Syst Parasitol (2015) 90:67–79
DOI 10.1007/s11230-014-9535-y
Molecular analysis of the genera Hapalotrema Looss, 1899
and Learedius Price, 1934 (Digenea: Spirorchiidae) reveals
potential cryptic species, with comments on the validity
of the genus Learedius
Phoebe A. Chapman • Thomas H. Cribb • David Blair •
Rebecca J. Traub • Myat T. Kyaw-Tanner • Mark Flint
Paul C. Mills
•
Received: 9 October 2014 / Accepted: 3 November 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract Adult blood flukes of the genera Hapalotrema Looss, 1899 and Learedius Price, 1934 were
collected from turtles off Queensland and the Hawaiian
Islands. Specimens were identified as Hapalotrema
pambanensis Mehrotra, 1973, H. synorchis Luhman,
1935, H. postorchis Rao, 1976 and Learedius learedi
Price, 1934 on the basis of morphology. No major
morphological differences were found between specimens from this study and previously published descriptions. DNA was also extracted and sequences obtained
using custom spirorchiid-specific primers for the ITS2
and 28S rDNA regions, in order to confirm species
P. A. Chapman (&) M. T. Kyaw-Tanner M. Flint P. C. Mills
School of Veterinary Science, The University of
Queensland, Gatton, Queensland, Australia
e-mail: [email protected]
T. H. Cribb
School of Biological Science, The University of
Queensland, St Lucia, Queensland, Australia
D. Blair
School of Marine and Tropical Biology, James Cook
University, Townsville, Queensland, Australia
R. J. Traub
Faculty of Veterinary and Agricultural Sciences,
University of Melbourne, Parkville, Victoria, Australia
M. Flint
School of Forest Resources and Conservation, University
of Florida, The Florida Aquarium’s Center for
Conservation, Apollo Beach, Florida, USA
identification and investigate phylogenetic relationships. Intraspecific genetic variation was generally
low. However the ITS2 region of H. postorchis and to
a lesser extent that of L. learedi showed considerable
variation between specimens from the Pacific and
Atlantic oceans. Further studies will be required to
determine whether this variation should be considered
inter- or intra-specific. Maximum likelihood phylogenetic analyses were completed for both sequenced
genes. Learedius learedi was unequivocally nested
among species of Hapalotrema, suggesting that the
status of the genus Learedius may need to be reassessed.
Introduction
Spirorchiid flukes inhabit the cardiovascular systems and
organs of marine turtles and are a factor in strandings and
mortalities worldwide (Aguirre et al., 1998; Flint et al.,
2010; Stacy et al., 2010). In Queensland, Australia,
spirorchiids can be found in 75–98% of green sea turtles
(Chelonia mydas Linneaus) presented for necropsy (Flint
et al., 2010; Gordon et al., 1998) and their pathogenicity
has been shown to cause up to 41% of green sea turtle
deaths at some localities (Flint et al., 2010). To date,
molecular characterisation and phylogenetic analysis of
the Spirorchiidae Stunkard, 1921 has received little
attention, despite the potential use of this information in
development of DNA-based diagnostic tests. Currently,
diagnosis of spirorchiid infection relies on faecal examination (lacking in specificity and sensitivity) or invasive
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68
procedures such as biopsy or necropsy. Evidence exists
for the presence of cryptic speciation within currently
recognised spirorchiid species (Stacy, 2008) and this may
be relevant in the development of diagnostic tests. Here,
we identify species that occur in marine turtles from
Queensland waters, explore their phylogenetic relationships and expand the limited sequence database for
spirorchiids. We focus on the genera Hapalotrema Looss,
1899 and Learedius Price, 1934 and compare sequences
from specimens sampled off Queensland and Hawaii
with published sequences from other geographical
regions (i.e. Atlantic Ocean). Available sequences are
used to investigate geographical variation and potential
cryptic speciation.
