Morphological and molecular characterization of the genus Coolia (Dinophyceae) from Bahía de La Paz, southwest Gulf of California

Abstract

The genus Coolia A. Meunier 1919 has a global distribution and is a common member of epiphytic dinoflagellate assemblages in neritic ecosystems. Coolia monotis is the type species of the genus and was the only known species for 76 years. Over the past few decades, molecular characterization has unveiled two species complexes that group morphologically very similar species, so their limits are often unclear. To provide new knowledge on the biogeography and species composition of the genus Coolia, 16 strains were isolated from Bahía de La Paz, Gulf of California. The species were identified by applying morphological and molecular approaches. The morphometric characteristics of all isolated Coolia species were consistent with the original taxa descriptions. Phylogenetic analyses (large subunit [LSU] rDNA D1 / D2 and internal transcribed spacer [ITS] 1 / 5.8S / ITS2) revealed a species assemblage comprising Coolia malayensis, C. palmy-rensis, C. tropicalis, and the C. cf. canariensis lineage. This is the first report of Coolia palmyrensis and C. cf. canariensis in Mexico and C. tropicalis in the Gulf of California. Our results strengthen the biogeographical understanding of these potentially harmful epiphytic dinoflagellate species.

Full text

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2
Open Access
Research Article
Copyright © 2022 The Korean Society of Phycology 185 http://e-algae.org pISSN: 1226-2617 eISSN: 2093-0860
Morphological and molecular characterization of the genus Coolia
(Dinophyceae) from Bahía de La Paz, southwest Gulf of California
Lourdes Morquecho1,*, Ismael Gárate-Lizárraga2 and Haifeng Gu3
1Programa de Planeación Ambiental y Conservación, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Calle
IPN #195, Col. Playa Palo de Santa Rita Sur, La Paz, Baja California Sur 23096, Mexico
2Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas (CICIMAR-IPN), Avenida Instituto Politéc-
nico Nacional s/n, Col. Playa Palo de Santa Rita, La Paz, Baja California Sur 23096, Mexico
3Department of Marine Biodiversity, ird Institute of Oceanography, Ministry of Natural Resources, Xiamen, Fujian,
361005, China
e genus Coolia A. Meunier 1919 has a global distribution and is a common member of epiphytic dinoagellate as-
semblages in neritic ecosystems. Coolia monotis is the type species of the genus and was the only known species for 76
years. Over the past few decades, molecular characterization has unveiled two species complexes that group morpho-
logically very similar species, so their limits are often unclear. To provide new knowledge on the biogeography and spe-
cies composition of the genus Coolia, 16 strains were isolated from Bahía de La Paz, Gulf of California. e species were
identied by applying morphological and molecular approaches. e morphometric characteristics of all isolated Coolia
species were consistent with the original taxa descriptions. Phylogenetic analyses (large subunit [LSU] rDNA D1 / D2 and
internal transcribed spacer [ITS] 1 / 5.8S / ITS2) revealed a species assemblage comprising Coolia malayensis, C. palmy-
rensis, C. tropicalis, and the C. cf. canariensis lineage. is is the rst report of Coolia palmyrensis and C. cf. canariensis
in Mexico and C. tropicalis in the Gulf of California. Our results strengthen the biogeographical understanding of these
potentially harmful epiphytic dinoagellate species.
Keywords: Bahía de La Paz; Coolia species; epiphytic dinoagellates; Gulf of California; ITS1 / 5.8S / ITS2; LSU rDNA;
morphology; taxonomy
INTRODUCTION
Epibenthic dinoagellates are globally distributed in
intertidal and estuarine ecosystems, particularly in shal-
low, sandy, and well-illuminated environments (Fraga
et al. 2012). ey inhabit the interstitial spaces of marine
sediments or live epiphytically on macroalgae and sea-
grasses (Hoppenrath et al. 2014). e epiphytic commu-
nity assemblage is usually comprised (in order of preva-
lence) of species belonging to the genera Prorocentrum
Ehrenberg 1834, Gambierdiscus Adachi & Fukuyo 1979,
Ostreopsis J. Schmidt 1901, Coolia Meunier 1919, Fukuyoa
Qiu, Lopes & Lin 2015, Amphidinium Claperède & Lach-
mann 1859, Bysmatrum M. A. Faust & K. A. Steidinger
1998, Sinophysis Nie & C. Wang 1944, and Cabra Shauna
Murray & D. J. Patterson 2004, among others less preva-
lent genera (Hoppenrath et al. 2014, Rhodes and Smith
2018).
Epibenthic dinoagellates produce toxins that aect
human and environmental health (Bomber and Aikman
Received January 24, 2022, Accepted September 2, 2022
*Corresponding Author
E-mail: lamorquecho@cibnor.mx
Tel: +52-612-1238490, Fax: +52-612-1238484
is is an Open Access article distributed under the terms
of the Creative Commons Attribution Non-Commercial
License (http://creativecommons.org/licenses/by-nc/3.0/) which permits
unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 186
& Usup 2010 (Leaw et al. 2010), C. tropicalis, C. palmy-
rensis Karafas, Tomas & York 2015 (Karafas et al. 2015),
and C. santacroce Karafas, Tomas & York 2015 (Karafas
et al. 2015) have been reported to be toxic, with biotoxin
production conrmed through cytotoxicity bioassays,
hemolytic assays, and chemical analyses (Holmes et al.
1995, Karafas et al. 2015, Wakeman et al. 2015, Leung et
al. 2017, Tibiriçá et al. 2020).
Currently, the species in the genus Coolia have been
grouped into two species complexes: the “Coolia mono-
tis” complex, including C. monotis C. malayensis, C.
palmyrensis, and C. santacroce; and the “C. canariensis”
complex, comprising C. canariensis S. Fraga 2008 (Fraga
et al. 2008), C. tropicalis, C. areolata, and C. cf. canariensis
(Karafas et al. 2015). Species are dierentiated primarily
based on the shape of the epitheca; the size of some hy-
potheca plates and the ornamentation of the theca also
distinguish species from one another. e size, shape, and
position of apical pore plate (Po) and plates 1', 2', 4', 6", 7",
and 3"' are traits used for species dierentiation. e posi-
tion and shape of plate 1' dierentiate at the species com-
plex level, the size of plate 3"' dierentiate between very
similar species, such as C. malayensis and C. monotis, and
the shape of plate 4' and the size of plate 6' separates C.
tropicalis from C. malayensis (de Queiroz-Mendes et al.
2019). Additional morphometric and tabular character-
istics used to dierentiate C. malayensis from C. monotis
are cell size, plate 3' shape, and Po length (Mohammad-
Noor et al. 2013). C. malayensis is generally smaller than
C. monotis (Leaw et al. 2016), although there is some size
range overlap, and C. malayensis is usually narrower. Plate
3' is pentagonal in C. monotis and quadrangular or pen-
tagonal in C. malayensis (Mohammad-Noor et al. 2013).
In C. malayensis, Po length is shorter and its postcingu-
lar plate 3"' is the largest in the hypotheca; in C. monotis,
plate 3"' can be either equal (as shown in Aligizaki and
Nikolaidis 2006, David et al. 2014, Leaw et al. 2016, Lewis
et al. 2018) or larger (as indicated in Meunier 1919, Penna
et al. 2005, Dolapsakis et al. 2006, Laza-Martínez et al.
2011) than plate 4"'. Both size variations in postcingular
plates 3"' and 4"' were observed by Abdennadher et al.
(2021) in C. monotis strains sampled in Southern Tunisia
(Mediterranean Sea).
In Mexico, research on dinoagellates has focused
mainly on planktonic species from the Mexican Pacic,
and taxonomic studies are limited to the assessment of
morphological traits (Okolodkov and Gárate-Lizárraga
2006). A few studies on species composition and distri-
bution of benthic dinoagellates have been conducted
in North Atlantic coastal ecosystems in the southern Gulf
1989). Benthic harmful algal blooms (BHABs) are becom-
ing more frequent and severe, probably due to anthropo-
genic inuences and global climate change (Tester et al.
2020). As of today, approximately 48 taxa are recognized
as harmful (Lundholm et al. 2009) and associated with
ciguatera sh poisoning (CFP) (Bomber and Aikman
1989, Karafas et al. 2015, Caruana and Amzil 2018), as
well as respiratory and dermatologic syndromes (Lewis
et al. 2018).
Fourteen Gambierdiscus, 13 Prorocentrum, 7 Ostreop-
sis, and 3 Fukuyoa species have been recognized as toxic
(Akselman and Fraga 2022, Hoppenrath 2022), and there
is a high probability that toxins produced by some Coolia
species also play a central role in harmful events (GEO-
HAB 2012). Also, Coolia blooms may impact the ecol-
ogy through high mucilage production or lead to anoxic
events (GEOHAB 2012).
e taxonomy of epibenthic dinoagellates is currently
under revision due to multiple similarities among the
taxa described. is is being conducted through a mor-
phological assessment, and new species have been dis-
covered using molecular methods only (GEOHAB 2012).
Signicant progress has been made in clarifying the tax-
onomy of epibenthic Gonyaulacales over the past two de-
cades. Seventeen new taxa were classied in the genera
Gambierdiscus (Litaker et al. 2009, Fraga et al. 2011, 2016,
Fraga and Rodríguez 2014, Nishimura et al. 2014, Smith et
al. 2016, Kretzschmar et al. 2017, Rhodes et al. 2017, Jang
et al. 2018), Coolia (Ten-Hage et al. 2000, Fraga et al. 2008,
Leaw et al. 2010, Karafas et al. 2015, David et al. 2020) and
Ostreopsis (Accoroni et al. 2016, Verma et al. 2016).
ere are eight currently accepted species in the ge-
nus Coolia (Guiry and Guiry 2021). Coolia monotis was
the rst taxon described (Meunier 1919) and has been
considered predominant. However, since the beginning
of this century, molecular sequencing and phylogenetic
analyses have revealed that this species actually com-
prises a species complex, and its distribution is not as
cosmopolitan as previously thought (Momigliano et al.
2013). e description of Coolia areolata L. Ten-Hage, J.
Turquet, J. P. Quod & Couté 2000 (Ten-Hage et al. 2000)
was based only on morphometric and tabular examina-
tions (Ten-Hage et al. 2000), without being genetically
characterized so far. Coolia guanchica H. David, Laza-
Martínez, F. Rodríguez & S. Fraga (David et al. 2020) is the
most recently described taxon; so far, C. tropicalis M. A.
Faust (Faust 1995) is the only species recognized as toxic
(Holmes et al. 1995, Tibiriçá et al. 2020) in the Taxonomic
Reference List of Harmful Microalgae (Akselman and
Fraga 2022). Nevertheless, C. malayensis Leaw, P. -T. Lim

