Invertebrate Biology 127(2): 121–138.
r 2008, The Authors
Journal compilation r 2008, The American Microscopical Society, Inc.
DOI: 10.1111/j.1744-7410.2007.00119.x
Cotylea (Polycladida): a cladistic analysis of morphology
Kate A. Rawlinson and Marian K. Litvaitisa
Department of Zoology and Center for Marine Biology, University of New Hampshire,
Durham, New Hampshire 03824, USA
Abstract. Polyclad flatworms are acoelomate bilaterians found in benthic communities
worldwide, predominantly in marine environments. Current polyclad systematics is unstable, with two non-concordant classification schemes resulting in a poor understanding of
within-group relationships. Here we present the first phylogenetic framework for the suborder Cotylea using a morphological matrix. Representatives of 34 genera distributed among all
cotylean families (except four, excluded due to their dubious taxonomic status) were investigated. The number of families included ranges from a conservative eight to a revisionary 11.
Outgroup analysis indicated that the suborder is monophyletic and defined by the presence of
a ventral adhesive structure, a short posteriorly positioned vagina, and cement glands. Of the
eight to 11 families included, we confirmed that three were monophyletic: Boniniidae, Prosthiostomidae, and Pseudocerotidae. Boniniidae was consistently recovered as the sister group
to other Cotylea, based on the retention of the plesiomorphic Lang’s vesicle. The clade consisting of Anonymus, Marcusia, and Pericelis is sister to the Boniniidae and the rest of the
Cotylea. Above this clade there is little resolution at the base of the sister group. The
Euryleptidae are found to be paraphyletic and give rise to the Pseudocerotidae. Neither classification scheme received unequivocal support. The intrafamilial relationships of the diverse
Pseudocerotidae and Euryleptidae were examined. Color pattern characters (used for species
identification) were highly homoplasious but increased cladogram resolution within genera.
The monophyly of seven genera within the Pseudocerotidae and Euryleptidae was not supported and many genera showed no autapomorphies, highlighting the need for taxonomic
revision of these families.
Additional key words: phylogeny, character evolution, Pseudocerotidae, Euryleptidae
The Cotylea are free-living flatworms belonging to
one of two suborders of the Polycladida (Platyhelminthes). Within marine environments, their distribution is global, but they are most prominent,
colorful, and diverse on tropical reefs. Although relatively rare, they may play an important ecological
role as predators of sessile benthic organisms. Cotyleans are free living, unlike some acotylean species
that live in association with echinoderm or molluscan
hosts (e.g., Wheeler 1894; Doignon et al. 2003). However, the biology and ecology of this suborder, and of
polyclads in general, have received very little attention.
Polyclad flatworms are morphologically quite homogeneous with taxonomic characters relying on de-
a
Author for correspondence.
E-mail: [email protected]
tails of the reproductive system and patterns of eyes,
both showing plasticity during maturation. As a result, two non-concordant systematic schemes are in
use (Faubel 1983, 1984; Prudhoe 1985). Still, both
schemes recognize two suborders based on the presence (Cotylea) or the absence (Acotylea) of a ventral
adhesive structure. Taxonomic descriptions of cotyleans began in 1815 (Montagu 1815) with the description of Planaria vittata MONTAGU 1815, which is
recognized today as Prostheceraeus vittatus
MONTAGU 1815 LANG 1884. Risso (1818) extended
the known distribution of cotyleans by describing
two species of Thysanozoon from the Mediterranean; however, he placed them erroneously into the
molluscan genus Tergipes LANG 1884. To date,
B400 cotyleans have been described worldwide.
These species have been grouped within ten to 15
families according to the type and position of the
pharynx, the presence of tentacles, the presence and
122
form of the prostatic vesicle, and the direction of
male apparatus and the uterine canals.
Faubel’s (1984) classification, which includes many
monotypic families, suggested non-monophyly in the
Cotylea. As a result, he erected four superfamilies,
two of which are monospecific (Ophisthogeniidae,
now considered dubious, and Ditremageniidae, repositioned in the Tricladida where Palombi (1928) had
initially placed them; A. Faubel, pers. comm.) and
two, Pseudocerotoidea and Euryleptoidea, which are
species rich. The superfamily Pseudocerotoidea was
based on the synapomorphous presence of pseudotentacles (tentacles being marginal folds) and the following general characteristics: (1) pair of ventral eyes
anterior to the brain, (2) plicate ruffled pharynx extending perpendicularly into the pharyngeal cavity,
(3) mouth opening in varying positions in the midventral line of the body, (4) Lang’s vesicle lacking,
and (5) uterine vesicles present. Euryleptoidea was
united by the synapomorphy plicate cylindrical pharynx, plus a true, free, prostatic vesicle and male copulatory apparatus, in the anterior body half.
Faubel (1984) presented a dendrogram of the hypothesized relationships of his polyclad superfamilies, complete with suggested character evolution.
However, these relationships have yet to be tested
by phylogenetic analysis. Prudhoe’s (1985) classification is more conservative, with ten families described
within a monophyletic Cotylea. Character evolution
was speculated upon but no attempt at assessment of
evolutionary relationships was made.
Within the two most species-rich families, Pseudocerotidae and Euryleptidae, generic classifications are
based on the male and female reproductive systems
(number of gonopores, presence of uterine vesicles),
the alimentary system (presence of anal pores and
intestinal vesicles), and external characters (dorsal
papillae, tentacles, and tentacular eyes). Species identification is generally reliant on color pattern (e.g.,
Hyman 1954, 1955a,b, 1959a,b; Prudhoe 1989; Newman & Cannon 1994, 2000).
Despite centuries of alpha taxonomy, the phylogenetic relationships of polyclads in general, and cotyleans in particular, have received little attention; this
changed when recent species- and generic-level assessments were made using molecules (Litvaitis &
Newman 2001) and morphology (Doignon et al.
2003). To explore relationships within the suborder,
we have developed a morphological data set for
Cotylea. Using morphological and anatomical data
already available from the literature, augmented by
direct observations in recently collected specimens,
the monophyly of Cotylea was tested; an attempt was
also made to reconstruct the ground pattern and to
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Rawlinson & Litvaitis
examine in-group relationships. We also formulated
hypotheses about evolutionary character transformations within the suborder. Furthermore, within-family relationships were examined for the species-rich
Pseudocerotidae and Euryleptidae.
Our overall intent was not to reorder cotylean systematics at this point, but to examine the validity of
the included families and genera using cladistic methodology, and to propose future directions for inquiry
that might lead to better systematic and phylogenetic
resolution.
Methods
Specimens examined
Polyclad descriptions have been based on morphological and anatomical characters as seen at the light
microscope level; as yet, very little work has been
carried out using electron microscopy. Characters
were obtained from the literature, direct observation
of recently collected specimens, and photographs of
live animals, and were coded either as binary or multistate. The data set for Cotylea included 51 characters and 75 species from all families, except
Ditremagenidae, Opisthogeniidae, Dicteroidae, and
Stylochoididae. Whenever possible, multitypic higher-level taxa (family and genus) were represented by
more than one exemplar species, to incorporate within-group diversity. Two non-cotylean outgroup taxa
were included in the present analysis: Notoplana queruca MARCUS & MARCUS (1968) and Pleioplana atomata MULLER 1776. These taxa were chosen because
they show characteristic acotylean traits (lack of
sucker and marginal tentacles, and long forwardlooping vagina) and we had access to freshly collected specimens.
Characters were coded from five sources: gross
morphology, sensory organs, alimentary system, reproductive system, and color patterns. A full list of
characters with sources, coding, and explanations is
given in Appendix 1.
Phylogenetic analysis
Maximum parsimony analysis was carried out using the heuristic search option (100 random replicates, tree bisection-reconnection branch-swapping
algorithm with a collapsing zero-branch length option) of PAUP version 4.0b10 (Swofford 2002).