Materials and methods
Sample collection
Dead turtles were obtained for examination through
government agencies (Queensland Parks and Wildlife
Service, QPWS) or wildlife rehabilitation facilities
including Underwater World (Mooloolaba) and Australia Zoo (Beerwah). Hawaiian turtles were obtained
through the United States Geological Survey National
Wildlife Health Centre. Necropsies were undertaken
using standard procedures (Flint et al., 2009). Major
organs and associated vasculature, including the heart
and great vessels, brain, gastrointestinal tract, liver,
lungs and spleen, were systematically searched for
spirorchiids. Spirorchiids were generally dead on collection and were fixed in 70% ethanol or 10% formalin.
Morphological identification
Spirorchiids for morphological identification were
stained with Mayer’s haematoxylin and destained in
1% hydrochloric acid followed by neutralisation in 1%
ammonia. Specimens were then dehydrated using a
graded ethanol series and cleared with methyl salicylate prior to mounting in Canada balsam. The
drawing was made with the aid of a drawing tube.
Measurements were taken using a calibrated eyepiece
micrometer and are reported in micrometres unless
otherwise stated and presented as the range followed
by the mean in parentheses.
Syst Parasitol (2015) 90:67–79
corresponding whole adults were used. DNA was
extracted using the DNeasy Blood and Tissue kit
(Qiagen, Hilden, Germany) according to manufacturer’s instructions. The 28S and ITS2 regions of the
ribosomal genome were amplified by PCR using familyspecific primers designed by aligning spirorchiid
sequences obtained from GenBank. The 28S region
was amplified using the forward primer L3F (50 -TCG
GGT TGT TTG TGA ATG CAG-30 ) and the reverse
primer L2R (50 -ATC GAT TTG CAC GTC AGA ATC
G-30 ), while the ITS2 region was amplified using the
forward primer IF1 (50 -GAT ATC CTG TGG CCA
CGC CTG-30 ) and the reverse primer IR1 (50 -CGG GTT
GTT TGT GAA TGC AGC-30 ). PCR was performed in
a 25 lL reaction volume, comprising 50ng DNA, 4 lL
dNTP (Qiagen) at a final concentration of 1.25 mM
each, 2.5 lL 109 PCR buffer, 2.5 lL of each forward
and reverse primer at a final concentration of 10 mM,
and 1.25 units HotStar Taq (Qiagen) with the remainder
of the volume made of nuclease-free water. Cycling
conditions to amplify the 28S comprised an initial
activation step of 94°C for 5 min, followed by 40 cycles
of 94°C for 30 s, 60°C for 30 s and 72°C for 2 min with a
final extension step of 72°C for 10 min. The same
conditions were used for ITS2, however with an
annealing temperature of 57°C replacing 60°C.
PCR products were purified and sequenced in both
directions by the Animal Genetics Laboratory (AGL)
within the School of Veterinary Science, The University of Queensland. Where multiple bands occurred,
the desired bands were cut from agarose gels and
purified using the MinElute Gel Extraction Kit
(Qiagen) according to the manufacturer’s recommendations prior to submission to the AGL for sequencing.
Sequence chromatograms were read and analysed
using the software program Finch TV v1.4.0 (Geospiza Inc., Seattle, WA), aligned using BioEdit v7.0.9.0
(Hall, 2005) and Maximum Likelihood trees constructed using MEGA v6 (Tamura et al., 2013) with
one thousand bootstrap replicates specified. The
Kimura 2 (ITS2) and General Time Reversible (28S)
models were used following the use of the MEGA v6
model test function.
Results
Molecular characterisation and phylogeny
Portions of adult worms were removed to provide
genetic material prior to staining, or additional
123
Turtles were obtained from various locations within
Queensland and Hawaiian coastal waters (Table 1).
