The Windblown: Possible Explanations for Dinophyte DNA in Forest Soils

Journal of Eukaryotic Microbiology ISSN 1066-5234

SHORT COMMUNICATION The Windblown: Possible Explanations for Dinophyte DNA in Forest Soils

Marc Gottschlinga , Lucas Czechb,c ,Fred eric Mahed,e , Sina Adlf & Micah Dunthorn*g,h a Department Biologie, Systematische Botanik und Mykologie, GeoBio-Center, Ludwig-Maximilians-Universitat€ Munchen,€ Munich, D-80638, Germany b Computational Molecular Evolution Group, Heidelberg Institute for Theoretical Studies, Heidelberg, D-69118, Germany c Department of Plant Biology, Carnegie Institution for Science, Stanford, California, 94305, USA d CIRAD, UMR BGPI, Montpellier, F-34398, France e BGPI, Universite de Montpellier, CIRAD, IRD, Montpellier SupAgro, , Montpellier, France f Department of Soil Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, SK, S7N 5A8, Canada g Eukaryotic Microbiology, Faculty of Biology, Universitat€ Duisburg-Essen, Essen, D-45141, Germany h Centre for Water and Environmental Research (ZWU), Universitat€ Duisburg-Essen, Essen, D-45141, Germany

Keywords ABSTRACT Biodiversity; biogeography; dinoﬂagellates; environmental DNA; phylogeney. Dinophytes are widely distributed in marine- and fresh-waters, but have yet to be conclusively documented in terrestrial environments. Here, we evaluated *Correspondence the presence of these protists from an environmental DNA metabarcoding M. Dunthorn, Eukaryotic Microbiology, Fac- dataset of Neotropical rainforest soils. Using a phylogenetic placement ulty of Biology, Universitat€ Duisburg-Essen, approach with a reference alignment and tree, we showed that the numerous Universitatsstrasse€ 5, D-45141 Essen, Ger- sequencing reads that were phylogenetically placed as dinophytes did not cor- many relate with taxonomic assignment, environmental preference, nutritional mode, Telephone number: +49-(0)-201-183-2453; or dormancy. All the dinophytes in the soils are rather windblown dispersal FAX number: +49 (0)201 183 3768 units of aquatic species and are not biologically active residents of terrestrial e-mail: [email protected] environments.

Received: 23 July 2020; revised 12 October 2020; accepted October 28, 2020. Early View publication 10 December, 2020 doi:10.1111/jeu.12833

ENVIRONMENTAL high-throughput sequencing (HTS) et al. 2020; Decelle et al. 2018; Elferink et al. 2017; Giner studies of protists have now been performed for over a et al. 2020; Gottschling et al. 2020; Le Bescot et al. 2016; decade (Santoferrara et al. 2020). During that time, a large Lentendu et al. 2018; de Vargas et al. 2015). HTS studies diversity of dinophyte DNA sequences has been uncov- have also detected DNA of dinophytes in terrestrial envi- ered. Dinophytes are an ecologically and economically ronments (Bates et al. 2013; Geisen et al. 2015; Mahe important group of protists that exhibit many types of life et al. 2017; Venter et al. 2017; Voss et al. 2019), although styles and nutritional modes, including phototrophic, mixo- they are not expected to be there. trophic, and heterotrophic forms as well as some being Aquatic protists can sometimes be detected in terres- parasitic (Saldarriaga and Taylor 2017). All known dino- trial environments, notably riparian soils, such as foramini- phytes are from marine or freshwater environments (Adl fera (Lejzerowicz et al. 2010; Meisterfeld et al. 2001) and et al. 2019), except maybe the unarmored (athecate) possibly haptophytes (Mahe et al. 2017). However, that Esoptrodinium (Fawcett and Parrow 2014). As dinophytes does not mean that the normally aquatic protists are bio- constitute a considerable fraction of the plankton and play logically active in soils or other drier environments (Geisen an important role in the global aquatic ecosystem, HTS et al. 2018). In the absence of observing putative soil dino- studies have detected their DNA from waters sampled phytes using direct microscopic observations, here we from the polar regions through to the tropics (Annenkova used Mahe et al.’s (2017) metabarcoding data from three

