Molecular Phylogeny of Coral-Reef Sea Cucumbers (Holothuriidae:
Aspidochirotida) Based on 16S Mitochondrial Ribosomal DNA
Sequence
Alexander M. Kerr,
1
* Daniel A. Janies,
2,

Ronald M. Clouse,
2,
Yves Samyn,
3
Jeni Kuszak,
2
Junhyong Kim
1,§
1
Department of Ecology and Evolutionary Biology, Osborn Zoo¨logical Laboratories, Yale University, New Haven, CT 06520, U.S.A.
2
Department of Invertebrate Zoology, American Museum of Natural History, New York, NY 10024, U.S.A.
3
Global Taxonomy Initiative, Royal Belgian Institute of Natural Sciences, Vaurierstraat 29, B-1000 Brussels, Belgium
Received: 4 February 2004 / Accepted: 5 May 2004 / Online publication: 30 December 2004
Abstract
Members of the Holothuriidae, found globally at low
to middle latitudes, are often a dominant component
of Indo–West Pacific coral reefs. We present the first
phylogeny of the group, using 8 species from the 5
currently recognized genera and based on approxi-
mately 540 nucleotides from a polymerase chain
reaction–amplified and conserved 3¢ section of 16S
mitochondrial ribosomal DNA. Parsimony and
likelihood analyses returned identical topologies,
permitting several robust inferences to be drawn.
Several points corroborated the Linnean classifica-
tion. Actinopyga and Bohadschia each appear
monophyletic and Pearsonothuria is sister to Boh-
adschia. Other aspects of our phylogeny, however,
were not in accord with the taxonomy of Holothu-
riidae or previous speculations about the group’s
evolutionary history. Most notably, the genus Hol-
othuria appears paraphyletic. Actinopyga and Boh-
adschia, sometimes held to be closely related to one
another because of certain morphologic similarities,
are only distantly related. The morphologically dis-
tinct Labidodemas, even thought to warrant sepa-
ration at the family level, is nested well within
Holothuria. A maximum parsimony reconstruction
of ancestral ossicle form on the phylogeny indicated
that, in addition to a probable bout of elaboration in
ossicle form (the modification of rods or rosettes to
holothuriid-type buttons), at least 2 rounds of ossicle
simplification also transpired in which buttons re-
verted to rods or rosettes. Cuvierian tubules, defen-
sive organs unique to numerous members of
Holothuriidae, were probably present before the
initial radiation of the family, but the reconstruction
is ambiguous as to their ancestral function.
Key words: phylogeny — 16S rDNA — mtDNA —
Holothuriidae — Isostichopus — Stichopus
Introduction
Sea cucumbers, or holothuroids, in the large, glob-
ally distributed family Holothuriidae primarily in-
habit the tropical eulittoral, usually coral reefs and
adjacent sandy areas, though temperate shallows and
deep water (to approx. 1 km) harbor a few species.
Holothuriids are primarily deposit feeders, in con-
trast to the suspension feeding by dendrochirote
holothuroids that prevails at higher latitudes. Like
nearly all other sea cucumbers, holothuriids have a
reduced skeleton of isolated, microscopic ossicles
embedded in a pliable body wall. And, like other
groups of soft-bodied organisms, their fossil history
is threadbare, though button ossicles and articulated
calcareous-ring elements characteristic of holothu-
riids are known from the Upper Jurassic (Gilliland,
1993) and the Middle Triassic (Reich, 2004), respec-
tively. The provenance of these few finds and the
family’s apparent Tethyan distribution, even at the
level of subgenera (Rowe, 1969; Clark and Rowe,
1971; Hendler et al., 1995; Massin, 1999), suggests
the antiquity of several groups within the family.
*Present address: Centre for Coral Reef Biodiversity, Department
of Marine Biology, James Cook University, Townsville QL 8411,
Australia


Present address: Department of Biomedical Informatics, The
Ohio State University, Columbus, OH 43210, U.S.A.

Present address: Department of Organismic and Evolutionary
Biology, Harvard University, Cambridge, MA 02138, U.S.A.
§
Present address: Department of Biology, University of Pennsyl-
vania, Philadelphia, PA 19104-6018, U.S.A.
Correspondence to: Alexander M. Kerr; E-mail: alexander.
kerr@jcu.edu.au
DOI: 10.1007/s10126-004-0019-y
Volume 7, 53–60 (2005)
 Springer Science+Business Media, Inc. 2005 53