Morquecho et al. Coolia in Southwest Gulf of California
187 http://e-algae.org
Espiritu Santo and Isla La Partida to the east (Fig. 1). Isla
San José is the largest island o the east coast of Baja Cali-
fornia Sur; its southwestern end (24°52'32" N, 110°33'30"
W) is characterized by extensive beaches and a small la-
goon bordered by mangrove forest and a narrow sand bar.
Macroalgae assemblages in Bahía de La Paz usually com-
prise the phyla Rhodophyta, Ochrophyta-Phaeophyceae,
and Chlorophyta, with the families Rhodomelaceae and
Ceramiaceae being the most abundant (Piñón-Gimate et
al. 2020).
Collection and preliminary handling of macroal-
gae samples
Macroalgae were collected from 5 selected sites in
Bahía de La Paz, both by scuba diving or snorkeling and
by hand (Table 1, Fig. 1). At site 1, the macroalgae sample
was directly collected from a dock at 0.5 m depth. In Bal-
andra beach (sites 3 and 4), macroalgae samples were
collected 5–15 m from the coastline at 1.5 m depth, and
in Isla San José lagoon at 5 m depth (site 5). Macroalgae
were sampled individually by detaching from the sub-
strate and placed separately in a resealable plastic bag
or ask. ese plastic bags or asks were lled with the
surrounding seawater, sealed tightly, and transported in a
cooler to the laboratory for processing.
Once in the laboratory, macroalgae samples were pro-
cessed following the protocol by Reguera et al. (2011) as
follows: samples were vigorously shaken (2 min) to de-
tach the cells. Macroalgae were removed, and the water
suspension was ltered and washed with ltered seawa-
ter (TCLP glass ber lters, 0.7 µm; Pall Laboratory, Port
of Mexico and the Yucatán Peninsula (Okolodkov et al.
2007, Almazán-Becerril et al. 2015).
Currently, Coolia cf. areolata (Irola-Sansores et al.
2018), C. malayensis (Sepúlveda-Villarraga 2017, Ramos-
Santiago 2021), C. monotis (Okolodkov and Gárate-
Lizárraga 2006, Okolodkov et al. 2007, Almazán-Becerril
et al. 2012, Gárate-Lizárraga et al. 2019), and C. tropicalis
(Almazán-Becerril et al. 2015) have been found and char-
acterized in Mexican ecosystems. In the Gulf of Mexico
and the Mexican Caribbean, Coolia species display no
particular anity for any macrophyte substrate. In the
Veracruz reef zone, C. monotis has been found associ-
ated with the seagrass alassia testudinum K. D. Koenig
1805, which is the most abundant and permanent host
species (Okolodkov et al. 2007). However, the seagrasses
Halodule wrightii Ascherson 1868 and Syringodium li-
forme Kützing 1860 are also dominant species in the Gulf
of Mexico, along with mainly green macroalgal species,
such as Caulerpa ashmeadii Harvey 1858, C. paspaloides
(Bory) Greville 1030, C. prolifera (Forsskål) J. V. Lamour-
oux 1809, Halimeda incrassata (J. Ellis) J. V. Lamouroux
1816, and Udotea abellum (J. Ellis & Solander) M. Howe
1904 (Okolodkov et al. 2007). In the Mexican Caribbean,
Coolia species have been found associated with Amphi-
roa J. V. Lamouroux, 1812 and Dictyota, J. V. Lamouroux,
1809; particularly, C. tropicalis is a very conspicuous spe-
cies on a variety of macroalgae along the coast (Almazán-
Becerril et al. 2015). In Bahía de La Paz, southern Gulf of
California, Coolia malayensis was isolated from Sargas-
sum horridum Setchell & N. L. Gardner 1924 (Sepúlveda-
Villarraga 2017). As far we know, there are no records of
harmful Coolia blooms in Mexico, and the toxicity of this
genus has not been investigated.
We isolated several Coolia strains from Bahía de La Paz
in the southwestern Gulf of California, which yielded new
information on the biogeography and biodiversity of the
genus Coolia in North Pacic coastal ecosystems in Me-
xico. We used morphological and molecular techniques
(DNA sequencing and phylogenetics) to determine the
taxonomic identity (species) of these strains.
MATERIALS AND METHODS
Site description
Bahía de La Paz is located on the Gulf of California
western coast (24°28'27" N, 110°33'2" W), delimited by
Isla San José to the north (24°58'23" N, 110°36'52" W), the
Baja California Peninsula to the west and south, and Isla
A B
Fig. 1. Maps of the Gulf of California and Bahía de La Paz indicating
the study area (A) and the location of the ve sampling sites (B). 1,
Ensenada de La Paz; 2, Isla Gaviota; 3 & 4, Balandra; 5, Isla San José.

