Coccolith mass and morphology of different Emiliania huxleyi morphotypes: A critical examination using Canary Islands material.

Different morphotypes of the abundant marine calcifying algal species Emiliania huxleyi are commonly linked to various degrees of E. huxleyi calcification, but few studies have been done to validate this assumption. This study investigated therefore whether E. huxleyi morphotypes can be related to coccolithophore calcification and coccolith mass. Samples from January (high productivity) and September (low productivity) 1997 at an open ocean and a coastal site near the Canary Islands were analysed using a combination of thickness measurements (Circular Polarizer Retardation estimates (CPR) method), Scanning Electron Microscope imaging, and Markov Chain Monte Carlo (MCMC) models. Mean E. huxleyi coccolith mass varied from a maximum of 2.9pg at the open ocean station in January to a minimum of 1.7pg in September at both stations. In contrast, overall calcite produced by E. huxleyi (assuming 23 coccoliths/cell) varied from a maximum of 2.6 μgL-1 at the coastal station in January to a minimum of 0.5 μgL-1 in September at the open ocean site. The relative abundance of "Overcalcified" Type A, Type A, Group B and malformed coccoliths was determined from SEM images. The mean coccolith mass of "Overcalcified" Type A was 2.0pg using the CPR-method, while mean mass of Type A and Group B coccoliths was determined using coccolith length measurements from SEM images and MCMC models relating thickness measurements to morphotype relative abundance. Type A cocccolith mass varied from a 1.6pg to 2.6pg and Group B coccolith mass varied from 1.5pg to 2.0pg. These results demonstrate that the coccolith mass of Type A, "Overcalcified" Type A, and Group B do not differ systematically and there is no systematic relationship between relative abundance of a morphotype and the overall calcite production of E. huxleyi. Therefore, morphotype appearance and relative abundance can not be uniformly used as reliable indicators of E. huxleyi calcification or calcite production.

Introduction

Since the industrial revolution, anthropogenic CO2 release into the atmosphere has led to a ~0.1 decrease in surface ocean pH in a process called ocean acidification [1]. While CO2 continues to increase in the atmosphere and surface ocean, pH may drop by another 0.4 units by the end of the century [2] and the CaCO3 saturation state of the ocean may drop by 2.0 units [3]. Because of the drop in pH and CaCO3 saturation state, calcifying organisms are thought to be particularly affected by ocean acidification [2-4], including coccolithophores [5-8].

Coccolithophores are an abundant group of marine algae that play an important part in the marine carbon cycle as both primary and calcite producers [9]. Much research has therefore focused on the effects of ocean acidification on calcification in coccolithophores, particularly the species Emiliania huxleyi (e.g. [5–7, 10–14]). The research has focused on E. huxleyi because it is the most abundant coccolithophore species in the modern ocean [9, 15]. In addition, E. huxleyi is relatively easy to culture in a laboratory [9]. However, studies on calcification in E. huxleyi are non-conclusive, with different studies reporting decreased (e.g. [5, 6]), increased (e.g. [10, 12]), or unchanged (e.g. [13, 14]) calcification with decreasing pH. The non-conclusive results may be due to methodological issues [16, 17], or strain-specific responses (e.g. [11]), compounded by the non-standardised use of the term calcification. Calcification is a rather loosely used term which encompasses the entire calcite production process from the transport of ions to precipitation of calcite [18]. The amount of calcite produced per cell (often in relation to organic carbon content, the PIC/POC ratio) or the calcite production rate are usually the specific parameters of interest in laboratory studies (e.g. [5, 10]). These parameters can readily be measured in cultured samples using various established methodologies [19-23], but these methodologies are not appropriate for species-specific data from field studies where numerous different calcitic organisms are present. Several studies have therefore relied on the weight/mass estimation of single coccoliths as a proxy for coccolithophore calcification (e.g. [8, 24, 25]). Different methods have been used for estimating coccolith mass, including the volumetric model of [26] based on coccolith length measurements (e.g. [27, 28]), mass measurements based on interference colour (e.g. [8, 29]), measurements of central tube thickness (e.g. [30, 31]), and the qualitative analysis of coccolith morphology from SEM images (e.g. [32, 33]).

The latter approach typically relates different morphotypes of E. huxleyi to different degrees of calcification based on visual impression. E. huxleyi is a morphologically diverse species with several recognized morphotypes [15, 34, 35], potentially representing distinct genetic varieties [36]. Different E. huxleyi morphotypes display environmental preferences over both seasonal (e.g. [33, 37]) and regional (e.g. [8, 15, 32, 38–40]) gradients, and might furthermore differ in their sensitivity to ocean acidification [41]. E. huxleyi morphotypes differ in robustness of distal shield elements and degree of central area closure, which is often interpreted as differences in degrees of calcite production or differences in coccolith mass between the morphotypes. For example, [32] related the relative abundance of E. huxleyi Type B/C morphotypes to the degree of calcite production in the Southern Ocean, and reported based on the abundance of Type B/C that E. huxleyi calcification had not changed over 12 years in this region. Meanwhile, [33] reported an increase in E. huxleyi calcite production with decreasing pH in the Bay of Biscay. This was based on the increased relative abundance of “Overcalcified” Type A with decreasing pH, assuming that the “Overcalcified” morphotype is systematically heavier than the “normally calcified” Type A. Moreover, the dominance of Type A in North Atlantic E. huxleyi blooms and Type B/C in E. huxleyi blooms at the Patagonian Shelf have been suggested to explain differences in calcite production between these blooms [42], while [8] linked increasing E. huxleyi coccolith mass in the Chilean upwelling zone with an increasing relative abundance of “Overcalcified” E. huxleyi morphotypes.

In contrast to these studies, [26] found similar coccolith mass (relative to size) for Type A and Type B coccoliths, despite the more delicate appearance of Type B coccoliths. Morever, a recent laboratory study reported that only one out of two strains of “Overcalcified” E. huxleyi morphotypes had a higher calcite content per cell than other E. huxleyi strains [39], and [43, 44] found that high degrees of malformation did not affect cellular calcite content in E. huxleyi.

The usefulness of E. huxleyi morphotypes as proxies for coccolith mass or degree of calcite production in E. huxleyi is unclear and needs to be validated. The goal of this study is thus to investigate.

whether coccoliths of different E. huxleyi morphotypes differ systematically in thickness and mass,

whether changes in relative abundance of E. huxleyi morphotypes between samples can be used as a proxy for changes in mean E. huxleyi coccolith mass between samples,

whether relative abundance of E. huxleyi morphotypes in a sample can be used as a proxy for calcite production at the sampled site.

Oceanographic setting

This study was conducted using samples collected near the Canary Islands, off the northwestern African coast during the two cruises F.S. METEOR 37/2b [45] and F.S. POSEIDON 233a [46] as part of the CANIGO project [47]. The Canary Islands are situated around 28-29°N in the North Atlantic in the southwest-moving Canary Current in a transitional zone between the northwest African coastal upwelling region and the oligotrophic region of the North Atlantic subtropical gyre [48]. Samples were collected at two stations during both cruises (Figs 1 and 2): EBC, situated near the coast at 28°42.5’N and 13°09.3’W, and LP, situated further from the coast at 29°45.7’N and 17°57.3’W [49, 50]. The EBC station is thus located within an upwelling area, while LP is located in an oligotrophic area [51, 52]. [53] identified two distinct seasons with regards to the upwelling in this region, with April-September giving the most favourable upwelling conditions leading to maximal upwelling intensity in July. The other season from October to March gives less favourable upwelling conditions and a minimum in upwelling intensity in December and January. Sea surface temperature varies more in the upwelling region, ranging from as low as 17°C in winter and early spring to as high as 24°C in summer and fall, while temperatures generally range between 20 and 24°C offshore near LP [51, 53]. Generally temperature differs between the coastal upwelling region and offshore by around 3-4°C [51, 53], though the difference can decrease to less than 1°C during winter [51]. Salinity has been reported to vary by up to ~0.5 units between coast and offshore [54]. During upwelling, filaments of upwelling waters can bring cold upwelling water and organic matter several hundred kilometers offshore [48, 51]. However, [51] found that while these filaments did at times reach LP as evidenced by reduced SST, primary productivity did not change at these times, indicating that the filament water may be nutrient depleted by the time it reaches LP.

Fig 1

Location of sample sites LP1 and EBC2 (red circles) in the Canary Islands region off the coast of Northwest Africa.

Map was made with Natural Earth.

Fig 2

Temperature profile in January and September 1997 (in °C) along the 29°latitude transect.

Black squares: sampled depths during cruises M37/2b and P233a; Black squares with white inlet: sampled depths investigated in this study. Figure modified from [55].

Biological activity is very high in the coastal area near EBC, and chlorophyll α and primary productivity values are significantly higher at EBC compared to LP [50, 51]. [51] reported for example chlorophyll α values ranging from 0.03-0.25mgm-3 at LP between September 1997 and March 1999, while chlorophyll α values at EBC over the same time period ranged from ~0.07-0.89mgm-3. Meanwhile, primary productivity ranged from 0.2-0.7gCm-2d-1 at LP over the September 1997 to March 1999 time period, while it ranged from 0.4-1.2gCm-2d-1 at EBC [51]. Primary production is typically high during late winter and early spring both at EBC and LP, due to a yearly phytoplankton bloom during this time period [51]. At EBC upwelling also leads to additional primary productivity peaks throughout the year [51].

Materials and methods

Sea water was collected in ten litre Niskin bottles and filtered onto 47mm diameter Nucleopore filters with 0.8 μm diameter pores. The filters were rinsed after filtration with NH4OH-buffered distilled water with a pH of 8.5 (see [56] for details). Sample depths for this study (15m, 50m, and 100m at the open ocean site in January, 10m at the coastal site in January, 10m at the open ocean site in September, and 75m at the coastal site in September) were chosen based on E. huxleyi cell densities from [55] (Fig 3). Specifically, the samples with the highest cell density at each site was chosen for analysis, plus samples at 15m and 50m at the open ocean site in January to evaluate potential changes with depth. The top most sample from each location was chosen for further MCMC analysis (see below for details).

