Vertical distribution of planktic foraminifera through an Oxygen Minimum Zone: how assemblages and test morphology reflect oxygen concentrations

15 Oxygen-depleted regions of the global ocean are rapidly expanding, with important implications for global biogeochemical cycles. However, our ability to make projections about the future of oxygen in the ocean is limited by a lack of empirical data with which to test and constrain the behavior of global climatic and oceanographic models. We use depth-stratified plankton tows to demonstrate that some species of planktic foraminifera 20 are adapted to life in the heart of the pelagic Oxygen Minimum Zone (OMZ). In particular, we identify two species, Globorotaloides hexagonus and Hastigerina parapelagica, living within the Eastern Tropical North Pacific OMZ. The tests of the former are preserved in marine sediments and could be used to trace the extent and intensity of low-oxygen pelagic habitats in the fossil record. Additional morphometric 25 analyses of G. hexagonus show that tests found in the lowest oxygen environments are larger, more porous, less dense, and have more chambers in the final whorl. The association of this species with the OMZ and the apparent plasticity of its test in response to ambient oxygenation invites the use of G. hexagonus tests in sediment cores as potential proxies for both the presence and intensity of overlying OMZs. 30

The goals of this study are to describe and quantify the abundance of living planktic 105 foraminifera above and within a modern OMZ, to test: 1) whether modern planktic foraminifera are present within the OMZ; 2) whether specific species are preferentially or exclusively living within the OMZ; and 3) whether morphological traits of OMZ-dwelling foraminifera reflect oxygenation levels in the environments from which they are recovered 110

The Eastern Tropical North Pacific Oxygen Minimum Zone
The Eastern Tropical Pacific is home to the world's largest OMZ, fueled by a combination of high coastal and equatorial productivity and poorly ventilated subthermocline waters (Paulmier and Ruiz-Pino, 2009;Fiedler and Talley, 2006). The OMZ 115 in the Eastern Tropical North Pacific (ETNP) is associated with both a deep particle maximum and a secondary nitrite maximum, indicative of reduction of nitrate to nitrite within the OMZ (Garfield et al., 1983;Buchwald, et al., 2015;Medina Faull et al., 2020).
The region sampled here is located west of the Baja peninsula and removed from the regions of greatest surface productivity, towards the northern reaches of the low oxygen 120 tongue of the ETNP OMZ ( Fig. 1; Supplemental Fig. 1).

Plankton Tow Collections
Day and night vertically stratified and horizontal MOCNESS (Multiple 125 Opening/Closing Net and Environmental Sensing System) tows were taken onboard the R/V Sikuliaq. An updated MOCNESS system, 1 m 2 in diameter, with 222 µm mesh nets and a Sea-Bird SBE911 CTD with updated software in place of the original sensors was used (see Wishner et al., 2018). All tows were carried out within relatively close proximity to one another (21° N, 117° W) between January 26 th and February 7 th 2017 130 (Wishner et al, 2018(Wishner et al, , 2020a(Wishner et al, , 2020b. This study utilized a total of 8 tows, with each tow including the deployment of eight to nine nets to sample a defined depth interval. We use six depth-stratified vertical profiles (#716, #718, #720, #721, #722, #725) that sampled portions of the 0 -1000 m water column, and two horizontal tows that sampled the OMZ at ~425 m depth (#724, #726) (Wishner et al. 2018(Wishner et al. , 2020a(Wishner et al. , 2020b. Vertical strata 135 sampled by each net were 25 m to 200 m thick, depending on the tow and depth (see Supplemental Table 1 or Wishner et al. 2019Wishner et al. , 2020b for net strata depths and volume filtered for each net in). In horizontal tows, each net sampled a distance of about 1 km (Wishner et al. 2018). Environmental data were collected with the MOCNESS CTD sensors simultaneous with plankton collections. For oxygen, a Sea-Bird SBE43 sensor 140 was used. All plankton samples were stored in sodium borate-buffered seawater and formalin at sea. Isolation of foraminifera from samples occurred in 2017-2019 at the University of Rhode Island. Between 3/10 ths and 1/125 ths of material in a net was examined, depending upon abundance of foraminifera, and all intact tests were isolated from the split. 145 Foraminifera were identified to the species level by light microscope at the University of South Carolina and Yale University. Some tests (9% of the total observed) were either damaged or, more rarely, appeared to be juvenile forms, such that no species-level identification could be assigned. Due to excellent tissue preservation, the presence or absence of foraminiferal cytoplasm was identifiable, and foraminifera were classified as 150 either "live," based on the presence of cytoplasm, or "dead" in the absence of cytoplasm ( Fig. 2). Although preservation was excellent in most tows, some dissolution was observed in 3 shallow (< 100 m) nets. These have been excluded from further analyses, to prevent skewing assemblages towards more dissolution-resistant taxa. We note that these 3 nets were exceptionally high in organic matter and that organic matter degradation was 155 the likely cause of dissolution despite buffering and a relatively short storage interval.
The organic matter concentration and preservation concerns in these 3 nets do not apply to the other nets considered in this study.

