Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

The Synonymy of the Toxic Dinoflagellates Prorocentrum

mexicanum and P. rhathymum and the Description of
P. steidingerae sp. nov. (Prorocentrales, Dinophyceae)

meza
Fernando Go , Dajun Qiub & Senjie Linc,d
a Carmen Campos Panisse 3, Puerto de Santa Mar
a, E-11500, Spain
b CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Science, 164
West Xingang Road, Guangzhou 510301, China
c Marine Biodiversity and Global Change Research Center, Xiamen University, Xiamen 361101, China
d Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, Groton, Connecticut 0634, USA

Keywords
Benthic Dinoflagellata; Caribbean Sea;
epiphytic Dinophyta; harmful algal blooms;
microalgae; molecular phylogenetics; new
species; red tides.
Correspondence

mez, Carmen Campos Panisse 3,
F. Go
Puerto de Santa Mar
a E-11500, Spain
Telephone number: +34 628460564;
FAX number: +1 860 4059153;
e-mail: fernando.gomez@fitoplancton.com
Received: 11 November 2016; revised 9
February 2017; accepted February 10, 2017.
Early View publication March 10, 2017
doi:10.1111/jeu.12403
ABSTRACT
Prorocentrum mexicanum and P. rhathymum are toxicologically important
dinoflagellates, but their relationship is not well defined. We investigated strains
from Puerto Rico and Brazil by light and scanning electron microscopies. We pro-
vide molecular data from a strain isolated near the type locality of P. rhathymum,
and the first morphological and molecular data from the South Atlantic Ocean.
The rRNA gene (rDNA) sequences of the Puerto Rican and Brazilian strains were
identical, and their morphologies fit the description of P. rhathymum. In the
molecular phylogenies, the globally distributed populations under the names
P. mexicanum and P. rhathymum are intermixed and branched together, except
for several divergent strains from Florida and Cuba. We examined the original
descriptions and iconotypes of the species Prorocentrum maximum, P. brochii,
P. mexicanum, and P. rhathymum. We conclude that P. maximum sensu Schil-
lers figure 41a corresponds to the earlier description of this species; the split of
P. mexicanum and P. rhathymum was based on a misidentification because
P. mexicanum sensu Corte
s-Altamirano & Sierra-Beltr
an corresponds to P. tex-
anum var. cuspidatum; and P. rhathymum is a junior synonym of P. mexicanum.
Several Floridian and Cuban strains correspond to a new species, which we
describe as Prorocentrum steidingerae sp. nov.

PROROCENTRUM is a distinctive genus of thecate
dinoflagellate that is markedly compressed laterally, and
the theca consists of two large valves joined together at
their marginal edges. Prorocentrum species are found
worldwide; they can be benthic, planktonic, tychoplank-
tonic, and vary widely in size and shape (Faust and Gul-
ledge 2002; Steidinger and Tangen 1997). The epiphytic or
epibenthic dinoflagellates received little attention until
Yasumoto et al. (1977) found that some species produce
toxins responsible for human diseases such as the ciguat-
era fish poisoning. Some species of Prorocentrum are
nontoxic but others are able to produce different toxins
(Steidinger 1983).
The earlier observations of the epiphytic dinoflagellates
were often based on resuspended cells found in the plank-
ton samples. Schiller (1933) proposed the new combination
P. maximum (Gourret) J. Schiller for the taxon described as
Postprorocentrum maximum Gourret 1883, and proposed
P. brochii as a synonym. Osorio-Tafall (1942) described
several new species of Prorocentrum from plankton sam-
ples that were collected in the Mexican Pacific. He
reported cells identified as P. maximum and described a
closely related species as P. mexicanum from Salina Cruz,
Oaxaca. These earlier descriptions were mainly based on
the valve shape, presence and shape of an apical spine,
and the general valve ornamentation, with scarce detail in
the type and distribution of valve pores (Osorio-Tafall 1942;
Schiller 1933). From 1970s, the scanning electron micro-
scopes (SEM) allowed detailed studies of the valve orna-
mentation. Loeblich et al. (1979) described Prorocentrum
rhathymum from a strain isolated from the surface waters
of the Caribbean Sea at Cinnamon Bay, St. John, U.S. Vir-
gin Islands, and subsequently reported from plankton in the
Mediterranean Sea (Estrada 1979) and from macroalgae in
coral reefs of the Eastern Pacific Ocean (Fukuyo 1981).
Currently, P. rhathymum is reported worldwide primarily in
tropical and subtropical waters (see Lim et al. 2013), and

rard-Therriault et al. 1999; Levasseur
also in cold waters (Be

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668 Journal of Eukaryotic Microbiology 2017, 64, 668677

