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Journal of Eukaryotic Microbiology ISSN 1066-5234


The Synonymy of the Toxic Dinoflagellates Prorocentrum
mexicanum and P. rhathymum and the Description of
P. steidingerae sp. nov. (Prorocentrales, Dinophyceae)

Fernando Go , Dajun Qiub & Senjie Linc,d
a Carmen Campos Panisse 3, Puerto de Santa Marıa, 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

Benthic Dinoflagellata; Caribbean Sea;
epiphytic Dinophyta; harmful algal blooms; Prorocentrum mexicanum and P. rhathymum are toxicologically important
microalgae; molecular phylogenetics; new dinoflagellates, but their relationship is not well defined. We investigated strains
species; red tides. 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,
Correspondence and the first morphological and molecular data from the South Atlantic Ocean.
mez, Carmen Campos Panisse 3,
F. Go The rRNA gene (rDNA) sequences of the Puerto Rican and Brazilian strains were
Puerto de Santa Marıa E-11500, Spain identical, and their morphologies fit the description of P. rhathymum. In the
Telephone number: +34 628460564; molecular phylogenies, the globally distributed populations under the names
FAX number: +1 860 4059153; P. mexicanum and P. rhathymum are intermixed and branched together, except
e-mail: for several divergent strains from Florida and Cuba. We examined the original
descriptions and iconotypes of the species Prorocentrum maximum, P. brochii,
Received: 11 November 2016; revised 9 P. mexicanum, and P. rhathymum. We conclude that P. maximum sensu Schil-
February 2017; accepted February 10, 2017. ler’s figure 41a corresponds to the earlier description of this species; the split of
Early View publication March 10, 2017 P. mexicanum and P. rhathymum was based on a misidentification because
P. mexicanum sensu Corte s-Altamirano & Sierra-Beltran 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 several new species of Prorocentrum from plankton sam-
dinoflagellate that is markedly compressed laterally, and ples that were collected in the Mexican Pacific. He
the theca consists of two large valves joined together at reported cells identified as P. maximum and described a
their marginal edges. Prorocentrum species are found closely related species as P. mexicanum from Salina Cruz,
worldwide; they can be benthic, planktonic, tychoplank- Oaxaca. These earlier descriptions were mainly based on
tonic, and vary widely in size and shape (Faust and Gul- the valve shape, presence and shape of an apical spine,
ledge 2002; Steidinger and Tangen 1997). The epiphytic or and the general valve ornamentation, with scarce detail in
epibenthic dinoflagellates received little attention until the type and distribution of valve pores (Osorio-Tafall 1942;
Yasumoto et al. (1977) found that some species produce Schiller 1933). From 1970s, the scanning electron micro-
toxins responsible for human diseases such as the ciguat- scopes (SEM) allowed detailed studies of the valve orna-
era fish poisoning. Some species of Prorocentrum are mentation. Loeblich et al. (1979) described Prorocentrum
nontoxic but others are able to produce different toxins rhathymum from a strain isolated from the surface waters
(Steidinger 1983). of the Caribbean Sea at Cinnamon Bay, St. John, U.S. Vir-
The earlier observations of the epiphytic dinoflagellates gin Islands, and subsequently reported from plankton in the
were often based on resuspended cells found in the plank- Mediterranean Sea (Estrada 1979) and from macroalgae in
ton samples. Schiller (1933) proposed the new combination coral reefs of the Eastern Pacific Ocean (Fukuyo 1981).
P. maximum (Gourret) J. Schiller for the taxon described as Currently, P. rhathymum is reported worldwide primarily in
Postprorocentrum maximum Gourret 1883, and proposed tropical and subtropical waters (see Lim et al. 2013), and
P. brochii as a synonym. Osorio-Tafall (1942) described rard-Therriault et al. 1999; Levasseur
also in cold waters (Be

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
668 Journal of Eukaryotic Microbiology 2017, 64, 668–677
mez et al.
Go Prorocentrum mexicanum, P. rhathymum, and P. steidingerae

