Embryonic, Larval, and Juvenile Development of the Sea
Biscuit Clypeaster subdepressus (Echinodermata:
Clypeasteroida)
Bruno C. Vellutini
1,2
*, Alvaro E. Migotto
1,2
1 Centro de Biologia Marinha, Universidade de Sa˜o Paulo, Sa˜o Sebastia˜o, Sa˜o Paulo, Brazil, 2 Departamento de Zoologia, Instituto de Biocieˆncias, Universidade de Sa˜o
Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil
Abstract
Sea biscuits and sand dollars diverged from other irregular echinoids approximately 55 million years ago and rapidly
dispersed to oceans worldwide. A series of morphological changes were associated with the occupation of sand beds such
as flattening of the body, shortening of primary spines, multiplication of podia, and retention of the lantern of Aristotle into
adulthood. To investigate the developmental basis of such morphological changes we documented the ontogeny of
Clypeaster subdepressus. We obtained gametes from adult specimens by KCl injection and raised the embryos at 26
0
C.
Ciliated blastulae hatched 7.5 h after sperm entry. During gastrulation the archenteron elongated continuously while
ectodermal red-pigmented cells migrated synchronously to the apical plate. Pluteus larvae began to feed in 3 d and were
*20 d old at metamorphosis; starved larvae died 17 d after fertilization. Postlarval juveniles had neither mouth nor anus
nor plates on the aboral side, except for the remnants of larval spicules, but their bilateral symmetry became evident after
the resorption of larval tissues. Ossicles of the lantern were present and organized in 5 groups. Each group had 1 tooth, 2
demipyramids, and 2 epiphyses with a rotula in between. Early appendages consisted of 15 spines, 15 podia (2 types), and 5
sphaeridia. Podial types were distributed in accordance to Love´n’s rule and the first podium of each ambulacrum was not
encircled by the skeleton. Seven days after metamorphosis juveniles began to feed by rasping sand grains with the lantern.
Juveniles survived in laboratory cultures for *9 months and died with v500 mm wide, a single open sphaeridium per
ambulacrum, aboral anus, and no differentiated food grooves or petaloids. Tracking the morphogenesis of early juveniles is
a necessary step to elucidate the developmental mechanisms of echinoid growth and important groundwork to clarify
homologies between irregular urchins.
Citation: Vellutini BC, Migotto AE (2010) Embryonic, Larval, and Juvenile Development of the Sea Biscuit Clypeaster subdepressus (Echinodermata:
Clypeasteroida). PLoS ONE 5(3): e9654. doi:10.1371/journal.pone.0009654
Editor: Christoph Winkler, National University of Singapore, Singapore
Received November 26, 2009; Accepted February 16, 2010; Published March 22, 2010
Copyright:  2010 Vellutini, Migotto. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Master’s degree scholarship to BCV funded by FAPESP - Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo http://www.fapesp.br/ (06/01898-7)
and CAPES - Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior http://www.capes.gov.br/. AEM is funded by CNPq - Conselho Nacional de
Desenvolvimento Cientı´fico e Tecnolo´gico http://www.cnpq.br/ (305608/2006-1). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: organelas@gmail.com
Introduction
Sea biscuits and sand dollars are representatives of the order
Clypeasteroida, the latest major branch of irregular echinoids to
evolve. Clypeasteroids diverged from a cassiduloid-like ancestor
around the Paleocene [1–3] and by Middle Eocene (49-37 million
years ago) the suborders of this group were already established [2]
and present in oceans worldwide [1]. Morphological changes
during the early evolution of clypeasteroids were tightly associated
with the occupation of shifting sand beds and involved the
retention of juvenile traits into adulthood [2]. Strong evidence of
this phenomenon is, for example, the presence of a lantern of
Aristotle in adult clypeasteroids, while in the remaining extant and
fossil irregular echinoids, including in the ancestral lineage of
Clypeasteroida (a branch of Cassiduloida – see [3] for an analysis
of cassiduloid-clypeasteroid relationships), the lantern atrophies
before adult life [2]. Besides being the only irregular echinoids
with the lantern of Aristotle and perignathic girdles in adult
individuals, clypeasteroids exhibit more than one podium per
ambulacral plate. This feature accounts for the drastic increase on
the number of nonrespiratory tube feet and allows greater
efficiency in the collection of food [2]. In addition, a recent study
established the relatively large anus, shortening of primary spines,
and flattening of the body as synapomorphies of Clypeasteroida
[4].
The sea biscuit Clypeaster subdepressus (Gray, 1825) belongs to the
suborder Clypeasterina, sister group of Scutellina, and to the
Clypeasteridae, a single-genus family of Clypeasteroida and sister
group of the Arachnoididae. C. subdepressus is found in the
Caribbean and East Coast of America, from North Carolina to
Rio de Janeiro [5] and Sa˜o Paulo [6,7]. Adult individuals live
semi-buried in coarse biogenic sand of coastal waters and feed on
organic matter present in the sediment [5,8]. Sand particles are
collected by accessory podia of the oral surface, taken to the mouth
region, and then crushed by the lantern teeth [9].
The larval development and early juvenile growth of C.
subdepressus was first described in Caribbean specimens [10]. The
study compared the development of C. subdepressus and C. rosaceus
PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9654

showing that the former has an obligatory planktotrophic larva in
contrast to the facultative feeding larva of the latter. Since this
study focused on the characterization of C. rosaceus development,
the timing of early development and a detailed ontogeny of
morphological traits of C. subdepressus were not provided.
Given the unique morphological evolution, a complete fossil
record [11], and readily feasible developmental studies [12],
clypeasteroids can be a valuable group for evolutionary analyses.
Furthermore, C. subdepressus has been used in recent studies related
to development [13–16], posing the need for a detailed
developmental table in order to comprehend the evolution of
echinoid morphology. The present work provides a complete
description of C. subdepressus development including stage timing,
cleavage and gastrulation dynamics, and post-metamorphic
development of juveniles.
Results
The morphological changes occurring during the embryonic,
larval, and juvenile development of C. subdepressus are depicted in
Table 1.
Spawning
Adult males and females of C. subdepressus had 5 gonopores at the
apex. A long (2–3 mm) genital papilla (Fig. 1A,B) and up to 3
shorter (v1 mm) accessory papillae were present at the distal end
of each gonoduct. During spawning induced by KCl injection one
or two gonopores released gametes continuously for several
minutes (maximum of 30 min with specimen underwater); the
flux of gametes through a genital papilla varied between 7 and
120
eggs
s
(Video S1). Accessory papillae of males released small
quantities of sperm under low intensity flux (Fig. 1A; Video S2). A
spontaneous spawning event was observed in the laboratory tanks
on March 8, 2007 at 17 h 30 min. Five individuals moved the
sand away from the apex and initiated spawning through the five
gonopores simultaneously (Fig. 1C). Spawning ceased after 10 min
and the apex was again covered with sand grains by action of
surrounding spines. After a few minutes it was uncovered and
gamete release re-initiated; this sequence was repeated at least 3
times for each specimen.
Table 1. C. subdepressus developmental table.