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 188
40 µmol m-2 s-1 photon irradiance (12 : 12 h Light : Dark),
which are the standard conditions dened for the Marine
Dinoagellate Collection (CODIMAR, for its acronym in
Spanish). Clonal cultures were deposited in CODIMAR:
https://www.cibnor.gob.mx/investigacion/colecciones-
biologicas/codimar.
Strain identication
Microscopy. eca examination and morphometric
measurements of specimens of each strain were carried
out using a phase-contrast microscope (BX41TF; Olym-
pus, Tokyo, Japan) equipped with a U-ECA 2 magnica-
tion changer and an ocular micrometer. Photographic
records were captured with a digital camera (CoolSNAP-
Pro; Media Cybernetics, Bethesda, MD, USA) and an
image processing software (Image-Pro Plus 4.1 for Win-
dows; Media Cybernetics, Silver Spring, MD, USA). Cells
were observed live or xed with 4% (v/v) formaldehyde
or acidic Lugol’s solution. All morphometric measure-
ments of each strain were made with xed samples, and
a variable number from 6 to 25 specimens was measured
with the ocular micrometer to determine cell length, cell
width, and dorsoventral (DV) depth. For the plate pat-
tern analysis, cells were dissected by pressing the cov-
erslip with a needle to squash the theca, adding sodium
hypochlorite solution as needed. e kofoidian plate
tabulation was used for plate designation (Kofoid 1909).
Washington, NY, USA) through overlapping sieves (250,
150, and 20 µm) to remove large particles and concen-
trate the epiphytic dinoagellates (20–150 µm fraction).
Finally, the cell concentrates were transferred to sterile
plastic tubes (50 mL).
Cell isolation and strain establishment
Coolia strains were isolated from macroalgae of the
phyla Chlorophyta and Rhodophyta; besides, some sam-
ples were collected with a plankton net (20 μm, vertical
trawls) starting from 20 and 7 m depth at sites 2 and 5,
respectively. Cells were isolated with the micropipette
technique under an inverted microscope (Axiovert 100;
Carl Zeiss USA, ornwood, NY, USA). Isolated cells
were washed three times with sterile seawater and indi-
vidually transferred to 24-well tissue culture plates (Fal-
con, Corning, NY, USA), previously lled with either GSe
(Doblin et al. 1999), L1 (Guillard and Hargraves 1993), or
f/2 (Guillard and Ryther 1962) culture media. e latter
was modied by adding H2SeO3 (10-8 M) and reducing the
concentration of CuSO4 to 10-8 M (Anderson et al. 1984)
because the absence of selenium can limit dinoagellates
growth (Mitrovic et al. 2004) while copper can inhibit it
(Herzi et al. 2013). e seawater salinity used to prepare
culture media varied between 37 and 39 psu. Considering
the prevailing in-vitro temperature range during sample
collection, cultures were grown at 20 ± 2 or 25 ± 2°C, with
Table 1. General information on Coolia strains isolated from Bahía de La Paz, southern Gulf of California
Species CODIMAR
code
Collection
date
Isolation
date
Culture
medium
Incubation
temperature
(°C)
Substrate Locality Coordinates
C. malayensis CAPV-1 06/15/16 17/06/16 f/2 20 ± 2 Spyridia
lamentosa
Balandra3,4 24°19'08" N,
110°19'16" W
CAPV-2 06/25/16 22/06/16 GSe 25 ± 2 Laurencia sp.
Ceramium sp.
24°19'24.93" N,
110°19'44.60" W
CAPV-3 01/18/18 22/01/18 L1 20 ± 2 Ulva sp. Ensenada
de La Paz124°08'25" N,
110°21'08" W
CAPV-4 f/2
CAPV-5
CAJV-1 11/28/14 29/11/14 f/2 25 ± 2 Data not recorded
due to loss of
sample
Isla San José524°52'31.82" N,
110°33'27.28" W
CAJV-2
CAJV-3 GSe
C. palmyrensis CLJV-1 09/29/11 30/09/11 GSe 25 ± 2 Water column
CLJV-2 09/29/11 30/09/11 f/2
C. tropicalis CTJV-1 11/28/14 01/12/14 f/2 25 ± 2 Data not recorded
due to loss of
sample
CTJV-2 11/28/14 01/12/14 f/2
C. cf. canariensis CMPV-1 02/2003 02/2003 GSe 20 ± 2 Water column Isla Gaviota224°17'12.66" N,
110°20'31.92" W
CCJV-1 04/08/14 11/04/14 f/2 25 ± 2 Halimeda
discoidea
Isla San José524°52'31.82" N,
110°33'27.28" W
CCJV-2
CCJV-3 GSe
Superscript numbers indicate the location of sampling sites on the map (see also Fig. 1).

Morquecho et al. Coolia in Southwest Gulf of California
189 http://e-algae.org
et al. 1996). ermocycler conditions with D1R and D2C
were as follows: initial denaturation at 95°C for 1 min,
followed by 35 cycles of 95°C for 30 s, 45°C for 30 s, and
72°C for 1 min 45 s, with a nal extension period at 72°C
for 5 min. ermocycler conditions with ITSA and ITSB
were as follows: initial denaturation at 94°C for 3.5 min
followed by 35 cycles at 94°C for 50 s, 47°C for 60 s, and
72°C for 80 s, with a nal extension period at 72°C for
10 min. e amplication products were puried and
Sanger sequenced (Macrogen, Seoul, Korea), both for-
ward and reverse.
LSU rDNA and ITS sequence analysis and phyloge-
netic reconstruction. e LSU rRNA gene and ITS1-5.8S-
ITS2 region sequences obtained in this study were aligned
with Coolia sequences downloaded from the GenBank
database (https://www.ncbi.nlm.nih.gov/genbank/) (Be-
nson et al. 2013). Ostreopsis cf. ovata was selected as the
outgroup. Sequences were aligned online using MAFFT
v7.110 (Katoh and Standley 2013) with the default set-
ting values. Complete alignments were then checked
using BioEdit v7.0.5 (Hall 1999). e nal alignment con-
sisted of 935 (LSU, 70 taxa) and 539 (ITS, 45 taxa) base
pairs, including the introduced gaps. The best molecular
evolution model (GTR + G) was selected by jModelTest
(Posada 2008) based on the Akaike Information Criterion
for both Bayesian inference and maximum likelihood
(ML) analyses. Bayesian inference was performed us-
ing MrBayes 3.2 (Ronquist and Huelsenbeck 2003). Four
Markov chain Monte Carlo chains were run for 2,000,000
generations with sampling every 1,000 generations. e
rst 10% of burn-in trees were discarded. The Bayesian
posterior probability of each clade was determined. ML
analyses were conducted online (Boc et al. 2012) using
RaxML v7.2.6 (Stamatakis 2006). Bootstrap support was
assessed with 1,000 replicates.
Pairwise genetic distances based on LSU rDNA (D1–
D2) and ITS region sequences were calculated using the
PAUP*4b10 software (Swoord 2002).
RESULTS
We isolated 16 Coolia strains from the macroalgae Ce-
ramium sp., Halimeda discoidea Decaisne 1842, Lauren-
cia sp., Spyridia lamentosa (Wulfen) Harvey 1833, and
Ulva sp. (Table 1). From these, we identied the following
species using morphological and molecular techniques:
Coolia tropicalis (2 strains), C. palmyrensis (2 strains), C.
malayensis (8 strains), and the genetic lineage C. cf. ca-
nariensis (4 strains) (Table 2). Coolia cf. canariensis and
Specic theca measurements, such as Po length and plate
7" length (L) : width (W) ratio, were made using micro-
graphs captured with the magnication marker and mea-
surement tools of the image analysis software. To test for
signicant dierences between strains, cell size measure-
ments and Po length of each species were analyzed using
a one-way ANOVA.
Cells were processed for scanning electron micros-
copy (SEM). First, cells were washed with ltered sea-
water (0.2 μm, Supor 200 Membrane Disc Filters 60301;
Pall Laboratory) supplemented with antibiotics and then
treated with 10% or 15% Triton X-100 solution (X100;
Sigma-Aldrich, St. Louis, MO, USA) to remove external
membranes. Cell xation was accomplished with 2%
(v/v) glutaraldehyde solution (16020; Electron Micros-
copy Sciences, Delray Beach, FL, USA) for 1 h, followed
by post-xation with 1% (w/v) osmium tetroxide (O5500;
Sigma-Aldrich) overnight at 4°C. en, cells were washed
three times to remove xatives, rst with a 1 : 1 solution
of ltered seawater (0.22 μm) and distilled water, then
three times with distilled water. Finally, cells were col-
lected on polycarbonate lters (8 μm, Nuclepore 110414;
Whatman, Maidstone, Kent, UK) and dehydrated by im-
mersion in ethanol of progressive concentration (277649;
Sigma-Aldrich) from 30 to 100% in eight steps. Filters
were critical-point dried with CO2 (Samdri PVT3B; Tousi-
mis Research, Rockville, MD, USA), glued onto stubs, and
sputter-coated with palladium (vacuum desk II; Denton
Vacuum, Moorestown, NJ, USA). An additional batch of
samples was xed with 2% (v/v) glutaraldehyde only and
dehydrated as described above but dried with HMDS
(44091; Sigma-Aldrich), placing the specimens on poly-
carbonate lters (3 μm, Nuclepore 110412; Whatman).
Filters were examined under two scanning electron mi-
croscopes (S-3000N; Hitachi, Tokyo, Japan and JSM 6360
LV; Jeol Ltd., Tokyo, Japan).
DNA extraction, PCR amplication, and sequencing.
DNA was extracted from 25-mL cultures in the logarith-
mic growth phase using the FastDNA SPIN Kit for Soil
(Catalog # 6560-200; MP Biomedicals, Solon, OH, USA).
PCR amplications were performed in a 50-μL reaction
volume using a GoTaq Flexi DNA Polymerase kit accord-
ing to the manufacturer’s protocol (Promega, Madison,
WI, USA). e large subunit (LSU) rDNA D1 / D2 region
was amplied using D1R (5'-ACCCGCTGAATTTAAGCA-
TA-3') and D2C (5'-CCTTGGTCCGTGTTTCAAGA-3')
primers (Scholin et al. 1994) and the internal transcribed
spacer (ITS) 1-5.8S-ITS2 region of the rDNA using the
ITSA (5'-CCTCGTAACAAGGHTCCGTAGGT-3') and ITSB
(5'-CAGATGCTTAARTTCAGCRGG-3') primers (Adachi

