Dinoflagellate macroevolution: some considerations based on an integration of molecular, morphological and fossil evidence

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Dinoflagellate macroevolution: some considerations based
on an integration of molecular, morphological and
fossil evidence
L. K. MEDLIN1* & R. A. FENSOME2*
1
Marine Biological Association of the UK, The Citadel, Plymouth PL1 2PB, UK
2
Geological Survey of Canada (Atlantic), Bedford Institute of Oceanography,
PO Box 1006, Dartmouth, Nova Scotia, Canada B2Y 4A2
*Corresponding authors (e-mail: lkm@mba.ac.uk and rfensome@nrcan.gc.ca)
Abstract: Dinoflagellates have been regarded as bizarre products of evolution. They belong to
one of the most strongly supported macrolineages among the protists, the superphylum/
kingdom Alveolata, which contains three main phyla: the Dinoflagellata, the Apicomplexa and
the Ciliata. These organisms all have cortical alveoli and micropores. Until the early 1990s,
living and fossil dinoflagellates were classified separately and both relied almost exclusively on
morphological characters. During the early 1990s, fossil and living taxa were brought together
in a detailed morphological classification that emphasized tabulation. Since that time, molecular
studies have supported many morphological groups, but have shown others to be paraphyletic.
Our understanding of phylogenetic relationships within the dinoflagellates has changed as
more taxa have been described and more genes have been analysed. Relationships among the
lineages also vary widely with the algorithm used to analyse the data. A highly unusual and
notable feature of dinoflagellates is the variety of plastid types that they have acquired by second-
ary and even tertiary symbiosis; indeed, they possess the most diverse array of plastids of any
eukaryotic lineageand they are truly the kings of symbioses. Genome rearrangements have
taken place as the plastids evolved. The genes that have moved to the nucleus in dinoflagellates
with peridinin plastids are different from those moved in all other eukaryotes; moreover the few
genes left behind in the peridinin plastid have become uniquely arranged into mini-circles.
Where tertiary endosymbiosis has taken place, the plastid genome was rearranged again. Mito-
chondrial modifications in the dinoflagellates are also unique among the eukaryotes. While
study of these factors remains critical in understanding dinoflagellate phylogeny, the fossil
record continues to contribute by presenting morphologies that are unrepresented (or under-
represented) among extant taxa; such observations can suggest relationships to be tested by
molecular analyses.
Dinoflagellates are protists that typically possess
a unique type of nucleus in which the chromoso-
mes are permanently condensed and in which the
motile cell typically possesses two distinctive, dis-
similar flagella. Other aspects of dinoflagellates
are also very unusual; for example, their metabolic
processes rarely follow the norm and the origin of
their plastids is enigmatic. Unlike many related
protistan groups, dinoflagellates have left a signifi-
cant (albeit incomplete) fossil record, mainly in
the form of resistant organic-walled or calcareous
resting cysts. In this review, we will discuss dino-
flagellate origin and evolution as evidenced by
both molecular methods and morphology, the lat-
ter including extensive observations from fossils.
We will also review the relationship between dino-
flagellates and other protists, and the evolution of
dinoflagellate plastids and mitochondria.
Origin and relationships with other
protists
Fensome et al. (1993) developed a phylogenetic tree
for dinoflagellates and their closest allies based
on ultrastructure (Fig. 1). It shows that dinofla-
gellates (dinokaryotes and syndinians) share the
possession of cortical vesicles (alveolae) with api-
complexans and ciliates, a grouping of protists
classified together as the alveolates (see also Adl
et al. 2005). According to this tree, alveolates in
turn share the possession of tubular mitochondrial
cristae, an intranuclear spindle (which in dinofla-
gellates purportedly became secondarily extranuc-
lear) and rod trichocysts with chromophytes. At
the base of the lineage to dinokaryotes (dinoflagel-
lates ‘proper’) and their sister group, the syndinians,
From:Lewis, J. M., Marret,F.&Bradley, L. (eds) 2013. Biological and Geological Perspectives of Dinoflagellates.
The Micropalaeontological Society, Special Publications. Geological Society, London, 263– 274.
#The Micropalaeontological Society 2013. Publishing disclaimer: http://www.geolsoc.org.uk/pub_ethics

Fensome et al. (1993) noted the development
of the characteristic dinoflagellate flagella and
redevelopment of an extranuclear mitotic spindle.
In the dinokaryote clade, Fensome et al. (1993)
interpreted the development of the temporary
dinokaryon (as in Noctilucales, which only have
condensed chromosomes in the gamete stage, and
Blastodiniales) as a basal trait, followed by the deve-
lopment of a permanent dinokaryon in all other
dinoflagellates. Saunders et al. (1997, fig. 4) built
upon this model by interpreting the peduncle mode
of feeding (myzotosis) as a basal trait leading to
free-living autotrophic dinoflagellates.
