Broad Phylogenetic and Functional Diversity Among Mixotrophic Consumers of Prochlorococcus

bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Broad phylogenetic and functional diversity among mixotrophic consumers of

Prochlorococcus

Qian Li1,2,3, Kyle F. Edwards2 , Christopher R. Schvarcz2,3, Grieg F. Steward2,3

1 School of Oceanography, Shanghai Jiao Tong University, Shanghai, China

2Department of Oceanography, School of Ocean and Earth Science and Technology (SOEST),

University of Hawaiʻi at Mānoa, Honolulu, Hawaiʻi

3Daniel K. Inouye Center for Microbial Oceanography: Research and Education, School of

Ocean and Earth Science and Technology (SOEST), University of Hawaiʻi at Mānoa, Honolulu,

Hawaiʻi

Corresponding author. Email: [email protected]

Competing interests

All authors declare that they have no competing interests related to this work.

Funding resource

This work was supported by National Science Foundation awards OCE 15-59356 and RII Track-

2 FEC 1736030, and a Simons Foundation Investigator Award in Marine Microbial Ecology and

Evolution (to K.F.E.).

Running title: Mixotrophic consumers of Prochlorococcus

Keywords: picoeukaryotes, bacterivory, grazing, haptophyte, chrysophyte, dictyochophyte

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Abstract

Small eukaryotic phytoplankton are major contributors to global primary production and

marine biogeochemical cycles. Many taxa are thought to be mixotrophic, but quantitative studies

of phagotrophy exist for very few. In addition, little is known about consumers of

Prochlorococcus, the abundant cyanobacterium at the base of oligotrophic ocean food webs.

Here we describe thirty-nine new phytoplankton isolates from the North Pacific Subtropical Gyre

(Station ALOHA), all flagellates ~2–5 um diameter, and we quantify their ability to graze

Prochlorococcus. The mixotrophs are from diverse classes (dictyochophytes, haptophytes,

chrysophytes, bolidophytes, a dinoflagellate, and a chlorarachniophyte), many from previously

uncultured clades. Grazing ability varied substantially, with specific clearance rate (volume

cleared per body volume) varying over ten-fold across isolates and six-fold across genera.

Slower grazers tend to create more biovolume per prey biovolume consumed. Using qPCR we

found that the haptophyte Chrysochromulina was most abundant among the isolated mixotrophs

at Station ALOHA, with 76–250 cells mL-1 across depths in the upper euphotic zone. Our results

show that within a single ecosystem the phototrophs that ingest bacteria come from many

branches of the eukaryotic tree, and are functionally diverse, indicating a broad range of

strategies along the spectrum from phototrophy to phagotrophy.

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Introduction

Small eukaryotic phytoplankton in the ‘pico’ (< 3µm) and ‘nano’ (2–20 µm) size classes are

often dominant contributors to phytoplankton biomass and primary production in open oceans

[1][2][3] and are major drivers of nutrient cycling [4][5]. At the same time, many flagellate and

ciliate taxa are known to be mixotrophic, capable of obtaining nutrition through combined

photosynthesis (autotrophy) and phagocytosis (heterotrophy) [6][7]. Mixotrophs have been

observed in sunlit habitats throughout the ocean [8][9] and are estimated to contribute about two

thirds of total bacterivory in open-ocean Atlantic ecosystems [10]. Phototrophic and

heterotrophic prokaryotes are themselves major contributors to pelagic biomass and production

[11][12][13], and therefore the bacterial grazers that also photosynthesize may play a key role in

regulating productivity, element cycling, and food web dynamics. To understand how these

systems function it is necessary to quantify fundamental parameters such as grazing kinetics and

growth efficiencies of the main bacterial grazers, and it may be particularly consequential if the

main grazers are mixotrophs, because models predict that mixotrophic consumers increase

primary production and carbon export, and decrease nutrient remineralization, relative to

heterotrophic consumers [14][15].

Although the aggregate importance of pigmented flagellates for bacterial grazing has been

documented [16][17], much less is known about which taxa are the major grazers, and how the

broad phylogenetic diversity among these organisms translates into diverse ecological roles

[8][18]. Progress in this area has been impeded in part by a paucity of representative cultured

mixotrophs relative to the complexity found in natural communities [18][19], which often

contain haptophytes, chrysophytes, dictyochophytes, chlorophytes, bolidophytes, cryptophytes,

and dinoflagellates [20].

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The North Pacific Subtropical Gyre (NPSG) is a chronically oligotrophic environment,

where prokaryotic and eukaryotic phototrophs are dominated by Prochlorococcus [21] and small

flagellates [22], respectively. Some of the likely grazers of Prochlorococcus in this habitat were

identified by stable isotope probing by addition of 13C- and 15N-labeled Prochlorococcus MED4

cells to natural communities [23]. The 18S rRNA of various haptophytes, dictyochophytes,

bolidophytes and dinophytes became significantly labeled, but most of the specific phylotypes

identified in that study have not been isolated, and controlled lab studies of their grazing

capabilities have been lacking. There has thus been no confirmation that most of these organisms

directly ingest Prochlorococcus and no quantitative assessment of how ingestion rates and

functional responses vary among them.

One exception is a recent study of the grazing ecophysiology of a phagotrophic mixotroph

Florenciella (strain UHM3021; class Dictyochophyceae) isolated from the NPSG [24]. Given

sufficient light, rapid growth of Florenciella was sustained by feeding on bacteria

(Prochlorococcus, Synechococcus, or a heterotrophic bacterium) as the primary nutrient source,

suggesting mixotrophy is an effective strategy for nutrient acquisition. The rate at which prey

were ingested by this Florenciella strain was relatively low compared to heterotrophic flagellates

of similar size, and prey ingestion was suppressed by high concentrations of dissolved nutrients.

This was the first detailed characterization of Prochlorococcus consumption by a mixotrophic

flagellate, and it remains unclear whether other small, open-ocean mixotrophs possess similar

physiology. For example, Florenciella may be a relatively autotrophic mixotroph that relies on

prey primarily for limiting nutrients [25]; other taxa may be more voracious grazers that rely on

prey as a major energy source [26][27].

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To broaden our understanding of how mixotrophs feed on one of the most important

primary producers in the ocean, we isolated diverse mixotrophs (dictyochophytes, haptophytes,

chrysophytes, bolidophytes, a dinoflagellate, and a chlorarachniophyte) from Station ALOHA,

an open ocean site in the NPSG, 100 km north of the island of Oʻahu. By characterizing their

grazing capabilities using Prochlorococcus as prey, and by quantifying their in situ abundances,

we reveal functional diversity among the mixotrophs in this ecosystem and their contributions to

Prochlorococcus mortality in situ.

Methodology

Isolation and cultivation

In total, 39 mixotrophic flagellates were investigated (Table 1). Thirty-three isolates were

enriched and isolated from euphotic zone samples at Station ALOHA (22° 45' N, 158° 00' W) in

February and May 2019. To select for mixotrophic grazers of Prochlorococcus, whole seawater

was amended with K medium [28] (1/20 final concentration), and live Procholorococcus

(MIT9301) was added as prey (~5×106 cells mL-1 final concentration). Enriched seawater

samples were incubated under ~70 µM photons m-2 s-1 irradiance on a 12 h:12 h light:dark cycle

and monitored by microscopy daily up to five days. Each day, samples were serially diluted to

extinction (9 dilution steps, 12 replicates per dilution) in 96-well plates in nutrient-reduced K

medium (1/20 concentration) with a constant background of Prochlorococcus cells. Wells at the

highest dilution showing growth of putative grazers were subjected to 3–6 further rounds of

dilution to extinction. Four additional mixotrophs were isolated in full K medium using water

from earlier cruises, and two (dictyochophyte strains UHM3021 described in [24] and UHM3050)

were enriched in K minus nitrogen medium (K-N) without Prochlorococcus enrichment. All

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isolates were rendered unialgal, but not axenic, and maintained at 24 °C in K-N medium (~0.2

µM N) amended with Prochlorococcus prey, under the same light conditions as above.

Prochlorococcus fed to the cultures was grown in Pro99 medium [29]. Dense cells in late

exponential-early stationary phase were harvested and concentrated through gentle centrifugation

at 2,000 RCF for 5 minutes and resuspended in fresh K-N medium.