© 2020 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists Journal of Eukaryotic Microbiology 2021, 68, e12833 1of6 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Dinophytes in Soils Gottschling et al. lowland Neotropical rainforest soils to ask whether the workflow are published in a GitHub code repository presence of dinophytes in those soils correlates with taxo- (https://github.com/lczech/dinoflagellate-paper). nomic assignment, environmental preference, nutritional mode, or dormancy. RESULTS AND DISCUSSION For the dinophyte reference alignment and tree, we MATERIALS AND METHODS included a broad representative taxon sample covering the known DNA sequence diversity with comprehensive Environmental sampling and data sequence information, similar to previous strategies Sampling and sequencing of tropical soils originally took (Chacon and Gottschling 2020; Gottschling et al. 2012, place in lowland rainforest in Costa Rica, Panama, and 2020; Gu et al. 2013). The alignment was 7,270 bp long Ecuador (Mahe et al. 2017). While the methodologies and and had 3,753 parsimony informative sites (52%, 15.7 per bioinformatics can be found in detail in Mahe et al. (2017), terminal taxon). The ML tree had many bipartitions with here in brief: the extracted soil DNAs were amplified for high if not maximal bootstrap values. The Dinophyceae the hypervariable V4 region of the SSU-rRNA locus using was inferred here to be monophyletic, and it contained general eukaryotic primers (Stoeck et al. 2010); this short the well-known subclades: Dinophysales, Gonyaulacales, region has relatively strong phylogenetic signal, although it Gymnodiniales, Peridiniales, Prorocentrales, †Suessiales is not as strong as the full-length SSU-rRNA (Dunthorn as well as Amphidomataceae, Brachydiniaceae, and Tovel- et al. 2014; Gottschling et al. 2020). Illumina sequencing liaceae. Only 207 OTUs from the Mahe et al.’s (2017) that reads were clustered into OTUs using swarm v2 (Mahe were assigned to the dinophytes, phylogenetically placed et al. 2015) and taxonomically assigned to the Protist Ribo- here across the reference tree with high likelihood weight somal Reference database v203 (Guillou et al. 2013) using scores (Fig. 1, File S5). VSEARCH v1.6.0 (Rognes et al. 2016). The 269 OTUs that There was no exclusive correlation with taxonomic were assigned to the dinophytes by Mahe et al. (2017) assignment. Some of the dinophyte OTUs formed distinct were extracted and used here for phylogenetic place- clades of sequences that were unknown until the present ments (File S1). study. However, the proportion of such undescribed diversity is low compared to other microbial lineages such as the Fungi (Bass et al. 2018; Jones et al. 2011). They Reference tree placed onto early branches, which comprise heterotrophic, From GenBank (Sayers et al. 2020), 228 ingroup dino- mainly parasitic species (Bachvaroff et al. 2012; Gomez phytes, plus 10 outgroups, were downloaded, then aligned et al. 2009; Gu et al. 2013; Saldarriaga et al. 2003), but with MAFFT v6.624b (Katoh and Standley 2013) using the also within the Peridiniales. Most of the OTUs, though, —auto option (File S2). Based on previous analyses placed within already known lineages of the Gymnodini- (Chacon and Gottschling 2020; Gottschling et al. 2012, aceae, Peridiniales, and †Suessiales, a truly heteroge- 2020; Zerdoner Calasan et al. 2019), the full sequences of neous set of dinophytes including unarmored and thecate each species were used without excluding ambiguously algae as well. There is no morphological trait that the aligned positions sites. Phylogenetic inferences of the ref- OTUs would therefore necessarily share and that would erence alignment were carried out by using Maximum unite them with these different taxa. Some thecate Likelihood (ML), as described in detail by Gottschling et al. groups such as Dinophysales, Gonyaulacales, Prorocen- (2012, 2020), as implemented RAxML v8.2.10 (Stamatakis trales, and Protoperidiniaceae (Peridiniales) did not include 2014) with the GTR + G substitution model. To determine any of the OTUs obtained from the environmental sam- the best-fitted ML tree, we executed 10-tree searches ples. from distinct random stepwise addition sequence Maxi- There was no exclusive correlation with habitat prefer- mum Parsimony starting trees and performed 1,000 non- ence. Some of the dinophyte OTUs placed onto branches parametric bootstrap replicates. Reference alignment and that contain freshwater species such as the Gymnodini- tree are available as Supporting Information (Files S3-S4). aceae (Kretschmann et al. 2015; Romeikat et al. 2020), Tovelliaceae (Lindberg et al. 2005), and peridinialean Naia- dinium comprising a freshwater lineage within otherwise Phylogenetic placement of environmental OTUs marine Scrippsiella s.l. (Kretschmann et al. 2014; Luo The OTU representative sequences obtained from Swarm et al. 2016). This placement of OTUs within freshwater were aligned against the reference alignment using clades was similar to what was shown for the haptophyte PaPaRa v2.0 (Berger and Stamatakis 2011) and phyloge- OTUs from the same rainforest soils (Mahe et al. 2017). netically placed onto the ML reference tree using the Evo- However, none of the OTUs placed with the Peridini- lutionary Placement Algorithm (Berger et al. 2011) as aceae, which is one of the most prominent freshwater implemented in RAxML. Next, all OTUs that were placed dinophyte lineages (Gottschling et al. 2020; Moestrup and with at least 95% probability (combined likelihood weight Calado 2018), but some OTUs placed on branches that ratios) in the dinophyte clade were extracted and visual- contain just marine species, such as the Amphidomat- ized, using Gappa v0.1.0 (Czech et al. 2020). For details aceae (Tillmann et al. 2014) and Brachydiniaceae (Berg- on the extraction, see Czech et al. (2018); details of the holtz et al. 2006; Henrichs et al. 2011). Groups such as

© 2020 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists 2of6 Journal of Eukaryotic Microbiology 2021, 68, e12833 Gottschling et al. Dinophytes in Soils

number of placed reads 10000 Protoperidiniaceae Blastodiniaceae Kryptoperidiniaceae Zooxanthellaceae Peridiniales Peridiniaceae

1000 Pent

“

Un Krypto A Ensiculif

Vulcano apharsodi m

†

E Arc

ruhd Amp Calcic

“ p

nsiculifera P

“

h P

i h A

peridinium o d

rid

Amp

inium a

t h

mphidiniopsi

o Is

Unruhdinium j e

era id

arpinum b in U p

di i p

Scrippsiell l

andinium

e in

e nruh iu

n p

Durins hidiniops

r er Peridiniopsidaceae ” cf. ri io

iu s i m

Zooxant m Huia

um dalei d

i A i

m rugo i P d

” ”

inim i

a minutum rchaeperidinium P n

Du din Blastod s P

r a c

Blas ff. i n er i

Scri is

im Peri u o erid

cf. me

Herdm Peri . u

t c

bo m idini kia oculat k

ium s m

um loe P

ariensis m a a

a ivalvum m

100 P P

p o

p in

t eri s r s spica rica

scquodinium r di

hella nutric rique t p is

”

e e P eridinium cinctu eridinium S re

psie a odin e

um bipes um iu

sum di

blichi a e

iulongens ni

Gloeodiniaceae ridin

fu n pe di

c f Hui r

er nutum

inium otundata ur

m b m

f. a

l w

Scr rippsi n

Thoracosphaeraceae QZ- a um

SC Peri n

u k n

Scrippsie Scri ani i

acuminata P n a

oensis r di

lla acuminata v um

i Per u

c i

n a

iu ci um

i trum c a

Sc c

a arv l

a ipes

i ium m

y CAP e ni i rvo

pps 2 i

i Peridi h

d gat rd u H

Scrippsie m dinium

a a

0 o

ppsi IFR10- n

el conto um wil um

r i

m o BI as

N 12 HBI:F di G i

ip i GeoB*230

s f

ii din

GeoB*220 A

ol di

i l m f

cras

ai unense

a e 0 ni is

ell e cf.