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 190
Table 2. Morphometric characteristics of Coolia strains isolated from Bahía de La Paz, Gulf of California
Species Strain Cell size (µm) Measurements of specic plates Distinctive morphology
Length Width Dorsoventral
depth
Po length
(µm)
7" L : W ratio
C. malayensis CAPV-1 29 ± 2
26–31 (12)
30 ± 2
28–36 (12)
29 ± 2
26–33 (14)
9 ± 2
6–11 (10)
1 ± 0
1–2 (8)
Rounded cell
1' o-centered: hexagonal or heptagonal; narrow and elon-
gated
3' pentagonal
6" is the largest epitheca plate
3"' is the largest hypotheca plate
Numerous round pores (0.31 ± 0.03 µm, n = 30)
with poroids irregularly scattered
CAPV-2 30 ± 2
28–33 (22)
33 ± 2
28–36 (22)
31 ± 2
26–33 (12)
7 ± 1
6–9 (11)
1 ± 1
1–1 (11)
CAPV-3 31 ± 3
26–36 (16)
30 ± 3
26–36 (16)
31 ± 1
28–33 (11)
7 ± 1
6–8 (17)
1 ± 0
1–2 (16)
CAPV-4 32 ± 3
26–39 (26)
33 ± 3
28–39 (26)
32 ± 4
28–39 (12)
7 ± 0.4
6–7 (5)
1 ± 0
1–1 (6)
CAPV-5 33 ± 5
23–44 (25)
31 ± 5
23–39 (25)
29 ± 2
26–31 (12)
7 ± 0.8
6–9 (17)
1 ± 0.2
1–2 (13)
CAJV-1 31 ± 2
28–36 (26)
32 ± 2
28–36 (26)
30 ± 3
26–36 (32)
7 ± 0.6
6–8 (12)
1 ± 0
1–1 (13)
CAJV-2 32 ± 3
28–39 (26)
31 ± 2
28–33 (26)
31 ± 2
28–33 (12)
7 ± 1
5–9 (12)
1 ± 0.2
1–2 (11)
CAJV-3 33 ± 4
26–39 (13)
31 ± 2
26–33 (13)
31 ± 2
28–33 (7)
7 ± 1
6–8 (11)
1 ± 0.2
1–1 (11)
C. palmyrensis CLJV-1 26 ± 3
23–31 (25)
25 ± 2
21–28 (25)
23 ± 1
21–26 (15)
6 ± 1
5–8 (6)
2 ± 0.4
1–2 (6)
Nearly rounded cell
1' o-centered, hexagonal, narrow and elongated
3' pentagonal
6" is the largest epitheca plate
3"' is the largest hypotheca plate
Few round pores (0.32 ± 0.07 µm, n = 11) with poroids
CLJV-2 27 ± 3
23–33 (25)
25 ± 3
21–31 (25)
24 ± 2
20–26 (20)
6 ± 1
5–8 (21)
1 ± 0.5
1–2 (13)
C. tropicalis CTJV-1 35 ± 3
31–44 (25)
38 ± 3
31–44 (25)
38 ± 2
36–41 (25)
9 ± 1
7–12 (25)
3 ± 0.6
2–4 (11)
Nearly rounded cell
1' central, pentagonal, large, widest towards the ventral
side
3' pentagonal
1' the largest epitheca plate
3"' is the largest hypotheca plate
Moderately covered with scattered round pores
(0.28 ± 0.04 µm, n = 10) with poroids
CTJV-2 36 ± 4
31–44 (25)
40 ± 5
31–46 (25)
39 ± 3
36–44 (8)
9 ± 1
7–11 (9)
3 ± 0.7
2–4 (3)
C. cf. canariensis CMPV-1 34 ± 3
28–39 (26)
34 ± 3
28–39 (26)
32 ± 2
31–36 (20)
9 ± 1
7–10 (13)
2 ± 0.2
2–2 (11)
Rounded cell
1' central, hexagonal, large, widest towards the ventral side
3' pentagonal
1' is the largest epitheca plate
4"' is the largest hypotheca plate
Numerous round pores (0.32 ± 0.05 µm, n = 20)
Postcingular plates with concavities
CCJV-1 34 ± 2
31–39 (18)
34 ± 2
31–39 (18)
33 ± 2
28–36 (18)
10 ± 1
8–11 (17)
2 ± 0.1
1–2 (9)
CCJV-2 33 ± 3
28–39 (23)
34 ± 2
31–39 (23)
33 ± 2
31–39 (12)
10 ± 1
7–13 (19)
2 ± 0.2
1–2 (9)
CCJV-3 32 ± 3
26–39 (39)
32 ± 3
28–39 (39)
30 ± 3
26–33 (12)
9 ± 1
8–11 (20)
2 ± 0
2–2 (8)
Values are presented as average ± standard deviation (above) and range (n) (below).