Fig 3

E. huxleyi cell density at each station measured during cruises M37/2b (January 1997) and P233a (September 1997).

Data from [55].

SEM analysis

A triangular piece was cut from each filter membrane, mounted on a stub and coated with platinum using a Leica SCD500 Metal Coater (Leica Microsystems, Wetzlar, Germany). Up to 1500 images per sample were then automatically captured at 3000x magnification with 1024 x 768 pixel resolution using a Zeiss Supra VP55 SEM (Carl Zeiss, Oberkochen, Germany). E. huxleyi coccoliths were counted and classified into various morphotypes and/or morphological groups. Type A and “Overclassified” Type A were classified according to [57] and [58]. Coccoliths belonging to Type C or Type B/C were grouped together and counted as Group B according to [58]. Malformed coccoliths, where distal shields were irregularly formed, often with disconnected distal shield elements and/or a missing/minimal central tube, were counted as malformed (Fig 4). The lengthand width of all counted coccoliths were measured using ImageJ 1.52a after calibration of the SEM using spherical beads with a 2 μm ±0.1 μm diameter (DYNO Particles, Lillestrøm, Norway). Broken or partially covered coccoliths were counted and measured if their length could be accurately determined.

Fig 4

E. huxleyi morphotypes.

A: Type A. B: “Overcalcified” Type A. C: Group B. D: malformed coccoliths. Horizontal bar: scale = 1 μm.

The length and width were measured for each counted coccolith in the shallowest samples at each site (Samples: M37/2b-31-10m—Coastal January, M37/2b-50-15m—Open ocean January, P233a-600-75m—Coastal September, and P233a-582-10m—Open ocean September). The central tube width along the length axis for up to 30 Type A coccoliths in each sample was measured as well (Fig 5). The central tube width was measured along the length axis (similar to [30]). To account for variation in central tube width, central tube width was measured in the present study on both sides of the central area, and then averaged to give a mean central tube width. The central tube width was then divided by coccolith length to obtain a size-independent central tube width to length ratio (CT:L).

Fig 5

SEM image of E. huxleyi Type A coccolith.

Red line shows the axis of distal shield (DL), the yellow line shows central tube width (CT). CT was measured at both sides of the central area along the DL axis and averaged.

Coccolith mass and thickness analysis

Coccoliths were transferred from filters onto glass slides and mounted using NOA 61 adhesive. Coccolith images were then captured using a Zeiss Axio Imager Z1 light microscope (Zeiss, Oberkochen, Germany) equipped with a Benford plate for circular polarization [59], a 1.6x optovar, neutral density filters, a Plan-Apo 100x, 1.4 NA oil objective, a 0.9 NA universal condenser, and a Canon 60D DSLR camera (Canon Inc., Tokyo, Japan) with a 5194 x 3457 resolution (a pixel resolution of 0.0003 μm-2) for light microscope (LM) analysis. Images were captured in RAW and converted to TIFF in sRGB colour space with gamma 2.2 according to [60]. Size calibration was done using a S8 Stage micrometer (02A00404 from PYSER-SGI Ltd., Edenbridge, UK) with steps 10 μm apart along a line with an overall length of 1000 μm ±1 μm. Microscope illumination and camera sensitivity were calibrated for accurate retardation / thickness estimation using polymer films with known retardations of 31nm and 129nm according to the Circular Polarizer Retardation estimates (CPR) method [61]. The condenser was partly closed to avoid polarization aberrations [62, 63]. Grey values were related to an sRGB Michel-Lévy chart from [60] using the ImageJ function [Calibrate…] in ImageJ 1.52. Subsequently, coccolith length, mass, and thickness were measured in ImageJ according to the CPR-method [61]. Coccoliths were separated from the background using a Canny-Deriche edge detection algorithm [64, 65] with an α of 0.5, before the ImageJ function [Analyze particles…] was used to obtain coccolith length, mass, and thickness. The standard uncertainty at a 95% confidence level of the LM measurements is ±0.2 μm for length, ±0.007 μm for thickness, and ±~14-15%/~0.3-0.4pg (depending on size) for mass.

“Overcalcified” Type A coccoliths could be recognized in the light microscope due to its closed central area (Fig 6, see also [66]). “Overcalcified” Type A coccolith thickness and mass could therefore be measured directly. However, Type A, Group B, and malformed coccoliths could not be distinginguished in the light microscope, and the mean thickness of these morphotypes had to be estimated from a Markov Chain Monte Carlo (MCMC) analysis (see below for details). Sample sizes were adjusted to obtain approximately 30 coccoliths of the rarest morphotype (see Table 1). Please note that only 75 coccoliths were measured from sample P233a-582-10m (Open ocean September) due to its coccolith scarcity.

Fig 6

E. huxleyi coccoliths captured under circular polarized light.

Note the different appearance of their central areas. A: E. huxleyi coccolith with a normal central area. B: E. huxleyi coccolith with an overgrown central area.

Table 1

Coccolith morphotype counts, relative abundance, length measurements, and central tube: Length ratio (CT:L) for the most shallow sample at each site measured in this study.

All measurements and counts were obtained from SEM images. Mean length and mean CT:L are shown including the 95% confidence interval (CI 95%). N: Number of counted coccoliths. Rel. ab. (%): Relative abundance of morphotypes. Morphotype abbreviations: A: Type A, OA: Overcalcified Type A, B: Group B, M: Malformed coccoliths.

Sample	Morphotype	N	Rel. ab.	Length (μm)		Width (μm)		Aspect ratio		CT:L
			(%)	Mean	CI 95%	Mean	CI 95%	Mean	CI 95%	Mean	CI 95%
M37/2b-31-10m	A	231	77	3.36	0.04	2.79	0.03	1.21	0.01	0.07	0.01
	B	67	22	3.18	0.09	2.66	0.09	1.20	0.02	-	-
	M	2	1	2.77	-	2.23	-	1.24	-	-	-
	Total	300	100	3.32	2.75	0.03	0.04	1.21	-	-	-
M37/2b-50-15m	A	264	88	3.41	0.04	2.84	0.04	1.20	0.01	0.07	0.01
	B	36	12	3.16	0.15	2.63	0.12	1.20	0.01	-	-
	Total	300	100	3.38	0.04	2.81	0.04	1.20	0.00	-	-
P233a-600-75m	A	108	36	3.14	0.06	2.61	0.06	1.21	0.01	0.06	0.01
	OA	56	19	3.00	0.08	2.51	0.06	1.21	0.01	-	-
	B	114	38	2.95	0.07	2.44	0.06	1.20	0.01	-	-
	M	22	7	2.98	0.14	2.39	0.13	1.25	0.04	-	-
	Total	300	100	3.03	0.04	2.51	0.04	1.21	0.01	-	-
P233a-582-10m	A	24	8	3.23	0.12	2.62	0.11	1.25	002	0.04	0.00
	B	2	1	2.94	-	2.50	-	1.17	-	-	-
	M	262	91	3.20	0.04	2.59	0.03	1.24	0.01	-	-
	Total	288	100	3.21	2.59	0.03	0.04	1.24	0.01	-	-

Statistical analysis

All statistical analyses in this study were done using R version 3.4.3 [67] in RStudio version 1.1.383. Mann-Whitney U-tests were performed to evaulate differences in coccolith length, mass, and thickness between months and stations while a Kruskal-Wallis test was performed to evaluate differences in coccolith length, mass, and thickness with depth. t-tests were performed to evaluate differences in length and central tube width of morphotypes between stations and seasons.

Markov chain monte carlo analysis

A Markov Chain Monte Carlo (MCMC) model was used to estimate mean thickness values for different E. huxleyi morphotypes at each site. Assuming that the thickness of each individual E. huxleyi morphotype population is normally distributed in each sample, the measured E. huxleyi coccolith thickness represents a mixed distribution of a number of normal distributions equal to the number of distinct morphotypes. The contribution (mixture weight) of each individual normal distribution in the mixed distribution would then correspond to the relative abundance of the individual morphotypes. The task of the MCMC analysis was therefore to estimate the mean and standard deviation values for the individual normal distributions using the relative abundance (mixture weight) and the total (mixed) distribution as input.

In other words, mean thickness for each morphotype was estimated by fitting the frequency distribution of thickness measurements to the relative abundance of E. huxleyi morphotypes. This was done using a Bayesian mixed normal distributions model built with a Gibbs sampler MCMC algorithm [68] using JAGS version 4.3.0 in R with the package R2Jags [69] and a template written by [70]. The template represent a model with K number of components (i.e. morphotypes), where each component is a normal distribution with a sampled mean and standard deviation. The model is initiated with prior distributions specified for each of the parameters mean, precision (defined as [71]), and relative abundance. Standard deviation is then calculated from the estimated precision of the model. The template of [70] was modified as follows:

The model samples for precision rather than standard deviation to keep with normal practice for the model software [71]. Standard deviation is then calculated from the precision as

The precision of the prior distribution of the mean parameter was altered to sample the mean parameter more efficiently.

Standard deviation/precision is sampled for each individual component, rather than sampling one constant standard deviation value for all components.

A gamma distribution is used instead of a t-distribution for the precision prior distribution.

Probability of component assignment for each data item is determined from a gamma distribution with the shape parameter α determined according to a dirichlet prior distribution and scale parameter θ set to 1 (see [71] for details). To avoid infinity issues during sampling of the four components model a minimum limit of 0.01 was set for the gamma values. Alpha hyperpriors for the dirichlet prior distribution are the prior relative abundance parameters, and were set as 20 and 80 for the two components models and 10, 35, 35, and 20 for the four components model.

No sorting of the mean parameter (two-component models only).