Table 1. Mixotrophic flagellates surveyed in this study.

Class Genus1 Isolates Source Collection ESD2 Tested Tested (#) depths dates (µm) grazing3 functional (m) (yyyy/mm) (#) response4 (#) Dictyochophyceae Florenciella 19 25, 75, 2011/03 3.0–4.3 12 1 100 2014/09 2019/02 2019/05 Rhizochromulina 4 25, 75 2019/05 3.0–3.2 4 1

DictyX 6 25, 100 2015/02 4.0–5.3 5 1 2019/02 2019/05 Bolidophyceae Triparma 2 25, 100 2019/05 2.5–2.7 2 0

Chrysophyceae ChrysoH 3 25 2019/05 2.2–2.4 3 1

Haptophyceae Chrysochromulina 2 5, 45 2012/01 3.8–4.3 2 1 2015/08 Hap2 1 25 2019/02 4.5 1 1

Chlorarchniophyceae ChloraX 1 n/a 2011/03 3.8 1 0

Dinophyceae Pelagodinium 1 25 2019/05 4.0 1 0

1DictyX, Hap2, ChrysoH and ChloraX were abbreviations for undescribed or environmental clades of Dictyochophyceae-X, Haptophyceae-2, Chrysophyceae-H and Chlorarachniophyceae- X, respectively. 2ESD = equivalent spherical diameter. 3Number of individual isolates of each type for which grazing rates were measured at least one prey concentration. 4Number of isolates of each type for which grazing rates were measured at multiple prey concentrations to determine the grazing functional response and estimate grazing parameters Cmax and Imax.

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18S rDNA sequencing and phylogenetic analysis

Cells were harvested by centrifuging 25–50 mL dense cultures at 3,000 RCF for 10 min at

4 °C. Genomic DNA was extracted from the pellets using the ZymoBIOMICS DNA Kit (Zymo

Research, Irvine, CA, USA). A near-full-length section of the eukaryotic small-subunit

ribosomal RNA gene (18S rDNA) was amplified by PCR with the Roche ExpandTM High

Fidelity PCR System (Sigma-Aldrich, St. Louis, MO, USA) using either forward primer 5'-

ACCTGGTTGATCCTGCCAG-3' and reverse primer 5'-TGATCCTTCYGCAGGTTCAC-3'

[30], or Euk63F 5'-ACGCTTGTCTCAAAGATTA-3' and Euk1818R 5'-

ACGGAAACCTTGTTACGA-3' [31]. Amplicons were purified using spin columns (DNA

Clean & Concentrator-25; Zymo Research, Irvine, CA, USA) and sequenced (Sanger) using the

same PCR amplification primers and an additional reverse primer 1174R, 5'-

CCCGTGTTGAGTCAAA-3' [32], when necessary to connect two ends. For phylogenetic

analyses, similar sequences were retrieved from the PR2 database [33] based on BLAST

similarity, and two environmental homologs (GenBank Acc. FJ537342 and FJ537336) were

retrieved from NCBI GenBank for the undescribed haptophyte taxon, which was not affiliated

with any reference sequence from the PR2 database. Sequence alignments including 39 isolates,

29 reference and 2 outgroup taxa were created with MAFFT v7.450 using the G-INS-i algorithm

[34] in Geneious R11.1.5 (http://www.geneious.com) [35]. Terminal sites that lacked data for

any of the sequences were trimmed and any sites with greater than 25% gaps were removed from

the alignment, which generated a total sequence length of 1617 bp. Phylogenetic analysis was

performed using MrBayes v3.2.6 in Geneious R11.1.5 [36] with two runs of four chains for

1,000,000 generations, subsampling every 200 generations with burn-in length 100,000, under

the GTR substitution model. The Bayesian majority consensus tree was further edited within

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iTOL v5 [37]. All 18S rDNA sequences were deposited in GenBank with accession numbers

MZ611704–MZ611740; MN615710–MN615711.

Microscopic observation

The average diameter of flagellates in the exponential growth phase (n = 20 cells per strain)

was measured by transmitted light microscopy using image analysis software (NIS-Elements AR,

Nikon, Minato City, Tokyo, Japan) calibrated with a stage micrometer. Equivalent spherical

diameter (ESD) and biovolumes were calculated assuming spherical cells. Chloroplasts were

visualized by autofluorescence under epifluorescence microscopy. An average ESD of 0.64 µm

was used for Prochlorococcus prey [24].

Visual evidence of phagocytosis was obtained by adding fluorescent beads (0.5 µm YG

Fluoresbrite Microspheres; Polysciences) to each culture. Samples post incubation (~2 h) were

fixed with an equal volume of 4% ice-cold glutaraldehyde, and subsamples (20 µL) were

mounted on a glass slide under a coverslip. Paired images captured using epifluorescence and

transmitted light microscopy (Olympus BX51 with Leica DFC 7000 T color digital camera) were

overlain to identify cells with ingested beads.

Grazing experiments

Long-term grazing experiments were conducted for all 39 grazers following [24] and 31

were used to quantify grazing rates based on rates of disappearance of Prochlorococcus cells,

which persist but do not readily grow in K-N medium. Rates were not calculated for eight

isolates because they were sampled at a lower frequency. Briefly, flagellate grazers reinoculated

from exponentially growing cultures were mixed with live, unstained prey in fresh K-N medium

at final concentrations of 102–103 flagellates mL-1 and 1.5–2×106 Prochlorococcus mL-1 and

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incubated for 3–8 days, depending on how fast prey were ingested. Cell concentrations of prey

and grazers were measured every 12–24 hours by flow cytometry on glutaraldehyde-fixed

samples (Attune NxT; Thermo Fisher Scientific, Waltham, Massachusetts, USA, and CytoFLEX;

Beckman Coulter Life Sciences, Indianapolis, IN, USA). Populations were distinguished based

on light scatter and pigment autofluorescence and occasionally confirmed with DNA

fluorescence (stained post-sampling with DNA stain SYBR Green I). Ambient bacteria

concentrations, monitored using DNA stains, were ≤ 10% of the added Prochlorococcus at the

start and during most periods of all grazing experiments. Eleven isolates representing all genera

(or approximately genus-level clades) were examined in more detail by replicating grazing

experiments three times (marked in bold in Supplementary Table S1), while twenty additional

isolates were replicated once to better survey within-genus variation.

Grazing rates and biovolume conversion efficiency

We calculated ingestion rates, I (prey grazer-1 h-1) for each grazer as:

# -# 1 ! = $ $&' (1) ()*+(-$&'--$)

-1 where1 !"1 and !"#$ are the prey abundance at sampling interval t and t+1 (cells mL ), Gavg is the

-1 arithmetic mean grazer abundance (cells mL ) over the time interval,1 and ("#$%-"#) is the time

(h) between two sampling intervals. Clearance rates (C, nL grazer-1 h-1) were calculated by

dividing the ingestion rate by average prey concentration over the same interval, and specific

clearance rate (body volume grazer-1 h-1) was calculated by dividing the clearance rate by cellular

biovolume (µm3) of each grazer. Final clearance rates reported in the Results were averaged over

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all sampling intervals (4–8 intervals, 12–24 h for each interval) prior to grazers reaching

stationary density.

Six grazers representing three classes (three dictyochophytes, two haptophytes, and one

chrysophyte) were further investigated to determine functional grazing responses using a wide

range of initial prey densities (105–107 cells mL-1). Functional responses were modeled using the

#$%&' Holling type II curve,1 ! = ) , where I is the ingestion rate over a sampling interval (eqn. 1), '( $%& *$%&

Imax is the maximum ingestion rate, Cmax is the maximum clearance rate and P is the arithmetic

mean prey density between two sampling points. This curve was fit to ingestion rate data using

maximum likelihood with R package bbmle [38].

For 31 isolates we calculated the amount of grazer biovolume created per prey biovolume

consumed, using data from the same grazing experiments used to calculate grazing rates. It was

calculated based on the following formula:

($ -$ ) 1 ! = % ' *+ (2) ()'-)%) *,

where Ff and Fi are the final and initial flagellate concentrations, Pi and Pf are initial and final

prey concentrations in each culture, and BF and BP are the cellular biovolume of prey and grazer.

