TEXAS TECH UNIVERSITY

Natural Science Research Laboratory

Occasional Papers

Museum of Texas Tech University

Number 336 3 November 2015

Emendation of Glyphobothrium and Glyphobothriumzwerneri
(Tetraphyllidea) Collected from Rhinoptera bonasus (Myliobatiformes:
Myliobatidae) in Campeche, Mexico, with Details of the Bothridial Suckers

Scott Monks, Griselda Pulido-Flores, and Scott L. Gardner

Abstract

During a study of the helminth parasites of stingrays from the coast of the Yucatan Pen¬
insula of Mexico, four specimens of Glyphobothrium zwerneri were collected from Rhinoptera
bonasus , Cownose Ray, off the Gulf of Mexico coast of Campeche, Mexico and examined. The
scolex of these worms had bothridia with an anterior sucker on each, a feature not reported in
the original description but verified as present on type material. The discovery of this feature
necessitated an emended diagnosis of the genus, an emendment of the description of the species,
and establishment of the family Glyphobothriidae fam. nov. for Glyphobothrium and Dupliciboth-
rium. The details of the anterior sucker and the scolex that are common to Glyphobothrium and
Duplicibothrium and the possible relationships of Tiarabothrium and Echeneibothrium with
Glyphobothrium and Duplicibothrium are presented.

Key words: Campeche, Cownose Ray, Duplicibothrium , Glyphobothriidae new family,
Glyphobothrium zwerneri , Gulf of Mexico, Mexico, Rhinoptera bonasus

Resumen

Durante el estudio de los helmintos de rayas de la costa de la Peninsula de Yucatan, Mexico,
se colectaron cuatro ejemplares de Glyphobothrium zwerneri, parasitos de Rhinoptera bonasus
en la costa de Campeche, en el Golfo de Mexico. El escolex de estos cestodos tiene botridios
con una ventosa anterior cada uno. Esta caracteristica no fue reportada en la descripcion origi¬
nal, pero fue verificada en los ejemplares de la serie tipo. Por el hallazgo de esta caracteristica
se realizo una enmienda a las diagnosis del genero, la descripion de la especie. Se estableco la
familia Glyphobothriidae fam. nov. para Glyphobothrium y la Duplicibothrium. Los detalles
de la ventosa anterior y del escolex son caracteristicas comunes para Glyphobothrium y Du¬
plicibothrium ; ademas, se presenta la posible relacion de Tiarabothrium y Echeneibothrium con
Glyphobothrium y Duplicibothrium.

Palabras claves: Campeche, Duplicibothrium , Glyphobothriidae nueva familia,
Glyphobothrium zwerneri , Golfo de Mexico, Mexico, Rhinoptera bonasus

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Occasional Papers, Museum of Texas Tech University

Introduction

As an initial step of a study of the parasites
of stingrays off the coast of the Yucatan Peninsula,
Mexico, potential hosts were collected and examined
for helminths; partial results have been published by
Pulido-Flores and Monks (2005, 2008, 2014). How¬
ever, many of the cestodes that were collected are only
now being processed and studied. In this material,
specimens of Glyphobothrium zwerneri Williams and
Campbell, 1977 had been collected from one of nine
individuals of the Cownose Ray, Rhinoptera bonasus
(Mitchill, 1815)(Myliobatiformes: Myliobatidae) (see
Pulido-Flores and Monks 2014). The description of G.
zwerneri was based on 53 specimens taken from four of
11 individuals of R. bonasus collected in Chesapeake
Bay, Virginia, U.S.A. (Williams and Campbell 1977).
Since that date, the species has been mentioned in
publications by Brooks and Barriga (1995), Brooks
and Evenhuis (1995), Caira et al. (1999a), Olson and
Caira (1999), Ruhnke et al. (2000), and Ruhnke (2011),
among others, and some of the original material was
reexamined using scanning electron microscopy (SEM)
(Caira et al. 1999a), but collection of new specimens
has not been reported.

This species is particularly interesting because of
the structure of the scolex, which is described as globu¬
lar with four superficial bothridia (i.e., on the surface
of the scolex proper) divided into three longitudinal

rows of loculi that are separated by narrow longitudinal
fissures (Williams and Campbell 1977). Furthermore,
in the original description, those authors mentioned
the presence of a small “pit” that resembled a vestigial
sucker, but they did not include in their figures nor did
they mention the presence of bothridial suckers. This
condition, lack of bothridial suckers, has been noted
by subsequent authors (cited above) and is important
as support for its placement in Serendipeidae.

Contrary to our expectations, specimens of G.
zwerneri that we collected from Mexican stingrays
each had a well-developed accessory sucker on the
apical end of the bothridia. Paratypes of G. zwerneri
(Howard W. Manter Laboratory; HWML-20875) were
examined and the bothridial suckers were found on
each specimen, confirming that this was a character¬
istic of the species and not just of the specimens from
Campeche (see collection locality definition below).
Finding these structures necessitated emendation of
the original description and a reappraisal of family-
level systematics of this group. Herein we provide
new information about the scolex, knowledge of which
made necessary the emendation of the concept of the
genus and the description of the species to include that
information, and the establishment of a new family for
the genus and its congeners.

