Hydrobiologia (2007) 592:439–454
DOI 10.1007/s10750-007-0789-0
PRIMARY RESEARCH PAPER
A revision of the systematics of panther worms
(Hofstenia spp., Acoela), with notes on color variation
and genetic variation within the genus
Matthew Hooge Æ Andreas Wallberg Æ Christiane Todt Æ
Aaron Maloy Æ Ulf Jondelius Æ Seth Tyler
Received: 28 July 2006 / Revised: 12 May 2007 / Accepted: 17 May 2007 / Published online: 31 July 2007

Springer Science+Business Media B.V. 2007
Abstract Species of the genus Hofstenia are
voracious predators and among the largest and
most colorful of the Acoela. They are known
from Japan, the Red Sea, the North Atlantic
Handling editor: K. Martens
Electronic supplementary material The online version of
this article (doi:10.1007/s10750-007-0789-0) contains
supplementary material, which is available to authorized
users.
M. Hooge (&)
S. Tyler
Department of Biological Sciences, The University of
Maine, Orono, ME 04469-5751, USA
e-mail: hooge@umit.maine.edu
A. Wallberg
Department of Systematic Zoology, Evolutionary
Biology Centre, Uppsala University, Norbyvägen
18D, 752 36 Uppsala, Sweden
C. Todt
Department of Biology, University of Bergen,
Thormøhlensgate 55, 5007 Bergen, Norway
A. Maloy
Centre of Applied Marine Biotechnology,
Letterkenny Institute of Technology, Letterkenny,
County Donegal, Ireland
U. Jondelius
Department of Invertebrate Zoology, Swedish
Museum of Natural History, POB 50007, 104 05
Stockholm, Sweden
islands of Bermuda and the Bahamas, and the
Caribbean and in a variety of habitats includ-
ing the rocky intertidal, among Thalassia sea
grass, on filamentous algae and decaying man-
grove leaves. Certain color morphs associated
with each of these habitats seem to have
confused the taxonomy of the group. While
brown-and-white banding and spotting patterns
of Hofstenia miamia and Hofstenia giselae are
distinctive for species associated with mangrove
leaves and Thallasia sp. and are likely to be
cryptic for these specific environments, we find
some evidence to suggest that the coloration is
mimicry of a nudibranch with aposematic
coloration. The common plan in these patterns
is one with three variously solid or spotted
lighter cross bands on a dark background. Our
examination of museum type material and live
specimens of Hofstenia collected from Baha-
mas, Belize, Bermuda, and Panama revealed
no internal morphological differences between
the Hofstenia species occurring in the Carib-
bean. Similarly, our analyses of 18S and 28S
molecular sequence data revealed no significant
differences among specimens. Accordingly, we
declare that Hofstenia giselae is a junior syn-
onym of Hofstenia miamia, the three-banded
panther worm.
Keywords Platyhelminthes
Turbellaria

Mangrove
Caribbean
Intraspecific variation
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Introduction
Species of the genus Hofstenia are among the
largest and most distinctive of the Acoela. They
are voracious predators of micrometazoans, suck-
ing in prey with a capacious muscular pharynx at
the anterior tip of the body. Mature specimens
are typically 4–9 mm in length and, rather than
being colorless and having the teardrop body
shape of most acoel species, Hofstenia species are
darkly pigmented, with patterns of white bands
and spots, and nearly cylindrical in shape, often
with a small pointed tail (Fig. 1). Their carnivory
and color patterns, reminiscent of panthers in
general, inspires their common name.
The large, muscular, anteriorly directed phar-
ynx simplex is one-third to one-half the length of
the entire worm (Fig. 2) and is used to suck up
copepods, ostracods, and turbellarians. Also dis-
tinctive of Hofstenia is its anteriorly positioned
male copulatory organ (Fig. 2). Located ventral
to the pharynx, the copulatory organ is composed
of a highly muscular, eversible penis equipped
Fig. 1 Dorsal aspect of eight specimens of Hofstenia miamia from Curaçao (from Corrêa, 1963)
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Hydrobiologia (2007) 592:439–454
with sclerotized needles. Species of Hofstenia lack
a female gonopore, but the sharp needles of the
everted penis can penetrate the body wall of the
receiving worm and deposit sperm directly
beneath its epidermis (Bock, 1923); sperm then
apparently migrate through the parenchymal
tissue to the oocytes.
Such distinctive features made it difficult for
early systematists to place Hofstenia among other
platyhelminths. The first described species, Hof-
stenia atroviridis Bock, 1923, attracted consider-
able attention for the primitive features of its
nervous system and gut relative to those of other
platyhelminths and for features of its reproduc-
tive system that seemed to be intermediate
between those of other primitive and the more
derived flatworms (Bock, 1923; Steinböck, 1924;
Bresslau, 1933; Karling, 1940). At first, similarity
of its reproductive organs and pharynx to those of
prolecithtophoran and some lecithoepitheliate
turbellarians was weighed heavily in placing it
among such so-called ‘‘alloeocoels,’’ even though
its affinity to the Acoela was recognized (Stein-
Hydrobiologia (2007) 592:439–454
Fig. 2 Sagittal histological section of Hofstenia miamia
from Belize. cs—Digestive central syncytium, e—egg,
gv—granule vesicle, m—mouth, ma—male antrum,
böck, 1924; Meixner, 1938; Karling, 1940). Not
until a quarter century after its discovery was its
true nature as an acoel established (Papi, 1957).
Currently there are four described species of
Hofstenia (see Tyler et al., 2006). The type
species, H. atroviridis, is a dark, blackish green
species from the coast of Japan where it is found
associated with coralline algae in tide pools and
on the holdfasts of Laminaria at subtidal depths
(Bock, 1923). H. miamia Corrêa, 1960, was
described from a single specimen found in algae
at Miami, Florida (Corrêa, 1960); however, addi-
tional specimens were subsequently found in
Antigua as well as Curaçao (Corrêa, 1963) and
were used to document the variation in the dark-
brown, white-banded color patterns within the
species (Fig. 1). Soon thereafter, Steinböck
(1966) published a monograph on the Hofstenii-
dae in which he established two new species: H.
beltagii Steinböck, 1966, from specimens collected
in the Red Sea and first identified by Beltagi
(1958) as H. atroviridis, and H. giselae Steinböck,
1966, collected from a Thalassia bed in the
Bahamas. With his specimens of H. giselae, which
were lighter-colored than the reported pigmenta-
tion of H. miamia, Steinböck experimented
extensively on their regenerative capabilities
(Steinböck, 1966, 1967).
Over a 1-year period from April 2004 to May
2005, we collected living specimens of Hofstenia
from Bahamas, Belize, Bermuda, and Panama
(Table 1). Some of these were light-colored and
associated with Thalassia and filamentous algae,
which we identified as H. giselae, and others dark
and associated with decaying mangrove leaves,
which we identified as H. miamia. We also
441
mgp—male gonopore, phm—pharynx musculature,
st—penis stylets, sv—seminal vesicle
acquired the type material for H. atroviridis, H.
giselae, and H. miamia (Table 2) for comparison.
We report here the results of our comparative
morphological study of this material. In addition,
we document the range in color patterns in
Caribbean specimens, and propose systematic
revision of the genus Hofstenia. Acoels have
been attributed to possess unusually fast evolving
nuclear ribosomal genes (Carranza et al., 1997;
Ruiz-Trillo et al., 1999), which may make these
molecular markers useful for studying closely
related acoel lineages (e.g. Tekle et al., 2005). We
therefore used 18S and 28S rDNA genes acquired
from several specimens of Hofstenia from all four
collection sites in an attempt to reconstruct a
molecular phylogeny of this taxon and test the
traditional classification.
Materials and methods
Collection and observation of living specimens
Approximately 150 living specimens of Hofstenia
were collected from sites in Bahamas, Belize,
Bermuda, and Panama (Table 1). The majority of
specimens we collected were taken from sub-
merged, decaying mangrove leaves found in piles
at the base of living mangroves, and had dark
coloration that appeared most similar to Corrêa’s
(1963) H. miamia specimens (Fig. 1). A single
specimen of H. miamia was also found on
Penicillus attached to a mangrove root at Man-
atee Cay, Belize. We collected lightly colored
specimens of Hofstenia from a Thalassia sp. bed
at Carrie Bow Cay, Belize, and from filamentous
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Table 1 Sampling information for specimens of Hofstenia used in this study

