Relating Divergence in Polychaete Musculature to
Different Burrowing Behaviors: A Study Using
Chris J. Law,
Kelly M. Dorgan,
* and Greg W. Rouse
Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093-0202
Dauphin Island Sea Laboratory, Dauphin Island, Alabama 36528
Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, California 95062
ABSTRACT Divergent morphologies among related
species are often correlated with distinct behaviors and
habitat uses. Considerable morphological and behav-
ioral differences are found between two major clades
within the polychaete family Opheliidae. For instance,
Thoracophelia mucronata burrows by peristalsis,
whereas Armandia brevis exhibits undulatory burrow-
ing. We investigate the anatomical differences that
allow for these distinct burrowing behaviors, then
interpret these differences in an evolutionary context
using broader phylogenetic (DNA-based) and morpho-
logical analyses of Opheliidae and taxa, such as
Scalibregmatidae and Polygordiidae. Histological three-
dimensional-reconstruction of A. brevis reveals bilat-
eral longitudinal muscle bands as the prominent
musculature of the body. Circular muscles are absent;
instead oblique muscles act with unilateral contraction
of longitudinal muscles to bend the body during undu-
lation. The angle of helical ﬁbers in the cuticle is con-
sistent with the ﬁbers supporting turgidity of the body
rather than resisting radial expansion from longitudi-
nal muscle contraction. Circular muscles are present in
the anterior of T. mucronata, and they branch away
from the body wall to form oblique muscles. Helical
ﬁbers in the cuticle are more axially oriented than
those in undulatory burrowers, facilitating radial
expansion during peristalsis. A transition in muscula-
ture accompanies the change in external morphology
from the thorax to the abdomen, which has oblique
muscles similar to A. brevis. Muscles in the muscular
septum, which extends posteriorly to form the injector
organ, act in synchrony with the body wall muscula-
ture during peristalsis: they contract to push ﬂuid
anteriorly and expand the head region following a
direct peristaltic wave of the body wall muscles. The
septum of A. brevis is much thinner and is presumably
used for eversion of a nonmuscular pharynx. Mapping
of morphological characters onto the molecular-based
phylogeny shows close links between musculature and
behavior, but less correlation with habitat. J. Morphol.
000:000–000, 2013. V
C2013 Wiley Periodicals, Inc.
KEY WORDS: hydrostatic skeleton; muscle; polychaetes;
functional morphology; locomotion
Polychaete annelids are an abundant and mor-
phologically diverse group of organisms that inhabit
a wide range of habitats, with behaviors ranging
from sessile tube-dwelling to active burrowing
(Rouse and Pleijel, 2001). Even among motile poly-
chaetes, the frequency and duration of movements
vary considerably, and locomotory gaits differ
among and sometimes within taxa, including para-
podial crawling, undulation, and peristalsis, as well
as several swimming gaits (Clark, 1964; Fauchald
and Jumars, 1979). Investigation of the differences
in morphological and muscular function is impor-
tant for further understanding of differences in
locomotory behaviors, which affect organismal dis-
tribution, performance, ﬁtness, and habitat adapta-
tion (Arnold, 1983; Irschick and Garland, 2001;
Wainwright et al., 2008). Understanding of func-
tional morphology underlying these burrowing
behaviors has been limited by difﬁculty in observing
infaunal organisms in situ (cf. Dorgan et al., 2006).
Body movements in many polychaetes, like in
other soft-bodied animals, are achieved using a
hydrostatic skeleton in which a muscular body
wall surrounds a constant volume of tissues and
extracellular ﬂuids. Because ﬂuid-ﬁlled hydrostats
maintain constant volume, any change in one
dimension (1D) will cause a compensatory change
Additional Supporting Information may be found in the online ver-
sion of this article.
Contract grant sponsor: NSF OCE; Contract grant number: OCE-
1029160; Contract grant sponsor: NSF OCE (G.W.R. and L.
Levin); Contract grant number: OCE-0826254 and OCE-
0939557; Contract grant sponsor: NSF Polar Programs (G.W.R.,
N. Wilson, and R. Burton); Contract grant number: 1043749; Con-
tract grant sponsor: Carlsberg Foundation; Contract grant number:
*Correspondence to: KM Dorgan; Dauphin Island Sea Laboratory,
Dauphin Island, AL, 36528. E-mail: firstname.lastname@example.org
Received 29 July 2013; Revised 9 October 2013;
Accepted 17 November 2013.
Published online 00 Month 2013 in
Wiley Online Library (wileyonlinelibrary.com).
C2013 WILEY PERIODICALS, INC.
JOURNAL OF MORPHOLOGY 00:00–00 (2013)
in at least one other dimension, and different mus-
cle groups act antagonistically to generate body
movements of elongation, shortening, bending, and
torsion (Kier and Smith, 1985). In a cylindrical,
worm-shaped body, muscle ﬁbers perpendicular
(circular, transverse, oblique) and parallel (longitu-
dinal) to the long axis control the diameter and
length, respectively (Kier, 2012). Locomotion by
peristalsis, well-documented in earthworms and
other vermiform animals, involves either alternat-
ing or simultaneous waves of contractions of longi-
tudinal and circular muscles in the body wall, in
which contraction of longitudinal muscles expands
the body radially and contraction of circular
muscles elongates and extends the body anteriorly
(Gray and Lissmann, 1938; Trueman, 1966; Sey-
mour, 1976; Elder, 1980).
A growing number of polychaete taxa, however,
have been found to have body walls inconsistent
with the traditionally described (e.g., Lanzavecchia
et al., 1988; Gardiner, 1992) outer layer of circular
muscles and inner layer of longitudinal muscles.
Rather, many polychaetes lack circular muscle
ﬁbers along part, or even all of the body (Tzetlin
and Filippova, 2005; Purschke and M€
Some of these taxa also exhibit nonperistaltic loco-
motory behaviors such as undulation (Clark and
Clark, 1960; Clark and Hermans, 1976; Dorgan
et al., 2013). Bending movements do not require
circular musculature and, instead, are achieved by
unilateral contraction of longitudinal muscles.
Unilateral longitudinal contraction alone would
result in shearing of the body; some mechanism of
resisting radial expansion is necessary to prevent
an asymmetrical increase in body thickness and
resultant longitudinal shortening (Kier, 2012). In
the polychaete Nephtys (Nephtyidae), dorsal-
ventral muscles act to prevent radial expansion and
enable bending (Clark and Clark, 1960), whereas in
the nematode Ascaris lumbricoides, a helical array
of inextensible ﬁbers in the cuticle serves a similar
function (Harris and Crofton, 1957; Fig. 1).
Considerable behavioral differences are found
between the two major clades within the poly-
chaete family Opheliidae, where species in Opheli-
ninae move by undulation and those in Opheliinae
use peristaltic locomotion (Rouse and Pleijel,
2001). These behavioral differences are accompa-
nied by clearly distinctive morphologies (Fig. 2)
and habitats. The two clades are represented in
this study by Armandia brevis and Thoracophelia
mucronata, respectively. A. brevis both burrows
and swims using undulatory movements (Clark
and Hermans, 1976; Dorgan et al., 2013) and has
a smooth, rigid body with ventral and lateral
grooves extending along the entire length. It is
found in surﬁcial (<3 cm) heterogeneous sedi-
ments (Woodin, 1974; Hermans, 1978). Most mac-
rofaunal burrowers in muddy sediments use
eversible mouth parts or muscular anterior
regions to apply dorsoventral forces to burrow
walls and extend the burrow by fracture (Dorgan
et al., 2005, 2006). A. brevis, however, lacks the
morphological features consistent with this mecha-
nism and, instead, uses body undulations (Fig. 2A)
to plastically rearrange sediments (Dorgan et al.,
Fig. 1. (A) Unilateral longitudinal muscle contraction (red arrows indicate muscle contraction) with no mechanism of resisting
radial expansion results in shearing of the body and longitudinal shortening (black arrows indicate body shape changes). Dorsal-
ventral muscles (magenta) in Nephtys (B) and radially-oriented helical cuticle ﬁbers (black) in A. lumbricoides (C) serve to resist
radial expansion and thus resist asymmetrical longitudinal shortening and facilitate bending. Axially-oriented helical cuticle ﬁbers
(D) do not resist radial expansion, and thus longitudinal shortening can occur.
2 C. J. LAW ET AL.
Journal of Morphology
2013). This mechanism is likely limited to uncom-
pacted, surface sediments, consistent with habitat
descriptions for A. brevis.T. mucronata (Fig. 2B)
is found in the high intertidal on sandy beaches,
in distinct zones of high abundance (McCon-
naughey and Fox, 1949). It burrows by direct peri-
stalsis, with the wave of contraction traveling
anteriorly, and has a body divided into distinct
regions: 1) an anterior cephalic region consisting
of the prostomium and ﬁrst two chaetigers; 2) a
swollen thoracic region; and 3) a tapering posterior
region with ventral and lateral grooves. A lateral
notopodial ridge separates the thoracic and poste-
rior regions at the 10th chaetiger (Blake, 2000;
Santos et al., 2004; Law et al., 2013).
The substantial differences between these ophe-
liid species in external morphology, behavior, and
habitat suggest a divergence in underlying muscu-
lature as well. A. brevis has longitudinal and
oblique muscles, but lacks circular muscles, and
has an open body cavity with two to –three ante-
rior septa (Clark and Hermans, 1976; Tzetlin and
Zhadan, 2009). The posterior region of T. mucro-
nata has similar general musculature to A. brevis
oder, 1958; Clark and Hermans,
1976), whereas circular muscles have been
described in the anterior region (Hartmann-
oder, 1958). T. mucronata also has an open
body cavity, but with anterior septa that extend
over the esophagus to form the “injector organ”
(McConnaughey and Fox, 1949). Here, we directly
compare musculature of A. brevis and T. mucro-
nata and relate muscle structure to locomotory
function for each species. We focus speciﬁcally on
1) the change in musculature at the transition
region from the thorax of T. mucronata to the
abdomen, over which circular muscles disappear,
2) the anterior septa, and 3) the oblique muscles
in the posterior of T. mucronata and the entire
body of A. brevis.
Inextensible helical ﬁbers in the cuticle of hydro-
stats resist changes in body shape, with more cir-
cumferentially oriented ﬁbers resisting radial
expansion caused by longitudinal muscle contrac-
tion (facilitating undulatory movement) and ﬁbers
oriented at small angles from the longitudinal
body axis resisting elongation (facilitating peristal-
sis), with an angle of 54!440intermediate between
the two (Kier, 2012; Wainwright et al., 1976; Fig.