Outliers, defined as coccoliths with thickness >1.5 times the interquartile range, were removed from each sample before running the MCMC, and morphotypes with relative abundances <1% in a given sample were ignored for the model. The model for each sample was run for 500,000 iterations with a 50,000 burn-in period and a thinning interval of 100. Convergence was confirmed visually using a trace plot (S1, S2, S3 and S4 Figs) and formally using the Gelman-Rubin diagnostic. Estimated mean and standard deviation of each morphotype was determined from the median of each sampled parameter with a credible interval showing the region around the median which 68% of the parameter samples fell within (a 68% credible interval is roughly analogous to a 1 standard deviation margin of error/confidence interval [72]).

The accuracy and efficiency of the models were evaluated on eight groups of simulated samples (Figs 7 and 8). The samples were created according to different scenarios (Table 2) using a random number generator. Three simulated samples were generated for each two-component scenario, while one simulated sample was generated for each four-component scenario. This process allowed for the model to be calibrated and evaluated under various conditions where the actual mean and standard deviation values for each component are known.

Fig 7

Density bar plots showing the distribution of simulated values for the two-component samples.

Black curve: density curve for each sample; vertical red stapled lines: the position of the mean of each component (simulated morphotype) Kn. K1: component/morphotype 1. K2: component/morphotype 2. μ: the component mean. σ: the component standard deviation. p: the relative abundance.

Fig 8

Density bar plots showing the distribution of simulated values for the four-component samples.

Black curve: the density curve for each sample, vertical red stapled lines: the position of the mean of each component (simulated morphotype) Kn. K1: component/morphotype 1. K2: component/morphotype 2. K3: component/morphotype 3. K4: component/morphotype 4. μ: the component mean. σ: the component standard deviation. p: the relative abundance.

Table 2

Conditions for each simulated sample as assigned to a random number generator.

1 to 3 samples were generated according to conditions in each row. Sim.: Number of simulated samples generated in each group; N: sample size; k: number of components (morphotypes); RAn: Relative abundance of each component n; Tn: Mean thickness of each simulated component n; SD: standard deviation of the sample distribution.

Group	Sim.	N	k	RA1 (%)	RA2 (%)	RA3 (%)	RA4 (%)	T1	T2	T3	T4	SD
1	3	200	2	25	75	-	-	0.100	0.120	-	-	0.020
2	3	200	2	10	90	-	-	0.100	0.120	-	-	0.020
3	3	200	2	25	75	-	-	0.110	0.110	-	-	0.020
4	3	200	2	10	90	-	-	0.110	0.110	-	-	0.020
5	1	500	4	10	35	35	20	0.085	0.100	0.110	0.125	0.02
6	1	500	4	35	10	35	20	0.085	0.100	0.110	0.125	0.02
7	1	500	4	35	35	10	20	0.085	0.100	0.110	0.125	0.02
8	1	500	4	10	35	35	20	0.080	0.100	0.120	0.140	0.02

Morphotype mass estimation

Assuming an elliptical coccolith shape, the mean coccolith mass of different coccolith morphotypes was approximated according to this formula: where m is coccolith mass, l is coccolith length, w is coccolith width, t is coccolith thickness, and d is calcite density (= 2.71g cm-3).

Calcite concentration estimation

Total E. huxleyi calcite concentration at each site was estimated from E. huxleyi cell density and coccolith mass according to the following equation: where Ca is E. huxleyi calcite concentration in μgL-1, CD is cell density, m is mean coccolith mass, and N is the number of coccoliths per cell. Cell density data was obtained from [55], while N was taken from [73] and [74]. Three different coccoliths per coccosphere numbers were used to give a probable range: 23 (mean number in both [73] and [74]), 10 (minimum in [74]), and 48 (maximum in [74]). The number of coccoliths per coccosphere was for simplicity assumed to be the same for all morphotypes.

Results

Overall E. huxleyi coccolith length, thickness, and mass was determined using light microscope (LM) images of a total of 1091 E. huxleyi coccoliths (Fig 9, Table 3). Mean coccolith length from LM images ranged from a maximum of 3.4 μm at the open ocean site in January to a minimum of 3.1 μm at the coastal site in September (Fig 9, Table 3). These length values compare well with the corresponding sample coccolith lengths measured from SEM images (Table 1). Coccolith thickness differed significantly from a maximum of 0.150 μm at the open ocean site in January to a minimum of 0.093 μm at the open ocean site in September (Fig 9, Tables 3 and 4). Mean E. huxleyi coccolith mass varied from a maximum of 2.6pg at the open ocean station in January to a minimum of 1.7pg at both stations in September.

Fig 9

Boxplots showing length, mass, and thickness of E. huxleyi coccoliths measured on the light microscope.

January represents samples from 10m at EBC (coastal) and 15m at LP (offshore) during METEOR cruise 37/2b, while September measurements represent samples from 75m at EBC (coastal) and 10m at LP (offshore). A: length. B: mass. C: mean thickness per coccolith. Horizontal white lines indicate the median, while yellow diamonds are the average values for each sample. Black dots represent outliers (values greater than 1.5 times the interquartile range).

Table 3

Mean values for length, mass, and mean thickness obtained on a light microscope.

N: Sample size; CI 95%: 95% confidence interval for population mean. Numbers in italics represent subsets of either “open” central area E. huxleyi coccoliths or “closed” central area E. huxleyi in the sample were both were present.

Sample	N	Length (μm)		Mass (pg)		Mean thickness (μm)
		Mean	CI 95%	Mean	CI 95%	Mean	CI 95%
M37/2b-31-10m	143	3.2	0.1	2.4	0.2	0.126	0.005
M37/2b-50-15m	250	3.4	0.1	2.7	0.1	0.133	0.003
M37/2b-50-50m	61	3.3	0.1	2.9	0.3	0.150	0.007
M37/2b-50-100m	61	3.3	0.0	2.5	0.3	0.126	0.008
P233a-600-75m	424	3.1	0.0	1.7	0.1	0.108	0.002
P233a-600-75m “open”	359	3.1	0.0	1.7	0.1	0.104	0.002
P233a-600-75m “Overcalcified”	70	3.1	0.1	2.0	0.2	0.124	0.006
P233a-582-10m	75	3.3	0.1	1.7	0.1	0.093	0.003

Table 4

Mann-Whitney U-test comparing length, central tube width to Coccolith Length ratio (CT:L—Type A only), mass and thickness at the shallowest samples at each site.

Italicized font indicates p-values that could not be computed exactly due to ties and were instead computed according to a normal approximation.

Length
Samples compared	N1	N2	U	p
M37/2b-31-10m (Coastal January) and P233a-600-75m (Coastal September)	300	300	65256	<0.01
M37/2b-50-15m (Open ocean January) and P233a-582-10m (Open ocean September)	300	288	55946	<0.01
M37/2b-31-10m (Coastal January) and M37/2b-50-15m (Open ocean January)	300	300	39996	0.02
P233a-600-75m (Coastal September) and P233a-582-10m (Open ocean September)	300	288	30265	<0.01
CT:L
Samples compared	N1	N2	U	p
M37/2b-31-10m (Coastal January) and P233a-600-75m (Coastal September)	30	30	600	0.03
M37/2b-50-15m (Open ocean January) and P233a-582-10m (Open ocean September)	30	24	27	<0.01
M37/2b-31-10m (Coastal January) and M37/2b-50-15m (Open ocean January)	30	30	419	0.65
P233a-600-75m (Coastal September) and P233a-582-10m (Open ocean September)	30	24	9	<0.01
Mass
Samples compared	N1	N2	U	p
M37/2b-31-10m (Coastal January) and P233a-600-75m (Coastal September)	143	424	43242	<0.01
M37/2b-50-15m (Open ocean January) and P233a-582-10m (Open ocean September)	250	75	15661	<0.01
M37/2b-31-10m (Coastal January) and M37/2b-50-15m (Open ocean January)	143	250	14898	0.01
P233a-600-75m (Coastal September) and P233a-582-10m (Open ocean September)	424	75	15537	0.75
Mean thickness
Samples compared	N1	N2	U	p
M37/2b-31-10m (Coastal January) and P233a-600-75m (Coastal September)	143	424	41909	<0.01
M37/2b-50-15m (Open ocean January) and P233a-582-10m (Open ocean September)	250	75	17426	<0.01
M37/2b-31-10m (Coastal January) and M37/2b-50-15m (Open ocean January)	143	250	15452	0.03
P233a-600-75m (Coastal September) and P233a-582-10m (Open ocean September)	424	75	23192	<0.01

Total calcite concentration of the E. huxleyi population was estimated assuming 10, 23, or 48 coccoliths per cell (Fig 10). At 23 coccoliths per cell, total E. huxleyi calcite ranged from 0.05 to 2.26μgL-1, while with 10 coccoliths per cell it ranged from 0.02 to 0.98μgL-1 and with 48 coccoliths per cell the range was 0.10 to 4.72μgL-1 (Table 5). E. huxleyi calcite concentration was higher in January than September at both sites. At the coastal site total E. huxleyi calcite concentration decreased from 2.26μgL-1 to 0.65μgL-1, while at the open ocean site it decreased from 0.74μgL-1 to 0.05μgL-1.

Fig 10

Estimated E. huxleyi calcite concentration.

A: January. B: September. Blue: Open ocean site at 10 or 15m depth. Orange: Coastal site at 10 or 75m depth. Numbers of coccoliths per cell were taken from [73] and [74].

Table 5

Estimated E. huxleyi calcite concentration at each site.

CD: Cell density; mass: Mean coccolith mass; TC23: Total E. huxleyi calcite concentration assuming 23 coccoliths per cell [73, 74]; TC10: Total E. huxleyi calcite concentration assuming 10 coccoliths per cell [74]; TC48: Total E. huxleyi calcite concentration assuming 48 coccoliths per cell [74].

Station	Month	Depth (m)	CD (cell/L)	mass (pg)	TC23 (μgL-1)	TC10 (μgL-1)	TC48 (μgL-1)
Coastal	January	10	41003	2.4	2.26	0.98	4.72
Open ocean	January	15	11983	2.7	0.74	0.32	1.55
Coastal	September	75	16632	1.7	0.65	0.28	1.36
Open ocean	September	10	1228	1.7	0.05	0.02	0.10

Coccolith length, thickness, and mass obtained from LM were also evaluated along a depth transect from 10 to 100 meters depth at the open ocean station (LP) in January. Thickness and mass varied statistically significantly with depth (Kruskal-Wallis p <0.05, Tables 3 and 6), though only thickness varied by a degree larger than the standard uncertainty of measurements (see Methods section). Thickness was greatest at 50m depth (0.149 μm), while thickness was similar at 15m and 100m depth (0.133 μm and 0.126 μm, respectively).