Multistate characters were treated as unordered and
all characters were weighted equally. Clade support
was estimated by 1000 ‘‘full heuristic’’ bootstrap
replicates in PAUP and the calculation of Bremer
Cladistic analysis of cotylean polyclads
support values (Bremer 1994) using TREEROT
(Sorenson 1996). Variation in levels of homoplasy
between character suites was tested using MacClade
4.06 (Maddison & Maddison 2003) to calculate
the RC for each character and to average them across
all most parsimonious cladograms (MPCs). A Kruskal–Wallis test was performed on arcsine-transformed data to determine whether the mean RC
was significantly different among the five character
suites.
MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003)
was used for Bayesian phylogenetic inference, and
run under the DATATYPE 5 standard option that
activates the M2 model in MrBayes. The M2 model
(Huelsenbeck & Ronquist 2001) was used as implemented in MrBayes version 3.0b4, and likelihood was
corrected for the scoring bias (only parsimony-informative characters were scored). All parameters were
associated with diffuse priors. The data set was run
for 2,000,000 generations. A tree was sampled every
200 generations, resulting in 10,000 trees.
Chain stationarity was achieved after 200,000 generations (burn-in) and 1000 trees were subsequently
discarded. To calculate the posterior probability of
the analysis, we constructed a 50% majority-rule
consensus tree from these remaining trees, and the
percentage of times a clade occurred among this sampling of trees was interpreted as its posterior probability. Three independently repeated analyses
resulted in similar tree topologies, and comparable
clade probabilities and substitution model parameters, suggesting that reasonable estimates of the posterior probability distributions were obtained.
Hypothesis testing
Constraint analyses were performed to test whether the phylogenetic signal within the data set was
compatible with existing classifications. Tree topologies reflecting competing classifications were
used as backbones to find equally most-parsimonious solutions; trees were saved and solutions were
determined for each competing hypothesis by comparing saved trees against unconstrained solutions.
Non-parametric Templeton tests were used as implemented in PAUP to test for statistical difference
(Larson 1994).
Character evolution
The most parsimonious explanation for the histories of morphological characters was inferred and visualized using MacClade under default settings. To
account for both phylogenetic and mapping uncer-
123
tainty in character evolution, Bayesian methods were
used to infer ancestral states at ancestral nodes of
supported monophyletic clades. Each node was constrained in turn and the Bayesian analysis was run in
MrBayes according to the settings described above.
A Kruskal–Wallis test was performed on the probability of each character state over the 10,000 trees to
determine whether the mean was significantly different among the states of each character.
Results
Phylogeny and hypothesis tests
This first inference of cotylean relationships included 75 taxa and 51 characters (46 parsimony-informative characters: 29 binary and 22 multistate).
An initial maximum parsimony heuristic search using
equal weights for each character found 42000 equally MPCs (length 5 174, CI 5 0.49, RI 5 0.85). The
strict consensus (Fig. 1) represents a preliminary
phylogenetic hypothesis of family and genera relationships within Cotylea. For Bayesian analysis, a
burn-in of 200,000 generations was found to be sufficient, and posterior probabilities of clade support
were plotted on Fig. 1. There was no difference in
tree topology between parsimony and Bayesian analyses.
From these analyses, monophyly of Cotylea was
supported (with bootstrap values only). Within the
in-group, Boniniidae and Prosthiostomidae formed
well-supported monophyletic families (98% and 82%
bootstrap support, respectively), with Boniniidae positioned as a sister group to the other Cotylea, and
Prosthiostomidae being more derived, positioned
among a paraphyletic Euryleptidae. The Pseudocerotidae form a supported derived clade (55% bootstrap support). A clade consisting of all cotyleans
except the Boniniidae was supported (53% bootstrap
support). The sister relationship of Pericelis cata
MARCUS & MARCUS 1968 and Marcusia ernesti
HYMAN 1953 was supported (93% bootstrap support)
but is controversial and will be discussed later. All
other family-level relationships were unresolved.
Only three generic-level classifications were bootstrap supported: Maritigrella (59%), Cycloporus
(61%), and Pseudoceros (54%).
Support for families (according to both classifications) and genera are given in Table 1. Non-monophyly was found in the following taxa: the
superfamilies Pseudocerotoidea and Euryleptoidea,
the families Euryleptidae (sensu Faubel), Amyellidae,
Chromoplanidae, Anonymidae, and the genera
Prostheceraeus, Acerotisa, Stylostomum, Eurylepta,
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Rawlinson & Litvaitis
Cladistic analysis of cotylean polyclads
125
Table 1. Genus assignment to cotylean family groupings according to two classifications, and support for family- and
genus-level relationships from the strict consensus of MPCs (—, not tested; MPCs, most parsimonious cladograms).
Classification
Genus
Phrikoceros
Maiazoon
Pseudobiceros
Thysanozoon
Nymphozoon
Pseudoceros
Acanthozoon
Tytthosoceros
Bulaceros
Yungia
Anonymus
Faubel (1984)
Prudhoe (1985)
Superfamily
Family
Family
Pseudocerotoidea
(paraphyletic)
Pseudocerotidae
(monophyletic)
Pseudocerotidae
(monophyletic)
Anonymidae
(paraphyletic)
Anonymidae
Pericelidae
(synonymous)
Marcusia
Pericelidae
Diposthidae
(monophyletic)
Pericelis
Diposthus
Asthenoceros
Chromoplana
Chromoplanidae
Chromoplanidae
(unresolved)
Amyellidae
(unresolved)
Amyella
Chromyella
Boninia
Paraboninia
Maritigrella
Cycloporus
Prostheceraeus
Eurylepta
Euryleptodes
Oligocladus
Oligoclado
Acerotisa
Stylostomum
Euryleptides
Laidlawia
Euprosthiostomum
Lurymare
Enchiridium
Diposthidae
(monophyletic)
Euryleptoidea
(paraphyletic)
Unresolved
Monotypic
Paraphyletic
Monophyletic
Monotypic
Monophyletic
—
Unresolved
—
—
—
Monotypic
—
—
Monotypic
Monotypic
Monotypic
Boniniidae
(monophyletic)
Boniniidae
(monophyletic)
Euryleptidae
(paraphyletic)
Euryleptidae
(polyphyletic)
Euryleptididae
Laidlawidae
Prosthiostomidae
(monophyletic)
Prosthiostomidae
(monophyletic)
Tytthosoceros, Phrikoceros, and Pseudobiceros. Topological constraints assessed with the non-parametric Templeton’s test rejected null hypotheses for the
taxonomic groups Pseudocerotoidea, Euryleptoidea,
Genus-level
relationships
Monotypic
—
Monotypic
Monophyletic
Monophyletic
Paraphyletic
Polyphyletic
Monotypic
—
Monotypic
Paraphyletic
Paraphyletic
Monotypic
—
—
—
—
Euryleptidae, and Prostheceraeus (Table 2), but
found no significant difference in tree lengths between constrained and unconstrained topologies for
the remaining taxa.
Fig. 1. Strict consensus of all most parsimonious cladograms (42000) (tree length 5 174, CI 5 0.49, RI 5 0.94). The left
score above the branch is the MP bootstrap support value (%), the right score above the branch is the Bremer support
value, and the score below the branch is the Bayesian posterior probability (%). The bars to the right of the species names
indicate the four major families and two superfamilies of the Cotylea.
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126
Rawlinson & Litvaitis
Table 2. Levels of homoplasy within character suites calculated by median rescaled consistency index (RCI). Kruskal–Wallis test revealed significant differences (H 5 16.43,
po0.05) in homoplasy between character suites.
Character suite
Gross morphology
Sensory organs
Alimentary system
Reproductive system
Color patterns
Cotylea
# of characters
Median RCI
6
7
8
18
11
0.77
0.50
0.59
0.40
0.03
There were significant differences in the levels of
homoplasy among the five character suites, with
gross morphology being the most phylogenetically
informative and color patterns the least (Table 3).