mez et al.
Go
et al. 2003). Strains of P. rhathymum have been reported
to produce several toxins (An et al. 2010; Caillaud et al.
2010; Nakajima et al. 1981; Naves et al. 2006; Pearce et al.
2005). Prorocentrum maximum and P. rhathymum have
been considered synonyms of P. mexicanum (Faust 1990;
Faust and Gulledge 2002; Steidinger 1983; Steidinger and
Tangen 1997).
The taxonomical controversy in the synonymy has
remained for over two decades. Corte
s-Altamirano and
Sierra-Beltr
an (2003) reinstated P. rhathymum as an inde-
pendent species based on plankton samples collected dur-
ing red tides in the Gulf of California and the Mexican
Pacific. They reported that the main differences between
P. rhathymum and P. mexicanum concern the fact that the
latter is a planktonic, lenticular in lateral view, has a centrally
located pyrenoid, an areolated valve surface, and a large,
winged, and two- or three-pointed apical spine, while
P. rhathymum is a benthic/epiphytic species with smooth
valve surface bearing trichocysts pores and a simple apical
spine, and lacking the pyrenoid (Corte
s-Altamirano and
Sierra-Beltr
an 2003). However, despite the important mor-
phological differences reported by these authors, the avail-
able sequences named as P. mexicanum or P. rhathymum
branched together and the species split is not supported
(as P. mexicanum for Cohen-Fern
andez et al. 2010; Daugb-
jerg et al. 2000; Grzebyk et al. 1998; Murray et al. 2007;
Scorzetti et al. 2009; Stern et al. 2010; as P. rhathymum
for Caillaud et al. 2010; Herrera-Sepu
lveda et al. 2013,
2015; Lim et al. 2013; Mohammad-Noor et al. 2007a; Mur-
ray et al. 2009; Pearce and Hallegraeff 2004; Su-Myat and
Koike 2013).
In this study, we document the morphological and
molecular data of the taxon that unequivocally corre-
sponds to Loeblich et al.s P. rhathymum. We provide
morphological and molecular data from Puerto Rico, close
to the type locality, and also the first morphological and
molecular data of the species from the South Atlantic
Ocean. The main objective was to combine the morpho-
logical and molecular data and verify the relationships
between Prorocentrum maximum, P. brochii, P. mexi-
canum, and P. rhathymum, and reexamine the earlier
descriptions and iconotypes of these taxa. From these
data, we also identified a taxon previously misidentified as
P. mexicanum, which we propose as a new species.
MATERIALS AND METHODS
See Appendix S1 in the Supporting Information.
RESULTS
Morphology
Few individuals of P. mexicanum/rhathymum were
observed as epiphyte in the leaves of marine plant Thalas-
sia testudinum in the coast of Puerto Rico (Fig. 1). At Bra-
zil, this dinoflagellate was responsible of conspicuous
discolorations in the moist sands and pools during the low
water (Fig. 2A). Prorocentrum mexicanum/rhathymum
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Journal of Eukaryotic Microbiology 2017, 64, 668677
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae
constituted the dominant dinoflagellate species (> 99%),
accompanied by Fukuyoa paulensis and Amphidiniopsis
spp., among others. The species was also observed from
macroalgae collected from rocky surfaces. In the recently
collected samples, the cells reached 38-lm long (Fig. 2B, C),
while 1 mo later the cultured cells rarely exceeded 32-lm
long (Fig. 2DF).
The Puerto Rican and Brazilian strains showed the same
behavior in cultures. During the exponential growth phase,
the cells tended to swim actively, while in the stationary
phase, the cells were not actively motile and remained
embedded in mucus. Under a light microscope, living cells
were oval or ellipsoidal in apical and antapical views, with
the widest part of the cell either in the middle or the upper
half, but not in the lower half (Fig. 1AG, K, M, 2DF). The
anterior end was slightly concave in the middle and con-
tained the periflagellar area (Fig. 1E,F, 2E,F). From the right
valve emerged a periflagellar collar that has often been
reported as a simple, flange, or wing, and termed as apical
spine in the generic type, P. micans (Fig. 1A, B). The cell
often showed a pusule located in the anterior half of the
cell (Fig. 1AC) and a circular nucleus in the posterior half
of the cell (Fig. 1A). Under LM, the empty valve was
smooth with several tens of pores (Fig. 1E, F). Under bright
field microscopy, the entire cell was covered by a chloro-
plast and the pyrenoid was not discernible (Fig. 1AC).
Under epifluorescence microscopy, each valve showed a
reticulate chloroplast (Fig. 1GN). The chloroplast showed
a structure with a circular contour in the middle of the valve
that corresponded to a single central pyrenoid (Fig. 1L, N)
that was hardly visible based on bright-field optics.
The surface of the valves was smooth and perforated
by pores of three different sizes (Fig. 1OS, 2GP). On
the posterior half, clumps of 35 small pores of about
~0.1 lm in diameter were observed near the sagittal
suture (Fig. 1O), while single small pores (~0.2 lm in
diameter) were irregularly scattered in the rest of the
valve (Fig. 1P). Other types of pores were larger (~0.5 lm
in diameter) and in some cases had a long ejectosome
body or trichocyst. There were ~60 trichocyst pores in
each valve (Fig. 1QR, 2GK). They lied in shallow circular
depressions and were located in 68 rows of 36 pores
radially arranged and perpendicular to the valve margin
near the posterior, as well as a few pores scattered else-
where (Fig. 1QR, 2HI). The center of the valve appeared
to be devoid of trichocyst pores. In the anterior end of the
valve near the periflagellar area, a line of 58 pores was
observed in both the right and left valves (Fig. 1P, S,
2JK). In the anterior right valve, the line (apical row) was
composed of 67 trichocyst pores surrounding the peri-
flagellar area (Fig. 1S, 2J, L). In the anterior left valve, a
straight line was composed of 45 trichocyst pores more
distant from the periflagellar area and formed a ~45 angle
with the intercalary band edge (Fig. 1S, 2K, M). Both
valves were convex in lateral view (Fig. 1S). In dividing
cells, the intercalary band was wide and smooth without
pores, and horizontally striated (Fig. 2G, N).
The periflagellar area was a small, broad, V-shaped shal-
low depression located in the right valve (Fig. 1S, 2LP).
669
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae
mez et al.
Go