Materials and Methods

Seven stingrays, Rhinoptera bonasus (Mitchill,
1815) (Myliobatiformes: Myliobatidae) (the Cownose
Ray), were collected by local fishermen (for open sea
localities see Pulido-Flores and Monks 2005); five from
north of Ciudad del Carmen (open ocean side), Isla del
Carmen, Campeche (18°83'N, 91°49'W) (collected
May 2000), one from Champoton, Campeche (19°21'N,
90°54'W) (February 1999), and one from Isla Contoy,
Quintana Roo (20°48'N, 86°47'W) (February 1999).
Subsequent to these, four stingrays were collected in
April 2005 from south of Ciudad del Carmen, Isla del
Carmen, Campeche, in Laguna de Terminos (center
of lagoon located at 18°36'N, 91°33'W). Individual
stingrays were killed and maintained on ice until

necropsied; the intestinal tract was removed and
examined according to Monks et al. (1996). All ecto-
and endohelminths were fixed and then transferred to
70% ethyl alcohol. The Monogenea (ectohelminths)
were reported by Pulido-Flores and Monks (2005),
but the endohelminths remained unprocessed until
the present study. One of the nine stingrays (a male
collected in May 2000) from the region of Ciudad
del Carmen, Campeche, was infected with four
specimens of G. zwerneri. Worms were stained using
Mayer’s carmalum, cleared in methyl malicylate, and
mounted in Canada balsam for examination as whole
mounts. Specimens of G. zwerneri were deposited
in the Coleccion Nacional de Helmintos, IBUNAM,

Monks et al.—Emendation of Glyphobothrium zwerneri

3

Mexico (CNHE-8838) and the Harold W. Manter
Laboratory of Parasitology, University of Nebraska-
Lincoln, U.S.A (HWML-49760). Specimens of
species discussed herein that had been deposited in the
United States National Parasite Collection (USNPC)
were not available for loan and could not be studied.
Measurements are included only when they depart
from those of the original descriptions. Taxonomic
identification of the hosts were based on current
morphological characteristics, although we note that
molecular studies have identified potential cryptic
species in each taxon that have, to date, not been
described formally (Naylor et al. 2012).

The evolutionary transitions leading to
Glyphobothrium Williams and Campbell, 1977
were not presented on the cladogram of Caira et al.
(1999a), so the data matrix from Caira et al. (1999b)
and the cladogram (Caira et al. 1999a; their Fig. 87)
were entered manually into Mesquite (Maddison and
Maddison 2015). The new states for the characters of
the taxa discussed herein were changed, as discussed
below, in the original data set and the cladogram
presented herein was produced using Mesquite.

Results and Descriptions

Based on the type material and the specimens of
Glyphobothrium collected from the waters of the Gulf
of Mexico around Ciudad del Carmen, Campeche,
Mexico, the diagnoses of the genus and of the species,
G. zwerneri , are herein emended and a new family is
established.

Glyphobothrium Williams and Campbell, 1977,
emended

Fig. 1

Diagnosis (following Williams and Campbell
1977 in all details other than the scolex).—Tetraphylidea.
Scolex globular, with four sessile superficial bothridia
(i.e., on the surface of the scolex proper) with a single
column of horizontal loculi, each loculus secondarily
subdivided into three sections by longitudinal muscle
bundles; each bothridium surmounted by anteriorly
directed sucker. Lacking apical organ {sensu Caira et
al. 1999a). Ovary consists of multiple lobes radiating
in all directions from a central ovarian isthmus. Type
and only known species: Glyphobothrium zwerneri
Williams and Campbell, 1977.

Glyphobothrium zwerneri Williams and Campbell,
1977, emended
Fig. 1

Description (following Williams and Campbell
1977 in details other than the scolex).—Tetraphylidea.
Scolex spherical (Fig. 1), with four sessile superficial

bothridia with a single column of 10-12 horizontal
loculi. Each loculus horizontal, secondarily subdi¬
vided into three superimposed quadrangular sections
by parallel longitudinal muscle bundles {sensu Caira
et al. 1999a) (Fig. 1). Horizontal septal borders with
distinct external flaps, continuous, not divided by lon¬
gitudinal muscle bundles. Bothridia surmounted by
single anteriorly positioned accessory sucker, width
112-180 pm (mean = 146 pm ±21 pm; n =13) (Fig.
1). Lacking apical organ but with remnants of vestigial
sucker {sensu Williams and Campbell 1977; Caira et
al. 1999a). Ovary consists of multiple lobes radiating
in all directions from a central ovarian isthmus.

Taxonomic Summary

Host.—Rhinoptera bonasus (Mitchill, 1815)
(Myliobatiformes: Myliobatidae), the Cownose Ray.

Type locality. —Chesapeake Bay, Virginia.