Embryos Larvae Juveniles
Events t Events t Events t
Sperm entry 0 PMC 10 h Lantern rudiments 0 d
Vitelline envelope elevation 30–40 s Red-pigmented cells 12 h Resorption of larval tissues 1 d
Fertilization membrane 2–6 min SMC 13 h Aboral plates 2 d
Pro-nuclei migration 5 min Archenteron invagination 14–24 h Gut differentiation 2–7 d
Pro-nuclei fusion 18 min Larval skeleton 15 h Anus 2 d
2 cells (96%) 80 min Prism 24 h Teeth ornamentation 4 d
4 cells (88%) 100 min Coeloms 24 h Aboral miliary spines 4 d
8 cells (74%) 120 min Tri-parted gut 24 h Pyramids 7 d
16 cells (88%) 160 min Pluteus 2-arms 2 d Mouth opening 7 d
32 cells (92%) 180 min Feeding 3 d Feeding 7 d
56–60 cells 3.2 h Pluteus 4-arms 4 d Ophicephalous pedicellariae 14 d
108 cells 3.5 h Hidropore opening 5 d Tridentate pedicellariae 30 d
Blastula formation 3.5–8 h Vestibule 6–10 d
Vegetal pole thickening 7.5–10 h Pluteus 6-arms 6 d
Cilia formation 7.5 h Pluteus 8-arms 10 d
Hatching 8 h Rudiment 10–23 d
Metamorphosis 23 d
Timing of morphological changes during embryonic, larval, and juvenile development of C. subdepressus at 26
0
C. t~time after fertilization (Embryos and Larvae);
t~time after metamorphosis (Juveniles).
doi:10.1371/journal.pone.0009654.t001
Figure 1. C. subdepressus genital papillae and spawning. A Male
genital papilla (arrow) releasing sperm and adjacent accessory papilla
(white arrow). B Female genital papilla (arrow) releasing eggs. C Adult
male releasing sperm during a spontaneous spawning event in March 8,
2007. Scale bars = 1 mm (A, B); 20 mm (C)
doi:10.1371/journal.pone.0009654.g001
Development of C. subdepressus
PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9654

Fertilization
After sperm entry, the male pro-nucleus was pushed by
microtubules towards the center of the egg (Fig. 2; Video S3). It
moved at a constant speed of 0:1
mm
s
. When touched by
microtubules, the female pro-nucleus was rapidly pulled towards
the male pro-nucleus. The fusion took place approximately 12 min
30 s after sperm entry. The cytoplasmic movements increased and
the cell surface acquired an irregular aspect. Video footage
accelerated 625| revealed that the deformations were dynamic
and that the membrane was vibrating (Video S3). Immediately
before the first cleavage the membrane ceased the vibration, the
cell surface became regular, and the hialine layer thickened.
Cleavages
Cell divisions initiated 80 min after fertilization (Table 2) and
were holoblastic. First and second cleavages were meridional,
dividing the embryo in 4 equal blastomeres (Fig. 3A–C). The third
division was equatorial, separating animal and vegetal blastomeres
at 120 min; the latter were 30% smaller in diameter (Fig. 3D).
Micromeres originated unequally from vegetal blastomeres while 8
mesomeres were formed by a meridional cleavage of animal
blastomeres (Fig. 3E–H). Equatorial division of mesomeres,
meridional division of macromeres, and unequal micromere
division formed embryos with 32 cells 180 min after fertilization
(Fig. 3I,J). Sixty-cell embryos were formed when blastomeres went
through an equatorial division while the 4 micromeres experi-
enced a meridional division. The seventh cleavage occurred
without micromere division resulting in embryos with 108 cells
(Fig. 3L).
Blastulae
Cells acquired a polygonal shape during the consolidation of the
epithelium between 3.5 and 6.5 h post fertilization (hpf) (Fig. 4).
The vegetal plate thickened and cilia were formed 7.5 hpf,
immediately before hatching.
Gastrulae
Primary mesenchyme cells (PMC) detached from the vegetal
pole, became spherical, and aggregated in an unipolar manner on
the vegetal pole; ingression to the blastocoel took place
approximately 10 hpf (Fig. 5A–C). PMC migrated through the
blastocoel forming a ring connected by thin filopodial pseudopodia
on the posterior end (Fig. 6A).
Red-pigmented cells differentiated on the vegetal pole 12 hpf
and migrated through the epithelium, simultaneously with PMC,
towards the apical plate 13 hpf (Fig. 5D). Secondary mesen-
chyme cells (SMC) originated on the vegetal pole, extending
cytoplasm projections towards the blastocoel during archenteron
invagination. The archenteron invagination began at 14 hpf
(Fig. 5E).
Figure 2. Migration and fusion of C. subdepressus pronuclei. Montage of a single zygote during a period of 12 min 30 s after sperm entry
(top). The movements of male (black dashed line) and female (white dashed line) pronuclei were outlined (bottom). Frames taken every 58 s. Scale
bar = 50 mm
doi:10.1371/journal.pone.0009654.g002
Table 2. Dynamics of C. subdepressus cell divisions.
t 0 20 40 60 80 100 120 140 160 180
Eggs 100.0 4.1 4.9 5.1 1.4 1.7 2.0 0.4 2.1 2.2
Zygote - 95.9 95.1 92.9 0.8 1.0 0.7 0.4 0.3 0.6
2 cells - - - 1.9 95.7 7.9 1.0 - - -
4 cells - - - - 2.2 88.0 21.9 1.5 - -
8 cells - - - - - 1.4 74.4 45.2 9.7 0.3
16 cells - - - --52.5 87.9 5.0
32 cells - - - -- --91.8
n 304 1145 411 311 369 291 301 259 331 317
Relative amount (%) of cell stages for each timespan during early cleavages. n~total embryos counted; t in min.
doi:10.1371/journal.pone.0009654.t002
Development of C. subdepressus
PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9654

PMC formed two aggregates 15 hpf (Fig. 5F) and initiated the
secretion of a calcareous triradiate spicule (Fig. 6B). SMC were
composed of different cell types including cells with filopodial and
lobopodial pseudopodia, ameboid cells without cytoplasm projec-
tions, and cells with red-pigmented granulae. SMC on the
archenteron reached the anterior pole 16,5 hpf (Fig. 5G) while
red-pigmented epithelial cells reached the anterior pole 19 hpf,
when the blastocoel was occupied by SMC (Fig. 5H). Epithelial
red-pigmented cells were not present on the ventral (oral) region of
the embryo at prism stage. The surface of the embryo at prism
stage was covered by cilia with an apical tuft on the anterior pole
and a ciliated ring around the anus (Fig. 6C).
The embryo height decreased during gastrulation from
257+9 mm at 12 hpf to approximately 225 mm from 16.5 hpf to
20 hpf; the width had a slight increase of 20 mm (Fig. 7). The
archenteron elongated continuously on a linear manner from 13.5
hpf (Fig. 7) and reached 84+10% of the blastocoel height (Fig. 7B).
Pluteus
In 48 hpf two coelomic pouches were present next to the
esophagus (Fig. 8A). At this stage the mouth opened, but the larvae
were unable to feed; microalgae captured by the larval arms were
carried towards the mouth, but were deflected away possibly by an
opposing current (Video S4). The gut was not functional, but
already had three portions identified as esophagus, stomach, and
intestine (Fig. 8A). Muscles of the esophagus began to contract 70
hpf (Video S5); the stomach grew in diameter while its epithelium
became thinner. The gut became functional and the larvae began
to feed 3 d post-fertilization (dpf) (Fig. 8B).