Morquecho et al. Coolia in Southwest Gulf of California
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ese particular strains were susceptible to damage from
SEM treatments, which precluded documenting all their
characteristics. e dierences in cell length (F1, 48 = 0.19;
p = 0.89), width (F1, 48 = 1.5; p = 0.23), DV depth (F1, 33 =
0.15; p = 0.70), and Po length (F1, 25 = 1.1; p = 0.31) were not
signicant.
Coolia tropicalis M. A. Faust (Fig. 4). Cell shape round;
size variation between strains: 31–44 µm long, 31–46 µm
wide, and 36–44 µm DV depth (Table 2, Fig. 4D, F & G).
Epitheca slightly smaller than hypotheca (Fig. 4D & G).
eca surface smooth and moderately covered with scat-
tered round pores (Fig. 4B, C & E) of an average diameter
of 0.28 ± 0.04 µm. Po o-centerer beside apical plate 2'
(Fig. 4A, G & H), 7–11 µm in length (Table 2). Apical plate
1' located centrally in the epitheca; it is the widest on the
ventral side (Fig. 4B & D); plate 3' pentagonal (Fig. 4A &
B). Width-to-length ratio of the seventh precingular plate
(7") from 2–4 across strains (Table 2, Fig. 4E). Equatorial
deep cingulum bordered by list with smooth edges (Fig.
4D & G). Sulcus deep not reaching the cell antapex (Fig.
4D & F). A narrow list partially covering the sulcus on ei-
ther side (Fig. 4D), and a narrower list located on the an-
tapical side. In the hypotheca, postcingular plates 3"' and
4"' equally large (Fig. 4C & F). Plates 2"' and 5"' smaller,
and 1"' is the smallest plate (Fig. 4C & F). Antapical plate
1"" very small and plate 2"" quadrangular (Fig. 4C & F).
e dierences in cell length (F1, 48 = 1.5; p = 0.23), width
(F1, 48 = 2.7; p = 0.11), DV depth (F1, 31 = 3.3; p = 0.08), and Po
length (F1, 48 = 0.06; p = 0.8) were not signicant.
Coolia cf. canariensis (Fig. 5). Cell shape also round;
size variation across strains: 26–39 µm long and 28–39 µm
wide and in DV depth (Table 2). ecal plates with nu-
merous scattered pores (Table 2, Fig. 5C, D, F & I) of an
average diameter of 0.32 ± 0.05 µm. Hypotheca with more
plate pores than epitheca (Fig. 5D & E). Po length 7–13 µm
(Table 2), o-centered (Fig. 5A, B & F) beside apical plate
2', easy to observe under light microscopy (Fig. 5A–C).
e hexagonal 1' is the largest plate of the epitheca (Table
2), with a central position (Fig. 5A, C & F). Plate 2' narrow
and elongated, in direct contact with Po along its left and
dorsal sides (Fig. 5C, F & G). Plate 3' is pentagonal (Table
2) and contacts plates 1', Po, 2', 4", 5", and 6" (Fig. 5B, C
& F). e pentagonal 6" is the largest of the precingular
plates (Fig. 5C & H). Width-to-length ratio of the seventh
precingular plate (7") from 1–2 across strains (Table 2, Fig.
5E). In the hypotheca, rst postcingular plate (1"') triangu-
lar and small; 2"', 3"', 4"', and 5"' large and elongated (Fig.
5D, H & I). Antapical plate 1"" elongated and narrow, par-
allel to the sulcus and bordering the posterior plate (Sp)
and precingular plates 1"' and 2"'; plate 2"" pentagonal,
C. palmyrensis had not been documented previously in
Mexican coastal waters, and C. tropicalis was found in the
Gulf of California for the rst time. Coolia species, par-
ticularly at Isla San José, shared their habitat with other
epiphytic dinoagellates of the genera Amphidinium, Fu-
kuyoa, Gambierdiscus, Ostreopsis, and Prorocentrum.
Species descriptions
Coolia malayensis Leaw, P. -T. Lim & Usup (Fig. 2).
Cell shape round; size variation across strains: 23–44 µm
long, 23–39 µm wide, and 29–39 µm DV depth (Table 2).
Epitheca slightly smaller than hypotheca (Fig. 2E). e-
cal plates smooth and irregularly scattered with round
pores of an average diameter of 0.31 ± 0.03 µm (Table 2,
Fig. 2A, E & G), and 3–6 poroids inside each pore (Fig.
2I). Po 5–11 µm long, narrow (Table 2, Fig. 2B, E & F), and
o-centered, neighboring the apical plate 2' (Fig. 2E & F).
First apical plate (1') narrow and oblong; plate 2' narrow,
elongated, and in direct contact with Po along its left and
dorsal sides (Fig. 2B, E & F). Plate 3' pentagonal (Table 2,
Fig. 2B & E), contacting plates 1', Po, 2', 4", 5", and 6" (Fig.
2E & F). Precingular 6" is the largest plate in the epithe-
ca (Fig. 2A, B & D). Width-to-length ratio of the seventh
precingular plate (7") from 1–2 between strains (Table 2).
In the hypotheca, postcingular plates 3"' and 4"' are the
largest (Fig. 2C, G & H), the former quadrangular and the
latter triangular. Plates 2"' and 5"' smaller; plate 1"' is the
smallest (Fig. 2C, G & H). e dierences in cell length
(F7, 158 = 3.2; p = 0.003), width (F7, 158 = 2.3; p = 0.03), and
Po length (F7, 87 = 3.6; p = 0.002) were signicant across
strains, while dierences in DV depth were not signicant
(F7, 104 = 1.3; p = 0.25).
Coolia palmyrensis Karafas, Tomas & York (Fig. 3).
Cell shape round; size variation between strains: 23–31
µm long, 21–31 µm wide, and 20–26 µm DV depth (Table
2, Fig. 3A, C & F). eca surface smooth and sparsely dot-
ted with pores of an average diameter of 0.32 ± 0.07 µm
(Table 2, Fig. 3C & F–H), and 3–6 poroids inside each pore
(Fig. 3I). Po short in length (5–8 µm), slightly curved in
apical view (Fig. 3D & E), and o-centered, abutting api-
cal plate 2' (Fig. 3G). Plate 1' hexagonal and narrow (Fig.
3B, C & G); precingular 6" is the largest plate in the epi-
theca (Fig. 3C & G). Width-to-length ratio of the seventh
precingular plate (7") from 1–2 between strains (Table
2, Fig. 3C). In the hypotheca, postcingular 3"' is the larg-
est plate, followed by plates 2"' and 4"', which are almost
equal in size (Fig. 3F & H). Plate 2"' medium-sized; 1"' is
the smallest plate (Fig. 3F & H). Antapical plate 1"" larger
than plate 2"" and the two contact each other (Fig. 3F).

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 192
Fig. 2. Coolia malayensis. Phase-contrast light microscopy (A–C) and scanning electron microscopy (D–I). (A) Epitheca with apical 1' and precin-
gular 6" plates (strain CAPV-1). (B) Epitheca with plate tabulation (strain CAPV-1). (C) Hypotheca with plate tabulation (strain CAPV-5). (D) Epitheca
in lateral view with apical 1' and precingular 6" and 7" plates (strain CAJV-1). (E) Theca in dorsal view with apical pore plate (Po), apical 2' and 3',
precingular 2", 3", and 4", and postcingular 2"' and 3"' plates (strain CAJV-1). (F) Po plate (strain CAJV-1). (G & H) Hypotheca with postcingular plates
(strain CAPV-5). (I) Poroids; arrows mark the view from the inner side of the plate (strain CAPV-5). Scale bars represent: A–E, G & H, 20 µm; F, 5 µm; I,
1 µm.
AC
D
B
E
G
F
H I