Figure 1 Puerto Rican strain of Prorocentrum mexicanum. (AG, I, K, M) Bright field. (H, J, L, N) Epifluorescence. (OS) Scanning electron micro-
graphs. (AC) Note the anterior and posterior nucleus. (DF). Empty valve, note the different shape in the anterior region. (GN) Bright-field and epi-
fluorescence images of the chlorophyll red autofluorescence. (L, N) Note the central pyrenoid. (O) Antapical view showing an oval cell with the
sagittal suture and intercalary bands. The arrow heads point aggregations of small pores in both valves. (P) Oblique apical view. Note the smooth
valve surface with randomly scattered small pores and large trichocyst pores located in shallow circular depression. (QR) Smooth valve surface
with large trichocyst pores and randomly scattered small pores. (S) Apical view and periflagellar area. a = apical spine, wing of the platelet a;
IB = intercalary bands; LV = left valve; nu = nucleus; PFA = periflagellar area; pu = pusule; py = pyrenoide; RV = right valve; sp = small pore;
ss = sagittal suture; tp = trichocyst pore; tr = trichocyst. Scale bars = 10 lm.

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Go Prorocentrum mexicanum, P. rhathymum, and P. steidingerae

Figure 2 Brazilian strain of Prorocentrum mexicanum. (AF) Bright field. (GP) Scanning electron micrographs. (AC) Wild cells. (DQ) Cultured
cells. (D) Note the flagella. (E) Left valve with slightly concave anterior end. (F) Empty theca. (G) Several cells. (HI) Right valve. (JK) Detail of
the apical right and left valve. (L) Detail of the periflagellar area and apical row of trichocyst pores in the right valve. (M) Detail of the periflagellar
area of the right valve. N. Apical view. (O) Periflagellar area. (P) Dividing (megacytic) cell with a wide intercalary band of striated smooth surface.
(Q) Apical view. a = apical spine, wing of the platelet a; AP = auxiliary pore; FP = flagellar pore; IB = intercalary bands; LV = left valve; PFA = peri-
flagellar area; ri = ridge in the platelet h; RV = right valve; ss = sagittal suture; tp = trichocyst pore; tr = trichocyst. Periflagellar platelets (a, b, c,
d, e, f, g, h). Scale bars = 10 lm (AK), 2 lm (LQ).

The periflagellar area consisted of eight platelets (a, b, c, d, apical collar adjacent to the auxiliary pore and a protuberant
e, f, g, h), an auxiliary pore (AP), and a flagellar pore (FP) apical plate adjacent to the flagellar pore (Fig. 2OQ). Under
(Fig. 2O). The periflagellar area showed a prominent curved the light microscope, the curved apical plate resembled a

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Prorocentrum mexicanum, P. rhathymum, and P. steidingerae
spine, while under SEM, it appeared as ear-shaped
(Fig. 2Q). Platelet a, which looked like a collar, was the lar-
gest of the platelets projected and appeared as a spine
under the light microscope (Fig. 2O, Q). Platelet b was
located to the right of platelet a and was surrounded by the
auxiliary pore (AP), platelet d, and platelet e. Platelet c was
surrounded by the flagellar pore (FP), AP, and platelets d, f,
and e (Fig. 2N, O). Platelets d and f were located on the left
valve side, while platelets e and g were on the right valve
side (Fig. 2O). Platelet h, which was the second largest pla-
telet, was located at the right end. There was a ridge (ri)
between platelet h and platelets f and g (Fig. 2O).
The living cells of the Puerto Rican strain ranged from
29 to 34 lm in length (average 31.8 lm, n = 10), and 19
24 lm in width. The average length/width ratio was 1.48
(1.421.58). The same strain after fixation and observed
under SEM ranged from 23 to 27 lm length (average
25.1, n = 11) and 1417 lm in width, with an average
length/width ratio of 1.60 (1.441.69). The fixation shrank
the cells ~21% in length and ~27% in width.
We compared the morphology of strains from locations
separated for ~6,000 km (Fig. 1, 2). We did not find signifi-
cant differences. The shape of the cells of the Puerto
Rican and Brazilian strains was similar to that in the origi-
nal descriptions of P. mexicanum and P. rhathymum. The
pore arrangement is similar to that in the original descrip-
tion of P. rhathymum. Unfortunately, at the time of the
description of P. mexicanum, the pore arrangement was
not noted. The size of P. mexicanum in the original
description was larger than in our cultured cells. We
observed that the cells reduced their size under culture
conditions and the size was also reduced after fixation.
Our morphological comparison indicated that our strains
corresponded to the species first described as P. mexi-
canum and later as P. rhathymum.
Molecular phylogeny
Our SSU rDNA sequences obtained from the Puerto Rican
and Brazilian strains of P. mexicanum were identical. In the
SSU rDNA phylogeny, the new sequences clustered
together with sequences identified as P. mexicanum
from Mexican Pacific (its type locality) and P. rhathymum
from the Caribbean Sea (its type locality), and sequences
from Asia, Polynesia, and Indian Ocean, together with
P. koreanum and a strain misidentified as P. micans. A sis-
ter clade was composed of two sequences of P. mexi-
canum from Florida (strains CCMP687 and USFDA), along
with sequences of P. texanum and P. micans (Fig. 3). Out-
side these two main clades was a sequence identified as
P. mexicanum (DQ174089) from Vietnam (Fig. 3). The
micrograph of this strain corresponded to Prorocentrum
compressum. The LSU rDNA phylogeny showed a similar
topology to the SSU phylogeny. The sequences identified
as P. mexicanum and P. rhathymum branched together into
two clades (Fig. S1). The main clade included strains of
P. mexicanum and P. rhathymum from the Mexican Pacific,
Caribbean Sea, Asia, Australia, Indian Ocean, NE Atlantic
and Brazil. There was not a clear geographical cut in the
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672