Character evolution
Parsimony analysis suggested that there were 40
uniquely derived characters and character states, 19
of which were synapomorphies (Fig. 2). At the base
of the tree, three synapomorphies distinguish the
Table 3. Topological constraints assessed with the nonparametric Templeton’s test. Additional steps are counted
between the best tree overall and the best tree constrained
to be consistent with the hypothesized topology. A po0.05
indicates a significant difference in the number of steps and
leads to a rejection of the hypothesized constraint.
Constraint (monophyly)
Additional steps
p
S.F. Pseudocerotoidea
S.F. Euryleptoidea
F. Euryleptidae (Faubel)
F. Amyellidae
F. Chromoplanidae
F. Anonymidae
G. Eurylepta
G. Prostheceraeus
G. Acerotisa
G. Stylostomum
G. Tytthosoceros
G. Phrikoceros
G. Pseudobiceros
98
73
22
0
1
1
2
29
1
2
2
2
1
o.01
o.01
o.01
4.05
4.05
4.05
4.05
.01
4.05
4.05
4.05
4.05
4.05
Cotylea from the acotylean outgroup: (a) the presence of an adhesive structure on the ventral surface
(Ch. 1: 0-1), (b) a short vagina directed posteriorly
(Ch. 31: 0-1), and (c) the presence of well-defined
cement glands (Ch. 36: 0-1). Plesiomorphic features
of the cotyleans that may constitute the ground pattern for the order include the following ancestral
states: (a) gross morphology: adhesive disc on ventral surface absent, oval body shape, smooth dorsal
surface; (b) sensory organs: no tentacles, marginal
and tentacular eyes absent, cerebral eyes in two clusters; (c) digestive system: simply folded ruffled pharynx in middle to posterior third of body with
anastomosing gut; (d) reproductive system: one
male and one female pore, male apparatus anterior
to male pore, and directed caudal. Male pore is in
posterior half of body, with stylet and interpolated
prostatic vesicle, one prostatic vesicle, no prostatoids,
long anteriorly looping vagina, Lang’s vesicle absent,
uteri absent, uterine canals extending anteriorly from
vagina, and a sac-like cement gland chamber; and (e)
color pattern: even color and translucent dorsal
surface.
Within the Cotylea, parsimony analysis suggests a
true sucker (Fig. 3A,C,D) evolved once in the sister
group to the Boniniidae (which have an adhesive
disc, Fig. 3B).
Marginal tentacles are diverse (Fig. 4) in form and
arose five times, and were lost at least once. Pharynx
form and position has evolved from the plesiomorphic ruffled and centrally positioned pharynx (Fig.
5A), to tubular/cylindrical in the Prosthiostomidae
(Fig. 5B) and Euryleptidae, and to ruffled and anteriorly positioned in the Pseudocerotidae to complexly
folded in the Pseudoceros1Acanthozoon clade.
Only four taxonomic groups were supported by all
three support methods (bootstrap, Bremer, and posterior probabilities) and by synapomorphies: Boniniidae, Prosthiostomidae, Pseudocerotidae, and
Pseudoceros. Ancestral states for the ancestral nodes
of these taxa were reconstructed using Bayesian inference and are shown in Table 4.
Discussion
Phylogeny of the Cotylea
This is the first inference of phylogeny for the suborder Cotylea. It uses existing morphological data and
reveals that neither of the current classification
Fig. 2. Phylogram depicting uniquely derived morphological characters mapped onto the strict consensus of the MPCs.
Note that the families Amyellidae and Chromoplanidae, plus the genera Phrikoceros, Tytthosoceros, and Stylostomum,
were constrained into monophyletic groups ().
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Cladistic analysis of cotylean polyclads
127
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Rawlinson & Litvaitis
Fig. 3. Cotylean
suckers.
A.
Ventral
view
of Pseudobiceros
pardalis showing two male (2
mp) and one female (fp) gonopores,
and
true sucker (su).
Scale bar, 5 mm.
B–D. Sagittal sections
through
ventral adhesive
structures. Scale
bar, 250 mm. B.
Adhesive disc of
Boninia
divae.
C. True sucker
of
Enchirdium
periommatum. D.
True
sucker
of Phrikoceros
mopsus.
schemes is unequivocally supported. Monophyly of
the Cotylea is supported by bootstrap analysis only.
The position of the root indicated that the Boniniidae
are sister to all other cotyleans. The clade consisting
of Anonymus, Marcusia, and Pericelis is sister to the
Boniniidae and the rest of the Cotylea. Above this
clade, there is little resolution at the base of the sister
group. The Euryleptidae is found to be paraphyletic
and gives rise to the Pseudocerotidae (Fig. 1; Table
2).
Interfamilial relationships
Prudhoe (1985) offered no hypotheses on relationships among cotylean families. Faubel (1984), however, erected two superfamilies distinguished from
one another by a ruffled or a cylindrical pharynx,
and other characters (see ‘‘Introduction’’). However,
our data suggest that taxa with ruffled pharynges
form a polyphyletic group, and those with cylindrical/tubular pharynges are paraphyletic; as such, neither superfamily is supported here as a natural taxon
(Fig. 1; Table 2).
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Boniniidae. Boniniidae is a morphologically distinct family consisting of five species from three genera. Our analysis clearly supported monophyly and
resolved Boniniidae as the most basal lineage. Some
morphological and reproductive characters are
shared with the acotyleans, such as the presence of
Lang’s vesicle and an adhesive disc (similar to that
found in cestoplanids; Prudhoe 1985). Other characters, such as a short, posteriorly directed vagina and
marginal tentacles, are found only in cotylean species. The two valid autapomorphies for the family are
the presence of an adhesive disc and fine tentacles on
lateral margins. Field observations of polyclads in
situ will facilitate the description of further
autapomorphies in the form of ecological and behavioral characters, such as mode of locomotion and
stress response. Boninia divae MARCUS & MARCUS
1968, for example, has been observed to secrete a
purple substance when stressed, possibly representing
a defense response common to the family.
Prosthiostomidae. Prosthiostomidae is similarly
delineated by both classification schemes, and
is a strongly supported monophyletic family in our
Cladistic analysis of cotylean polyclads
129
Fig. 4. Variations in cotylean marginal tentacles. A. Eurylepta sp., showing well-developed, pointed tentacles.
B. Pseudobiceros pardalis, with pseudotentacles. C. Boninia divae, showing fine, lateral tentacles on either side of the
head. D. Cycloporus sp., with marginal bumps. Scale bar, 0.5 mm.
analysis, defined by the synapomorphic presence
of two prostatic vesicles per male complex. Withinfamily relationships were not explored.
Chromoplanidae and Amyellidae. Chromoplanidae
sensu Prudhoe, and Amyellidae sensu Faubel, are unresolved, species-poor, and relatively unknown taxa.
As such, their taxonomic status remains dubious.
Diposthidae. There is a weakly supported sister
taxa relationship between the genera Diposthus and
Asthenoceros. This grouping is consistent with the
family designation Diposthidae of both classification
schemes (Faubel 1984; Prudhoe 1985). However,
there are no synapomorphies supporting the clade.
Anonymidae and Pericelidae. The Pericelis1Marcusia and Anonymous clade was characterized by
male copulatory apparatus enclosed in a massive
bulb (Ch. 38: 0-1). The sister taxa relationship of
Pericelis cata and Marcusia ernesti was well support-
ed (93% bootstrap support). An autapomorphy of
Marcusia was a common genital atrium and gonopore, although this may be an artifact of preservation
(Faubel 1984). According to Prudhoe (1985), Pericelidae is a monogeneric family, with Pericelis synonymous with Marcusia, therefore accepting the
common gonopore as an artifact. He also states
that Pericelis has no distinct prostatic organ, but
that the proximal region of the ejaculatory duct is
lined with granular gland cells when the male phase
of the worm is fully active. In contrast, Faubel (1984)
accepts Marcusia as its own genus and places it in the
family Anonymidae, with Anonymus and Simpliciplana (not included here), all three taxa lacking prostatic vesicles or glands. He places Pericelis in its own
family and considers it to have an interpolated prostatic vesicle. However, the original description of P.
cata (Marcus & Marcus 1968) shows no indication of
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Rawlinson & Litvaitis
Fig. 5. Cotylean body shapes
and pharynges. A. Ventral
view of oval-shaped Pericelis cata, showing ruffled
pharynx (ph) and uteri (u).
