Reviewer (lines 173).\n9. Line 128: Reword to avoid the words “subtropical” and “tropical”. The location of the\ntropics has changed through time, so it is better to discuss the location of biogeographic patterns\nrelative to static latitudinal markers.\nWe have reworded the sentence as suggested (lines 176).\n10. Line 128: Say “shift” rather than “noticeable shift”. I agree it’s present, but it’s a slight and\nqualitative signal rather than jumping off the page. Also remove the word “significant” from the\nfollowing sentence unless it is associated with a statistical test result.\nWe have reworded the sentence as suggested (lines 175).\n11. Line 136: This study calculates relative occurrence frequency, not species’ niche breadth or\nposition. Changes in relative occurrences could be due to niche occupancy changes but could\nalso result from changes in the spatial extent of species’ habitats or other factors. Please reword\nto avoid the phrase “ecological niche expansion” here. Similarly, please remove the phrase\n“reconfiguration of ecological niches” in the caption to fig 3.\nWe have reworded the sentence as suggested by the Reviewer (lines 186-189) and revised the\ncaption for Figure 3 to eliminate the phrase \"reconfiguration of ecological niches,\" addressing\nyour concern (see supplementary figure 2).\n12. Line 150: Environment is presumably stable along both vertical and latitudinal axes, right?\nYes, that’s correct.\n13. Line 167: The in-text results in the sentence beginning “The highest rate of change…” don’t\nappear match what I see in Fig 4. It’s unclear that ecogroup diversity did drop after cCAS in the\nNorth Atlantic, and I certainly wouldn’t call it a “sharp decline”. Rather, the green line for the N\nAtlantic barely drops (not significant) in the first three time bins proceeding from the cCAS, and\nit then drops at the onset of the mPWP (Fig 4B). The “highest rate of change in ecogroup\ndiversity” instead looks like it might be in the North Pacific around 3.4 Ma.\n14. Line 171: As in comment #13, the text description here appears to differ from what I see in\nthe figures. Ecogroup diversity does not consistently increase in the Northern Pacific (second\npanel of Fig 4B) as the sentence beginning on this line asserts (“a consistent increase in ecogroup\ndiversity is evidence across all basins in the Northern Hemisphere”). I am also unconvinced that\nthe changes happened exactly at the iNHG, in either fig 3 or 4, as stated in the captions there and\nin line 172. The onset of a directional trend could be argued to originate anytime between the\nmPWP and iNHG from figs 3 and 4, and it is ultimately a qualitative call rather than a\nquantitative break-point estimate.\nReply to both point 13 and 14: Thank you for your detailed feedback regarding Fig. 4B. We have\ncarefully reviewed the patterns and revised the text accordingly. Our analysis indicates that the\nlargest changes in ecogroup diversity occurred in both the North Atlantic and North Pacific,\nthough during different periods. In the North Atlantic, ecogroup diversity increased immediately\nafter the mPWP (around 3 Ma), while in the North Pacific, the increase occurred earlier, between\n3.7 and 3.2 Ma.\nThe magnitude of change in both basins was similar, around 0.8 corrected sampled-in-bin\ndiversity (SQS diversity). We have clarified these points in the text and adjusted the interpretation\nto reflect the timing and magnitude of these changes more accurately (lines 220ff).\n15. Consider discussing limitations of unequal sample size between hemispheres and basins\nThank you for your suggestion. We have included a discussion of the statistical tests performed to\naddress the limitations of unequal sample sizes between hemispheres and basins. Specifically, we\nincorporated resampling analyses and sampling coverage to ensure the robustness of our findings.\nThese analyses confirm that observed patterns are not artifacts of sampling bias (lines 133-139).\nDiscussion\n16. Line 177: I would suggest an additional reference here as in the Introduction, as this timing\nhas been a contentious topic.\nWe revised the text to as suggested by the Reviewer.\n17. Sentence beginning 182: I’m unable to follow the reasoning of this sentence. The evidence\ncited in the preceding sentence seems to be from around the closure at 2.8 Ma, but also implies\nthis study found matching changes beginning at 3.6. Those timings don’t match up perfectly. In\naddition, the “global stability…prior to 3.6 Ma” mentioned here isn’t previously mentioned in\nthe Results and is based on only 2 data points (between 3.6 and 3.9 Ma). I agree that the closing\nof the CAS was a major biogeographic event, and it is worth mentioning in the Discussion. That\nsaid, looking at the figures, e.g., Fig 3 and 4, I struggle to see any features of the data around\nthen that couldn’t be attributable to white-noise variation (no rapid directional changes between\n3.5 and 3.9 Ma). I suggest walking back the strength of the claims in this paragraph. If there were\nmajor shifts from before vs. after the cCAS (entirely possible and even expected!), the time\nseries of the data analyzed here unfortunately don’t extend back long enough to document it.\nThere is still plenty of discussion material without this point.