Counting and Statistics 160
Total counts of foraminifera were adjusted for both the tow split analyzed as well as the total water volume filtered and are presented as individuals m -3 or as relative abundance. Diversity was calculated using the 'diversity' function and Shannon index in the R 'vegan' package (Oksanen, et al., 2013). All other statistics were carried out in the 165 base package in R (R Core Team, 2017).

Morphological Analyses
All individuals of the species G. hexagonus were weighed on a Mettler Toledo ultramicrobalance (± 1 μg) in the Yale Analytical and Stable Isotope Center and imaged 170 on a Leica DM6000 light microscope at Yale University. Measurements were made in ImageJ by identifying a flat section of the F (final/ultimate) or F-1 (penultimate) chamber minimally affected by glare and measuring the total area of the section and the total area of section excluding pores. All other morphometric measurements were made using the AutoMorph software (Hsiang et al., 2016). 175 Porosity is reported as the percentage of test surface area comprised of pores. Sizenormalized weight was assessed using the area density method described by Marshall et al. (2013), with the weight of each test normalized to its 2-dimensional surface area. The compactness of tests was assessed as the ratio of the 2-dimensional surface area to the area of a circle (the most compact possible geometry) of the same perimeter. The aspect 180 ratio was defined as the ratio between the height (longest dimension) and width (perpendicular to the longest dimension) as measured in the AutoMorph software (Hsiang et al., 2016). Test size was ascertained by length, surface area, and test perimeter. As surface area and test perimeter were used in deriving compactness and size-normalized weights, respectively, and all parameters are interrelated, we refer to the longest test 185 dimension when referring to size.
Micro CT-scans were generated at the Naturalis Biodiversity Center using a Zeiss Xradia 520 Versa micro-CT scanner aiming at a voxel size of 0.627 μm; realized resolution varied from 0.4-0.7 μm. Scans were made at 90 kV using 20X optical magnification, and were reconstructed using the Zeiss software. Micro CT scans were

Hydrological Data from Tows 195
Plankton tows sampled depths between 0 and 1000 m, across dissolved oxygen levels between 0.03 and 4.93 ml L -1 and temperatures ranging from 4.5 to 22.9° C. Although small-scale oxygen features and their depth relative to the oxycline and OMZ varied somewhat (Wishner et al., 2018(Wishner et al., , 2020b, the overall structure of the water column was consistent across tows. A warm, oxygenated surface mixed layer overlaid an oxygen 200 depleted OMZ, with gradual cooling at increasing depth below the thermocline. The upper oxycline (the zone of rapidly decreasing oxygen) was located between 150 and 250 m water depth, with its upper boundary at the thermocline ( Fig. 3-5). Categorization of oxygen levels follows the discussion of Hofmann et al. (2011) and Moffitt et al. (2015).