1C,D). Circumferentially oriented cuticle ﬁbers
("75!from the body axis) in the nematode, A.
lumbricoides, prevent radial expansion so that
unilateral longitudinal muscle contraction results
in bending (Harris and Crofton, 1957; Fig. 1C).
Clark and Hermans (1976) found that cuticle
ﬁbers in the undulatory-moving opheliid, Ophelina
sp., have angles "55!from the longitudinal body
axis, and they suggested that bending is enabled
by oblique muscles rather than the cuticle ﬁbers.
We compare cuticle ﬁber angles between the
undulatory-burrowing and peristaltic-burrowing
opheliids, hypothesizing that cuticle ﬁber angles
will be smaller than 54!440in the anterior of T.
mucronata to enable radial expansion during
We also construct an opheliid phylogeny based
on DNA sequences to generalize the morphologies
and behaviors of A. brevis and T. mucronata
described in this study across Opheliidae. The
morphologically similar Polygordiidae and the
closely related Scalibregmatidae were incorporated
for broader comparison. Polygordiids have been
suggested to be close to or part of Opheliidae
based on similar morphological characteristics
such as cuticle, muscular organization, and undu-
latory locomotion (McIntosh, 1875; Clark and Her-
mans, 1976; Giard, 1880) and Travisia was
recently moved from Opheliidae to Scalibregmati-
dae (Paul et al., 2010), reﬂecting similar morpho-
logical characters such as body shape and
epidermal rugosity that have linked Travisia with
Fig. 2. Live adult specimens of (A)Armandia brevis (Ophelini-
nae), found in surﬁcial heterogenous sediments and exhibits
undulating locomotion, and (B)Thoracophelia mucronata (Ophe-
liinae), found in high intertidal sandy beaches and exhibits peri-
staltic locomotion. Each species represents one of the two clades
within Opheliidae. Scale bar 51 mm.
3OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
Scalibregmatidae for over a century (Ashworth,
1901). All three families share the presence of a
ventral groove and mostly nonseptate bodies.
Using the phylogeny, we generate here, we map
morphological characters to examine broader rela-
tionships among external morphologies, muscula-
ture, and burrowing behavior and habitat. Several
additional taxa, notably Terebellidae (Nogueira
et al. 2010) and Pisionidens (Sigalionidae) (Aiyar
and Alikunhi, 1940; Tzetlin, 1987; Norlinder et al.,
2012), exhibit ventral or dorsal grooves, suggesting
that broader analyses of these characteristics
across annelids may be useful, but as this study
focuses on morphological divergence, we limit our
analysis to taxa closely related to opheliids (cf.
Struck et al. 2006, 2011). Moreover, most Terebelli-
dae are sessile tube dwellers rather than active
burrowers (Fauchald and Jumars, 1979), and the
ventral groove in this group may serve another
In this study, we use morphological data from
histology, 3D-reconstructions of thin sections, live
microscopy, and cuticle ﬁber angle measurements,
as well as DNA-based phylogenetic analyses to 1)
describe the musculature and morphological fea-
tures used for locomotion within Opheliidae, using
A. brevis and T. mucronata as representatives of
the two major opheliid clades; 2) relate these mus-
cular and morphological features to disparate
forms of burrowing and behavior; and 3) investi-
gate broader morphological comparisons among
Opheliidae, Scalibregmatidae, and Polygordiidae.
MATERIALS AND METHODS
A. brevis (Moore, 1906) and T. mucronata (Treadwell, 1914)
were collected from Mission Bay, San Diego, California on June
9, 2011 and La Jolla Shores Beach, California on May 4, 2012,
respectively. Specimens (10) of each species were relaxed in
and ﬁxed in 4% glutaraldehyde buffered with 0.2
sodium cacodylate with 0.3 mol l
sucrose for 24 h. The
specimens were then rinsed in buffer and dehydrated in a
graded series of ethanol rinses and embedded in low viscosity
Spurr’s resin, following manufacturer’s instructions (Spurr,
1969). Semithin serial cross-sections of specimens were pre-
pared using a Histo diamond knife (DiATOME) on a PowerTome
Ultramicrotome (Boeckeler Instruments) and stained with Tolui-
dine blue. A total of four specimens, two each of A. brevis and T.
mucronata, were sectioned at a thickness of 1.5 mm for histologi-
cal 3D-reconstruction, and two A. brevis specimens were sec-
tioned sagittally at a thickness of 3 mm to visualize anterior
septa. Serial sections were photographed using either a Canon
Powershot G9 camera attached to a Leica DMR microscope or a
Canon T1i camera attached to an Olympus CX41 microscope.
Selected sections were viewed with a Zeiss AxioObserver Z1
microscope with DIC ﬁlters and AxioVision software to photo-
graph ﬁner details of the connectivity and transitions between
circular and oblique musculature in both worms.
3D-Reconstruction and Visualization
AMIRA 5.4 (Visage Imaging) running on MAC OS v.10.6.8
was used for all 3D-reconstructions, following procedures modi-
ﬁed from Ruthensteiner (2008). Every other section, equaling 3-
mm increments, of the A. brevis specimen was photographed,
and every fourth section, equaling 6-mm increments, of the T.
mucronata specimen was photographed. Before importing into
AMIRA for 3D-reconstruction, section images were reduced in
size, converted to grayscale, contrast enhanced, and color
inverted using Adobe Photoshop CS5; color inversion is neces-
sary for volume rendering in AMIRA. A 3D-reconstruction of
the region of four anterior chaetigers of A. brevis (excluding the
head region) targeted the following morphologies and muscula-
ture: dorsal longitudinal muscles, ventral longitudinal muscles,
oblique muscles, and ventral nerve cord. For T. mucronata, 3D-
reconstructions of Chaetigers 2–9 focused on the body cavity
and septum/injector organ and of Chaetigers 9–14 on the body
cavity and lateral ridge. The least-squares alignment mode was
initially used to align the sections, followed by manual adjust-
ments when necessary. The Segmentation Editor was used to
create the 3D-images of structures. Labeling of structures was
done by hand on every third slice followed by interpolation to
connect intermediate slices. Resampling and separation of the
structures, labeled in Amira as “materials”, were performed
prior to surface rendering to decrease ﬁle output size. Surface
rendering was performed with the SurfaceGen module under
unconstrained smoothing at default settings followed by the
SmoothSurface module to improve surface quality with itera-
tions of >80. The Volren module was used to visualize external
features of both specimens. Dimensions were adjusted so 1
model unit equaled 1 mm.
The 3D-model was embedded into a PDF ﬁle with the 3D
Reviewer and 3D-Toolkit extensions found in Adobe Acrobat 9
Pro Extended running on Windows XP. Each 3D-object was
saved as a separate Wavefront OBJ-ﬁle in AMIRA and recom-
bined as one model in the 3D, Reviewer by importing one OBJ-
ﬁle at a time. Objects were color edited and transferred to the
3D toolkit as a PDF for further editing of orientation, rendering
style, background color, and lighting.
Fiber Angle Measurements
Cuticle ﬁber angles were measured in anterior, mid-body,
and posterior regions, on dorsal and ventral sides of T. mucro-
nata and Ophelina acuminata (€
Orsted, 1843), a species morpho-
logically and behaviorally similar to A. brevis but larger and
much easier to dissect. O. acuminata were collected from ﬁne-
grained subtidal sediments in Friday Harbor, Washington, and
T. mucronata from La Jolla Shores Beach, California. Four O.
acuminata and three T. mucronata were anesthetized in 7.5%
, ﬁxed in a phosphate-buffered mixture of 3% glutaralde-
hyde and 3% paraformaldehyde, then rinsed in distilled water
overnight, frozen at 220!C overnight to facilitate separation of
cuticle from muscle tissue (Murray et al., 1981), and thawed in
distilled water. In addition, ﬁve unpreserved T. mucronata were
anesthetized in the freezer, frozen overnight, and thawed in
distilled water. The cuticle was removed from different regions
of the body, mounted on slides with a drop of distilled water,
and visualized with polarized microscopy (Fig. 3). Angle of
ﬁbers from the longitudinal axis of the body was measured as
half of the total angle between crossed ﬁbers. Orientation of the
cuticle was obvious from circumferential grooves (visible as
lines) separating segments.
To observe the movements of the septum and injector organ
corresponding with the peristaltic wave, live T. mucronata were
placed in tunnels in a thin layer of seawater gelatin between a
microscope slide and cover slip. Tunnels were created by allow-
ing the gelatin to set around straight pieces of ﬁshing line,
which were then pulled out of the set gelatin. Small worms
with diameter close to that of the ﬁshing line were positioned
with the anterior at the entry of the tunnel and encouraged to
move. Videos were recorded using a Canon T3i attached to a
Leica DMR microscope with polarizing ﬁlters (see Supporting
Information, S-Movie). Crossed polarizers were used to view
4 C. J. LAW ET AL.
Journal of Morphology
muscle ﬁbers, which are birefringent. Movements of internal
structures and musculature as the worm moved through the
tunnel were described, with emphasis on the synchrony
between the body wall peristaltic wave and the musculature of
the septum and injector organ.
DNA Ampliﬁcation and Sequencing
Forty one specimens of 33 species were used for phylogenetic
analyses: 25 opheliids, four polygordiids, and 10 scalibregmatids.
The two outgroup taxa, a capitellid Notomastus sp. and an areni-
colid A. marina were chosen based on Struck et al.’s (2011)
annelid phylogeny (Table 1) with Notomastus sp. being used as
the root terminal. Newly collected specimens [from Beaufort, NC
(Ophelina sp1.); Costa Rica (Ophelina sp3.); Friday Harbor, WA
(Notomastus sp., O. acuminata,Polygordius sp., and Scali-
bregma inﬂatum); Greenland (O. acuminata, O. cylindricaudata,
and O. limacina); La Jolla, CA (A. brevis,Polyophthalmus sp.,
and T. mucronata); Lizard Island, Australia (Armandia sp1.);
and off the Oregon coast (Ophelina sp2.)] were relaxed in 7.5%
and ﬁxed in 95% ethyl alcohol. Sequences for the remain-
ing 26 species were accessed through GenBank (Table 1).
A Qiagen DNeasy tissue kit was used to extract genomic
DNA from specimens according to the manufacturer’s instruc-
tions. Approximately 500 base pairs of the mitochondrial small
subunit ribosomal DNA (16S) were ampliﬁed using the primers
16SarL and 16SbrL (Palumbi, 1996) with temperature proﬁles
of 95!C for 3 min, followed by 40 cycles of 95!C for 40 s, 48!C
for 40 s, 68!C for 50 s, and ﬁnal extension at 68!C for 5 min
(see Supporting Information, Table S1).