Table 6

Kruskal-Wallis test results comparing length, mass and thickness at the depths 15m, 50m, and 100m at the open ocean station LP in January.

Measurements for all parameters were obtained from the light microscope images. df = degrees of freedom.

Parameter	N15m	N50m	N100m	Chi-Square	df	p
Length	250	61	61	4.213	2	0.12
Mass	250	61	61	8.645	2	0.01
Mean thickness	250	61	61	27.120	2	<0.01

Individual E. huxleyi morphotypes measurements

In order to analyze the relationship between E. huxleyi morphotypes and coccolith mass, the relative abundance of E. huxleyi morphotypes at each site was determined from SEM images (Fig 11). At least four different morphotypes were identified from a total of 1188 E. huxleyi coccoliths: Type A, “Overcalcified” Type A, Group B, and malformed coccoliths (Fig 4, Table 1). January samples consisted of Type A and Group B coccoliths, while September samples also contained significant numbers of malformed coccoliths at both sites, and “Overcalcified” Type A at the coastal site. Type A dominated both sites in January (relative abundance >50%), but not in September. The open ocean site was dominated by malformed coccoliths in September, while the coastal site was not dominated by any single morphotype. The most common morphotype in September at the coastal site was Group B with a relative abundance of 38%, followed by Type A (36%), “Overcalcified” Type A (19%), and malformed coccoliths (7%) (Table 1).

Fig 11

Relative abundance of E. huxleyi morphotypes at each station.

Error bars show margin of error of counts with a 95% confidence level. Overall: Mean value of all E. huxleyi coccoliths in a sample. L: Coccolith length. T: Coccolith thickness. M: Coccolith mass.

Morphometric analysis of the same 1188 coccoliths on SEM images revealed that overall E. huxleyi coccolith length differed statistically significantly (Mann-Whitney p <0.05) at both sites between January and September (Table 4). E. huxleyi coccoliths were 0.29 μm longer in January at the coastal site and 0.17 μm longer in January at the open ocean site. Length furthermore differed significantly between the two sites both in January and September. Coccoliths at the open ocean site were 0.06 μm longer than coccoliths at the coastal site in January and 0.18 μm longer in September.

Type A coccolith length ranged from 3.14 μm to 3.41 μm (Table 1). Type A coccoliths were significantly longer in January than September at the coastal site (difference of 0.22 μm, t-test p <0.05). However, there were no statistically significant differences between the two sites in either January or September. “Overcalcified” Type A coccoliths were only found at the coastal site in September where they were on average 3.00 μm long.

Group B coccolith length ranged from 2.95 μm to 3.18 μm (Table 1) and they were significantly longer (0.23 μm) in January than in September at the coastal site. Malformed coccoliths were significantly larger at the open ocean site than at the coastal site, with a coccolith length difference of 0.22 μm.

An MCMC analysis of thickness measurements of E. huxleyi coccoliths was used to estimate the thickness of individual morphotypes, as Type A, Group B, and malformed coccoliths could not be visually identified in the light microscope for direct thickness/mass measurements (Fig 6). Type A coccolith thickness ranged from 0.089 μm to 0.131 μm (Table 7). Type A coccoliths were thicker in January than September at both sites, with a thickness difference of 0.033 μm or 0.020 μm at the coastal site and 0.042 μm at the open ocean site.

Table 7

Markov Chain Monte Carlo (MCMC) analysis results for estimated thickness of E. huxleyi morphotypes in each sample.

μ is the estimated mean thickness, σ is the estimated standard deviation of thickness, and CI lower, median, and CI upper shows the 16th, 50th and 84th quartiles of the sampled parameters, giving the 68% Credible Interval for each parameter. Morphotypes: A: Type A; B: Group B; OA: Overcalcified Type A; M: malformed coccoliths. Note that in sample P233a-600-75m the MCMC model could not confidently assign a thickness to Type A or Group B.

Sample	Morphotype	μ(μm)			σ (μm)
		CI lower	Median	CI upper	CI lower	Median	CI upper
M37/2b-31-10m	A	0.127	0.131	0.134	0.024	0.027	0.029
	B	0.091	0.097	0.107	0.014	0.018	0.024
M37/2b-50-15m	A	0.129	0.131	0.133	0.018	0.019	0.020
	B	0.110	0.120	0.130	0.018	0.019	0.020
P233a-600-75m	A or B	0.094	0.098	0.103	0.008	0.011	0.015
	A or B	0.108	0.111	0.113	0.006	0.008	0.014
	OA	0.123	0.128	0.130	0.010	0.012	0.014
	M	0.083	0.086	0.090	0.009	0.011	0.013
P233a-582-10m	A	0.079	0.089	0.099	0.015	0.019	0.027
	M	0.089	0.091	0.094	0.013	0.014	0.016

Group B coccolith thickness ranged from 0.097 μm to 0.120 μm (Table 7) and coccoliths were 0.023 μm thicker at the open ocean site than at the coastal site in January. Meanwhile, coastal Group B coccolith thickness were similar in January and September (Fig 12). Malformed coccoliths were 0.091 μm thick at the open ocean site and 0.086 μm thick at the coastal site. While malformed coccoliths were thinner than other morphotypes at the coastal site, Type A and malformed coccoliths were similar in thickness at the open ocean site.

Fig 12

Histograms of coccolith mean thickness measured at each site.

The height of the bars represent the relative frequency of the bin (density). The black curve shows the density distribution for coccolith thickness in each sample. Coloured vertical bars represent the 68% credible interval for the MCMC-sampled mean coccolith thickness for each morphotype, while the vertical stapled line represent the median sampled mean value. Red bars/lines: Type A. Blue bars/lines: Group B. Grey bar/line: “Overcalcified” Type A. Brown bars/lines: malformed coccoliths.

Because “Overcalcified” Type A coccolith mass could be determined directly from LM images (Fig 6), direct measurements were done of “Overcalcified” Type A at the coastal site in September. These measurements could be compared to LM measurements of the remaining sample, representing an aggregate of all other E. huxleyi morphotypes present in the sample (Table 3). They could also be compared to coccolith thickness and mass of other morphotypes as estimated from MCMC models and Eq 2 (Figs 11 and 12, Table 7). “Overcalcified” Type A was both heavier and thicker than non-“Overcalcified” Type A morphotypes at the coastal site in September. The same “Overcalcified” Type A coccoliths were, however, on average lighter than the mean measured E. huxleyi coccolith mass at both sites in January, which contained no “Overcalcified” Type A (Fig 11, Table 3). In fact, mass approximations done using volumetric calculations (Eq 2) show that Type A coccoliths at the two sites in January were heavier than “Overcalcified” Type A coccoliths in this study. Type A mean coccolith mass in January was 2.7pg at the open ocean site and 2.6pg at the coastal site (Fig 11). Group B coccoliths at the open ocean site in January had similar coccolith mass (2.2pg) as the coastal “Overcalcified” Type A coccoliths found in September (Fig 11).

As a quality control of the morphotype thickness and mass estimations, “Overcalcified” Type A coccolith thickness and mass was also estimated the same way as other morphotypes and compared with the direct LM measurements. Measured “Overcalcified” Type A coccolith thickness (0.124 μm, Table 3) compared well with estimated coccolith thickness from the MCMC model (0.128 μm, Table 7). Similarly, coccolith mass compared well between direct measurements (2.0pg, Table 3) and volumetric model estimation using Eq 2 (2.1pg).

MCMC accuracy evaluation

Using a MCMC model for E. huxleyi morphotype analysis is a novel approach which allowed for a more complete understanding of intra-morphotypic variation in coccolith morphology and mass in E. huxleyi. The accuracy of the MCMC model was evaluated using simulated sample compositions (Table 2). A random number generator was used to create samples with mixtures of two or four morphotypes (two or four components). The mixed values were generated from normal distributions with mean, standard deviation, and weight (relative abundance) set as shown in Table 2. The total number of 16 simulated samples (twelve with two morphotypes and four with four morphotypes) was chosen as a balance between covering a sufficient range of different scenarios and computing time, as each individual MCMC model analysis is time-consuming. Subsequently, MCMC analyses were done based on these simulated thickness distributions and the relative abundances chosen for each simulated morphotype. This approach allowed for the evaluation of the accuracy of the MCMC models on samples resembling the real coccolith samples but where the (simulated) mean thickness is known independently of the MCMC model (S3 Table).

Using the MCMC model it was possible to estimate the mean thickness for the simulated morphotypes with an accuracy of ±0.01 units. The only exceptions were one morphotype in each of the two samples: Group 3: Simulation 3 and Group 4: Simulation 3 (Fig 7). The simulated mean thickness also fell within the 68% credible interval of the MCMC models (for details see Methods section), except for the same two morphotypes in the simulated samples mentioned above (Group 3: Simulation 3 and Group 4: Simulation 3). In total the simulated morphotype mean thickness fell within the 68% credible interval in the case of 95% of the simulated morphotypes (Fig 13). The 68% credible interval thus appears to give a reliable margin of error for the estimated thickness from the MCMC analysis.

Fig 13

Scatter plots of actual mean thickness versus mean thickness estimated by the MCMC (A) and actual standard deviation versus standard deviation estimated by the MCMC (B) for each simulated component (simulated morphotype) in the various two- and four-component simulated samples.

Each dot represents the median of each MCMC sampling with the error bars representing the 68% Credible Interval. Each color represents a different simulation group (see subsection on Markov Chain Monte Carlo analysis in the Methods section in text for details for each group).