Morquecho et al. Coolia in Southwest Gulf of California
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Fig. 3. Coolia palmyrensis. Phase-contrast light microscopy (A, B, D, E, G & H) and scanning electron microscopy (C, F & I). (A) Theca in ventral
view showing cingulum and sulcus (strain CLJV-1). (B) Epitheca with the apical 1' and precingular 6" and 7" plates (strain CLJV-1). (C) Theca in ven-
tral view showing apical 1' and most of the precingular plates (strain CLJV-2). (D & E) Epitheca with apical pore plate (Po) in apical (strain CLJV-2)
and dorsal (strain CLJV-1) view. (F) Hypotheca with plate tabulation (strain CLJV-2). (G) Epitheca with plate tabulation (strain CLJV-2). (H) Hypothe-
ca with plate tabulation (strain CLJV-1). (I) Zoom of pores with a dissimilar number of poroids (arrows). Scale bars represent: A–H, 20 µm; I, 1 µm.
AC
D
B
E
G H
I
F

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 194
in the ITS1 / 5.8S / ITS2 tree, these strains were separated
into a subgroup but shared similarities with both Atlantic
and Indo-Pacic strains. Coolia palmyrensis from Isla San
José was grouped with strains from Palmyra atoll (in the
Central Pacic), Dominican Republic, and Brazil. A sepa-
rate group was composed of three closely related species:
C. guanchica, C. canariensis, and C. cf. canariensis, each
contacting the Sp and postcingular plates 2"', 3"', 4"', and
5"' (Fig. 5D). Postcingular plates with concavities giving
them a rough appearance (Fig. 5E & I). e dierences in
cell width (F3,102 = 5.7; p = 0.001), and DV depth (F3, 58 = 7.9;
p = 0.0002) were signicant across strains, while the dif-
ferences in cell length (F3,102 = 2.3; p = 0.08), and Po (F3, 64 =
2.1; p = 0.1) were not signicant.
Molecular characterization
Fourteen Coolia strains were genetically character-
ized based on LSU rDNA (D1 / D2 region) and nine based
on ITS1 / 5.8S / ITS2 sequencing (Table 3). e Bayes-
ian phylogenetic reconstruction showed well-supported
clades representing seven of the eight currently accept-
ed Coolia species (Figs 6 & 7). Bayesian posterior prob-
abilities and the ML bootstrap values strongly supported
clade clustering, dening three groups. Strains were
grouped into clades, corresponding to C. malayensis (8
strains), C. palmyrensis (1 strain), C. tropicalis (1 strain),
and the C. cf. canariensis linage (4 strains). A large group
was composed of clades C. malayensis, C. monotis, C.
santacroce, and C. palmyrensis. In the LSU (D1–D2 re-
gion) tree, Coolia malayensis from Bahía de La Paz was
grouped mainly with strains from the Indo-Pacic region;
Fig. 4. Coolia tropicalis. Phase-contrast light microscopy (A–C) and scanning electron microscopy (D–H). (A) Epitheca with apical pore plate (Po)
and apical plate 3' (strain CTJV-2). (B) Epitheca with plate tabulation (strain CTJV-1). (C) Hypotheca with plate tabulation (strain CTJV-1). (D) Theca
in ventral view with plate tabulation (strain CTJV-1). (E) Precingular plate 7" (strain CTJV-1). (F) Theca in ventral-antapical view showing hypotheca
tabulation (strain CTJV-1). (G) Theca in dorsal view with plate tabulation (strain CTJV-1). (H) Po plate (strain CTJV-1). Scale bars represent: A–D, F & G,
20 µm; E, 10 µm; H, 5 µm.
AC D
B
E H
GF
Table 3. GenBank accession numbers assigned to the obtained
Coolia sequences
Species CODIMAR
code
GenBank accession No.
LSU rDNA
(D1 / D2)
ITS1 / 5.8S /
ITS2
C. malayensis CAPV-1 MW865385 -
CAPV-2 MW865386 ON943078
CAPV-3 MW865387 -
CAPV-4 MW865388 -
CAPV-5 MW865389 ON943079
CAJV-1 MW865390 ON943077
CAJV-2 MW865391 -
CAJV-3 MW865392 -
C. palmyrensis CLJV-1 MW865393 ON943084
C. tropicalis CTJV-2 MW865396 ON943085
C. cf. canariensis CMPV-1 ON943083
CCJV-1 MW865382 ON943080
CCJV-2 MW865383 ON943081
CCJV-3 MW865384 ON943082
LSU, large subunit; ITS, internal transcribed spacer.

Morquecho et al. Coolia in Southwest Gulf of California
195 http://e-algae.org
C. malayensis and C. palmyrenis (<0.06) but high in C.
canariensis (0.19), which was comparable to interspecic
distances (0.11–0.41). Genetic distances based on ITS
region sequences (Table 5) showed that intraspecic
values were low in C. malayensis and C. tropicalis (<0.09)
but high in C. canariensis (0.19). e latter was also
comparable to interspecic distances (0.19–0.50).
establishing a well-supported monophyletic clade. Coo-
lia cf. canariensis from Bahía de La Paz was grouped with
isolates from Spain (Mediterranean Sea) and Korea (Figs
6 & 7). In a third group, C. tropicalis showed a monophy-
letic origin, and the strain CTJV-2 was closely related to
Australian, Atlantic, and Indo-Pacic strains.
Genetic distances based on LSU (D1–D2) sequences
(Table 4) showed that intraspecic values were low in
AC
D
B
E
G
F
H I
Fig. 5. Coolia cf. canariensis. Phase-contrast light microscopy (A–D), and scanning electron microscopy (E–I). (A) Epitheca with apical pore plate
(Po) and apical plate 1' (strain CCJV-1). (B) Epitheca with apical plate 3' and Po (strain CCJV-1). (C) Epitheca with plate tabulation (strain CCJV-1). (D)
Hypotheca with plate tabulation (strain CCJV-2) and posterior plate (Sp). (E) Theca in apical view showing epitheca tabulation and postcingular 5"
plate (arrow) with concavities that give a rough appearance (strain CCJV-1). (F) Epitheca showing Po and apical plates 1'–3' (strain CCJV-1). (G) Po
plate (strain CCJV-1). (H) Theca in lateral view with plate tabulation (strain CCJV-2). (I) Hypotheca with plate tabulation; concavities also apparent
(strain CCJV-2). Scale bars represent: A–F, H & I, 20 µm; G, 5 µm.

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 196
Fig. 6. Molecular phylogeny of Coolia using Bayesian inference based on partial large subunit rRNA (D1−D2 region) gene sequences. New se-
quences are marked in red. Eight species and one lineage were labeled and indicated with vertical lines. The scale bar indicates the number of
nucleotide substitutions per site; branch length is shown to scale. Numbers adjacent to each branch indicate the statistical support (left: Bayesian
posterior probabilities; right: maximum likelihood bootstrap support values), to avoid overlap some numbers are located outside the branches
and their position is indicated by arrows. Bayesian posterior probabilities lower than 0.9 and bootstrap support values lower than 50 are not
shown. Dotted line means a half-length. Asterisks indicate maximum likelihood bootstrap support value of 100% and a posterior probability of 1.0.

Morquecho et al. Coolia in Southwest Gulf of California
197 http://e-algae.org
Fig. 7. Molecular phylogeny of Coolia using Bayesian inference based on the internal transcribed spacer (ITS) 1-5.8S-ITS2 rDNA gene sequences.
New sequences are marked in red. Eight species and one lineage were labeled and indicated with vertical lines. The scale bar indicates the num-
ber of nucleotide substitutions per site; branch length is shown to scale. Numbers adjacent to each branch indicate the statistical support (left:
Bayesian posterior probabilities; right: maximum likelihood bootstrap support values), to avoid overlap some numbers are located outside the
branches and their position is indicated by arrows. Bayesian posterior probabilities lower than 0.9 and bootstrap support values lower than 50 are
not shown. Asterisk indicate maximum likelihood bootstrap support value of 100% and a posterior probability of 1.0.