Subsequent molecular data confirm that the
dinoflagellates are a monophyletic group, with the
so-called pre-dinoflagellate Oxyrrhis lying outside
core dinoflagellates. In the Fensome et al. (1993)
tree, Oxyrrhis would diverge between ciliates and
syndinians prior to development of dinokont fla-
gella and an extranuclear spindle. Molecular stud-
ies have confirmed dinoflagellates as the sister
group to the apicomplexans (perkinsid flagellates)
with high bootstrap support, which are in turn sis-
ter to the ciliates, again with high bootstrap support
(Leander & Keeling 2004). A less robust sister
relationship (,50%) has been commonly recovered
between alveolates (apicomplexans) and the stra-
menopiles (¼heterokont organisms) (Leander &
Keeling 2004), leading to a grouping of the alveo-
lates with the cryptomonads and haptophytes to
form the chromalveolates. The chromalveolates
have the least bootstrap support and lack firm mol-
ecular evidence, but are nevertheless still thought
to be monophyletic. Recent re-analysis of 108
genes from nuclear, plastid and mitochondrial
genomes have failed to recover a well-supported
host-cell lineage for the chromalveolates (Baurain
et al. 2010).
A date for the divergence of stramenopiles
(heterokonts) and alveolates at 950 Ma has been
determined by molecular clocks using fossils other
than dinoflagellates for calibration (Douzery et al.
2004). Using fossil dinoflagellates to calibrate
the clock, Medlin (2008, 2011) dated the divergence
of the dinoflagellates from the apicomplexans at
650 Ma. The presence of triaromatic dinosteroids,
assumed to be derived from dinoflagellates, has
been recorded in some acritarchs (organic-walled
microfossils of uncertain origin on morphologi-
cal grounds) of pre-Carboniferous age (Moldowan
et al. 1996; Moldowan & Talyzina 1998). These bio-
geochemical markers could therefore reflect the
existence of ancestral members of the dinoflagellate
lineage. An alternative suggestion could be that at
least some acritarchs represent one or more extinct
lineages with no surviving descendants.
Although biogeochemical evidence of the exist-
ence of pre-Mesozoic dinoflagellates is compel-
ling, it is not backed up by fossil morphological
evidence either because pre-Mesozoic members of
the dinoflagellates did not leave a preservable
record, or because pre-Mesozoic dinoflagellate
remains are not clearly recognizable as such. The
few claims based on morphology for dinoflagel-
late affinity among pre-Mesozoic fossils have not
been convincing (see Fensome et al. 2000). The
most famous of these is the Silurian fossil Arpy-
lorus, which Le Herisse et al. (2012) have con-
vincingly shown not to be a dinoflagellate but part
of a more complex biological structure possibly
associated with eurypterids. One recent intriguing
observation by Servais et al. (2009) was that some
Ordovician calcareous microfossils display wall
structures that are surprisingly similar to those of
calcareous dinoflagellate cysts. All confirmed
calcareous cysts are peridinioids, a group that on
compelling evidence aside from crystal structure
originated in the Jurassic. Speculation by Servais
et al. (2009) that these fossils could represent the
ancestors of dinoflagellates, if borne out, would
therefore be a fundamental challenge to current
ideas on the pattern and timing of dinoflagellate
evolution.
APICOCOMPLEXANS
SYNDINIANS
DINOKARYOTES
CILIATES
CHROMOPHYTES
6
9
7
11
10
8
13
4
5
123
12
Fig. 1. A phylogenetic tree for dinoflagellates
and their allies based on ultrastructure as proposed by
Fensome et al. (1993). The length of the lines is not
significant. Derived character states (indicated by
numbers): 1, tubular mitochondrial cristae; 2,
intranuclear spindle; 3, rod trichocysts; 4, compound
flagellar hairs; 5, cortical (amphiesmal) vesicles
(alveolae); 6, apical complex; 7, polykineties; 8, nuclear
dimorphism; 9, donokont flagella; 10, extranuclear
spindle; 11, dinokaryon (temporary); 12, dinokaryon
(permanent); 13, dinokaryon (temporary); loss
of trichocysts.
L. K. MEDLIN & R. A. FENSOME264

Plastid evolution
A series of endosymbiotic events has given rise
to all photosynthetic organisms. Initially a hetero-
trophic ancestor engulfed a cyanobacterium and
transformed it into a plastid to form the first photo-
synthetic cell. This single primary endosymbiosis
resulted in the red, green and glaucophyte algae
(see reviews in Archibald & Keeling 2002 and
Sanchez-Puerta & Delwiche 2008). Molecular ana-
lyses have shown that the host and the plastid
lineages in this event are both monophyletic, so this
event happened only once. Another heterotrophic
organism then engulfed either a red or green alga
from the first endosymbiosis to form the secondary
event. If plastids in a particular alga are surrounded
by two membranes, then the alga was derived from
the primary endosymbiosis; if three to four mem-
branes surround the plastid, then the alga is derived
from a secondary endosymbiosis (Keeling 2004).
The hypothesis that this secondary endosymbiosis
also happened only once has been controversial
(Yoon et al. 2002); the recent re-analysis of multiple
genes from multiple genomes that failed to recover a
monophyletic origin for all plastids from the sec-
ondary endosymbiosis (Baurain et al. 2010) has res-
urrected the hypothesis of multiple secondary
endosymbiosis events for each plastid in the second-
ary endosymbiosis.