The skeleton of postoral and posterodorsal arms was fenestrated
while the anterolateral and preoral pairs were non-fenestrated (see
Fig. S1 for the arrangement of larval arms). The hydropore
formed above the cardiac sphincter approximately 3 dpf by an
extension of the left coelomic pouch and opened on the surface 5
dpf (Fig. 8A,B detail). The vestibule appeared between 4 and 5 dpf
on the left side of larvae between the postoral and postero-dorsal
arms (Fig. 9A). The vestibule fused with the left coelom (Fig. 9B,C)
forming the rudiment (Fig. 9D) between 6 and 10 dpf.
The rudiment developed podia and spines, which became active
still inside the larval body (Fig. 9D). No pedicellariae were formed on
the surface of the larval body, as commonly observed in competent
larvae of regular echinoids. Some rudiments were oriented 45
0
,
and not perpendicular, to the
antero
posterior
axis of the larva.
Starved. Compared to fed larvae, starved ones formed the
same structures at similar developing times during the first week
post-fertilization. Differences were noticed only during vestibule
invagination. The vestibule of starved larvae began to invaginate,
but did not reach the left coelom; larvae died 17 dpf.
Figure 3. Early cleavages of C. subdepressus under light microscopy. A Two nuclei (arrows) during cariocinesis of the first cell division. B
Embryo with two cells before the second cleavage; hialine layer is visible between cells (arrow). C Animal pole view of an embryo with 4 cells. D
Lateral view of an embryo with 8 cells; blastomeres on the vegetal pole are smaller (bottom). E Micromeres (arrow) on the vegetal pole of an embryo
with 16 cells. F Lateral view of an embryo with 16 cells. G Arrangement of micromeres and macromeres on the vegetal pole of a 16 cell embryo. H 16
cell embryo showing the mesomere arrangement on the animal pole. I Fifth division cycle showing child-micromeres (arrow) on the vegetal pole;
embryo with 32 cells. J Lateral view of an embryo with 32 cells. K Vegetal pole of an embryo with 56 cells. L Lateral view of an embryo with 108 cells.
Scale bars = 30 mm
doi:10.1371/journal.pone.0009654.g003
Development of C. subdepressus
PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9654

Metamorphosis
Competent larvae exhibited a typical substrate-test behaviour
which consisted of swimming near the bottom and exposing the
vestibule pore with protruding podia (type I, see below) by
movingtheleftarms(Fig.10A;VideoS6).Larvaewereableto
fully evert the rudiment by opening the arms 180
0
posteriorly,
attach to the substrate, return to the larval conformation and
resume swimming. Metamorphosis occurred when larvae
attached firmly to the bottom with the protruding podia and
the larval tissues began to regress and accumulate on the aboral
surface of the rudiment (Fig. 10B). During this process larval
spicules became exposed and broke off; fragments of tissue
were lost (Fig. 11A). Larval tissues accumulated on the aboral
surface of the rudiment forming a globoid structure (Fig.
11B). Metamorphosis took approximately 1 h 30 min from
attachment to the complete regression of the larval tissues
(Video S7).
Juvenile
Metamorphosis is followed by the resorption of larval tissues
and the development of juvenile structures. The body of
metamorphosing and early postlarval juveniles did not exhibit
clear evidence of bilateral symmetry when seen from the aboral
surface (Fig. 11B). However, a close look at the oral surface under
polarized light revealed hints of bilateral symmetry identifiable by
the shape of the body and position of the rudiments of the lantern
of Aristotle (Fig. 12A). The bilateral symmetry passing through the
III-5 plane (Love´n’s axis) became evident after the resorption of
larval tissues. The bilaterality of postlarval juveniles was identified
by the ovoid body shape, the longer interambulacrum-5 pair of
posterior spines, the anus opening posteriorly, and the ambula-
crum-III always pointing in the moving direction of the juvenile.
Appendages of 1-day-old postlarval juveniles were disposed in
two rows along the ambitus, an infracoronal (oral) and a
supracoronal (aboral) row. The infracoronal row had one spine
Figure 4. Compactation of ectodermic cells during blastula formation. A and B show different optical sections of embryos sampled 3.5, 5.0,
and 6.5 hpf. A Ectodermic cells acquired a polygonal shape and became smaller during the division cycles. B Epithelium became more uniform and
cells lost the globoid shape. Scale bar = 30 mm
doi:10.1371/journal.pone.0009654.g004
Development of C. subdepressus
PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9654

for each interambulacral zone and one sphaeridium (a modified
spine of uncertain function) between a pair of accessory podia for
each ambulacral zone, totalling 5 sphaeridia, 5 spines, and 10
podia on the oral surface (Fig. 13). There were two types of
suckered podia in postlarval juveniles; no buccal podia were
observed. Type I had a narrow tip without an internal spicule
(Fig. 11F, left) while type II had an expanded tip and a ring-shaped
spicule inside (Fig. 11F, right). The former originated in the outer
margin of the peristome (inner margin of the skeleton of the oral
surface), but not embraced by the skeleton at ambulacral positions
Ib, IIb, IIIa, IVb, and Va (bbaba; see Fig. 13 and 14A); podia was
positioned in a concavity of the skeleton (Fig. 14B,C). The latter,
podial type II, emerged from a skeleton pore at the complemen-
tary ambulacral columns Ia, IIa, IIIb, IVa, and Vb (aabab). Podial
type I occupied an inner (closer to the mouth) ambulacral position
than podial type II on the infracoronal row of juveniles
(Fig. 14B,C). At this stage, sphaeridia were equal in size bearing
a birefringent oval tip 20 mm in diameter (Fig. 11E), but they were
smaller during metamorphosis (width between 3 and 17 mm; see
Fig. 11A). The supracoronal row had a pair of spines for each
interambulacral area and a type I podium for each ambulacral
zone. The postlarval juvenile had 15 spines, 15 podia, and 5
sphaeridia in total; miliary spines were occasionally present on
ambulacral zones and no pedicellariae were observed at this stage.
Primary spines and podia of C. subdepressus were permanent and
did not regress during the observed period.
Early postlarval juveniles had no skeleton on the aboral surface,
except for the remnants of larval rods (Fig. 11D). The gut was not
yet formed and neither mouth nor anus were present. During the
resorption of larval tissues the rudiments of the lantern of Aristotle
were visible in the oral region under polarized light. Teeth were
positioned on the interambulacral zones between a pair of
demipyramids and a pair of epiphyses (Fig. 12A). Each set of 5
independent ossicles was intercalated by a single ossicle, a
rudiment rotula (Fig. 13). Except for the teeth, the skeleton had
the trabecular structure typical of stereom.
Two days after metamorphosis the demipyramids were larger
and still separated by the tooth slide (Fig. 12B). Tips of the teeth
were narrow without ornamentation (Fig. 12B) and became robust
with a complex surface 4 dpm. The digestive tract became visible
through the aboral surface two days after metamorphosis; the gut
tube exits the lantern of Aristotle onto the right side of the cavity
and towards the anterior region; it turned counterclockwise
towards the left side of the cavity, into the posterior region, and
then again reaching the right side. The tube then bent 180
0
left,
pointing posteriorly, and opened on the posterior portion of the
aboral surface below an anal plate (Fig. 15E).