Chelonia mydas
Chelonia mydas
Hapalotrema
postorchis
Hapalotrema
pambanensis
Eretmochelys
imbricata
Chelonia mydas
Hapalotrema
synorchis
Learedius
learedi
Eretmochelys
imbricata
Host species
Spirorchiid species
Coolum, QLD
Moreton Bay, QLD
Kailua Bay, HI
Waianae, HI
Hauula, HI
Kaneohe Bay, HI
GT7811
SVS11257
UQ1203
UQ1206
UQ1207
UQ1209
Waialua, HI
Waianae, HI
UQ1206
Coolum, QLD
GT7811
UQ1202
Boyne Island, QLD
Buddina Beach, QLD
HBT6711
GT1011
Redland Bay, QLD
HBT5911
Buddina Beach, QLD
Quoin Island, QLD
GT6311
HBT6711
Moreton Bay, QLD
GT5811
Hauula, HI
UQ1207
Boyne Island, QLD
Quoin Island, QLD
Coolum, QLD
GT6311
GT7811
GT1011
Moreton Bay, QLD
Locality
GT5811
Host ID
2012
2012
2011
2011
2011
2011
2011
2012
2012
2012
2012
2011
2011
2011
2011
2011
2012
2011
2011
2011
Year
–
–
43.2
–
35.4
42.5
–
–
–
–
–
68.7
43.2
45.1
90.2
–
–
45.1
43.2
90.2
Host CCL
(cm)
Heart
5
1
1
Lung
Heart
1
1
Heart
Heart
5
2
1
Heart
1
1
2
–
1
2
Lung
Body cavity
Heart
Heart
Heart
Heart
Heart
Heart
3
1
Thrombus
Heart
2
Heart
2
1
Body cavity
Heart
2
7
Pulmonary aorta
1
1
6
2
1
No. of
specimens
Heart
Heart
Heart
Aorta
Thrombus
Pulmonary aorta
Site in host
Table 1 Location and host details of spirorchiid specimens collected from Queensland coastal waters and Hawaii
KM652625
–
KM652619
–
KM652619
KM652618
KM652618
KM652618
–
–
KM652624
KM652624
KM652624
KM652624
KM652616
–
–
–
KM652616
KM652616
KM652616
–
–
KM652617
KM652617
KM652617
GenBank
accession
number
ITS2
KM652626
–
KM652623
–
KM652623
KM652622
KM652622
KM652622
KM652622
–
KM652627
KM652627
KM652627
KM652627
KM652620
–
KM652620
–
KM652620
KM652620
KM652620
–
–
KM652621
KM652621
KM652621
28S
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69
123
*Denotes Queensland Museum slide accession number. Abbreviations: CCL, curved carapace length; QLD, Queensland; HI, Hawaii
–
–
–
1
1979
QM226107*
Dalrymple Island, QLD
–
Heart
–
–
–
1
Heart
1979
–
1979
Badu Island, QLD
QM226106*
QM226104*
Badu Island, QLD
–
Heart
1
KM652626
–
–
–
–
KM652625
1
2
Heart
Heart
1979
1979
–
–
2012
Badu Island, QLD
Badu Island, QLD
QM226102*
QM226103*
UQ1210
Haleiwa, HI
–
Heart
1
KM652626
KM652625
KM652625
1
1
Heart
Heart
–
Kaneohe Bay, HI
–
2012
2012
Waialua, HI
UQ1209
UQ1208
GenBank
accession
number
ITS2
No. of
specimens
Site in host
Host CCL
(cm)
Year
Locality
Host ID
Host species
Spirorchiid species
Table 1 continued
123
KM652626
Syst Parasitol (2015) 90:67–79
28S
70
On the basis of morphology, three species of Hapalotrema and one species of Learedius were identified
from examination of 32 C. mydas, three Eretmochelys
imbricata L., and one Caretta caretta L. (Table 1).
Further L. learedi specimens, originating from islands
off Northern Queensland, Badu (10°060 04.000 S,
142°060 50.700 E)
and
Dalrymple
(9°360 4500 S,
0
00
143°18 16 E), were borrowed from the Queensland
Museum (Table 1). Prevalence of each species is
provided in Table 2. Where identification to species
level was not possible due to poor specimen quality,
infection is noted as unidentified Hapalotrema/Learedius sp. No spirorchiids were recovered from the
single C. caretta examined.
Family Spirorchiidae Stunkard 1921
Genus Hapalotrema Looss, 1899
Hapalotrema postorchis Rao, 1976
Type-host: Chelonia mydas Linneaus.
Type-locality: Pamban, Gulf of Mannar, India.
New records
Host: Chelonia mydas Linnaeus.
Localities: See Table 1.
Sites in host: Pulmonary aorta, right aorta, heart.
New specimens deposited: QM G234563–G234572.
Sequence data: KM652617 (ITS2); KM652621 (28S).