Recently, Moustafa et al. (2009) have uncovered
unusual evidence from whole genome and expres-
sed sequence tag (EST) analyses, which suggests
that a sequence of unusual endosymbiotic events
led to the chromalveolate lineage. Traces of green
genes can be found in this lineage, which today is
a red algal plastid lineage. This has been inter-
preted to mean that algae in the lineage first had
green plastids, but this green plastid type was sub-
sequently replaced by a red plastid. Falkowsky
et al. (2004) hypothesized that red plastids had
an adaptive advantage only after the end-Permian
mass-extinction event at 251 Ma. At that time,
oceans became anoxic and their trace-metal chem-
istry changed such that iron became abundant. The
red plastid-bearing microalgae have iron-containing
cytochrome c6 in their photosynthetic electron
carrier complex instead of the copper-containing
plastocyanin of the photosystem of green plastid-
bearing algae.
If chromalveolates underwent multiple second-
ary endosymbioses (Baurain et al. 2010), then each
lineage should have acquired a red algal plastid
and dumped the green one at different times.
Medlin (2011) constructed a molecular clock to
determine if the radiations in the different chromal-
veolates lineages relate to the end-Permian event.
She found that it corresponded to different taxo-
nomic levels of radiation in the various lineages,
and that this supported the idea that the green plas-
tid was replaced by the red plastid multiple times.
Dinoflagellates have many different types of
plastid. The predominant peridinin plastid is
believed to have originated from a red algal second-
ary endosymbiosis. Supposedly, a tertiary endosym-
biosis occurred in the dinoflagellates when the
original plastid from the secondary endosymbiosis
(this should be the peridinin plastid) was replaced
by another plastid from the secondary endosym-
biotic algae. Cryptophyte, prasinophyte and hapto-
phyte plastids, which are each originally derived
from a red algal secondary endosymbiosis, were
therefore incorporated into the dinoflagellate line-
ages by various species (see Saldarriaga et al. 2001).
However, the peridinin lineage does not appear
independent in any of the plastid gene trees in dino-
flagellates as it does in the heterokont, cryptophyte
and haptophyte plastid lineages (Yoon et al. 2005;
Verbruggen 2011; A. Moustafa, pers. comm., 2012).
In contrast, the peridinin lineage is either (1)
embedded in the heterokont lineage as sister to the
diatoms (Yoon et al. 2005) or, with better taxon
sampling, as sister to the chrysophytes/synurophyte
lineage, which is sister to the diatoms (Verbrug-
gen 2011); or (2) embedded in the green lineage.
Evidence therefore suggests that all dinoflagellate
plastids are the result of at least four tertiary endo-
symbioses (heterokont ¼diatom, cryptophyte, hap-
tophyte and peridinin ¼heterokont chrysophytes).
This means that in the dinoflagellates, there was
no transformation of either a red or a green plastid
into the peridinin plastids and all modern plastids
in the dinoflagellates are the result of tertiary endo-
symbioses. Furthermore, it also suggests that it was
the green plastid that was eliminated at the time of
the four tertiary endosymbiosis. If the timing of
the end-Permian event is placed over the dinoflagel-
late tree using a molecular clock, this extinction
event corresponds to radiation at the generic level
in the dinoflagellates, at the order and family level
radiation in the haptophytes and at the phylum
level radiation in the heterokonts (Medlin 2011).
Generic level radiation of the extant dinoflagellates
also is also supported by their early Mesozoic fossil
record (Fensome et al. 1996). After the end-Permian
mass extinction, the dinoflagellates likely evolved
from a heterotrophic lineage to an autotrophic or
mixotrophic lineage by only tertiary endosymbioses
of several different algal groups replacing the orig-
inal green plastid. However, it makes no evolution-
ary sense to have re-engulfed a green algal cell if
the green algal plastid was a disadvantage at this
time; it is more likely that the dinoflagellate/green
lineage represents a relict green plastid that was
originally present in the entire alveolate lineage.
There is evidence that the last common ancestor of
dinoflagellates, apicomplexans and ciliates likely
CONSIDERATIONS OF DINOFLAGELLATE MACROEVOLUTION 265

had a green plastid, because there are traces of green
plastid genes in all of the host lineages (Takishita
et al. 2003; Hackett et al. 2004; Patron et al. 2006;
Moustafa et al. 2009). The common mode of feed-
ing by a peduncle in the ancestral dinoflagellates
would mean that the host cell membrane was left
behind after the dinoflagellate finished sucking out
its contents, which would result in typically only
three membranes around the dinoflagellate plastid
instead of the four membranes found around the
plastid in the other algal groups involved in the
secondary endosymbiosis.