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Prorocentrum rhathymum PRJJ2 Korea HF565182
Prorocentrum rhathymum NMN016 Malaysia FJ842096
Prorocentrum rhathymum PRJJ1 Korea HF565181
Prorocentrum mexicanum Puerto Rico KY426836
Prorocentrum rhathymum PRJJ3 Korea HF565183
Prorocentrum mexicanum Brazil KY426837
Prorocentrum mexicanum PMOO04 Polynesia Y16232
Prorocentrum koreanum KP711344 Prorocentrum
Prorocentrum koreanum KP711350 mexicanum / koreanum
Prorocentrum rhathymum S7 Florida EU287487
Prorocentrum rhathymum PXPV-1 Mexican Pacific JQ616822
Prorocentrum micans China DQ004735
Prorocentrum triestinum EF492512
Prorocentrum gracile AY443019
Prorocentrum triestinum DQ004734
Prorocentrum triestinum AB183673
Prorocentrum mexicanum CCMP687 Florida EU287485
Prorocentrum micans EF492511
0.01
Prorocentrum texanum JQ390504 Prorocentrum micans / texanum
Prorocentrum micans AJ415519 +
Prorocentrum micans JN717145 2 divergent strains of P. mexicanum
100
Prorocentrum micans AY803739
Prorocentrum micans EU780638
Prorocentrum mexicanum USFDA Florida EF492510
Prorocentrum mexicanum sp3 Vietnam DQ174089 (=P. compressum)
94 Prorocentrum minimum AY803740
80 Prorocentrum minimum AY421791
Prorocentrum donghaiense AJ841810
95
Prorocentrum donghaiense DQ336054
Prorocentrum dentatum AY803742
Prorocentrum emarginatum Y16239
100 Prorocentrum fukuyoi EU196420
Prorocentrum emarginatum EU196418
Alexandrium tamarense JF521641
Figure 3 SSU rDNA-based phylogeny of Prorocentrum. Sequences
obtained in this study are bold-typed. Support of nodes is based on
bootstrap values of ML/NJ with 1,000 and 1,000 resamplings, respec-
tively. See methods in Appendix S1. Only values greater than 60 are
shown. Alexandrium tamarense was used as outgroup.
sequences of P. mexicanum/P. rhathymum. The second
clade included some strains identified as P. mexicanum and
P. rhathymum from Florida (strain CCMP687) and Cuba
(strain PXHV-1) (Fig. S1). In the ITS phylogeny, the
sequences of P. mexicanum/P. rhathymum were divided
into two clades. One main clade included the sequences
from the Mexican Pacific, Gulf of Mexico, Caribbean Sea,
Atlantic, Indian, and Western Pacific Oceans (Fig. 4). The
Floridian and Cuban strains CCMP687 and PXHV-1, respec-
tively, formed a clade that branched separately from the
main clade of P. mexicanum/P. rhathymum (Fig. 4).
Sequences from the type localities, the Mexican Pacific
and the Caribbean Sea, identified as both P. mexi-
canum and P. rhathymum, branched together in the same
clade, and there was no molecular support for the split
between P. mexicanum and P. rhathymum. The strains
CCMP687, USFDA, FIU25, and PXHV-1 from Florida and
Cuba are distantly related to the clade of P. mexicanum/
P. rhathymum. These sequences assigned to P. mexicanum/
P. rhathymum belong to an independent species (Fig. 3, 4).
DISCUSSION
The synonymy of Prorocentrum mexicanum and
P. rhathymum

s-Altamirano and Sierra-Beltr

Since Corte an (2003) pro-
posed the split of P. rhathymum and P. mexicanum,
Journal of Eukaryotic Microbiology 2017, 64, 668677

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Go Prorocentrum mexicanum, P. rhathymum, and P. steidingerae

Prorocentrum rhathymum PRJJ1 Korea HF565181
Prorocentrum mexicanum Brazil KY426837
Prorocentrum rhathymum PRJJ2 Korea HF565182
Prorocentrum rhathymum PXPV-1J Mexican Pacific Q616822
type locality of P. rhathymum. The morphology and the
behavior of the cells of the Puerto Rican strain fit with
P. rhathymum as reported by Loeblich et al. (1979). No
reliable distinction can be found from the morphological

Prorocentrum mexicanum
Prorocentrum mexicanum Vietnam AY886763
88/83 Prorocentrum rhathymum PRJJ3 Korea HF565183