K-11 :

t ps Y

Geo nctum

i o SD2

orali n

b P ul so A 8

ad e oB 28 TY

“ iu -

um rma

Scr ell ma

Jiulo GeoB 459 n

collini Pa

Scrippsiell a swee 0 2 lla Y arv

i i um

i a c i

p m r iu

ella wille

in 0 u

# rot

-2

i at

027 a

a k

Scrippsi t

naceus M*66 sum

Chim e r le

rtum 1

1 0

PS r l m heca4

aff. 2 l

o m

ium polonicu i o

00 v

oc cf

l e

NIE

pp 0

s 1

6 s

l sp. i 8 c

ngji g 1 r odin

B J20 dinium

a bi i

GeoB*20 4 1

. . -1 1 batum

lo 4

sp c

BSR JL01 cultatum

Thel imb

sie u

batum

Dinophysales

onodinium a ell_1

GeoM*778

bo S

n T m

ang 2 4 te

PGDA cumina C

“ . GeoB 1 Gl

c mar ium

Per

Sc K

e 5

L a

Geo VS 10 Palati lla b

ar C

ella l nn atu

su oeodini

eodini yae u

† a 32 007

GeoB 2 o

r A

” t

Calciodinip i 1 i

10 n

ra n G M

diniops C sp. m d

p 3 al

B m

psi at 427

e 9

1 1 1

01 w

r -

p † a n

eca oM*5 1

8 G

us api us

Pern ch m ta a 3 ls p

lomnic u in

el *

* c

m 0

um 35 N leno HY

m s

iniak pl

l 2

rymosa ST 99 C

a 83 I su

81 A

kii kii

ria ES495

GeoB CM2 Dinophysis

HBI:M i C N *

mon

” c JA

a * s

ellu 3 mph

-D Geo Sto d 2 alcispo *

bor

m d CMA2 AC0002

3 c

9 in

0 k ongha ii

bu 6 1 GeoM*753

ii 6

Ge 94 3

e m 80 * l isole

va atu M* tanum 1

gia G20 4 gei

94 G ke ope 0

o 11 * sis

27 eoM*70

r 7

. GeoB J 0 s

r 98

rum 76 6 tub

95 Din A nia *

ia wi i 082

e Dino

1 8 O 0 acuminat

d 6

r st 0 PB

n *

GeoM*

c 1 rnithocercus uncod e osum

2 ei

hangw *

rz er is b

3a ph

62 CCAC006

SK-A ide

2 p

ejs o *

S 9

M * 67 hy

54 ii

toe sa CS 85 ysis

P ntata

in sis Ph

k 762

5 69 *

o ii NIES

c - a

ium glandulu ca Borghiellaceae

Pfi SZN741 5 al

ker ne

Ge 7

6 9

7 c GeoM*7 1

est

8 ud D

nsis m

6 u

oB*6 4

i *

roma roma

a AR

min 59 1 L

eria shumwa agn

al 5 E392 His

Pf ta

Luciella masanensis g 5 U

1 a

-2013

SS ifi ici cf.

est t ta

15 Ph

oneis oneis

L cus F

uc da M

TL6 rotundatu

er S a l m 2 clone

T ie acr

Ty yran ia piscicida 080 *

l S 9 rann la CB sp.

y a1 97 72 o

a ma rapa ma

s 6 T

a C4L7 n ld

ogul

Am o o p. e Y i

nia a nia

din dinium D m

yloodini FTL

Borghi

Flori a

ium edax No j 6

ga-S 2

55 au

d 78 77

L12 a acty

ella dodgei ella

e a L C

Cryptheum ocellatum da NC

iensis

UTE C4L 1 ucy lo

x MA303 Borghiella Asulcocephalium miricentonis Asulcocephalium diniu

Apo H 6 *

FJ167681 9 X1

c BI

c o pterobelotu m

alathiu :H 5 56

Ap din 5 greenGS

B20 9 2

o iu 9 C †Suessiales

ca sp

m N 10 5 CAC007

lath m aci .

s ngd 10a *

89 *

ium p. Geo

cul e1 Yi 5 m

Fusiperidinium hel

m CA 4 M*5

ife

almog A 12 * yeosuensi la

† vnd 4

Posoni rum E-CL2 Polarell 2

iense 97 5

PA -

a glac a ella kt mi1 *

E S y

wi 1 mbiodinium mbiodinium

tri R1 s -8

c s SHTV1 ialis

† ari kt

Leonella con YY140

ne sinens

Thoracos l NCMA1

loi Effre

des 5 Suessiaceae s

grani p.

e mium 383

phaer fera P NCMA2456 T vorat 94

a LY01 * Durusdinium Cladocop

heimii G u

eoB 38 m

Tin SvF sp. sp.

tinnophagusCCC acutus go ium L1

M670 Fuga

Rhinodini rea

Heterocapsaceae P1-05

i iu

NCMA

Heterocapsa arctica k u a

m bro waguti

Heterocapsa pseu 466 7 i

omeense 9 Fu NCM

tium tium A246

sp. sp.

NCMA44 Brevi

dotriquetra NCMA

olum minutum olum

5 24 55 9

Heterocapsa steinii GeoB 2 *

Breviolum minutum Breviolum *

CMA830

Heterocap 222 63 52

U * M

T 1 f

sa pygmaea KG7 bre Biecheleria .05b.01

1C6

UTE isulc

cinnum a

X ta 9

1653

rd27

nium con nium

9 -kt

Azadi Ansanel

la la

granifera

TIO256

por

* AGSW

m po m u

i 10

Azadi

Bie

le rio

s i

s s

a d D1

ria 2

A tica

t r d27 trinitat 8- k t nium nium * adi z

Pe l a g

d in iu

m m

s p . .

E P - TF6 2

1 5 SHE

nosum spi

4 nium nium 9 zadi A

87 65

Azadi

nium de nium x teroporum

Amphidomataceae 1D12

95 5 Ceratium furcoides HBI:SC201002a

zadin * ium

caudat 3

var

cauda

t um

IF R

1 1

91 * Ceratium cf. hirundine A lla

zadinium c zadinium 51 WA28-13

audatum

r . .