The transformation of the heterokont plastid
into the peridinin plastid after it was engulfed
provides some of the most intriguing genomic rear-
rangement known in the eukaryotic world. Only
about 12 genes coding for plastid function are left
behind following the massive transfer of genes
from the plastid into the nucleus. These 12 genes
form mini-circles of genes of different sizes in
different species of the same genus but the mini-
circles from different species within a genus share
nearly identical spacer regions between the genes
(Zhang et al. 2002). This same massive gene trans-
fer and gene rearrangement did not happen in the
haptophyte and cryptophyte plastid-bearing dino-
flagellates, which adds more evidence that the ter-
tiary endosymbioses are independent events and
that the red algal plastid in each of these lineages
came from a different, independent secondary endo-
symbiosis. The genes encoded by the plastid pos-
sess another different feature. The normal poly A
tail of the messenger ribosomal nucleic acids
(mRNA) in the plastid is replaced by a poly T tail
(Wang & Morse 2006), so when converting mRNA
to complementary Deoxyribonucleic acid (cDNA)
all plastid-encoding ESTs from dinoflagellates can
be differentially separated from other plastids.
Evolution within the dinoflagellates:
morphological background
Prior to the late 20th century, little effort was made
to develop a view of dinoflagellate evolution that
accommodated evidence from both fossils and
modern forms. The first concerted attempt to relate
the two realms was that of Evitt (1985), who made
detailed and extensive comparisons of tabulation
patterns reflected on fossil cysts with the tabulation
patterns of living taxa. However, Evitt (1985) made
little attempt to couch his comparisons in phyloge-
netic terms; that was left to Fensome et al. (1993)
who, building on Evitt’s work, produced the first
modern phylogenetically based classification that
incorporated both fossil and living dinoflagellates.
A key feature of dinoflagellate anatomy is
tabulation. As alveolates all dinoflagellates have
vesicles in the cortex, beneath the cell’s outer cell
membrane. In dinoflagellates, this cortical region
is referred to as the amphiesma, and the vesicles
are known as amphiesmal vesicles. Amphiesmal
vesicles may remain empty or they may contain cel-
lulosic plates, termed thecal plates. Collectively,
thecal plates form a cell wall known as a theca.
The arrangement of amphiesmal vesicles (with or
without thecal plates) is called tabulation. (The
term paratabulation is sometimes used to refer to
‘reflected’ tabulation on cysts.) Arrangement of
the thecal plates varies to form tabulation patterns,
many elements of which tend to be consistent and
stable within related taxa or lineages. Tabulation
and tabulation patterns are therefore important in
unravelling phylogenetic relationships among many
groups of dinoflagellates. Fensome et al. (1993)
recognized six basic tabulation pattern types
(Fig. 2):
(1) gymnodinioid, in which amphiesmal vesicles
(usually without thecal plates) are numerous
and arranged randomly or in more than ten
latitudinal series
(2) suessioid, in which amphiesmal vesicles
(usually with thecal plates) are arranged into
seven to ten latitudinal series and a cingulum
that tends not to be distinctly differentiated
(3) gonyaulacoid-peridinioid, in which amphies-
mal vesicles (with thecal plates) are arranged
in five or six latitudinal series, with a clearly
differentiated cingulum
(4) dinophysioid, in which amphiesmal vesicles
can be attributed to four latitudinal series
with a distinctive cingulum, and in which a
sagittal (longitudinal) suture, perpendicular
to the plane of the cingulum, divides the
theca into left and right halves
(5) nannoceratopsioid, in which the hyposome
(that part of the cyst posterior to the cingulum)
is ‘dinophysioid’ and the episome (anterior to
the cingulum) is ‘gonyaulacoid-peridinioid’
(6) prorocentroid, in which the vesicles are
arranged into two very large plates separated
by a sagittal suture and an area of small
plates surround the flagellar apertures.
The earliest confirmed fossil dinoflagellates are
from the (possibly) Middle–Late Triassic, 240 –
200 Ma. Distinctive among these were forms with
a suessioid tabulation pattern, although a variety
of tabulation patterns were represented by Late Tri-
assic and Early Jurassic forms. From the Middle
Jurassic, most fossil dinoflagellates have a clearly
gonyaulacoid-peridinioid tabulation pattern. In an
early attempt to make sense of the dinoflagellate
fossil record, Bujak & Williams (1981) proposed
three possible evolutionary models: the plate
increase model, in which early tabulation involved
L. K. MEDLIN & R. A. FENSOME266

relatively few plates, the number of plates tending
to increase with time; the plate reduction model
with the reverse trend (a model which would make
sense if forms such as Suessia were indeed early-
derived); and the plate fragmentation model, which
would involve early, perhaps prorocentroid-like,
forms with few plates leading to later multi-plated
forms. As has become apparent with hindsight and
armed with molecular data, these models are too
simplistic. They did provide a timely stimulus for
tying fossils and living forms together from an evol-
utionary perspective, however.
A question among fossil dinoflagellate research-
ers in the latter decades of the 20th century was
whether the appearance of dinoflagellates during
the later Triassic– Early Jurassic reflected a real
evolutionary event, or perhaps the ‘switching on’,
of preservable cyst-forming capability in one or
more lineages with a long pre-Mesozoic ancestry.
Researchers at the time were concerned about the
incompleteness of the fossil record and worried
that this might hamper anything meaningful being
determined from fossils about the evolution of the
group (Evitt 1981). However, Fensome et al.