Prorocentrum rhathymum VGO761 Spain EU244465

-/- Prorocentrum rhathymum Iran JN020163
66/- Prorocentrum rhathymum VGO893 Malaysia EU244464
99/99 Prorocentrum rhathymum NMN16 Malaysia FJ155840
Prorocentrum rhathymum CCMP2933 Florida EU927561
73/-
-/- Prorocentrum rhathymum TIO93 China KY010248
87/84
Prorocentrum rhathymum TIO29 China KY010246
89/76
Prorocentrum rhathymum TIO55 China KY010247
99/99 Prorocentrum mexicanum CCMP687 Florida EU927554
Prorocentrum rhathymum PXHV-1 Cuba JQ638938
-/- 60/- Prorocentrum micans FJ823584
66/86 Prorocentrum micans EU244467
94/93 Prorocentrum micans EU780638
95/89 Prorocentrum micans
99/-
Prorocentrum micans DQ485145
Prorocentrum micans EU927531
98/90 99/97 Prorocentrum micans EU927521
Prorocentrum texanum JQ390505
99/99 Prorocentrum koreanum KP711348
Prorocentrum koreanum KY010231
81/83 Prorocentrum donghaiense JN595869
99/99 Prorocentrum dentatum FJ823581
Prorocentrum rostratum EU244471
99/98
99/99 Prorocentrum minimum KY290717
Prorocentrum minimum FJ823587
Karena brevis AF352823
Figure 4 ITS1-5.8S-ITS2-based phylogeny of Prorocentrum. Sequence
obtained in this study is bold-typed. Support of nodes is based on
bootstrap values of NJ/ML with 1,000 and 1,000 resamplings, respec-
tively. See methods in Appendix S1. Only values greater than 60 are
shown. Karenia brevis was used as outgroup.
nearly all the SEM studies have identified it as P. rhathy-
mum and showed a smooth valve surface (Aligizaki et al.
2009; Gul and Saifullah 2011; Laza-Mart
nez et al. 2011;
Lim et al. 2013; Mohammad-Noor et al. 2007b; Su-Myat
and Koike 2013). From strains isolated near the type local-
ity of P. mexicanum, Cohen-Fern
andez (2011) identified
the sequences as P. mexicanum, but the SEM micro-
graphs corresponded to P. rhathymum, cells with smooth
valve surface as illustrated by Loeblich et al. (1979). After
Corte
s-Altamirano and Sierra-Beltr
an (2003), there have
been no further SEM pictures of the bloom-forming spe-
cies P. mexicanum. The earlier sequences were submitted
as P. mexicanum (Cohen-Fern
andez et al. 2010; Daugbjerg
et al. 2000; Grzebyk et al. 1998; Murray et al. 2007;
Scorzetti et al. 2009; Stern et al. 2010), and after 2003
also submitted as P. rhathymum (Caillaud et al. 2010;
Herrera-Sepu
lveda et al. 2013, 2015; Lim et al. 2013;
Mohammad-Noor et al. 2007a; Murray et al. 2009; Pearce
and Hallegraeff 2004; Scorzetti et al. 2009). Some authors
submitted their sequences using both names, and without
data to illustrate the morphological differences between
the species (Murray et al. 2007, 2009; Scorzetti et al.
2009). This can cause confusion in the research commu-
nity, as has been experienced in the study of Alexandrium
tamarense complex until in-depth molecular phylogeny
helped clear up the ambiguities in its morphology-based
taxonomy (e.g. Miranda et al. 2012; Wang et al. 2014).
In this study, we provide morphological and molecular
data of a strain collected from Puerto Rico, close to the
characteristics between this and P. mexicanum as
reported in the original description by Osorio-Tafall (1942)
(see Appendix S2 in the Supporting Information). By
molecular phylogenetic analyses, we show unequivocally
that the Puerto Rican strain represents P. rhathymum. We
also obtained the first morphological and molecular data of
this species from the South Atlantic Ocean. This extends,
sp. nov.
even more, the wide geographical distribution of this
important toxic species. The current knowledge reveals
that sequences identified as P. mexicanum/P. rhathymum
are a common species in shallow protected areas of warm
and temperate inshore waters (Fig. 3, 4 and Fig. S1, see
P. texanum map in Lim et al. 2013). It is striking that this globally dis-
P. koreanum tributed and bloom-forming species had not been reported
before 1942 or 1979. A taxonomical account is reported in
the Appendix S2. The first illustration of P. mexicanum/
P. rhathymum corresponded to the figure 41a of P. maxi-
mum from the Adriatic Sea (Fig. 5A, Schiller 1933), and
0.02
later reported in the same location as P. mexicanum,
P. venetum, and P. rhathymum (Appendix S2).
Loeblich et al. (1979) proposed the new species
P. rhathymum ignoring the description of P. mexicanum by
Osorio-Tafall (1942). Prorocentrum rhathymum and P. mexi-
canum were soon considered synonyms with priority for
the earlier name (Faust 1990; Faust and Gulledge 2002;
Steidinger 1983; Steidinger and Tangen 1997). Corte
s-Alta-
mirano and Sierra-Beltr
an (2003) reinstated P. rhathymum
and P. mexicanum as separate species describing signifi-
cant differences between both species. However, the spe-
cies that they attributed to P. mexicanum did not
correspond in shape, surface ornamentation, and other
details (i.e. apical spine) to the description of P. mexicanum
in Osorio-Tafall (1942) (Fig. 5BD and Table 1;
Appendix S2). The original description of P. mexicanum
reported an ellipsoidal cell, with symmetrical valve margin
and a rounded antapex (Fig. 5C, Osorio-Tafall 1942). In con-
trast, P. mexicanum sensu Corte
s-Altamirano & Sierra-
Beltr
an possesses asymmetric valve margins and a slightly
pointed antapex (Fig. 5D). Prorocentrum mexicanum was
A B C D E
Figure 5 Line drawings of Prorocentrum mexicanum and allied
species. (A) P. maximum, redrawn from Schiller (1941, fig. 41a).
(B) P. rhathymum, redrawn from Lim et al. (2013). (C) P. mexicanum,
redrawn from Osorio-Tafall (1942). (D) P. mexicanum, redrawn from
Corte
s-Altamirano and Sierra-Beltr
an (2003, fig. 1c). Note the scat-
tered larger pores on the poroid surface. (E) P. texanum, redrawn
from Henrichs et al. (2013, fig. 2f). The large scattered pores are illus-
trated, while the poroid surface is omitted.