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Locality Latitude (N) Longitude (W) Date Habitat Depth (m) Specimens

Bahamas
Lee Stocking Island 2346¢26† 7606¢26† April 2004 filamentous algae <1 ~10
Belize
Manatee Cay 1639¢ 8811¢ April 2004 Penicillus on mangrove root 1 1
Twin Cays, Grouper Garden 1649¢46† 8806¢10† April 2004 Submerged mangrove leaves 1 ~25
Carrie Bow Cay, North side 1643¢9† 8804¢54† April 2004 Sediment underlying Thalassia <1 4
Panama
Bocas del Toro, STRI field station 921¢ 8215¢ August 2004 Submerged mangrove leaves 1 ~30
Bermuda
Walsingham Pond, East side 3220¢46† 6442¢32† May 2005 Submerged mangrove leaves 1 ~90

Table 2 Comparison of histological sections of Hofstenia, including those shown in Fig. 4

Specimen Location Habitat Color Body Pharynx Pharynx/ Mouth Stylet Stylet
length length body length number
(mm) (mm) ratio (lm)

Hofstenia atroviridis
Lectotype Japan High-energy intertidal/subtidal Dark green 4.2 2.1 0.5 Subterminal 70 ~25
SMNH 2508a assoc. with Laminaria, Corallina
Hofstenia giselae
Lectotype Bahamas Low-energy White/brown 3.7 1.0 0.4 Subterminal 70 ~25
NMW
12, III, 5
SMNH Belize Low-energy assoc. with Thalassia White/brown assoc. 3.9 1.7 0.4 Subterminal 65 ~25
89913 with Thalassia
Hofstenia miamia
Paratype Curaçao Low-energy among Brown/white 2.5 1.0 0.4 Subterminal 40 ~20
SMNH Thalassia and algae
74878/9
USNM Belize Low-energy submerged Brown/white 4.2 1.3 0.3 Subterminal 75 ~25
1096778 mangrove leaves
Hydrobiologia (2007) 592:439–454
Hydrobiologia (2007) 592:439–454
algae at the surface of a saltwater pond on Lee
Stocking Island, Bahamas. These specimens
matched the coloration described for Hofstenia
giselae by Steinböck (1966).
Hofstenia giselae from Thalassia were ex-
tracted by placing a large quantity of Thalassia
sp. (~3 L), including the roots and underlying
sediment, in a bucket of sea water. Specimens
crawled out of the Thalassia sp. to the water
surface over a period of 3–8 h. Mangrove-dwell-
ing specimens of H. miamia were similarly
extracted by placing submerged mangrove leaves
and associated detritus in buckets of sea water.
These specimens crawled to the water surface
over a period of 1–5 h.
Several specimens from each collection site
were photographed with a digital camera at-
tached to a dissecting microscope. The specimens
reacted poorly to isotonic magnesium chloride or
magnesium sulfate, often undergoing convulsions
and thereby distorting their natural habitus. As an
alternative, drops of ethanol were added to a
small dish of seawater until the specimens slowed
enough for photography.
Histology
For histological study, unrelaxed specimens were
squirted directly into hot Stefanini’s fixative
(Stefanini et al., 1967), washed in phosphate
buffer (Millonig’s, 0.1 M), fixed in phosphate-
buffered 1% (v/v) osmium tetroxide, dehydrated
in an acetone series, and embedded in EMBed/
Araldite epoxy resin. Dehydration was quickened
by microwave radiation according to Giberson &
Demaree (1995). Serial 2-lm-thick sections were
made according to Smith & Tyler (1984) using a
Diatome diamond knife mounted in a Butler
trough (Butler, 1979), and stained with toluidine
blue.
DNA analyses
In addition to Hofstenia miamia and H. giselae,
our molecular sequence analyses used other acoel
terminals as well as several non-acoel outgroups
(Table 3), including two specimens of an uniden-
tified species of Hofsteniidae that were found in
the bay near the Marine Biological Station of ‘‘A
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Graña’’ (Ferrol, Galiza, Spain) in August 2006 in
mud sediments obtained by dredging with an
Ockelmann dredge at 6–10 m depth.
Genomic DNA was extracted from most spec-
imens using the Qiagen DNeasy Tissue Kit
following the manufacturer’s default protocol.
PCR amplification of the small and large
nuclear ribosomal subunit genes (18S and 28S)
were carried out using Amersham Biosciences
PuReTaqTM Ready-To-GoTM PCR Beads and an
Eppendorf Mastercycler Gradient thermal cycler.
18S was amplified using a two-step nested PCR
approach first using primers TIMA-TIMB, fol-
lowed by combining two overlapping fragments
for a total length of about 1,750 nucleotides
(primers S30-5FK and 4FB-1806R). 28S was
amplified using a single step approach combining