The input order for relative abundance was not important for the two-morphotypes MCMC models, but in the four-morphotypes models the morphotype mean thickness values were sorted during analysis to avoid label switching (a phenomenon resulting in the averaging out of individual component mean values—see [75] for details). Because the morphotype mean thickness values in the four-morphotypes models were sorted, the input order for relative abundance may influence results as the first morphotype, for example, is now both given prior information about its relative abundance (first relative abundance input) and its mean thickness relative to the other morphotypes (least thickness). Tests done on the simulated samples revealed that the MCMC models were able to correct for the influence of the input order.

Type A central tube width

The ratio of the central tube width to coccolith length (CT:L) was measured in the SEM for 30 Type A coccoliths at each site as a potential measure of calcification. CT:L was equal at both sites in January at 0.07 ±0.01, while CT:L was 0.06 ±0.01 in September at the coastal site and 0.04 ±0.00 in September at the open ocean site. The variation between January and September was statistically significant at both sites, though only at the open ocean site was the difference outside the margin of error.

Discussion

Relating coccolith morphology to coccolith mass is a challenging task, as only “Overcalcified” Type A can be distinguished in a light microscope (Fig 6). The CPR-method is able to estimate coccolith thickness with high accuracy [61], but differences between most morphotypes, for example Type A and Group B coccoliths, can not be directly measured in a light microscope. Using morphotype relative abundance obtained from SEM images and sample distributions of measured E. huxleyi coccolith thickness obtained from the CPR-method as input, a Markov Chain Monte Carlo method was therefore used to get accurate thickness estimations for each E. huxleyi morphotype (generally ±0.01 μm or less (Table 7)). Together with these morphotype thickness estimates, morphotype length measurements from SEM images (Table 1) can be used to get reliable estimates of coccolith mass per morphotype (e.g. Eq 2, see also [61]). It was thus possible to obtain insights into differences in coccolith mass between morphotypes, revealing that coccolith morphology and mass of E. huxleyi morphotypes vary unsystematically and inconsistently (Figs 11 and 14). “Overcalcified” Type A were, for example, heavier than Type A coccoliths in some samples, but lighter than Type A coccoliths in other samples. Similarly, “Overcalcified” Type A were either heavier or same mass as Group B coccoliths. In fact, no morphotype was consistently heavier or lighter than another morphotype when compared across different samples (Fig 14A). E. huxleyi cell density also varied between samples, resulting in significantly different calcite production by E. huxleyi even when relative abundance of morphotypes were similar (Fig 15B). In effect, the relative abundance of an individual morphotype may not be used as a proxy for either mean coccolith mass or E. huxleyi calcite production (Fig 15).

Fig 14

A: Mean coccolith mass of individual E. huxleyi morphotypes estimated using a volumetric model (Eq 2). B: Calcite production by individual E. huxleyi morphotypes. Calcite concentration was estimated using measured coccolith mass in A, cell density, and assuming 23 coccoliths per cell. Colour of circle denotes the morphotype, while the size of the circle indicates the relative abundance of the morphotype in each sample. Note that in sample P233a-600-75m Type A and Group B coccoliths had each two possible mean coccolith thickness values obtained from the MCMC model. Therefore, two alternative coccolith mass and calcite concentration estimates are given for each of these morphotypes.

Fig 15

A: E. huxleyi morphotype relative abundance versus mean coccolith mass. Mean coccolith mass was measured directly from LM images using the CPR-method. Error bars represent the 95% confidence interval of the mean. B: E. huxleyi morphotype relative abundance versus estimated calcite concentration of E. huxleyi. Calcite concentration was estimated using measured coccolith mass in A, cell density, and assuming 23 coccoliths per cell. Colour of circle denotes the morphotype.

These results are in contrast with studies that have relied on SEM imaging and an a priori assumption of coccolith mass differences between morphotypes to make inferences on the impacts of ocean acidification on E. huxleyi calcite production (e.g. [32, 33, 37]). These studies solely rely on the distal shield appearance of morphotypes to make inferences about relative coccolith mass differences between different morphotypes, but other aspects of the coccolith also appear to be significant. The greater thickness of Type A coccoliths in January than “Overcalcified” Type A in September may, for example, be due to other factors influencing coccolith thickness and mass. Proximal shield closure or central tube height, which are not obvious from SEM images, might be equally important. This could also explain the similar PIC per cell in the “Overcalcified” Type A E. huxleyi strain C352 in [39] compared to normal Type A E. huxleyi strains in the same study.

In summary, the term “Overcalcified” appears to be a poor term with potentially misleading implications. Referring to Group B coccoliths as “lightly calcified” likewise appears to be misleading. Group B coccoliths at the open ocean site in January appear to be thicker than Type A in September at the coastal site (Table 7), despite similar coccolith size (Table 1). Referring to Group B coccoliths as lightly or less calcified compared to Type A is therefore unjustified because it is based only on the distal shield appearance.

Malformed coccoliths

Several studies have linked ocean acidification to increased malformation in culture experiments (e.g. [5, 76, 77]), though [78] reported that this response is strain-specific in E. huxleyi. Furthermore, in recent culture experiments calcite content per cell did not decrease in E. huxleyi even when more than 95% of coccoliths were malformed [43, 44]. In line with the findings of [43, 44], coccolith mass in the present study was the same at both study sites in September, despite the high (>90%) proportion of malformed coccoliths at the open ocean site (Table 3). Malformed coccoliths appear therefore to be a poor indicator of coccolith mass, in the same way that different morphotypes are.

Central tube width

Type A central tube width have been linked to different degrees of calcification in E. huxleyi [30, 40]. In the present study CT:L ratio followed Type A coccolith mass, as both decreased together from January to September at both sites. Central tube width is a factor likely contributing to coccolith mass, but CT:L alone does not seem to explain the full variation in coccolith mass seen within Type A in the present study. For example, the CT:L ratio decreased by ~15% at the coastal site from January to September, but coccolith mass decreased by 23-35% (Fig 11). Moreover, Type A calcite concentration decreased by ~86-88%, showing that variation in Type A CT:L is not a suitable indicator of E. huxleyi calcite production either.

MCMC assumptions

The MCMC model relies on the assumption that the samples can be described by two or four morphotypes with approximately normal distributions of morphological parameters. The number of components/morphotypes in the model reflects the number of morphotypes counted in the SEM, with the exception of very rare morphotypes (<1% relative abundance). Previous studies have reported several different morphotypes within both Type A [40] and Group B (e.g. [34, 35]), and the presence of different sub-varieties of these morphotypes may invalidate this assumption. However, there is no evidence of bimodality in the Type A or Group B coccoliths, suggesting they are well represented as one morphological unit in the present study. While the main difference between Group B morphotypes is size [57], the length of Group B coccoliths in this study is normally distributed in all samples according to a Shapiro-Wilk’s test. Group B coccoliths in the present study therefore seem to represent a single morphotype, at least within an individual sample. Shapiro-Wilk’s tests also revealed that coccolith length and CT:L is normally distributed in Type A, with the exceptions of Type A coccolith length in the Open ocean January sample (Shapiro Wilk’s p = 0.048) and Type A CT:L in the Coastal January sample (Shapiro Wilk’s p = 0.013). These are due to the influence of two or one outliers, respectively. If the outliers are removed, the Shapiro Wilk’s p >0.05, and the distributions are normal as well. Therefore, Type A coccolith length and CT:L measurements are considered to be normally distributed, and Type A coccoliths seem to represent one normally distributed unit within each individual sample.

A potential exception to the assumptions is seen in the Open ocean sample in September. The three Markov chains used for the parameter sampling by the MCMC model appeared to converge in this sample (S3 Fig), but the mean values sampled by the model were often obviously wrong for the Type A component in the Open ocean September sample. They were often smaller than the lowest measured value, and sometimes even negative. This was not seen for the other models, and may mean that the two components chosen for this sample model are wrong. Either malformed and non-malformed coccoliths in this sample may not be separated from each other in terms of thickness, or Type A coccoliths in this sample are too rare to contribute significantly to the sample thickness distribution. Unfortunately, it was not possible to obtain more coccoliths even after preparing a second slide from the same sample, because the sample is so sparse in coccoliths.

Comparison with other studies

Other studies on morphotype-specific differences in E. huxleyi coccolith mass have combined relative abundance estimates with coccolith mass estimates obtained from their birefringence in a polarized light microscope (e.g. [8, 40]). These studies did not use MCMC analysis or other tools to estimate coccolith mass per morphotype, but rather correlated changes in E. huxleyi (or Noelrhabdaceae) coccolith mass with changes in morphotype relative abundance. However, this approach appears to be not suitable to disentangle the effects of changes in relative abundance from intra-morphotypic changes in coccolith mass. For example, in the present study, the ~25-36% decrease in Type A coccolith mass at the coastal site from January to September is not obvious from looking at the changes in relative abundance over the same time period (Fig 14). Moreover, while Type A coccolith size and thickness both decreased at the coastal site from January to September, Group B coccoliths decreased in size but not in thickness (Tables 1 and 7). Similarly, Group B coccoliths were thicker at the open ocean site in January compared to the coastal site, while Type A coccolith thickness did not change. The complex mass variation patterns among morphotypes in the present study is consistent with culture studies showing that environmental responses in E. huxleyi are both strain-specific [11, 78, 79] and morphotype-specific [39, 41, 80, 81], and is unlikely to be unique to the Canary Islands region. On the other hand, the present study appears to contradict, for example, [8], who reported that E. huxleyi coccolith mass was driven by variation in E. huxleyi morphotype relative abundance. The reason for this contradiction lies in part in the applied method of [8], which has been shown to have significant flaws detailed in [61, 82–86]. In addition, [8] measured only coccolith mass at a family level (Gephyrocapsa oceanica plus E. huxleyi) without analyzing the coccolith mass of individual E. huxleyi morphotypes. The present study, however, demonstrated that coccolith mass variation within individual morphotypes may be an important factor in explaining coccolith mass differences between samples.