margalefii Tripos lo * ng

IFR ipe 11

A s 9 mphidoma mphidoma

0 NCMA1770

langui T Ceratiaceae

da ripo

2A fur 11 ca Pr * or

oce

ntr

um dong um Gram

66 m *

haiens * atodi

Pro

e ni ro um tongy ce

tru * Frag

m min m ilidi eonginum

Prorocent um

imu d

m L

96 up IM

-PS2 * l

D * Py o

-127 cam 33

rum rum rocy pan 4

mexi

P st aefo

r is

or 8 noct rm canum 2

ocen Py iluca e Fd-LO Pyrophacaceae

t rro

H NM

m tria A

Proroc N B

C N16 E

st d M 01

e in A732

* i

di G iu

n m e ge

8 a

4 m p 96

t ra

ru b o

Pseu l

e y m m i

e e

r d

mic d ricum

C * iscu MA6 da Fu

de s

87 ku

s G

Adeno

no 1 yo

N ro

i * a

d 6 l * MA1 in

es es 5 Fuk sp ia

ides elud ides n 5 . ko

8 u

* uyoa

9 D s Prorocentrum hoffmannianum Prorocentrum f

o Q-

i NOA

di 2015

r A6 uetzl

PSE6 9

ens e _

9 x 7

P an _1

or 8 dr

A 3 A i oc

DE 9 le ium SK 2

e 7 xandrium

P LMP

ro mar

um * 8 _

roce 6 Alexa g S0

l al 4

Pr 4

im efi

o t

a n a i

ru Al

r d ndersonii A o

m m r

N e i M

c u

CMA * xand C N

lima m

ak C Q

M -1

t Alexan i

9 r

a A n 1

370 6

sue ya 73 ri

8 m le m

3 u

N N

a m

CM tum C

rlo A MA

v dr mi

A68 l

is ex acr 2222 *

din

Br *

ium n

9 * NC

5 an utu

NCM a

ot 9 A

iu * M

chi mi

r l d m

m exa A2

K r

A26

ch i

Prorocentrales v 08 d

a * Alexan um os nu NC

i r e

a 2

n n

e tum 9 n

i d M

eficum 9

5 ri A1 M

i 7 96

a u

0 A

C7 3 te 1 r

ae ae 3 en

b lex nf AM 2

Ka dri

e aff e

-D5

sp. sp.

a a A a

vis l

* um di

r i N ndr

e 9 l n mi *

Brach exandriu i

n e

MA1

catene GrA I ia

k Al i MR_

NC u

i A

G e m C

97 r0

MA7

e xandri - V_

y Ale ca idi 1

toi llifo

mno *

* 06

“ * l

nium la 1 5

G m tene

Alexanx r

N ymnodi

mis andriu ca A 0 L 7 *

xa u

C l CC

ep m c

M la

ca * Alex te

nthe

A4 *

99 n

Gyro

id C

pi ell NCM

2 Al ate

A dr m c

tatum

W a ni a

ll W

din e i

a a ndri nel

D u

um * Ale x A di a 80

79 m N 1

s 54 an

Nematodi tene 846 i rnowi la C p.

n u

” Ale u me MA

i m x s

el dri m

Barru andrium NC m

virid * lla 1

l C di

u x u t 7 a

8 * 1

“ a MA m

a a o a m t

ydae 9

Gyr e

9 Co o n m S shiw

s r 5

e 17

P aust a r PE1

fe l d

p. i a

97 C a canari ren 1

nium ol r n

9 ta respl ta

“

91 eum

l o 0-3

d G p

y C i 57

haens GS ral

BC ol a m acifi s inium k

Gymnodinium

ym ool e

rik sp.

8 “ ia S i

6 p en

s Al

Gymnodiniaceae Gymnodiniaceae

W10

5 2 * a A

p. mo cum S o no Lin

* * e ia e cif se T Z e Gy s gemi s en

SW01

” Ling xa m nsi N

* N ic din 01 i

UBC g mp no

de “ QAIF1 u

Brachydiniaceae mnod nd A

* T ul alaye s ymn

G m A T

ium * * -

n T t C BB0 1

udicum h odi i

3 ulod Gymnod

s s

ymnod he r NQ P

n Thec e iu

* A P Gy

a 0 1

” ca n m xsp o Im 0

GM N 3

t n AIF

in cadi

Nusut 1 cat

u i Pyrocystaceae

d 7 Im i um ” s C

baicalens d n

m ium * mnodin

Nus -K

inium a inium p is M

17 8 Go i iu h 2

e ad

N * pa agi n i A1 5 0

iniu † pol r 2 5 na n m G i

Nusu * um a u

* P Sp i iu N 3

ini ny a

Nusuttod * n

u n p 92

suttod gidi s QAIF354 t

todi

1 Protoc r d

u Nusut m o 5

tt m m iu cp

p Cer otocera in y

um fusc um ” o

m reo

Spinif i aul i

* ed

odi Bysm n ya

u m lye

Spin

iu i

B ko

e i niu u S n

ttodinium ttodinium A f r NIE

m fu m

Spi

lum er ax m

GnC y ium ac ium um

as Amphi a c shi

nium nium d piniferodinium palustre piniferodinium

Bis f

ol J mp smat o Akash f. in LM0802

c Es to i m cas r S Akashi

todinium po todinium e

u T ad um

t t idii

Lev 61

i T mae ero

icum elonga

M e ko

iu l

t01 ferodi

n N atr r

ti o Moestrupia Moestrupia co

Amo

Moestrupi s

p sp

i A o t

um unculti ati

v s Perk hi

E 2

nium op

T V

i T T Geo i

m v

c wi

oestr o

f um re f

ino v

moebo p

aerugi oi erodiniu o te

u ry ra udubo e d

er um

h h i

dini r . i

ande ct u S L

ae e n

MUCC2 ie dotum di m gi um

d lli i d PC r

e C

B s

lli n s m m i

c e k il

dinium angelaceum dinium n

wo od Ge i

a e e MUCC2

TIO82 i i a a i

ni i 2

wo sanguinea wo

l i

uca scintillans

l ta

i a h

l e Q1 r reticulatum

o u o u p ns m s

desymbi e

e A

n b 32 ium

l uginosum

upia upia

LO2 a o ti n

um lim um cf. sp ap c su o

m m ubsalsum Th P

l vated marine alveolat marine vated inium

r o NCMA18 e sus

san

nosum a ph

ri c B se

aponi r

i K

us 8

W8e a a b

p r s

galeifor u cyst . s

na na phr s

4 6 . p ca id 1 c

ec - 281

cqu r h m pa m Gonyaulacales

id l

m 007 f 15 Gaci-J ma a a atum a 82D p ave l .k- a a

rya a a

sp. sp. K i a

gui rt KC5

ilo n a H

sp. 0

a n

fissa

sp. sp. y 6

-0 na C17 2 2

tl 4

n e

n s r

oid BI

n s

a s

ell 90

o a v

c g G

d ire

i p. rae

eticum s

nea c

l t

hroum s :

n a 1

a nti o

ustr s e G a

r SC ar B

a a a es

pan p

tu a e

AW o

me n p. aer-Japan aer-Japan n

CGRL1 A Gaer

G4M . B t

wsi c ti

sp. sp. o

m si K

W22- C a 2

M P

CCA i NCMA us

e 1 i

r 01 C

NCMA124

2 CP2 RP

i s

Aam m H 8

2-15 32

g A 3 AB

G 1

J A M483394

Fukau

C M

i V-

u P GeoM*719 0 ap

s C C

nS 12 n T

GeoM PA

Ho SER

g MA18 AL 1

48339

p- 92 1

k S 0 J CAUT

a u a

431 J1 a

# 0 g

J n

kuto JL01

a N

N G1

H r a 21 0 #

ra a

299+ D

C3 pa C

2 G23

51 37 -

A 5 H n #b n

e 9

A 8–4a

A †Lingulodiniacaceae

B 1 FBB2 921300 921300 5

Gymnodiniaceae 5 Thecadiniacaceae

Gymnodiniales Gonyaulacaceae Ceratocoriaceae

Amphidiniaceae Tovelliaceae

Figure 1 A molecular reference tree recognizing major groups of dinophytes. Maximum likelihood (ML) tree of 228 representative dinophyte sequences (with strain number information), plus 10 outgroups, as inferred from a SSUrRNA nucleotide alignment (3,767 parsimony informative positions). Numbers on branches are ML bootstrap (above) and Bayesian support values (below) for the clusters (*maximal support values; values < 50 not shown).