Holothuriidae is the second largest family of sea
cucumbers, with about 185 species, or about 11% of
the diversity of living holothuroids (Smiley, 1994).
The family comprises 5 genera, the largest of which
is Holothuria, with about 150 species. This diverse
genus has dominated comprehensive reviews of the
family (Pearson, 1914; Panning, 1929–1935; Deich-
mann, 1958; Rowe, 1969) because the remaining
genera are small, well delimited, and easily diag-
nosed. Consequently, little attention has been paid
to the evolutionary relationships in the family as a
whole. Pearson (1914) believed that 2 genera, Ac-
tinopyga and Bohadschia, were closely related and
primitive compared with Holothuria. Deichmann
(1958) suggested that evolution in Holothuria had
proceeded via progressive simplification of the ossi-
cles from tables and buttons characteristic of many
Holothuria to less elaborate rods or rosettes, like
those found in the Holothuria subgenera Selen-
kothuria and Semperothuria. In contrast, Rowe
(1969) proposed that the genus had proceeded via the
opposite trend, from simple ossicles to complex
ones, and on the basis of this trend, he provided a
tentative phylogeny of subgenera in Holothuria that
would require the genus to be monophyletic. More
recently, Levin (1999), in partial agreement with
Deichmann (1958), argued that the evolution of
Holothuriidae proceeded from a form similar to the
burrowing Labidodemas, which has tables and
sometimes buttons, to vagile-exposed forms and to
suspension feeders, some of which have rods and
rosettes.
In this study we tested the utility of a conserved
3¢ section of 16S mitochondrial ribosomal DNA to
clarify relationships between the genera in Holo-
thuriidae, as well as performing an initial test for
monophyly of the largest genus Holothuria. We then
used the estimate of the relationships to make
inferences about the evolution of morphology and
ecology in the group.
Materials and Methods
DNA Preparation. We sampled one specimen from
one to 2 species in each of the 4 small and mor-
phologically well-circumscribed genera of holothu-
riids. The fifth and largest genus Holothuria consists
of several subgenera, from which we selected 2
putatively divergent (morphologically disparate)
species. As outgroups to root the holothuriid tree, we
used 2 species from different genera within Sticho-
podidae (Table 1). This latter family was selected
largely because of its sister status to the study group
Holothuriidae as inferred from a maximum parsi-
mony analysis of morphologic characters (Kerr and
Kim, 2001).
To prepare DNA for sequencing, approximately
100 mg of gonad, muscle, or tentacle tissue pre-
served in ethanol or DMSO was air-dried and ground
to a powder in liquid nitrogen, then whole genomic
DNA was extracted using the CTAB method of Ar-
ndt et al. (1996) or the EZ-DNA genomic DNA iso-
lation reagent (Morwell MD Biosciences). We
amplified a portion of approximately 540 nucleotides
from the conserved 3¢ end of the mitochondrial gene
coding for the 16S-like, large ribosomal RNA sub-
unit using the echinoderm-specific universal prim-
ers 16Sar (5¢-CGCCTGTTTATCAAAAACAT-3¢) and 16Sbr
(5¢-CTCCGGTTTGAACTCAGATCA-3¢), then performed
double-stranded polymerase chain reaction (PCR)
under standard conditions (an initial 30-seconds
denaturation at 95C, followed by 40 cycles of 95C
for 30 seconds, 50 to 55C for 30 seconds, and 72C
for 1 minute, then a final 4-minute extension at 72)
Table 1. Taxa Used in This Study
Family and Species
Museum
voucher number
Genbank
accession number Locality
Holothuriidae
Actinopyga agassizi (Selenka, 1867) A110110 AY338413 Long Key, Florida
A mauritiana (Quoy and Gaimard, 1833) E51760 AY338414 Guam, Mariana Islands
Bohadschia argus Jaeger, 1833 A113273 AY338416 Guam, Mariana Islands
B. marmorata Jaeger, 1833 E51759 AY338417 Guam, Mariana Islands
Holothuria (Platyperona) excellens (Ludwig, 1875) A113261 AY338418 Guam, Mariana Islands
H. (Mertensiothuria) leucospilota (Brandt, 1835) E51763 AY338419 Guam, Mariana Islands
Labidodemas semperianum Selenka, 1867 E53083 AY338420 Guam, Mariana Islands
Pearsonothuria graeffei (Semper, 1868) E51761 AY338421 Guam, Mariana Islands
Stichopodidae
Isostichopus macroparentheses (HL Clark, 1922) E47524 AY338415 Guana Island, British
Virgin Islands
Stichopus chloronotus Brandt, 1835 E47517 AY338422 Guam, Mariana Islands
a
Note: E indicates U.S. National Museum of Natural History echinoderm collection; A, American Museum of Natural History inver-
tebrate tissue collection.
54 ALEXANDER M. KERR ET AL.: HOLOTHURIID SEA CUCUMBERS