(1996) reasoned that the early Mesozoic record of
dinoflagellates bore clear hallmarks of a true
evolutionary radiation, with highly variable ‘exper-
imental’ morphologies (at least as reflected by
tabulation) over a few tens of millions of years
during the later Triassic and Early Jurassic, before
stabilization into a few ‘standard’ tabulation types
from about 175 Ma to the present day (again in
terms of mostly gonyaulacoid and peridinioid tabu-
lation patterns). Although multi-plated Suessia,
as well as forms such as Rhaetogonyaulax with
a plate-rich gonyaulacoid-peridinioid tabulation,
were prominent during the Late Triassic, Valvaeo-
dinium, a gonyaulacoid-peridinioid with below-
average plate numbers for the group, was also a
member of the Late Triassic assemblage. Also pre-
sent among Jurassic assemblages was Nannocera-
topis, a strange combination of dinophysioid and
gonyaulacoid-peridinoid tabulation features (Piel
& Evitt 1980).
In their intra-dinoflagellate tree, Fensome
et al. (1993, p. 206; slightly modified as Fig. 1 here)
placed the Noctilucales and Blastodiniales as
basal groups because of their ‘part-time’ dinokar-
yon, with the Gymnodiniales basal to the remainder
of the dinoflagellates, followed in order of deriva-
tion by the Suessiales, Gonyaulacales, Peridin-
iales, Dinophysiales and Prorocentrales. No major
advances based on dinoflagellate morphology have
been proposed since this scheme was introduced.
Below we will explore how this scheme holds up
against subsequently established molecular trees.
(a)
(d)(e)(f)
(b)(c)
Fig. 2. Dinoflagellate tabulation types: (a) gymnodinioid, (b) suessioid, (c) gonyaulacoid-peridinioid, (d) dinophysioid,
(e) nannoceratopsioid, (f) prorocentroid.
CONSIDERATIONS OF DINOFLAGELLATE MACROEVOLUTION 267

Evolution of the dinoflagellates: molecular
results
Unfortunately, most molecular trees for dinoflagel-
lates have concentrated on closely related genera
or groups of species rather than the entire range
of the group. An exception was the tree produced
by Saldarriaga et al. (2004). Using ciliates as the
outgroup, parasitic and atypical taxa such as Amoe-
bophrya and Cryptosporidium diverge early, as pre-
dicted by their morphological features, before the
core dinoflagellates radiate. In this radiation, gym-
nodinioid clades (in which all taxa are athecate or
naked) alternate in divergence with clades that con-
tain peridinioid taxa. Although most thecate genera
were well supported and monophyletic in the var-
ious peridinioid clades, Gymnodinium was paraphy-
letic and relationships between the gymnodinioid
clades were not supported. The Gonyaulacales were
in the final divergence and the Prorocentrales were
paraphyletic.
A maximum likelihood analysis of 1246 dinofla-
gellate 18S rRNA gene sequences has not improved
the resolution but has added a few new surprises
(Fig. 3). The core dinoflagellates diverge simul-
taneously into four major clades. The first major
clade contains a mixture of gymnodinioid and peri-
dinioid taxa; Amphidinium commonly occurs as a
basal divergence in a peridinioid clade; prorocen-
troids are split into benthic and planktonic clades
not too distantly related; and dinophysioids are a
basal not derived divergence. The second major
but smaller clade is a gymnodinioid clade. The
third major clade also contains a mixture of peri-
dinioid and gymnodinioid taxa; surprisingly, the
Noctilucales are embedded in this clade, as also
seen by Hoppenrath & Leander (2010) in their
heat shock protein tree, as are the Blastodiniales.
Gonyaulacoids are a final divergence in this clade.
The fourth major clade is primarily composed of
naked forms, with the extant suessioids as a final
divergence. Most clades consist of either thecate
or non-thecate forms, the exception being in the
first major clade, in which Amphidinium spp. are
at the base of some peridinioid lineages. Basically,
this tree shows that the taxa conventionally attri-
buted to the Gymnodiniales and Peridiniales
have evolved multiple times and only a few con-
ventional orders, such as the Gonyaulacales, are
monophyletic.
More recent trees with more genes show a
similar pattern of divergences from the ciliates to
the core dinoflagellates along with other significant
relationships. Using three genes and a reduced
taxon sampling, Zhang et al. (2007) found that
Amphidinium was at the base of the entire dinofla-
gellate lineage. In the tree of Zhang et al. (2007),
the core dinoflagellates diverge into two major
well-supported clades: an endosymbiotic clade
and a free-living clade in which the gonyaulacoids
are monophyletic with one exception. Zhang et al.
(2007) contended that the prorocentroids are a
monophyletic group, as also recovered by Hop-
penrath & Leander (2010) using the heat-shock
protein gene.
Toward a reconciliation of morphological
and molecular approaches
We can now test the tree proposed by Fensome
et al. (1993) (slightly modified as in Fig. 4) against
molecular results. Fensome et al. (1993) consid-
ered a temporary dinokaryon (a dinoflagellate-
style nucleus that occurs only in part of the life
cycle) to be a primitive feature and so placed the
Noctilucales and Blastodiniales at the base of the
tree, derived early from the core dinoflagellates.