Three nuclear loci were also sequenced. The small subunit
ribosomal DNA (18S) was ampliﬁed using three primer sets: 1)
1F and 5R; 2) 3F and bi; and 3) a2.0 and 9R (Giribet et al.,
1996, 1999). Temperature proﬁles for the 1F/5R and a2.0/9R
primer sets were 95!C for 3 min, followed by 40 cycles of 95!C
for 30 s, 52!C for 30 s, 72!C for 90 s, and ﬁnal extension at
72!C for 8 min. The temperature proﬁle for the 3F/bi primer
set was 95!C for 3 min, followed by 40 cycles of 95!C for 30 s,
49!C for 30 s, 72!C for 90 s, and ﬁnal extension at 72!C for 8
min. Approximately 930 base pairs of the large subunit ribo-
somal DNA (28S) were ampliﬁed using the primers Po28F1 and
Po28R4 (Struck et al., 2006), and "360 base pairs of the
nuclear protein coding gene Histone H3 were ampliﬁed using
the primers H3aF and H3aR (Colgan et al., 1998). Both genes
were ampliﬁed using the same temperature proﬁles of 94!C for
2 min, followed by 35 cycles of 94!C for 45 s, 48!C for 60 s,
72!C for 90 s, and ﬁnal extension at 72!C for 10 min.
Ampliﬁcation reactions (25 ml) were conducted containing 2
ml of DNA template, 1 ml of forward and reverse primers, 12.5
ml GoTaq Green Master Mix (Promega), and 8.5 mlH
ExoSAP-IT (Affymetrix) was used to purify PCR products.
Sequencing was done by either Retrogen (San Diego, CA) or
Euroﬁns MWG Operon (Louisville, KY). Sequences were edited
using Geneious 5.5.6 (www.geneious.com) and aligned with
MAFFT 3.8 (Katoh and Kuma, 2002) under default settings
with no manual alterations. The combined molecular dataset
consisted of 3,955 total characters, 1,075 of which were parsi-
mony informative and 436 were uninformative.
Parsimony analyses on the combined genes (16S, 18S, 28S,
and H3) were conducted in PAUP* 4.0b10 (Swofford, 2002)
using a heuristic search with random stepwise addition of the
terminals for 1,000 replicates, with tree bisection and reconnec-
tion. The character matrix was equally weighted, and gaps
were treated as missing data. Clade support was assessed using
jackkniﬁng with 37% deletion of sites over 1,000 replicates with
10 random additions per iteration. Maximum likelihood analy-
ses were performed in RAxML 7.2.8 (Stamatkis, 2006) as a
four-gene partitioned dataset and under the General Time
Reversible 1Gamma (GTR 1G) model. Bootstrap (thorough
Fig. 3. Polarized images of cuticle from the anterior region of (A)Thoracophelia mucronata and (B)Ophelina acuminata with
intersegmental groove (ISG) indicated. The body axis, indicated with the double-arrowed white line, is perpendicular to the interseg-
mental grooves. Crossed helical ﬁbers of the cuticle are visible between intersegmental grooves, with one ﬁber traced with a dotted
line on either side of the body axis line and ﬁber angle indicated as a. Scale bar 550 mm.
5OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
TABLE 1. GenBank and voucher accession numbers
Taxon Specimen origin Voucher 16S 18S 28S H3
Arenicola marina, Linnaeus
2AY532328 AF508116 AY612629 DQ779718
Notomastus sp., Hendel
Friday Harbor, WA, USA A3421 KF511858 KF511859 KF511860 KF511880
2DQ779604 DQ779641 DQ779676 DQ779719
Armandia brevis, Moore
La Jolla, CA, USA A3411 KF511804 KF511818 KF511838 KF511861
Armandia brevis, Moore
Friday Harbor, WA, USA
2HM746708 EU418854 HM746736 HM746752
Armandia maculata, Webster
Twin Cayes, Belize
22 2 HM746737 HM746753
Armandia sp. Lizard Island, Great
A3412 KF511806 KF511820 KF511843 KF511866
Ophelia bicornis, Savigny
Ophelia limacina, Rathke
Greenland A3403 KF511817 KF511829 KF511850 KF511868
Ophelia neglecta, Schneider
Ophelia rathkei, Mcintosh
North Sea island of Sylt,
Ophelina acuminata (CA),
Southern CA, USA A3413 KF511810 KF511825 KF511839 KF511869
Ophelina acuminata (Eur),
2HM746716 HM746735 HM746744 HM746761
Ophelina acuminata (Eu),
Europe A3414 KF511811 KF511826 KF511840 2
Ophelina acuminata (FH),
Friday Harbor, WA, USA A3404 KF511812 KF511827 KF511842 KF511870
Ophelina acuminata (GR),
Greenland A3415 KF511813 KF511828 KF511841 KF511871
(NE), Hansen (1878)
New England, USA
2HM746717 HM746730 HM746746 HM746763
(GR), Hansen (1878)
Greenland A3416 2KF511824 KF511848 KF511865
Ophelina sp1. Beaufort, NC, USA A3417 KF511814 KF511834 KF511849 KF511876
Ophelina sp2. Oregon, USA 2KF511807 KF511822 KF511845 KF511862
Ophelina sp2. Oregon, USA A3418 KF511808 KF511821 KF511846 KF511863
Ophelina sp3. Costa Rica 2KF511809 KF511823 KF511847 KF511864
Lemon Tree Passage,
22 AB106267 AF185171 AF185259
Polyophthalmus sp. La Jolla, CA, USA A3419 KF511805 KF511819 KF511844 KF511867
Hutchings and Murray (1984)
22 2 AB106266 2
_ _ HM746725 HM746738 HM746755
(LJ), Treadwell (1914)
La Jolla, CA, USA A3409 2KF511831 KF511852 KF511873
North Sea Island
22 AY525629 EU418872 2
Polygordius jouinae, Ramey
et al. (2006)
Beach Haven Ridge,
New Jersey, USA
22 DQ153064 22
Polygordius lacteus, Schnei-
2DQ779633 DQ779669 DQ779707 DQ779757
Polygordius sp Friday Harbor, WA, USA KF511815 KF511835 KF511855 KF511879
Hyboscolex paciﬁcus, Moore
Santa Barbara, CA,
2HM746712 AB106268 HM746740 HM746757
22 AF508120 22
Neolipobranchius sp., Gulf of Maine, USA
22 AY612616 AY612626 2
6 C. J. LAW ET AL.
Journal of Morphology
option) values were estimated using 100 pseudoreplicates under
the same model.
Characters for Transformations
A behavioral and morphological character matrix was com-
piled to relate burrowing mode with distinctive morphology and
musculature across the DNA-generated phylogeny (Tables 2
and 3). We constructed nine characters based on key morpho-
logical and behavioral features that underlie the different loco-
motory behaviors exhibited by A. brevis and T. mucronata. The
nine characters, with character states given in brackets, are
shown below as a brief outline for each feature. Characters
were only assigned states based on direct evidence found in the
literature or on observations from this study with exception of
Characters 1–3 (burrowing), where unknown burrowing states
were generalized over genera. Characters with unknown states
are indicated with a “?”. Characters that were inapplicable for
a given terminal are indicated by “-“ (treated the same as “?”).
Justiﬁcations and references for the scoring of each terminal
are provided in Supporting Information, Appendix A. The bur-
rowing behavioral and morphological characters were traced
onto the tree generated by the maximum likelihood analysis
using most parsimonious transformations implemented in Mes-
quite 2.75 (Maddison and Maddison, 2011).
Burrowing. Burrowing mode [(0) peristaltic (1)
undulatory]. Many polychaetes with diverse morphologies
burrow by peristalsis, in which a wave of muscular contraction
moving anteriorly or posteriorly results in movement of the
body (Trueman, 1978). Some polychaetes, such as A. brevis and
O. acuminata, use undulatory body movements rather than
peristalsis to move (Clark and Hermans, 1976; Dorgan et al.,
Type of peristalsis [(0) direct (1) retrograde].
Peristaltic locomotion can be categorized into two general types:
retrograde peristalsis, in which the peristaltic wave travels in
the opposite direction of locomotion, and direct peristalsis, in
which the peristaltic wave travels in the same direction as loco-
motion (Elder, 1980). For direct peristalsis to result in forward
movement, simultaneous contractions of longitudinal and circu-
lar musculature must move ﬂuid through the body cavity.
Direct peristalsis is thus limited to animals with open body cav-
ities such as T. mucronata (e.g., Wells, 1961; Elder, 1973). Ret-
rograde peristalsis, on the other hand, can occur both in
segmented animals divided by septa and those with open body
cavities, (e.g., Seymour, 1976).
Proboscis use during burrowing [(0) absent (1)
present]. For worms burrowing in muds, eversion of a phar-
ynx or proboscis applies a dorsoventral force on the burrow
walls that is ampliﬁed at the crack tip, resulting in burrow
extension by fracture (Dorgan et al., 2005; Murphy and Dorgan,
2011). Arenicolids evert their axial nonmuscular proboscises
(Tzetlin and Purschke, 2005) for initial penetration into the
sediment and further deepening of their burrows (Trueman,
1966). Both A. brevis and T. mucronata have nonmuscular
pharynges that are, however, not used during burrowing.
Musculature. Circular muscles [(0) absent (1)
present but restricted to anterior (2) present along
entire body]. Polychaete musculature has traditionally
been described as consisting of an outer layer of circular
muscles between the epidermis and longitudinal muscles (Lan-
zavecchia et al., 1988; Gardiner, 1992). Opheliids, however, lack
circular muscle in part or all of the body (Hartmann-Schroder,
1958; Clark and Hermans, 1976), Polygordiids also have
Table 1. (continued).
Taxon Specimen origin Voucher 16S 18S 28S H3
Polyphysia crassa, Orsted
2HM746719 HM746731 HM746748 HM746765
Scalibregma inﬂatum (Eu),
2AY532331 AF448163 AY612624 DQ779764
Scalibregma inﬂatum (FH),
Friday Harbor, WA, USA A3420 KF511816 KF511837 KF511857 KF511877
Gulf of Maine, USA
22 AY612615 AY612623 2
Travisia brevis, Moore (1923) Friday Harbor, WA, USA
2HM746721 AY966901 HM746749 HM746767
Travisia kerguelensis, McIn-
Antarctica 2KF511836 KF511856 KF511878
Travisia pupa, Moore (1906) Bamﬁeld, Canada
2HM746722 HM746733 HM746750 HM746768
GenBank numbers in bold indicate new sequences.
Rousset et al. (2007).
Paul et al. (2010).
Bleidorn et al. (2003).