Live Foraminiferal Assemblages
Assemblages of live foraminifera, described using the definitions of oxygen outlined 215 above, can be divided into three categories: those living in oxic conditions (minimum [O2] within a net > 2.45 ml L -1 ), OMZ conditions (maximum [O2] within a net < 1.4 ml L -1 ), and transitional (nets sampling between these two concentrations). The oxic group was the shallowest, with the deepest tow included in this category extending to only 150 m water depth. These tows had the highest standing stock of foraminifera with 3.4 220 individuals m -3 and the greatest diversity with a mean Shannon index value of 1.3 (ranging from 1.2 to 1.5 across 5 nets). In this relatively shallow, oxic environment, the assemblage was dominated by Trilobatus sacculifer (74.6%) followed by bulloides were found in low abundance (<1%) (Table 1; Fig. 6). The OMZ assemblages were also the least diverse, with a mean Shannon index value of 0.9 (ranging from 0.8 to 1.0 in 54 nets).
The transitional assemblages primarily represented depths between 100 and 250 m 240 and had the lowest standing stock of foraminifera with 0.1 individuals m -3 . There was one net that sampled 800 to 1000 m and would also fall into this oxygen categorization, but was excluded from analyses as it contained only a few G. ruber (< 0.01 individuals m -3 ) which were likely dead, and cannot be readily compared to the upper oxycline habitat of other transitional samples. The transitional assemblage was nearly as diverse as the oxic 245 assemblage with a Shannon index of 1.2 (ranging from 1.1 to 1.2 across 4 nets). It was (3.9%), G. conglobatus (2.4%), and S. dehiscens (1.6%). A few other species, H. parapelagica, C. nitida, G. ruber, and P. obliquiloculata, were found in abundances < 250 assemblage (Table 1). While every species occurring with cytoplasm was also found without cytoplasm, two species, Hastigerina digitata and Neogloboquadrina dutertrei, were identified in low abundances without cytoplasm, but were not observed with cytoplasm. 265

Porosity
Porosity of the most recent chamber in G. hexagonus was highly variable among individuals and among tows, ranging from 1.7% to 19.4% of the surface area measured 270 by light microscope. Porosity decreased as oxygen increased, with the clearest relationship between the log of porosity and log of dissolved oxygen (R 2 = 0.38, p-value < 0.001). We chose to focus porosity measurements on the most recent chamber as it was the chamber most likely to have formed under the conditions recorded at collection; however as the foraminifera analyzed had not yet reproduced, it is not possible to know 275 whether this chamber would also have been the terminal chamber, analogous to the final chamber in a fossil shell.
A comparison between porosity of the most recent chamber, measured by CT scan and light microscope, showed that CT measurements consistently demonstrated higher porosities (Fig. 7). This methodology allowed for non-destructive imaging of the 280 inner test unobscured by later calcite growth, the ability to manipulate test orientation to reduce artifacts of test curvature, as well as higher resolution, and should be considered a more accurate measure of test porosity. A direct comparison of the two methods carried out on a subset of tests (n = 31) showed that the results from the two approaches are capturing a similar trend, the approaches are distinct enough that measurements by one method (light microscopy) are not sufficient to predict porosity as measured by another (CT scan). Final chamber porosity increased linearly with the size across individuals (R 2 = 0.33, p-value < 0.001), and with ontogeny within individuals (Fig. 8), demonstrating a possible relationship between size, ontogeny, and porosity. 295

Size and Chamber Number
Size decreased with the log of oxygen (Spearman's ρ = -0.64; p-value < 0.001).
The largest change in size, as well as the largest change in size-normalized weight and chamber number, was a step change occurring at oxygen levels between 0.1 and 0.2 ml L -300 1 (Fig. 9). The number of chambers visible in the final whorl ranged between 4 and 7 (net means between 4.8 and 6.1) and the largest change in mean chamber number also occurred between 0.1 and 0.2 ml L -1 O2, with tests having a greater number of chambers in the final whorl in low oxygen tows (correlation of chamber number to log of average oxygen: Spearman's ρ = -0.68; p-value < 0.001; Fig. 9). 305

Compactness and Aspect Ratio
We further tested the utility of test compactness and aspect ratios as potentially diagnostic of the morphological gradient observed. Although test compactness increased linearly with oxygenation (R 2 = 0.03 p-value = 0.04) and aspect ratio decreased linearly with the log of oxygen (R 2 = 0.09 p-value < 0.001), oxygenation accounted for very little 330 of the variance in either parameter and they were not considered further.