We refer to the quantity E as the biovolume conversion efficiency, and we use it as an indicator

of physiological differences among diverse mixotrophs. Note that biovolume conversion

efficiency can be greater than 1, if prey have greater nutrient:biovolume than the grazer.

Quantitative PCR

Real-time, quantitative PCR (qPCR) was performed to quantify the 18S rDNA gene

abundances of representative mixotroph groups discriminated at approximately the genus level,

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including Florenciella, Rhizochromulina and another undescribed clade within the class

Dictyochophyceae; Chrysochromulina and another undescribed clade within the class

Haptophyceae; Chrysophyceae clade H in the class Chrysophyceae; and certain species of

Triparma in the class Bolidophyceae (including T. eleuthera and T. Mediterranea but not T.

Pacifica). Partial environmental 18S rDNA sequences (700–800 bp) of putative grazers

identified in [23] were included for primer design to maximize the potential of targeting various

mixotrophic species from each group (Supplementary Fig. S1). Specific primers were designed

targeting a short region of 18S rDNA genes for each group ranging between 95–176 bp

(Supplement Table S2) and were synthesized by Integrated DNA Technologies (IDT Inc.,

Coralville, IA, USA).

In situ gene abundances were quantified with water samples collected from Sta. ALOHA by

Niskin bottles at 5, 25, 45, 75, 100, 125, 150, and 175m, during HOT cruises #259 (Jan), #262

(Apr), #264 (Jul), and #266 (Oct) of 2014. Seawater (ca. 2 L) was filtered through 0.02 µm pore-

sized aluminum oxide filters (Whatman Anotop, Sigma Aldrich, Saint Louis, MO, USA) and

stored at -80°C. Genomic DNA of both grazer cultures and environmental samples were

extracted (MasterPure Complete DNA and RNA Purification Kit; Epicentre) according to [39].

Four replicated PCR reactions (10 µL) were carried out for each sample except for Triparma

(duplicates) and consisted of 5 µl of 2× PowerTrack SYBR Green Master Mix (Thermo Fisher

Scientific, USA), 10 ng environmental DNA, 500 nM of each primer, and nuclease-free water.

Reactions were run on an Eppendorf Mastercycler epgradient S realplex2 real-time PCR

instrument. Each run contained fresh serial dilutions (1–6 log gene copies µL-1) of target-specific,

750-bp synthetic standards (gBlocks, IDT) prepared in triplicate. The cycling program included

an initial denaturation step of 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 55 °C

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for 30 s. Specificity of amplification was checked with a melting curve run immediately after the

PCR program and gel electrophoresis. Amplification efficiencies ranged from 97% to 104% for

all the primers.

To allow conversion from gene copies to cell numbers, the 18S rDNA gene copy number

per cell was determined for the seven targeted clades. Known amounts of cultured cells (106–107

cells) were pelleted at 4,000 RCF for 15 min at 4 °C, DNA was extracted, and the gene copy

numbers were determined with the group-specific primers. Cell abundances in both original

cultures and supernatants were monitored by flow cytometry to account for losses. Various

isolates from each group were selected for this purpose and a mean cellular 18S rDNA gene copy

number was calculated for each genus. To estimate contributions of each isolate to the local

community of small phytoplankton at Station ALOHA, calculated in situ cell abundances were

compared to counts of photosynthetic picoeukaryotes from the Hawaiʻi Ocean Time-series

(https://hahana.soest.hawaii.edu/hot/hot-dogs/).

Global distribution revealed through Tara Oceans 18S rDNA metabarcodes

To estimate the relative abundance of the OTUs closely related to our diverse isolates on a

broader geographic scale, we searched the 18S rDNA-V9 sequence data from the 0.8–5 µm

fraction of surface water sampled at 40 stations by the Tara Oceans project (http://taraoceans.sb-

roscoff.fr/EukDiv/). Reads for a ‘Tara lineages’ with highest similarity (E-value < 10-15) to each

of our targeted clades (Supplementary Table S1) were expressed as a fraction of total reads

excluding dinoflagellates but included all other Tara Oceans phytoplankton ‘taxogroups’:

Bacillariophyta, Bolidophyceae, Chlorarachnea, Chlorophyceae, Chrysophyceae/Synurophyceae,

Cryptophyta, Dictyochophyceae, Euglenida, Glaucocystophyta, Haptophyta, Mamiellophyceae,

Other Archaeplastida, Other Chlorophyta, Pelagophyceae, Phaeophyceae, Pinguiophyceae,

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Prasino-Clade-7, Pyramimonadales, Raphidophyceae, Rhodophyta and Trebouxiophyceae.

Dinoflagellates were excluded because of the difficulty in assigning phototrophic vs.

heterotrophic status to all taxa, and because nearly all dinoflagellate reads were from a single,

poorly annotated OTU that was also highly abundant in larger size fractions.

Results

Phylogenetic diversity

Diverse Prochlorococcus-consuming mixotrophic flagellates ranging in size from 2–5 µm

were isolated from oligotrophic, open-ocean waters of the NPSG (Table 1). The isolates include

species from nine genera (or approximately genus-level clades, referred to hereafter as genera for

brevity) and six classes (Fig. 1a; Supplementary Table S1): one Pelagodinium isolate in class

Dinophyceae; one isolate in an undescribed clade within class Chlorarachniophyceae (hereafter

referred to as ChloraX); two Chrysochromulina isolates and one environmental HAP-2 clade

isolate (hereafter Hap2) [40] in class Haptophyceae; three environmental clade H isolates in class

Chrysophyceae (hereafter ChrysoH) [41]; two Triparma isolates in class Bolidophyceae; and

twenty-nine isolates in class Dictyochophyceae: nineteen Florenciella isolates, four

Rhizochromulina isolates, and six isolates in an undescribed clade (hereafter DictyX). The

Florenciella isolates cluster with two cultivated strains (Florenciella parvula, GenBank Acc.

AY254857; Florenciellales sp. NIES 1871, GenBank Acc. AB518483) and one environmental

sequence (GenBank Acc. AY129059). The three chrysophyte isolates are closely related to an

environmental Chrysophyceae clade H sequence (GenBank Acc. EF172998). The closest

relatives of the two Triparma isolates are Triparma eleuthera and Triparma mediterranea, and

the two Chrysochromulina isolates are most closely related to an environmental

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Chrysochromulina (GenBank Acc. AF107083) and Chrysochromulina sp. RCC400 (GenBank

Acc. KT861300), respectively. All isolates showed evidence of phagocytosis (e.g., Fig. 1b) and

maintained permanent chloroplasts.

a b Engelmanniella mobilis macronuclear AF164134 ciliate outgroup Strombidinopsis acuminata FJ790209 (Spirotrichea) Pelagodinium sp EF492490 0.8 Pelagodinium beii strain RCC1491 KF422623 Pelagodinium Hap2 Pelagodinium beii strain clone 7963 JF791066 0.8 UHM4000 (Dinophyceae) 0.9 Pelagodinium beii Globigerinoides U37365 Minorisa minuta clone SSRPE06 EF173002 UHM2000 ChloraX Gymnochlora stellata AF076171 Chlorarachnion reptans CCMP238 U03477 (Chlorarachniophyceae) 0.7 Chlorarachnida XX sp UEPAC33p4 DQ369018 Uncult Euk clone Biosope T65 100FJ537342 Uncult haptophyte clone Biosope T58 080 FJ537336 Hap2 (Haptophyceae) UHM4150 Chrysochromulina sp clone N10E02 EF172967 Chrysochromulina Chrysochromulina sp AF107083 Tree scale: 0.1 UHM4110 Chrysochromulina throndsenii L12 AJ246277 Chrysochromulina acantha UIO024 AJ246278 Chrysochromulina Chrysochromulina sp MBIC10513 AB199882 Chrysochromulina sp AF166376 0.9 Chrysochromulina sp RCC400 KT861300 UHM4100 Chrysophyceae Clade-H X sp clone UEPAC48p3 AY129063 Chrysophyceae Clade-H X sp clone Q2B03N10 EF172974 ChrysoH UHM3502 UHM3501 (Chrysophyceae) ChrysoH UHM3500 Chrysophyceae Clade-H X sp clone SSRPE02 EF172998 Triparma mediterranea RCC238 AF123596 UHM3600 Triparma Triparma pacifica clone OLI51104 AF167157 Triparma laevis clone DH114 3A83 FJ032653 (Bolidophyceae) Triparma Triparma eleuthera RCC212 AF167155 UHM3610 UHM3060 UHM3051 UHM3050 DictyX UHM3054 DictyX 0.9 UHM3053 0.9 UHM3052 Rhizochromulina marina U14388 Rhizochromulina sp AJ402337 UHM3073 UHM3071 Rhizochromulina UHM3072 (Dictyochophyceae) UHM3070 UHM3011 Rhizochromulina Florenciellales NIES1871 AB518483 UHM3012 UHM3010 UHM3002 UHM3005 UHM3004 0.9 UHM3003 UHM3001 UHM3000 Florenciella parvula AY254857 UHM3022 Florenciella Florenciellales X sp AY129059 Florenciella UHM3028 UHM3029 UHM3024 UHM3023 UHM3021 UHM3026 UHM3027 UHM3025 5 µm Fig. 1 UHM3020 Fig. 1. Phylogenetic diversity among the isolates and visual evidence of phagotrophy. (a) A