Another widely used approach to understand intraspecific coccolith mass variation in E. huxleyi has been to estimate coccolith mass from SEM images using the ks model (e.g. [26, 28, 87]). In this model mass is estimated using measured coccolith length and a species or morphotype-specific ks value. However, the ks values for both Type A and Type B were defined based on a very limited number of coccoliths (four and three, respectively [26]). Moreover, a recent culture study found that coccolith thickness did not vary proportionally with coccolith length in several Type A E. huxleyi strains [88]. Similarly, in the present study Group B coccoliths varied in thickness, but not length, between the coastal and open ocean site in January. Intra-morphotypic coccolith mass variation is purely a reflection of variation in coccolith length in the ks model. Therefore the model may present an incomplete picture of the true variation in coccolith mass.

Relationship between E. huxleyi coccolith mass and calcite concentration

The present study demonstrated that the relationship between coccolith mass and E. huxleyi morphotype relative abundance is not straightforward. The relationship between coccolith mass and the total amount of calcite produced by E. huxleyi appears to be also not straightforward, as illustrated by the estimated E. huxleyi calcite concentrations. Please note that the calcite concentration estimations presented here are not intended as accurate representations of calcite produced by E. huxleyi at each site. Instead, the intention of these estimations is to highlight the importance of factors other than simply coccolith mass when determining coccolithophore calcite production. Considering coccolith mass alone, and assuming coccolith mass is closely related to coccolithophore calcite mass [8] (i.e. the number of coccoliths per coccolithophore cell is similar in different samples), one might be tempted to conclude that calcite production is similar at the two sites in the present study (Table 5). However, when cell density is taken into account, it is obvious that calcite production at the coastal site is much greater than at the open ocean site because of the differences in cell density (Fig 10, Table 5). At the same time, the greater E. huxleyi coccolith mass at the open ocean site in January gives greater calcite production compared to the coastal site in September, despite the lower cell density (Table 5). Neither cell density or coccolith mass should be ignored for accurate calcite production estimation in the ocean.

Accurate calcite production estimates require accurate knowledge of the number of coccoliths per cell as well. In the present study, 10, 23, and 48 coccoliths per cell (taken from [73] and [74]) were used to illustrate the importance of coccolith numbers for overall calcite concentration estimation. [89] reported even greater variation in number of coccoliths per cell from samples collected at different cruises along the Portuguese coast in the North Atlantic, where they varied from 4 to 135 coccoliths per cell (excluding samples where no coccospheres/cells were seen). Furthermore, E. huxleyi is known to shed coccoliths during growth, particularly during bloom conditions [20], which may also impact calcite concentration estimations.

Following the same reasoning, relative contributions of individual morphotypes to E. huxleyi calcite production does not only depend on the coccolith mass of the individual morphotypes, but also on the cell density and coccolith production of the morphotypes. Type B coccoliths have, for example, been reported to produce more coccoliths per cell than Type A in a culture study, accompanied by greater calcite production [80]. Whether studying calcite production at species or sub-species level, neither cell density nor coccolith mass and coccoliths per cell should be ignored for accurate estimates.

Conclusion

This study combined thickness measurements from the CPR-method and E. huxleyi morphotype relative abundance from SEM images with MCMC analysis to estimate coccolith thickness and mass of individual E. huxleyi morphotypes in different sampling sites and seasons near the Canary Islands. This analysis revealed that thickness and/or mass can not be used to characterize a specific E. huxleyi morphotype nor to describe the relations between different morphotypes (e.g. characterizing “Overcalcified” Type A coccoliths as heavier than Group B coccoliths). Coccolith length and thickness of morphotypes vary both seasonally and regionally, so that changes in relative abundance of morphotypes can not be used as a proxy for either E. huxleyi coccolith mass or calcite production. Furthermore, this study revealed that the terms “Overcalcified” and “lightly calcified” are misleading; “Overcalcified” Type A coccoliths in this study were lighter and thinner than some “normally calcified” Type A coccoliths and similar in thickness to some “lightly calcified” Group B coccoliths. This study highlights the challenges in trying to estimate coccolithophore calcite production from coccolith habitus, and uncritically using coccolith habitus as a proxy for calcification appears to be ill-advised.

Traceplot for the MCMC model for the open ocean site in January.

Blue, green and red lines each represent an individual chain for the sampling.

(TIFF)

Click here for additional data file.

Traceplot for the MCMC model for the coastal site in January.

Blue, green and red lines each represent an individual chain for the sampling.

(TIFF)

Click here for additional data file.

Traceplot for the MCMC model for the open ocean site in September.

Blue, green and red lines each represent an individual chain for the sampling.

(TIFF)

Click here for additional data file.

Traceplot for the MCMC model for the coastal site in September.

Blue, green and red lines each represent an individual chain for the sampling.

(TIFF)

Click here for additional data file.

SEM Counts and measurements from the open ocean and coastal sites in January and September 1997.

A: Type A; B: Group B; OA: “Overcalcified” Type A; M: Malformed.

(CSV)

Click here for additional data file.

LM coccolith measurements from samples used in the study.

Images were captured in RAW and converted to TIFF in sRGB with 2.2 gamma. Coccoliths were segmented from the image background using a Canny-Deriche edge detection algorithm.

(CSV)

Click here for additional data file.

Mean thickness, standard deviation, and relative abundance of simulated samples generated with a random number generator.

RA: Relative abundance; SD: Standard deviation. Note that samples are simulated with no units.

(CSV)

Click here for additional data file.

1 Oct 2019

PONE-D-19-23241

Coccolith mass and morphology of different Emiliania huxleyi morphotypes

PLOS ONE

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Reviewer #1: Emiliania huxleyi without doubt is the best studied calcifying nanoplankton. However from studying different global strains/populations it has become clear this organism represents an enormous physiological and genetic diversity, potentially deserving at least discrimination of several varieties (Medlin 1994). As a first approximation researchers therefore have tried to discriminate between "morphotypes" such as A, A overcalcified, B/C etc. These ecotypes roughly represent differing sensitivities to ocean acidification (Muller et al. 2005 MEPS) and , at least in the Southern Ocean, have different temperature tolerance and ranges (Cubillos 2007, MEPS) and light responses. The present work makes a valid point that over time, these morphotypes , sometimes carelessly called "calcification morphotypes", have become equated with calcite density. This is something that requires more careful measurements using a combination of SEM measurements, birefringence polarising microscopy (Beaufort method), but also (not conducted here, because working with field samples, not clonal cultures) coulter counter sizing of coccosphere diameter and number of coccoliths and sizes.

The present work on a mixed assemblage from the Canary Islands claims that A, A overcalcified, B did not significantly differ in mass and thickness. This conclusion contradicts previous work by Young & Ziveri 2000. Deep-Sea Research 47,1679-1700 (2000),Poulton et al. 2013, Global Biochemical Cycles 27, 1023-1033, Poulton et al. 2011, Mar. Ecol.-Prog. Ser., 443, 1–17, Charalampopoulou et al. 2016, Biogeosciences, 13, 5917–5935; Beaufort et al. 2011. Nature. etc

The reasons for this discrepancy need to be carefully explored. Either the present authors' conclusion (no differences between morphotypes and mass/thickness) are correct but relate only to the particular Canary Island coccolithophorid population studied (which I find hard to believe). OR , this is due to differences in methodology or concepts used. The criticisms against the Beaufort birefringence method [line 486] are too easily raised however and lack detail. Another complication is that different morphotypes have different coccosphere diameters and produce different numbers of coccoliths of differing size as well as calcite mass. Note that coccosphere diameter is not considered here and number of liths is largely ignored as well (line 301, only guessed ). Thickness and mass (line 23) cannot be estimated from SEM and were estimated here from LM and a Markov Chain Monte Carlo analysis. I must admit that I have difficulty following the assumptions using this approach, but whatever, this needs to be better explained.

The authors raise a good point of warning against carelessly equating morphotypes with calcification fluxes, BUT their results contradict a significant body of literature, and they need to better explain differences in concepts (not coccosphere, not number of liths), but also of methods (MCMC) and materials used.

Reviewer #2: This study mainly combined the CPR-method and MCMC analysis to estimate the coccolith thickness and calcite mass of two different Emiliania huxleyi morphotypes sampled off the northwestern African coast. The results suggest that the coccolith length and thickness did not necessarily correlate with the characteristics of different E. huxleyi morphotypes. Therefore, the study provides some insights on further understanding the complex implication of the existence of different morphotypes of the cosmopolitan coccolithophore species E. huxleyi on calcite production. However, some of the important details are missing, the results were poorly discussed and the structure of the discussion section needs to be reorganized. I would suggest the manuscript resubmitted for review after some severe revisions.

My specific comments are listed below.

Abstract: The present abstract is poorly written. It seems lacking important information of the study. Please add some brief description of the methodology (i.e. how the morphotypes, mass and thickness of coccoliths were examined), the major results/findings and the oceanographic implications of the study.

Introduction

Line 22: I think the word “organic” should be revised to “inorganic”, that is the carbon pool produced by calcification.

Lines 26-28: It says “sever studies…”, however, there is only one reference [8] cited here.

Oceanographic settings:

Lines 84-86: Please specify how the primary productivity was measured.

Materials and Methods:

Lines 93-96: Please specify how the sampling depths of this study were determined. It is mentioned in the text that “ the depths… were chosen based on E. huxleyi cell densities”. Were they the depths where the maximum E. huxleyi cell abundances observed? Were there any other coccolithophore species observed? According to the cited reference [45] (Bollmann J, Cortés MY. Distribution of Living Coccolithophores North of the Canary Islands: Vertical Seasonal and Interannual Variations. In: American Geophysical Union, Fall Meeting 2007; 2007. p. OS11A–0189), the dominant coccolithophore species in the upper photic zone was Gephyocapsa ericsonii.

Lines 103-108: Please specify how the Type A coccoliths were defined and grouped.

Results:

Lines 219-222: This paragraph should be moved to the “Materials and Methods” section.

Lines 251- 256: Please add some details on the specific differences in thickness between the open ocean and coastal sampling sites.