et al. 2003). Strains of P. rhathymum have been reported constituted the dominant dinoflagellate species (> 99%),
to produce several toxins (An et al. 2010; Caillaud et al. accompanied by Fukuyoa paulensis and Amphidiniopsis
2010; Nakajima et al. 1981; Naves et al. 2006; Pearce et al. spp., among others. The species was also observed from
2005). Prorocentrum maximum and P. rhathymum have macroalgae collected from rocky surfaces. In the recently
been considered synonyms of P. mexicanum (Faust 1990; collected samples, the cells reached 38-lm long (Fig. 2B, C),
Faust and Gulledge 2002; Steidinger 1983; Steidinger and while 1 mo later the cultured cells rarely exceeded 32-lm
Tangen 1997). long (Fig. 2D–F).
The taxonomical controversy in the synonymy has The Puerto Rican and Brazilian strains showed the same
remained for over two decades. Corte s-Altamirano and behavior in cultures. During the exponential growth phase,
Sierra-Beltran (2003) reinstated P. rhathymum as an inde- the cells tended to swim actively, while in the stationary
pendent species based on plankton samples collected dur- phase, the cells were not actively motile and remained
ing red tides in the Gulf of California and the Mexican embedded in mucus. Under a light microscope, living cells
Pacific. They reported that the main differences between were oval or ellipsoidal in apical and antapical views, with
P. rhathymum and P. mexicanum concern the fact that the the widest part of the cell either in the middle or the upper
latter is a planktonic, lenticular in lateral view, has a centrally half, but not in the lower half (Fig. 1A–G, K, M, 2D–F). The
located pyrenoid, an areolated valve surface, and a large, anterior end was slightly concave in the middle and con-
winged, and two- or three-pointed apical spine, while tained the periflagellar area (Fig. 1E,F, 2E,F). From the right
P. rhathymum is a benthic/epiphytic species with smooth valve emerged a periflagellar collar that has often been
valve surface bearing trichocysts pores and a simple apical reported as a simple, flange, or wing, and termed as apical
spine, and lacking the pyrenoid (Corte s-Altamirano and spine in the generic type, P. micans (Fig. 1A, B). The cell
Sierra-Beltran 2003). However, despite the important mor- often showed a pusule located in the anterior half of the
phological differences reported by these authors, the avail- cell (Fig. 1A–C) and a circular nucleus in the posterior half
able sequences named as P. mexicanum or P. rhathymum of the cell (Fig. 1A). Under LM, the empty valve was
branched together and the species split is not supported smooth with several tens of pores (Fig. 1E, F). Under bright
(as P. mexicanum for Cohen-Fernandez et al. 2010; Daugb- field microscopy, the entire cell was covered by a chloro-
jerg et al. 2000; Grzebyk et al. 1998; Murray et al. 2007; plast and the pyrenoid was not discernible (Fig. 1A–C).
Scorzetti et al. 2009; Stern et al. 2010; as P. rhathymum Under epifluorescence microscopy, each valve showed a
for Caillaud et al. 2010; Herrera-Sepu lveda et al. 2013, reticulate chloroplast (Fig. 1G–N). The chloroplast showed
2015; Lim et al. 2013; Mohammad-Noor et al. 2007a; Mur- a structure with a circular contour in the middle of the valve
ray et al. 2009; Pearce and Hallegraeff 2004; Su-Myat and that corresponded to a single central pyrenoid (Fig. 1L, N)
Koike 2013). that was hardly visible based on bright-field optics.
In this study, we document the morphological and The surface of the valves was smooth and perforated
molecular data of the taxon that unequivocally corre- by pores of three different sizes (Fig. 1O–S, 2G–P). On
sponds to Loeblich et al.’s P. rhathymum. We provide the posterior half, clumps of 3–5 small pores of about
morphological and molecular data from Puerto Rico, close ~0.1 lm in diameter were observed near the sagittal
to the type locality, and also the first morphological and suture (Fig. 1O), while single small pores (~0.2 lm in
molecular data of the species from the South Atlantic diameter) were irregularly scattered in the rest of the
Ocean. The main objective was to combine the morpho- valve (Fig. 1P). Other types of pores were larger (~0.5 lm
logical and molecular data and verify the relationships in diameter) and in some cases had a long ejectosome
between Prorocentrum maximum, P. brochii, P. mexi- body or trichocyst. There were ~60 trichocyst pores in
canum, and P. rhathymum, and reexamine the earlier each valve (Fig. 1Q–R, 2G–K). They lied in shallow circular
descriptions and iconotypes of these taxa. From these depressions and were located in 6–8 rows of 3–6 pores
data, we also identified a taxon previously misidentified as radially arranged and perpendicular to the valve margin
P. mexicanum, which we propose as a new species. near the posterior, as well as a few pores scattered else-
where (Fig. 1Q–R, 2H–I). 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 5–8 pores was
See Appendix S1 in the Supporting Information. observed in both the right and left valves (Fig. 1P, S,
2J–K). In the anterior right valve, the line (apical row) was
composed of 6–7 trichocyst pores surrounding the peri-
flagellar area (Fig. 1S, 2J, L). In the anterior left valve, a
straight line was composed of 4–5 trichocyst pores more
distant from the periflagellar area and formed a ~45° angle
Few individuals of P. mexicanum/rhathymum were with the intercalary band edge (Fig. 1S, 2K, M). Both
observed as epiphyte in the leaves of marine plant Thalas- valves were convex in lateral view (Fig. 1S). In dividing
sia testudinum in the coast of Puerto Rico (Fig. 1). At Bra- cells, the intercalary band was wide and smooth without
zil, this dinoflagellate was responsible of conspicuous pores, and horizontally striated (Fig. 2G, N).
discolorations in the moist sands and pools during the low The periflagellar area was a small, broad, V-shaped shal-
water (Fig. 2A). Prorocentrum mexicanum/rhathymum low depression located in the right valve (Fig. 1S, 2L–P).