Distinct OMZ Community of Planktic Foraminifera
Live foraminifera obtained from vertical profiles with depth-stratified nets in the 335 ETNP form three distinct pelagic assemblages associated with differing oxygen levels.
The OMZ community, living at the lowest oxygen level, was typified by the presence and high relative abundance of the foraminifer G. hexagonus.
menardii was the most abundant species. A slightly shallower thermocline (compare Fig.   3 to Fig. 4 and 5) and deep chlorophyll maximum may be partially responsible for differing abundances. However, there may also be a lunar-associated reproductive response affecting abundance patterns. Tow #716 was taken during a waning moon, but 350 tows #721 and #725 were taken during a waxing moon (USNO, accessed 10/10/2019).
Trilobatus sacculifer reproduces on a lunar cycle, with the largest sizes reached just prior to reproduction during the full moon Erez et al., 1991;Kawahata et al., 2002;Lin et al., 2010;Jonkers et al., 2015;Venancio et al., 2016). As a result, more individuals large enough (> 222 µm) to be sampled in our nets may have been present 355 just prior to a full moon (tows #721 and #725).
The OMZ assemblage was dominated by the species G. hexagonus, followed by T. it is unlikely that this species, which has photosymbionts and a relatively shallow, photic zone habitat (Fairbanks et al., 1982;Ravelo & Fairbanks, 1992;Schiebel et al., 2004;Regenberg, et al., 2009;Birch et al., 2013;Rebotim et al., 2017), was resident in the deep across 4 tows; Fig. 3-5). The low density of foraminifera in the oxycline is an interesting contrast to the vertical distributions of many metazoan species that often peak in abundance in the upper oxycline and decline in the core of the OMZ (Maas et al. 2014, 385 Wishner et al., 1995, 2020b. Based on the mixed assemblage and low densities, we hypothesize that planktic foraminifera are largely absent from the upper oxycline, with populations restricted to either the oxygenated photic zone habitat above or the OMZ below. Whether this distributional pattern is related to physiological constraints, food resources, predation pressure, physical oceanographic mechanisms, or other 390 Deleted: density environmental parameters is unknown and future sampling at higher vertical resolution through the oxycline is required to test these hypotheses.

Globorotaloides hexagonus as an OMZ Indicator Species 395
Globorotaloides hexagonus was consistently found within our low oxygen nets, though individuals were sparsely distributed (mean density of 0.2 individual m -3 ), with peak abundances between 300-500 m depth in the core of the OMZ ( Globorotaloides hexagonus has previously been associated with deep, low oxygen 405 water masses across the Indo-Pacific, including the Eastern North Pacific (Sautter & Thunell, 1991;Ortiz et al., 1996;Davis et al., 2016), Equatorial Pacific (Fairbanks et al., 1982;Rippert et al., 2016;Max et al., 2017;Rippert et al., 2017), the Peru-Chile margin (Marchant et al., 1998), and the Indian Ocean (Rao et al., 1989;Schiebel et al., 2004;Birch et al., 2013). The species is sometimes assumed to be extinct in the Atlantic, with 410 recent identifications of G. hexagonus in Atlantic sediments explicitly used to date sediments as pre-Holocene or ascribed to taxonomic error (e.g., Kucera et al., 2005;Siccha & Kucera, 2017). However, the assumption of a basin-wide extinction appears poorly supported, and G. hexagonus tests were isolated from deep (500 -3200 m)  (Smart et al., 2018). We hypothesize that G. hexagonus occupies low-oxygen mid-waters globally (i.e., in the Atlantic as well as the Indo-Pacific), but that its deep habitat and low abundance have biased observations away from identifications of G. hexagonus in the modern Atlantic. However, additional 420 evidence, such as molecular genetics, may be required to finally resolve this question.
Altogether, the geographic distribution, presence of cytoplasm-bearing G. hexagonus in OMZ tows, and scarcity of G. hexagonus above the oxycline, strongly suggest that G. hexagonus lives preferentially, or even exclusively, within the OMZ. This species can be considered an indicator of an OMZ habitat and may be useful as an OMZ marker in 425 sedimentary records.
We also found a second, less abundant, species, H. parapelagica, in association with low oxygen waters. This same morphology was previously observed in situ in low oxygen waters by Hull et al. (2011), and more recently by Gaskell et al. (2019), referred to as "Hastigerina spp." by the former and "Hastigerina pelagica" by the latter. Given 430 the depth distribution and morphological variation observed here for H. parapelagica, we suspect that it is synonymous with the globally distributed "Hastigerina pelagica" genotype IIa, described by Weiner et al. (2012) and use the name Hastigerina parapelagica (Saito et al., 1976) as the senior synonym of Hastigerina pelagica genotype IIa (Weiner et al. 2012). 435