three overlapping fragments for a total maximum
length of about 3,000 nucleotides (U178-L1642,
1200F-R2176, U1846-L3449). The typical PCR
regime for each gene and fragment was: 95C for
5 min, 35· (94C for 30 s, 50C for 30 s, 72C for
30 s), 72C for 10 min. Samples were sent to
Macrogen, Inc. (Seoul, Korea) for sequencing.
Total genomic DNA of the Proporus carolin-
ensis specimens was extracted using the QIA-
amp DNA Micro Kit (Qiagen). Two nearly
equal lengths of the 18S rRNA gene were PCR-
amplified primers 1F-5R and 5F-9R (Carranza
et al., 1997). Reaction conditions included 1 ll of
template DNA, 2.5 mM MgCl2, 0.75 lM of each
primer, 200 lM of each deoxynucleoside triphos-
phate, 1.25 U Taq polymerase (Invitrogen Corp.,
Carlsbad, Calif.), and 2.5 ll 10X PCR buffer. The
remaining volume was completed with nuclease-
free water. PCR regime was: 95C for 3 min, 35·
(95C for 30 s, 55C for 30 s, 72C for 30 s), 72C
for 7 min. Amplicons were filter-purified (Mon-
tageTM, Millipore) and directly sequenced at the
University of Maine’s DNA sequencing facility
using an ABI model 3730 DNA Analyzer
(Applied Biosystems, Foster City, Calif.) with
the BigDye Version 3.1 Cycle Sequencing Kit per
manufacturer’s instructions.
The sequences were assembled using the GNU/
Linux-version of the program Staden v1.6.0 (Sta-
den, 1996); suite enhanced by the Phred/Phrap
base calling/assembly modules (Ewing et al., 1998;
Ewing & Green, 1998) and manually edited at
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Table 3 Taxa used in molecular analyses, GenBank sequence accession numbers, and location of newly collected material
Taxon 18S 28S Location

Chordata
Mus musculus MMRNA18 –
Ctenophora
Mnemiopsis leidyi MNESSRRNA –
Cnidaria
Nematostella vectensis AF254382 –
Pennatula sp. AY838561 –
Nemertodermatida
Meara stichopi AF100191 –
Acoela
Childiidae Dörjes, 1968
Childia brachyposthium AY297952 AJ849499
Childia crassum AJ845242 AJ849497
Childia cycloposthium AF329178 AJ849494
Childia groenlandica 1 AY078367 –
Childia groenlandica 2 CGR012529 AY157603
Childia groenlandica 3 AM701805 AM701806 Galiza, Spain
Childia macroposthium AY297953 AJ849500
Childia submaculatum AY297953 AJ849496
Childia trianguliferum AY297950 AJ849495
Childia vivipara AY297954 AJ849498
Hofsteniidae Bock, 1923
Hofstenia giselae 1 AM701807 AM701808 Lee Stocking Island, Bahamas
Hofstenia giselae 2 AM701809 AM701810 Lee Stocking Island, Bahamas
Hofstenia giselae 3 AM701811 AM701812 Carrie Bow Cay, Belize
Hofstenia miamia 1 AM701813 AM701814 Walsingham Pond, Bermuda
Hofstenia miamia 2 AM701815 AM701816 Bocas del Toro, Panama
Hofstenia miamia 3 AM701817 AM701818 Twin Cays, Belize
Hofstenia sp. 1 AM701819 AM701820 Galiza, Spain
Hofstenia sp. 2 AM701821 AM701822 Galiza, Spain
Paratomellidae Dörjes, 1966
Paratomella rubra AF102892 AY157604
Paratomella unichaeta AY078379 –
Proporidae Graff, 1882
Proporus carolinensis 1 AM701825 – Carrie Bow Cay, Belize
Proporus carolinensis 2 AM701826 – North Carolina, USA
Solenofilomorphidae Dörjes, 1968
Myopea ‘‘callaeum’’ sp. AY078380 –
Oligofilomorpha interstitiophilum AM701823 AM701824 Gullmarsfjorden, Sweden
Accession numbers in bold denote newly acquired sequences

some sites. Small alignments, including only Hof-
stenia sequences, were inferred using the default
settings of the Muscle v3.6 progressive alignment
software (Edgar, 2004). These datasets were con-
catenated. Additional 18S/28S sequences were
downloaded from NCBI Genbank and were
aligned using the same methods; the alignments
were not refined manually. A complete list of
included sequences is provided in Table 3.
Phylogenetic trees with branch support were
first inferred from each gene separately using the
parsimony criterion and Bremer support
(Bremer, 1994) assessment as implemented in
the program TNT v1.0 (Goloboff et al., 2000),
where trees up to 20 steps longer than the most
parsimonious tree were included to calculate the
support. The Hofstenia-only datasets were ana-
lyzed using the branch-and-bound algorithm,
while the large datasets were analyzed using 10
random additions, each with TBR branch swap-
ping holding up to 10 trees. Phylogenies were also
inferred with the Bayesian method as imple-
mented in the program MrBayes (Huelsenbeck &
Ronquist, 2001; Ronquist & Huelsenbeck, 2003).