B. Dorsal view of elongated
Prosthiostomum sp. showing
tubular pharynx (ph). Scale
bar, 2 mm.
a prostatic vesicle. This is also the case in other species of the genus (e.g., Pericelis hymanae POULTER
1974). We have therefore coded prostatic vesicle as
being absent in Pericelis in our analysis. Anonymidae
sensu Faubel is paraphyletic; this same family sensu
Prudhoe is monogeneric with three well-defined
autapomorphies all related to its multiple male pores
and their positioning (Fig. 2).
Euryleptidae. Euryleptidae is a large heterogeneous
group. Faubel (1984) created individual families for
the genera Laidlawia (Laidlawidae) and Euryleptides
(Euryleptididae), which, according to our analysis,
are early divergent members of the Euryleptidae
1Prosthiostomidae complex or at least share a common ancestry with it. Euryleptididae is distinguished
from Euryleptidae by the presence of prostatoid
organs and uterine vesicles. Laidlawidae is supported by the autapomorphic presence of a genitointestinal duct.
Euryleptidae sensu Prudhoe is polyphyletic as it
includes the genera Euryleptides and Laidlawia. The
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subfamilies Euryleptinae and Laidlawiinae, erected
by Hallez (1913), were maintained by Prudhoe
(1985), with Euryleptides grouped in Euryleptinae
together with Cycloporus, Oligoclado, Eurylepta,
Prosthecereaus, and Stylostomum. This subfamily is
also polyphyletic. The monophyly of the Laidlawiinae was not tested here, as information on the
other genera Leptoteredra and Enterogonimus was
not sufficient. The polyphyletic nature of the
Euryleptidae and Euryleptinae sensu Prudhoe is
supported here (Fig. 1, Table 2). Further evidence
from ultrastructural and molecular characters will
need to confirm this polyphyly.
Pseudocerotidae. The validity of the family Pseudocerotidae has now been confirmed using morphology and nucleotide sequence data (Litvaitis &
Newman 2001). In this analysis, the Pseudocerotidae
form a monophyletic-derived clade supported by the
presence of pseudotentacles (Ch. 7: 2-1). Within
this species-rich family, there is considerable variation in pseudotentacle form, eye arrangement,
0:99
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47
46
45
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1:99
1:99
0:99
0:99
1:99
1:99
1:99
0:99
0:89
0:99
0:99
0:99
1:99
1:99
33
32
31
30
29
28
27
26
25
3:57
0:99
1:99
1:99
39
38
36
34
35
Character
Taxon
Boniniidae
Prosthiostomidae
Pseudocerotidae
Pseudoceros
41
40
1:99
0:99
1:80
2:99
1:99
0:99
1:94
1:99
2:96
2:99
2:65
1:99
0:99
0:99
3:62
2:99
0:99
0:99
4:45
3:99
0:99
0:99
1:99
1:99
0:55
2:99
0:99
0:99
0:99
0:99
0:36
1:99
3:99
0:99
1:99
1:99
0:99
0:99
0:99
0:99
0:96
0:99
1:99
1:99
1:96
0:99
0:99
0:99
0:98
1:99
1:99
1:99
0:95
1:99
1:99
1:99
1:99
1:99
1:99
1:99
Boniniidae
Prosthiostomidae
Pseudocerotidae
Pseudoceros
0:99
0:99
0:99
0:99
43
42
44
1:99
0:99
1:99
1:99
1:99
0:99
0:99
0:99
3:99
2:99
3:97
3:99
0:99
0:99
0:99
0:99
51
0:99
0:99
0:99
0:99
0:99
1:99
0:99
0:99
0:99
0:99
0:97
0:99
0:99
0:99
0:99
0:99
0:99
0:99
0:99
0:99
23
22
21
20
19
18
17
16
15
14
13
12
11
1
2
3
4
5
6
7
8
9
10
Character
Taxon
Table 4. Inferred ancestral state (1–4): its probability (%), at well-supported ancestral nodes inferred from Bayesian analysis. The posterior probability of
these character states being present at the nodes was significantly greater (95% confidence level) than that for other character states. Uninformative characters
(#24, 37, 48, 49, and 50) have been excluded.
Cladistic analysis of cotylean polyclads
131
number of male and female gonopores, and color
pattern. The reconstructed ancestral character states
(Table 4) suggest that the ancestral pseudocerotid
had a smooth dorsal surface, pseudotentacular eyes
scattered between the tentacles on the dorsal surface
and in two dense clumps on the ventral surface, cerebral eyes in two clusters, and a simply folded ruffled
pharynx. It also possessed one female and one male
gonopore, had a penis armed with a stylet, paired uteri, lacked anal pores, and was cryptically colored
probably with a mottled background and marginal
bands.
Intrafamilial relationships
Within the Pseudocerotidae there were two derived
clades, but little resolution among the remaining
taxa. One clade consisted of Acanthozoon and Pseudoceros, genera supported by a complexly folded
pharynx and ventral pseudotentacular eyes in two
loose clusters. Within this clade, Pseudoceros formed
a well-supported taxon with two synapomorphies:
pseudotentacles as simple folds in the anterior margin (Ch. 8: 5-1) and dorsal pseudotentacular eyes in
two to three lines (Ch. 11: 2-3).
The second clade consisted of Pseudobiceros,
Maiazoon, Nymphozoon, and Thysanozoon, supported
by the presence of two male pores (Ch. 23: 0-1) and
four dense clusters of ventral pseudotentacular eyes
(Ch. 12: 3-4). Only two genera were monophyletic,
Pseudoceros and Thysanozoon, although constraint
analysis of Pseudobiceros, Phrikoceros, and Tytthosoceros resulted in trees that were not significantly
longer (Table 3). The species-rich Pseudobiceros is
paraphyletic in this analysis, and there is variation in
pseudotentacle form (square vs. earlike), color pattern, and function. Analysis of pseudocerotid
relationships using the D3 expansion segment of the
28S rDNA gene also highlighted the heterogeneous
nature of Pseudobiceros (Litvaitis & Newman
2001:fig. 1) although, again, a constrained analysis
was not significantly different. Therefore, it is suggested that this recently differentiated genus (Faubel
1984) be the subject of further investigation. The
monophyly of Bulaceros, Yungia, and Acanthozoon
was not tested.
Based on morphology, the Euryleptidae is suggested to be an unnatural taxon and there are few morphological synapomorphies supporting intrafamilial
relationships. Maritigrella species form a sister clade
to the more derived species belonging to a clade consisting of Acerotisa, Stylostomum, Prosthecereaus,
Cycloporus, Eurylepta, Oligocladus, and Oligoclado,
Invertebrate Biology
vol. 127, no. 2, spring 2008
132
of which only Cycloporus is considered to be monophyletic.
According to this analysis, the newly erected genus
Maritigrella (Newman & Cannon 2000) is well
supported but has no synapomorphies. Acerotisa
and Stylostomum are supported as sister taxa, and
monophyly of these genera is possible (Table 3). The
monophyly of Stylostomum is supported by a combined mouth and male gonopore (Fig. 2) and is consistent with Faubel (1984). However, Prudhoe (1985)
suggested synonymy of Stylostomum and Acerotisa
due to a thin fold seen to separate the gonopore and
mouth, but this is considered to be a preservation artifact (Holleman 2001).