Jordens et al. (2004).
Hall et al. (2004).
Stuck et al. (2008).
Ramey et al. (2006).
Persson and Pleijel (2005).
TABLE 2. Summary of morphological characters
1. Burrowing mode: (0) peristaltic; (1) undulatory.
2. Type of peristalsis: (0) direct; (1) retrograde.
3. Proboscis use during burrowing: (0) absent; (1) present.
4. Circular muscles: (0) absent; (1) present, but restricted
to anterior; (2) present, along entire body.
5. Oblique muscles: (0) absent; (1) present.
6. Septa: (0) along the entire body; (1) 3 25 anterior septa;
(2) 1—2 anterior septa.
7. Modiﬁed anterior septa: (0) absent; (1) present.
8. Sand/mud habitat distribution: (0) sand; (1) mud.
9. Ventral groove (0) absent; (1) present, but restricted to
posterior; (2) present, along the entire length of body.
7OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
traditionally been described with absent circular muscles (Frai-
pont, 1887); however, a recent study shows “minute” circular
muscles occur in Polygoridus appendiculatus (Lehmacher et al.,
Oblique muscles [(0) absent (1) present]. Oblique
muscles are present in some polychaete groups, running from
the midventral line on either side of the ventral nerve cord to
the midlateral region (Rouse and Pleijel, 2001).
Septa. Septa [(0) along the entire body (1) 3–5
anterior septa (2) 1–2 anterior septa]. Septa are uni-
form throughout the body in most polychaetes (Fauchald and
Rouse, 1997). However, some polychaetes are unusual in having
only anterior septa and reduced or absent posterior septa,
which seals off the head from the remaining undivided body
cavity (Ashworth, 1904; Dales, 1962; Hunter et al., 1983; this
Modiﬁed anterior septa [(0) absent (1) present].
We deﬁne a modiﬁed septum as a muscularized anterior sep-
tum that is associated with anterior eversible structures. In
Ophelia and Thoracophelia, these anterior septa extend toward
the posterior to form the injector organ (Brown, 1938; McCon-
naughey and Fox, 1949; Harris, 1994; this study). Similarly, a
muscularized septum extends posteriorly in arenicolids and
capitellids to form the gular membrane (Eisig, 1887; Wells,
1954; Dales, 1962).
Habitat distribution. Sand/mud habitat distri-
bution [(0) sand (1) mud]. Mechanical responses of
granular sands and elastic cohesive muds to forces applied by
burrowers differ (Dorgan et al., 2006). Habitat is characterized
based on personal observations or literature descriptions.
External morphologies. Ventral groove [(0)
absent (1) present but restricted to posterior (2)
present along entire body]. Opheliids are characterized
by the presence of a ventral groove along the entire length of
the body or restricted to just the posterior (Blake, 2000). Poly-
gordiids also exhibit a ventral groove along the entire length of
the body (Rota and Carchini, 1999).
Morphology and Musculature
A. brevis (and O. acuminata). The body is
not divided into distinct body regions and shows
deep ventral and lateral grooves along the entire
length (Figs. 2A,4A). Internally, large dorsal and
ventral longitudinal muscle bands lie directly
beneath the epidermis (Fig. 4B,C). The ventral
longitudinal muscles form two well-developed ven-
tral bundles that shape the ridges of the ventral
groove and are separated by the ventral nerve
cord (Fig. 4B,G). The dorsal longitudinal muscle
bands become thinner middorsally but do not sep-
arate completely. No circular muscle ﬁbers are
found between the epidermis and longitudinal
muscles, but four bands of oblique muscle occur
per segment (Fig. 4D,E). Oblique muscle bands
extend from just dorsal of the ventral nerve cord
and attach to the lateral epidermis between the
dorsal and ventral longitudinal muscles (Fig. 4F–
The only septa present occur in the anterior
region, where two septa occur just posterior to the
pharynx (Fig. 4I). The remaining body cavity is
undivided by septa, allowing coelomic ﬂuids to
ﬂow freely during body movements.
The angle between the helical ﬁbers of the cuticle
and the longitudinal axis in O. acuminata is not sig-
niﬁcantly different from 54!440in the anterior (t-
test, P>0.05) and only slightly lower in the posterior
(52.4 60.2!(mean 6s.d.); t-test, P50.002; Fig. 5).
T. mucronata.The body is divided into three
distinct regions, i.e., the head (prostomium, peristo-
mium, and chaetigers 1–2), thorax (Chaetigers 3–
10), and abdomen (Chaetigers 11–38); a pair of lat-
eral ridges occur at Chaetiger 10 and a ventral
groove is present only along the abdomen (Figs. 2B,
6A, and 7). Dorsal and ventral longitudinal muscles
run along the entire length of the body. The ventral
nerve cord separates the ventral longitudinal
TABLE 3. Character matrix
1 2 3456789
Arenicola marina 0 0 1201100
Notomastus sp. 0 1 1 2 0 0 1 1 0
Armandia bilobata 1—0? ? ? ? 02
Armandia brevis (SD) 1—0012012
Armandia brevis (FH) 1—0012012
Armandia maculata 1—0????12
Armandia sp. 1 — 0 ? ? ? ? ? 2
Ophelia bicornis 0? ?112101
Ophelia limacina 0? ? 11? ? 01
Ophelia neglecta 0? ?112101
Ophelia rathkei 0? ?11??01
Ophelina acuminata (CA) 1 — 0 0 1 ? ? 1 2
Ophelina acuminata (EU) 1 — 001??12
Ophelina acuminata (FH) 1 — 001? ? 12
Ophelina acuminata (GER) 1 — 001? ? 12
Ophelina acuminata (GR) 1 — 0 ????12
Ophelina cylindricaudata (GR) 1 — 0 ????12
Ophelina cylindricaudata (NE) 1 — 001??12
Ophelina sp1. 1 — 0 ????12
Ophelina sp2. 1 — 0 ????12
Ophelina sp2. 1 — 0 ????12
Ophelina sp3. 1 — 0 ????12
Polyophthalmus pictus 1—001?? 02
Polyophthalmus sp. 1 — 0 0 1 ? ? 0 2
Thoracophelia bibranchia 000?1??01
Thoracophelia ezoensiss 000?1??01
Thoracophelia mucronata 0 0 0112101
Polygordius appendiculatus 1—0210002
Polygordius jouinae 1—0?? ? ? 02
Polygordius lacteus 1—0010002
Polygordius sp 1—0?10002
Hyboscolex paciﬁcus 0 0 ?????10
Lipobranchius jeffreysii 0 0 ?????1?
Neolipobranchius sp. 0 0 ? ? ? ? ? 1 ?
Polyphysia crassa 0 0 0211?10
Scalibregma inﬂatum (EU) 0 0 ? 211011
Scalibregma inﬂatum (FH) 0 0 ? 211011
Sclerobregma branchiata 0 0 ??????0
Travisia brevis 0? ?21??10
Travisia kerguelensis 0? ?????10
Travisia pupa 0? ? 211010
8 C. J. LAW ET AL.
Journal of Morphology
muscles; dorsal longitudinal muscles become thin-
ner middorsally but are not completely divided.
The posterior region of the body has musculature
similar to A. brevis, with longitudinal muscle bands
directly beneath the epidermis and no circular
muscles in between. Oblique muscles extend from
the ventral nerve cord to the lateral epidermis
between the dorsal and ventral longitudinal
muscles (Fig. 6J–N). The oblique muscles attach
more ventrally than those of A. brevis (Fig. 4B),
below the ventral nerve cord (Fig. 6J). In addition,
in T. mucronata, a secondary, more ventral band of
oblique muscle extends from the body wall ventral
of the ventral nerve cord to either the lateral
epidermis or the parapodial muscle complex (Fig.
6K,L). Longitudinal muscle bands are much
smaller (Fig. 6J) than those of A. brevis (Fig 4B).
In the head, chaetigers, thorax, and lateral
ridges of the anterior region, a thin, nearly contin-
uous layer of circular muscle lies beneath the epi-
dermis (Fig. 6B–G). Circular muscle is also found
in the transitional region between the thorax and
abdomen, becoming less continuous more posteri-
orly: circular muscle gradually disappears ven-
trally in Chaetiger 9 of the thorax and becomes
completely absent ventrally in Chaetiger 10 (Fig.
6F,H). Circular muscles are present dorsally (Fig.
6F,G) and in the lateral ridge (Fig. 6I) until the
transitional Chaetiger 11 (Fig. 6J,M). Oblique
muscles are also found in the anterior region of
Fig. 4. Musculature of Armandia brevis (A) Schematic drawing of Armandia brevis (lateral view). (B) 1.5-mm semithin cross sec-
tion. (C) 3D-reconstruction of body musculature over four anterior segments excluding the head (front-lateral view). (D) 3D-
reconstruction of oblique muscles and ventral longitudinal muscles, shown in dorsal view. (E) Polarized image of ventral view of the
body, revealing four bands of oblique muscle per segment; segments distinguished by eyespots and parapodia. (F) Semithin cross sec-
tion, oblique muscle attaching to the epidermis (between parapodia). (G) Semithin cross section, oblique muscles attaching dorsal to
the ventral nerve cord. (H) Semithin cross section, oblique muscle attaching to epidermis dorsal to parapodium. (I) Longitudinal cross
section revealing two anterior septa. dlm, dorsal longitudinal muscles; es, eyespots; g, gut; obm, oblique muscles; pp, parapodia; sp,
septa; vlm, ventral longitudinal muscles; vnc, ventral nerve cord. To activate the interactive 3D mode, view PDF in Adobe Reader
and click on the image plate.
9OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
the body, ﬁrst apparent anterior to the septum in
Chaetiger 3 (cf. Fig. 8). Anterior oblique muscles
connect dorsal and ventral circular muscles,
attaching lateral to the ventral nerve cord, and
are much thinner than in the posterior (Fig. 6B–
F). Circular muscle in the anterior region bifur-
cates on both sides of the ventral nerve cord, with
one branch extending away from the body wall to
form oblique muscle (Fig. 6B,D,F,H). These oblique
bands extend lateral-dorsally through the body
cavity and reconverge with circular muscle in the
lateral body wall between the epidermis and dor-
sal longitudinal muscles (Fig. 6B,C,F,G). Gaps in
the longitudinal muscle, both ventrally and later-
ally, allow oblique muscle to branch from circular
muscle (which lies between the longitudinal mus-
cle, where present, and the epidermis) into the
coelomic cavity (Fig. 6).
A single muscular septum separates the anterior
of body cavity between the third and fourth
chaetigers of the thorax (Fig. 8A). The septum
encapsulates the pharynx and extends over the
esophagus to form the “injector organ” that in this
specimen extends from the 6th to 9th chaetigers.