2017 The Author(s) Journal of Eukaryotic Microbiology 2017 International Society of Protistologists
Journal of Eukaryotic Microbiology 2017, 64, 668677 673
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae
Table 1. Comparison of the morphology of the Prorocentrum mexicanum (=P. rhathymum), P. steidingerae sp. nov. based on Faust (1990) and
P. texanum based on Corte
s-Altamirano and Sierra-Beltr
an (2003) and Henrichs et al. (2013): ornamentation of the valve surface; excavation of
the periflagellar indentation, number of trichocyst pores per valve; number of tangential rows of trichocyst pores per valve; number of trichocyst
pores under the periflagellar indentation of the right and left valves; shape of the apical flange
Taxa Surface Excavation #TP
P. mexicanum Smooth Deep < 70
P. steidingerae Partially poroid Shallow 80100
P. texanum Poroid Shallow < 70
TP = trichocyst pore; RV = right valve; LV = left valve.
described with a tiny apical spine in Osorio-Tafall (1942),
while for Corte
s-Altamirano and Sierra-Beltr
an, showed a
prominent three-horned (sometimes two-horned) spine.
Osorio-Tafall described P. mexicanum with a smooth valve
surface with disperse large pores (Fig. 5C). Corte
s-Altamir-
ano and Sierra-Beltr
an reported the valves fully covered by
poroids, and other dispersed type of pores (Fig. 5D). It is
expected that the important morphological differences
reported in Corte
s-Altamirano and Sierra-Beltr
an (2003) will
be associated with genetic differences between P. rhathy-
mum and P. mexicanum. However, in the molecular phylo-
genies, the sequences from distant oceans identified as
P. rhathymum and P. mexicanum are intermixed and
branched together, with the exception of several strains
from the Gulf of Mexico (Fig. 3, 4 and Fig. S1). The micro-
graphs associated with sequences identified as P. rhathy-
mum or P. mexicanum did not correspond to the cells
reported as P. mexicanum by Corte
s-Altamirano and Sierra-
Beltr
an (2003). This means that nobody has obtained yet the
sequence of the bloom-forming species illustrated by
Corte
s-Altamirano and Sierra-Beltr
an (2003) or that the
sequence of P. mexicanum sensu Corte
s-Altamirano &
Sierra-Beltr
an has been reported under other species name.
Prorocentrum mexicanum and P. texanum
Corte
s-Altamirano and Sierra-Beltr
an (2003) misidentified
P. mexicanum with the planktonic species P. texanum that
was later described from the Gulf of Mexico (Fig. 5D, E,
Henrichs et al. 2013). The shape and size of P. texanum
var. cuspidatum (Fig. 5E) are similar to that of P. mexi-
canum sensu Corte
s-Altamirano & Sierra-Beltr
an (Fig. 5D).
The valves of both taxa were covered by poroids and
trichocyst pores. Both taxa have 56 recessed, trichocyst
pores below the periflagellar platelets on the right valve.
Other distinctive feature of both taxa is the 23 crest ser-
rated apical flange (Corte
s-Altamirano and Sierra-Beltr
an
2003; Henrichs et al. 2013). In the SSU rDNA and ITS
phylogenies, P. texanum is well separated from the clade
of P. mexicanum/rhathymum and close to P. micans
(Fig. 3, 4). This is coherent with the morphological differ-
ences (Table 1 and Appendix S2). Prorocentrum texanum
was previously overlooked as morphotypes of P. micans
(Henrichs et al. 2013). Prorocentrum micans is a common
and nontoxic species and the blooms have received less
attention. In the Mexican Pacific, P. texanum (=P. mexi-
canum sensu Corte
s-Altamirano & Sierra-Beltr
an) could be
2017 The Author(s) Journal of Eukaryotic Microbiology 2017 International Society of Protistologists
674