\nThank you for your feedback. We have revised the text to address your concerns. Specifically, we\nhave softened the statement about \"global stability prior to 3.6 Ma\" to avoid overinterpretation\n(lines 234-239).\n18. Paragraph 205: The events postulated here may all have occurred. We cannot know for\ncertain that this is exactly how history played out, however. Please add in some qualifiers to\nindicate that this might have been what happened. For example: “ocean cooling could have\nenhanced water column stratification….cooling also might have contributed to the decrease….as\na result, more organic material would have reached a mesopelagic zone”.\nWe have modified the text to include qualifiers, as recommended, to reflect the uncertainty\nsurrounding the proposed events (Lines 275-278).\n19. Line 214: As commented in point #11, this study doesn’t estimate species’ niche breadth or\nposition. From the data presented, it isn’t possible to distinguish the effect of changes in niche\noccupancy from changes in the spatial extent (vertical and/or latitudinal) of habitats representing\nthe original (potentially unchanged) niche. A mechanism-agnostic reframe of this sentence\nwould be, “An elevated nutrient availability might have enabled subthermocline populations to\ngrow”.\nWe have modified the text as suggested (lines 278-281).\n20. Line 215: As commented in point #14, the exact onset of the change in ecogroup and/or\nspecies diversity is hard to pin down as perfectly corresponding to the iNHG – in Fig 4, it looks\nlike it could have started even has far back as 2.8 or 2.9 Ma in the Northern Atlantic. Rather than\ndeclaring that “the intensification of the Northern Hemisphere Glaciation catalyzed a\nreconfiguration of latitudinal diversity and equitability gradients,” perhaps the authors would\naccept a softening to something like, “there is a possible mechanistic pathway for the iNHC to\nhave catalyzed a reconfiguration…”.\nWe modified the text as suggested (lines 282ff).\n21. Line 248: There is only one prior point in the manuscript with any mention of results for the\nIndian Ocean (line 129). I know there are few data for the northern hemisphere component,\nbecause it has so little geographic area, but it would be nice to give the (southern) Indian Ocean\nmore comparable page space as the Pacific throughout the article.\nWe have made an effort to incorporate more information about the Indian Ocean where\nappropriate in the manuscript. However, the Indian Ocean basin, particularly its Northern\nHemisphere component, has limited data coverage and does not exhibit significant patterns\ncomparable to those observed in the Atlantic or Pacific basins. As such, the scope for discussion\nis naturally more constrained. Nonetheless, we have aimed to ensure that the Indian Ocean is\nrepresented as fully as possible given the available data (see lines 175-177; 264-269; 274-277;\n318-321).\nMethods\n22. Line 489: Define kyr at first use for non-geology/paleontology readers\nWe have revised the text as suggested by the Reviewer (line 93).\n23. State how many species were studied\nWe now include the number of studied species (line 340) and include a table with all the studied\nspecies in the supplementary material (Supplementary Table 1-2).\n24. State how many time bins there were\nWe have revised the text as suggested by the Reviewer (line 345).\n25. Line 509: Is sampling coverage quantified as Good’s u? That seems to be what is described,\nbut it needs to be specified if so – or if not, how else coverage is calculated\nSampling coverage in the iNext package is indeed based on Good’s U estimator. We have\nclarified this in the text and provided additional details as needed (lines 366-368).\n26. Is the ESI weighted by ecogroup diversity? It sounds like it is calculated by the relative,\nraw occurrence counts of the different ecogroups, but since there are many more species in some\ngroups than others my naïve guess would be that changes in depauperate ecogroups (mainly the\npolar one) would under-influence ESI. Or maybe the point of null models is to mitigate this for\nresults interpretation? This gets back to needing more text (perhaps even a whole paragraph) of\nthorough explanation about what bipartite analysis is and how exactly ESI is computed.\nThe ESI is calculated using the relative occurrence counts of ecogroups within each\npaleolatitudinal band, ensuring that the contributions of depauperate ecogroups, such as the polar\ngroup, are proportionate to their representation. While larger ecogroups inherently have more\ninfluence due to higher occurrence counts, the use of relative frequencies mitigates this bias.\nAdditionally, null models are employed to account for expected distributions under random\nconditions accounting for statistical variations and sampling errors, allowing us to interpret\ndeviations with more statistical rigor. We have expanded the manuscript to include a detailed\nexplanation of the bipartite analysis, the calculation of ESI, and the role of null models in\naddressing any potential biases (see lines 371ff).