The aboral surface was occupied by calcareous plates 2 dpm
(Fig. 15A). Eight miliary spines (with a crown-like tip) grew on the
supracoronal row and 4 appeared on the aboral surface between 2
and 4 dpm (Fig. 15B,C); older juveniles also had miliary spines
between other appendages, including ambulacral zones. Each pair
of demipyramids were sutured together into one pyramid 7 dpm
(Fig. 12C). No other skeletal element was formed on the oral
surface of the juvenile during this period.
The mouth opened on the centre of the oral surface 7 dpm and
juveniles began to feed (Fig. 15F,G). At this stage the peristomial
membrane and the lantern of Aristotle were actively moving while
the esophagus exhibited peristaltic contractions (Video S8).
Exotrophic juveniles frequently had food in their gut and
eventually in the rectum; fecal pellets were observed (Fig. 15E).
Type II podia significantly increased in number and seemed to
Figure 5. Sequential images of C. subdepressus embryos during gastrulation. Vegetal pole is bottom. A Post-hatching swimming blastula
with thickened vegetal pole epithelium 9 hpf. B Initial ingression of PMC into the blastocoel 10 hpf. C Ingressed PMC aggregated on the posterior
end 11 hpf. D Initial migration of PMC through the inner side of ectoderm 13 hpf; red-pigmented cells are present on the vegetal pole (not visible). E
Initial invagination of the archenteron 14 hpf. F SMC ingress the blastocoel from the archenteron tip 15 hpf; PMC form lateral aggregates (arrows) and
begin to secrete the skeleton. G SMC reaching the anterior pole 16.5 hpf (arrow); red-pigmented cells reached the middle region of the embryo
through the ectoderm. H Final stage of archenteron invagination with SMC touching the anterior pole 19 hpf; blastocoel is populated with PMC and
SMC and red-pigmented cells reached the anterior pole. Scale bars = 30 mm
doi:10.1371/journal.pone.0009654.g005
Development of C. subdepressus
PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9654

play a major role in locomotion. Juveniles moved quickly among
sand grains, extending the anterior type II podia forward more
than a body length ahead (Fig. 15H; see Video S9 for locomotory
examples).
One-month-old juveniles exhibited a digestive tract with
three distinct parts besides the esophagus. The stomach and
intestine were wider, opaque, and brownish while the rectum
was covered by red-pigmented cells (Fig. 15E). Four ophice-
phalous pedicellariae appeared on the ambitus of the posterior
region 14 dpm (Fig. 15D). Tridentate pedicellariae appeared
at the posterior region 30 dpm; they were approximately one
quarter of the size of the posterior spines with a stem two
thirds of the length. This kind of pedicellaria was also seen on
the middle region of the juvenile, but not on the anterior
portion.
Juveniles did not eat free living benthic organisms, but
apparently rasped organic material off larger sand grains.
Smalerparticlesweremanipulatedbypodiaandheldin
position by the peristomial membrane while the lantern rubbed
the surface of the grain (Video S10). They survived in
laboratory cultures for 8 months and 20 days after fertilization.
Post-metamorphic juveniles were initially 200–270 mm wide and
300–330 mm in length and reached *480 mm in diameter and
*570 mm in length. Juveniles were active and showed no sign of
food privation, except for the last 3 weeks when they became
less active and more pale. A single sphaeridium per ambula-
crum was still visible (not internalized) by the time of juvenile
death.
Discussion
Direct observations of the behavior of C. subdepressus during
spawning revealed that specimens covered by a layer of sand
(not completely buried) repeatedly exposed the apex, released
gametes, and covered the apex by moving the grains adapically.
Apex exposure before spawning was observed previously in
burrowed individuals of Arachnoides placenta [17], but intermittent
Figure 7. Gastrulation of C. subdepressus.ATime-series plots with
fitted smooth curve of 4 morphometric measurements during
gastrulation. The height, width, blastocoel height, and archenteron
length were measured every 30 min for 8 h (12–20 hpf, except 12.5
hpf). Height and blastocoel height decreased during the period while
the width showed a slight increase; archenteron elongation is
continuous. B Relative amount of archenteron elongation during
gastrulation calculated by the ratio between archenteron length and
blastocoel height. n~99 for each timespan.
doi:10.1371/journal.pone.0009654.g007
Figure 6. PMC and SEM of prism larval stage of C. subdepres-
sus.APMC forming a ring near the posterior end 14 hpf; cells are
connected by cytoplasmic bridges (arrow). B Triradiate calcareous
spicule secreted by PMC initiating the formation of the larval skeleton
15 hpf. C The apical tuft (at) and future mouth (m) are present at the
anterior region. On the posterior end the postoral arms (po) begin to
extend and the blastopore is visible (bp). Scale bars = 30 mm (A, C);
10 mm (B)
doi:10.1371/journal.pone.0009654.g006
Development of C. subdepressus
PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9654

gamete release during spontaneous spawning was previously
undescribed for clypeasteroids. Until there are field observations
to better comprehend the external fertilization dynamics of
clypeasteroids, their reproductive behavior will remain largely
unknown.
Although it was suggested that long papillae, swollen during
spawning, could disperse gametes across a thin layer of sand [17],
our observations showed that C. subdepressus avoid contact of
gametes with sand grains by clearing the apex during spawning.
Thus, the genital papillae seem to play a role in allowing gametes
to be released well above the spines, consequently decreasing the
number of eggs and sperm trapped within the skin mucus [17] and
avoiding mechanical stress caused by the movement of spines and
pedicellariae.
Cleavages of C. subdepressus were similar to other echinoids with
planktotrophic larvae [10,18–20], except that macromeres were
approximately 30% smaller than mesomeres. The timing of the
formation and hatching of C. rosaceus blastulae took longer (12 h at
27
0
C [10]) than in C. subdepressus (7 h at 26
0
C). Developmental
timing of later stages did not differ between Brazilian and
Caribbean specimens of C. subdepressus at 26
0
C and 27
0
C,
respectively [10].
Data on the gastrulation of C. subdepressus fits the correlation
between the type of gastrulation and the pattern of migration of
Figure 8. Coeloms, gut, and hydropore formation in C. subdepressus.ADifferentiation of a three-part gut has begun and coelomic sacs (c)
were formed next to the archenteron 48 hpf. Detail shows the initial extension of the hydropore channel (arrow) from the left coelom. B
Differentiated gut, mouth (m), esophagus (e), and stomach (s), after 3 d. Hidropore channel (arrow) next to the larval epithelium 3 dpf. Scale
bars = 30 mm
doi:10.1371/journal.pone.0009654.g008
Figure 9. Vestibule and rudiment formation in C. subdepressus larvae. A Vestibule invagination 5 dpf (arrow). B Detail of vestibule reaching
the left coelomic sac. C Fusion of vestibule and left coelom (arrow). D Posterior region of a competent pluteus larva showing a well-developed
rudiment (dashed line). Appendages (podia and spines – arrowheads) are present and active; developing rudiment occupied most of the body and
displaced the larval gut (s). Scale bars = 50 mm (A, D); 30 mm (B, C)
doi:10.1371/journal.pone.0009654.g009
Development of C. subdepressus
PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9654

red-pigmented cells in echinoids [21]; red-pigmented cells
originate on the vegetal pole and migrate through the ectoderm
to the apical plate while the archenteron elongation is continuous.