mez et al.
Go
Rows TP in apical RV TP in apical LV Flange
68 67 45 Nonserrated, pointed
812 4 No, 912 TP in periphery Nonserrated, curved
811 56 No, 57 TP in periphery Serrated (26 crests)
mistaken for P. micans, and this species has not received
attention in the molecular surveys (Cohen-Fern
andez et al.
2010; Herrera-Sepu
lveda et al. 2013). We conclude that the
species with a smooth valve surface P. maximum sensu
Schiller (1933, his fig. 41a), P. rhathymum, and P. venetum
are synonyms of P. mexicanum. The planktonic species
with a rugose valve surface assigned as P. mexicanum by
Corte
s-Altamirano and Sierra-Beltr
an (2003) corresponds to
P. texanum (Table 1 and Fig. 5).
The genetically divergent Floridian and Cuban strains
Osorio-Tafall (1942) and Loeblich et al. (1979) described
P. mexicanum with a smooth valve surface, while P. tex-
anum (=P. mexicanum sensu Corte
s-Altamirano & Sierra-
Beltr
an) has a rugose valve surface covered by poroids.
Prorocentrum mexicanum was described with a nonser-
rate apical flange, while P. texanum showed a serrated
flange with crests (Corte
s-Altamirano and Sierra-Beltr
an
2003; Henrichs et al. 2013). Prorocentrum mexicanum is
more commonly reported as an epiphytic or tychoplank-
tonic species, while P. texanum is planktonic. Despite the
considerable morphological and ecological differences, the
existence of a third species has contributed to the contro-
versy on the synonymy of P. mexicanum and P. rhathy-
mum. This third species shares the same habitat and
distribution of P. mexicanum in the Gulf of Mexico and
Caribbean Sea, and it shows intermediate morphological
characteristics between P. mexicanum and P. texanum.
In one of the earlier studies on epiphytic dinoflagellates in
the Gulf of Mexico, Bomber (1985) isolated the strain of
Prorocentrum sp. FIT#182 from the Florida Keys. Bomber
(1985) did not report illustrations and reported the strain as
P. rhathymum (syn. mexicanum) in pages 4 and 103, and
as P. mexicanum in page 22. Up to date, that species identi-
fied as P. mexicanum is maintained as strain CCMP687 at
the National Center for Marine Algae and Microbiota at
Bigelow Laboratory, East Boothbay, Maine, USA. Other
strains from the Florida Keys (CCMP2933, CCMP2976) are
named P. rhathymum (https://ncma.bigelow.org/). The
strain CCMP687 is genetically characterized using several
molecular markers (Murray et al. 2007, 2009; Scorzetti
et al. 2009; J.F. Ferrell, M.J. Beaton, unpublished). Ferrell
and Beaton provided LM and SEM pictures of the strain
CCMP687 that showed a valve surface was partially cov-
ered by poroids. Most of their pictures corresponded to
cells under division, when the parent cell is splitting in two
Journal of Eukaryotic Microbiology 2017, 64, 668677

mez et al.
Go
with each theca forming a new companion theca. In these
young cells, the valve is partially smooth, while the rugose
surface appears in the mature cells. A characteristic of
P. mexicanum as described by Osorio-Tafall (1942) and Loe-
blich et al. (1979) is the smooth surface of the entire thecae.
The valve ornamentation of the strain CCMP687 is closer to
relatives such as P. micans, P. texanum, and P. koreanum.
Other strains of this species were isolated from Florida and
identified as the P. mexicanum strain USFDA (Evens et al.
2008), Prorocentrum sp. strain FIU25 (An et al. 2010), and
from Cuba identified as P. rhathymum strain PXHV-1 (Her-
rera-Sepu
lveda et al. 2013). Toxicity analyses revealed that
this species is toxic (An et al. 2010). Currently, ITS phy-
logeny has been used to support the proposal of new spe-
cies such as P. texanum and P. koreanum (Han et al. 2016;
Henrichs et al. 2013). The sequences of these Floridian and
Cuban strains, especially in the ITS phylogeny, revealed that
this species formed a sister group of the clade of P. mexi-
canum (Fig. 4). Faust (1990) reported the detailed descrip-
tion of this species by SEM from wild epiphytic cells
collected in the coral reefs of Belize. She remarked that it
was the most abundant epiphytic species. Faust (1990) illus-
trated the cells with a rugose valve surface covered by por-
oids, a nonserrate round apical flange, deep indentation of
the periflagellar area in the right valve, as well as an arrange-
ment of the trichocyst pores that does not correspond to
P. mexicanum or P. texanum (Fig. 6). As Faust (1990)
assigned P. mexicanum to this species, Corte
s-Altamirano
and Sierra-Beltr
an (2003) may assume that P. mexicanum
has a valve surface covered by poroids, and they mistook
P. mexicanum for planktonic cells of P. texanum, while they
assigned P. rhathymum for epiphytic cells with a smooth
valve surface. The identity of the species reported by Bom-
ber (1985) and Faust (1990) as well as isolates by several
authors in Florida and Cuba needs to be clarified as a neces-
sary step to resolve the controversy in the synonymy of
P. mexicanum/P. rhathymum. We propose here that taxon
as a new species based on the existing morphological and
molecular data.