near-full length 18S rDNA-based Bayesian majority consensus tree, showing the phylogenetic

positions of 39 mixotrophic grazers isolated from the NPSG (in bold). Genera are shaded with

different colors and labeled with genus and class (in parentheses) names, and the six grazers

investigated for grazing functional responses are marked with black diamonds. Most branches

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have complete support (posterior probability = 1.0) and only those with lower support (<1.0) are

labeled with support values. The scale bar indicates 10% divergence. (b) Digital micrographs of

grazers consuming 0.5-µm fluorescent beads were captured using transmitted light and

epifluorescence microscopy. The scale bar is 5 µm.

Grazing capability and growth efficiency

All 39 isolates were confirmed to consume Prochlorococcus and grew when

Prochlorococcus was the sole added prey and primary source of nitrogen. Within a five-day time

course, consumption of 1–2×106 Prochlorococcus mL-1 supported grazer growth of 103–104 cells

mL-1 for the different isolates (Supplementary Fig. S2). Grazing rates varied across phylogenetic

groups, with >10-fold and ~6-fold variation in both clearance rate (volume cleared per cell) and

specific clearance rate (volume cleared per body volume) across species and genera, respectively

(Fig. 2a–b). Isolate UHM3050 of clade DictyX possessed the highest raw clearance rate of ~10

nL grazer-1 h-1 and strain UHM3501 of clade ChrysoH displayed the highest specific clearance

rate of ~5×105 body volumes grazer-1 h-1. On the genus level, ChrysoH also had the highest

specific clearance rate (3.6×105 body volumes grazer-1 h-1), followed by three clades of Triparma,

Rhizochromulina, and DictyX (1.6–1.9×105 body volumes grazer-1 h-1) and Chrysochromulina

(1.2×105 body volumes grazer-1 h-1). Florenciella are among the lowest in terms of both raw and

specific clearance rates (1.2 nL grazer-1 h-1, 0.5×105 body volumes grazer-1 h-1). The remaining

flagellates showed clearance rates in between these extremes, with significant variation in rates

-4 2 explained by genus (ANOVA on log-transformed clearance rates: F9,20 = 8.8, p < 10 , R = 0.80;

-4 2 ANOVA on log-transformed specific clearance rates: F9,20 = 7.6, p < 10 , R = 0.77).

Biovolume conversion efficiency also varied among genera, with the highest mean value

observed for Florenciella (2.7), followed by the other two dictyochophyte clades,

15 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a Rhizochromulina (1.9) and DictyX (1.4). The 12

) lowest efficiencies were exhibited by the two -1

h 8 -1 6 Triparma (0.5), three ChrysoH (0.7) and three 4 Clearance rate (nL grazer (nL 2 haptophyte isolates (0.8) (Fig. 2c). Strains

0 with higher specific clearance rates tended to b )

-1 5 h

-1 have lower growth efficiencies (r = -0.64, p < 4 -3 3 10 ; Fig. 2d). There was substantial variation

2 in these traits even within the 1 body volumes grazer Specific clearance rate 5 0 Dictyochophyceae, with the highest (10 c 5 conversion efficiencies seen in Florenciella

4 strains that possessed the lowest clearance 3 olume

v rates (e.g., UHM3029 and UHM3023), and

o 2 Bi ersion efficiency v n 1 co some of the lowest conversion efficiencies 0 found in strains with higher clearance rates ma aX r r ulina ysoH ulina r Hap2 m DictyX ipa m r Chlo T Ch Florenciella (e.g., Rhizochromulina UHM3071 and ochro elagodinium z P ysochro r Rhi Ch d DictyX UHM3052). 5 Dictyochophyceae Bolidophyceae 4 Chrysophyceae Haptophyceae Chlorarachniophyceae 3 Dinophyceae

olume v

o 2 Bi ersion efficiency v n 1 co

0 1 2 3 4 5 Specific clearance rate 5 -1 -1 Fig. 2 (10 body volumes grazer h ) Fig. 2. Comparison of grazing rates and growth efficiencies among NPSG mixotrophs. Panels

show (a) clearance rates or (b) specific clearance rates among isolates in different genera and

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biovolume conversion efficiencies for isolates (c) grouped by genus or (d) as a function of

specific clearance rates. Individual classes were denoted with different colors and symbols in all

four panels (legend in panel d), and genera that have multiple strains included an arithmetic

mean shown in black asterisk. Note that for some isolates (marked in bold in Supplementary

Table S1) replicated grazing experiments were conducted and the means are presented in this

figure.

Functional responses

Functional responses were estimated for six strains representing different classes and genera

(Fig. 3; strains denoted in Table 1 and Fig. 1). Maximum ingestion rates (Imax) ranged from 2

cells grazer-1 h-1 (ChrysoH) to 92 cells grazer-1 h-1 (Hap2), with varying prey saturation

concentrations between 105 cells mL-1 for ChrysoH, 106 cells mL-1 for Rhizochromulina and 107

-1 cells mL for the remaining strains (Fig. 3). Specific Imax (volume ingested per body volume)

ranged from 0.04 body volumes grazer-1 h-1 (ChrysoH) to 0.43 body volumes grazer-1 h-1

5 -1 (Chrysochromulina), respectively and specific Cmax ranged from 0.9×10 body volumes grazer

h-1 (Florenciella) to 1.9×106 body volumes grazer-1 h-1 (ChrysoH) (Supplementary Table S3).

Substantially higher growth of grazers and faster removal of prey were supported by higher

Prochlorococcus densities (e.g., 5×106–5×107 cells mL-1), compared to treatments with lower

Prochlorococcus densities (< 1×106 cells mL-1) (Supplementary Fig. S3).

17 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a b c ) ) 2 ) 2 2 -1 -1 -1 10 Florenciella 10 Rhizochromulina 10 DictyX

h h h -1 -1 -1

10 10 10

1 1 1

10-1 10-1 10-1 Ingestion (cells grazer Ingestion (cells grazer Ingestion (cells grazer

104 105 106 107 104 105 106 107 104 105 106 107 Prochlorococcus (cells mL-1) Prochlorococcus (cells mL-1) Prochlorococcus (cells mL-1) d e f ) ) ) 2 2 2 -1 -1 -1

10 ChrysoH 10 Chrysochromulina 10 Hap2

h h h -1 -1 -1

10 10 10

1 1 1

10-1 10-1 10-1 Ingestion (cells grazer Ingestion (cells grazer Ingestion (cells grazer

104 105 106 107 104 105 106 107 104 105 106 107 Fig. 3 Prochlorococcus (cells mL-1) Prochlorococcus (cells mL-1) Prochlorococcus (cells mL-1)

Fig. 3. Functional responses of six representative grazers from different genera. Ingestion rates

of Prochlorococcus are plotted as a function of prey concentration. Holling type II curves were

fitted to the data to estimate Cmax (maximum clearance rate, the initial slope of the curve) and

Imax (maximum ingestion rate, the asymptote of the curve). Grazer and prey trajectories from

experiments used to estimate functional responses are shown in Supplementary Fig. S3.