Remarks
Measurements of major morphological features are
provided in Table 3. Hapalotrema postorchis was
described from the heart of C. mydas in India by Rao
(1976) and has subsequently been reported and
described from Hawaiian (Dailey et al., 1993),
Queensland (Cribb & Gordon, 1998) and Brazilian
waters (Werneck et al., 2014). It has been reported on
further occasions, but without detailed descriptions, off
Queensland (Gordon et al., 1998; Platt & Blair, 1998),
Costa Rica (Santoro et al., 2006, 2007), Florida (Stacy,
2008; Stacy et al., 2010) and Taiwan (Chen et al.,
2012). Hapalotrema postorchis can be distinguished
from other species in the genus by the restriction of the
testes to the posterior half of the body. The testes are
also divided into roughly even pre- and post-ovarian
groups. Examination of nine newly-collected specimens (Table 1) revealed no significant morphological
Syst Parasitol (2015) 90:67–79
71
Table 2 Numbers of infected/examined hosts and prevalence (in %) of Hapalotrema spp. and Learedius spp. in the host species
studied
Chelonia
mydas (QLD)
Eretmochelys
imbricata (QLD)
Chelonia
mydas (HI)
Hapalotrema postorchis
3/22 (13.6)
0/3 (0)
1/10 (10)
Hapalotrema pambanensis
10/22 (41.7)
1/3 (33.3)
5/10 (50)
Hapalotrema synorchis
0/22 (0)
2/3 (66.7)
0/10 (0)
Learedius learedi
9/22 (37.5)
0/3 (0)
5/10 (50)
Unidentified Hapalotrema/ Learedius sp.
7/22 (29.2)
1/3 (33.3)
–
Overall infection rate Hapalotrema/ Learedius sp.
14/22 (58.3)
2/3 (66.7)
7/10 (70)
Abbreviations: QLD, Queensland; HI, Hawaii
differences relative to the description given by Cribb &
Gordon (1998); dimensions are generally similar
(Table 3). However, when compared with the measurements reported by Rao (1976) and Dailey et al.
(1993), our specimens are on average smaller and have
fewer testes. The morphometric data published by
Werneck et al. (2014) from off Brazil are based on a
single specimen; the measurements provided are
smaller than those for our specimens in some instances
(e.g. body width, ventral sucker). One new specimen
was examined from off Hawaiian Islands and revealed
no noteworthy differences relative to those from off
Queensland, although not all features could be assessed
due to specimen quality. When compared with the
description of Dailey et al. (1993), the Hawaiian
specimen was shorter (7.9 vs 10.3 mm) but similar in
width.
Molecular characteristics
Two sequences of the ITS2 and partial 28S regions were
obtained for this species (Table 1). No variation was
observed, and the ITS2 was identical to a published
sequence derived from a specimen from off Hawaii
(Stacy, 2008). However, they differed at 6 of 259 bases
relative to the ITS2 sequence for H. postorchis available
on GenBank (GU937895.1, sourced from the northwest Atlantic/Caribbean, USA).
Hapalotrema pambanensis Mehrotra, 1973
Syns Hapalotrema mehrai Rao, 1976; Hapalotrema
dorsopora Dailey, Fast & Balazs, 1993
Type-host: Chelonia mydas Linneaus.
Type-locality: Gulf of Mannar, India.
New records
Host: Chelonia mydas Linnaeus; Eretmochelys imbricata Linnaeus.
Localities: See Table 1.
Sites in host: Heart including ventricles, pulmonary aorta,
vasculature of body cavity and gastrointestinal tract.
New specimens deposited: QM G234538–G234562.
Sequence data: KM652616 (ITS2 – QLD);
KM652620 (28S – QLD); KM652624 (ITS2 – HI);
KM652627 (28S – HI).