Dinophysales, Gonyaulacales, Prorocentrales, and Pro- the dinophytes (Fawcett and Parrow 2014; Jeong et al. toperidiniaceae (Peridiniales) are primarily marine and did 2012), but there is no phylogenetic signal for this trait at not include any of the OTUs obtained from the environ- high taxonomic levels. Additionally, some OTUs placed mental samples. onto early branches that include many parasitic species There was no exclusive correlation with nutritional (Bachvaroff et al. 2012; Gomez et al. 2009; Saldarriaga mode. Some of the dinophyte OTUs placed onto branches et al. 2003), and they placed onto later branches that also that predominantly contain phototrophic species, such as include parasites, such as the Gymnodiniaceae (Gomez Gymnodiniaceae, Peridiniales, and †Suessiales. Other et al. 2009; Kretschmann et al. 2015; Romeikat et al. OTUs placed with the heterotrophic species such as in 2020) and Peridiniales (Coats et al. 2010; Gottschling and Thoracosphaeraceae (i.e. Pﬁesteria and relatives), but not Sohner€ 2013). in the consistently heterotrophic Dinophysales and Pro- There was no exclusive correlation with dormancy prac- toperidiniaceae (Peridiniales). Transitions between pho- tice. In addition to ﬂagellated trophic cells, coccoid stages totrophic and heterotrophic modes are thought to occur in are integral part in the life-history of many dinophytes

© 2020 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists Journal of Eukaryotic Microbiology 2021, 68, e12833 3of6 Dinophytes in Soils Gottschling et al. from marine and freshwater environments. Coccoid cells LITERATURE CITED are particularly abundant in the Gonyaulacales where no OTUs placed, or in the Peridiniales where many OTUs Adl, S. M., Bass, D., Lane, C. E., Lukes, J., Schoch, C. L., Smir- placed (Dale 1983; Evitt and Davidson 1964; Fensome nov, A., Agatha, S., Berney, C., Brown, M. W., Burki, F., Carde- et al. 1993; Wall 1971). The exact ecological functions of nas, P., Cepicka, I., Chistyakova, L., Campo, J., Dunthorn, M., coccoid cells are not worked out rigorously for more than Edvardsen, B., Eglit, Y., Guillou, L., Hampl, V., Heiss, A. A., a handful of dinophyte species, but may frequently corre- Hoppenrath, M., James, T. Y., Karnkowska, A., Karpov, S., Kim, E., Kolisko, M., Kudryavtsev, A., Lahr, D. J. G., Lara, E., Le Gall, spond to resting and/or dormancy stages (Fensome et al. L., Lynn, D. H., Mann, D. G., Massana, R., Mitchell, E. A. D., 1993). Deposited in sediments, coccoid cells have the Morrow, C., Park, J. S., Pawlowski, J. W., Powell, M. J., Rich- potential to preserve the local biodiversity like diaspores ter, D. J., Rueckert, S., Shadwick, L., Shimano, S., Spiegel, F. in a seed bank (Dale 1983). They can be considered W., Torruella, G., Youssef, N., Zlatogursky, V. & Zhang, Q. either as old remnants from formerly aquatic, but now 2019. Revisions to the classification, nomenclature, and diver- currently terrestrial environments (Boere et al. 2011; Søn- sity of eukaryotes. J. Eukaryot. Microbiol., 66:4–119. stebø et al. 2010) or the result of random dispersal Annenkova, N. V., Giner, C. R. & Logares, R. 2020. Tracing the (Foissner 2006, 2011) by birds (Kretschmann et al. 2018; origin of planktonic protists in an ancient lake. Microorganisms, Tesson et al. 2018) or by wind. However, the difference 8:543. between the taxonomic assignments of the rainforest soil Bachvaroff, T. R., Kim, S., Guillou, L., Delwiche, C. F. & Coats, D. W. 2012. Molecular diversity of the syndinean genus Eudubosc- samples (see above) may indicate that the ability to form quella based on single-cell PCR analysis. Appl. Environ. Micro- coccoid cells during life history is not decisive for their biol., 78:334–345. terrestrial occurrence. An ecological group that has been Bass, D., Czech, L., Williams, B. A. P., Berney, C., Dunthorn, M., receiving more interest in the past years and that may Mahe, F., Torruella, G., Stentiford, G. D. & Williams, T. A. 2018. also be considered for the evaluation of the terrestrial Clarifying the relationships between Microsporidia and Crypto- samples is benthic dinophytes living in the intertidal (Hop- mycota. J. Eukaryot. Microbiol., 65:773–782. penrath et al. 2014); phylogenetically, it is a heteroge- Bates, S. T., Clemente, J. C., Flores, G. E., Walters, W. A., Par- neous assemblage recruiting members particularly from frey, L. W., Knight, R. & Fierer, N. 2013. Global biogeography – Gonyaulacales and Peridiniales. But the bigger question of highly diverse protistan communities in soil. ISME J., 7:652 remains, as to whether there are any soil dinophytes at 659. Berger, S. A., Krompass, D. & Stamatakis, A. 2011. Performance, all, or we are simply detecting windblown cells or lost accuracy, and web server for evolutionary placement of short dormant cells. sequence reads under maximum likelihood. Syst. Biol., 60:291– 302. CONCLUSION Berger, S. A. & Stamatakis, A. 2011. Aligning short reads to reference alignments and trees. Bioinformatics, 27:2068–2075. The presence of dinophyte DNA sequences in the Bergholtz, T., Daugbjerg, N., Moestrup, Ø. & Fernandez-Tejedor, Neotropical rainforest soils—as an exemplar of a terrestrial M. 2006. On the identity of Karlodinium veneficum and descrip- environment—did not correlate with taxonomic assign- tion of Karlodinium armiger sp. nov. (Dinophyceae), based on ment, environmental preference, dormancy practice during light and electron microscopy, nuclear-encoded LSU rDNA, and – life history, or nutrition mode/organismal interaction. The pigment composition. J. Phycol., 42:170 193. Boere, A. C., Rijpstra, W. I. C., de Lange, G. J., Malinverno, E., reason for their presence thus remains to be identified. Sinninghe Damste, J. S. & Coolen, M. J. L. 2011. Exploring pre- We have to keep in mind that presence of DNA does not served fossil dinoflagellate and haptophyte DNA signatures to necessarily indicate biological activity. The environmental infer ecological and environmental changes during deposition of DNA sequences identified here are scattered unevenly sapropel S1 in the eastern Mediterranean. Paleoceanography, across the classification, but it may represent more easily 26:PA2204. dispersed surface algae. To completely resolve this para- Chacon, J. & Gottschling, M. 2020. Dawn of the dinophytes: a dox, microscopy on soil samples with dinophyte DNA, first attempt to date origin and diversification of harmful algae. especially with fluorescence in situ hybridization probes Harmful Algae, 97:101871. https://doi.org/10.1016/j.hal.2020. designed from the molecular OTUs, needs to be per- 101871 formed to verify their presence; however, dinophytes have Coats, D. W., Kim, S., Bachvaroff, T. R., Handy, S. M. & Del- wiche, C. F. 2010. Tintinnophagus acutus n. g., n. sp. (Phylum never been conclusively observed in soils even after two Dinoflagellata), an ectoparasite of the ciliate Tintinnopsis cylin- centuries of observations. As suggested for other protists drica Daday 1887, and its relationship to Duboscquodinium col- (Ehrenberg 1849), the most likely explanation for dino- lini Grasse 1952. J. Eukaryot. Microbiol., 57:468–482. phyte DNA in soils is that they were passively dispersed Czech, L., Barbera, P. & Stamatakis, A. 2018. Methods for auto- there—they are the windblown. matic reference trees and multilevel phylogenetic placement. Bioinformatics, 35:1151–1158. Czech, L., Barbera, P. & Stamatakis, A. 2020. Genesis and Gappa: ACKNOWLEDGMENTS processing, analyzing and visualizing phylogenetic (placement) – We thank Matthew Parrow and Gerard Versteegh for help- data. Bioinformatics, 36:3263 3265. ful edits, and Alexandros Stamatakis for computational Dale, B. 1983. Dinoflagellate resting cysts: “Benthic plankton”. In: Fryxell, G. A. (ed.), Survival Strategies of the Algae. Cambridge support. Funding came from the Deutsche Forschungsge- University Press, Cambridge. p. 69–136. meinschaft (grant DU1319/5-1) to MD.