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 198
2015) than in our specimens (23–31 and 20–26 µm, re-
spectively) (Table 2). Similarly, in the original description
of C. tropicalis, the cell length and width ranges of 23–40
and 25–39 µm, respectively, were somewhat wider than
in our specimens (31–44 µm length and 31–46 µm wide).
However, DV depth, which reached 65 µm according to
Faust (1995), did not exceed 44 µm (Table 2) in our speci-
mens. e description of Coolia cf. canariensis was simi-
lar to the original C. canariensis description by Fraga et
al. (2008). e distinctive features of this taxon, i.e., mean
cell size (27.2–38.4 µm long and 25.6–40 µm wide), Po
length (8 µm), the large size and central position of plate
1', and plate 7" size overlap with those of our specimens
(Table 2).
e taxonomic classication of the genus Coolia is
further supported by combining morphometric analysis
with molecular characterization techniques. Research
using both methods over the past decade has improved
the delimitation of morphometric characteristics that
were also supported phylogenetically. In particular, the
shape and position of the rst apical plate have been
valuable for dierentiating the two major Coolia spe-
cies complexes (Karafas et al. 2015). e strains included
DISCUSSION
is study focused on isolating and characterizing the
species composition of the genus Coolia in Bahía de La
Paz, southwestern Gulf of California. Species were char-
acterized using morphometric and molecular analyses.
e 16 well-established strains comprised the species
Coolia malayensis, C. palmyrensis, C. tropicalis, and the
genetic lineage C. cf. canariensis.
Our results document for the rst time the presence
of Coolia cf. canariensis and C. palmyrensis in Mexico
and also report C. tropicalis in the Gulf of California for
the rst time. A previous molecular characterization of
a Coolia strain from Bahía de La Paz with the 28S rDNA
marker also demonstrated the presence of C. malayensis
(Sepúlveda-Villarraga 2017, Ramos-Santiago 2021).
Except for subtle morphometric dierences, the Coolia
strains found in the present study are comparable to the
original taxa descriptions. Leaw et al. (2010) reported a
Po length of 5 µm in C. malayensis, while our specimens
measured 5–11 µm (Table 2). In the original description
of C. palmyrensis, cell length (16–26 µm) and DV depth
(21–30 µm) ranges were slightly wider (Karafas et al.
Table 4. Pairwise genetic distances between Coolia species / strains based on LSU rDNA (D1−D2) sequences
No. Species GenBank No. / strain 1 2 3 4 5 6 7 8 9
1C. cf. canariensis MW865382 / CCJV-1 -
2C. canariensis AM902738 / VGO787 0.19 -
3C. guanchica KU514007 / TNW 0.22 0.26 -
4C. malayensis MW865385 / CAPV-1 0.38 0.39 0.37 -
5C. malayensis MW865386 / CAPV-2 0.37 0.37 0.36 0.03 -
6C. palmyrensis MW865393 / CLJV-1 0.33 0.34 0.32 0.18 0.19 -
7C. palmyrensis MH319015 / UTSHI3C5 0.33 0.33 0.33 0.19 0.19 0.06 -
8C. santacroce KT288057 / Cos1503-1 0.36 0.37 0.35 0.13 0.14 0.14 0.15 -
9C. monotis KX845010 / CMBZT14 0.37 0.39 0.35 0.14 0.14 0.17 0.18 0.11 -
10 C. tropicalis MW865396 / CTJV-2 0.38 0.41 0.37 0.39 0.39 0.37 0.38 0.40 0.39
LSU, large subunit.
Table 5. Pairwise genetic distances between Coolia species / strains based on ITS sequences
No. Species GenBank No. / strain 1 2 3 4 5 6 7 8 9 10
1C. malayensis ON943077 / CAJV-1 -
2C. malayensis KP172273 / Cm1303-1 0.09 -
3C. malayensis KR605275 / wzd01 0.09 0.05 -
4C. monotis KF896873 / Dn202EHU 0.20 0.20 0.19 -
5C. santacroce MT295348 / LM123 0.23 0.22 0.24 0.22 -
6C. palmyrensis MT295347 / LM112 0.29 0.31 0.32 0.30 0.33 -
7C. cf. canariensis ON943080 / CCJV-1 0.45 0.41 0.43 0.41 0.39 0.43 -
8C. canariensis MZ801757 / G17 0.40 0.40 0.42 0.39 0.37 0.41 0.19 -
9C. guanchica MK945760 / TnW 0.45 0.43 0.42 0.42 0.41 0.45 0.31 0.35 -
10 C. tropicalis ON943085 / CTJV2 0.49 0.48 0.48 0.47 0.47 0.49 0.46 0.46 0.40 -
11 C. tropicalis KR605303 / DPR_5 0.50 0.48 0.49 0.47 0.47 0.49 0.45 0.46 0.40 0.03
ITS, internal transcribed spacer.

Morquecho et al. Coolia in Southwest Gulf of California
199 http://e-algae.org
the work based on the LSU rDNA and ITS1 / 5.8S / ITS2
phylogenetic analyses (Laza-Martínez et al. 2011, Jeong
et al. 2012, Momigliano et al. 2013, David et al. 2014,
2020, Leaw et al. 2016, de Queiroz-Mendes et al. 2019,
Nascimento et al. 2019). David et al. (2014) consider that
the degree of separation is still insucient to delineate
clades. However, they hypothesized that there are likely
two cryptic species within the C. canariensis clade; giv-
en the insuciency of genetic data, they proposed that
this should be referred to as C. cf. canariensis. Jeong et al.
(2012) and Leaw et al. (2016) highlight the need to con-
duct additional comparative morphological and DNA se-
quence (in particular, ITS) analyses to assess whether or
not they are cryptic species.
Based on LSU and ITS rDNA phylogenies, Nascimen-
to et al. (2019) separate the C. canariensis complex into
three phylogroups. Our results reinforce the cryptic-spe-
cies hypothesis proposed by David et al. (2014) and the
separation delineated by Nascimento et al. (2019) since
our strains were grouped within the phylogroup III. Al-
though our strains are morphologically very similar to
the C. canariensis type species, we found dierences in
cell size relative to the phylogroup II. Cell size within the
strain UNR-25 from Brazil was 24.9 ± 2.3 μm long, with a
DV depth of 27.5 ± 2.4 μm and a Po of 7.8 ± 1.1 μm long;
our strains had a size variation of 26–39 µm long, 28–39
µm wide and in DV depth, and a Po length pf 7–13 µm
(Table 2). However, additional comparative morpho-
metric, genetic, and life-cycle characterization analyses
are required to determine whether or not they are cryp-
tic species. For example, the slight anteroposterior cell
compression in the strain UNR-25 found by Nascimento
et al. (2019) was also identied in some of the specimens
studied in the present study (Fig. 5F). is feature may be
a morphological variation in cell growth, theca develop-
ment, or associated with a life cycle stage.
Genetic distances based on LSU (D1–D2) and ITS
region sequences appear to support that C. canariensis
is a cryptic species. Genetic distances within the C. ca-
nariensis strains are similar or even greater than those
among Coolia species (Tables 4 & 5).
e strain CMPV-1 (Tables 1 & 2) was morphologically
(CODIMAR) (Morquecho and Reyes-Salinas 2004) and
genetically (JQ638943) (Herrera-Sepúlveda et al. 2013)
identied as C. monotis. In this study, however, we de-
termined that this strain belongs to the C. cf. canariensis
clade (Figs 6 & 7). e sequence JQ638943 retrieved from
GenBank was previously used by David et al. (2014), Gó-
mez et al. (2016), and Nascimento et al. (2019), who also
concluded that this strain, attributed initially to C. mono-
in the present study were mainly grouped within the C.
monotis and C. canariensis species complexes based on
the LSU rDNA and ITS1 / 5.8S / ITS2 phylogenetic analy-
ses; these ndings are consistent with other related stud-
ies (Mohammad-Noor et al. 2013, Karafas et al. 2015,
Gómez et al. 2016, Leaw et al. 2016, Lewis et al. 2018, Lars-
son et al. 2019, Nascimento et al. 2019, David et al. 2020,
Abdennadher et al. 2021). e results of the LSU and ITS
analyses strongly suggest that C. santacroce is closely re-
lated to C. monotis and that C. palmyrensis emerges ear-
lier than both C. monotis and C. malayensis (Figs 6 & 7).
is same clade arrangement was observed by Karafas et
al. (2015) and Nascimento et al. (2019). In the C. canar-
iensis complex (Figs 6 & 7), 1' is the largest plate and has a
central position in the epitheca, whereas in the C. mono-
tis complex (Figs 6 & 7), this plate is oblong and narrow,
in a central position or to the left (Karafas et al. 2015, and
references therein). e shape of plate 4' and the size of
plate 6" separate C. tropicalis from C. malayensis, while
the thecal surface ornamentation dierentiates C. tropi-
calis from C. canariensis (de Queiroz-Mendes et al. 2019).
It is also worth noting that morphometric features have
been proposed to discriminate closely related Coolia spe-
cies, although with no phylogenetic support. ese in-
clude cell size, Po size, pore size and density, presence of
poroids, W : L ratio of plate 7', L : L ratio of the APC (apical
pore complex) and 2', size and shape of plates 1' and 3',
W : L ratio of 7" and 6", and size and shape of 3"' (Fraga et
al. 2008, Karafas et al. 2015, Leaw et al. 2016, de Queiroz-
Mendes et al. 2019).
Based on morphometric and theca traits, we initially
considered that our strains corresponded to C. canarien-
sis. However, the phylogenetic analysis showed that these
strains belong to the C. cf. canariensis lineage, specically
grouped within the phylogroup III dened by Nascimen-
to et al. (2019) for the C. canariensis complex (Figs 6 & 7).
is lineage has also been reported for strains from the
Canary Islands, Mediterranean Spain, Korea, Australia,
China, and Brazil (Laza-Martínez et al. 2011, Jeong et al.
2012, Momigliano et al. 2013, David et al. 2014, Leung et
al. 2017, de Queiroz-Mendes et al. 2019, Nascimento et
al. 2019). e lineage corresponding to the holotype (C.
canariensis sensu stricto) had not been observed previ-
ously outside the Canary Islands (David et al. 2020). Coo-
lia canariensis was described by Fraga et al. (2008) based
on three strains that were split phylogenetically into two
sister lineages with no morphological dierences be-
tween them. In this work, we found that the C. canariensis
strains were clustered in a lineage that excludes the type
species (strain VGO787, AM902738), which agrees with