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MrAIC v1.4.2 (Nylander, 2004) and Phyml v2.4.4
(Guindon & Gascuel, 2003) were used to select
one of the evolutionary models available for the
Bayesian analysis. The method consistently sug-
gested the two models GTR + G and GTR +
I + G with very similar likelihoods (see Lanave
et al., 1984; Tavaré, 1986 for formal description of
the GTR model). We used GTR+G for the small
datasets and GTR + I + G for the large datasets.
All analyses used four independent runs of four
MCMC chains of several millions of generations.
Average standard deviation of split frequencies of
0.005 or lower were used to deduce when param-
eters had been adequately sampled from the
stationary distribution. The burn-in fraction was
set to 50%. Trees were saved every 500th gener-
ation. The two small Hofstenia-only datasets were
also concatenated and analyzed using the same
methods. The majority rule consensus tree result-
ing from this analysis was rooted using the two
Hofstenia specimens from Galiza as outgroups.
The patristic distances between terminals
inferred by the bayesian method were calculated
by summing the branch lengths separating termi-
nals and inspected to try addressing within/
between species divergence.
Type material
We examined all the type material available for
the genus Hofstenia from the Swedish Museum of
Natural History (SMNH) and the Naturhistoris-
ches Museum Wien (NMW). For H. atroviridis
we examined Bock’s (1923) original material
including the lectotype (SMNH 2508, 2508a), as
well as several paratypes (SMNH 74901-74915).
For H. miamia we examined SMNH 2744, 2744a.
These slides were designated ‘‘Holotype’’ by
Dörjes & Karling (1975). However, the slides
are marked ‘Antigua’, M56 and M57 whereas the
type specimen collected in Virginia Key, Miami
Florida was originally marked M2 (Corrêa, 1960).
We therefore regard SMNH 2744 and 2744a as
the neotype of Hofstenia miamia designated by
Dörjes & Karling (1975). The specimen is prob-
ably one of those collected by PW Hummelinck
as mentioned by Corrêa (1963). We could not
locate the original holotype specimen from
Miami, Florida. Also for H. miamia, we examined
445
SMNH 74869-74880, which bear markings from
the University of São Paulo, Brazil, and are likely
Corrêa’s (1963) specimens from Curaçao. For H.
giselae, we examined Steinböck’s (1966) material,
including NMW 12-III-4, 5, 6, 7. There is appar-
ently no deposited type material of H. beltagii
(see Steinböck, 1966).
Results
Morphological analysis
Mature specimens of Hofstenia collected from
Bahamas, Bermuda, Belize, and Panama were
approximately 4 mm long when contracted, but
could elongate up to 6–7 mm. The coloration of
H. miamia specimens collected from mangroves
varied considerably, and no two specimens had
exactly the same pattern of coloration (Fig. 3).
All specimens of H. miamia were predominately
brown to blackish brown, but they varied with
regard to the occurrence of additional white
coloration. Most common was the pattern of
three white cross bands that were either solid or
somewhat discontinuous. Other white dapples or
flecks of white often occurred, particularly at the
anterior tip of the animal and also along the
dorsal midline. While the coloration of speci-
mens collected from Panama were predomi-
nately spotted (Fig. 3: 1–8) and those from
Belize were mostly striped (Fig. 3: 25–28), we
observed a whole gamut of coloration in Ber-
muda, from specimens that nearly lacked any
white coloration, to those with solid white
stripes and additional white blotches (Fig. 3: 9–
24).
Specimens of Hofstenia giselae collected from
Thalassia at Carrie Bow Cay, Belize (Fig. 3: 30–
31), and from filamentous algae from a saltwater
pond on Lee Stocking Island, Bahamas (Fig. 3:
32–33) had a distinctly different coloration than
specimens of H. miamia. These were predomi-
nately covered with overlapping white flecks, so
that they appeared mostly white. In the posterior
half of the body was a wide brown band that
covered the lateral regions of the dorsal side but
did not cross over the dorsal midline, so as to
leave a white stripe in that region.
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Fig. 3 Color variation in Hofstenia miamia and Hofstenia
giselae from four geographic locations. All specimens of
H. miamia (1–29) were collected from submerged man-
grove leaves. Specimens of H. giselae were found in
Despite differing external body coloration,
specimens of Hofstenia have matching internal
morphology. Our comparison of the internal
morphology in serial histological sections of
Hofstenia atroviridis, H. giselae, and H. miamia
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Hydrobiologia (2007) 592:439–454
sediment with Thalassia (30–31) and on filamentous algae
(32–33). (1–8) Bocas del Toro, Panama. (9–24) Walsingham
Pond, Bermuda. (25–29) Twin Cays, Belize. (30–31) Carrie
Bow Cay, Belize. (32–33) Lee Stocking Island, Bahamas
revealed only minor differences (Fig. 4, Table 2),
none of which appear to be useful diagnostically.
The mouth in all specimens examined was posi-