The septum separates the head from the main
body cavity (Fig. 8). The septum/injector organ
complex also consists of septal longitudinal and
circular muscle ﬁbers (Fig. 8C,E,G).
Cuticle ﬁber angles were not signiﬁcantly differ-
ent between the two methods of anesthetizing T.
mucronata, with MgCl
before ﬁxation in glutaral-
dehyde (n53) and with cold, placing worms in the
freezer without ﬁxation (n55; ANOVA, P>0.05).
Results were therefore combined (n58). Fiber
angles in both the thorax and abdomen were <54!
440(t-test, P<0.01), with anterior ﬁber angles sig-
niﬁcantly smaller than posterior (ANOVA multiple
comparison test, P<0.05; Fig. 5). Thoracic ﬁber
angles were signiﬁcantly smaller for T. mucronata
than O. acuminata (ANOVA multiple comparison
test, P<0.01), but abdominal ﬁber angles were
not signiﬁcantly different between the two species
(ANOVA multiple comparison test, P>0.05).
Functional Morphology of the Anterior of T.
mucronata during Peristaltic Burrowing
Direct peristaltic movement in T. mucronata
involves not only anteriorly-traveling waves of
contraction of circular and longitudinal body wall
muscles, but considerable movement of the sep-
tum, injector organ, and coelomic ﬂuid (Fig. 9;
Supporting Information, S-Movie). As the peristal-
tic wave moves forward into the head region, con-
traction of body wall circular and longitudinal
muscles and relaxation of the septum pushes the
pharynx backwards and forces coelomic ﬂuids
from the head region into the injector organ (Fig.
9A–E). Subsequent contraction of the septal circu-
lar and longitudinal muscles forces the pharynx
and coelomic ﬂuid back into the head region,
expanding the head radially (Fig. 9F–J).
The maximum parsimony (MP) and maximum
likelihood (ML) analyses for the combined molecu-
lar data produced similar results, though the ML
topology is shown here (Fig. 10). There were differ-
ences in the placement of Arenicola between the
two analyses (see below), and there were two most
parsimonious trees of length 4652 steps that only
differed from each other in the placement of Ophe-
lia. rathkei and O. bicornis. Monophyly of Ophelii-
dae was well supported (ML bootstrapping 5BS:
100%; MP jackkniﬁng 5JK: 100%), as were the
two subfamilies Opheliinae and Ophelininae. How-
ever, paraphyly was found for several genera and
for one species-level taxon. Within Ophelininae,
Ophelina was paraphyletic. The specimens of O.
acuminata formed a clade that was the sister
group to a well-supported clade comprised of the
remaining ophelinins. The two specimens identi-
ﬁed as O. cylindricaudata (New England, USA
Fig. 5. Cuticle ﬁber angle (mean6s.d.) of the anterior and
posterior of Thoracophelia mucronata (n58) and Ophelina acu-
minata (n54). A dotted line is drawn at 54!44’. Schematic of
anterior ﬁber angles shown for each species to illustrate
10 C. J. LAW ET AL.
Journal of Morphology
Fig. 6. Musculature of Thoracophelia mucronata (A) Schematic drawing of Thoracophelia mucronata with septum/injector organ
shown in red (lateral view). (B) Semithin cross section of Segment 8. (C) Close up from B (lateral-dorsal). Band of oblique muscles
(obm) merge with a continuous band of circular muscles (cm) between the epidermis and dorsal longitudinal muscles (dlm). (D) Close
up from B (lateral-ventral). Thin layer of circular muscles lies between the epidermis and ventral longitudinal muscles (vlm), merg-
ing with a band of oblique muscles that continues dorsally. (E) Schematic drawing of a cross section (thorax region) showing the gen-
eral pattern of oblique muscles and circular muscles. Circular muscles underlie the epidermis and bands of oblique muscles connect
circular ﬁbers ventrally, laterally, and dorsally. (F) Semithin cross section of Segment 10. No circular ﬁbers present between epider-
mis and vlm. Dorsal and ventral circular muscles are connected by oblique muscle. (G) Close up of the right side of the section shown
in F (lateral). Band of circular muscles between the epidermis and dorsal longitudinal muscles (dlm) extend away from the body wall
and thicken to form oblique muscles (obm). (H) Close up from F (ventral). Band of oblique muscle merges with circular ﬁbers
between ventral nerve cord and epidermis. No circular ﬁbers are present between epidermis and vlm. (I) Close up of lateral ridge
from F (lateral). Circular muscle ﬁbers are visible beneath the epidermis of the lateral ridge. (J) Semithin cross section of Segment
14. (K) Close up of nonparapodial side from J (ventral). Circular muscle ﬁbers are absent, and multiple oblique muscle bands attach
to epidermis. (L) Close up of parapodial side from J (ventral). Oblique muscle bands attach to epidermis. Secondary oblique muscle
bands attach to parapodial complex. (M) Close up from J (dorsal). No circular ﬁbers present between dlm and epidermis. (N) Close
up from L (ventral). No circular ﬁbers present between vlm and epidermis. dlm, dorsal longitudinal muscles; ep, epidermis; g, gut; io,
injector organ; obm, oblique muscles; vlm, ventral longitudinal muscles; vnc, ventral nerve cord.
11OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
and western Greenland) did not form a clade, with
several specimens of unidentiﬁed Ophelina (from
the eastern Paciﬁc Ocean; Oregon and Costa Rica)
forming a clade with the Greenland specimen of
O. cylindricaudata.Ophelina was further found to
be paraphyletic in that Ophelina sp. 1 (from the
western Atlantic Ocean; North Carolina) formed a
well-supported clade with Armandia and Polyoph-
thalmus.Armandia was also found to be paraphy-
letic, with Polyopthalmus well nested inside this
group. Within Opheliinae, Ophelia was paraphy-
letic with O. limacina recovered as the sister
group to Thoracophelia under ML, rather than
grouping with the other Ophelia specimens,
though with poor support. In contrast, the MP
analysis showed Thoracophelia to be paraphyletic
with respect to Ophelia (not shown), also with
Our data supported a clade comprising Ophelii-
dae and Polygordius, though with very low sup-
port under ML, in contrast to the strong support
under MP (BS 46; JK 99). Scalibregmatidae was
found to be sister to Opheliidae/Polygordius under
MP (not shown), with strong support (JK 99),
though under ML Arenicola was the sister-group
to Scalibregmatidae (Fig. 10), albeit with low sup-
port. The monophyly of Scalibregmatidae, includ-
ing Travisia, was well supported (BS 91; JK 100).
Travisia formed a clade with Neolipobranchius,
and this clade is sister to the remaining
For characters with multiple most parsimonious
reconstructions (MPRs), only two of the possible
reconstructions were used: using an accelerated
transformation (ACCTRAN), where changes are
assigned as close to the root as possible and rever-
sals are favored, and a delayed transformation
(DELTRAN), where changes are assigned as far
away from the roots as possible and convergence
is favored. The ML tree topology shown in Figure
10 was used for the transformations.
Burrowing mode (Character 1) showed two
MPRs. Both showed that peristaltic burrowing is a
plesiomorphic for the ingroup (Fig. 11A), and
under ACCTRAN there was a transition from peri-
staltic to undulatory burrowing for the Opheliidae/
Polygordius clade, with a subsequent reversal
back to peristaltic burrowing for Opheliinae (Fig.
11A). Under DELTRAN, peristaltic burrowing was
the plesiomorphic condition for Opheliidae with
undulatory burrowing appearing twice, once for
Polygordius and once for Ophelininae. This ambi-
guity is complicated by the poor support for the
clade comprising Polygordius as sister to Ophelii-
dae. Of our sampled taxa, only Notomastus was
scored with retrograde peristalsis (Character 2).
The change from retrograde to direct peristalsis
therefore occurred either below or at the ingroup
node, and thus it is unclear as to whether retro-
grade peristalsis or direct peristalsis is the plesio-
morphic condition for our terminal taxa. Direct
peristalsis was, however, shared among Scalibreg-
matidae, Arenicola, and Opheliidae.
Only the terminals Notomastus and Arenicola
were scored with proboscis use during burrowing
(Character 3), which consisted of ﬁve MPRs.
Under ACCTRAN, loss of proboscis use occurred
once for the ingroup, with a subsequent reappear-
ance in Arenicola. Under DELTRAN, a loss of pro-
boscis use appeared independently for the
scalibregmatid clade and also the Opheliidae/Poly-
gordius clade. Various MPRs occurred owing to
the unknown states for many of the scalibregmatid
and Travisia terminals.
The multistate character pertaining to circular
musculature (Character 4) consisted of seven MPR
(Fig. 11B). Under ACCTRAN, circular muscle
bands were lost in the Opheliidae/Polygordius
clade, with a reappearance of circular muscles in
P. appendiculatus and a second reappearance,
restricted to the anterior region of the body, in
Opheliinae. Under DELTRAN, loss of circular
muscles appeared independently in the Ophelini-
nae clade and in P. lacteus. In addition, the loss of
circular muscles in the posterior region appeared
in the Ophelininae clade. There were three MPRs
for the character based on oblique musculature
(Character 5). Under ACCTRAN, the presence of
oblique muscles appeared at the ingroup node
with a subsequent loss in Arenicola whereas,
under DELTRAN, oblique muscles appeared inde-
pendently in Scalibregmatidae and the Opheliidae/
Fig. 7. 3D-reconstruction of Chaetigers 9–14 in Thoracophelia
mucronata, showing the external morphological transition from
thorax to abdomen (front-lateral view). lr, lateral ridge; vb, ven-
tral bundle; vg, ventral groove. To activate the interactive 3D-
mode, view PDF in Adobe Reader and click on the image plate.
12 C. J. LAW ET AL.
Journal of Morphology
The multistate character pertaining to septa
(Character 6) only consisted of one MPR (Fig.
11C). Of the ingroup taxa, only Polygordius exhib-
ited the outgroup condition of septa along the
entire body. Loss of body septa along the body
occurred twice, once for the clade of scalibregma-
tids and Arenicola and once for Opheliidae. Noto-
mastus,Arenicola, and the opheliid subfamily
Opheliinae were scored with the presence of modi-
ﬁed anterior septa (Character 7), which resulted
in eleven MPRs, owing to the large number of ter-
minals with unknown states. Under ACCTRAN,
appearance of one or more altered anterior septa
occurred once for the ingroup, with a subsequent
disappearance in Scalibregmatidae, Polygordius,
and Ophelininae. Under DELTRAN, 1 or more
altered anterior septa appeared independently in
Notomastus,Arenicola, and Opheliinae.