This prediction is supported by the Saldarriaga et al.
(2004) tree but it is not supported in the tree in
Figure 3 or in the heat-shock protein tree by Hop-
penrath & Leander (2010). This suggests that the
temporary dinokaryon is not (consistently) a sym-
plesiomorphic feature but can be re-instated from
time to time among different lineages.
The most recent molecular trees suggests that
the athecate (naked, unarmoured) condition tradi-
tionally grouped as the Gymnodiniales is para-
phyletic, indicating that loss and (re)gain of thecal
plates has happened repeatedly throughout dinofla-
gellate evolution. This makes sense when consider-
ing that the athecate state is not the equivalent of
gymnodinioid tabulation pattern, and presumably
gymnodinioids in the sense of athecate forms may
arise in lineages with non-gymnodinioid tabulation
patterns.
Molecular trees (Saldarriaga et al. 2004; Zhang
et al. 2007, fig. 3) show that modern dinoflagel-
lates attributed to the Suessiales (primarily Sym-
biodinium) appear to be derived, suggesting that
the modern forms are not closely related to the
Triassic fossil taxon Suessia. This is perhaps no sur-
prise as Fensome et al. (1993) based the grouping
of Symbiodinium with fossils, such as Suessia, gen-
erally on a particular range in the number of plate
series and not on tabulation details. However, the
indication that Symbiodinium and Suessia are not
closely related does make the suggestion that
Suessia was in some way related to the rise of
scleractinian corals through symbiosis less compel-
ling, even though the first appearance of Suessia
broadly coincides with the time of origin of the
modern corals in the Triassic.
The separation of gonyaulacoids and peridi-
nioids as a fundamental split has long been recog-
nized among palaeontologists. The molecular
L. K. MEDLIN & R. A. FENSOME268

results (Saldarriaga et al. 2004; Zhang et al. 2007,
Fig. 3) support a strong gonyaulacoid clade but
indicate that modern peridinioids are paraphyletic.
In the tree published by Saldarriaga et al. (2004,
fig. 1), within the gonyaulacoids, goniodomoids
and ceratioids fall out very clearly as separate
clades. The goniodomoids are represented by the
clade at the top of the diagram that connects
species of Pyrocystis,Fragilidinium,Alexandrium
and Pyrodinium. Below these in the same tree,
species of Ceratium, the modern representative of
an important and coherent fossil-rich family, fall
neatly into a single lineage. Below these, with
common derivation, are clades containing other
forms that would generally be considered gonyau-
lacacean on the basis of morphology occur on the
remaining major gonyaulacoid clade. The molecu-
lar evidence thus accords well with a gonyaulacoid
lineage that, based on fossils, has been separate for
the past 175– 180 Ma. Interestingly, Saldarriaga
et al. (2004) indicated that part of the grouping that
Fensome et al. (1993) had assembled as the Phy-
todiniales is molecularly associated with Gonyaulax
spinifera, the type of Gonyaulax. Other forms
considered by Fensome et al. (1993) to be phyto-
dinialeans fell out in very different lineages. Like
the presence of an athecate amphiesmal and a part-
time nucleus, the inclusion of a prominent coccoid
life-cycle stage is therefore a feature developed or
retained by several separate lineages, not a synapo-
morphy that can be used to define high-level taxa.
In all trees so far published (e.g. Saldarriaga
et al. 2004; Zhang et al. 2007, Fig. 3) peridinioids
resolve into several clades and thus the morpho-
logically based Peridiniales is shown to be poly-
phyletic. This is surprising from a palaeontological
perspective because the tabulation among fossil
peridinioids is so consistent and apparently stable,
especially from the Late Jurassic to the Miocene,
far more so than the gonyaulacoid fossil record.
To understand this discordance between the fos-
sil and molecular evidence, it may help to delve
a little deeper into the nature of the former for
peridinioids. Fensome et al. (1993) designated a
‘standard’ peridinioid tabulation involving 4 api-
cal plates, 3 anterior intercalaries symmetrically
arranged on the dorsal surface, 7 precingulars, 5
postcingulars and 2 symmetrically disposed ant-
apicals. Especially characteristic and commonly
recognizable through archeopyle formation, even
if the rest of the tabulation is not reflected, is the
mid-dorsal second anterior intercalary plate (2a),
which is most often 6-sided. These tabulation char-
acteristics are present in the vast majority of peridi-
nioid genera including calcareous forms from the
Middle Jurassic to the Palaeogene, and the stability
of this tabulation pattern despite other types of
morphological variation (overall shape, cyst-wall
composition, archeopyle details) strongly suggests
a single clade, even though this can never be con-
firmed by molecular evidence.
From primarily fossil evidence, Fensome et al.
(1993) (and palaeontologists in general over recent
decades) recognized a protoperidinioid lineage
separate from the peridinioid mainstream from the
Late Cretaceous. Partly recognized by cyst-wall
characteristics and by a distinctive cingular tabula-
tion evidenced by modern forms (cingular details
are almost always not discernible on fossils), fossil
and modern protoperidinioids are characterized by
a variation in episomal tabulation, especially dor-
sally; this is in contrast to the stability of this
feature among fossil peridinioids. As illustrated by
the molecular tree published by Saldarriaga et al.