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
Journal of Eukaryotic Microbiology 2017, 64, 668–677 669
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae mez et al.

Figure 1 Puerto Rican strain of Prorocentrum mexicanum. (A–G, I, K, M) Bright field. (H, J, L, N) Epifluorescence. (O–S) Scanning electron micro-
graphs. (A–C) Note the anterior and posterior nucleus. (D–F). Empty valve, note the different shape in the anterior region. (G–N) 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. (Q–R) 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.

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
670 Journal of Eukaryotic Microbiology 2017, 64, 668–677
mez et al.
Go Prorocentrum mexicanum, P. rhathymum, and P. steidingerae

Figure 2 Brazilian strain of Prorocentrum mexicanum. (A–F) Bright field. (G–P) Scanning electron micrographs. (A–C) Wild cells. (D–Q) Cultured
cells. (D) Note the flagella. (E) Left valve with slightly concave anterior end. (F) Empty theca. (G) Several cells. (H–I) Right valve. (J–K) 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 (A–K), 2 lm (L–Q).

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. 2O–Q). Under
(Fig. 2O). The periflagellar area showed a prominent curved the light microscope, the curved apical plate resembled a

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
Journal of Eukaryotic Microbiology 2017, 64, 668–677 671
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae mez et al.

spine, while under SEM, it appeared as ear-shaped Prorocentrum rhathymum PRJJ2 Korea HF565182
Prorocentrum rhathymum NMN016 Malaysia FJ842096
(Fig. 2Q). Platelet a, which looked like a collar, was the lar- Prorocentrum rhathymum PRJJ1 Korea HF565181
gest of the platelets projected and appeared as a spine Prorocentrum mexicanum Puerto Rico KY426836
Prorocentrum rhathymum PRJJ3 Korea HF565183
under the light microscope (Fig. 2O, Q). Platelet b was
Prorocentrum mexicanum Brazil KY426837
located to the right of platelet a and was surrounded by the Prorocentrum mexicanum PMOO04 Polynesia Y16232
auxiliary pore (AP), platelet d, and platelet e. Platelet c was Prorocentrum koreanum KP711344 Prorocentrum
Prorocentrum koreanum KP711350 mexicanum / koreanum
surrounded by the flagellar pore (FP), AP, and platelets d, f, Prorocentrum rhathymum S7 Florida EU287487
and e (Fig. 2N, O). Platelets d and f were located on the left Prorocentrum rhathymum PXPV-1 Mexican Pacific JQ616822
Prorocentrum micans China DQ004735
valve side, while platelets e and g were on the right valve
Prorocentrum triestinum EF492512
side (Fig. 2O). Platelet h, which was the second largest pla- Prorocentrum gracile AY443019
telet, was located at the right end. There was a ridge (ri) Prorocentrum triestinum DQ004734
Prorocentrum triestinum AB183673
between platelet h and platelets f and g (Fig. 2O). Prorocentrum mexicanum CCMP687 Florida EU287485
The living cells of the Puerto Rican strain ranged from Prorocentrum micans EF492511