Habitat distribution (Character 8) showed eight
MPRs. Under ACCTRAN, a shift from mud-
dwelling to sand-dwelling occurred at the ingroup
node, with mud-dwelling reappearing twice, in
Scalibregmatidae and in the opheliid subfamily
Ophelininae (Fig. 11D). Sand-dwelling secondarily
reappeared in Armandia bilobata and in the Poly-
ophthalmus clade along with a subsequent second-
ary reappearance of mud-dwelling in A. brevis
(Fig. 11D). Alternatively, ﬁve independent changes
from mud-dwelling to sand-dwelling occurred
under DELTRAN: once in Arenicola, once in
Fig. 8. Septum and injector organ of Thoracophelia mucronata (A) 3D-reconstruction of septum and injector organ (lateral view).
(B) Semithin cross section from segment 4. (C) Close up of B showing septum with septal longitudinal and septal circular muscles.
(D) Semithin cross section from segment 6. (E) Close up of D showing septum/injector organ transition with septal longitudinal and
circular muscles. (F) Semithin cross section from segment 8. (G) Close up of F showing injector organ with septal longitudinal and
circular muscles. cm, circular muscles; dlm, dorsal longitudinal muscles; g, gut; io, injector organ; slm, septa longitudinal muscles;
sp, septum; scm, septal circular muscles. To activate the interactive 3D-mode, view PDF in Adobe Reader and click on the image
13OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
Polygordiidae, once in the opheliid subfamily
Opheliinae, once in A. bilobata, and once in the
The multistate character pertaining to ventral
groove (Character 9) consisted of one MPR (Fig.
12A). The transformation shows a ventral groove
along the whole body as plesiomorphic for the
Opheliidae/Polygordius clade before transforming
to being restricted to the posterior end in Ophelii-
nae. The presence of a ventral groove restricted to
the posterior end also appeared independently in
Scalibregma (Fig. 12A). The presence of a ventral
groove has been attributed to attachment of
oblique muscles (Clark and Hermans, 1976), and
the MPR for oblique muscles corresponds well to
that of the presence of a ventral groove (data not
shown). The ventral groove MPR also showed
some interesting congruence with the MPR for the
circular muscle character (Fig. 12B). The absence
or restriction of circular muscles was coincident
with presence of a ventral groove in the Ophelii-
dae/Polygordius clade. The exception was the ven-
tral groove (restricted to the posterior end) found
in Scalibregma where circular muscles are present
along the body.
Functional Morphology of Undulatory
Burrowing in A.brevis
Undulating movements of A. brevis resemble
those of nematodes both in qualitative behavior
and in body shape, characterized by the ratio of
amplitude to wavelength (Dorgan et al., 2013).
Like nematodes, A. brevis has thick bands of longi-
tudinal muscle that contract unilaterally for undu-
latory bending. Bending during undulatory
burrowing requires unilateral contraction of longi-
tudinal muscles simultaneously with a mechanism
to resist radial expansion and axial shortening on
the side of muscle contraction. As the wave of con-
traction passes posteriorly, longitudinal muscles
on the nonbending side contract, extending the
contracted longitudinal muscles and serving as a
Fig. 9. Images with schematic drawings of the burrowing
mechanism in T. mucronata.(A) Relaxed anterior showing sep-
tum (s) in gold in the drawing, pharynx (p) drawn in blue, and
injector organ (io) drawn in red. Scale bar 5200 mm (all images
at same scale). (B) 0.27 s, peristaltic wave moving anteriorly. (C)
0.6 s, peristaltic wave approaches septum. (D) 0.9 s, peristaltic
wave close to septum contact with body wall, pharynx moving
posteriorly, septum relaxed and extending, injector organ inﬂat-
ing. (E) 1.23 s, peristaltic wave moving anterior of septum,
septum-body wall contact moving forward, pharynx posterior of
septum-body wall contact, septum relaxed, injector organ
inﬂated. (F) 1.63 s, head moving forward, septum-body wall con-
tact moved forward, pharynx still posterior of septum contact
but moving forward, injector organ inﬂated. (G) 1.87 s, pharynx
moving forward and septum muscles contracting, injector organ
muscles starting to contract, anterior close to or at full distance
travelled. (H) 2.0 s, Septum mostly contracted, pharynx anterior
of septum contact, injector organ contracting, anterior has
reached full distance travelled. (I) 2.47 s, Septum fully con-
tracted, head fully expanded, injector organ fully contracted. (J)
3.03 s, Septum and injector organ relaxed, injector organ par-
14 C. J. LAW ET AL.
Journal of Morphology
restoring force. The contracted oblique muscles
presumably also extend when the body reaches
the opposite curvature, although contraction of the
ventral longitudinal muscle would likely extend
relaxed oblique muscles as well. In the nematode
A. lumbricoides, radially-oriented cuticle ﬁbers
prevent unilateral radial expansion, enabling lon-
gitudinal muscle contraction to bend the body
(Fig. 1C). We found that helical ﬁbers in the
cuticle of O. acuminata, a closely related species to
A. brevis with very similar undulatory behavior
and morphological and muscular features (Law
and Dorgan, unpublished data), have ﬁber angles
much lower than that of A. lumbricoides, consist-
ent with ﬁndings by Clark and Hermans (1976).
This suggests that the cuticle of A. brevis does not
resist radial expansion in the same way as that of
A. lumbricoides, rather that the oblique muscles
contract on the same side as the longitudinal
muscles to enable bending (Fig. 13).
Fig. 10. Phylogenetic results of maximum likelihood tree from combined molecular data. Sup-
port values are shown as bootstrap from maximum likelihood and jackknife from maximum par-
simony analyses, respectively, separated by /. * indicates 100% bootstrap support.
15OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
Helical ﬁbers not only resist expansion or elon-
gation depending on their angle, but they also con-
trol the maximum volume in a cylinder and the
extent to which body shape can change. At an
intermediate ﬁber angle of 54!440, a cylinder
reaches its maximum volume, and, if turgid, the
ﬁbers will resist both expansion and elongation
(Kier, 2012). The cuticle ﬁber angle in O. acumi-
nata is not signiﬁcantly different from 54!440, cor-
responding to the maximum volume of a circular
cylinder, consistent with observations of rigid-
bodied live worms. The cuticle, therefore, appears
to function to prevent both radial expansion and
axial elongation and may facilitate both bending
and axial forcing against the substratum during
Functional Morphology of Peristaltic
Burrowing in T.mucronata
Peristalsis in burrowers with segments sepa-
rated by muscular septa, such as the earthworm
Lumbricus terrestris, is described as a nearly
simultaneous wave of circular and longitudinal
muscle contractions of the body wall traveling in
the opposite direction of locomotion (Gray and
Lissmann, 1938; Clark, 1964). For direct peristal-
sis, in which waves of contraction of the body wall
travel in the direction of movement to result in
forward movement, ﬂuid must be able to travel
away from the region of contraction and thus
requires an open body cavity (Clark, 1964; Elder,
1980). T. mucronata has an open body cavity con-
sistent with direct peristalsis, but we show that
activity of muscles of the septum and injector
organ accompany anteriorly-traveling waves of
contraction of circular and longitudinal muscles in
the body wall. Muscle contractions in the septum/
injector organ complex force coelomic ﬂuid into the
head region following passage of the peristaltic
wave along the body wall (Fig. 9). This expansion
of the anterior is likely important both in burrow
construction and in anchoring to allow the remain-
ing posterior body to be pulled forward into the
burrow. Analogous structures for anterior expan-
sion are found in other direct peristaltic burrowers
as well: Arenicola marina has a modiﬁed anterior
septum, the gular membrane, that is important in
pharynx eversion (Wells, 1954), and the priapulid
Priapulus caudatus has an open body cavity but
Fig. 11. Character transformations using parsimony reconstruction methods. Tree topologies
identical to tree seen in Figure 9. (A) Burrowing mode. (B) Presence of circular muscles. (C)
Presence of septa. * indicates modiﬁed anterior septum. (D) Sand/mud habitat distribution.
16 C. J. LAW ET AL.
Journal of Morphology
uses an eversible praesoma to expand the anterior
(Elder and Hunter, 1980).
The difference in angle of helical ﬁbers in the
cuticle between T. mucronata and O. acuminata is
consistent with their different locomotory behav-
iors. More axially-oriented ﬁbers in the anterior of
T. mucronata resist axial elongation of the anterior
so that an increase in internal pressure causes
radial expansion (Clark and Cowey, 1958). The
lower cuticle ﬁber angle indicates that the volume
of ﬂuid is less than the maximum, consistent with
observations of a less turgid body in T. mucronata
than in A. brevis and O. acuminata (cf. Clark and
Cowey, 1958). Cuticle ﬁber angles are not signiﬁ-
cantly different in the posterior of the two species,
which has more similar musculature as well.
As with other members of Opheliinae (e.g.,
Ophelia rathkei; Brown, 1938), the posterior
region of T. mucronata resembles the body plan of
A. brevis in lacking circular muscle (Fig. 6J–N).
However, compared to the thick and robust
musculature of A. brevis, both longitudinal and
oblique muscles in T. mucronata appear much
thinner (Fig. 4B,6J). Whereas undulations occur
along the entire length of A. brevis, the posterior
of T. mucronata is much less active during burrow-
ing. Rather than simply being dragged passively
behind the anterior however, the posterior appears
to be pulled along in discrete anterior movements
(Dorgan, unpublished data), presumably by simul-
taneous contraction of longitudinal muscles on
both sides of the body. Rather than having a single
thick band of oblique muscle as in A. brevis,T.
mucronata has two thinner bands, with a more
ventral second band that attaches laterally at the
parapodia or just ventral of the lateral groove
(Fig. 6M,N). We suggest that this secondary mus-
cle band may both assist in parapodial control and
also enable greater control of changes in body
shape around the ventral ridge. Observations of
live worms show considerable anterior-posterior
movement of coelomic ﬂuid through the ventral
Fig. 12. Character transformations using parsimony reconstruction methods. Tree topologies
identical to tree seen in Figure 9. (A) Presence of ventral groove. (B) Presence of circular
17OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
ridges on either side of the ventral groove. Con-
traction of posterior oblique muscles may reduce
the diameter of the body during forward move-
ment, potentially reducing frictional resistance
along the abdomen.
A. brevis and T. mucronata respectively belong
in each of the two main clades within Opheliidae;
the former in the undulatory Ophelininae and the
latter in the peristaltic Opheliinae. Although
monophyly of the opheliid subfamilies was well
supported, paraphyly of genera was found within
both subfamilies. Although a clade of two speci-
mens of Ophelina cylindricaudata, two specimens
of Ophelina sp. 2, and a single specimen of Ophe-
lina sp. 3 was recovered, they likely belong to four
different species (Fig. 10). In addition, Ophelina
formed a grade, with Ophelina sp.1 forming a
clade with Armandia and Polyophthalmus (Fig.