Remarks
Hapalotrema pambanensis was described by Mehrotra (1973) from the heart of C. mydas in India. The
original description was brief, providing only body
dimensions and a limited list of key features. A more
complete description of the type-material was supplied by Gupta & Mehrotra (1981). Hapalotrema
pambanensis has subsequently been reported from
Florida (Stacy, 2008; Stacy et al., 2010). Hapalotrema mehrai was described from C. mydas in India
by Rao (1976) and has also been reported from off
Queensland by Cribb & Gordon (1998), who determined that H. pambanensis and H. dorsopora
(described from off Hawaii by Dailey et al. (1993)
were junior synonyms of H. mehrai, a proposition
supported by Platt & Blair (1998) on the basis that the
original description of H. pambanensis by Mehrotra
(1973) did not meet the criteria for availability under
the ICZN. However, Platt (2002) argued that H.
pambanensis was valid and therefore the senior
synonym. Upon examination of the first use of H.
pambanensis by Mehrotra (1973), it appears that
although the description provided is brief, it qualifies
as a description under the ICZN, thereby making H.
pambanensis the senior synonym.
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123
343–514 (457)
314–714 (557)
229–400 (314)
286–457 (350)
Ventral sucker width
Oesophagus length
Ovary length
Ovary width
Abbreviations: QLD, Queensland; HI, Hawaii
343–514 (486)
86–114 (95)
Cirrus-sac width
Ventral sucker length
200–257 (219)
Cirrus-sac length
143–229 (167)
6–8 (8)
No. of post-ovarian testes
114–200 (155)
714–1,543 (965)
7–8 (7)
Width
No. of pre-ovarian testes
Oral sucker width
5.20–9.40 (7.79)
Length (mm)
Oral sucker length
QLD
(n = 9)
Locality
Sample size
–
–
–
–
–
–
–
–
–
–
1,143
–
7.57
HI
(n = 1)
Hapalotrema postorchis
Character / Species
229–600 (441)
229–571 (392)
429–829 (633)
429–543 (489)
343–571 (471)
229–371 (302)
257–400 (324)
57–171 (95)
171–37 (284)
35–54 (45)
429–971 (680)
41–53 (47)
5.31–9.43 (7.06)
QLD
(n = 20)
343–514 (429)
314–514 (448)
543–629 (581)
429–514 (471)
429–486 (457)
314–371 (343)
314–371 (333)
86–114 (100)
229–371 (305)
(51)
486–600 (543)
(53)
5.23–7.09 (6.08)
HI
(n = 5)
Hapalotrema pambanensis
Table 3 Morphometric data for Hapalotrema spp. and Learedius learedi
400–829 (529)
314–457 (386)
1,000–1,457 (1,186)
457–600 (526)
457–600 (531)
229–343 (286)
229–343 (281)
57–86 (76)
200–257 (229)
(27)
914–1,714 (1,333)
29–35 (32)
5.86–7.66 (6.45)
QLD
(n = 9)
Hapalotrema synorchis
200–371 (276)
114–237 (181)
474–1,200 (709)
(237)
(237)
174–284 (214)
142–286 (197)
45–77 (59)
86–348 (174)
0
506–886 (692)
28–36 (33)
1.57–3.71 (2.35)
QLD
(n = 10)
Learedius learedi
143–289 (221)
114–229 (164)
800–1,314 (986)
343–486 (414)
400–514 (440)
143–314 (233)
229–343 (265)
–
–
0
543–857 (711)
27–32 (29)
2.46–4.46 (3.08)
HI
(n = 9)
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Syst Parasitol (2015) 90:67–79
73
Hapalotrema pambanensis can be distinguished
from other species of Hapalotrema by the presence of
well-defined testes which occupy the posterior twothirds of the body, and are present in larger numbers
anterior to rather than posterior to the ovary. Twenty
specimens identified as H. pambanensis were examined from Queensland waters. These specimens were
on average longer than reported for H. mehrai by
Cribb & Gordon (1998) (mean 7.06 vs 5.89 mm,
Table 3), with a correspondingly longer oesophagus
(mean 633 vs 401 lm). They are similar in width to
those reported by Rao (1976) (0.43–0.97 vs 0.69–0.92
mm) however have a smaller oral sucker. No other
notable differences were observed. Measurements of
other major morphological features are provided in
Table 3.
Five Hawaiian specimens were examined (Table 3)
and compared with the description of H. dorsopora by
Dailey et al. (1993). The latter were reported as larger in
most respects. However Cribb & Gordon (1998) reexamined the type-material and found the actual measurements to be smaller than reported, and in accordance
with the values reported here. Hawaiian specimens
prepared and examined during this study were on
average smaller than those from Queensland waters
(Table 3), with an average length of 6.08 mm; however,
no notable structural differences were detected.