29 to 34 lm in length (average 31.8 lm, n = 10), and 19– 0.01
Prorocentrum texanum JQ390504 Prorocentrum micans / texanum
Prorocentrum micans AJ415519 +
24 lm in width. The average length/width ratio was 1.48 Prorocentrum micans JN717145 2 divergent strains of P. mexicanum
(1.42–1.58). The same strain after fixation and observed 100
Prorocentrum micans AY803739
Prorocentrum micans EU780638
under SEM ranged from 23 to 27 lm length (average Prorocentrum mexicanum USFDA Florida EF492510
25.1, n = 11) and 14–17 lm in width, with an average Prorocentrum mexicanum sp3 Vietnam DQ174089 (=P. compressum)
94 Prorocentrum minimum AY803740
length/width ratio of 1.60 (1.44–1.69). The fixation shrank 80 Prorocentrum minimum AY421791
the cells ~21% in length and ~27% in width. Prorocentrum donghaiense AJ841810
We compared the morphology of strains from locations 95
Prorocentrum donghaiense DQ336054
Prorocentrum dentatum AY803742
separated for ~6,000 km (Fig. 1, 2). We did not find signifi- Prorocentrum emarginatum Y16239
cant differences. The shape of the cells of the Puerto 100 Prorocentrum fukuyoi EU196420
Prorocentrum emarginatum EU196418
Rican and Brazilian strains was similar to that in the origi- Alexandrium tamarense JF521641
nal descriptions of P. mexicanum and P. rhathymum. The
pore arrangement is similar to that in the original descrip- Figure 3 SSU rDNA-based phylogeny of Prorocentrum. Sequences
tion of P. rhathymum. Unfortunately, at the time of the obtained in this study are bold-typed. Support of nodes is based on
description of P. mexicanum, the pore arrangement was bootstrap values of ML/NJ with 1,000 and 1,000 resamplings, respec-
not noted. The size of P. mexicanum in the original tively. See methods in Appendix S1. Only values greater than 60 are
description was larger than in our cultured cells. We shown. Alexandrium tamarense was used as outgroup.
observed that the cells reduced their size under culture
conditions and the size was also reduced after fixation. sequences of P. mexicanum/P. rhathymum. The second
Our morphological comparison indicated that our strains clade included some strains identified as P. mexicanum and
corresponded to the species first described as P. mexi- P. rhathymum from Florida (strain CCMP687) and Cuba
canum and later as P. rhathymum. (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
Molecular phylogeny
from the Mexican Pacific, Gulf of Mexico, Caribbean Sea,
Our SSU rDNA sequences obtained from the Puerto Rican Atlantic, Indian, and Western Pacific Oceans (Fig. 4). The
and Brazilian strains of P. mexicanum were identical. In the Floridian and Cuban strains CCMP687 and PXHV-1, respec-
SSU rDNA phylogeny, the new sequences clustered tively, formed a clade that branched separately from the
together with sequences identified as P. mexicanum main clade of P. mexicanum/P. rhathymum (Fig. 4).
from Mexican Pacific (its type locality) and P. rhathymum Sequences from the type localities, the Mexican Pacific
from the Caribbean Sea (its type locality), and sequences and the Caribbean Sea, identified as both P. mexi-
from Asia, Polynesia, and Indian Ocean, together with canum and P. rhathymum, branched together in the same
P. koreanum and a strain misidentified as P. micans. A sis- clade, and there was no molecular support for the split
ter clade was composed of two sequences of P. mexi- between P. mexicanum and P. rhathymum. The strains
canum from Florida (strains CCMP687 and USFDA), along CCMP687, USFDA, FIU25, and PXHV-1 from Florida and
with sequences of P. texanum and P. micans (Fig. 3). Out- Cuba are distantly related to the clade of P. mexicanum/
side these two main clades was a sequence identified as P. rhathymum. These sequences assigned to P. mexicanum/
P. mexicanum (DQ174089) from Vietnam (Fig. 3). The P. rhathymum belong to an independent species (Fig. 3, 4).
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
The synonymy of Prorocentrum mexicanum and
two clades (Fig. S1). The main clade included strains of
P. rhathymum
P. mexicanum and P. rhathymum from the Mexican Pacific,
Caribbean Sea, Asia, Australia, Indian Ocean, NE Atlantic s-Altamirano and Sierra-Beltr
Since Corte an (2003) pro-
and Brazil. There was not a clear geographical cut in the posed the split of P. rhathymum and P. mexicanum,

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
672 Journal of Eukaryotic Microbiology 2017, 64, 668–677
mez et al.
Go Prorocentrum mexicanum, P. rhathymum, and P. steidingerae