tioned subterminally. Nearly all of the sectioned
specimens we examined were approximately
Hydrobiologia (2007) 592:439–454
Fig. 4 Photomicrographs of histological sections of Hof-
stenia, showing morphology of male copulatory organ (left
side), and close-up of penis stylets (right side). (a) H.
atroviridis, sagittal section, Lectotype SMNH 2508a,
Misaki, Japan. (b) H. miamia, frontal section, Paratype
SMNH 74878, Piscadera Baai, Curaçao. (c) H. giselae,
sagittal section, Lectotype NMW 12, III, 5, North Bimini,
4 mm long and had a pharynx that was one-third
to one-half the body length. There was also
similarity with regard to the penis stylets, with
most specimens having approximately 25 stylets
that were 65–75 lm in length. The exception to
these measurements was H. miamia from Cura-
çao, which had a shorter body length, 2.5 mm,
was proportionally smaller with regard to pharynx
and stylet length, and had fewer stylet needles
(~20) (Fig. 4b, Table 2).
447
Bahamas. (d) H. miamia, sagittal section, Carrie Bow Cay,
Belize from Thalassia. (e) H. miamia, sagittal section,
Twin Cays, Belize from mangrove leaves. Scale bars at
bottom of figure apply to all images on right and left sides
respectively. gv—granule vesicle, ma—male antrum,
pb—penis bulb, phe—pharynx epithelium, sp—penis
sperm, st—penis stylets, sv—seminal vesicle
Behavior
Living specimens of H. miamia collected from
mangroves in Bermuda were successfully kept in
the laboratory in Petri dishes at room tempera-
ture for as long as four weeks. During this time
the specimens tended to lie unmoving at the
bottom of the dish unless disturbed by vibrations
or light. No changes in the coloration of the
specimens could be discerned during this period.
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448
Squeeze preparations and histological sections
revealed that the worms fed upon copepods and
ostracods. Additionally, we observed the animals
feeding upon other prey items we provided them,
including small acoels, rhabdocoel turbellarians,
and polychaetes. Using the dissecting microscope
we also observed some copulation events. The
worms occasionally everted their penes in the
presence of another specimen, and there ap-
peared to be a short period of penis ‘probing’
before the penis stylets were finally stabbed into
the epidermis at the posterior end of the receiving
worm.
Molecular analysis
Analysis of both 18S and 28S sequences support a
monophyletic Hofstenia clade as the sister taxon
to members of the Solenofilomorphidae (Figs. 5,
6). Sequences of H. miamia and H. giselae form a
well-supported group within that clade, clearly
1.00/4
1.00/20+
1.00/4
1.00/20+
1.00/5
1.00/9 Pennatulasp
Nematostella vectensis
Mnemiopsis leidyi
0.1
Fig. 5 Phylogenetic analyses of nuclear ribosomal small
subunit 18S rDNA sequences indicate that Hofstenia is
closely related to Solenofilomorphidae. Topology, branch-
lengths and bayesian posterior probabilities (bold values
above branches) were estimated from 24,000 trees col-
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Hydrobiologia (2007) 592:439–454
separated from the unidentified Hofstenia
sequences; but neither H. miamia or H. giselae
are recovered as distinctly separate species when
the two genes are analyzed separately (Figs. 5, 6)
or concatenated (Fig. 7). Instead, the H. miamia
and H. giselae sequences are mixed with respect
to the traditional classification. The 18S alignment
limited to only Hofstenia sequences recovers a
slightly different topology compared to the com-
plete 18S alignment (Fig 5), where the two
Hofstenia specimens from Belize make up the
sister group to the other H. miamia and H. giselae
(not shown). The corresponding 28S alignment
recovers the same phylogeny (not shown)
as the complete and concatenated alignments
(Figs. 6, 7).
Analyses of the concatenated genes recover
two sister groups: an ‘‘Atlantic’’ clade uniting
specimens from Bahamas and Bermuda, and a
‘‘Caribbean’’ clade of specimens from Panama
and Belize (Fig. 7). There are very few variable or
1.00/13 Childia macroposthium
1.00/3 Childia brachyposthium
0.98/3 Childia vivipara
0.99/4 Childia crassum
Childia groenlandica1 USA
1.00/20+
1.00/3 Childia groenlandica 3 Spain
Childia groenlandica 2 Europe
1.00/20+ 0.68/0 Childia submaculatum
Childia trianguliferum
1.00/5
Childia cycloposthium
1.00/20+ Proporus carolinensis 1 Belize
Proporus carolinensis 2 USA
0.68/0 Hofstenia giselae 1 Bahamas
1.00/3 Hofstenia giselae 2 Bahamas
1.00/1 Hofstenia miamia 1 Bermuda
1.00/4
Hofstenia miamia 2 Panama
1.00/17
Hofstenia miamia 3 Belize
1.00/20+ Hofstenia giselae 3 Belize
1.00/20+ Hofstenia sp 1 Spain
Hofstenia sp 2 Spain
1.00 /20+ Myopea sp “callaeum”
Oligofilomorpha interstitiophilum
1.00/20+ Paratomella unichaeta
Paratomella rubra
Mus musculus
Meara stichopi
lected from four runs of 6 million MCMC generations
using the GTR+I+G model in MrBayes. Bremer support
(italic values above branches) for the corresponding most
parsimonious tree(s) (2 trees, 2,961 steps, CI = 0.55,
RI = 0.78) were calculated using TNT
Hydrobiologia (2007) 592:439–454 449