Algae 2022, 37(3): 185-204
https://doi.org/10.4490/algae.2022.37.9.2 200
changing global climate. Núñez-Vázquez et al. (2019) re-
ported that CFP is the second-most prevalent form of hu-
man seafood poisoning in Mexico, after paralytic shellsh
poisoning. From 1984 to 2013, 52% of the 464 ciguatera
cases occurred in Baja California Sur, where this study
was carried out. Interestingly, ciguatera human poisoning
and ciguatoxins have been reported on the small rocky is-
land El Pardito (Núñez-Vázquez et al. 2013), near the San
José Lagoon, where several Coolia species were isolated.
Coolia species can contribute to mucilage production
and formation of benthic blooms leading to the develop-
ment of mucilaginous aggregates, which can have seri-
ous environmental eects, including lower water quality,
substrate deposits that foster the growth and transport of
other harmful microorganisms, benthic fauna mortality,
and contact dermatitis in humans and terrestrial fauna
(Lewis et al. 2018).
In summary, this is the rst comprehensive taxonomic
report of the Coolia species composition in the southwest
Gulf of California. e phylogenetic analysis revealed a
diverse and heterogeneous Coolia species assemblage.
e Coolia cf. canariensis lineage and C. palmyresis had
not previously been reported along the Mexican coasts,
and their geographic range now stretches to subtropi-
cal coasts of the North American Pacic. Given the great
morphological similarity among taxa, the reports of C.
monotis in the Mexican Pacic most likely correspond to
C. malayensis. e occurrence of the toxic C. tropicalis in
the Gulf of California had not been reported previously.
Given the current context of global climate change, fur-
ther studies are needed to delimit the species composi-
tion of the genus Coolia and assess the factors that aect
their growth to determine the potential for harm and tox-
icity of this genus and other epiphytic dinoagellates.
ACKNOWLEDGEMENTS
Jorge Angulo-Calvillo and Enrique Calvillo-Espinoza
provided technical assistance in the eld as divers and
boat drivers. Amada Reyes-Salinas, Alejandra Mazar-
iegos-Villarreal and Ariel Cruz-Villacorta provided tech-
nical assistance. elma Castellanos, Angel Carrillo and
Goretty Caamal provided technical advice and infrastruc-
ture of the Molecular Microbial Ecology Laboratory. All
are at CIBNOR. Yuri Okolodkov (Universidad Veracru-
zana) assisted during the 2016 macroalgae sampling.
Laura E. Gómez-Lizárraga (SAMEB, ICMyL-UNAM) also
provided technical assistance. is study was supported
by the CIBNOR project 20014, and the CONACYT A1-S-
tis, actually belongs within the C. cf. canariensis clade.
Based on our results, we suggest that this sequence in the
GenBank be revised and reclassied.
It is also worth noting that our C. malayensis strains in
the ITS tree were grouped separately, suggesting a certain
degree of diversication. Nascimento et al. (2019) also
hypothesize that this species contains a high molecular
diversity. To conrm this apparent genetic diversication
in the southern Gulf of California populations, it will be
necessary to isolate strains from other locations in the
Mexican Pacic to gather complete ITS sequences.
Our study documented four of the eight currently ac-
cepted Coolia species. is species composition has also
been found in the Great Barrier Reef, Australia (Momi-
gliano et al. 2013), China (Leung et al. 2017), and Brazil
(Nascimento et al. 2019, Tibiriçá et al. 2020). ese nd-
ings suggest that the genus Coolia is widespread in the
Mexican Pacic, with a predominance of C. malayensis.
Given the high morphological similarity among taxa, it
is likely that the records of C. monotis in studies on phy-
toplankton species composition in the Mexican Pacic
(Okolodkov and Gárate-Lizárraga 2006) correspond to C.
malayensis. is is currently recognized as the most wide-
spread species, occurring between latitudes 36° S and 34°
N, while C. monotis appears to be restricted to the North
Atlantic and the Mediterranean Sea (Larsson et al. 2019).
As described recently, C. canariensis (Fraga et al. 2008)
and C. palmyrensis (Karafas et al. 2015) still lack global
distribution patterns. Together with C. areolata and C.
tropicalis, these species likely occur along the tropical
and subtropical Pacic coastlines. e latter two species
have been reported in Isla del Coco National Park, Costa
Rica (Vargas-Montero et al. 2012).
e Coolia species assemblage found in this study is
potentially toxic. e production of moderately toxic bio-
active compounds by C. malayensis and C. palmyrensis
has been reported (Karafas et al. 2015, Wakeman et al.
2015, Li et al. 2020). However, C. tropicalis and C. malay-
ensis are the only two species with a more accurate toxi-
cological characterization and are recognized due to the
production of cooliatoxins, which have not been chemi-
cally identied yet (Holmes et al. 1995), and two 44-meth-
yl-gambierone isomers (Murray et al. 2020), previously
limited to Gambierdiscus spp. (Tibiriçá et al. 2020). ere-
fore, further research is needed to characterize the toxic-
ity of Coolia species and their inuence on ecotoxicology
in Bahía de La Paz and the Gulf of California.
While Coolia BHABs have not been recorded in the
Gulf of California, the potential threat from these species
cannot be overlooked, particularly given the projected

Morquecho et al. Coolia in Southwest Gulf of California
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CONFLICTS OF INTEREST
e authors declare that they have no potential con-
icts of interest.
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