Prorocentrum rhathymum PRJJ1 Korea HF565181 type locality of P. rhathymum. The morphology and the
Prorocentrum mexicanum Brazil KY426837
Prorocentrum rhathymum PRJJ2 Korea HF565182
behavior of the cells of the Puerto Rican strain fit with
Prorocentrum rhathymum PXPV-1J Mexican Pacific Q616822 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
characteristics between this and P. mexicanum as
-/- Prorocentrum rhathymum Iran JN020163 reported in the original description by Osorio-Tafall (1942)
66/- Prorocentrum rhathymum VGO893 Malaysia EU244464 (see Appendix S2 in the Supporting Information). By
99/99 Prorocentrum rhathymum NMN16 Malaysia FJ155840
Prorocentrum rhathymum CCMP2933 Florida EU927561
molecular phylogenetic analyses, we show unequivocally
-/- Prorocentrum rhathymum TIO93 China KY010248 that the Puerto Rican strain represents P. rhathymum. We
Prorocentrum rhathymum TIO29 China KY010246
89/76 also obtained the first morphological and molecular data of
Prorocentrum rhathymum TIO55 China KY010247
99/99 Prorocentrum mexicanum CCMP687 Florida EU927554 this species from the South Atlantic Ocean. This extends,
sp. nov.
Prorocentrum rhathymum PXHV-1 Cuba JQ638938 even more, the wide geographical distribution of this
-/- 60/- Prorocentrum micans FJ823584 important toxic species. The current knowledge reveals
66/86 Prorocentrum micans EU244467
94/93 Prorocentrum micans EU780638 that sequences identified as P. mexicanum/P. rhathymum
95/89 Prorocentrum micans
Prorocentrum micans DQ485145 are a common species in shallow protected areas of warm
Prorocentrum micans EU927531
98/90 99/97 Prorocentrum micans EU927521 and temperate inshore waters (Fig. 3, 4 and Fig. S1, see
Prorocentrum texanum JQ390505 P. texanum map in Lim et al. 2013). It is striking that this globally dis-
99/99 Prorocentrum koreanum KP711348
P. koreanum tributed and bloom-forming species had not been reported
Prorocentrum koreanum KY010231
81/83 Prorocentrum donghaiense JN595869
before 1942 or 1979. A taxonomical account is reported in
99/99 Prorocentrum dentatum FJ823581 the Appendix S2. The first illustration of P. mexicanum/
Prorocentrum rostratum EU244471
99/98 P. rhathymum corresponded to the figure 41a of P. maxi-
99/99 Prorocentrum minimum KY290717
Prorocentrum minimum FJ823587 mum from the Adriatic Sea (Fig. 5A, Schiller 1933), and
Karena brevis AF352823 later reported in the same location as P. mexicanum,
P. venetum, and P. rhathymum (Appendix S2).
Figure 4 ITS1-5.8S-ITS2-based phylogeny of Prorocentrum. Sequence
Loeblich et al. (1979) proposed the new species
obtained in this study is bold-typed. Support of nodes is based on
P. rhathymum ignoring the description of P. mexicanum by
bootstrap values of NJ/ML with 1,000 and 1,000 resamplings, respec-
Osorio-Tafall (1942). Prorocentrum rhathymum and P. mexi-
tively. See methods in Appendix S1. Only values greater than 60 are
canum were soon considered synonyms with priority for
shown. Karenia brevis was used as outgroup.
the earlier name (Faust 1990; Faust and Gulledge 2002;
Steidinger 1983; Steidinger and Tangen 1997). Corte s-Alta-
nearly all the SEM studies have identified it as P. rhathy- mirano and Sierra-Beltr an (2003) reinstated P. rhathymum
mum and showed a smooth valve surface (Aligizaki et al. and P. mexicanum as separate species describing signifi-
2009; Gul and Saifullah 2011; Laza-Martınez et al. 2011; cant differences between both species. However, the spe-
Lim et al. 2013; Mohammad-Noor et al. 2007b; Su-Myat cies that they attributed to P. mexicanum did not
and Koike 2013). From strains isolated near the type local- correspond in shape, surface ornamentation, and other
ity of P. mexicanum, Cohen-Fernandez (2011) identified details (i.e. apical spine) to the description of P. mexicanum
the sequences as P. mexicanum, but the SEM micro- in Osorio-Tafall (1942) (Fig. 5B–D and Table 1;
graphs corresponded to P. rhathymum, cells with smooth Appendix S2). The original description of P. mexicanum
valve surface as illustrated by Loeblich et al. (1979). After reported an ellipsoidal cell, with symmetrical valve margin
Cortes-Altamirano and Sierra-Beltran (2003), there have and a rounded antapex (Fig. 5C, Osorio-Tafall 1942). In con-
been no further SEM pictures of the bloom-forming spe- trast, P. mexicanum sensu Corte s-Altamirano & Sierra-
cies P. mexicanum. The earlier sequences were submitted Beltran possesses asymmetric valve margins and a slightly
as P. mexicanum (Cohen-Fernandez et al. 2010; Daugbjerg pointed antapex (Fig. 5D). Prorocentrum mexicanum was
et al. 2000; Grzebyk et al. 1998; Murray et al. 2007;
Scorzetti et al. 2009; Stern et al. 2010), and after 2003 A B C D E
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.
Figure 5 Line drawings of Prorocentrum mexicanum and allied
2009). This can cause confusion in the research commu- species. (A) P. maximum, redrawn from Schiller (1941, fig. 41a).
nity, as has been experienced in the study of Alexandrium (B) P. rhathymum, redrawn from Lim et al. (2013). (C) P. mexicanum,
tamarense complex until in-depth molecular phylogeny redrawn from Osorio-Tafall (1942). (D) P. mexicanum, redrawn from
helped clear up the ambiguities in its morphology-based Cortes-Altamirano and Sierra-Beltr an (2003, fig. 1c). Note the scat-
taxonomy (e.g. Miranda et al. 2012; Wang et al. 2014). tered larger pores on the poroid surface. (E) P. texanum, redrawn
In this study, we provide morphological and molecular from Henrichs et al. (2013, fig. 2f). The large scattered pores are illus-
data of a strain collected from Puerto Rico, close to the 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, 668–677 673
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae mez et al.