Other localities. —North of Ciudad del Car¬
men, Isla del Carmen, Campeche, Mexico (18°83 5 N,
91°49’ W).

Type specimens. —United States National Parasite
Collection USNPC-74513 (holotype), USNPC-74512
(paratypes); HWML-20875 (paratypes).

Additional specimens. —CNHE-8838 and
HWML-49760 (vouchers; this study).

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Occasional Papers, Museum of Texas Tech University

Figure 1. Glyphobothrium zwerneri Williams and Campbell, 1977 from Rhinoptera bonasus. A. Drawing of
the scolex of a specimen of G. zwerneri (CNHE 8838). B. Photo of the scolex of a specimen of G. zwerneri
(CNHE 8838). Scale bar = 100 pm.

Differential Diagnosis

Williams and Campbell (1977) described Gly¬
phobothrium zwerneri , the only known member of
the genus, and assigned it to the Phyllobothriidae.
This species, from elasmobranchs, has a singular type
of scolex that is spherical with four bothridia that are
fused to the scolex, each divided into horizontal loculi
(Fig. 1). The specimens we collected conform to the
description of G. zwerneri established by Williams and
Campbell (1977) in this and the other main features
included in their description.

Brooks and Barriga (1995) established Serendipi-
dae (now Serendipeidae Brooks and Evenhuis, 1995)
for a putative clade containing Serendip deborahae
Brooks and Barriga, 1995, and species in the genera
Duplicibothrium Williams and Campbell, 1978, and
Glyphobothrium , which they proposed as sister group
to Dioecotaenia Schmidt, 1969. The members of that
putative clade all have fusion of the bothridia (to each
other or to the scolex), testes arranged in two layers in
the proglottids, and some testes that are postovarian
(Brooks and Barriga 1995). Of these three genera, Gly¬
phobothrium is distinguishable from Serendip Brooks
and Barriga, 1995 by having bothridia divided into
loculi rather than bothridia that are entire, scolex that is
spherical rather than composed of flat bothridia directed
anteriorly, and horizontal septal borders rather than
radial septal borders (discussed further by Monks et al.

2015). It should be noted that these relationships have
not been tested yet in a formal phylogenetic analysis.

Given the observations discussed above and the
results of Caira et al. (1999a), Glyphobothrium is most
similar to Duplicibothrium. Duplicibothrium was origi¬
nally diagnosed as having bothridia fused anteriorly
into dorsal-ventral pairs and without pedicles. Both
taxa possess testes that overlap the ovary and extend
into the postovarian space, follicular vitellaria that are
joined dorsally and ventrally (i.e., form a continuous
sleeve around the reproductive organs, sensu Williams
and Campbell, 1978), and a weakly craspedote strobila.
The ovary of Duplicibothrium minutum Williams and
Campbell, 1978 was described as bilobed in dorso-
ventral view, which it clearly is not, although they
went on to say it was “...subdivided into digitiform
processes radiating from ovarian isthmus... ” (Williams
and Campbell, 1978; pg. 836), but clarified this by say¬
ing “The long digitiform lobes of the ovary combined
with its short isthmus made it virtually impossible to
determine if the species has a tetralobed or bilobed
ovary.” (Williams and Campbell, 1978; pg. 837). This
is consistent with their figures and with both Caira et
al. (1999a), who coded the species as having an ovary
that consists of multiple lobes radiating in all direc¬
tions from a central ovarian isthmus (Character 108,
state “4”), and Ruhnke et al. (2000). Glyphobothrium
and Duplicibothrium share these putative synapomor-
phies, but the two can be distinguished by the form of

Monks et al.—Emendation of Glyphobothrium zwerneri

5

the scolex (spherical or bothridia fused into two pairs
forming two earlike outgrowths, respectively).

Until this account, G. zwerneri has not been
reported since the original description and no other
species in the genus have been discovered or described.
Williams and Campbell (1977) tentatively assigned G.
zwerneri to Phyllobothriidae because it had multilocu-
lated bothridia, even though they did not have pedicles.
However, Williams and Campbell (1977) also com¬
mented that the species had features in common with
Dioecotaenia , a similarity that has not gone unnoticed
by recent systematists (see works cited above), but a
relationship not supported by recent cladistic analyses
(Caira el al. 1999a; Caira el al. 2001).

Brooks and Barriga (1995) suggested that Phyl¬
lobothriidae should be divided into two groups: spe¬
cies that have bothridial accessory suckers but lack
bothridial loculi, and species that have bothridial
loculi but lack bothridial accessory suckers. Thus, as
described originally, Glyphobothrium could have been
assigned to the second group. They further noted that
the Triloculariidae was comprised of species that have
bothridial accessory suckers and others without acces¬
sory suckers, and members of Dioecotaeniidae have
bothridial loculi but lack accessory suckers. However,
instead of assigning Glyphobothrium to either of these,
they assigned it to a new family, Serendipeidae, along
with Duplicibothrium and Serendip, because each has
bothridia fused to each other, to the scolex, or to both,
among other putative synapomorphies.