mez, D. Qiu & Senjie
Prorocentrum steidingerae F. Go
Lin, sp. nov
Diagnosis. Cells are oval in valve view (3040 lm long
and 2025 lm wide), subovate, and convex in side view.
The periflagellar area is located in a depression in the
right valve. The cells show an apical spine with a curved
shape. The valve surface of mature cells is rugose, cov-
ered by poroids. The valves show two types of pores.
The small pores (~0.2 lm in diameter) are circular and
unevenly distributed along the valve. No clumps of small
pores were observed in the apical margins of the valve.
There are 80100 trichocyst pores in each valve, some
unevenly distributed and most of them forming tangen-
tial rows. There are 812 rows of 36 trichocyst pores
in depressed furrows in the margin of the anterior and
medium parts of the valve. There is a row of four tri-
chocyst pores in the right valve adjacent to the periflag-
ellar area.
2017 The Author(s) Journal of Eukaryotic Microbiology 2017 International Society of Protistologists
Journal of Eukaryotic Microbiology 2017, 64, 668677
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae
A C
RV RV
B D
RV
RV
LV
LV
Figure 6 Line drawings of Prorocentrum mexicanum and P. stei-
dingerae sp. nov. (A) Right valve and (B) Apical view of P. mexicanum
based on this study. (C) Right valve and (D) Apical view of P. stei-
dingerae sp. nov. based on Faust (1990, her fig. 5, 7).
This new species shares the habitat with and has been
mistaken for P. mexicanum in the Gulf of Mexico and Carib-
bean Sea. The diagnostic characters are reported in the
Table 1 and Fig. 6. The excavation of the periflagellar area in
the right valve is deeper in P. steidingerae than in P. mexi-
canum. The platelet a forms a prominent curved apical collar
that is acuter in P. mexicanum. The new species has a valve
surface rugose covered by poroids, while the valve is smooth
in P. mexicanum. Prorocentrum steidingerae has more than
80 trichocyst pores per valve, while P. mexicanum has less
than 70 trichocyst pores. The new species shows 812 tan-
gential rows of trichocyst pores, while usually six rows in
P. mexicanum. This new species shows a row of four tri-
chocyst pores in the apical right valve, while P. mexicanum
shows a row of 67 trichocyst pores. Prorocentrum stei-
dingerae does not have the clumps of 35 small pores in the
valve apical margins that are a characteristic feature of
P. mexicanum (Table 1 and Fig. 6).
Iconotype. Figure 6C.
Type locality. Gulf of Mexico, off Knight Key, Florida
(24420 29.88N, 8170 30W). Isolated by Bomber (1985).
Habitat. Epiphytic in macrophytes, detritus and sediments
in tropical coasts. Currently known from Belize, Cuba, and
USA at Florida.
Etymology. In honor of Karen A. Steidinger, who first
denoted the synonymy between P. mexicanum and
P. rhathymum, and carried out numerous studies on toxic
dinoflagellates, especially in Florida where this new species
is commonly reported.
675
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae
Synonyms. Prorocentrum mexicanum sensu Faust (1990,
her fig. 510), P. mexicanum sensu Steidinger and Tangen
(1997, p. 421, plate 8); Prorocentrum mexicanum
CCMP687 (=PM200A, =FIT#182) from Florida Keys (Bom-
ber 1985) (accession numbers DQ336183, EU287485,
EU165318, EU916907, EU927554); P. mexicanum USFDA
from Florida (accession number EF492510); Prorocentrum
sp. FIU25 from Florida (accession number EU165283);
P. rhathymum PXHV-1 from Havana, Cuba (accession
number JQ638938).
Zoobank registration. urn:lsid:zoobank.org:act:28ADC0
EA-114C-4A08-B0F6-6BCA01D342C9.
TAXONOMICAL CONSIDERATIONS
Prorocentrum maximum (Gourret) J. Schiller (1933, fig.
41c). Basionym: Postprorocentrum maximum Gourret
(1883, fig. 50)
Non-Prorocentrum maximum sensu Schiller (1933, fig.
41bc), nec P. maximum sensu Osorio-Tafall 1942. This
species is probably a junior synonym or confused with
P. micans Ehrenberg.
Prorocentrum triestinum J. Schiller (1918, fig. 1a). Syno-
nyms: Prorocentrum brochii J. Schiller (1918, fig. 2),
P. maximum sensu Schiller (1933, fig. 41b).
Non-P. maximum sensu Schiller (1933, fig. 41a, c), nec
P. maximum sensu Osorio-Tafall (1942).
Prorocentrum mexicanum Osorio-Tafall (1942, fig. 11). Syno-
nyms: Prorocentrum maximum sensu Schiller (1933, fig.
41a), P. rhathymum A.R. Loeblich, J.L. Sherley & R.J. Sch-
midt 1979; P. venetum Tolomio & Cavolo 1985.
Non-P. mexicanum sensu Corte
s-Altamirano and Sierra-
Beltr
an (2003), nec P. mexicanum sensu Faust (1990).
Prorocentrum steidingerae F. Go
mez, D. Qiu & Senjie Lin
(fig. 6CD) . Synonyms: Prorocentrum mexicanum sensu
Faust (1990, fig. 510), P. mexicanum sensu Steidinger &
Tangen (p. 421, plate 8). See above a list of strains.
Non-P. mexicanum Osorio-Tafall, nec P. rhathymum
Loeblich, J.L. Sherley & R.J. Schmidt, nec P. texanum
Henrichs, Steidinger, P.S. Scott & L. Campbell.
Prorocentrum texanum Henrichs, Steidinger, P.S. Scott &
L. Campbell 2013. Synonyms: P. mexicanum sensu
Corte
s-Altamirano and Sierra-Beltr
an 2003, P. micans
strain CCMP2772.
ACKNOWLEDGMENTS
This work was supported by the Brazilian Conselho Nacio-
nal de Desenvolvimento Cient
fico e Tecnolo
gico (grant
number BJT 370646/2013-14 to F.G.), United States
National Science Foundation (EF-0629624 to S.L.), and
Science Technology Program Guangzhou of China
(201607010289 to D.Q.). We thank E. Otero-Morales and
B.M. Soler for the laboratory facilities and hospitality
extended during the sampling in Puerto Rico.
2017 The Author(s) Journal of Eukaryotic Microbiology 2017 International Society of Protistologists
676

mez et al.
Go
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in
the supporting information tab for this article:
Figure S1. (D1D3) LSU rDNA-based phylogeny of
Prorocentrum.
Appendix S1. Materials and methods.
Appendix S2. Taxonomy of Prorocentrum mexicanum
and allied species.
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