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-Beltran (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 Rows TP in apical RV TP in apical LV Flange

P. mexicanum Smooth Deep < 70 6–8 6–7 4–5 Nonserrated, pointed
P. steidingerae Partially poroid Shallow 80–100 8–12 4 No, 9–12 TP in periphery Nonserrated, curved
P. texanum Poroid Shallow < 70 8–11 5–6 No, 5–7 TP in periphery Serrated (2–6 crests)

TP = trichocyst pore; RV = right valve; LV = left valve.

described with a tiny apical spine in Osorio-Tafall (1942), mistaken for P. micans, and this species has not received
while for Corte s-Altamirano and Sierra-Beltran, showed a attention in the molecular surveys (Cohen-Fern andez et al.
prominent three-horned (sometimes two-horned) spine. 2010; Herrera-Sepu lveda et al. 2013). We conclude that the
Osorio-Tafall described P. mexicanum with a smooth valve species with a smooth valve surface P. maximum sensu
surface with disperse large pores (Fig. 5C). Corte s-Altamir- Schiller (1933, his fig. 41a), P. rhathymum, and P. venetum
ano and Sierra-Beltran reported the valves fully covered by are synonyms of P. mexicanum. The planktonic species
poroids, and other dispersed type of pores (Fig. 5D). It is with a rugose valve surface assigned as P. mexicanum by
expected that the important morphological differences Cortes-Altamirano and Sierra-Beltr an (2003) corresponds to
reported in Corte s-Altamirano and Sierra-Beltran (2003) will P. texanum (Table 1 and Fig. 5).
be associated with genetic differences between P. rhathy-
mum and P. mexicanum. However, in the molecular phylo-
The genetically divergent Floridian and Cuban strains
genies, the sequences from distant oceans identified as
P. rhathymum and P. mexicanum are intermixed and Osorio-Tafall (1942) and Loeblich et al. (1979) described
branched together, with the exception of several strains P. mexicanum with a smooth valve surface, while P. tex-
from the Gulf of Mexico (Fig. 3, 4 and Fig. S1). The micro- anum (=P. mexicanum sensu Corte s-Altamirano & Sierra-
graphs associated with sequences identified as P. rhathy- Beltran) has a rugose valve surface covered by poroids.
mum or P. mexicanum did not correspond to the cells Prorocentrum mexicanum was described with a nonser-
reported as P. mexicanum by Corte s-Altamirano and Sierra- rate apical flange, while P. texanum showed a serrated
Beltran (2003). This means that nobody has obtained yet the flange with crests (Corte s-Altamirano and Sierra-Beltr an
sequence of the bloom-forming species illustrated by 2003; Henrichs et al. 2013). Prorocentrum mexicanum is
Cortes-Altamirano and Sierra-Beltran (2003) or that the more commonly reported as an epiphytic or tychoplank-
sequence of P. mexicanum sensu Corte s-Altamirano & tonic species, while P. texanum is planktonic. Despite the
Sierra-Beltran has been reported under other species name. 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-
Prorocentrum mexicanum and P. texanum
mum. This third species shares the same habitat and
Cortes-Altamirano and Sierra-Beltran (2003) misidentified distribution of P. mexicanum in the Gulf of Mexico and
P. mexicanum with the planktonic species P. texanum that Caribbean Sea, and it shows intermediate morphological
was later described from the Gulf of Mexico (Fig. 5D, E, characteristics between P. mexicanum and P. texanum.
Henrichs et al. 2013). The shape and size of P. texanum In one of the earlier studies on epiphytic dinoflagellates in
var. cuspidatum (Fig. 5E) are similar to that of P. mexi- the Gulf of Mexico, Bomber (1985) isolated the strain of
canum sensu Corte s-Altamirano & Sierra-Beltran (Fig. 5D). Prorocentrum sp. FIT#182 from the Florida Keys. Bomber
The valves of both taxa were covered by poroids and (1985) did not report illustrations and reported the strain as
trichocyst pores. Both taxa have 5–6 recessed, trichocyst “P. rhathymum (syn. mexicanum)” in pages 4 and 103, and
pores below the periflagellar platelets on the right valve. as P. mexicanum in page 22. Up to date, that species identi-
Other distinctive feature of both taxa is the 2–3 crest ser- fied as P. mexicanum is maintained as strain CCMP687 at
rated apical flange (Corte s-Altamirano and Sierra-Beltr an the National Center for Marine Algae and Microbiota at
2003; Henrichs et al. 2013). In the SSU rDNA and ITS Bigelow Laboratory, East Boothbay, Maine, USA. Other
phylogenies, P. texanum is well separated from the clade strains from the Florida Keys (CCMP2933, CCMP2976) are
of P. mexicanum/rhathymum and close to P. micans named P. rhathymum ( The
(Fig. 3, 4). This is coherent with the morphological differ- strain CCMP687 is genetically characterized using several
ences (Table 1 and Appendix S2). Prorocentrum texanum molecular markers (Murray et al. 2007, 2009; Scorzetti
was previously overlooked as morphotypes of P. micans et al. 2009; J.F. Ferrell, M.J. Beaton, unpublished). Ferrell
(Henrichs et al. 2013). Prorocentrum micans is a common and Beaton provided LM and SEM pictures of the strain
and nontoxic species and the blooms have received less CCMP687 that showed a valve surface was partially cov-
attention. In the Mexican Pacific, P. texanum (=P. mexi- ered by poroids. Most of their pictures corresponded to
canum sensu Corte s-Altamirano & Sierra-Beltran) could be cells under division, when the parent cell is splitting in two