To support placement of Glyphobothrium within
the Phyllobothriidae, Williams and Campbell (1977)
argued that having multiloculated bothridia was more
important in this aspect than having pedunculated
bothridia. Confidence in Phyllobothriidae as a natural
group remained until at least the studies of Brooks
and McLennan (1993), who recognized that the fam¬
ily was not monophyletic. Caira et al. (1999a, 1999b)
empirically tested that hypothesis and also could not
support the monophyly of Phyllobothriidae, nor was
it supported by Healy et al. (2009) or Ruhnke (2011).
Although the idea of what it means to be a “phylloboth-
riid” is still with us, we are at the point, as declared by
Ruhnke (2011; pg. 1), that “Of the valid genera histori¬
cally associated with the family, only Phyllobothrium is
considered to be an unambiguous member of the fam¬

ily.” Caira et al. (1999a) did not assign Glyphobothrium
to that family, although the study at least did identify
it as a “tetraphyllidean (phyllobothriid)” (Caira et al.
1999a; Fig. 87). Based on the new information pre¬
sented herein and the discussions above, we are unable
to assign Glyphobothrium to that family.

With the discovery that Glyphobothrium has a
anterior bothridial sucker, the suite of bothridial char¬
acters of the genus does not fit in Serendipeidae, and
even a cursory comparison with specimens of Serendip ,
suggests that, while Serendipeidae is ideal for Serendip,
the other members of that family now seem rather di¬
vergent from the type genus {Serendip). The members
of Serendip have a scolex that is plate-like, comprised
of four triangular bothridia without pedicels, not fused
to the scolex, bothridia subdivided by septa extending
radially but not dividing the bothridia into distinct
loculi (Brooks and Barriga 1995; Monks et al. 2015).
Furthermore, Dioecotaenniidae, with the single mem¬
ber Dioecotaenia cancellata (Linton, 1890) Schmidt
1969, proposed as putative sister group to Serendipei¬
dae by Brooks and Barriga (1995), has a scolex with
oval bothridia on short pedicels, each divided into 21
loculi, and lacking accessory suckers (Schmidt 1969).
Based on the diagnosis and the suite of characters that
Glyphobothrium possesses, we are convinced that it
now is inappropriate to place G. zwerneri within that
family; as well, a close relationship between Dioeco¬
taenia Schmidt 1969 and Glyphobothrium has not been
supported in phylogenetic analyses (Caira et al. 1999a;
Caira et al. 2001). Particularly, although scolex types
always were divergent in Serendipeidae, the presence
of bothridial suckers marks Glyphobothrium as unac¬
ceptable for that family. We therefore, create a new
family for this group.

Glyphobothriidae fam. nov.

Diagnosis. —Tetraphyllidea. Scolex comprising
four elongate bothridia, bothridia fused lengthwise to
spherical scolex or dorsal and ventral bothridia fused
into two pairs. Each bothridium subdivided into loculi
by horizontal septal borders, not subdivided. Longi¬
tudinal muscle bundles run continuously throughout
the length of acetabulum {sensu Caira et al. 1999a).
Scolex lacking myzorhynchus, hooks, and apical or¬
gan; accessory sucker on bothridia present. Cephalic

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Occasional Papers, Museum of Texas Tech University

peduncle present or absent. Strobila weakly craspedote.
Testes distributed in two irregular dorso-ventral fields,
overlapping with ovary throughout entire length and ex¬
tending into postovarian field. Genital pore in anterior
third of proglottid, marginal to submarginal, irregularly
alternating. Genital atrium present, deep, with vagina
and cirrus sac joined to atrium medially. Cirrus weakly
developed in terminal proglottids. Ovary consists of
multiple lobes radiating in all directions from a central
ovarian isthmus. Uterus weakly developed in terminal
proglottids. Vitellaria follicular, converging in dorsal
and ventral fields, except at level of ovary and cirrus-
sac. Parasites of cownose stingrays (. Rhinoptera spp.).

Type genus.—Glyphobothrium Williams and
Campbell, 1977.

Other genera.—Duplicibothrium Williams and
Campbell, 1978

Differential Diagnosis

In the description of G. zwerneri , Williams and
Campbell (1977) noted that the apex of the scolex had
a “small pit” that resembled a vestigial sucker, but no
other suckers were described or depicted in the figures.
No evidence of a pit or vestigial sucker could be seen on
any of the specimens examined. However, an anterior
accessory sucker is evident on each bothridium of the
paratypes deposited in the HWML and our specimens
from Campeche (Fig. 1). The bothridia of some of
our specimens appeared to be slightly detached at
the anterior end, slightly more than the bothridia of
G. zwerneri and of Prosobothrium sp. in the figures of
Caira et al. (1999a; their figures 59-60). It is unclear

The usefulness of phylogenetic trees as predic¬
tive tools has come to the point of being common
knowledge. Cladograms, as phylogenetic hypotheses,
may be used more often for predictions of biographic
distributions and for identifying potentially important
species in the field of health and the problem of emerg¬
ing infectious diseases (Brooks 2000; Brooks and Ho-
berg 2006; Brooks et al. 2014), but they also contain
predictions about the characters of the taxa included
therein. However, morphologists often do not make use

whether this condition (slightly spread apart) in our
specimens is normal or if it was caused by a possible
but unintentional compression of the scolex during
collection and processing.