Two unnatural genera have been highlighted:
Eurylepta, with considerable variation in all morphological character suites, and Prostheceraeus, with
many shared similarities with Oligoclado. Although
constraint analysis of Eurylepta did not lead to significantly longer trees, there are no synapomorphies
uniting this genus. Indeed, Eurylepta multicelis
HYMAN 1955 is a supported sister species to Cycloporus due to the shared presence of intestinal vesicles.
The exclusion of Oligoclado from the Prostheceraeus
clade leads to significantly longer trees. These genera
are in need of taxonomic revision using new data sets.
Evolution of morphology
The primary goal of this study was to reconstruct
the phylogeny of the Cotylea. However, early inferences into the evolution of some characters are discussed below. In this analysis, gross morphological
characters were most phylogenetically informative
(Table 2). In general, polyclad taxonomy has relied
on three gross morphological characters: pharynx
shape, sucker presence, and tentacular presence and
form. The distribution of these characters and their
states can be visualized on this preliminary hypothesis of cotylean relationships.
Although most polyclads and cotyleans have a
ruffled pharynx, some have tubular- or cylindricalshaped pharynges. Cotyleans with a cylindrically
shaped pharynx have traditionally been considered
to be a natural group (Faubel 1984; Prudhoe 1985).
Our data indicate that these taxa do not form a natural taxon (Fig. 1, Table 3), and that the cylindricalpharynx morphology has evolved once and been subsequently lost in a derived clade. More information
on musculature and ultrastructure of pharynx types
is needed to assess whether ruffled pharynges at either end of the trees are homologous, and indeed
whether the cylindrical pharynx is homogeneous in
structure among the taxa in the middle of the tree.
Invertebrate Biology
vol. 127, no. 2, spring 2008
Rawlinson & Litvaitis
The true sucker is a diagnostic character for cotyleans, and the most parsimonious explanation for the
history of its origin is that it arose once in the sister
clade to the Boniniidae. Despite the superficial resemblance of the Boniniidae adhesive disc to other
cotylean ‘‘true suckers,’’ Prudhoe (1985) maintains
that they are structurally different and that similar
adhesive structures are found in the acotylean family
Cestoplanidae. Again, further histological and ultrastructural information will help elucidate homology
between these structures. Marginal tentacular form is
diverse within the Cotylea with five morphologies
(Ch. 7). Two forms (states 4 and 5) appear to have
arisen independently several times, and tentacles are
suggested to have been secondarily lost within some
members of the Euryleptidae complex (some Acerotisa and Stylostomum species).
Whether or not the character to be mapped should
be included in the phylogenetic analysis is an old argument and one that has plagued comparative biologists (de Queiroz 2000). Including characters of
interest in the analysis is often seen as logically problematic. One solution is to exclude the character of
interest if it affects tree topology; a second solution is
to reconstruct the tree based on an independent data
set, most often a molecular one. However, due to the
relatively weak support for the Cotylea in this study,
we will wait until a phylogeny for the order has been
proposed before carrying out a more comprehensive
evaluation of polyclad character transformations.
Phylogenetic signal and characters
Polyclads are extremely delicate and tend to autolyze during fixation. However, a novel fixation technique (Newman & Cannon 1995) and observations of
live animals have greatly increased our ability to examine morphological details and color patterns of
polyclads. Unfortunately, specimens in museum collections are poorly preserved, and the majority of illustrations and descriptions in the taxonomic
literature are often incomplete and superficial. This
has led to a paucity of reliable morphological characters, as fixation has led to artifacts found in all
character suites from gross morphology (e.g., marginal ruffling), to sensory organs (marginal tentacle
shape and marginal eye distribution, Prudhoe 1985),
to reproductive systems (e.g., common gonopores).
In spite of this, our study has produced some informative and interesting results that could not at present be addressed using molecular techniques, due to
the lack of suitably preserved material.
Within the cotyleans and in particular Pseudocerotidae and Euryleptidae, color patterns are relied
Cladistic analysis of cotylean polyclads
upon heavily for species-level taxonomy because of
their relative morphological homogeneity. However,
the utility of color features for phylogenetic reconstruction is generally avoided because of concerns
over their plasticity. The color pattern characters included in our analysis have low RCI values (Table 2),
suggesting that coloration does not provide a strong
source of phylogenetic signal for generic relationships. As expected, removing these color pattern
characters resulted in trees that were less well resolved within the genera.
The biological role of the color pattern has been
shown to influence the phylogenetic signal of color
characters (Areekul & Quicke 2006), with those asso-
133
ciated with mimicry and aposematism being more evolutionary, and therefore phylogenetically, constrained,
often resulting in conflict with the morphological signal. Therefore, the use of such color patterns in phylogenetic construction is cautioned against. Without
more ecological data on polyclads, elucidating the
role of color pattern is difficult for some species.
Our analyses provide a working framework for
future systematic investigations, and afford an opportunity to understand character distribution and
transformation series within the suborder. Better hypotheses of character evolution will be achieved once
phylogenetic reconstruction of the order has been
completed. Of particular interest will be the taxa
Table 5. Matrix of 51 characters and 77 species (including two outgroup taxa) used in the analysis of cotylean relationships. Character numbers and character states correspond to the list of characters in Appendix 1. ?, unknown.
Character
Taxon
Phrikoceros fritillus
Phrikoceros baibaiye
Phrikoceros galaticus
Phrikoceros mopsus
Maiazoon orsaki
Pseudobiceros pardalis
Pseudobiceros flowersi
Pseudobiceros hymanae
Pseudobiceros mikros
Pseudobiceros sp. 1
Pseudobiceros damawan
Pseudobiceros kryptos
Pseudobiceros murinus
Pseudobiceros uniarborensis
Pseudobiceros brogani
Thysanozoon flavotuberculatum
Thysanozoon californicum
Thysanozoon sp.1
Pseudoceros bicolor
Pseudoceros depiliktabub
Pseudoceros heronensis
Pseudoceros paralaticlavus
Pseudoceros bolool
Pseudoceros imperatus
Pseudoceros sapphirinus
Nymphozoon bayeri
Acanthozoon albopapillosum
Tytthosoceros inca
Tytthosoceros nocturnus
Tytthosoceros lizardensis
Bulaceros porcellanus
Yungia saskii
Boninia divae
Paraboninia caymanensis
1
1
2
3
4
5
0
0
0
0
0
111010130123111031000000110110101001000000000010000
111010160123111031000000110110101001000000000010000
111010160123111031000000110110101001000000000010000
111010130123111031000000110110101001000000000010000
111010130124311031000011210110101001000100001000000
111010150124111031000010210110101001000100000000100
111010130124111031000010210110101001000100001000000
111010150124111031000010210110101001000100010000000
111010150124111031000010210110101001000000000010000
111010150124111031000010210110101001000000000000000
111010130124111031000010210110101001000000000010000
111010130124111031000010210110101001000000000010000
111010130124111031000010210110101001000000000010000
111010130124111031000010210110101001000100010000000
111010150124111031000010210110101001000100001000000
111011150124111031000010210110102001000001000000000
111011150124111031000010210110102001000000000010000
111011150124111031000010210110102001000001000000000
111010110132112031000000110110101001000100000010000
111010110132112031000000110110101001000100001000000
111010110132112031000000110110101001000000000010000
111010110132112031000000110110101001000100001000000
111010110132112031000000110110101001000100000000000
111010110132112031000000110110101001000100000000000
111010110132112031000000110110101001000100010000000
11?010150????11031000012311110102001000100001000000
111011150122112031000000?10110102001000000000010000
111010150143111031000000110110101001000000000001000
111010150143111031000000110110101001000001000000000
111010150143111031000000110110101001000000000010000
111010140143211031000000110110101001000000000010000
11101012011?211031001000111110101001000000000010000
100100302000211131000000301001111111000001000000000
100100300000411131000000001001111111000001000000000
Invertebrate Biology
vol. 127, no. 2, spring 2008
134
Table 5.