10). Ophelina sp.1 lacked the eyespots that occur
in Armandia and Polyophthalmus and so was cor-
rectly assigned to this genus, though further
assessment is clearly required. Polyophthalmus
nested within a grade of Armandia. Whether
Armandia Filippi, 1861 should be synonymized
with Polyophthalmus Quatrefages, 1850 in future
taxonomic revisions requires additional investiga-
tion. The absence of branchiae currently distin-
guishes Polyophthalmus from Armandia and
Ophelina (Blake, 2000).
The MP and ML analyses showed incongruent
topologies for the subfamily Opheliinae. The MP
result showed Ophelia to be within a paraphyletic
Thoracophelia, whereas ML (Fig. 10) showed
Ophelia to be paraphyletic with respect to Thora-
cophelia. In each, the support for these topologies
Fig. 13. Schematic drawing of musculature used for bending in Armandia brevis and Ophelina acuminata from frontal dorsal (A)
and frontal lateral (B) views. Bending (yellow arrows) is achieved by the unilateral contraction of dorsal and ventral longitudinal
muscles (red arrows) simultaneously with the antagonistic contraction of oblique muscles on the same side (red arrows). Contraction
of oblique muscles acts to resist radial expansion and axial shortening, and longitudinal muscles on the opposite side serves as the
restoring force. Inextensible helical ﬁbers in the cuticle are oriented at an intermediate ﬁber angle (between radially and axially ori-
ented) corresponding to the maximum volume of a circular cylinder and may help prevent radial expansion as well as axial elonga-
tion (black arrows). (C) Schematic of cross-section of Thoracophelia mucronata shown for comparison. Oblique muscles attach below
the ventral nerve cord, a position likely to be less effective in resisting radial expansion. dlm, dorsal longitudinal muscles; obm,
oblique muscles; vlm, ventral longitudinal muscles; vnc, ventral nerve cord.
18 C. J. LAW ET AL.
Journal of Morphology
was poor. The two genera have traditionally been
distinguished by the difference in body regions:
Ophelia has two distinct regions and Thoracophe-
lia has three (Blake, 2000).
Similar morphological characteristics such as
the presence of a ventral groove, undulatory bur-
rowing behavior, and lack of circular muscles have
linked polygordiids with opheliids such as Arman-
dia and Polyophthalmus for over a century (McIn-
tosh, 1875; Fraipont, 1887; Rouse and Pleijel,
2001). Our parsimony analysis did recover a clade
consisting of Opheliidae and the morphologically
similar Polygordius with strong support, though it
was markedly lower with ML (Fig. 10), suggesting
further investigation is required. Additionally, the
only previous molecular-based analysis on a
broader scale (Rousset et al. 2007) that included
these taxa found no close relationship for Ophelii-
dae and Polygordius. A recent phylogenetic study
suggested that Scalibregmatidae and Opheliidae
are sister groups (Paul et al., 2010), but this was
not found by Persson and Pleijel (2005) or Rousset
et al. (2007), and Struck et al. (2008) found that
Scalibregmatidae was closer to Fauveliopsis and
Sternaspis (neither included in our analysis) than
Our results showing that Travisia belongs with
Scalibregmatidae, rather than Opheliidae, was
consistent with ﬁndings ﬁrst shown by Persson
and Pleijel (2005) and then corroborated by Paul
et al. (2010). The placement of Travisia into Scali-
bregmatidae conﬁrms century-old discussions of
morphological similarities between the two taxa
and suggestions that there may have been prob-
lems with the placement of Travisia in Opheliidae
(Ashworth, 1901; Blake, 2000; Rouse and Pleijel,
2001). Paul et al. (2010) found that their two spe-
cies of Travisia formed a grade with respect to
Neolipobranchius, suggesting that Neolipobran-
chius Hartman and Fauchald, 1971 should be syn-
onymized with Travisia Johnston, 1840 in future
taxonomic revisions. Our inclusion of a third spe-
cies of Travisia (T. kerguelensis) also found that
Travisia includes Neolipobranchius.
Evolution of Musculature
It has been well-documented that peristaltic
burrowing behavior is common in polychaetes and
involves both circular and longitudinal muscles
(e.g. Arenicola marina,Polyphysia crassa,L. ter-
restris,T. mucronata; Trueman, 1966; Elder, 1973;
Seymour, 1976). We found that the loss of circular
muscles, in part of all of the body, coincided with a
switch from peristaltic to undulatory burrowing in
Polygordius (with the exception of P. appendicula-
tus; Lehmacher et al., in press) and some Ophelii-
dae (Fig. 12A,B).
The reappearance of anterior circular muscula-
ture was found for Opheliinae, which are peristaltic
burrowers. The presence of circular musculature
anteriorly is consistent with our analysis showing
that T. mucronata exhibits peristaltic movements
in only the anterior region of the body, in contrast
to other direct peristaltic burrowers for which the
wave travels the entire length of the body (e.g., P.
crassa; Elder, 1973).
The recent discovery of “minute” circular
muscles in P. appendiculatus (Lehmacher et al., in
press) is interesting as polygordiids exhibit undu-
latory behavior (Dorgan, unpublished data),
whereas circular muscles are generally used in
peristaltic burrowing. They also have oblique
muscles, similar to A. brevis, which likely simi-
larly act with unilateral contraction of longitudinal
muscles to bend the body during undulation. It
seems feasible that the circular muscles may act
in conjunction with the oblique muscles to prevent
radial expansion and enable bending, although
their function during undulatory burrowing and
whether circular musculature may occur in other
polygordiids requires additional study.
With the exception of the outgroups Notomastus
and Arenicola, all our terminal taxa (where
known), both undulatory and peristaltic bur-
rowers, showed oblique muscles that extend from
the midventral line to the midlateral body wall.
Oblique muscles either appear at the ingroup
node, with a subsequent reversal in Arenicola
(ACCTRAN), or appear independently in Scali-
bregmatidae and the Opheliidae/Polygordius clade
(DELTRAN). Broader taxon sampling is needed to
distinguish between these two alternatives. Better
resolution of the position of Polygordius is particu-
larly important in determining the evolution of
undulatory burrowing among these taxa.
The function of oblique muscles appears to differ
between undulatory and peristaltic burrowers.
Oblique muscles are important during locomotion
in A. brevis, acting with longitudinal muscles to
achieve lateral bending (Fig. 13). The presence of
similarly large oblique muscles as well as large
longitudinal muscles in other undulatory Ophe-
lina,Polyophthalmus (Purschke and M€
Fig 2B), and Polygordius suggests similar mecha-
nisms during undulatory behaviors. The oblique
muscles of the peristaltic burrower, T. mucronata
(Fig. 6K,L) occur as multiple thinner bands that
attach at distinct positions along the body wall,
and oblique muscles of the related Ophelia sp.
appear to be similar (Brown, 1938). We suggest
that these secondary muscle bands likely contract
bilaterally rather than unilaterally as in A. brevis
and may help contract the body to reduce friction
as it is pulled forward with the longitudinal
The branching of circular muscles to form
oblique muscles in the anterior of T. mucronata
(Fig. 6C,D,G) suggests that oblique muscles may
have been derived from circular muscles, although
19OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
our phylogeny leaves the plesiomorphic state of
circular muscles in Opheliidae ambiguous, and we
cannot discount the possibility that circular
muscles may instead be derived from oblique
muscles. This is further complicated by the poor
support (ML) for a Polygordius/Opheliidae clade
and by the presence of circular muscles in P.
appendiculatus and uncertainty about other Poly-
gordius. Transformations showing polygordiids
lacking circular muscles (e.g., ACCTRAN), and the
fact that oblique muscles are also shared by Poly-
gordius and the Ophelininae clade, suggests that
circular muscles may be secondarily derived from
oblique muscles in T. mucronata and other Ophe-
liinae (Fig. 12). An alternative MPR (DELTRAN),
which showed polygordiids as having circular
muscles, suggests that circular muscles were only
lost once in Ophelininae. In this case, oblique
muscles, present in the posterior of Thoracophelia
and Scalibregmatidae, and presumably functioning
to lift the ventral posterior and reduce friction as
the posterior is dragged along, may have moved
more proximally in Armandia to more effectively
prevent radial expansion from longitudinal muscle
contraction during bending. Further research on
the development of musculature is needed to test
Function of Septa in Burrowing
Of all our taxon terminals, only the outgroup
taxon, Notomastus, and Polygordius spp. were
scored with the presence of septa along the entire
body (Fig. 12C). Scoring for Polygordius sp., P.
appendiculatus, and P. lacteus was based on Frai-
pont’s (1887) anatomical study of various polygor-
diid species in which he stated that the body
cavity is separated by septa; presence of septa in
P. jouinae remains unknown. Similarly, Rota and
Carchini (1999) show the presence of intersegmen-
tal septa in the post oesophageal region based on
serial sectioning of Polygordius antarcticus. Under
the MPR shown in Figure 12C, body septa were
lost twice, once in the clade of scalibregmatids and
Arenicola and once for Opheliidae (Fig. 12C). Like
earthworms, the fully septate Notomastus exhibits
retrograde peristalsis (Dorgan, unpublished data).
Polygordius exhibits undulatory rather than peri-
staltic movements (Clark and Hermans, 1976),
similar to aseptate Armandia, but this is clearly
not incompatible with the presence of septa along
the body. Polygordius exhibits more complex move-
ments than Armandia, and it is possible that
septa may provide additional control for maneu-
vering through pore spaces in coarse sands and
gripping grains to prevent being washed out of the
With the exception of Polygordius, the majority
of our ingroup taxa, both peristaltic and undula-
tory, have body cavities that are open and lacking
septa, suggesting that an aseptate body form is
not directly correlated with undulatory vs. peri-
staltic behavior. The function of anterior septa,
however, does differ between the undulatory and
peristaltic burrowers in our study. In the undula-
tory A. brevis, anterior septa are thin and not
muscular. Anterior septa in Armandia likely func-
tion in anterior hydrostatic pressure changes nec-
essary for proboscis eversion (Tzetlin and Zhadan,
2009). Unlike some muscular proboscises (e.g.,
nereids, glycerids), that of A. brevis is not used
during burrowing (Dorgan et al., 2013). The ante-
rior septum in T. mucronata, however, has both
septal circular and longitudinal muscles that con-
tract to inﬂate the head region and are synchron-
ized with the direct peristaltic wave in the body
wall (Fig. 9). Our anatomical analyses reveal that
only one anterior septum/injector organ occurs in
T. mucronata, which differs from the two anterior
septa suggested by McConnaughey and Fox
(1949). A possible explanation for McConnaughey
and Fox’s (1949) description of a second septum is
that the oblique muscles, previously considered
absent in the anterior region (Clark and Hermans,
1976), could easily be misinterpreted as a septum
in serial sections (cf. Fig. 6).