The body volume-specific Cmax and Imax of our isolates cover nearly the whole range of

values previously reported in the literature for nano-sized heterotrophic and mixotrophic

flagellates, which are from <104 to >106 body volumes grazer-1 h-1, and <0.1 to >1 body volumes

grazer-1 h-1, respectively (Fig. 4; Supplementary Table S3). It is noteworthy that the ChrysoH

strain studied here, which is the smallest of our isolates (2–3 µm), demonstrated a higher specific

Cmax than any protistan grazer studied to date. For the one isolate previously studied and re-

examined here (Florenciella UHM 3021), grazing rates were somewhat faster than previously

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measured, but both prior and current Cmax estimates for this strain are substantially lower than the

other five isolates analyzed (Supplementary Table S3), consistent with the general pattern of low

clearance rates for this genus (Fig. 2).

a b

) Chrys 1 − 10 ) Dicty 1 h 1

− Hapt 1 h −

r HNF 1 − e r z e x a x a z r a m a g m

1 r I C

s g 0.1

c e c i s i f f i m i e c c u l e m e o p u p l v s

s o y

0.1 v

d y

o 0.01

d Euk b

5 HB b

( this study Pro 10 ( 0.01 literature Syn

3 5 8 15 3 5 8 15 grazer ESD (µm) grazer ESD (µm)

Fig. 4. Grazing rate parameters as a function of size (equivalent spherical diameter, or ESD) for

heterotrophic (HNF) and mixotrophic nanoflagellates that have been studied in culture. (a)

Specific Cmax (maximum volume cleared per body volume) and (b) specific Imax (maximum cell

volume ingested per body volume), for flagellates grazing on Prochlorococcus (Pro),

Synechococcus (Syn), heterotrophic bacteria (HB), or eukaryotic prey (Euk). In both panels

(legend in panel b), grey-filled symbols indicate HNF grazers, and other color-filled symbols

indicate mixotrophic grazers (both from this study and literature), including chrysophytes

(Chrys), dictyochophytes (Dicty) and haptophytes (Hapt). Symbol shapes indicate the type of

prey (source data in Supplementary Table S3). Large symbols indicate data from this study.

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Abundance in the field

a 6

5 Estimates of 18S rDNA gene copy numbers -1 4 indicate that all isolates tend to have low copy numbers

3 per cell, with approximately one copy cell-1 for 2 Gene copies cell ChrysoH and Triparma, one to three copies cell-1 for 1

-1 0 Florenciella and Rhizochromulina, two copies cell for

na i lina u Hap2 -1 DictyX Chrysochromulina, three copies cell for Hap2, and Triparma ChrysoH Florenciella

Rhizochromul -1 b Chrysochrom five copies cell for DictyX (Fig. 5a). 18S rDNA gene 6 Florenciella Rhizochromulina DictyX 5 Triparma copy number correlated with cell size among these ChrysoH Chrysochromulina -1 4 Hap2 genus mean isolates either at the strain level (r = 0.78, p < 0.001),

3 or when grouped by genus (r = 0.90, P < 0.01) (Fig. 2 Gene copies cell 5b). 1

0 2 3 4 5 6 Fig. 5 ESD (µm)

Fig. 5. 18S rDNA gene copy numbers per cell, shown for (a) each of seven genera or (b) as a

function of equivalent spherical diameter (ESD) of the cells. Each colored symbol in both panels

denotes the mean of a single isolate (averaged over triplicate measurements) and asterisk

symbols in panel (b) are mean values for each genus (averaged over isolates). Least-squares

linear regression line is plotted for isolate means (R2 = 0.61).

Average abundance of 18S rDNA gene copies in the euphotic zone at Sta. ALOHA varied

from 1×102 to 5.3×105 copies L-1 across groups, and estimated cell concentrations varied from

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1×102 to 2.5×105 cells L-1 (Fig. 6). All groups were more abundant at upper euphotic depths (5–

100 m), with abundance typically peaking at 45 m, except for Florenciella (75 m) and

Chrysochromulina (100 m). Chrysochromulina was most abundant at every depth, peaking at

5.3×105 copies L-1 (2.5×105 cells L-1) at 100 m. The Hap2 clade had the second highest gene

abundance (1.2×105 copies L-1 at 45 m), but estimated cell abundances of Hap2, Florenciella and

ChrysoH clades were all very similar with maxima of 3.3–3.6×104 cells L-1 at 45–75 m. Lower

abundances were seen for Triparma, Rhizochromulina, and DictyX clades, which had maxima

ranging between 1.1–1.5×104 copies L-1 (3×103 –1.3×104 cells L-1) at 45 m.

a b 0 0

50 50

100 100 Depth (m) Depth (m)

Florenciella 150 Rhizochromulina 150 DictyX Triparma ChrysoH Chrysochromulina 200 Hap2 200 Chrysochromulina Chrysochromulina 0 100 200 300 400 500 600 0 50 100 150 200 250 all others all others 0 20 40 60 80 100 120 0 10 20 30 40 50

Gene copies x 103 L-1 Cells x 103 L-1 Fig. 6

Fig. 6. Average in situ abundance as a function of depth of (a) 18S rDNA gene copies and (b)

the corresponding inferred cell abundances. Results are the averages from four depth profiles

(one collected in each of four seasons during 2014). Complete source data for each season shown

in Supplementary Fig. S4.

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Estimated contributions of each clade to the total photosynthetic picoeukaryotes in upper

euphotic depths (0–100 m) were 0.1–1% (DictyX, Rhizochromulina and Triparma), 1–4%

(ChrysoH, Florenciella and Hap2) and 9–23% (Chrysochromulina) (Supplementary Fig. S4a). In

total these targeted clades accounted for a relative abundance between 14%–31% in upper (0–

100 m) and 7–14% in lower euphotic depths (125–175 m), respectively. Group abundances

varied over time (Supplementary Fig. S4b–h), with DictyX (Supplementary Fig. S4d) and Hap2

(Supplementary Fig. S4g) clades more abundant during October, while Triparma

(Supplementary Fig. S4e) and ChrysoH (Supplementary Fig. S4f) clades were more abundant

during April .

Analysis of Tara Oceans OTUs closely related to our isolates indicates that these taxa are

widespread across surface ocean samples (Fig. 7 a-c), with median relative abundances ranging

from < 0.1% to around 5% (Fig. 7d). Dictyochophyte and haptophyte each constituted 10-25% of

the community at over 10 stations, and their most abundant groups of Florenciella parvula and

Chrysochromulina_X sp. alone accounted for > 10% at 5 stations (Fig. 7 a-b). In total the focal

OTUs accounted for 5–36% (median 15%) of non-dinoflagellate phytoplankton in the 0.8–5 µm

size fraction (Fig. 7d).

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a 25% 10% 1%

Florenciella parvula Florenciellales_X sp. Rhizochromulina marina Dichtyochales_X sp. b

Chrysochromulina_X sp. Chrysochromulina Prymnesiophyceae_XXX sp. c

Bolidomonas mediterranea Clade-H_X sp. d Chlorarachnida_X 35

15 10

Relative 5 1 abundance (%)

Clade-H_X sp. Summed OTUs Chrysochromulina Chlorarachnida_X Florenciella parvula Florenciellales_X sp. Dichtyochales_X sp. Rhizochromulina marinaChrysochromulina_X sp. Fig. 7 Bolidomonas mediterranea Prymnesiophyceae_XXX sp.

Fig. 7. Global patterns of Tara Oceans OTUs closely related to our mixotroph isolates. Relative

abundances of ten OTUs were grouped into (a) Dictyochophyceae, (b) Haptophyceae, and (c)

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others, and are represented with pie charts at each station or (d) presented as box and whisker

plots of the percent contributions for each targeted OTU (n = 40). Outliers determined by the

1.5×IQR rule are shown as filled circles. Pie chart areas are proportional to OTU relative

abundances expressed as a percentage of total phytoplankton OTUs (excluding dinoflagellates)

in the 0.8-5 µm size fraction. OTUs are labeled with their lowest-level taxonomic annotations

from the Tara Oceans dataset (full Tara lineages, numeric IDs, and corresponding isolates are

presented in Supplementary Table S1).

Discussion

Phylogenetically diverse and globally common mixotrophs

Marine protists are incredibly diverse and much of this diversity remains uncultured, even

for the relatively well-studied phytoplankton, which makes it challenging to interpret the

functional significance of environmental sequence data. The flagellates isolated and

characterized in this study appear to be the only mixotrophs for which consumption of

Prochlorococcus has been confirmed and quantified in culture-based lab experiments.