Rawlinson & Litvaitis
(cont’d.)
Taxon
Character
1
Maritigrella crozieri
Maritigrella eschara
Maritigrella stellata
Maritigrella ocellata
Cycloporus gabriellae
Cycloporus atratus
Cycloporus harlequin
Cycloporus venetus
Acerotisa alba
Acerotisa arctica
Acerotisa californica
Acerotisa bituna
Acerotisa leuca
Oligoclado floridanus
Prostheceraeus bellostriatus
Prostheceraeus boucheti
Prostheceraeus floridanus
Prostheceraeus flavomaculatus
Stylostomum spanis
Stylostomum sanjuania
Stylostomum lentum
Eurylepta tuma
Eurylepta rugosa
Eurylepta aurantiaca
Eurylepta multicelis
Euryleptodes insularis
Oligocladus sanguinolentus
Oligoclado floridanus
Lurymare utarum
Enchiridium periommatum
Euprosthiostomum mortenseni
Diposthus popeae
Asthenoceros woodworthi
Pericelis cata
Marcusia ernesti
Anonymus multivirilis
Amyella lineata
Chromyella saga
Chromoplana bella
Laidlawia polygenia
Euryleptides brasiliensis
Notoplana queruca
Pleioplana atomata
1
2
3
4
5
0
0
0
0
0
111010201000200001000000110110100001000000000000001
111010201000200001000000110110100001000000000000001
111010201000200001000000110110100001000000000000010
111010201000200001000000110110100001000000000000000
111010500200200001110000110110101021000100000010000
111010500200200001110000110110101021000000000010000
111010500200200001110000110110101021000100000010000
111010500200200001110000110110101021000100001000000
111010000200200000100000110110101011000000000000000
111010000200200000100000110110101011000001000000000
111010000200200001100000110110101011000001000000000
111010000200200000100000110110101011000000000100000
111010000200200000100000110110101011000001000000000
111010201100300021000000010110101001000100001000000
111010201100200001000000110110101021000000000000000
111010201100200001000000110110101021000100001000000
111010201100200001000000110110101021000000000010000
111010201100200001000000110110101021000100000000000
111010000200200000100100110110101011000010000000000
111010000200200000100100110110101001000001000000000
111010500200200000100100110110101011000000000100000
111010201100200010000000510110101011000000100000000
111012500200200001000000110110101011000001000000000
111010200100200001000000110110101011000010000100000
111010500200200001010000110110101011000000000100000
1110100000002000010000001101101030?1000?00000000000
111010201200200000101000110110101011000000000000000
111010201100300021000000010110101001000100001000000
111000002000200001000000110120101001000?00100000000
111000002000200001000000110120101001000000000000000
111000002000200001000000010120101001000000000000000
112010400100211131000000301110100011000001000000000
111010000000011131000000101?1010001?000?0?000000000
112010403100211131000000001000101111010000000100000
112010403100211131000200001000101101010?00000100000
111010003000211131000420321000101001010001000000000
11101000200021113000000010100010000?000000100000000
11101000200021113100030010100010000?000000100000000
1110100020002110300000001?1210100001002000100000000
111010502000200110000000101110100001100?0?000000000
111010002000200011000000100111101011000?0?000000000
000010000000211131000000000210010100001011000000000
000010000100211130000000000210010100001001000000000
that form a link between the cotyleans and acotyleans. Many character sources have yet to be explored, such as trends within the nervous and
musculature systems, ultrastructural information of
reproductive systems, and molecular and embryological data, all of which are necessary to produce a fully
resolved phylogeny of the Polycladida. It is hoped
Invertebrate Biology
vol. 127, no. 2, spring 2008
that this study will provide the foundation and impetus for future studies of relationships within the
Polycladida.
Acknowledgments. We thank Sigmer Quiroga, Marcela
Bolaños, and Marcin Liana for help with collecting live
Cladistic analysis of cotylean polyclads
specimens and with character coding, and for providing
data from museum collections. We also thank two
anonymous referees for helpful suggestions. This work
was supported by NSF grant DEB-0412932, and is
Scientific Contribution No. 2318 of the New Hampshire
Agricultural Experiment Station.
References
Areekul B & Quicke DLJ 2006. The use of colour characters in phylogenetic reconstruction. Biol. J. Linn. Soc.
88: 193–202.
Bremer K 1994. Branch support and tree stability. Cladistics 10: 29–304.
Cannon LRG 1986. Turbellaria of the World: A Guide to
Families and Genera. Queensland Museum, Brisbane,
Australia. 136 pp.
Doignon G, Artois T, & Deheyn D 2003. Discoplana malagasensis sp. nov, a new turbellarian (Platyhelminthes:
Polycladida: Leptoplanidae) symbiotic in an ophiuroid
(Echinodermata), with a cladistic analysis of the Discoplana/Euplana species. Zool. Sci. 20: 357–369.
Faubel A 1983. The Polycladida, Turbellaria proposal and
establishment of a new system. Part I the Acotylea. Mitt.
Hambg. Zool. Mus. Inst. Band 80: 17–121.
FFF 1984. The Polycladida, Turbellaria proposal and
establishment of a new system. Part II the Cotylea. Mitt.
Hambg. Zool. Mus. Inst. Band 81: 189–259.
Hallez MP 1913. Polylcades et Triclades maricoles. Deuxième expedition antarctique fran@aise (1908–1910). Masson et Cie (eds) Paris. 69 pp.
Holleman JJ 1998. Two new species of the genus Anonymus
from New Zealand (Polycladida, Cotylea). Hydrobiologia 383: 61–67.
FFF 2001. A review of the genus Stylostomum Lang,
1884 (Platyhelminthes, Polycladida) and the description
of a new species. Belg. J. Zool. 131: 227–229.
Huelsenbeck JP & Ronquist R 2001. MrBayes: Bayesian
inference of phylogenetic trees. Bioinformatics 17: 754–
755.
Hyman LH 1954. The polyclad genus Pseudoceros, with
special reference to the Indo-Pacific region. Pacific Sci. 8:
331–336.
FFF 1955a. Some polyclad flatworms from the West
Indies and Florida. Proc. US Natl. Mus. 104: 115–150.
FFF 1955b. Some polyclad flatworms from Polynesia
and Micronesia. Proc. US Natl. Mus. 105: 65–82.
FFF 1959a. A further study of Micronesian polyclad
flatworms. Proc. US Natl. Mus. 108: 543–597.
FFF 1959b. Some Australian polyclads. Rec. Australian
Mus. 25: 1–17.
Lang A 1884. Die Polycladen (Seeplanarien) des Golfes
von Neapel und der angrenzenden Meeresabschnitte,
Eine Monographie, Fauna Flora Golfes v Neapel, Leipzig.
Larson A 1994. The comparison of morphological and
molecular data in phylogenetic systematics. In: Molecu-
135
lar Ecology and Evolution: Approaches and Applications. Schierwater B, Streit B, Wagner GB, & DeSalle R,
eds., pp. 317–390. Birkhäuser Verlag, Basel.
Litvaitis MK & Newman LJ 2001. A molecular framework for the phylogeny of the Pseudocerotidae
(Platyhelminthes, Polycladida). Hydrobiologia 444:
177–182.
Maddison WP & Maddison DR 2003. MacClade Version
4.06. Sinauer Associates, Sunderland, MA.
Marcus E & Marcus E 1968. Polycladida from Cura@ao
and faunistically related regions. Stud. Fauna Cura@ao
other Caribb. Is. 26: 1–133.
Michiels NK & Newman LJ 1998. Sex and violence in hermaphrodites. Nature 391: 647.
Montagu G 1815. Description of several new or rare animals, principally marine, found on the South coast of
Devonshire. Trans. Linn. Soc. London 11: 25–26.