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
674 Journal of Eukaryotic Microbiology 2017, 64, 668–677
mez et al.
Go Prorocentrum mexicanum, P. rhathymum, and P. steidingerae

with each theca forming a new companion theca. In these A C
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 RV RV
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-Sepulveda 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 B D
Cuban strains, especially in the ITS phylogeny, revealed that
this species formed a sister group of the clade of P. mexi- RV
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- LV
oids, a nonserrate round apical flange, deep indentation of
the periflagellar area in the right valve, as well as an arrange- Figure 6 Line drawings of Prorocentrum mexicanum and P. stei-
ment of the trichocyst pores that does not correspond to dingerae sp. nov. (A) Right valve and (B) Apical view of P. mexicanum
P. mexicanum or P. texanum (Fig. 6). As Faust (1990) based on this study. (C) Right valve and (D) Apical view of P. stei-
assigned P. mexicanum to this species, Corte s-Altamirano dingerae sp. nov. based on Faust (1990, her fig. 5, 7).
and Sierra-Beltran (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 This new species shares the habitat with and has been
assigned P. rhathymum for epiphytic cells with a smooth mistaken for P. mexicanum in the Gulf of Mexico and Carib-
valve surface. The identity of the species reported by Bom- bean Sea. The diagnostic characters are reported in the
ber (1985) and Faust (1990) as well as isolates by several Table 1 and Fig. 6. The excavation of the periflagellar area in
authors in Florida and Cuba needs to be clarified as a neces- the right valve is deeper in P. steidingerae than in P. mexi-
sary step to resolve the controversy in the synonymy of canum. The platelet a forms a prominent curved apical collar
P. mexicanum/P. rhathymum. We propose here that taxon that is acuter in P. mexicanum. The new species has a valve
as a new species based on the existing morphological and surface rugose covered by poroids, while the valve is smooth
molecular data. 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 8–12 tan-
 mez, D. Qiu & Senjie
Prorocentrum steidingerae F. Go
gential rows of trichocyst pores, while usually six rows in
Lin, sp. nov
P. mexicanum. This new species shows a row of four tri-
Diagnosis. Cells are oval in valve view (30–40 lm long chocyst pores in the apical right valve, while P. mexicanum
and 20–25 lm wide), subovate, and convex in side view. shows a row of 6–7 trichocyst pores. Prorocentrum stei-
The periflagellar area is located in a depression in the dingerae does not have the clumps of 3–5 small pores in the
right valve. The cells show an apical spine with a curved valve apical margins that are a characteristic feature of
shape. The valve surface of mature cells is rugose, cov- P. mexicanum (Table 1 and Fig. 6).
ered by poroids. The valves show two types of pores. Iconotype. Figure 6C.
The small pores (~0.2 lm in diameter) are circular and Type locality. Gulf of Mexico, off Knight Key, Florida
unevenly distributed along the valve. No clumps of small (24°420 29.88″N, 81°70 30″W). Isolated by Bomber (1985).
pores were observed in the apical margins of the valve. Habitat. Epiphytic in macrophytes, detritus and sediments
There are 80–100 trichocyst pores in each valve, some in tropical coasts. Currently known from Belize, Cuba, and
unevenly distributed and most of them forming tangen- USA at Florida.
tial rows. There are 8–12 rows of 3–6 trichocyst pores Etymology. In honor of Karen A. Steidinger, who first
in depressed furrows in the margin of the anterior and denoted the synonymy between P. mexicanum and
medium parts of the valve. There is a row of four tri- P. rhathymum, and carried out numerous studies on toxic
chocyst pores in the right valve adjacent to the periflag- dinoflagellates, especially in Florida where this new species
ellar area. is commonly reported.