Quantitative data on the grazers of Prochlorococcus is important because these cyanobacteria are

major primary producers in oligotrophic waters and the most abundant autotroph on a global

scale [13][42]. Among the mixotrophic flagellates described here are representatives of four

genera (DictyX, ChrysoH, Hap2, ChloraX) from which no cultivated representatives have

previously been reported, but which appear frequently in molecular surveys. We have quantified

grazing behavior in these isolates, as well as isolates from three described genera

(Rhizochromulina, Triparma, and Pelagodinium) that had not previously been documented to

consume prey. We have also studied isolates of Chrysochromulina from the oligotrophic open

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ocean, whereas previous isolates were mostly from productive coastal environments and algal

bloom events. Our isolates are smaller than most previously studied mixotrophs, perhaps

reflecting their origins in the subtropical oligotrophic open ocean (Supplementary Table S4).

Comparison of our isolates to environmental sampling efforts suggests that

Prochlorococcus-consuming mixotrophs are common components of open ocean ecosystems,

with two genera of Florenciella and Chrysochromulina being ubiquitous in certain regions (Fig.

7a-b). The aggregate relative abundance of OTUs related to our isolates in surface Tara Oceans

contributed up to 36% and the median abundance is similar to qPCR-based relative abundance of

the targeted clades at Sta. ALOHA, i.e., 15% vs. 14% (Supplementary Fig. S4a; Fig. 7). This

suggests that our isolates may represent the most abundant populations in these clades, and

further efforts to estimate the absolute abundance of these globally-common mixotrophs are

necessary.

A broad spectrum of trophic strategies

The NPSG is one of the most oligotrophic ecosystems in the global ocean, with persistently

depleted surface nutrients and relatively stable microbial distributions [44]. The dominant

eukaryotic phototrophs are small, and the 2–5 µm size class alone contributes approximately half

of eukaryotic phytoplankton biomass and about one-fifth of total phytoplankton biomass [22].

This community includes non-trivial populations of haptophytes, dinoflagellates,

dictyochophytes, chrysophytes, prasinophytes, bolidophytes, cryptophytes, and pelagophytes

[1][43]. Our results suggest that the high diversity of these organisms may be attributable in part

to a broad spectrum of trophic strategies, including autotrophy (e.g., immotile prasinophytes) and

a variety of mixotrophic strategies, illustrated by the large variation in grazing abilities among

our isolates. Differences among genera in the traits we have measured suggests that the

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functional capacity of these communities may be somewhat predictable based on taxonomic

information alone. We hypothesize that fast grazers such as the ChrysoH clade are relatively

heterotrophic mixotrophs, acquiring more energy from prey and less from photosynthesis

compared to slower grazers such as Florenciella. Although we do not have data on autotrophic

capacities, a spectrum of autotrophy vs. heterotrophy is consistent with a tradeoff between

clearance rate and biovolume conversion efficiency (Fig. 2d), because relatively autotrophic

mixotrophs may incorporate more prey biomass into grazer biomass than relatively heterotrophic

mixotrophs that respire more prey biomass for energy [45][46]. Therefore, retention vs.

remineralization of prey biomass may vary substantially across mixotrophs that co-occur in the

same ecosystem, and the consequences for aggregate nutrient cycling and trophic transfer

efficiency will depend on which physiologies are most abundant.

Maximum ingestion rates of the NPSG mixotrophs are intermediate or high compared to

previously studied mixotrophs including haptophytes and chrysophytes, but are generally lower

than most heterotrophs (Fig. 4). These results are consistent with the expectation that mixotrophs

are constrained in their phagotrophic ability by tradeoffs with functions such as photosynthesis

and nutrient uptake. At the same time, maximum clearance rates of the NPSG mixotrophs are

intermediate-to-high compared to heterotrophs, suggesting that the two traits (Imax and Cmax) may

be constrained by different mechanisms. Cmax quantifies grazing performance when grazing is

encounter-limited, and it may reflect swimming speed or feeding currents that determine

encounter rate, or the efficacy of prey capture per encounter [47]. In contrast, Imax may reflect the

rate of vacuole formation or other processes limiting the rate of digestion. Comparison of such

traits among diverse mixotrophs, as well as phototrophic traits, would illuminate the physical

basis of differences in grazing ability.

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In situ abundances and potential grazing impacts

At Station ALOHA, a typical abundance of small pigmented flagellates is ~1000–2000 cells

mL-1 [22][48], and many of these photosynthetic picoeukaryotes may be mixotrophic based on

RNA stable isotope labeling experiments [23]. However, counts of individual taxa of small

protists are scarce [49]. Our qPCR estimates show that the haptophyte genus Chrysochromulina

was the most abundant of the groups we targeted, and the targeted groups together accounted for

up to ~30% of the flow cytometric counts of total photosynthetic picoeukaryotes. A recent study

at this location using fluorescently labeled bacterial prey estimated mixotrophs at 5–25 m to be

~100–200 cells mL-1 [17]. This is comparable to the sum of our targeted mixotroph taxa, which

suggests the targeted groups could account for most of the mixotrophs. However, use of labeled

prey may underestimate the total grazer population and our assays did not target all potential

mixotrophs, so the contribution of mixotrophs to total photosynthetic eukaryotes is likely > 30%.

Based on a previous metabarcoding study at this location [43], the remaining small, pigmented

eukaryotes likely include presumed autotrophs (immotile prasinophytes, coccolithophores,

Pelagomonas), but also other presumed mixotrophs (dinoflagellates, cryptophytes, additional

clades within the classes that our mixotrophs belong to) that we did not target (Supplementary

Fig. S1).

When combining estimated clade abundances with the maximal clearance rates of our

isolates (2.7–16.6 nL flagellate-1 h-1), we predict that the targeted mixotrophs could consume up

to 15% of Prochlorococcus produced daily in upper euphotic depths (0–100 m) at Sta. ALOHA,

using Prochlorococcus production estimates at this site from [50]. If we assume that all

pigmented picoeukaryotes in this system are mixotrophs, and that they graze with a clearance

rate of 6.6 nL flagellate-1 h-1 (weighted average of different mixotroph groups), then grazing

27 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

mortality by mixotrophs would account for 26–50% of daily Prochlorococcus production in

upper euphotic depths. These results suggest bacterial removal through mixotrophic grazing is an

important process for mortality. However, the true grazing contribution from mixotrophs will

depend on the relative abundance of autotrophs vs. mixotrophs, a number that remains elusive, as

well as mixotroph community structure, as we have found that grazing abilities vary substantially

among taxa. Furthermore, to better understand mixotrophy in ocean ecosystems it will be

important to test how the prevalence of different trophic strategies is related to environmental

conditions, and whether this leads to predictable gradients in how mixotrophs affect productivity

and nutrient cycling.

28 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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35 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Acknowledgments

This work was supported by National Science Foundation awards OCE 15-59356 and OIA 17-

36030, and a Simons Foundation Investigator Award in Marine Microbial Ecology and

Evolution (to K.F.E.). We thank the personnel in the Hawai’i Ocean Time-series program (NSF

award 12-60164) for assistance with water sampling. We thank Dr. Sallie W. Chisholm

(Massachusetts Institute of Technology) for providing the Prochlorococcus strains. We are also

grateful to Tina M. Weatherby at the Biological Electron Microscope Facility of the University

of Hawaiʻi at Manoa (UHM), as well as Dr. Karen E. Selph at the School of Ocean and Earth

Science and Technology of UHM for their assistance with microscopic imaging and flow

cytometry measurements.

Competing interests

All authors declare that they have no competing interests related to this work.