Williams and Campbell (1977) considered the
ovary of their specimens to be bi-lobed in dorsal view
and tetra-lobed (i.e., X-lobed) in cross section. How¬
ever, their figures and our observations do not support
that. Caira et al. (1999a) coded Glyphobothrium as hav¬
ing an H-shaped ovary (i.e., two lateral lobes connected
by a median isthmus) in dorsal view, but the figures of
Williams and Campbell (1977), our own observations
of the paratypes and of the new material, confirms that
the ovary consists of multiple lobes radiating in all
directions from a central ovarian isthmus (Character
108, state “4” of Caira et al. 1999a).

In addition to the fusion of the septate bothridia
with each other (in Dioecotaenia , Duplicibothrium ,
and Serendip ), Brooks and Barriga (1995) indicated
that having the testes arranged in two layers and
having post-ovarian testes are synapomorphies for the
Serendipeidae. In that diagnosis, Brooks and Barriga
(1995) established Serendipeidae as having X-shaped
ovaries with lobes that are digitiform—in Serendip ,
the cross-sections that they depicted in their figures
(Brooks and Barriga 1995; Fig. 9) could be interpreted
thusly, but they are better described as consisting of
multiple lobes radiating in all directions from a central
ovarian isthmus (also see Monks et al. 2015), the same
condition that is present in Glyphobothrium (Williams
and Campbell 1977; Fig. 4). As well, Glyphobothrium
also has testes arranged in two layers and post-ovarian
testes.

of this same predictive strength (Brooks et al. 2006) in
the search for and identification of characteristics that
have not been described for particular species.

Our placement of Duplicibothrium in Gly-
phobothriidae is based on the use of the predictive value
of the phylogenetic hypothesis presented by Caira et
al. (1999a) and Caira et al (2001). We first considered
potential synapomorphies for the putative family, and
for this we consulted the most recent and most com-

Monks et al.—Emendation of Glyphobothrium zwerneri

7

prehensive phylogenetic hypothesis for the tetraphyl-
lidean parasites of elasmobranchs using morphological
data, that of the aforementioned studies. Because their
study focused on higher-level relationships, Caira et
al. (1999a) did not discuss many of the features that
identify Glyphobothrium as sister to Duplicibothrium,
or those that might support the recognition of the two
genera in a new family.

In that hypothesis, Glyphobothrium and Du¬
plicibothrium are on a branch (Caira et al. 1999a; figure
87) supported by nine character changes, although these
cannot be demonstrated as synapomorphies because
the clade to which the two taxa are members is a soft
polytomy (unresolved because of lack of included data).
The character states, phrased as they were presented
in Caira et al. (1999a), shared by the two taxa are:
horizontally-oriented loculi that are not subdivided into
separate sub-loculi (Character 26; State 1); the number
of rows of loculi on the acetabulum greater than three
(27; 4); external horizontal septal borders between
loculi on acetabulum not present (29; 0); the cephalic
peduncle present (41; 1) (although more recently
Ruhnke et al. 2000, described D. paulum Ruhnke, Cur¬
ran, and Holbert, 2000 as lacking a cephalic peduncle);
armature on the cephalic peduncle not present (42; 0);
segmental (sic- i.e., proglottid) margins craspedote
only (83; 1); number of layers of testes in cross-section
anterior to cirrus-sac greater than one (91; 1); cirrus sac
shape straight (96; 0); and, the vitelline elements are
distributed circum-segmental (sic- i.e., proglottid, as
above). To these we add the presence of a bothridial
sucker (22; 1) (Fig. 2), longitudinal bundles of muscles
in the bothridia that run continuously throughout the
length of the acetabulum (30; 1) (Fig. 2), and the ovary
consisting of multiple digitiform lobes radiating in all
directions from a central ovarian isthmus (109; 2). Of
course, several of these features are shared with other
members of the putative clade and some with distantly
related taxa as homoplasies. The complete recognition
of synapomorphies for the family must await a study
that will resolve the relationships of the 14 members the
clade (Fig. 3); however, characteristics of the internode/
hypothetical ancestor of the family Glypobothriidae
fam. nov. are presented in Figure 3. The internode
before the branching off point that represents the hypo¬
thetical ancestor of Glypobothriidae (indicated by the
number 89), not an actual entity, is inferred to exhibit

the traits shared among the taxa above it (Glyphoboth¬
rium and Duplicibothrium) (Prendini 2001).