Newman LJ & Cannon LRG 1994. Pseudoceros and Pseudobiceros (Platyhelminthes, Polycladida, Pseudocerotidae) from eastern Australia and Papua New Guinea.
Mem. Queensl. Mus. 37: 205–266.
FFF 1995. The importance of the fixation of colour,
pattern and form in tropical Pseudocerotidae
(Platyhelminthes, Polycladida). Hydrobiologia 305:
141–143.
FFF 1996. New genera of pseudocerotid flatworms
(Platyhelminthes; Polycladida) from Australia and Papua New Guinean coral reefs. J. Nat. Hist. 30: 1425–1441.
FFF 2000. A new genus of euryleptid flatworm (Platyhelminthes, Polycladida) from the Indo-Pacific. J. Nat.
Hist. 34: 191–205.
Palombi A 1928. Report on the Turbellaria (Cambridge
Expedition Suez Canal 1924). Trans. Zool. Soc. London
22: 579–631.
Poulter JL 1974. A new species of the genus Pericelis, a
polyclad flatworm from Hawaii. In: Biology of the Turbellaria. Riser NW & Morse MP, eds., pp. 93–107. McGraw-Hill Book Company, New York.
Prudhoe S 1977. Some polyclad turbellarians new to the
fauna of the Australian coasts. Rec. Aus. Mus. 31: 586–
604.
FFF 1985. A Monograph on Polyclad Turbellaria. Oxford University Press, London, p. 259.
FFF 1989. Polyclad turbellarians recorded from African
waters. Bull. Br. Mus. Nat. Hist. (Zool.) 55: 47–96.
de Queiroz K 2001. Logical problems associated with including and excluding characters during tree reconstruction and their implications for the study of
morphological character evolution. In: Phylogenetic
Analysis of Morphological Data. Wiens JJ, ed., pp.
192–212. Smithsonian Institution Press, Washington,
DC.
Risso A 1818. Sur quelques gastropodes nouveaux, nudibranches et testibranches observes dans la mer de Nice.
J. Phys. Chim. Hist. Nat. 87: 368–376.
Ronquist F & Huelsenbeck JP 2003. Mr Bayes3: Bayesian
phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
Invertebrate Biology
vol. 127, no. 2, spring 2008
136
Rawlinson & Litvaitis
Sorenson MD 1996. TREEROT. University of Michigan,
Ann Arbor.
Swofford DL 2002. PAUP, Phylogenetic Analysis Using
Parsimony ( and Other Methods) Version 4. Sinauer
Associates, Sunderland, MA.
Wheeler WM 1894. Planocera inquilina, a polyclad inhabiting the branchial chamber of Sycotypus canaliculatus
Gill. J. Morphol. 9: 195–201.
Morphological character definitions used to reconstruct phylogenetic familial-level relationships within
Cotylea.
from the parenchyma. This structure lies very close to
the posterior end of the body in boniniid polyclads.
5. Body shape: (0) elongate; (1) oval.
The unsegmented, bilaterally symmetrical, dorsoventrally flattened body of a polyclad varies in shape
from broadly oval to elongate or ribbonlike. The two
body forms coded here are discrete among the cotylean species, with little overlap.
6. Dorsal surface: (0) smooth; (1) papillated.
In the cotylean genera Thysanozoon and Acanthozoon, the dorsal surface is raised into many papillae,
of undetermined function. This is considered to be
the apomorphic state.
Gross morphology
Sensory organs
Although there are few external features of polyclads, the most important in terms of classification is
the organ of attachment or sucker. There are three
forms of sucker: (a) the true sucker, (b) the adhesive
disc, and (c) the genital sucker (Prudhoe 1985); only
the first two are represented in cotyleans. A presence
or absence of a sucker on the ventral surface is used
to distinguish between suborders. The acotyleans
generally lack a sucker, whereas the cotyleans possess a sucker at varying positions along the median
line. There are, however, exceptions to this rule; six
cotylean species do not exhibit a sucker (although
descriptions were based on damaged specimens, these
species are not included in the analysis) and two
acotylean species, Leptoplana tremellaris MULLER
1774 and Itannia ornata MARCUS 1947, show genital
suckers. The adhesive disc is found in the boniniid
(cotyleans) and some cestoplanid (acotyleans) polyclads.
1. Adhesive structure on the ventral surface: (0)
absent; (1) present.
2. True sucker: (0) absent; (1) present.
This is known from many cotyleans and lies more
or less in the middle of the ventral surface. It consists
of a modified epithelium covering a basement membrane and a muscular lamella differentiated from the
parenchyma, originating from the longitudinal muscles of the body wall and from the dorsoventral muscles serving as retractor muscles.
3. True sucker position: (0) sucker absent; (1) middle third of body; (2) posterior end of body.
Most true suckers are positioned in the middle region of the ventral surface; however, in Pericelidae,
the sucker is positioned posteriorly, due to a centrally
positioned pharynx.
4. Adhesive disc: (0) absent; (1) present.
The adhesive disc is a shallow depression constructed similarly to a sucker but not differentiated
Sensory organs in polyclads have been used extensively for identification at all taxonomic levels. They
include tentacles, and eyes or photoreceptors. There
are three different areas where eyes are located: marginally, on the tentacles, and in the cerebral region.
Although eyes are systematically important, the
number and arrangement can change ontogenetically (Prudhoe 1977). Therefore, wherever possible,
phylogenetic characters have been coded from mature individuals.
7. Marginal tentacle form: (0) absent; (1) as folds in
anterior margin (pseudotentacles); (2) prominent,
pointed, well developed on anterior margin; (3) fine
tentacles on either side of head, lateral margin; (4)
widespread marginal folds with a ‘‘v’’ shape in between, on anterior margin; and (5) small marginal
bumps.
The presence of marginal tentacles is an apomorphy of Cotylea. Marginal tentacle form is generally a
character that distinguishes the cotylean families.
However, this character is polymorphic in Euryleptidae.
8. Pseudotentacle form: (0) absent; (1) simple folds;
(2) deep folds; (3) square form; (4) distal knobs; (5)
earlike; (6) deep lateral ruffles.
9. Marginal eyes: (0) absent; (1) present, anterior
body only; (2) present, around entire margin.
It is suggested that the presence of eyes on the
margins of the body is a primitive feature among
polyclads, having arisen from a radiate ancestor
(Prudhoe 1985).
10. Tentacular eyes: (0) absent; (1) present; (2) at
site of missing tentacles.
The arrangement of tentacular eyes is particularly
important in species recognition of pseudocerotids.
11. Dorsal pseudotentacular eyes: (0) absent; (1)
two clusters; (2) four clusters; (3) scattered lines; (4)
scattered between pseudotentacles.
Appendix 1
Invertebrate Biology
vol. 127, no. 2, spring 2008
Cladistic analysis of cotylean polyclads
12. Ventral pseudotentacular eyes: (0) absent; (1)
scattered; (2) two loose clusters; (3) two dense clusters; (4) four dense clusters.
13. Cerebral eyes: (0) absent; (1) single cluster; (2)
two clusters; (3) horse-shoe shape; (4) inverted ‘‘v’’;
(5) frontal, scattered in cerebral area.
Digestive system
A plicate pharynx and intestinal ramifications, often anastomosing, are synapomorphous characters
of Polycladida. Within the order, pharyngeal form
was one of the characteristics used by Faubel (1984)
to establish monophyletic superfamilies.
14. Pharynx: (0) tubular; (1) ruffled.
The tubular or cylindrical form of the pharynx is
found in the cotylean families Euryleptidae and Prosthiostomidae (also Laidlawiidae and Euryleptididae),
and may be derived.
15. Pharynx ruffled: (0) absent; (1) present, simple
pharyngeal folds; (2) present, complex pharyngeal
folds.
The ruffled pharynx is found in all other cotylean
families, with the pharynx of species belonging to the
genera Pseudoceros and Acanthozoon, having more
complexly folded lobes (Newman & Cannon 1994).