1.00/2 Hofstenia giselae 1 Bahamas

0.54/0 Hofstenia giselae 2 Bahamas
Hofstenia miamia 1 Bermuda
1.00/20+
0.97/1 Hofstenia giselae 3 Belize
0.99/1 Hofstenia miamia 3 Belize
1.00/20+
Hofstenia miamia 2 Panama
1.00/20+ 1.00/20+ Hofstenia sp 1 Spain
Hofstenia sp 2 Spain
Oligofilomorpha interstitiophilum
1.00/10 Childia macroposthium
1.00/6 Childia brachyposthium
Childia vivipara
0.92/--
0.83/1 Childia trianguliferum
0.99/--1.00/-- Childia cycloposthium
Childia submaculatum
1.00/5
Childia crassum
1.00/20 Childia groenlandica
Childia groenlandica Spain
Paratomella rubra
0.1

Fig. 6 Phylogenetic analyses of nuclear ribosomal large
subunit 28S rDNA sequences indicate that Hofstenia is
closely related to Solenofilomorphidae. Topology, branch-
lengths and bayesian posterior probabilities (bold values
above branches) were estimated from 32,000 trees col-
lected from four runs of 8 million MCMC generations
using the GTR + I + G model in MrBayes. Bremer
support (italic values above branches) for the correspond-
ing most parsimonious tree(s) (1 tree, 1,498 steps, CI
= 0.79, RI = 0.90) were calculated using TNT

1.00/2 Hofstenia giselae 1 Bahamas
0.96/2 Hofstenia giselae 2 Bahamas
Hofstenia miamia 1 Bermuda
1.00 /20+
1.00/3 Hofstenia giselae 3 Belize
1.00/3 Hofstenia miamia 3 Belize
Hofstenia miamia 2 Panama
Hofstenia sp 1 Spain
Hofstenia sp 2 Spain
0.01
Fig. 7 Phylogenetic analyses of concatenated ribosomal
data (nuclear small and large subunit 18S/28S rDNA) fail
to recover separate H. miamia and H. giselae clades.
Topology, branch-lengths and bayesian posterior proba-
bilities (bold values above branches) were estimated from
120,000 trees collected from four runs of 20 million
MCMC generations using the GTR + G model in
MrBayes. Bremer support (italic values above branches)
for the corresponding most parsimonious tree(s) (1 tree,
229 steps, CI = 0.98, RI = 0.97) were calculated using
TNT. The majority rule consensus tree resulting from this
analysis was rooted using the two Hofstenia specimens
from Galicia as outgroups
parsimony informative sites in the 18S and 28 S
sequences within the H. miamia / H. giselae clade
(Table 4). For instance, only 11 sites in the 18S
sequence are actually variable among the samples
and only 7 of those are parsimony informative;
they seem to have changed character states only
once inside the clade (CI = 1.0). In the longer
28S sequence, only 14 sites are parsimony infor-
mative out of 45 variable sites.
The average patristic distance or sequence
divergence between a member of one geographic
clade and a member of the other is low; only
0.0089 for 18S. Since analysis of that gene did not
recover the monophyletic ‘‘Caribbean’’ clade
when analyzing all data at once, its monophyly
was enforced as a topological constraint. The
corresponding average distance for 28S is 0.0098.
These distances translate into an expected
number of substitutions between the members
of different clades of approximately 15 in a
1,750-nucleotide long stretch of 18S and 27 in a
3,000-nucleotide long stretch of 28S. The molec-
ular divergence within the H. miamia/H. giselae
clade is thus lower than or about equal to that in
the Hofstenia sp. clade (18S: 0.0144; 28S: 0.0077),
higher than or about equal to that within the
Childia groenlandica clade (18S: 0.0030; 28S:
0.0099), lower than that within the Proporus
carolinensis clade (18S: 0.026212) and lower than
that between the two most closely related but
distinctly classified Childia species, Childia mac-
roposthium and Childia brachyposthium (18S:
0.0117; 28S: 0.0139).

123
450 Hydrobiologia (2007) 592:439–454

Discussion

generation; avg SD
Stationarity (from

<0.002
<0.005
<0.003
<0.005
<0.005
<0.001
of split freq.)
Distinguishing Hofstenia species

million;
million;
million;
million;
million;
million;
In his monograph of the Hofsteniidae, Steinböck
(1966) detailed the morphological differences

3
5
5
4
3
5
Bayesian inference

among species of all 6 species of the Hofsteniidae

he recognized, namely four Hofstenia species and
(generations)

10 million two species in monotypic genera, Hofsteniola

20 million

20 million
6 million

8 million
6 million
MCMC

pardii Papi, 1957, and Marcusiola tinga (Marcus,

1957). In his key to Hofstenia species, Steinböck
(1966: 137) distinguishes H. miamia and H. beltagi
from H. atroviridis and H. giselae by a propor-
Bremer support
(trees MPT+20

tionally shorter antrum and smaller body size, and

H. beltagi specifically by its antrum having a wider
distal part opening in a section perpendicular to
steps)

26406

7156

the ventral body surface. He distinguished

561

112
80
–

H. atroviridis from H. giselae by shape of the

penis bulb and pouch.
0.97 (0.96; 0.91)b
0.97(0.97; 0.91)b

Contrary to the findings of Steinböck (1966),

0.98 (1.0; 1.0)b
0.98 (1.0; 1.0)b

our investigation of the histological sections of

Hofstenia species revealed the morphology of the
Maximum parsimony inference
Consistency

copulatory organs to be quite similar (Fig. 4,

0.78

0.90
(CI; RI)

Table 2). We suggest that minor differences in the

0.55;
0.97;
0.97;
0.79;
0.97;
0.98;

length or shape of muscular structures such as the

penis pouch and male antrum are more likely due
to differences in contraction during fixation,
(trees; steps)

rather than true diagnostic differences. Most

specimens we examined had similar body length,
MPT(s)a

Topology constraint enforce according to the 18S + 28S topology

Table 4 Technical details for each phylogenetic analysis performed

2; 2961

1; 1489
1; 76
1; 77

2; 152
1; 229

pharynx length, stylet length, and stylet number

Number of steps only including parsimony informative sites

(Fig. 4, Table 2). Although the H. miamia spec-

imen from Curaçao is proportionally smaller with
regard to these features (Fig 4B), the holotype of
89; 73 (11; 7)b
89; 73 (11; 7)b

262; 144 (45; 14)b

351; 217 (56; 21)b
1080; 871

1473; 813

H. miamia was reported by Corrêa (1960) to be

Variable sites

informative)
(variable; of
which pars.

more similar at 4 mm long and with penis stylets

70 lm in length. We are, however, perplexed that
Inside the H. miamia + H. giselae clade

the H. miamia holotype was also reported to have

50 penis stylets (Corrêa, 1960), far more than any
of the specimens we examined (Table 2), even
Dimensions

those prepared by Corrêa (1963).