(2004; as also represented by unlabelled clade 5 in
Fig. 3), it is not surprising that protoperidinioids
resolve into a clade separate from other peridinioids.
What is unexpected from a palaeontological
perspective is that peridiniaceans (sensu stricto,
thus excluding protoperidiniaceans) resolve onto a
number of clades, some quite distant from others.
The youngest-known fossil organic-walled peridi-
niacean, Palaeocystodinium, made its last appear-
ance in the Miocene. Perhaps, non-calcareous
fossils with a standard peridinioid tabulation can
be considered part of a clade, the last representative
of which became extinct in the Miocene. The stable
tabulation of fossil peridiniaceans is most like
modern Peridinium bipes; indeed the symmetrical
dorsal episomal tabulation of fossil peridinioids is
sometimes referred to as ‘bipesioid’. Perhaps, the
fossil peridiniacean clade is related to Peridinium
bipes. However, fossil ‘bipesioids’ (peridiniaceans)
are all marine, like modern protoperidiniaceans,
whereas the modern peridiniaceans in the clade
containing Peridinium bipes are all non-marine.
Because the Early Jurassic peridinioids and a
few later ones such as Cretaceous Angustidinium
had five apical plates (in contrast to four in the vast
majority of later fossil peridinioids; see Below
1987), Fensome et al. (1993) placed these forms
with modern Heterocapsa in the suborder Hetero-
capsineae. From the molecular trees of Saldarriaga
et al. (2004) and Figure 3, Heterocapsa appears
to be a relatively derived peridinioid. The pre-
sence of five apical plates in early peridinioids
and Heterocapsa is a very broad similarity, but in
detail the apical thecal plates of the two entities
are not closely similar in arrangement, and the simi-
larity in apical plate number most probably rep-
resents convergence. Early fossil forms with five
apical plates are therefore probably not related to
Heterocapsa, but may well represent a group ances-
tral to other Jurassic– Miocene peridinioids.
Among dinoflagellates with calcareous life-
cycle stages (primarily if not always cysts),
CONSIDERATIONS OF DINOFLAGELLATE MACROEVOLUTION 269

L. K. MEDLIN & R. A. FENSOME270

Fig. 3. (a) Maximum likelihood analysis of 1296 SSU rRNA sequences currently held in the ARB database and
alignment by secondary structure. Black dots indicate the simultaneous divergence of four clades. (b) The opening of the
gonyaulacoid clade from A.
RHAETOGONYAULACINEAE
COMPARODINIACEAE
STEPHANELYTRACEAE
NANNOCERATOPSIALES
SYNDINIALES
NOCTILUCALES
DESMOCAPSALES
GYMNODINIINEAE
ACTINISCINEAE
PTYCHODISCALES
SUESSIALES
PHYTODINIALES
THORACOSPHAERALES
GONIODOMINEAE
GONYAULACINEAE
CERATIINEAE
CLADOPYXIINEAE
GLENODINIINEAE
HETEROCAPSINEAE
PERIDINIACEAE
PROTOPERIDINIACEAE
OXYTOXACEAE
DINOPHYSIALES
PROROCENTRALES
BLASTODINIALES
Fig. 4. Slightly modified phylogenetric tree with principle dinoflagellate taxa, originally developed by
Fensome et al. (1993).
CONSIDERATIONS OF DINOFLAGELLATE MACROEVOLUTION 271

Fensome et al. classified the Calciodinelloideae as a
subfamily of the Peridiniaceae and treated Thora-
cosphaera (which lacks indications of tabulation
and had been interpreted not to be a cyst) in its
own order, the Thoracosphaerales. The tree pub-
lished by Saldarriaga et al. (2004) clearly associ-
ated Thoracosphaera with Scrippsiella, which is a
modern representative of the calciodinelloids; the
separation of Thoracosphaera from other dinofla-
gellates with calcareous life-cycle stage is therefore
not upheld. However, neither is a close relationship
between calciodinelloids and Peridinium bipes.
The tabulation of the calciodinelloids, where it can
clearly be seen on fossil cysts, is consistently the
‘standard peridiniacean pattern’ and thus conforms
with that of most Jurassic – Miocene organic-walled
cysts. Other than cyst-wall composition, the most
significant difference between fossil peridiniaceans
with organic-walled cysts and those with calcareous
cysts (calciodinellods) is the archeopyle type. In
organic-walled forms, the archeopyle in almost all
genera is focused on the second anterior intercalary
plate; in calcareous forms it is generally apical
however, a fundamental difference going back into
the Mesozoic.
One aspect of the fossil record is that it provides
unique evidence for whole groups of taxa not rep-
resented today and of which we have no other
record. Nannoceratopsis, cited above, is an excel-
lent example; the basal position of the dinophysioids
in Figure 3 seems in accordance with the early pres-
ence of Nannoceratopsis in the fossil record.
Another exclusive fossil lineage is represented by
the wetzelielloids, a group of peridinioids with an
episomal tabulation that is basically bipesioid, but
with a four-sided rather than a five-sided 2a plate.