Also, the behaviour of red-pigmented cells matches the pattern
described for C. japonicus where these cells migrate through the
ectoderm, but are also seen at the archenteron tip [21]. Red-
pigmented cells can have a regulatory role and are known to
trigger gastrulation in Echinometra mathaei [22]. These cells might
participate on the morphological changes during prism formation
and early axis specification of plutei, because they were absent
from the ventral ectoderm of C. subdepressus; a region that remained
flat until the formation of the larval mouth.
Starved larvae of C. subdepressus neither develop a complete
vestibule invagination nor a rudiment, but the eggs provided
sufficient energy for the survival of a functional pluteus larva for
17 dpf. Based on larval skeleton measurements, the facultative
feeding period of larvae was estimated in 5 clypeasteroid species
[13]. The authors determined when fed and starved larvae
differed in size from fertilization, indicative of the exhaustion of
maternal provisioning. In C. subdepressus larvae diverged between
60 and 72 hpf. This range matches with the necessary timespan
for the differentiation of the larval gut, ciliated bands and
beginning of larval feeding, described in the present study. Thus,
we directly observed that the pre-feeding stage of C. subdepressus
larvae ends 3 dpf. The appearance of size differences between
larvae might not solely indicate when starved larvae stopped
growing, but also when fed larvae increased their growth rate
because of food ingestion. Since starved larvae reached the 8-arm
pluteus stage without qualitative morphological differences, the
exhaustion of maternal resources in C. subdepressus apparently
does not occur as early as suggested; even though the absence of
food immediately after the pre-feeding stage could directly affect
early growth rates.
Competent larvae of C. subdepressus exhibited a substrate-test
behaviour similar to other echinoid species [23–26]. Although
early postlarval juveniles resemble regular urchins with a
spherical body, bilateral symmetry could be identified soon after
the resorption of larval tissues and was probably determined
during rudiment formation. Several morphological features mark
the early bilateral symmetry of C. subdepressus such as elongated
body, longer posterior spines, and anus positioned posteriorly. In
other irregular urchins bilateral symmetry is recognized by
different characters. In the spatangoid Echinocardium cordatum, the
subanal fasciole is visible on the rudiment still inside the larvae
[26], the peristome is positioned anteriorly and the posterior
region is more developed [27]. The bilateral symmetry of the
sand dollar Echinarachnius parma is identified by the size and
number of plates in the ambulacra and by differences in size of
the sphaeridia [28].
The bbaba distribution at ambulacral columns and the inner
position of infracoronal type I podia suggests strict adherence to
Love´n’s rule of echinoid peristome development [29]. These
patterns indicate that C. subdepressus podial type I preceded type
II during ontogeny. Since we did not track the formation of
skeletal plates, the coordinance between the appearance of podia
and growth of skeletal plates has not been elucidated. We
observed that the first podium (type I) was not encircled by the
skeleton and occupied the edge of the peristome, while the
following podium on the ambulacrum (type II) protruded from a
pore in the calcareous mesh. At this early stage there were no
signs of disruption in the alternating pattern of podial
organization, which in other echinoids is a by-product of the
alternating deposition of plates known as the ‘‘ocular plate rule’’
[30]. Therefore additional data on juvenile skeletal growth
remains crucial to trace the plate formation and ontogenetic
fate of these podia. In addition, precise tracking of ambulacral
growth can identify the developmental basis behind a well-
known innovation of Clypeasteroida, the breakage of the one-
podium-per-plate rule and further increase in the number of
nonrespiratory podia [2].
Following metamorphosis C. subdepressus juveniles had 3 spines
per interambulacrum (15 total), 3 podia and one spheridium per
ambulacrum (15 podia and 5 sphaeridia total), and no pedicellar-
iae. In contrast, competent larvae of the regular urchins
Paracentrotus lividus and Strongylocentrotus franciscanus already have
pedicellariae during the late larval period and after metamorphosis
[25,31], while pedicellariae of S. purpuratus appear some time after
metamorphosis. Competent larvae of E. cordatum do not exhibit
spines or pedicellariae [26]. Differently from C. subdepressus, newly
metamorphosed juveniles of these regular sea urchins have 4
primary spines per interambulacrum (20 total) [25,31]. The
irregular echinoid E. cordatum has a greater number of primary
spines per interambulacrum after metamorphosis and also differs
from C. subdepressus by the presence of secondary spines and a
subanal fasciole with clavulae and 4 primary spines [26]. By
contrast, C. subdepressus uniquely displays 3 podia per ambulacrum
after metamorphosis while S. franciscanus and S. purpuratus [31], P.
lividus [25] and E. cordatum [26] have 1 podium per ambulacrum.
The two podial types identified in postlarval juveniles of C.
subdepressus did not clearly fit into any specific category previously
described for adult clypeasteroids [32]. Further podial specializa-
tion should occur associated with body growth and differentiation
of food grooves. The appendages of C. subdepressus did not regress
after metamorphosis as observed for E. cordatum, but the postlarval
period of C. subdepressus (7 d to exotrophy) was considerably longer
than observed for E. cordatum (3.5 d) [26]; regular echinoids have a
longer postlarval period (8–10 d) [25,31]. Differences in nutrient
Figure 10. Substrate-test behaviour and metamorphosis of C.
subdepressus.ACompetent larvae opening the arms and exposing the
vestibule pore. B Metamorphosis took approximately 1 h 30 min, from
attachment to the complete regression of larval tissues. Scale
bars = 100 mm
doi:10.1371/journal.pone.0009654.g010
Development of C. subdepressus
PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9654

storage and latent effects of larval life [33] might account for
variations in the postlarval period. Finally, the mouth of E. cordatum
opens before the anus while the mouth of C. subdepressus opened
after the anus.
The initial formation of the lantern of Aristotle of C. rosaceus was
identified between 24 and 48 h; although it became functional 7 d
later, its development was completed in approximately 12 d, when
the mouth opened [10]. In contrast, the rudiments of the lantern
of C. subdepressus were already present during metamorphosis, and
feeding began soon after (7 dpf). At this early stage, size obviously
poses limitations to the feeding mechanisms of juveniles. While
adult C. subdepressus systematically collects and grinds sand grains
[9], juveniles actively harvested individual grains using the lantern
of Aristotle as a rasping tool. The strength to crush sand grains and
the ability to collect many particles simultaneously depend on a
larger lantern of Aristotle and a greater number of podia available,
respectively; both are expected to be acquired with body growth.
In other words, size can directly shape the feeding habits of
developing sea biscuits.
Adult specimens of Cassiduloida and Oligopygoida, ancestral
lineages of the Clypeasteroida, have multiple open sphaeridia
per ambulacrum [2]. The Clypeasterina has 2 closed sphaeridia
per ambulacrum while the remaining clypeasteroids (Lagani-
formes and Scutelliformes) have only 1 closed sphaeridium.