(terminals;

1988
1773
1773
3445
2994
4767
Dataset

The lack of any available type material for

sites)

H. beltagii precludes us from verifying the validity

8;
8;

8;
8;
29;

19;

of this species. The diagnostic characters Stein-

böck (1966) provides – namely, in the shape of the
18S constrainedc

male antrum and canal – seem rather weak, since

agonistic contraction upon fixation could easily
cause such shape differences. He, himself, cau-
18S+28S
Gene

tions that the material is poorly preserved and

18S
18S

28S
28S

comprises just two sets of sections. In all other

b
a

123
Hydrobiologia (2007) 592:439–454
characters that Steinböck (1966) mentions (struc-
ture of the nervous system and pharynx, arrange-
ment of ovaries and testes) H. beltagii is like
H. miamia and H. giselae, and its color is
described as mottled brown, also perhaps like
that of H. miamia. Lacking type specimens and
adequately documented distinguishing features,
we declare H. beltagi to be a species inquirenda.
As for H. atroviridis, although its internal
morphology seems to match closely that of other
Hofstenia species, there are other characteristics
that seem to secure its position as a distinct
species. H. atroviridis can be superficially distin-
guished from other species by its dark blackish-
green coloration and by its lack of a tail (although
perhaps this feature was simply overlooked by
Bock, 1923). More important is the habitat
preference of H. atroviridis, which is distinct from
that of the Caribbean species. Bock (1923)
collected H. atroviridis from wave-washed inter-
tidal and subtidal sites where the oxygen content
of the water is frequently replenished. Bock
(1923) found that specimens kept in dishes in
the laboratory were often attached to the water
surface and only lived a short time; he hypothe-
sized that their deaths were due to a lack of an
adequate supply of oxygen. By contrast, H. mi-
amia and H. giselae are found in quiet subtidal
water in rather stagnant conditions among dead
and decaying vegetable material. We were able to
keep specimens of H. miamia alive in Petri dishes
for as long as a month, doing little to maintain
them other than occasionally changing their water
and feeding them specimens of the acoel Isodi-
ametra pulchra (Smith & Bush, 1991).
Synonomization of Hofstenia giselae under
Hofstenia miamia
In his description of H. giselae, Steinböck (1966)
reported many differences with H. miamia, stat-
ing that H. giselae was longer, wider, taller, had a
distinct tail that was lacking in H. miamia and was
without the distinct ‘‘head’’ of H. miamia. In
addition, Steinböck also mentioned differences in
mouth position, pharynx length, and characters of
the male copulatory organ. The findings of
Steinböck (1966) may have been askew in part
because his information on H. miamia came
451
solely from the descriptions of Corrêa (1960,
1963) and not from his own personal examination
of the type material. Our investigation of the
morphology of H. giselae and H. miamia revealed
virtually no difference between these two species
with regard to body size, shape, the position of
the mouth, or the morphology of the copulatory
organs (Fig. 4, Table 2).
Our molecular analysis also does not support
H. miamia and H. giselae being classified as
separate species. Specifically, the molecular phy-
logeny reconstructed here has a topology that is
incongruent with the traditional classification and
instead suggests a geographic partitioning of
lineages. Although such patterns should perhaps
be expected in benthic fauna with little known
capability of long distance pelagic dispersal, it has
not been demonstrated in acoels before.
A comparison of patristic distances in non-
Hofstenia acoel clades do not yield consistent
results regarding within-species or between-spe-
cies variation. However, the molecular sequence
data available for such studies in acoels are too
limited to bring any clear insight into these
matters at this point. Molecular divergence in
the H. miamia + H. giselae clade is very low,
about 1% in the two ribosomal genes, and a large
proportion of the variation is not parsimony
informative. We question whether this low vari-
ation and degree of molecular synapomorphies
approach that which might be randomly gener-
ated when base calling and assembling chroma-
tograms with some low level of ambiguity. Not all
chromatograms were perfect in our case, but it is
unlikely that a minor number of errors would
create such a phylogenetic bias that the two genes
independently recover very similar phylogenies,
assuming that chromatogram errors are randomly
distributed to begin with. Such errors would most
likely simply inflate the proportion of variable but
uninformative sites and we might therefore actu-
ally overestimate the molecular divergence in this
group, which in a way further supports the finding
of H. miamia and H. giselae not being separate
species. If an unknown biological mechanism is
indeed responsible for generating elevated evo-
lutionary rates in acoel sequences, its effect seems
to go unnoticed in this sample of geographically
separated Hofstenia specimens.
123
452
The phylogeny recovered within the H. miamia
+ H. giselae clade displays a curious geographic
pattern, but is based on very little informative
data and may be subject to change if more data is
added. We suggest a posteriori that further studies
of very closely related lineages of acoels focus on
more quickly evolving markers such as mitochon-
drial genes or the ITS-region, as done in many
other taxa.
Given the failure of our morphological and
molecular analyses to support H. miamia and
H. giselae being classified as separate species, we
herein designate H. giselae as a junior synonym of
H. miamia.
Systematic position of the Hofsteniidae
Although not widespread, species possessing
pharynges simplices do occur in several acoel
families, including Convolutidae (Oligochoerus),
Diopisthoporidae, Hallangiidae, Hofsteniidae,
Isodiametridae (Isodiametra helgolandica), Nadi-
nidae, Proporidae, and Solenofilomorphidae.