Morphological Variation in G. hexagonus Reflects Water Column Oxygenation
Globorotaloides hexagonus shares several morphological traits with low-oxygen associated benthic foraminifera including a flattened whorl maximizing its surface area/volume ratio at a given size and large pores (e.g., Bernhard, 1986). Both characters 440 could serve to increase gas exchange and fulfill metabolic requirements in an oxygenlimited environment (Leutenegger & Hansen, 1979;Corliss, 1985). Unlike some digitate planktic foraminifera previously associated with deep and oxygen depleted environments (Hull et al., 2011;Coxall et al., 2007;Gaskell et al., 2019), G. hexagonus is non-spinose, which may suggest that it is herbivorous or bacterivorous as described for other non-445 spinose foraminifera (Schiebel & Hemleben 2017;Bird et al., 2018), rather than dependent on live zooplankton as prey.
The tests of G. hexagonus in deeper, less oxygenated waters appeared more porous, larger, and less compact than those from shallower, more oxygenated environments. These observations, and the presence of G. hexagonus across a wide range 450 of depths and oxygenation levels, led us to quantify the environmental correlates of morphological variation in porosity, size-normalized weight, size, chamber number and shape as potential proxies in paleo-environmental reconstructions. A high test porosity and high pore density have been widely associated with low oxygen environments in benthic foraminifera (Bernhard, 1986;Perez-Cruz & Machain-Castillo, 1990;Glock et 455 al., 2011Glock et 455 al., , 2012Kuhnt et al., 2013Kuhnt et al., , 2014Rathburn et al., 2018) and in cultured planktic foraminifera (Kuroyanagi et al., 2013). These characteristics may play an important role in facilitating gas exchange (Leutenegger & Hansen, 1979;Corliss, 1985), and may represent a balance between the need for gas exchange and structural constraints (Richirt et al., 2019). However, increased porosity has also been associated with other parameters: 460 increasing temperature Burke et al., 2018), decreasing nitrate availability , and increasing test size (Burke et al., 2018). In the OMZ samples where G. hexagonus was found, porosity increased with both decreasing oxygen concentration and increasing test size, with the lowest oxygen conditions hosting the largest and most porous tests (Fig. 9). In contrast to this trend, porosity decreases 465 through ontogeny in G. hexagonus with the most recent chamber being less porous than earlier chambers (Fig. 8). While the presence of a relationship between porosity of G.
hexagonus and oxygenation is clear in our data set, any future efforts to quantify this relationship should target a population of exclusively post-reproductive individuals, using both light microscopy and CT imaging in addition to Scanning Electron Microscopy of 470 the inner test walls. Neither temperature nor nitrate availability (used by some benthic foraminifera as an alternative terminal proton acceptor in very low oxygen environments; Risgaard-Petersen et al., 2006;Hogsland et al., 2008;Pina-Ochoa et al., 2010;Bernhard et al., 2011Bernhard et al., , 2012aBernhard et al., , 2012bWoehle et al., 2018), are likely to drive the observed variation in porosity as temperature was nearly constant (7.7-8.5 °C) across samples and nitrate 475 availability increases with depth in the region (Podlaska et al., 2012;Buchwald et al., 2015;Medina Faull et al., 2020).
Tests collected at lower oxygen levels also had lower size-normalized weights, a property which negatively correlates with porosity. Size-normalized weight in planktic foraminifera has frequently been associated with changes in carbonate chemistry (i.e., 480 Bijma et al., 2002;Russell et al., 2004;Marshall et al., 2013). As oxygen and DIC (Dissolved Inorganic Carbon) depth profiles in the ocean are inversely related, the OMZ is also a region of exceptionally high DIC (Paulmier et al., 2008(Paulmier et al., , 2011. While no carbonate chemistry measurements are available in conjunction with our tows, calcite saturation state at equivalent latitudes in the Eastern Tropical South Pacific OMZ 485 Deleted: 2105 approaches 1, below which calcite dissolution is favored (Bates, 2018). Both an increase in porosity, as well as a decrease in size-normalized weight (whether due to porosity, a decrease in test thickness, or a combination of factors), is consistent with a reduction of overall calcification in low calcite saturation states associated with the OMZ, where 490 precipitation and maintenance of a test may be more metabolically expensive.
Tests collected from the lowest oxygen conditions were less compact with more chambers visible in the final whorl ( Figure 10). The addition of more lobes via increased chamber number have the effect of increasing the surface area/volume ratio for a given size, which could facilitate increased gas exchange via diffusion. However, the increase 495 in size with decreased oxygen availability is such that larger G. hexagonus in low oxygen environments would still have lower surface/volume ratios than smaller individuals from more oxygenated environments (Supplemental Fig. 6). It may be that increased porosity in larger individuals is able to partially compensate for this decrease in surface area/volume ratios. 500 Although the increase in size at low oxygen levels appears enigmatic, there are several potential reasons for this pattern. First, surface area increases with size, which could be beneficial for increasing encounters with food. Larger sizes could also result from delayed reproduction at lower oxygen levels. Alternatively, increased size (cell volume) has been associated with greater capacity for denitrification in some benthic 505 foraminifera (Glock et al., 2019). An inconsistent relationship between surface area/volume ratios and oxygenation has also been observed in several facultative anaerobic species of benthic foraminifera, with only two of the four species studied showing the expected decrease in size with decreasing oxygen levels (Keating-Bitonti and Payne, 2017). Whether G. hexagonus possesses physiological strategies that allow it to function as a facultative anaerobe cannot be determined at this point. However, the combination of increased size (potentially indicative of anaerobic strategies) and 520 increased porosity and morphologies apparently optimized for increasing aerobic capacity in low oxygen environments, suggest a complex physiology. A decrease in porosity with ontogeny could even hint at a shift in physiology over the lifespan of an individual (Fig. 8). Further unraveling the environmental pressures driving test morphology in G. hexagonus will require a greater understanding of the species' ecology. 525