© 2020 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists 4of6 Journal of Eukaryotic Microbiology 2021, 68, e12833 Gottschling et al. Dinophytes in Soils de Vargas, C., Audic, S., Henry, N., Decelle, J., Mahe, F., Loga- Thalassomyces Niezabitowski, 1913 form a monophyletic diver- res, R., Lara, E., Berney, C., Le Bescot, N., Probert, I., Carmi- gent clade within the Alveolata. Syst. Parasitol., 74:65–74. chael, M., Poulain, J., Romac, S., Colin, S., Aury, J.-M., Bittner, Gottschling, M., Chacon, J., Zerdoner Calasan, A., Neuhaus, S., L., Chaffron, S., Dunthorn, M., Engelen, S., Flegontova, O., Kretschmann, J., Stibor, H. & John, U. 2020. Phylogenetic Guidi, L., Horak, A., Jaillon, O., Lima-Mendez, G., Lukes, J., placement of environmental sequences using taxonomically reli- Malviya, S., Morard, R., Mulot, M., Scalco, E. Sian, O. R., Vin- able databases helps to rigorously assess dinophyte biodiversity cent, F., Zingone, A., Dimier, C., Picheral, M., Searson, S., Kan- in Bavarian lakes (Germany). Freshw. Biol., 65:193–208. dels-Lewis, S., Tara Oceans Coordinators, Acinas, S. G., Bork, Gottschling, M., Sohner,€ S., Zinßmeister, C., John, U., Plotner,€ J., P., Bowler, C., Gorsky, G., Grimsley, N., Hingamp, P., Iudicone, Schweikert, M., Aligizaki, K. & Elbrachter,€ M. 2012. Delimitation D., Not, F., Ogata, H., Pesant, S., Raes, J., Sieracki, M. E., Spe- of the Thoracosphaeraceae (Dinophyceae), including the cal- ich, S., Stemmann, L., Sunagawa, S., Weissenbach, J., careous dinoflagellates, based on large amounts of ribosomal Wincker, P.& Karsenti, E. 2015. Eukaryotic plankton diversity in RNA sequence data. Protist, 163:15–24. the sunlit ocean. Science, 348:1261605. Gottschling, M. & Sohner,€ S. 2013. An updated list of generic Decelle, J., Carradec, Q., Pochon, X., Henry, N., Romac, S., names in the Thoracosphaeraceae. Microorganisms, 1:122–136. Mahe, F., Dunthorn, M., Kourlaiev, A., Voolstra, C. R., Wincker, Gu, H., Kirsch, M., Zinßmeister, C., Sohner,€ S., Meier, K. J. S., Liu, P. & de Vargas, C. 2018. Worldwide occurrence and activity of T. & Gottschling, M. 2013. Waking the dead: morphological and the reef-building coral symbiont Symbiodinium in the open molecular characterization of extant †Posoniella tricarinelloides ocean. Curr. Biol., 28:3625–3633. (Thoracosphaeraceae, Dinophyceae). Protist, 164:583–597. Dunthorn, M., Otto, J., Berger, S. A., Stamatakis, A., Mahe, F., Guillou, L., Bachar, D., Audic, S., Bass, D., Berney, C., Bittner, L., Romac, S., de Vargas, C., Audic, S., BioMarKs Consortium, Boutte, C., Burgaud, G., de Vargas, C., Decelle, J., Del Campo, Stock, A., Kauff, F. & Stoeck, T. 2014. Placing environmental J., Dolan, J. R., Dunthorn, M., Edvardsen, B., Holzmann, M., next-generation sequencing amplicons from microbial eukary- Kooistra, W. H., Lara, E., Le Bescot, N., Logares, R., Mahe, F., otes into a phylogenetic context. Mol. Biol. Evol., 31:993–1009. Massana, R., Montresor, M., Morard, R., Not, F., Pawlowski, Ehrenberg, C. G. 1849. Passat-Staub und Blut-Regen: ein grosses J., Probert, I., Sauvadet, A. L., Siano, R., Stoeck, T., Vaulot, D., organisches unsichtbares Wirken und Leben in der Athmo- Zimmermann, P. & Christen, R. 2013. The Protist Ribosomal sphare;€ mehrere Vortrage.