Species of Duplicibothrium currently are diag¬
nosed as not having an apical bothridial sucker, but
there are similarities between the two genera (Gly¬
phobothrium and Duplicibothrium ), at least from the
published descriptions, that led to our reevaluation of
bothridial structures in those taxa. In both the drawings
and the SEM micrographs of Ruhnke et al. (2000) there
is what we interpret as a weakly developed anterior
sucker, and in the micrographs the vertical divisions of
the bothridia appear to be like those in Glyphobothrium.
We examined specimens of D. minutum , D. cairae
Ruhnke, Curran, and Holbert, 2000, and D. paulum
and observed weak but definite anterior suckers on the
bothridia of each specimen (Fig. 2).

Caira et al. (1999a) considered, and argued, that
in Glyphobothrium the division of the horizontal loculi
by the internal muscle bundles is not homologous with
the acetabular boundaries that demark each loculus; i.e.,
they are products of the internal muscle bands that uplift
the floor of the loculi, but are not true boundaries. The
scanning electron micrographs of Caira et al. (1999a)
are not very helpful in that aspect because the scolex
is not in its natural form, but our observation that the
horizontal septal borders in G. zwerneri are not divided
completely by the longitudinal muscles bands supports
that argument.

Ruhnke et al. (2000) described D. cairae and
D. paulum as having horizontal septal borders with
longitudinal divisions. However, the SEMs of Ruhnke
et al. (2000) suggest that the vertical divisions of the
horizontal loculi of species of Duplicibothrium also are
the result of longitudinal bands of muscle because they
do not completely divide the horizontal loculi as true
boundaries. Looking only at their figures (Ruhnke et al.
2000; Figs. 1-16), the resemblance between the both¬
ridia of the members of the two genera is remarkable,
despite the bothridia of one being almost completely
attached to the scolex and the others fused to each
other dorsally and ventrally. The structures dividing the
loculi vertically are difficult to draw in order to show
the same information as in the micrographs; our line
drawing fails completely in the elucidation of these
structures. This resemblance of Duplicibothrium to

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Occasional Papers, Museum of Texas Tech University

Figure 2. Three species of Duplicibothrium Williams and Campbell, 1978 showing the apical sucker and
the longitudinal muscle bands. A. Du. minutum Williams and Campbell, 1977 (HWML-20884); arrows
indicate the postermost limit of the apical sucker. Scale bar = 50 pm. B. Du. cairae Ruhnke, Curran, and
Holbert, 2000 (HWML-15276). C. Du.paulum Ruhnke, Curran, andHolbert, 2000 (HWML-15276); arrow
indicates lateral sucker/loculus, “L” indicates longitudinal muscle band. Scale bar for figure A = 50 pm,
scale bar for figures B and C = 200 pm.

Glyphobothrium provides another putative synapomor-
phy for the recognition of the two taxa as members of
Glyphobothriidae, although that has not been tested
empirically.

Call (2007), in an unpublished thesis, infor¬
mally redescribed D. minutum based on specimens
deposited in the HWML (HWML-20884) and the
USNPC (Holotype, USNPC-74724; paratypes,
USNPC-74725-74726); 13 voucher specimens
identified only as USNPC-00000, LRP-00000, and
KUNHM-00000) in which he confirmed the presence
of longitudinal muscle bundles, and they are depicted
in his SEMs (Call 2007; Fig. 11, pg. 48) (KUNHM=
Kansas University Natural History Museum; LRP =
Lawrence R. Penner Parasitology Collection, Depart¬
ment of Ecology and Evolutionary Biology, University
of Connecticut, Storrs, Connecticut, U.S.A.). The pres¬
ence of longitudinal muscle bundles was confirmed
by Call (2007) in two other species that he described
informally (D. karenae and D. mergacephalum ; nomen
nuda ), but which, unfortunately, have not yet been
published. Ruhnke et al. (2000) described D. cairae
and D. paulum as having longitudinal muscle bundles,
although those authors interpreted these as divisions
that formed longitudinal septal borders. However,
in their SEMs (Ruhnke et al. 2000; Figs. 6, 14) the
longitudinal divisions appear to be homologous with
those of G. zwerneri. Examination of specimens D.
cairae , D. minutum (HWML-20884), and D. pauium
(CNHE-3846-3849; HWML-15275-15278) support

the recognition of the presence of horizontal bothridial
loculi for each species that are undivided by septa and
the presence of longitudinal muscle bundles (Fig. 2),
all synapomorphies for Glyphobothriidae, as do the
figures presented in Ruhnke et al. (2000). The draw¬
ings and SEMs of Call (2007) depict the longitudinal
muscle bands in all of the species included in his
study, including D. minutum. Those figures suggest
that the divisions of the posteriormost loculi may be
divided by the same type of muscular bands rather than
locular walls, although cross-sections of the scoleces
of these species would be necessary for evaluation of
this characteristic.