Modiﬁed anterior septa also appear in other
closely related opheliids. Previous studies on
Ophelia rathkei by Brown (1938) and O. bicornis
by Harris (1994) reveal that two anterior septa are
present rather than just a single anterior septum
as exhibited by T. mucronata. Each of the two
septa gives rise to an injector organ, with one sac
being inside the other (Fig. 14). Harris (1994) sug-
gested that the injector organs are “passive,”
although still important in the maintenance of
prostomial coelomic ﬂuid pressure, as the walls of
the injector organs in O. bicornis lacked contract-
ile activity needed to inﬂate/deﬂate and push coe-
lomic ﬂuids from the organ into the head region.
Rather, pressure created by the inﬂation of the
blind capillaries of the prostomial plexuses against
the inelastic cuticle provides additional turgidity
during burrowing (Harris, 1994). Both the struc-
ture and function of the injector organ in T.
mucronata differ from Harris’ (1994) description of
the injector organ in O. bicornis, with that of
Ophelia appearing to be intermediate between the
thin septa of A. brevis and the more muscular and
extended injector organ of T. mucronata.
A modiﬁed anterior septum, termed a gular
membrane, has also been described in Arenicola
(Wells, 1954). Like in T. mucronata, a muscular
anterior septum extends posteriorly to form the
gular membrane, which regulates coelomic ﬂuid
pressure for proboscis eversion during burrowing
and feeding (Wells, 1954; Dales, 1962; Trueman,
1966). The gular membrane and modiﬁed septum
of Thoracophelia appear to be convergent, as other
taxa included in our analysis, including
20 C. J. LAW ET AL.
Journal of Morphology
scalibregmatids, lack such modiﬁed septa. Scali-
bregmatids such as Travisia pupa and S. inﬂatum
have three to –four anterior septa, the most ante-
rior of which is relatively muscular (Dales, 1962).
Dales (1962) suggests that these anterior septa
help regulate ﬂuid pressurization for proboscis
eversion, which does not appear to be used by S.
inﬂatum during burrowing (Dorgan, unpublished
Even though the mechanical responses of muds
and sands to burrowers are substantially different,
muds are elastic materials through which most
worms extend burrows by fracture, whereas sands
are noncohesive granular materials, suggesting
that morphologies and behaviors of burrowing ani-
mals might be distinct between these two habitats,
our data showed that habitat distribution is vari-
able and did not coincide well with burrowing
mode, musculature, or presence of septa (Fig. 11).
The nearly identical morphologies, musculature,
and undulatory burrowing behavior within Opheli-
ninae did not coincide with a single sediment dis-
tribution: A. bilobata and Polyophathalmus are
sand-dwelling whereas Ophelina and the remain-
ing of our Armandia species are mud-dwelling. In
muds, A. brevis does not extend burrows by frac-
ture like most mud-burrowers. Rather its body
undulations displace surﬁcial aggregates of muddy
sediment, a mechanism that seems just as feasible
in surﬁcial granular sands (Dorgan et al., 2013),
perhaps explaining this range of habitats for mor-
phologically similar species. However, even gener-
alizations based on similar morphologies and
musculature that appear to be convergent seem to
be an unreliable indicator of habitat distribution.
For instance, injector organs (or gular membranes)
are found in Arenicola (Wells, 1954) and the Thor-
acophelia/Ophelia clade, suggesting that this con-
vergent feature is an important characteristic for
sand burrowing; however, the presence of a gular
membrane in the mud-dwelling Notomastus (Eisig,
1887) does not follow this pattern. Moreover,
Thoracophelia live in noncohesive, granular beach
sands that differ mechanically from the heteroge-
neous sands in which arenicolids are found, where
hydraulic fracture can result from irrigation, indi-
cating that at least some of these sediments con-
tain enough organic material to behave elastically
(Matsui et al., 2011). Simple characterization of
sand vs. mud may therefore overgeneralize the
mechanical responses of sediments to burrowing
behaviors. Similarities in musculature, lack of
septa, and use of direct peristalsis by Scalibregma-
tidae and Thoracophelia/Ophelia suggest a similar
function and potentially similar habitat, yet mem-
bers of the former taxa inhabit muddy sediments,
while the latter inhabit sandy beach environ-
ments. Linking habitat distribution to morphologi-
cal characters is further complicated by the
presence of both undulatory and peristaltic poly-
chaetes in the same habitat (e.g., Woodin, 1974).
The high variability in habitat seen among our
sampled taxa would increase further with greater
taxonomic resolution. For example, whereas the
four species of Ophelia included in this study are
all found in clean sands, three species in that
genus are found in muds or muddy sands (Bellan
and Dauvin, 1991). Similarly, whereas most scali-
bregmatids are found in very ﬁne muds, Asclero-
cheilus beringianus is found in sandy silts and A.
kudenovi in the rocky intertidal (Blake, 2000), and
species in the genera Axiokebuita and Speleo-
bregma, not included in our analysis, crawl or
swim through coarse gravel and boulders in caves
(Martinez et al., in press). Future comparisons of
habitat and morphological characters across a
broader diversity of annelids are needed to deter-
mine whether these characters are correlated and
may also identify additional convergence events.
The ventral groove appears twice in the termi-
nals assessed here; once in S. inﬂatum and once
in the Opheliidae/Polygordius clade (Fig. 12A).
This ventral groove is restricted to the posterior
region of the body in Opheliinae and in S. inﬂa-
tum. The presence of the ventral groove coincides
well with the absence of circular muscles and the
presence of oblique muscles: in the entire body of
Ophelininae and Polygordius, the ventral groove is
present where circular muscles are absent and
where oblique muscles are present, and in Ophelii-
nae, the ventral groove is present only in posterior
region of the body that also lacks circular muscles
but contain oblique muscles. This trend is incon-
sistent in the peristaltic S. inﬂatum, where circu-
lar muscles remain present in posterior region
despite being characterized by a ventral groove
(Ashworth, 1901). The ventral groove is not as
prominent as that of opheliids, however, and scali-
bregmids do have oblique muscles, contraction of
Fig. 14. Schematic drawing of the anterior of Ophelia bicornis
showing two smaller injector organs (io) instead of just a single
septum/injector organ as seen in T. mucronata (Fig. 7). Adapted
from Harris (1994).
21OPHELIID POLYCHAETE MUSCULATURE AND BURROWING
Journal of Morphology
which has been suggested to form the ventral and
lateral grooves (cf. Clark and Hermans, 1976).
With the exception of some Travisia, e.g., T. fusi-
formis,T. gravieri, and T. hobsonae (Dauvin and
Bellan, 1994), scalibregmatids have not been
described as having a ventral groove. S. inﬂatum
now represents the only other scalibregmatid to be
scored with this feature, and whether other mem-
bers of Scalibregmatidae have ventral grooves
require additional anatomical study.
Interestingly, several other annelid taxa have
ventral grooves, including Terebellidae (Nogueira
et al. 2010), which are primarily tube-dwelling,
suggesting that oblique muscles likely have a func-
tion more similar to those of T. mucronata than A.
brevis.Pisionidens has oblique muscles that, like
those of Armandia, extend diagonally across the
coelomic cavity and create an externally visible
groove, although in Pisionidens the oblique
muscles connect dorsally rather than ventrally,
resulting in a dorsal groove (cf. Fig. 2 of Tzetlin,
1987). Pisionidens lack circular muscles and live
in sandy sediments, often interstitially, and their
morphology indicates that they move similarly to
Armandia. That oblique muscles appear to evolved
independently in several independent clades and
with clearly nonhomologous structures further
supports their important role in locomotion.
Examination of the musculature of A. brevis and
T. mucronata reveals a number of divergences
that lend insight into the functional morphology of
these two species. Our direct comparison identiﬁed
several functionally important differences in mor-
phologies, e.g., the attachment of oblique muscles,
and the orientations of the helical ﬁbers in the
cuticle, in addition to previously described pres-
ence vs. absence of circular muscles. Variability in
musculature that is closely tied to locomotory
function is seen broadly across the taxa included
in our phylogenetic analysis. Although most of our
ingroup taxa lack septa all along the body and use
direct peristalsis, the presence of septa in Polygor-
dius suggest that even this feature is not consist-
ent, but has been lost and regained even among
this limited sampling of polychaetes. Both Thora-
cophelia and Arenicola have modiﬁed anterior
septa that are important in burrowing, and these
appear to be convergent features. Most of our
characters show multiple equally parsimonious
transformations, yet musculature seems to be
closely tied to locomotory function and suggests
that muscle structure is quite variable evolutio-
narily and that divergence of muscle structure
may be key to evolving different behaviors. Sup-
plementary investigation of the associated motor
patterns, however, is required to fully understand
the evolution of both muscular and functional
change (Lauder, 1990). Habitat, characterized here
as sand vs. mud, showed very poor phylogenetic
consistency. This is unsurprising given the vari-
ability in musculature and that seemingly similar
behaviors, e.g., direct peristalsis, are used by bur-
rowers in both sands and muds.
Polychaetes are an abundant and morphologi-
cally diverse group of organisms and serve as
important members of benthic communities (Rouse
and Pleijel, 2001). Our results highlight the need
for better understanding of both the locomotory
functions of musculatures across a broader sam-
pling of polychaetes and of the interactions
between burrowing behaviors and habitat charac-
teristics, for example, comparison of direct peri-
stalsis in muds versus sands, in understanding
the evolution of burrowing behaviors. Linking dif-
ferences in morphologies between related taxa to
their behaviors and habitats will give us greater
context to the evolution and function of burrowing
animals. Uncovering these functional roles allows
better understanding of the relationship between
community dynamics and ecosystem function as
well as interpreting the importance of species
GWR thanks Reinhardt Kristensen, Martin
S!rensen, and Katrine Worsaae for the invitation
to join the Arctic Workshop 2010: “Exploration of a
cold trail: Arctic pieces to the puzzle of Evolution”
and the Board of the Arctic Station for logistical
support. Special thanks to Jos"
e Ignacio Carvajal
for assistance with DNA sequencing, Harim Cha
for accessioning the vouchers into the Scripps
Benthic Invertebrate Collection, and Martin Tres-
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