Morphological studies suggest that pharynges
were derived independently in multiple lineages
within the Acoela (Riedl, 1954; Doe, 1981), and
molecular sequence data supports this conclusion
(Hooge et al., 2002). Our finding that 18S and 28S
sequence data supports a close relationship
between Hofsteniidae and Solenofilomorphidae
may indicate that the pharynges in these groups
have a common origin. The male copulatory
organ of hofsteniids is distinctly different from
that of other acoels, including solenofilomorphids,
and does not appear to provide characters useful
for elucidating the phylogenetic position of the
Hofsteniidae within the Acoela. However, we
expect that on-going studies on body-wall mus-
culature, as well as sperm and pharynx ultrastruc-
ture may be helpful in this regard.
A full account of synonyms for the family
Hofsteniidae is provided in Electronic supple-
mentary material Appendix 1.
Patterns of body coloration
The color patterns of H. miamia are unusual in
part because most acoels do not have opaque
coloration, but also because intraspecific variation
123
Hydrobiologia (2007) 592:439–454
of body color is uncommon in the Acoela.
Intraspecific color variation comparable to that
of Hofstenia occurs in the Polycladida and also in
the Nudibranchia [e.g., Roboastra gracilis (Bergh,
1877)]. The polyclad genera Pseudoceros and
Pseudobiceros can be identified by their color
patterns, but in several species the details of the
coloration are inconstant among individuals (e.g.,
Pseudoceros dimidiatus von Graff, 1893; P. gratus
Kato, 1937; P. scintillates Newman & Cannon,
1994; P. zebra Leuckart, 1828). In Pseudoceros
dimidiatus, the body always has a black back-
ground with an orange margin and two yellow
longitudinal stripes; however, the thickness and
spacing of these longitudinal stripes are not fixed,
and some specimens may have additional trans-
verse yellow stripes that can be quite variable
(Newman & Cannon, 1994, 2003).
In the case of Pseudoceros dimidiatus, the
bright yellow and black coloration is thought to
be a classic example of aposematic coloration that
warns potential predators of the worms’ toxicity.
Polyclads also use mimicry to deter predators.
Some palatable species use Batesian mimicry to
match the coloration of noxious nudibranchs,
while some unpalatable species have coloration
closely matching other unpalatable organisms, so
called Müllerian mimicry (Newman & Cannon
2003). The contrasting white and brown banding
pattern in H. miamia may be a case of aposematic
coloration or a case of mimicry of the similarly
colored nudibranch Navanx aenigmaticus (Bergh,
1893), an unpalatable species that is sometimes
found in Thallasia sp. beds in the Caribbean
(Gundersen, 2003). Not all individuals are dis-
tinctly banded, however, and we have no exper-
imental data to show that H. miamia is
unpalatable to fish or other potential predators.
More than likely, the coloration of H. miamia
is cryptic for its particular habitat. From our
mangrove samples, which consisted of brown
leaves spotted with white patches of sulfur bac-
teria and the sessile ciliate Zoothamnium niveum
Ehrenberg, 1838, we found that several of the
associated small invertebrates had similar brown
and white banding, including a small amphipod
that appeared superficially quite similar to H.
miamia, particularly if viewed from the rostral
apex. If Hofstenia coloration is adapted to their
Hydrobiologia (2007) 592:439–454
particular habitat, this could explain the distinct
color differences we found between specimens
collected from mangrove leaves and Thalassia
beds.
True to its name, H. atroviridis is dark black-
green in color and relatively conspicuous among
the coralline algae in the tidepools from which it
was collected (Bock, 1923: 3, 4). Hofsteniola
pardii is reddish to pink in color, the most heavily
pigmented around the pharynx and paler caudally
(Papi, 1957: 133). The last hofsteniid, Marcusiola
tinga, is colorless (Marcus, 1957: 170).
Future studies
Our investigation shows Hofstenia miamia to be a
widespread and abundant species in the Carib-
bean, and we foresee its use in future ecological
and experimental investigations. That it has
apparently escaped any mention in studies of
Caribbean ecology, particularly the diversity of
mangrove assemblages, is surprising, especially
given its voracious appetite for other small
invertebrates. Its relatively large size compared
to that of other acoel species, along with its
apparent basal position in the Acoela, its ability
to regenerate (Steinböck, 1966, 1967), and its
potential for being easily cultured in the labora-
tory, make H. miamia a suitable candidate for use
in experimental biology, or perhaps even as a
model organism of basal bilaterians.
Acknowledgements We are grateful to Klaus Rützler for
the opportunity to work at Carrie Bow Cay and we thank
Wolfgang Sterrer and Jörg Ott for their help in collecting
specimens in Belize. We thank Rachel Collin of the
Smithsonian Tropical Research Institute (STRI) for
inviting us to be a part of the taxonomic survey at Bocas
del Toro, Panama and for providing financial assistance
and logistical support. We also extend our gratitude to
Wolfgang Sterrer of the Bermuda Natural History
Museum for his invaluable help in the collection of
specimens in Bermuda and to the highly altruistic staff of
the Perry Institute for Marine Science in Lee Stocking
Island, Bahamas. Type material was generously made
available to us by the Swedish Museum of Natural History
and the Naturhistorisches Museum Wien. This is
contribution no. 797 of the Caribbean Coral Reef
Ecosystems Program, National Museum of Natural
History, Smithsonian Institution, and contribution no.
125 of the Bermuda Biodiversity Project (BBP),
Bermuda Aquarium, Natural History Museum and Zoo.
This material is based upon work supported by the
453
National Science Foundation under grant no. 0118804
(MH, ST) and the Swedish Research Council (UJ).
Wallberg was kindly funded by Inez Johanssons stiftelse.
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