16. Position of pharynx: (0) anterior third of body;
(1) middle or posterior third of body.
A pharynx positioned centrally or posteriorly is an
acotylean feature; however, the boniniids and pericelids share this character state. All other cotyleans
have the pharynx positioned anteriorly.
17. Mouth position: (0) immediately posterior to
cerebral organ; (1) same level as the brain; (2) anterior to cerebral; (3) located centrally in the pharynx.
18. Intestine: (0) numerous radiating branches; (1)
anastomosing.
The intestinal branches ramify repeatedly toward
the periphery of the body, where they usually terminate blindly. In most cotylean forms, the intestinal
branches anastomose, to form an intricate network
of canals.
19. Anterior branch of intestinal trunk: (0) absent;
(1) present, over pharynx.
20. Intestinal vesicles: (0) absent; (1) present.
21. Anal pores: (0) absent; (1) present.
Reproductive system
Very little is known about the reproductive behavior of polyclads (Michiels & Newman 1998); yet it is
well established that the anatomy of the reproductive
system is of major significance in the classification of
flatworms (Cannon 1986).
137
22. Gonopore position: (0) male anterior to female;
(1) male pore and mouth combined; (2) common gonopore; (3) male and female gonopore and mouth
combined; and (4) peripheral male pores, medial female pore.
The male pore is almost invariably anterior to the
female pore; an exception is within the monotypic
cotylean family Opishtogeniidae (this taxon has been
excluded from the analysis as its anatomy is poorly
described). A combined mouth and male pore is an
autapomorphy of the euryleptid genus Stylostomum
(sensu Holleman 2001). Among the cotyleans, a common gonopore is an autapomorphy for Marcusia ernesti, although this may be an artifact of fixation. It
has been suggested that Marcusia is synonymous
with Pericelis (Prudhoe 1985), where the gonopores
are proximate but separate. A combined oral, male,
and female aperture is apomorphic for the genus
Chromyella, although its validity is uncertain. Peripheral male pores are apomorphic for the genus Anonymus.
23. Number of male pores: (0) 1; (2) 2; (3) multiple.
It is assumed that a single male pore is the plesiomorphic condition among cotyleans as well as polyclads, with multiple pores evolving independently in
diverse taxa. Two pores are most common among
pseudocerotid genera. Multiple pores are an apomorphy of the family Anonymidae sensu Prudhoe (1985).
24. Number of female pores: (0) 1; (2) 3–5; (2) 8.
A single female pore is the common form for Cotylea, and multiple pores are found in two pseudocerotid genera.
25. Orientation of male copulatory apparatus: (0)
positioned anterior to male pore and directed backwards; (1) positioned posterior to male pore and directed forwards; (2) positioned laterally to male pore,
directed medially; (3) directed perpendicular to long
axis of body; (4) posterior to pore directed backwards; and (5) anterior to pore directed forwards.
26. Position of male copulatory apparatus: (0) in
posterior half of body (1) in anterior half of body or
central; (2) in longitudinal rows on either side of the
pharynx; and (3) anterior to pharynx.
Descriptions of the sexual behavior of polyclads
are rare; however, from recent observations it is
hypothesized that the position of the male apparatus determines whether a taxon raises its head or
tail to copulate or transfer sperm. Among the outgroup and other Acotylea, the apparatus is posteriorly situated; this is also the case for the boniniids
and pericelids. Anonymus multivirilis HOLLEMAN 1998
and the family Anonymidae (sensu Prudhoe) have
multiple male pores in rows on either side of the
pharynx.
Invertebrate Biology
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138
27. Copulatory organ: (0) penis papilla with stylet;
(1) penis papilla without stylet.
The majority of cotylean species representing the
three most diverse families have penis papillae armed
with sclerotized stylets. Boniniid and pericelid polyclads lack a stylet.
28. Prostatic vesicle: (0) absent; (1) present, free;
(2) present, interpolated.
The prostatic tissue, its presence, its form, and its
distribution are useful taxonomic characters. However, their relative importance in classification is contentious. Faubel (1983, 1984) places great reliance on
the nature of the prostate tissue, especially in relation
to the lining of the prostatic vesicle. Prudhoe (1985),
on the other hand, reports that these characters are
unreliable as they are capable of change during the
development and maturation of some species.
Among most of the cotylean families, the prostatic
vesicle is free. Interpolated prostatic vesicles are
found in Chromoplanidae (sensu Faubel) and in the
outgroup.
29. Number of prostatic vesicles per male complex:
(0) 0; (1) 1; (2) 2.
The number of prostatic vesicles varies between
cotylean families; Boniniidae (and Anonymidae and
Amyellidae sensu Faubel) lack vesicles. Euryleptidae,
and Pseudocerotidae possess one vesicle, and Prosthiostomidae have two.
30. Prostatoid organs: (0) absent; (1) present.
Prostatoid organs are glands of prostatoid character entering the walls of the male atrium, they are
thought to be plesiomorphic (Faubel 1983), and are
found in Boniniidae.
31. Vagina: (0) long, looping toward male complex; (1) short, directed posterior.
A short posteriorly directed vagina is a synapomorphy of Cotylea.
32. Lang’s vesicle: (0) absent; (1) present.
It is probable that Lang’s vesicle is a seminal receptacle, and is found in the outgroup and Boniniidae only.
33. Uterus: (0) absent; (1) paired; (2) as median sac.
The presence and form of the uterus is variable
within cotylean families.
34. Uterine canals: (0) extending posteriorly from
vagina; (1) extending anteriorly from vagina.
The extension of the uterine canals is a function of
the position of the sexual apparatus. In the outgroup, Boniniidae, and Pericelidae, the gonopores
are positioned posteriorly and therefore the uterine
canals can only extend anteriorly.
Invertebrate Biology
vol. 127, no. 2, spring 2008
Rawlinson & Litvaitis
35. Uterine vesicles: (0) absent; (1) present.
Uterine vesicles are common to Boniniidae,
Pericelidae, and some Euryleptidae.
36. Cement chamber: (0) tubelike; (1) saclike.
The cement gland or mucous gland chamber is
considered to be a synapomorphy of Cotylea; the
lack of this gland is an autapomorphy of the genus
Chromyella. Amyellidae sensu Faubel is characterized by the lack of a cement chamber. However, this
character is dubious due to the protandrous nature of
the species and the stage of development of the individuals examined.
37. Ductus genitointestinalis: (0) absent; (1) present.
This connection between the female copulatory
apparatus and the intestine is an autapomorphy for
Laidlawia polygenia PALOMBI 1938. Although an uninformative character in the initial analysis, it was
included as an example of a character that may show
a phylogenetic signal if more taxa are included in
further analyses.
38. Male apparatus enclosed in a massive bulb: (0)
no; (1) yes.
This is a character common to Pericelis, Marcusia,
and Anonymus.
39. Connection between ejaculatory duct and prostatic vesicle: (0) absent or free prostatic vesicle; (1)
with projection; (2) without projection.
These structural differences of the interpolated
prostatic vesicle separate the outgroup from Chromoplana.
Color patterns
40. Color pattern function: (0) cryptic; (1) aposematic.
41. Translucent dorsal surface: (0) no; (1) yes.
42. Even color: (0) no; (1) yes.
43. Even color longitudinal stripe: (0) no; (1) yes.
44. Even color and marginal bands: (0) no; (1) yes.
45. Even color, with longitudinal stripes and marginal bands: (0) no; (1) yes.
46. Mottled background: (0) no; (1) yes.
47. Mottled background with marginal bands: (0)
no; (1) yes.
48. Mottled background with marginal bands and
longitudinal stripes: (0) no; (1) yes.
49. Spotted: (0) no; (1) yes.
50. Star pattern, with marginal band and longitudinal stripe: (0) no; (1) yes.
51. Transverse streaks: (0) no; (1) yes.