\n27. Line 525-528: Can you reword the explanation of the null model? I am especially confused by\nthe wording, “taking into account the number of occurrences within specific latitudinal bands\nacross all groups and within group temporal bin across all latitudes.” I know the journal may\nimpose strict word limits, but adding at least another sentence or even a whole paragraph would\nbe worth it to make this very clear to visualize and follow. For instance, how is the model iterated?\nNothing in the one-sentence overview of the model is described as probabilistic, but it must be for\nthe model to differ between runs. The figures of null model results (supplemental figs 10-12)\naren’t mentioned in the main text, so I’m also unclear how they feed into or are compared against\nthe main analysis.\nWe have now reworded the explanation of the null model to increase clarity and explicitly describe\nthe probabilistic nature of the model, including how iterations are performed. The null models\nexplicitly show whether the observed occurrences may happen by chance alone or not, given the\noccurrence data across the spatiotemporal bins. These models also inform us about the extent and\nthe direction of deviation from random chance, and this ensures a secondary validation of the ESI\nvalues we calculate from the observed occurrence values (see lines 390-401). We have also now\nmentioned the supplemental figures 10-12, which present a summary form of the results from our\nnull model analyses, in the main text.\n28. Line 536: Explain what “corrected sampled-in-bin” metric is and give a citation\nThe 'corrected sampled-in-bin' metric is used to account for sampling biases when estimating the\npresence of taxa within a specific time bin. It adjusts the observed data to better reflect true\necological or evolutionary signals by correcting for variations in sampling effort across different\nintervals. This approach is particularly useful in paleontological and ecological studies to avoid\nover- or under-representing taxa due to uneven sampling. For further details, please refer to\nAlroy (2010), who provides a comprehensive methodology for implementing these corrections:\nAlroy, J. (2010). Fair sampling of taxonomic richness and unbiased estimation of origination and\nextinction rates. Paleobiology, 36(2), 227-243.\nWe added the explanation of the “corrected sampled-in-bin” metric to the methods and provided\na citation as suggested (see lines 406-410).\n29. Line 538: What is the smallest sample coverage estimated from the empirical samples and\nhow does it compare to the value of 0.7 chosen as quorum value? The quorum shouldn’t exceed\ntwice the minimum coverage, and personally I prefer not to extrapolate at all (i.e., to set the\nquorum at or below the minimum attained by empirical samples). Please report the coverage and\nrevise it if appropriate.\nThe sample coverage for species diversity in our dataset ranges from 0.89 to 1.00, while the\ncoverage for ecogroup diversity is consistently 1.00. Based on your feedback, we reviewed our\nchosen quorum value. Initially, we had set a quorum of 0.7; however, we actually increased this\nto 0.8 during the analysis to better align with the observed sample coverage. For further details\non our sample coverage, please see the supplementary figures (Supplementary Fig.13-14 and 17).\nMajor methods comments\n30. Line 538: I am confused by the explanation of why to use 100 kyr time bins here when the\nstudy is conducted at 150 kyr resolution. What do you mean it’s because of “the possibility” of\nrunning one analysis at a finer resolution? In practice, from what I understand, both taxonomic\nand functional diversity analyses were run at the same resolution of 150 kyr. Unless there is a\nsimple explanation I am missing, I would ask the authors to repeat this at 150 kyr to match the\nanalyses. If the analyses are run at different resolutions (e.g., one at 100 kyr and one at 150 kyr),\nthen I would ask to repeat the one at 100 kyr again at 150 kyr, to be able to evaluate results on\nthe same temporal scale.\nTo avoid confusion and ensure consistency, we now conduct all analyses, including taxonomic and\nfunctional diversity, at the same resolution of 150 kyr. We appreciate your suggestion to improve\nthe clarity and rigor of our methodology.\n31. Line 542: SQS has assumptions and limitations like any method, which need to be addressed\nhere. I know that SQS is the go-to diversity estimation method in many quantitative paleobiology\nstudies, most of which poorly justify this choice, and I also know that there’s a widespread lack\nof understanding/lack of teaching about exactly how SQS works under the hood. My goal isn’t to\ntalk the authors out of SQS, but to provide some more information to help them make this\nmethods choice more robust. To begin, SQS is a type of rarefaction (it goes by Coverage-Based\nRarefaction or CBR in ecology literature), so it's incorrect to say it “offers a significant\nadvancement over traditional subsampling approaches like rarefaction.” Yes, CBR/SQS can lead\nto more accurate estimates than classical count-based rarefaction under certain conditions (cite\nboth Chao and Jost 2012 and Alroy 2010, Science). CBR/SQS can also produce wildly less\naccurate estimates if the data fail to meet its assumptions. One of the more challenging\nassumptions is that evenness is constant across time/samples. Here, then, are my suggestions:\no Reword the Species and Ecogroup Diversity subsection to explain more clearly how SQS\nworks. The supplement to Close et al. 2018 in MEE, “How should we estimate diversity in the\nfossil record,” is an accessible place to read about this besides Alroy’s original paper.