This may seem a subtle difference, but wetzeliel-
loids were a highly distinctive and prominent
group of Palaeogene dinoflagellates. They appear
to have left no descendants, despite early and now-
disproven claims that they were protoperidinioids.
Wetzelielloids appear to have had a peridiniacean
cingular tabulation and a tabulation overall that
was symmetrical and stable, like that of other
fossil peridiniaceans but in contrast to the variable
and commonly asymmetrical protoperidiniacean
tabulation.
The fossil record also reveals what can be
thought of as ‘iceberg’ taxa, ones with far greater
representation in the past that at the present time.
An example is the cladopyxioid lineage, repres-
ented by just two or three modern genera (including
Cladopyxis) but which has a significant, mainly
Jurassic–Cretaceous, fossil record. Indeed, from
tabulation details, the cladopyxoids appear to be
transitional between gonyaulacoids and peridi-
nioids, so a molecular analysis of a modern mem-
ber of the lineage would be very interesting.
Mitochondrial evolution
The mitochondria of dinoflagellates are another
example in which the group has taken a bizarre
evolutionary pathway. Lukes et al. (2009) docu-
mented that within the alveolate lineage, ciliates
have a normal-sized circular mitochondrial ge-
nome; apicomplexans and dinoflagellates how-
ever have a reduced genome with only three genes
and, after transcription, the mRNAs can be modified
to change the codons or the amino acid sequence
into other proteins. It therefore does not matter
that they have reduced their mitochondrial genome
to only three genes because they have post-
transcriptional modification of the mRNAs.
An unusual case of convergent evolution?
Although the dinoflagellates have evolved many
unusual features, similar features also occur in
the Euglenozoa (Lukes et al. 2009). Are such simi-
larities the result of convergent evolution? Shared
similarities include flagella with a paraflagellar rod;
a large nucleolus and permanently condensed
chromosomes, mucocysts or trichocysts ejected
through pores; cell walls composed of ‘protein-
aceous/cellulosic’ strips or plates; thylakoids with
three lamellae; and chloroplast endoplasmic reticu-
lum (CER) composed of three membranes. Another
comparison is that the dinoflagellate mitochond-
rion has only three genes and euglenoid mitochon-
dria have mini-circles with three genes. It is
tempting to suggest that the reduction of the mito-
chondrial genome to only three genes is the next
evolutionary step before the formation of the mini-
circles. Both dinoflagellates and euglenoids have
post-transcriptional editing of their mitochondrial
mRNA. The unique splice leaders to the mRNA
are conserved at the class level in dinoflagellates
and at the species level in euglenids. Both groups
have mRNAs transcribed with multiple genes. In
the dinoflagellates they are tandem repeats of the
same gene, but these are different genes in the eugle-
nophytes. Each of these strange features likely
conveys some evolutionary advantage in these two
very different lineages of eukaryotic microalgae.
It would therefore seem that, although the dino-
flagellates exhibit some rare and unusual cellular
features, they are not alone in having these features
and their bizarre evolutionary pathways are not as
unique as once believed.
Conclusions
As with the two ‘solitudes’ of fossil and extant
dinoflagellate studies until the 1980s and 1990s,
the morphological (primarily fossil) and molecular
L. K. MEDLIN & R. A. FENSOME272

approaches to dinoflagellate evolution have, until
now, largely developed separately in recent years.
Our attempt here to bring the two approaches
together will, we hope, pave the way for greater col-
laboration in the quest to understand dinoflagellate
macroevolution.
Current combined evidence suggests that dino-
flagellates separated from their apicomplexans
cousins some 650 Ma. Although they did not leave
a convincing morphological record until the Meso-
zoic, biogeochemical evidence provides some
evidence for the group during the Palaeozoic. Evi-
dence from molecular analyses and plastids sup-
ports the fossil evidence for an early Mesozoic
radiation of dinoflagellates, during which it seems
that the morphology of the group as we know it
today, with cingulum, sulcus and characteristic fla-
gella and tabulation patterns, first appeared. Some
lineages reflected by the fossil record, such as cera-
tioid gonyaualacaleans and protoperidiniod peridi-
nialeans, are strongly supported by molecular
evidence. Other relationships, such as that of the
Triassic fossil Suessia with modern Symbiodinium,
appear to be unfounded by molecular data. Molecu-
lar evidence is clearly our strongest indication for
phylogenetic relationships, but any overall evol-
utionary tree must accommodate morphologies rep-
resented only by fossils.
We thank Jane Lewis and the DINO 9 team for the invita-
tion to collaborate on this paper and Jane especially for
unwavering encouragement and help in completing the
task. We are grateful to Jim Riding and an anonymous
reviewer for helpful reviews. The second author would
like to thank Jennifer Galloway and James White for pro-
viding feedback on an early draft, and to many colleagues
over the years for discussions on dinoflagellate evolution,
most notably Jim Riding, Max Taylor and Graham Wil-
liams. This publication constitutes ESS Contribution no.
20130070. This work was supported in part by FP7 EU
MIDTAL, contract number 701924.
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