36 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

a Tree scale: 0.1

EU500104

1.0 Ciliophrys

0.5 1.0 1.0 Pedinellales AJ402337|Rhizochromulina_sp.|strain|NA|clone|NA 0.5 U14388|Rhizochromulina_marina|strain|NA|clone|NA 1.0 EU500153 0.8 EU500152 0.9 UHM3071 Rhizochromulina primer UHM3070 UHM3072 0.8 UHM3073 EF695207 1.0 EU500123 EF695204 0.9 EF695205 0.8 EU500108 EF695189 EU500107 EU500129 1.0 0.8 EF695202 EF695208 1.0 EU500117 1.0 EU500118 EU500121 1.0 EU500161 FJ537336 EU500067 1.0 0.9 EU500069 1.0 EU500066 EU500073 0.5 EU500074 EU500072 EU500076 1 EU500075 2 Supplementary Fig. S1. (Continued on next page) 3

37 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

b Tree scale: 0.1 outgroup EU500064 UHM4150 1.0 'Uncult Euk clone FJ537336' Hap2 primer EU500065

38 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

39 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

0.9 Clade F

1.0 Clade D

0.7 Clade B2 0.5

1.0 Clade C

0.8 Clade E

0.7 1.0 Clade G

0.6

1.0 Clade B1 10 11 12 Supplementary Fig. S1. Phylogenetic analysis of the isolates used in this study. Bayesian 13 majority consensus trees (with posterior probability shown on major branches) were inferred 14 from partial 18S rDNA sequence alignments of our mixotroph isolates (in red), reference 15 sequences from the PR2 database, and environmental sequences of putative grazers identified by 16 Frias-Lopez et al. (2009) (in bold) for (a) Dictyochophyceae, (b) Haptophyceae, (c) 17 Bolidophyceae and (d) Chrysophyceae. Six primer pairs theoretically target the majority, if not 18 all grazers in the genera (or genus-level clades) Florenciella, DictyX, Rhizochromulina, Hap2, 19 Chrysochromulina and ChrysoH. The Triparma primers target only some Triparma species, 20 including T. mediterranea and T. eleuthera. The scale bar indicates the fraction of divergence.

40 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

AL-45-004C AL-FL05 AL-TEMP-12 NF-H275-5m<3 PX-310-100-02 PX-310-100-03 PX-310-100-05 PX-310-100-07

2.0 1.5 1.0 0.5 0.0

PX-310-25-02 PX-310-25-03 PX-310-25-04 PX-310-25-06 PX-310-75-03 PX-310-75-05 PX-311-100-01 PX-311-100-02 )

1 2.0 − 1.5 1.0 0.5 0.0 cells mL cells

6 PX-311-100-03 PX-311-100-05 PX-311-25-01 PX-311-25-02 PX-311-25-03 PX-311-25-04 PX-311-25-05 PX-311-25-07

2.0 1.5 1.0 0.5 0.0 Prey (×10 Prey 0 50 100 150 200 PX-311-25-09 PX-311-25-11 PX-311-75-01 PX-311-75-04 PX-311-75-05 UA-265-01 UA-269-03

2.0 1.5 1.0 0.5 0.0 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 hour

AL-45-004C AL-FL05 AL-TEMP-12 NF-H275-5m<3 PX-310-100-02 PX-310-100-03 PX-310-100-05 PX-310-100-07

60 40 20 0

PX-310-25-02 PX-310-25-03 PX-310-25-04 PX-310-25-06 PX-310-75-03 PX-310-75-05 PX-311-100-01 PX-311-100-02 ) 1 − 60 40 20 0 cells mL cells 3 PX-311-100-03 PX-311-100-05 PX-311-25-01 PX-311-25-02 PX-311-25-03 PX-311-25-04 PX-311-25-05 PX-311-25-07

60 40 20 0

Grazer (×10 Grazer 0 50 100 150 200 PX-311-25-09 PX-311-25-11 PX-311-75-01 PX-311-75-04 PX-311-75-05 UA-265-01 UA-269-03

60 40 20 0 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 hour 21 Supplementary Fig. S2. Changes in abundance of prey (upper panels) and grazer (lower panels) 22 during grazing experiments. Prochlorococcus was the sole added prey and primary nitrogen 23 source in the experiments. In the two types of controls (prey with no grazers or grazers with no 24 prey) the prey and grazer abundances stayed roughly unchanged from day 1–5 at ~2×106 25 Prochlorococcus mL-1 or 100–1,000 grazers mL-1, respectively (data not shown). Replicated 26 experiments for 11 isolates (Supplementary Table S1) have mostly yielded similar results and 27 thus only data from one experiment each is shown here.

41 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

28 Supplementary Fig. S3. Grazer and prey trajectories (adjacent panels) of six isolates that were 29 investigated for functional responses. Symbol colors indicate the concentration of added 30 Prochlorococcus (cells mL-1) as shown in the legend.

42 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

Relative abundance (%) Florenciella (×103 cells per L) 0 5 10 15 20 25 0 20 40 60 80 100 0 0 a b 20 40 50 60 80 100 100 Depth(m) Depth(m) Florenciella 120 Rhizochromulina 140 150 DictyX H259-Jan Triparma 160 H262-Apr ChrysoH Hap2 180 H264-Jul Chrysochromulina H266-Oct 200 200 Rhizochromulina (×103 cells per L) DictyX (×103 cells per L) 0 10 20 30 0 5 10 15 0 0 c d 20 20 40 40 60 60 80 80 100 100 Depth(m) Depth(m) 120 120 140 140 160 160 180 180 200 200 Triparma (×103 cells per L) ChrysoH (×103 cells per L) 0 10 20 30 0 20 40 60 80 100 0 0 e f 20 20 40 40 60 60 80 80 100 100 Depth(m) Depth(m) 120 120 140 140 160 160 180 180 200 200 Hap2 (×103 cells per L) Chrysochromulina (×103 cells per L) 0 20 40 60 80 100 0 200 400 600 800 0 0 g h 20 20 40 40 60 60 80 80 100 100 Depth(m) 120 Depth(m) 120 140 140 160 160 180 180 31 200 200 32 Supplementary Fig. S4. Relative and absolute abundances of seven mixotroph populations as a 33 function of depth in the euphotic zone. (a) Annual average relative abundance as a function of 34 depth, calculated by dividing the average absolute abundance of each group by the average flow 35 cytometric counts of total pigmented eukaryotes over the same time period (data from at 36 https://hahana.soest.hawaii.edu/hot/hot-dogs/). (b–h) Variations in the abundance of seven 37 mixotrophic groups collected in four different seasons at Station ALOHA during Hawaii Ocean 38 Time-series cruises #259, #262, #264 and #266, which took place in Jan, Apr, Jul and Oct of 39 2014, respectively.

43 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

40 Supplementary Table S1. Taxonomy, culture IDs, and comparable Tara Oceans OTU information for isolates described in this study. Class Genus1 Species2 Strain3 Culture ID4 Tara lineage5 Tara ID6 Dictyochophyceae Florenciella F. parvula PX-310-100-01 UHM3000 Eukaryota|Stramenopiles|Stramenopiles_X| 146146 Dictyochophyceae|Dictyochophyceae_X|Florenciellales|F PX-310-100-02 UHM3001 lorenciella|Florenciella+parvula PX-310-100-03 UHM3002 PX-310-100-04 UHM3003 PX-310-100-05 UHM3004 PX-310-100-07 UHM3005 PX-311-25-03 UHM3010 PX-311-100-01 UHM3011 PX-311-100-03 UHM3012 sp. AL-45-004C UHM3020 Eukaryota|Stramenopiles|Stramenopiles_X| 73377 Dictyochophyceae|Dictyochophyceae_X|Florenciellales| UA-265-01 UHM3021 Florenciellales_X|Florenciellales_X+sp. PX-310-25-01 UHM3022 PX-310-25-04 UHM3023 PX-310-25-05 UHM3024 PX-310-75-01 UHM3025 PX-310-75-02 UHM3026 PX-310-75-03 UHM3027 PX-310-75-04 UHM3028 PX-310-75-05 UHM3029 Rhizochromulina sp. PX-311-25-09 UHM3070 Eukaryota|Stramenopiles|Stramenopiles_X| 67389 Dictyochophyceae|Dictyochophyceae_X| PX-311-75-01 UHM3071 Rhizochromulinales|Rhizochromulina| PX-311-75-04 UHM3072 Rhizochromulina+marina PX-311-75-05 UHM3073 DictyX sp. UA-269-03 UHM3050 Eukaryota|Stramenopiles|Stramenopiles_X| 46831 Dictyochophyceae|Dictyochophyceae_X|Dictyochales|Dic PX-310-25-02 UHM3051 tyochales_X|Dictyochales_X+sp. PX-310-25-03 UHM3052 PX-311-25-08 UHM3053 PX-311-100-05 UHM3054 PX-311-25-02 UHM3060