\nThank you for the suggestion. We have revised the text as suggested (see lines 403ff).\no Add error bars to the diversity estimates in Figure 4. Or, if this makes the figure too cluttered,\nmake it a separate supplemental figure that gets its own page to be a larger size. It’s easy to ask\nfor, say, a 90% confidence interval from iNEXT output\nThank you for the suggestion. We have added 90% confidence intervals to the diversity\nestimates, which are now presented in Supplementary Figure 18-19 for clarity.\no Calculate one or more evenness metrics and plot them as a time series in a supplemental figure,\nwith panels for each ocean basin. If they seem variable, or it’s hard to feel confident in them,\nrepeat the diversity analysis using either raw diversity or classical count-based rarefaction. Make\na new version of figure 4 with this different calculation of diversity, and put it in the supplement.\nThank you for your suggestion. We ran Pielou's Evenness (J) as the evenness metric, as it is\nparticularly well-suited for capturing species balance in our dataset, which spans different time\nbins and hemispheres with variable richness and the presence of rare species. The results are\nplotted as time series, with panels for each ocean basin, and can be seen in Supplementary Figure\n15-16 . Our sampling coverage is quite high, as well as the calculated evenness, so we have opted\nnot to repeat the diversity analysis using count-based rarefaction.\nRecurring comment throughout the text:\n32. Please check and revise the way in-line citations are formatted to match journal guidance.\nI noticed through the article that for multi-author papers, the format is “Jones et al. found…”\nexcept when one of the authors of the present study is the second author. Then, the format\nchanges to “Jones and Johnson et al. found…”. Only the first author should be named (or\nhowever journal formatting rules dictate).\nThe citation format for Woodhouse and Swain et al., 2023 is intentional, as both authors are listed\nas co-first authors. This format follows the journal's guidelines for acknowledging co-first\nauthorship. We have ensured that all other in-line citations are formatted according to the\njournal's rules. If further clarification is needed, we are happy to make additional adjustments.\n33. Cite version numbers for all individual software packages, e.g., for bipartite, divDyn,\niNEXT, etc.\nVersion numbers of all individual software packages were included in the manuscript.\nFigures\n34. Fig 2: Please separate the panels with white space\nWe separated the panels in Figure 2 with white space, as suggested.\n35. SM fig 1: Please change the color coding. The blue and green dots are difficult for me to\ndiscern and I expect will be inaccessible to people with color vision deficit. Coblis is a good tool\nto check for accessibility https://www.color-blindness.com/coblis-color-blindness-simulator/\nThank you for your suggestion. All figures in the manuscript use color schemes that are color-\nblind friendly, as defined by ColorBrewer, and were tested in a color blindness simulator. For SM\nFig. 1, We adjusted the colors based on your feedback and hope the new scheme improves\naccessibility. We appreciate your input to ensure the figures are inclusive.\n36. SM fig 2: Consider changing the definition of axis ticks from every 0.15 Ma to 0.1 or 0.2.\nConsider (optional) reversing the plot order of ecogroups 1 to 5, so group 1 is on bottom.\nWe have updated the axis ticks to intervals of 0.2 Ma as recommended and reversed the plot order\nof Ecogroups 1 to 5, placing Group 1 at the bottom. We appreciate your input in improving the\nfigure's readability.\n37. SM fig 3-7: Define iNHG, mPWP, and cCAS in captions. Add confidence interval shading\naround the linear fit lines so the viewer can tell whether they are significantly different from a\nzero-slope/horizontal line. I realize that might add visual clutter, in which case you could remove\nthe lines connecting points and just leave the points (not necessary, just a suggestion to try in\ncase it’s helpful).\nThe terms iNHG, mPWP, and cCAS have been defined in SM Fig. 2 caption. We also added 90%\nconfidence interval shading around the linear fit lines to help viewers assess whether they are\nsignificantly different from a zero-slope/horizontal line. See supplementary figures 18-19. We\nappreciate your feedback in improving the clarity of these figures.\n38. All plots: Convention differs by scientific field, but I often recommend authors consider\nflipping age axes so time moves from left (oldest) to right (youngest). It’s up to the authors, just\nmake sure to update the slope definitions on line 563 if changing this.\nWe understand the convention of flipping age axes in some scientific fields; however, we prefer\nto retain the current orientation of our axes, as it aligns with the conventions commonly used in\nNeogene vs Phanerozoic plots. We believe this approach maintains consistency and clarity for\nour intended audience. We appreciate your understanding."}]

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