Conclusions
Vertically-stratified plankton tows taken through the Eastern Tropical North Pacific show that distinct assemblages of planktic foraminifera live above and within the OMZ, and that a depauperate fauna occupies the upper oxycline. Two species, G. 530 hexagonus and H. parapelagica, were found living preferentially or exclusively within the OMZ. Several aspects of test morphology in G. hexagonus varied in response to ambient oxygen levels. Some morphological features may be associated with facilitating gas exchange (i.e., porosity, chamber arrangement) or decreasing expenditure on calcification (size-normalized weight, porosity) under the low oxygen and/or carbonate 535 saturation state conditions of the OMZ. The function of other morphological trends, like size, remain enigmatic. Abundance patterns and the co-variation of specific morphological features with oxygenation levels in G. hexagonus tests could be used to reconstruct changes in OMZ environments, providing an additional proxy record of the mid-water OMZ in which these foraminifera lived. As the species appears to be living primarily in the OMZ, recovery of G. hexagonus tests from sediments would be a strong indication of low-oxygen mid-waters. Moreover, large tests with high porosity, low sizenormalized weight and more chambers in the final whorl could be interpreted as having calcified closer to the core of the OMZ than their smaller, less porous conspecifics.

Data Availability
All data associated with this article is available in the supplement or has been previously published and archived on the BCO-DMO database found at http://lod.bcodmo.org/id/dataset/755088. Tables   Table 1. The relative abundance of planktic foraminifera within oxygen defined assemblages: the oxic assemblage (minimum O2 within a net O2 > 2.45 ml L -1 ), 570 transitional assemblage, and OMZ assemblage (maximum O2 within a net < 1.4 ml L -1 ).