Although a complete ontogeny of sphaeridia is known only for
isolated species, it is known that all irregular echinoids have a
single open sphaeridium per ambulacrum after metamorphosis
[2]. The spatangoid E. cordatum bears 5 sphaeridia on the
ambulacral zones after metamorphosis [26,34] like E. parma [28]
and C. subdepressus (present work). A subtle difference between
spatangoids and clypeasteroids is that sphaeridia of the latter
are not equal in size during the resorption of larval tissues
after metamorphosis. This might indicate that the rudiments of
spatangoids are more developed at metamorphosis. Regular
urchins exhibit calcified sphaeridia only 9 d after metamorphosis
in both S. franciscanus and S. purpuratus [31] and 6 d after
metamorphosis in P. lividus [25],eventhoughasphaeridialbud
grows after 4 d in the former. The sphaeridia remained open for
nearly 9 months showing that enclosure does not occur shortly
after metamorphosis in C. subdepressus as previously suggested for
Clypeasteroida [2]. We have no further evidence to suggest that
enclosure of sphaeridia occurs before or after the appearance of
the second sphaeridium, which is present in adult specimens of
Clypeasterina.
We found only 3 small-sized specimens and no individuals less
than 8 cm in diameter at sampling sites (personal observation).
Figure 11. Morphology of postlarval juveniles of C. subdepressus.ASide view of the final stage of larval tissue regression; fragments of tissue
were disrupted (arrow) near the skeleton of larval arms; a tiny sphaeridium (arrowhead) is visible between spines. B Side view showing the
accumulation of larval tissues in the aboral region before the resorption. C Aboral view of A; bilaterality is not yet clearly identified. D Remnants of
the larval skeleton (arrow) on the aboral surface under polarized light. E Distribution of sphaeridia (arrowhead) on the oral surface. F Different types
of podia present after metamorphosis; type I (left) and type II (right) podia; circular spicule (arrow and detail). Scale bars = 30 mm (F); 50 mm (A, E);
100 mm (D); 200 mm (B, C)
doi:10.1371/journal.pone.0009654.g011
Development of C. subdepressus
PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9654

The occasional nature of recruitment events of C. subdepressus
might affect their size distribution in the field. Also, the cryptic
habit, high mortality [35] and the minute size of newly meta-
morphosed juveniles greatly decreases the probability of finding
them in their natural habitat.
Exotrophic juveniles of C. subdepressus raised in laboratory
cultures for nearly 9 months did not grow more than 500 mm
across. Accordingly, post-metamorphic juveniles of C. subdepressus
and C. rosaceus from the Caribbean increased in diameter for 8 d
and then ceased growth at approximately 420 mm [10]. Although
C. subdepressus juveniles from Sa˜o Sebastia˜o survived considerably
longer than the 30 d observed for the Caribbean specimens, the
conditions of death were similar; juveniles progressively became
more transparent with slower movements. Since juveniles
frequently had food in the gut and released fecal pellets, they
could have suffered a progressive nutritional deficiency because of
the limited conditions of laboratory cultures. The absence of
essential nutrients and/or appropriate diet might have inhibited
their further development. To explain the minimal growth, one
could suggest that Clypeaster species have inherently slow growth
rates and a long life span. However, this hypothesis still needs to
be properly tested once we learn more about the feeding habits
and ideal habitat conditions for the growth and survival of
juveniles. Until then, a series of morphological changes occurr-
ing between the juvenile and adult life, such as the anus migration
to the oral surface, the appearance of the second sphaeridium,
the enclosure timing of sphaeridia within the test, and associ-
ated shape changes, will remain undocumented in these large
clypeasteroids.
Materials and Methods
Collecting adults
We collected adult specimens of Clypeaster subdepressus Gray,
1825 on sand beds 4–6 m deep in Sa˜o Sebastia˜o Channel,
Northern shore of State of Sa˜o Paulo, Brazil. We sampled between
January and March 2007 at two locations: Portinho (23
0
50’25
00
S;
45
0
24’22
00
W) and Parcel da Praia Grande (23
0
50’59
00
S; 45
0
24’59
00
W) (Fig. 16). We kept the specimens in 500 L tanks with
continuous flowing sea-water and sediment from collecting sites
at Centro de Biologia Marinha da Universidade de Sa˜o Paulo
(CEBIMar-USP).
Cultures
Embryos and larvae. We induced spawning by puncturing
the medial portion of one food groove (ambulacrum III) and
injecting 2–3 mL of 0,53 M KCl solution into the body cavity.
Normally, a second injection of 2 mL was required to trigger
gamete release. We poured the eggs through a 300 mm nylon mesh
and suspended them in a 500 mL beaker with filtered sea-water
(FSW). Eggs were fertilized with 1 mL solution of diluted sperm
and moved to a 4.5 L bowl with FSW. After fertilization we kept
cultures at 26
0
C and natural light. Approximately 10 h post
fertilization (hpf) we transferred swimming larvae to vessels of
100 mL with FSW and no aeration. We kept a maximum of
1
larva
mL
and inspected the cultures daily. During these observations
we removed dead larvae and changed 80% of the sea water with a
plastic pipette and a 60 mm nylon mesh, or transferred healthy
larvae to a new vessel.
Figure 12. Formation of the lantern of Aristotle of C. subdepressus.ARudiments of the lantern after the regression of larval tissues
at the end of metamorphosis. Each set of ossicles was intercalated by a rotula (not visible) and consisted of a central tooth (arrow),
two demipyramids (arrowheads), and two epiphyses (dashed line). Latter and rotula not visible because of orientation of polarized light.
Asterisks mark the epiphyses of the adjacent sets. B Demipyramids (d) were formed, but not sutured together 2 dpm; teeth had narrow tips
(arrow). C Demipyramids were tightly sutured into a pyramid (p) 7 dpm. D Mature teeth with ornamentation on the inner side (left). Scale
bars = 30 mm
doi:10.1371/journal.pone.0009654.g012
Development of C. subdepressus
PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9654

When larvae began to feed we added Dunaliella tertiolecta and
Rhodomonas sp. to the cultures daily at a final concentration
between 8|10
5
and 1|10
6
cells
mL
. Ten cultures were deprived of
food, but received a normal maintenance routine (i.e. water
changes).
Metamorphosis. We transferred advanced pluteus larvae to
petri dishes with FSW and added a few sand grains from the
sampled sites. Successfully metamorphosed larvae were kept in the
same container overnight and then moved to a different culture,
shown below.
Juveniles. We kept juveniles in the same 100 mL vessels used
for larvae, but with a monolayer of sand on the bottom. We
changed the water weekly and sealed the cultures with PVC film to
minimize water evaporation and subsequent rise in salinity.
During water changes we added 3 mL of a plankton sample or
biofilm of aquaria, in an attempt to keep food (organic content)
available for the juveniles, once we did not change the substrate of
these cultures.
Kinetics of cleavages
We homogenized the suspension of developing embryos and
collected 2 samples of 3 mL in 10% sea-water formalin every
20 min during a period of 180 min. Posteriorly, we counted the
total number of embryos (sum of samples) and estimated the
relative frequency of each stage, according to the number of cells
(1, 2, 4, 8, 16 and 32 cells), for each timespan.
Archenteron elongation during gastrulation
To verify if the gastrulation of C. subdepressus was continuous or
truncated we fixed samples of developing embryos every 30 min
between 12 and 20 hpf (except 12.5 hpf) in 1% paraformaldehyde.
For each period we transferred the embryos to glass slides,
photographed, and measured their width, height, blastocoel height
and archenteron length (n~99) with the image processing
software ImageJ [36]. The final degree of invagination was
calculated with the ratio between the archenteron length and
blastocoel height.
Figure 13. Postlarval appendages and lantern rudiments. Oral view representation of a postlarval juvenile after the regression of larval tissues.