Discussion:

Line 317: “This study characterizes E. huxleyi morphotyples using coccolith length and thickness”: I think this is confusing, since later in lines 341-344, the authors mentioned that the morphotypes were somehow determined by the appearance of the distal shield elements.

Lines 342-343: Again, please specify how the “overcalcified” and “normal” Type A coccoliths were defined and separated in detail. From Fig. 13, I only found two groups of different morphotypes (Type A with closed central area in red and Group B with an open central area in black). How was the conclusion “this study revealed that despite their appearance, “overcalcified” Type A coccoliths are not necessarily heavier or thicker than “normal” Type A coccoliths” drawn based on this figure?

Lines 355-357: The current expression is confusing. Similar thickness and length don’t necessarily imply similar coccolith mass.

Lines 362-363: It is mentioned here that “variable proximal shield slit closure, width of the central tube, thickness of distal shield elements and the size of the proximal shield related to the distal shield” may all contribute the “degree of calcification” of coccolith. However, the authors used a way too simplified model (Eq. 3) to estimate the coccolith mass by only considering the length, width and thickness of coccolith. Therefore, this would have generated rather biased results and further conclusions.

Lines 393-426: This paragraph should be moved to the “results” section.

Lines 463-467: How would these exceptions affect the outcome of the analysis?

Line 473: What is the assumption in reference #31? Please provide some details.

Lines 494-498: The expression is confusing. How can you extrapolate the trend for coastal site based on the observations for open ocean site?

In general, the discussion is rather vague at this stage. For example, were there any potential effects of the environmental conditions (such as seawater carbonate chemistry, nutrient concentrations, temperature and salinity) on the coccolith mass and calcite stock? Was there any difference observed between the coastal and open ocean site? These are important factors controlling the calcification process in coccolithophores and therefore will regulate the coccolith mass and calcite production of the cells. I think the authors should take these factors into consideration.

**********

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Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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29 Nov 2019

Response to reviewers

Reviewer #1: "Emiliania huxleyi without doubt is the best studied calcifying nanoplankton. However from studying different global strains/populations it has become clear this organism represents an enormous physiological and genetic diversity, potentially deserving at least discrimination of several varieties (Medlin 1994). As a first approximation researchers therefore have tried to discriminate between "morphotypes" such as A, A overcalcified, B/C etc. These ecotypes roughly represent differing sensitivities to ocean acidification (Muller et al. 2005 MEPS) and , at least in the Southern Ocean, have different temperature tolerance and ranges (Cubillos 2007, MEPS) and light responses. The present work makes a valid point that over time, these morphotypes , sometimes carelessly called "calcification morphotypes", have become equated with calcite density. This is something that requires more careful measurements using a combination of SEM measurements, birefringence polarising microscopy (Beaufort method), but also (not conducted here, because working with field samples, not clonal cultures) coulter counter sizing of coccosphere diameter and number of coccoliths and sizes.

The present work on a mixed assemblage from the Canary Islands claims that A, A overcalcified, B did not significantly differ in mass and thickness. This conclusion contradicts previous work by Young & Ziveri 2000. Deep-Sea Research 47,1679-1700 (2000),Poulton et al. 2013, Global Biochemical Cycles 27, 1023-1033, Poulton et al. 2011, Mar. Ecol.-Prog. Ser., 443, 1–17, Charalampopoulou et al. 2016, Biogeosciences, 13, 5917–5935; Beaufort et al. 2011. Nature. etc

The reasons for this discrepancy need to be carefully explored. Either the present authors' conclusion (no differences between morphotypes and mass/thickness) are correct but relate only to the particular Canary Island coccolithophorid population studied (which I find hard to believe). OR , this is due to differences in methodology or concepts used. The criticisms against the Beaufort birefringence method [line 486] are too easily raised however and lack detail. "

We agree with the reviewer that discrepancies between this study and other studies is not likely to be due to geographical peculiarities, but is rather methodological. Bollmann (2013,2014) outlined in detail the issues using the method proposed by Beaufort (2005). In addition, most studies have not captured a complete picture of variation within individual morphotypes (e.g. Young and Ziveri (2000), Beaufort et al. (2011), D’Amario et al. (2018). We have revised the discussion section to better explain this point.

"Another complication is that different morphotypes have different coccosphere diameters and produce different numbers of coccoliths of differing size as well as calcite mass. Note that coccosphere diameter is not considered here and number of liths is largely ignored as well (line 301, only guessed). "

Our main goal was to estimate the mass of single coccoliths of different morphotypes. However, in order to demonstrate the accumulated effect when coccoliths per cell are used for total calcite mass estimates of coccospheres we did some simple calculations based on published data on coccoliths per cell.

"Thickness and mass (line 23) cannot be estimated from SEM and were estimated here from LM and a Markov Chain Monte Carlo analysis. I must admit that I have difficulty following the assumptions using this approach, but whatever, this needs to be better explained."

We added a paragraph on MCMC assumptions in the Methods section and rewrote parts of the Discussions section on the MCMC assumptions to better explain the methodology.

"The authors raise a good point of warning against carelessly equating morphotypes with calcification fluxes, BUT their results contradict a significant body of literature, and they need to better explain differences in concepts (not coccosphere, not number of liths), but also of methods (MCMC) and materials used."

We modified the Discussions section and added more elaborated comparisons with other studies.

Reviewer #2: "This study mainly combined the CPR-method and MCMC analysis to estimate the coccolith thickness and calcite mass of two different Emiliania huxleyi morphotypes sampled off the northwestern African coast. The results suggest that the coccolith length and thickness did not necessarily correlate with the characteristics of different E. huxleyi morphotypes. Therefore, the study provides some insights on further understanding the complex implication of the existence of different morphotypes of the cosmopolitan coccolithophore species E. huxleyi on calcite production. However, some of the important details are missing, the results were poorly discussed and the structure of the discussion section needs to be reorganized. I would suggest the manuscript resubmitted for review after some severe revisions.

My specific comments are listed below.

Abstract: The present abstract is poorly written. It seems lacking important information of the study. Please add some brief description of the methodology (i.e. how the morphotypes, mass and thickness of coccoliths were examined), the major results/findings and the oceanographic implications of the study."

The abstract was rewritten and expanded.

"Introduction

Line 22: I think the word “organic” should be revised to “inorganic”, that is the carbon pool produced by calcification."

The word should be organic. The sentence was slightly revised to clarify.

"Lines 26-28: It says “sever studies…”, however, there is only one reference [8] cited here."

We added more references.

"Oceanographic settings:

Lines 84-86: Please specify how the primary productivity was measured."

We did not measure primary productivity. It is stated in the cited reference.

"Materials and Methods:

Lines 93-96: Please specify how the sampling depths of this study were determined. It is mentioned in the text that “ the depths… were chosen based on E. huxleyi cell densities”. "

Correct, highest cell densities of EHUX to obtain sufficient specimens for measurements. Two sentences were added to the materials section to clarify.

"Were they the depths where the maximum E. huxleyi cell abundances observed?"

The sample selection was mainly based on cell density, plus two other depths at one site to check whether depth has a significant effect on coccolith mass.

"Were there any other coccolithophore species observed? "

Yes (see Bollmann and Cortes (2007) and Abrantes et al. (2002) cited in the paper), but the presence of other coccolithophores species is not relevant to this study.

"According to the cited reference [45] (Bollmann J, Cortés MY. Distribution of Living Coccolithophores North of the Canary Islands: Vertical Seasonal and Interannual Variations. In: American Geophysical Union, Fall Meeting 2007; 2007. p. OS11A–0189), the dominant coccolithophore species in the upper photic zone was Gephyocapsa ericsonii."

That is correct.

"Lines 103-108: Please specify how the Type A coccoliths were defined and grouped."

They were defined according to Young et al. (2003). The text was slightly revised to clarify.

"Results:

Lines 219-222: This paragraph should be moved to the “Materials and Methods” section."

The start of the Results section was rewritten and restructured.

"Lines 251- 256: Please add some details on the specific differences in thickness between the open ocean and coastal sampling sites."

Details on the thickness difference between the coastal and open ocean site in January were added.

"Discussion:

Line 317: “This study characterizes E. huxleyi morphotyples using coccolith length and thickness”: I think this is confusing, since later in lines 341-344, the authors mentioned that the morphotypes were somehow determined by the appearance of the distal shield elements."

The Discussion section has been revised.

"Lines 342-343: Again, please specify how the “overcalcified” and “normal” Type A coccoliths were defined and separated in detail. From Fig. 13, I only found two groups of different morphotypes (Type A with closed central area in red and Group B with an open central area in black). How was the conclusion “this study revealed that despite their appearance, “overcalcified” Type A coccoliths are not necessarily heavier or thicker than “normal” Type A coccoliths” drawn based on this figure?"

Figure 13 showed “Overcalcified” Type A in red and all other, non-”Overcalcified” Type A morphotypes (i.e. Type A, Group B, and malformed) in black. After some consideration, Figure 13 doesn’t add much to the discussion, and here seems to cause some misunderstanding. It was therefore removed, and some relevant information instead added to Table 4. The relevant section in the Materials and Methods section was also slightly revised to clarify the identification.

"Lines 355-357: The current expression is confusing. Similar thickness and length don’t necessarily imply similar coccolith mass."

We are not sure what the reviewer means here. Coccolith mass is a function of volume and density of the coccolith. It can be assumed that coccolith density does not vary (as they consist of only one mineral, calcite). Volume varies depending on (mean) thickness and area. The area can be simply calculated from the coccolith length and width, assuming an elliptical shape. Nevertheless, the confusing sentence was changed in the Discussion revision.

"Lines 362-363: It is mentioned here that “variable proximal shield slit closure, width of the central tube, thickness of distal shield elements and the size of the proximal shield related to the distal shield” may all contribute the “degree of calcification” of coccolith. However, the authors used a way too simplified model (Eq. 3) to estimate the coccolith mass by only considering the length, width and thickness of coccolith. Therefore, this would have generated rather biased results and further conclusions."