In the literature there exists ample support for the
existence of the structures mentioned above. Exami¬
nation of specimens of D. minutum (HWML-20884),
D. cairae (CNHE-3846,3847; HWML-15275,15276),
and D. paulum (CNHE-3848, 3849; HWML-15277,
15278) confirmed the presence of a bothridial sucker
(Fig. 2). The SEMs and optical photomicrographs of
plerocercoids of D. minutum presented by de Buron et
al. (2013) (identity confirmed by their use of sequences
of 28S rRNA) makes their similarity with Glyphoboth¬
rium even more obvious. First, the bothridial suckers
(identified by previous authors as apical loculi) are
present (de Buron et al. 2013; Figs. 1-2), and in the
plerocercoid the four bothridia are completely fused
to the scolex rather that just fused in pairs, pointing
to another putative synapomorphy for the members
of the Glyphobothriidae. An accessory sucker, as dis-

Monks et al.—Emendation of Glyphobothrium zwerneri

9

Figure 3. Clade (unresolved; soft polytomy) containing the branch with Duplicibothrium and Glyphobothrium as
sister taxa. The internode/hypothetical ancestor of the family Glyphobothridae n. f. is indicated by the number 89
and the number 65 indicates the hypothetical ancestor of the clade that contains the new family (numbers assigned
by Mesquite). Characters and states, taken from Caira et al. (1999b), are given as the number of the character as
designated in that work, with the character state at the node as a superscript; * indicates values modified according
to the emended descriptions. Figure redrawn from Caira et al. (1999b).

Node 65: 1°, 2°, 3°, 4°, 5 9 , 6 9 , 7 9 , 8 9 , 9 9 , 10 9 , 11°, 12 9 , 13 9 , 14 9 , 15 9 , 16 9 , 17 9 , 18 9 , 19 3 , 20 1 ’ 3 , 21°, 22°, 23 9 , 24 9 , 25°> 9 ,
26°, 27°, 28 9 , 29 9 , 30°, 31 9 , 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42 9 , 43°, 44°, 45°, 46 9 , 47 9 , 48 9 , 49 9 , 50 9 , 51 9 ,
52 9 , 53 9 , 54 9 , 55 9 , 56 9 , 57 9 , 58°, 59 2 , 60 9 , 61 9 , 62 9 , 63 9 , 64 1 , 65 2 , 66 1 , 67 2 , 68 9 , 69 9 , 70 9 , 71 9 , 72 9 , 73 9 , 74 9 , 75 9 , 76 9 , 77 9 ,
78 9 , 79°, 80 2 , 81°, 82°, 83°, 84 2 , 85°, 86°, 87 1 , 88 1 , 89°, 90 2 , 91°, 92°, 93°, 94°, 95°, 96 1 , 97 1 , 98°, 99°, 100°, 101°, 102°,
103°, 104', 105°, 106°, 107°, 108°, 109 1 , 110 2 , 111 0 , 112 3 , 113 1 , 114°, 115 2 , 116°, 117 2 , 118°, 119 1 , 120°.

Node 89: 1°, 2°, 3°, 4°, 5 9 , 6 9 , 7 9 , 8 9 , 9 9 , 10 9 , 11°, 12 9 , 13 9 , 14 9 , 15 9 , 16 9 , 17 9 , 18 9 , 19 3 , 20 0 - 1 ’ 3 , 21°, 22 1 *, 23 9 , 24 9 , 25 9 ,
26 1 , 27 4 , 28 9 , 29°, 30 1 *, 31 0 - 1 ’ 9 , 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41 1 , 42°, 43°, 44°, 45°, 46 9 , 47 9 , 48 9 , 49 9 , 50 9 ,
51 9 , 52 9 , 53 9 , 54 9 , 55 9 , 56 9 , 57 9 , 58°, 59 2 , 60 9 , 61 9 , 62 9 , 63 9 , 64 0 - 1 , 65 2 , 66 1 , 67 2 , 68 9 , 69 9 , 70 9 , 71 9 , 72 9 , 73 0 ’ 9 , 74 2 ’ 9 , 75 9 ,
76 9 , 77 9 , 78 9 , 79°, 80 2 , 81°, 82°, 83 1 , 84 2 , 85°, 86°, 87 1 , 88 1 , 89°, 90 2 , 91 1 , 92°, 93°, 94°, 95°, 96°, 97 1 , 98°, 99°, 100°,
101°, 102°, 103°, 104 1 ,105°, 106°, 107°,108°, 109 2 *, HO 023 , 111 0 ,112 3 , T13 1 , 114 2 ,115 2 ,116°, 117 2 , 118°, 119 1 ,120°.

10

Occasional Papers, Museum of Texas Tech University

cussed above in adults of Glyphobothrium, also could
be identified. These larvae also are similar to adults of
Glyphobothrium in having a spherical scolex, with the
four bothridia completely attached in one specimen and
with the anterior portion extending above the spherical
scolex in another (their Figs 1 and 2, respectively). The
longitudinal muscle bundles are not obvious in these
immature specimens, but that may be because they only
become well developed in the adults.