Ossicles of the lantern of Aristotle ossicles were present at the center of the oral region. Teeth (t), demipyramids (d), epiphyses (ep), and rotulae (r).
Podia, spines, and sphaeridia are present on the calcified oral region (grey area). Dashed line delimits the second row of appendages positioned
above the ambitus. Miliary spines not shown.
doi:10.1371/journal.pone.0009654.g013
Development of C. subdepressus
PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9654

Light microscopy
We documented the development under differential interfer-
ence contrast (DIC) with a Nikon Coolpix 4500 for photomicro-
graphs and Sony DCR HC1000 digital video camera for videos.
Cameras were attached to a Zeiss Axioplan2 compound scope and
a Zeiss Stemi SV11 APO stereomicroscope. We visualized the
calcareous elements of larvae and juveniles under polarized light.
Pluteus larva reconstruction. After manually capturing
118 sequential focal planes of a pluteus larva under DIC we
imported the images into ImageJ [36]. The stack was converted to
8-bit, aligned, inverted, and the voxel depth set to 2mm.We
corrected the contrast/brightness so that only areas in focus of
each image remained visible. Finally, we created a maximum
intensity z-projection (Fig. S1A) and a red-cyan anaglyph of the
larva (Fig. S1B).
Scanning electron microscopy
We fixed samples in 2% glutaraldehyde, post-fixed in 1%
osmium tetroxide, and dehydrated in ethanol series until 100%
ethanol. Standard procedures of critical point and metal coating
were done at Instituto de Biocieˆncias da Universidade de Sa˜o Paulo.
Supporting Information
Figure S1 Frontal view of a pluteus larva of Clypeaster subdepressus
reconstructed from differential interference contrast image-
sequence. A Grayscale reconstruction showing the position of
the arms. B Red-Cyan 3D image of the same pluteus larva. Scale
bar = 50 mm
Found at: doi:10.1371/journal.pone.0009654.s001 (5.02 MB TIF)
Video S1 Female of the sea biscuit Clypeaster subdepressus releasing
eggs through the gonopore papilla.
Found at: doi:10.1371/journal.pone.0009654.s002 (1.43 MB AVI)
Video S2 Main and accessory papillae of a male sea biscuit
Clypeaster subdepressus releasing sperm.
Found at: doi:10.1371/journal.pone.0009654.s003 (1.38 MB AVI)
Video S3 Elevation of the fertilization membrane after sperm
entry, pronuclei migration, and initial clevages of Clypeaster
subdepressus.
Found at: doi:10.1371/journal.pone.0009654.s004 (5.36 MB AVI)
Video S4 Pre-feeding early pluteus larva rejecting microalgae.
Found at: doi:10.1371/journal.pone.0009654.s005 (0.35 MB AVI)
Video S5 Esophagus initial contractions of a pre-feeding early
pluteus larva.
Found at: doi:10.1371/journal.pone.0009654.s006 (0.89 MB AVI)
Video S6 Pluteus larvae with a well-developed rudiment
exhibiting the ‘‘substrate test behavior’’. Larvae open the arms
exposing the rudiment while podia touches the substrate. If
metamorphosis is not initiated larvae can resume swimming.
Found at: doi:10.1371/journal.pone.0009654.s007 (5.98 MB AVI)
Video S7 Metamorphosis of the sea biscuit Clypeaster subdepressus.
Found at: doi:10.1371/journal.pone.0009654.s008 (2.00 MB AVI)
Video S8 Movements of the lantern of Aristotle, the peristome,
and the esophagus of a Clypeaster subdepressus feeding juvenile.
Found at: doi:10.1371/journal.pone.0009654.s009 (2.60 MB AVI)
Figure 14. Ambulacra of C. subdepressus juveniles. A Oral view of a juvenile showing the distribution of podial types within ambulacral zones
(I–V in white circles); bbaba for type I and aabab for type II. The middle section of interambulacra are marked with dashed white lines. B Detail of
ambulacrum I of a juvenile during the resorption of larval tissues. Dashed white circles mark the position of the first two podia. Type I originated on
the ambulacral column Ib on the peristome edge and was not encircled by the skeleton; type II emerged from a pore in the skeleton on the
ambulacral column Ia. C Ambulacrum I of a 2 months old juvenile still showing the same pattern of the younger juvenile in B. Scale bars = 50 mm (A);
30 mm (B, C)
doi:10.1371/journal.pone.0009654.g014
Development of C. subdepressus
PLoS ONE | www.plosone.org 13 March 2010 | Volume 5 | Issue 3 | e9654

Video S9 Activity and locomotion of postlarval and feeding
juveniles.
Found at: doi:10.1371/journal.pone.0009654.s010 (5.24 MB AVI)
Video S10 Feeding juvenile manipulating a tiny sand grain with
podia and peristome.
Found at: doi:10.1371/journal.pone.0009654.s011 (2.99 MB AVI)
Acknowledgments
We thank CEBIMar-USP for providing the facilities and support for this
project [nu 2006/12], the graduate program of Departamento de Zoologia
do IBUSP, and MZUSP for the SEM. Laborato´rio de Cultura de
Microorganismos Marinhos do IOUSP for providing the cultures of
Dunaliella tertiolecta – CF and Rhodomonas sp. – USA. Rich Mooi for the
enthusiastic discussions on clypeasteroid morphology and comments on a
draft version of this manuscript.
Author Contributions
Conceived and designed the experiments: BCV AEM. Performed the
experiments: BCV. Analyzed the data: BCV AEM. Contributed reagents/
materials/analysis tools: AEM. Wrote the paper: BCV.
Figure 15. Aboral structures and mouth formation of C. subdepressus.ASkeletal plates on the aboral surface of a 2 d old juvenile under
polarized light. B Aboral view showing the distribution of miliary spines (arrow). C Detail of a miliary (left) and primary (right) spine. D Pair of
ophicephalous pedicellariae (arrows) at the posterior region of the juvenile. E Digestory track of the juvenile and fecal pellet (arrow) being released by
the anus. F Side view of the mouth (arrowhead) after the opening of the peristomial membrane. G Oral view showing the mouth and peristomial
membrane 7 dpm. H Juvenile 120 dpf during locomotion with podia extended anteriorly and the characteristic longer pair of posterior spines
(arrows). Scale bars = 30 mm (C, G); 50 mm (A, D, E, F); 100 mm (B); 250 mm (H)
doi:10.1371/journal.pone.0009654.g015
Figure 16. Collecting sites of adult C. subdepressus. Portinho and
Parcel da Praia Grande (dots) are located on the island shore of Sa˜o
Sebastia˜o Channel. CEBIMar-USP (arrowhead) is located on the
continent shore at the Northern region of Sa˜o Paulo State. Marks every
159. Brazil and Sa˜o Paulo vector images cortesy of Felipe Menegaz and
Raphael Lorenzeto de Abreu, respectively.
doi:10.1371/journal.pone.0009654.g016
Development of C. subdepressus
PLoS ONE | www.plosone.org 14 March 2010 | Volume 5 | Issue 3 | e9654

References
1. Kier P (1982) Rapid evolution in echinoids. Palaeontology 25: 1–9.
2. Mooi R (1990) Paedomorphosis, Aristotle’s lantern, and the origin of the sand
dollars (Echinodermata: Clypeasteroida). Paleobiology 16: 25–48.