€ Koniglichen€ Akademie der Wis- Reference database (PR2): a catalog of unicellular eukaryote senschaften, Berlin. small sub-unit rRNA sequences with curated taxonomy. Nucleic Elferink, S., Neuhaus, S., Wohlrab, S., Tobe,€ K., Voß, D., Gottschl- Acids Res., 41:D597–D604. ing, M., Lundholm, N., Krock, B., Koch, B. P., Zielinski, O., Henrichs, D. W., Sosik, H. M., Olson, R. J. & Campbell, L. 2011. Cembella, A. & John, U. 2017. Molecular diversity patterns Phylogenetic analysis of Brachidinium capitatum (Dinophyceae) among various phytoplankton size-fractions in West Greenland from the Gulf of Mexico indicates membership in the Kareni- in late summer. Deep Sea Res. Part I, 121:54–69. aceae. J. Phycol., 47:366–374. Evitt, W. R. & Davidson, S. E. 1964. Dinoflagellate studies. I. Hoppenrath, M., Murray, S. A. & Chomerat, N. 2014. Marine ben- Dinoflagellate cysts and thecae. In: Harbaugh, J. W. (ed.), Stan- thic dinoflagellates – Unveiling their worldwide biodiversity. Sch- ford University Publications in the Geological Sciences. Stanford weizerbart Science Publishers, Stuttgart. University Press, Stanford, CA. p. 1–12. Jeong, H. J., Yoo, Y. D., Kang, N. S., Lim, A. S., Seong, K. A., Fawcett, R. C. & Parrow, M. W. 2014. Mixotrophy and loss of Lee, S. Y., Lee, M. J., Lee, K. H., Kim, H. S., Shin, W., Nam, S. phototrophy among geographic isolates of freshwater Esoptro- W., Yih, W. & Lee, K. 2012. Heterotrophic feeding as a newly dinium/Bernardinium sp. (Dinophyceae). J. Phycol., 50:55–70. identified survival strategy of the dinoflagellate Symbiodinium. Fensome, R. A., Taylor, F. J. R., Norris, G., Sarjeant, W. A. S., Proc. Natl. Acad. Sci. USA, 109:12604–12609. Wharton, D. I. & Williams, G. L. 1993. A classification of living Jones, M. D. M., Forn, I., Gadelha, C., Egan, M. J., Bass, D., and fossil dinoflagellates. Micropaleontology. Special Publication Massana, R. & Richards, T. A. 2011. Discovery of novel inter- Number, 7:1–245. mediate forms redefines the fungal tree of life. Nature, Foissner, W. 2006. Biogeography and dispersal of micro-organ- 474:200–203. isms: a review emphasizing protists. Acta Protozool., 45:111– Katoh, K. & Standley, D. M. 2013. MAFFT multiple sequence 136. alignment software version 7: improvements in performance Foissner, W. 2011. Dispersal of protists: the role of cysts and and usability. Mol Biol. Evol., 30:772–780. human introductions. In: Fontaneto, D. (ed.), Biogeography of Kretschmann, J., Filipowicz, N. H., Owsianny, P. M., Zinßmeister, microscopic organisms: is everything small everywhere? Cam- C. & Gottschling, M. 2015. Taxonomic clarification of the unu- bridge University Press, Cambridge. p. 61–87. sual dinophyte Gymnodinium limneticum Wołosz. (Gymnodini- Geisen, S., Mitchell, E. A. D., Adl, S., Bonkowski, M., Dunthorn, aceae) from the Tatra Mountains. Protist, 166:621–637. M., Ekelund, F., Fernandez, L. D., Jousset, A., Krashevska, V., Kretschmann, J., Owsianny, P. M., Zerdoner Calasan, A. & Singer, D., Spiegel, F. W., Walochnik, J. & Lara, E. 2018. Soil Gottschling, M. 2018. The hot spot in a cold environment: Puz- protists: a fertile frontier in soil biology research. FEMS Micro- zling Parvodinium (Peridiniopsidaceae, Peridiniales) from the biol. Rev., 42:293–323. Polish Tatra Mountains. Protist, 230:206–230. Geisen, S., Tveit, A. T., Clark, I. M., Richter, A., Svenning, M. M., Kretschmann, J., Zinßmeister, C. & Gottschling, M. 2014. Taxo- Bonkowski, M. & Urich, T. 2015. Metatranscriptomic census of nomic clarification of the dinophyte Rhabdosphaera erinaceus active protists in soils. ISME J., 9:2178–2190. Kamptner, Scrippsiella erinaceus comb. nov. (Thoracosphaer- Giner, C. R., Pernice, M. C., Balague, V., Duarte, C. M., Gasol, J. aceae, Peridiniales). Syst. Biodivers., 12:393–404. M., Logares, R. & Massana, R. 2020. Marked changes in diver- Le Bescot, N., Mahe, F., Audic, S., Dimier, C., Garet, M.-J., Pou- sity and relative activity of picoeukaryotes with depth in the lain, J., Wincker, P., de Vargas, C. & Siano, R. 2016. Global pat- world ocean. ISME J., 14:437–449. terns of pelagic dinoflagellate diversity across protist size Gomez, F., Lopez-Garc ıa, P., Nowaczyk, A. & Moreira, D. 2009. classes unveiled by metabarcoding. Environ. Microbiol., 18:609– The crustacean parasites Ellobiopsis Caullery, 1910 and 626.