Sites in host: Heart, vasculature of lungs and body
cavity.
New specimens deposited: QM G234573–G234581.
Sequence data: KM652618 (ITS2); KM652622 (28S).
Molecular characteristics
Molecular characteristics
Eight ITS2 sequences and nine partial 28S sequences
were obtained from specimens off Queensland, along
with two and four sequences, respectively, from
Hawaiian samples (Table 1). All sequences for the
two regions were identical. The 28S H. mehrai sequence
available on GenBank (AY604708.1, sourced from
Hawaii, USA) was also identical with the sequences
from this study across the 893 compared positions.
Four ITS2 sequences and five partial 28S sequences,
all from turtles off the Queensland coast, were
obtained for this species (Table 1). Neither region
showed intraspecific variation. In maximum likelihood trees inferred for the ITS2 region, a sequence
originally reported as being for H. pambanensis from
C. caretta (GU937894.1; off Florida, USA) grouped
with specimens of H. synorchis collected during this
study. Personal communication with the author (Stacy,
2008) of GU937894.1 regarding these results resulted
in the voucher specimens associated with the sequence
being re-examined, and the GenBank entry amended
to appear as H. synorchis. The two species are
strikingly similar in morphology. However, H. synorchis has been reported only from C. caretta and E.
imbricata (see Smith, 1997b), whereas H. pambanensis is commonly found in C. mydas as well as in C.
caretta and E. imbricata.
Hapalotrema synorchis Luhman, 1935
Syn. Hapalotrema orientalis Takeuti, 1942
Type-host: Caretta caretta Linneaus.
Type-locality: Tortugas, Florida, USA.
New records
Host: Eretmochelys imbricata Linnaeus.
Localities: See Table 1.
Remarks
Hapalotrema synorchis was described from C.
caretta in Florida by Luhman (1935) and has
subsequently been reported from Queensland in C.
caretta by Platt & Blair (1998). This species is
distinguishable from other members of the genus
using the same features described above for H.
pambanensis. However, the testes form a compact
mass as opposed to the distinct testes observed in
H. pambanensis, and the intestinal bifurcation is
located closer to the ventral sucker. Examination of
nine specimens identified as H. synorchis from two
Eretmochelys imbricata collected off southern
Queensland (Table 1) revealed no notable morphological differences relative to the original description. Average length and width were slightly
smaller in this study, and a greater length range
was reported by Platt & Blair (1998). The width of
the cirrus-sac was greater in the present study,
whereas the dimensions of ovaries were smaller
(Table 3).
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Genus Learedius Price, 1934
Learedius learedi Price, 1934
Syn. Learedius europaeus Price, 1934
Type-host: Chelonia mydas Linneaus.
Type-locality: Washington DC, USA.
New records
Host: Chelonia mydas Linneaus.
Localities: See Table 1.
Sites in host: Heart.
New specimens deposited: QM G234582–G234599.
Sequence data: KM652619 (ITS2 – QLD);
KM652623 (28S – QLD); KM652625 (ITS2 – HI);
KM652626 (28S – HI).
Description (Fig. 1)
[Based on ten specimens collected off Queensland
(nine whole, one sectioned); measurements provided
in Table 3.] Body dorsoventrally flattened, elongate,
constricted in midbody, with width c.0.2–0.259 length
of body. Oral sucker terminal. Oesophagus long,
sinuous, extends just anterior to midbody constriction.
Pharynx absent. Oesophageal bulb present at oesophageal/caecal junction. Caeca loop anteriorly after
bifurcation and then extend to posterior end of body.
Ventral sucker large, pedunculate, at level of midbody
constriction, slightly posterior to caecal bifurcation.
Testes numerous, generally clearly distinct from each
other, entirely anterior to ovary. External seminal
vesicle between testes and ovary, transversely elongate. Cirrus-sac well developed; genital pore ventromedial and posterior to ovary. Ovary large and
irregularly lobed; oviduct arises from right side of
ovary. Vitelline follicles distributed from level of
anterior caecal loop to posterior extremity of body;
vitelline reservoir immediately posterior to genital
pore; Laurer’s canal posterior to vitelline reservoir on
right side. Single fusiform egg sometimes present.