3. Smith AB (2001) Probing the cassiduloid origins of clypeasteroid echinoids using
stratigraphically restricted parsimony analysis. Paleobiology 27: 392–404.
4. Sauce`de T, Mooi R, David B (2007) Phylogeny and origin of Jurassic irregular
echinoids (Echinodermata: Echinoidea). Geological Magazine 144: 333–359.
5. Hendler G, Miller J, Pawson D, Kier P (1995) Sea Stars, Sea Urchins, and Allies:
Echinoderms of Florida and the Caribbean. Washington: Smithsonian
Institution Press. 390 p.
6. Tommasi L (1966) Lista dos equino´ides recentes do Brasil. Contribuic¸o˜es
Avulsas do Instituto Oceanogra´fico da Universidade de Sa˜o Paulo 11: 1–50.
7. Netto L, Hadel V, Tiago C (2005) Echinodermata from Sa˜o Sebastia˜o Channel
(Sa˜o Paulo, Brazil). Revista de Biologı´a Tropical (International Journal of
Tropical Biology and Conservation) 53(Suppl.3): 207–218.
8. Mortensen T (1948) A Monograph of the Echinoidea IV.2. Clypeastroida.
Clypeastridae, Arachnoididae, Fibulariidae, Laganidae and Scutelidae. Copen-
hagen: C.A. Reitzelpublisher. 471 p.
9. Telford MJ, Mooi R, Harold AS (1987) Feeding activities of two species of
Clypeaster (Echinoides, Clypeasteroida): further evidence of clypeasteroid
resource partitioning. Biological Bulletin 172: 324–336.
10. Emlet RB (1986) Facultative planktotrophy in the tropical echinoid Clypeaster
rosaceus (Linnaeus) and a comparison with obligate planktotrophy in Clypeaster
subdepressus (Gray) (Clypeasteroida: Echinoidea). Journal of Experimental Marine
Biology and Ecology 95: 183–202.
11. Kier PM (1977) The poor fossil record of the regular echinoid. Paleobiology 3:
168–174.
12. Hart MW (2002) Life history evolution and comparative developmental biology
of echinoderms. Evolution & Development 4: 62–71.
13. Miner BG, McEdward LA, McEdward LR (2005) The relationship between egg
size and the duration of the facultative feeding period in marine invertebrate
larvae. Journal of Experimental Marine Biology and Ecology 321: 135–144.
14. Reitzel A, Heyland A (2007) Reduction in morphological plasticity in echinoid
larvae: relationship of plasticity with maternal investment and food availability.
Evolutionary Ecology Research 9: 109–121.
15. Smith MS, Zigler KS, Raff RA (2007) Evolution of direct-developing larvae:
selection vs loss. BioEssays 29: 566–571.
16. Zigler KS, Lessios HA, Raff RA (2008) Egg energetics, fertilization kinetics, and
population structure in echinoids with facultatively feeding larvae. Biological
Bulletin 215: 191–199.
17. Chia FS (1977) Structure and function of the genital papillae in a tropical sand
dollar, Arachnoides placenta (L.) with a discussion on the adaptive significance of
genital papillae in echinoids. Journal of Experimental Marine Biology and
Ecology 27: 187–194.
18. Strathmann MF (1987) Reproduction and development of marine invertebrates
of the northern Pacific coast: data and methods for the study of eggs, embryos,
and larvae. Seattle: University of Washington Press. 684 p.
19. Pearse JS, Cameron RA (1991) Echinoderms and Lophophorates, Pacific Grove:
Boxwood Press, volume VI of Reproduction of Marine Invertebrates,chapter
Echinodermata: Echinoidea. pp 513–662.
20. Wray GA (1997) Embryology: constructing the organism. Sunderland: Sinauer
Associates, Inc., chapter Echinoderms. pp 309–329.
21. Takata H, Kominami T (2004) Behavior of pigment cells closely correlates the
manner of gastrulation in sea urchin embryos. Zoological Science 21:
1025–1035.
22. Takata H, Kominami T (2004) Pigment cells trigger the onset of gastrulation in
tropical sea urchin Echinometra mathaei. Development, Growth & Differentiation
46: 23–35.
23. Caldwell JW (1972) Development, metamorphosis, and substrate selection of the
larvae of the sand dollar, Mellita quinquesperforata (Leske, 1778). Master’s thesis,
University of Florida.
24. Burke RD (1980) Podial sensory receptors and the induction of metamorphosis
in echinoids. Journal of Experimental Marine Biology and Ecology 47: 223–234.
25. Gosselin P, Jangoux M (1998) From competent larva to exotrophic juvenile: a
morphofunctional study of the perimetamorphic period of Paracentrotus lividus
(Echinodermata, Echinoida). Zoomorphology 118: 31–43.
26. Nunes CDAP, Jangoux M (2007) Larval growth and perimetamorphosis in the
echinoid Echinocardium cordatum (echinodermata): the spatangoid way to become a
sea urchin. Zoomorphology 126: 103–119.
27. Gordon I (1927) The development of the calcareous test of Echinocardium
cordatum. Philosophical Transactions of the Royal Society of London Series B,
Containing Papers of a Biological Character (1896–1934) 215: 255–313.
28. Gordon I (1929) Skeletal development in Arbacia, Echinarachnius and Leptasterias.
Philosophical Transactions of the Royal Society of London Series B, Containing
Papers of a Biological Character (1896–1934) 217: 289–334.
29. David B, Mooi R, Telford MJ (1995) The ontogenetic basis of Love´n’s Rule
clarifies homologies of the echinoid peristome. In: Emson RH, Smith AB,
Campbell AC, eds. Echinoderm Research 1995, Proceedings of the 4th
European Colloquium on Echinoderms, London. Rotterdam: Balkema. pp
155–164.
30. Mooi R, David B, Marchand D (1994) Echinoderm skeletal homologies:
Classical morphology meets modem phylogenetics. In: David B, Guille A, Fe´ral J,
Roux M, eds. Echinoderms Through Time. Rotterdam: Balkema. pp 87–95.
31. Miller BA, Emlet RB (1999) Development of newly metamorphosed juvenile sea
urchins (Strongylocentrotus franciscanus and S. purpuratus): morphology, the effects of
temperature and larval food ration, and a method for determining age. Journal
of Experimental Marine Biology and Ecology 235: 67–90.
32. Mooi R (1986) Non-respiratory podia of clypeasteroids (Echinodermata,
Echinoides): II. Diversity. Zoomorphology 106: 75–90.
33. Pechenik JA (2006) Larval experience and latent effects–metamorphosis is not a
new beginning. Integrative and Comparative Biology 46: 323–333.
34. Gordon I (1926) The development of the calcareous test of Echinus miliaris.
Philosophical Transactions of the Royal Society of London Series B, Containing
Papers of a Biological Character (1896–1934) 214: 259–312.
35. Cameron RA, Rumrill SS (1982) Larval abundance and recruitment of the sand
dollar Dendraster excentricus in Monterey Bay, California, USA. Marine Biology 71:
197–202.
36. Rasband WS (1997) ImageJ. U. S. National Institutes of Health, Bethesda,
Maryland, USA. URL http://rsb.info.nih.gov/ij/.
Development of C. subdepressus
PLoS ONE | www.plosone.org 15 March 2010 | Volume 5 | Issue 3 | e9654