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
Journal of Eukaryotic Microbiology 2017, 64, 668–677 675
Prorocentrum mexicanum, P. rhathymum, and P. steidingerae mez et al.

Synonyms. Prorocentrum mexicanum sensu Faust (1990,
her fig. 5–10), P. mexicanum sensu Steidinger and Tangen
(1997, p. 421, plate 8); Prorocentrum mexicanum Aligizaki, K., Nikolaidis, G., Katikou, P., Baxevanis, A. D. & Abat-
CCMP687 (=PM200A, =FIT#182) from Florida Keys (Bom- zopoulos, T. J. 2009. Potentially toxic epiphytic Prorocentrum
ber 1985) (accession numbers DQ336183, EU287485, (Dinophyceae) species in Greek coastal waters. Harmful Algae,
EU165318, EU916907, EU927554); P. mexicanum USFDA 8:299–311.
An, T., Winshell, J., Scorzetti, G., Fell, J. W. & Rein, K. R. 2010. Iden-
from Florida (accession number EF492510); Prorocentrum
tification of okadaic production in the marine dinoflagellate Proro-
sp. FIU25 from Florida (accession number EU165283); centrum rhathymum from Florida Bay. Toxicon, 55:653–657.
P. rhathymum PXHV-1 from Havana, Cuba (accession  rard-Therriault, L., Poulin, M. & Bosse
Be , L. 1999. Guide d’identifi-
number JQ638938). cation du phytoplancton marin de l’estuaire et du golfe du
Zoobank registration. Saint-Laurent incluant e galement certains protozoaires. Publ.
EA-114C-4A08-B0F6-6BCA01D342C9. Spe  c. Can. Sci. Halieut. Aquat., 128:141–240.
Bomber, J. W. 1985. Ecological studies of benthic dinoflagellates
with ciguatera from the Florida Keys. M.S. Thesis. Florida Insti-
TAXONOMICAL CONSIDERATIONS tute of Technology, Melbourne. 104 p.
Prorocentrum maximum (Gourret) J. Schiller (1933, fig. Caillaud, A., de la Iglesia, P., Campas, M., Elandaloussi, L.,
Fern andez, M., Mohammad-Noor, N., Andree, K. & Dioge  ne, J.
41c). Basionym: Postprorocentrum maximum Gourret
2010. Evidence of okadaic acid production in a cultured strain
(1883, fig. 50) of the marine dinoflagellate Prorocentrum rhathymum from
Non-Prorocentrum maximum sensu Schiller (1933, fig. Malaysia. Toxicon, 55:633–637.
41b–c), nec P. maximum sensu Osorio-Tafall 1942. This Cohen-Fern andez, E. J. 2011. Morfologıa externa del complejo
species is probably a junior synonym or confused with Prorocentrum micans, P. gracile y P. sigmoides y filogenia
P. micans Ehrenberg. molecular de Prorocentrum (Dinoflagellata) del Pacıfico Mexi-
cano. Ph.D. Universidad Auto  noma Metropolitana, Mexico City,
Prorocentrum triestinum J. Schiller (1918, fig. 1a). Syno- Mexico. 112 p.
nyms: Prorocentrum brochii J. Schiller (1918, fig. 2), Cohen-Fern andez, E. J., Pedroche, F. F., Rodrıguez-Palacios, M.,
P. maximum sensu Schiller (1933, fig. 41b). Hern andez, S. & Meave del Castillo, E. 2010. Molecular phy-
logeny of Prorocentrum (Dinoflagellata) from the Pacific coast of
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Mexico based on the parsimony analysis of fragments of LSU
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© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists
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