Caira et al. (1999a) coded G. zwerneri as lacking
an accessory organ (their character 11; 0), having ses¬
sile bothridia (char. 20; 0), possessing bothridia with a
single column of loculi (char. 26; 1) with longitudinal
muscle bundles (char. 30; 1), among other characters
not discussed herein. Their SEMs (Caira et al. 1999a,
Fig. 75) do not provide any other new information
about the structure of the scolex. Although the sagit¬
tal sections presented by Caira et al. (1999a, Fig. 59)
and Williams and Campbell (1977, Fig. 7) support the
recognition of an apical depression, the area between
the bothridia is more open in our specimens (Fig. 1)
but, as mentioned above, we don’t know if this is an
artifact of collection. However, histological studies of
better-fixed specimens would yield further information.

Specimens of Tiarabothrium javanicum Shipley
and Homell, 1906 {species inquirendum , sensu Ruhnke
2011) were depicted as having a spherical scolex and
bothridia with horizontal loculi that are superficially
divided by muscle bundles that begin in the peduncle
(Shipley and Hornell 1906; Plate IV, Figs. 65-68).
These specimens have been lost, but they clearly share
scolex characteristics with Glyphobothrium and should
be assigned to this family, with appropriate taxonomic
changes to the diagnosis of the family, if this can be
confirmed by the collection of new material. Young
(1956), however, thought the genus might be a syn¬
onym of Echeneibothrium van Beneden, 1850 (as did
Euzet 1994). Ruhnke et al. (2000) suggested that T.
javanicum might belong to Serendipeidae. However,

based on Euzet (1994), it is considered to be a species
incertae sedis and Ruhnke et al (2000) and Ruhnke
(2011), in his study of Phyllobothriidae, considered
T. javanicum to be a genus inquirendum. Shipley and
Horned (1906) described T. javanicum as having four
bothridia, mostly fused to the spherical scolex, with the
anterior ends able to separate slightly from the scolex
(i.e., “standing out” [sic]; Shipley and Horned 1906,
pg. 67), similar to members of Glyphobothriidae and
to the specimens of de Buron et al. (2013). Resolution
of the taxonomic status of this genus depends on the
recovery of the original specimens or the finding of
new material.

Of course, with the discovery of new stmctures
and the reinterpretation of known ones, taxa that have
or lack those characteristics should be emended or
redescribed. Some of the specimens of the species men¬
tioned herein that were deposited in museum collections
are not available for study at this time, or they have
been lost, although available material and published
descriptions were more than sufficient to confirm the
presence of the structures used to support our argu¬
ments. It is regrettable is that the specimens studied
by Cad (2007) have not been described formally. This
combination of events makes a full redescription of
Duplicibothrium difficult pending availability of fresh
material, particularly those specimens that have been
lost to science. However, this is not to say that reevalu¬
ation of taxonomic concepts and validity of species
cannot be performed based solely on publications; if we
cannot trust the publications of competent systematists
then there is no reason even to publish, and even less
for peer review, but there is no doubt that arguments
are stronger when specimens have been deposited. As
always, when systematists are struggling to understand
the totality of the concept of a taxonomic unit, the one
obvious conclusion, as least to the authors of this work,
is that ' L Ea qua scimus sunt pars minima eorum, qua
ignoramus' ’ (Linnaeus 1758; pg. 823).

Acknowledgments

Work on what was thought to be a simple range
extension of G. zwerneri began during a Postdoctoral
residency (2011) by SM and GP-F at the HWML, par¬

tially financed by the Patronato Universitario (Gerardo
Soza Castelan, President), Universidad Autonoma del
Estado de Hidalgo, and the Consorcio de Universidades

Monks et al.—Emendation of Glyphobothrium zwerneri

11

Mexicanans (CUMEX) and by the U.S. National Sci¬
ence Foundation Grant No. DBI-0646356 to SLG and
the J. Teague Self Fund of the University of Nebraska-
Foundation. Dr. Gabor R. Racz, Collection Manager,
HWMF, helped with photos and access to material of
that collection. Mutual examination via the internet of

some specimens by the authors was made possible by
Zeiss, USA. M.S. Francisco Zaragoza-Tapia provided
the line drawing of the scolex of G. zwerneri. The
authors thank the two anonymous reviewers for their
valuable contribution to improving the quality of this
work.

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13

Addresses of authors:

Scott Monks

Universidad Autonoma del Estado de Hidalgo
Centro de Investigaciones Biologicas
Apartado Postal 1-10, CP. 42001
Pachuca, Hidalgo, Mexico
scottmonks@hotmail. com

Griselda Pulido-Flores

Universidad Autonoma del Estado de Hidalgo
Centro de Investigaciones Biologicas
Apartado Postal 1-10, CP. 42001
Pachuca, Hidalgo, Mexico
gpflores6@hotmail. com

Scott Lyell Gardner

Harold W. Manter Laboratory of Parasitology,

University of Nebraska State Museum, and School of

Biological Sciences

W-529 Nebraska Hall

University of Nebraska-Lincoln

Lincoln, Nebraska, USA 68588-0512

slg@unl.edu

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