44 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456384; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

Dinophyceae Pelagodinium P. beii PX-311-25-11 UHM4000 NA NA 41 1 DictyX, Hap2, ChrysoH and ChloraX are abbreviations for undescribed or environmental approximately genus-level clades of Dictyochophyceae, 42 Haptophyceae, Chrysophyceae and Chlorarachniophyceae, respectively. 43 2 Species assignments are putative based high similarity of the near-full length 18S rDNA gene phylogeny of the isolate and known species 44 3 ‘Strain’ codes are the original unique identifiers used at the time of isolation. 45 4 Culture IDs are identifiers assigned to cultures of protists isolated by our group at University of Hawaiʻi at Mānoa (UHM####) that are reported in publications. 46 Grazing rates were measured at least once on all isolates and were measured in triplicate for the eleven isolates in bold font. Culture IDs in grey italic font were 47 found to graze on Prochlorococcus, but rates were not calculated, because of low sampling resolution. 48 5 ‘Tara lineage’ is the taxonomic annotation of the most similar OTU in the Tara dataset (E-value <10-15). 49 6 ‘Tara ID’ lists the identifier of the 18S rDNA V9 reference sequence of the OTU (http://taraoceans.sb-roscoff.fr/EukDiv/).

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Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

50 Supplementary Table S2. Detailed information of group-specific qPCR primers designed for targeting various mixotrophs in situ.

Clade or Genus Primer Sequence (5’–3’) Tm () Amplicon qPCR length (bp) efficiency Florenciella Florenciella-652F GGA TTT CTG GTG GGG CTG TC 58 132 1.04 Florenciella-784R GTA AAC GAC GAG CGT CAC TCC 58 Rhizochromulina Rhizochromulina-700F GTG TTG TCT CCG GTT CTA TTT CG 56 176 0.99 Rhizochromulina-876R GGC AGA ACC ACC AAA GTC CT 58 DictyX DictyX-1638F CGT CTT TCG TGA TAG GGA TAG 52 165 0.96 DictyX-1803R AAG CGG TAC TCC TCA TGT C 54 Chrysochromulina Chrysochromulina-291F TGC CGA TGG GTA CGC ACT G 60 95 0.97 Chrysochromulina-386R AGT AAA CGW TCC GTC TCC CGC C 61 Hap2 Hap2-291F TGC CGA TGG GTA TGC ACT G 58 95 0.99 Hap2-386R AGT AAA AGT CCT GTT TCC TAC GCC 57 ChrysoH ChrysoH-694F CAC CTC ACG GTG TCT GTA C 55 108 0.99 ChrysoH-802R AAT GAC GAG ATT CCC RAG C 54 Triparma Triparma-1632F AAC CTT GAC CGA GAG GTC TG 56 122 0.96 Triparma-1754R CAA TGC AGT CTG ATG AAC TGC 54

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Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

51 Supplementary Table S3. Maximum clearance rate and maximum ingestion rate parameters for 52 heterotrophic and mixotrophic nanoflagellates†. Species or Genus or Clade V I C Nutrition Prey Reference Culture ID max max Chrysochromulina ericina 108 0.02 0.06 mix euk [1] Chrysochromulina polylepis 2680 NA 0.04 mix hb [2] Paraphysomonas sp. 113 NA 0.27 het pro [3] Paraphysomonas sp. 113 0.07 1.99 het syn [3] Prymnesium patellliferum 275 0 0.01 mix hb [4] Pseudobodo sp. 14 NA 0.99 het pro [5] Pseudobodo sp. 14 0.15 5.08 het syn [5] Actinomonas mirabilis 75 0.86 11.0 het hb [6] Bodo designis 54 1.99 8.70 het hb [6] Ciliophrys infusionum 220 0.79 1.98 het hb [6] Codosiga gracilis 35 0.69 0.45 het hb [6] Diaphanoeca grandis 40 0.33 2.30 het hb [6] Jakoba libera 75 0.05 0.14 het hb [6] Monosiga sp. 20 0.81 0.98 het hb [6] Ochromonas sp. 50 0.42 1.70 mix hb [6] Ochromonas sp. 200 0.57 0.52 mix hb [6] Paraphysomonas imperforata 212 0.2 2.97 het hb [6] Paraphysomonas vestita 190 0.8 0.91 het hb [6] Pleoromonas jaculans 50 0.65 0.55 het hb [6] Pseudobodo tremulans 90 0.56 1.10 het hb [6] Pseudobodo sp. 34 0.41 3.90 het hb [6] Pseudobodo sp. 22 0.29 0.28 het euk [6] Spurnella sp. 65 0.09 0.25 het hb [6] Stephanoeca diplocostata 20 0.30 1.60 het hb [6] Stephanoeca diplocostata 83 0.30 4.58 het hb [6] Florenciella UHM3021 29 0.04 0.32 mix pro [7] Florenciella UHM3021 29 0.17 1.62 mix syn [7] Florenciella UHM3021 29 0.12 0.90 mix pro this study Rhizochromulina UHM3071 14 0.09 7.10 mix pro this study DictyX UHM3050 76 0.06 2.20 mix pro this study Chrysochromulina UHM4110 29 0.43 2.09 mix pro this study Hap2 UHM4150 48 0.27 1.70 mix pro this study ChrysoH UHM3501 7 0.04 18.5 mix pro this study † 53 Values were collected from published studies and this study (in bold). Volume (V) = grazer body volume in µm3, -1 -1 54 Imax = specific maximum ingestion rate (body volumes consumed grazer h ), Cmax = specific maximum clearance 55 rate (×105 body volumes cleared grazer-1 h-1), nutrition = mixotroph (mix) or heterotroph (het), prey = heterotrophic 56 bacteria (hb) or Prochlorococcus (pro) or Synechococcus (syn) or eukaryotic alga (euk). In some cases, Cmax and Imax 57 were not reported in the original study, and we estimated them by fitting a type II functional response to reported 58 data.

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Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

59 Supplementary Table S4. Nano-sized, non-dinoflagellate marine eukaryotes reported to have phago-mixotrophic capability†. Class Genus Size Primary evidence of mixotrophy Original (µm) publication Chlorarachniophyceae Cryptochlora >10 ingestion of eukaryotic prey [11] Lotharella >5 presence of food vacuole [12] ChloraX <5 consumption and growth on Prochlorococcus this study Chrysophyceae Ochromonas >5 ingestion of bacteria or FLB e.g. [13][14] Dinobryon >3 ingestion of latex-beads or FLB [15][16] ChrysoH ~2 consumption and growth on Prochlorococcus this study Cryptophyceae Geminigera >5 ingestion of eukaryotic prey or microsphere [17][18] Teleaulax >5 ingestion of bacteria and Synechococcus sp. [19] Haptophyceae Chrysochromulina >3 ingestion of bacteria or other prey e.g. [20][21] this study Prymnesium >5 ingestion of bacteria or other prey e.g. [22][4] Haptolina >5 ingestion of bacteria or other prey [23][1] Hap2 ~5 consumption and growth on Prochlorococcus this study Prasinophyceae Pyramimonas >10 ingestion of FLB, microspheres or live bacteria e.g. [24][18][25] Mantoniella <5 ingestion of microspheres or FLB [18][26] Cymbomonas >10 ingestion of live bacteria [27][25] Nephroselmis ~5 ingestion of live bacteria [9][25] Dolichomastix ~5 ingestion of live bacteria [25] Raphidophyceae Heterosigma >10 ingestion of Synechococcus [28] Dictyochophyceae Florenciella <5 consumption and growth on Prochlorococcu, Synechococcus and bacteria [7]; this study Rhizochromulina ~3 consumption and growth on Prochlorococcus this study DictyX 3-5 consumption and growth on Prochlorococcus this study Bolidophyceae Triparma ~3 consumption and growth on Prochlorococcus this study † 60 Taxa are mainly drawn from references [6][8][9] with original source references indicated. Specifically excluded are species that were isolated from benthic 61 rocks or freshwaters, are larger than 20 µm, or for which evidence of bacterivory has been disputed (Micromonas, [10]).

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Supplementary Material for Li et al., Broad phylogenetic and functional diversity among mixotrophic consumers of Prochlorococcus

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