Lejzerowicz, F., Pawlowski, J., Fraissinet-Tachet, L. & Marmeisse, Sønstebø, J. H., Gielly, L., Brysting, A. K., Elven, R., Edwards, R. 2010. Molecular evidence for widespread occurrence of For- M., Haile, J., Willerslev, E., Coissac, E., Rioux, D., Sannier, J., aminifera in soils. Environ. Microbiol., 12:2518–2526. Taberlet, P. & Brochmann, C. 2010. Using next-generation Lentendu, G., Buosi, P. R. B., Cabral, A. F., Segovia, B. T., de sequencing for molecular reconstruction of past Arctic vegeta- Meira, B. R., Lansac-Toha, F. M., Velho, L. F. M., Ritter, C. & tion and climate. Mol. Ecol. Resour., 10:1009–1018. Dunthorn, M. 2018. Planktonic protist biodiversity and biogeog- Stamatakis, A. 2014. RAxML version 8: a tool for phylogenetic raphy in lakes from four Brazilian river-floodplain systems. J. analysis and post-analysis of large phylogenies. Bioinformatics, Eukaryot. Microbiol., 66:592–599. 30:1312–1313. Lindberg, K., Moestrup, Ø. & Daugbjerg, N. 2005. Studies on Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M. D. M., woloszynskioid dinoflagellates I: Woloszynskia coronata re-ex- Breiner, H. W. & Richards, T. A. 2010. Multiple marker parallel amined using light and electron microscopy and partial LSU tag environmental DNA sequencing reveals a highly complex rDNA sequences, with description of Tovellia gen. nov. and eukaryotic community in marine anoxic water. Mol. Ecol., Jadwigia gen. nov. (Tovelliaceae fam. nov.). Phycologia, 19:21–31. 44:416–440. Tesson S. V. M., Weißbach A., Kremp A., Lindstrom€ A., Renge- Luo, Z., Mertens, K. N., Bagheri, S., Aydin, H., Takano, Y., Mat- fors K. (2018) The potential for dispersal of microalgal resting suoka, K., McCarthy, F. M. G. & Gu, H. 2016. Cyst-theca rela- cysts by migratory birds. Journal of Phycology, 54 (4), 518–528. tionship and phylogenetic positions of Scrippsiella plana sp. http://dx.doi.org/10.1111/jpy.12756 nov. and S. spinifera (Peridiniales, Dinophyceae). Eur. J. Protis- Tillmann, U., Elbrachter,€ M., Gottschling, M., Gu, H., Jeong, H. J., tol., 51:188–202. Krock, B., Nezan, E., Potvin, E., Salas, R. & Sohner,€ S. 2014. Mahe, F., de Vargas, C., Bas, S. D., Czech, L., Stamatakis, A., The dinophycean genus Azadinium and related species – mor- Lara, E., Singer, D., Mayor, J., Bunge, J., Sernaker, S., Sie- phological and molecular characterization, biogeography, and mensmeyer, T., Trautmann, I., Romac, S., Berney, C., Kozlov, toxins. In: Kim, H. G., Reguera, B., Hallegraeff, G. M., Lee, C. A., Mitchell, E. A. D., Seppey, C. V. W., Egge, E., Lentendu, G., K., Han, H. S. & Choi, J. K. (ed.), Harmful Algae 2012 (Proceed- Wirt, H. R., Trueba, G. & Dunthorn, M. 2017. Parasites domi- ings of the 15th International Conference on Harmful Algae). nate hyperdiverse soil protist communities in Neotropical rain- International Society for the Study of Harmful Algae. p. 149– forests. Nat. Ecol. Evol., 1:0091. 152. Mahe, F., Rognes, T., Quince, C., de Vargas, C. & Dunthorn, M. Venter, P. C., Nitsche, F., Domonell, A., Heger, P. & Arndt, H. 2015. Swarm v2: highly-scalable and high-resolution amplicon 2017. The protistan microbiome of grassland soil: diversity in clustering. PeerJ, 3:e1761. the mesoscale. Protist, 168:546–564. Meisterfeld, R., Holzmann, M. & Pawlowski, J. 2001. Morphologi- Voss, C., Fiore-Donno, A. M., Guerreiro, M. A., Persoh, D. & Bon- cal and molecular characterization of a new terrestrial allogro- kowski, M. 2019. Metatranscriptomics reveals unsuspected miid species: Edaphoallogromia australica gen. et spec. nov. protistan diversity in leaf litter across temperate beech forests, (Foraminifera) from Northern Queensland (Australia). Protist, with Amoebozoa the dominating lineage. FEMS Microbiol. 152:185–192. Ecol., 95:fiz142. Moestrup, Ø. & Calado, A. J. 2018. Freshwater flora of Central Wall, D. 1971. Biological problems concerning fossilizable Europe, Vol. 6: Dinophyceae. Springer Spektrum, Berlin. dinoflagellates. Geosci. Man, 3:1–15. Rognes, T., Flouri, T., Nichols, B., Quince, C. & Mahe, F. 2016. Zerdoner Calasan, A., Kretschmann, J. & Gottschling, M. 2019. VSEARCH: a versatile open source tool for metagenomics. They are young, and they are many: dating freshwater lineages PeerJ, 4:e2584. in unicellular dinophytes. Environ. Microbiol., 21:4125–4135. Romeikat, C., Knechtel, J. & Gottschling, M. 2020. Clarifying the taxonomy of Gymnodinium fuscum var. rubrum from Bavaria (Germany) and placing it in a molecular phylogeny of the SUPPORTING INFORMATION – Gymnodiniaceae (Dinophyceae). Syst. Biodivers., 18:102 115. Additional supporting information may be found online in Saldarriaga, J. F., McEwan, M. L., Fast, N. M., Taylor, F. J. R. & the Supporting Information section at the end of the article. Keeling, P. J. 2003. Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of Table S1. Voucher list. the dinoflagellate lineage. Int. J. Syst. Evol. Microbiol., 53:355– File S1. The 269 OTUs that were assigned to the dino- 365. phytes by Mahe et al. (2017) that were extracted and Saldarriaga, J. F. & Taylor, F. J. R. 2017. Dinoflagellata. In: Hand- used here for phylogenetic placements. book of the Protists, 2nd edn. Springer International Publishing File S2. Taxon sampling for the dinophyte reference align- AG, Cham. p. 625–678. Santoferrara, L., Burki, F., Filker, S., Logares, R., Dunthorn, M. & ment. McManus, G. B. 2020. Perspectives from ten years of protist File S3. Reference alignment for the dinophytes. studies by high-throughput metabarcoding. J. Eukaryot. Micro- File S4. Reference tree for the dinophytes. biol., 67:612–622. File S5. The 207 OTUs that were assigned to the dino- Sayers, E. W., Cavanaugh, M., Clark, K., Ostell, J., Pruitt, K. D. & phytes, which phylogenetically placed here across the ref- Karsch-Mizrachi, I. 2020. GenBank. Nucleic Acids Res., 48:D84– erence tree with high likelihood weight scores. D86.