The average thickness obtained from retardation measurements reflects the variation of proximal shield slit closure, width of the central tube, thickness of distal shield elements and the size of the proximal shield related to the distal shield and therefore the results are not biased.

"Lines 393-426: This paragraph should be moved to the “results” section."

The paragraph was moved to the Results section as suggested.

"Lines 463-467: How would these exceptions affect the outcome of the analysis?"

If these indications imply that the morphotypes are not picked from a normally distributed population, it might affect the results. However, the exceptions appear to be due to the influence of one or two outliers, and the p-values are relatively large as well (above 0.01), so we argue there is not strong enough evidence that the morphotypes do not represent normally distributed populations.

"Line 473: What is the assumption in reference #31? Please provide some details."

The assumption is that Overcalcified Type A is systematically heavier/more calcified than Type A, which this study showed is not a valid assumption. The text has been revised/changed in the Discussion section revision.

"Lines 494-498: The expression is confusing. How can you extrapolate the trend for coastal site based on the observations for open ocean site?"

We clarified that there is no systematic difference in mass of coccoliths with depth .

"In general, the discussion is rather vague at this stage. For example, were there any potential effects of the environmental conditions (such as seawater carbonate chemistry, nutrient concentrations, temperature and salinity) on the coccolith mass and calcite stock? "

There might be several environmental factors influencing coccolith mass and morphotype abundance. However, it was not the goal of our study to analyse these factors. We looked at the mass and relative abundance of different morphotypes to the test whether morphotypes can be related to overall calcification. The driving factors will the investigated in future studies and is outside of the scope of the present study.

"Was there any difference observed between the coastal and open ocean site? "

Yes there were. See introduction/oceanographic settings, plus Fig 2.

"These are important factors controlling the calcification process in coccolithophores and therefore will regulate the coccolith mass and calcite production of the cells. I think the authors should take these factors into consideration."

The goal of the study was to investigate whether different morphotypes indicate different degrees of calcification/coccolith mass. The driving environmental actors behind the coccolith mass/calcification is a separate issue that requires detailed data on the carbon chemistry of the water that are not available for all samples used in this study.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

10 Jan 2020

PONE-D-19-23241R1

Coccolith mass and morphology of different Emiliania huxleyi morphotypes

PLOS ONE

Dear Dr. Linge Johnsen,

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PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Overall I am happy with the authors responses, but there are a few minor things I like to see better clarified

Title: I remain unconvinced that this criticism of the use of coccolith morphotypes as a proxy for coccolith mass applies to all global Ehux populations. The work only examined Canary Island material, and hence I suggest a cautious subtitle:

"Coccolith mass and morphology of different Emiliania huxleyi morphotypes: a critical examination of Canary Island material"

In the abstract, explain that CPR stands for Circular Polarizer Retardation

Final sentence of abstract: ..cannot be uniformly used as reliable indicators...

"line 362: estimated the same way

line 423: to obtain insights

line 594-95: and uncritically using coccolith morphology as a proxy...

Reviewer #2: The authors have thoroughly revised and improved the manuscript, including having clarified some of the methodologies, expanded the abstract and re-organized the discussion section. However, I still find some of the discussion hard to follow and need to be improved for more clarity.

The study mainly estimated the coccolith thickness and calcite mass of coccolithophore Emiliania huxleyi collected off the northwestern African coast by combining the CPR-method and MCMC analysis. The major results indicate that the coccolith length and thickness did not necessarily correlate with the characteristics of different E. huxleyi morphotypes. The calcite mass was then calculated based on the coccolith length and thickness, and the authors concluded that the morphotype appearance and relative abundance can not be used as reliable indicators of E. huxleyi calcification or calcite production.

This conclusion is contradicted with many of the previously published research. For this reason, the authors have added two paragraphs of “comparison with other studies” to explain the discrepancy between the methods used in the present study and other studies (polarized microscopic and SEM imaging methods). However, I still think the comparison is missing some important point. My major concern is that the present study mainly used the unified calcite density and simplified coccolith volume (equation 2) to generate the final conclusion, and thereby the effects of different morphotypes are ignored based on my understanding – how is this assumption valid compared to other methods? For example, Beaufort et al. (2008, biogeosciences) used the relative lightness of crystals as indicators of calcite mass.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Feb 2020

Response to reviewers

Reviewer #1: Overall I am happy with the authors responses, but there are a few minor things I like to see better clarified

Title: I remain unconvinced that this criticism of the use of coccolith morphotypes as a proxy for coccolith mass applies to all global Ehux populations. The work only examined Canary Island material, and hence I suggest a cautious subtitle:

"Coccolith mass and morphology of different Emiliania huxleyi morphotypes: a critical examination of Canary Island material"

Reply:

We changed the title as suggested by the reviewer.

"In the abstract, explain that CPR stands for Circular Polarizer Retardation

Final sentence of abstract: ..cannot be uniformly used as reliable indicators...

line 362: estimated the same way

line 423: to obtain insights

line 594-95: and uncritically using coccolith morphology as a proxy... "

Reply:

We changed the lines and abstract as suggested.

Reviewer #2: The authors have thoroughly revised and improved the manuscript, including having clarified some of the methodologies, expanded the abstract and re-organized the discussion section. However, I still find some of the discussion hard to follow and need to be improved for more clarity.

The study mainly estimated the coccolith thickness and calcite mass of coccolithophore Emiliania huxleyi collected off the northwestern African coast by combining the CPR-method and MCMC analysis. The major results indicate that the coccolith length and thickness did not necessarily correlate with the characteristics of different E. huxleyi morphotypes. The calcite mass was then calculated based on the coccolith length and thickness, and the authors concluded that the morphotype appearance and relative abundance can not be used as reliable indicators of E. huxleyi calcification or calcite production.

This conclusion is contradicted with many of the previously published research. For this reason, the authors have added two paragraphs of “comparison with other studies” to explain the discrepancy between the methods used in the present study and other studies (polarized microscopic and SEM imaging methods). However, I still think the comparison is missing some important point. My major concern is that the present study mainly used the unified calcite density and simplified coccolith volume (equation 2) to generate the final conclusion, and thereby the effects of different morphotypes are ignored based on my understanding – how is this assumption valid compared to other methods? For example, Beaufort et al. (2008, biogeosciences) used the relative lightness of crystals as indicators of calcite mass.

Reply:

We agree with Reviewer Two that our results appear to contradict many previously published papers that utilised the method first published by Beaufort (2005). The contradictory results are not surprising because it has been demonstrated by several studies that the method published by Beaufort (2005) is flawed in many ways (see e.g. Bollmann (2013, 2014), Lochte (2014), Beaufort et al. 2014; Gonzalez-Lemos (2018). The method is not only inaccurate as it significantly overestimates coccolith weights, it also leads to irreproducible results because of a flawed calibration method (see publications above).

Having said this, the method used in our research is based on the same basic principles as reported by Beaufort (2005). It uses “the relative lightness of crystals as indicators of calcite mass” and image analysis techniques (thresholding) to detect the outline/area of a coccolith and measure the grey value of individual pixels (lightness) within the area as a proxy for coccolith thickness/mass.

Furthermore, using the average (unified according to Reviewer Two) grey values/lightness/thickness of a coccolith to infer the mass/weight of a certain length is also a technique that Beaufort has used in many publications (e.g. Beaufort et al. (2008), Biogeosciences; Beaufort et al. (2011), Nature). Here is one excerpt from Beaufort et al. (2008) Biogeosciences page 1105 where 4 pixel/0.6µm were added to the length of coccoliths and the average (unified according to Reviewer Two) grey value/lightness/thickness were extrapolated to calculate coccolith mass/weight:

Quote: “The Coccolith Analyser measures the grey level of objects, their diameter and surface, and tabulates the results. There is a bias of 0.6μm in the measurement of the diameter of small, and dim objects, such as coccoliths. This is because we apply a minimum Grey Level threshold above background. This threshold erodes 2 pixels of the periphery of dim objects. Each pixel is 0.15μm, and 4 pixels are eroded in total when the long diameter is measured. Thus we added 0.6μm to the measurement of coccolith length to compensate for this automatic loss. Another small bias in the measurements of the length of coccoliths of E. huxleyi exists, the distal shield being not detected in its entirety in cross-polarized light. There-fore the length of E. huxleyi presented here are slightly underestimated. We estimated this bias to a factor of 1.25 by comparing measures from the Coccolith analyzer and from SEM.”

Therefore, we do not agree with the following concern of Reviewer Two:

Quote “My major concern is that the present study mainly used the unified calcite density and simplified coccolith volume (equation 2) to generate the final conclusion, and thereby the effects of different morphotypes are ignored based on my understanding – how is this assumption valid compared to other methods? For example, Beaufort et al. (2008, biogeosciences) used the relative lightness of crystals as indicators of calcite mass.”

In contrast to Beaufort et al. (2008), we do not extrapolate grey values/thickness to missing pixels/length. We use actual grey value/thickness measurements of coccoliths and use the average grey value of a coccolith in the LM to estimate the weight of similar sized coccoliths from SEM observations. We have not only overcome the size bias introduced by unsuitable thresholding techniques used by Beaufort (2005) by applying the Canny edge detection technique, we have also increased the taxonomic resolution significantly by using circular polarised light. In contrast to Beaufort et. al. (2008, 2011) who were only able to identify coccoliths at the family level, we were able to distinguish between species within the Noelaerhabdaceae.

In conclusion, our data quality is significantly improved compared to any publication that has used the method by Beaufort (2005). Moreover, to the best of our knowledge, there is NO study that has been able or attempted to estimate the mass of different morphotypes at sub-species level on a light microscope. Therefore, it appears to be odd that Reviewer Two takes issue with our technique, results and interpretation with reference to other light microscope studies that were not even able to distinguish between species within the Noelaerhabdaceae.

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4 Mar 2020

Coccolith mass and morphology of different Emiliania huxleyi morphotypes: a critical examination using Canary Islands

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9 Mar 2020

PONE-D-19-23241R2

Coccolith mass and morphology of different Emiliania huxleyi morphotypes: a critical examination using Canary Islands material

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