Remarks
Learedius learedi was described by Price (1934) from
a C. mydas that died at the National Zoological Park,
Washington, USA. The source of the turtle was not
recorded. Smith (1972, 1997a, b) published reviews of
the sanguinicolids and spirorchiids, and noted that five
123
Fig. 1 Learedius learedi. Adult, ventral view, collected from
the heart of Chelonia mydas at Badu Island, Queensland
(QM226104). Scale-bar: 1 mm
species of Learedius had been named. Of these, Price
(1934) determined that L. europaeus Price, 1934 may
be synonymous with L. learedi, and Mehra (1939)
transferred L. similis Price, 1934 to the genus Monticellius Mehra, 1939. Learedius loochooensis
Syst Parasitol (2015) 90:67–79
Takeuti, 1942 differs from L. learedi only in the shape
of ovary and cirrus-sac, while L. orientalis Mehra,
1939 was considered by Fischthal & Acholonu (1976)
and Dyer et al. (1995a, b) to also be potentially
synonymous with L. learedi, differing only in testicular position and form. Based on these reports, it
appears possible that only one species of Learedius, L.
learedi, should be recognised.
Flukes identified as belonging to the genus Learedius have been previously reported from off northern
Queensland C. mydas by Glazebrook et al. (1989) but
no attempt was made to identify the species. No
detailed morphological characterisation of any Learedius spp. from the west Pacific/Oceania region has
been published, therefore a description has been
provided here.
Measurements from the ten specimens examined
here from Queensland waters (Table 3) did not differ
significantly relative to those reported by Price (1934).
The specimens resemble the description of L. learedi
from off Mexico (Pacific Ocean), published by
Inohuye-Rivera et al. (2004), but are smaller in most
dimensions (Table 3). No significant variations in
form were noted in the nine Hawaiian specimens
relative to those from Queensland waters; however,
Hawaiian specimens were generally larger (Table 3).
75
within the 28S were examined, with one difference
found in a sequence from a specimen off Queensland
and one in a sequence from a specimen off Hawaii.
The GenBank sequence AY604707.1, also originating
from off Hawaii, showed one further difference
relative to these sequences.
Phylogenetic relationships
After alignment of sequences, 259 nucleotide positions were available for analysis in the ITS2 region and
670 within the 28S. Maximum likelihood analysis of
datasets for both regions produced trees of similar
topology (Figs. 2, 3). Species of Hapalotrema and L.
learedi formed a monophyletic clade in which L.
learedi was nested among species of Hapalotrema in
both analyses. For both genes, the positioning of L.
learedi among the Hapalotrema species was supported by bootstrap values of 100, while the placement
of L. learedi as sister to H.pambanensis/H. synorchis
was supported by values of 80 and 87 respectively.
Discussion
Taxonomy
Molecular characteristics
Seven ITS2 and seven partial 28S sequences (two and
five from specimens collected off Queensland and
Hawaii, respectively) were obtained for this species
(Table 1). One base difference (supported by a strong
clear peak on the chromatogram) was noted in one of
the ITS2 sequences from Queensland waters; all other
sequences were identical across the 259 positions
examined. Sequences were compared with those
published by Stacy (2008), who identified two ITS2
variants among 45 sequences for L. learedi. Stacy’s
(2008) Variant A (GenBank accession GU937892.1)
was associated with specimens from off Florida and
the Caribbean and showed differences in two positions
relative to sequences from this study. Variant B of
Stacy (2008) was found in all Hawaiian specimens
sequenced by Stacy, as well as several from off
Florida. It was identical with the present sequences
from off Hawaii, whereas all sequences from specimens from Queensland waters differed from it by a
single base. Eight hundred and ninety-three positions
We found evidence for the presence of three species of
Hapalotrema and one of Learedius in Queensland
waters. All four species match previously published
descriptions in terms of morphology. In some cases,
slight differences in morphometric data were noted
between geographical regions.
Little or no genetic variation was observed
between morphologically identified species from
separate geographical regions in most cases. ITS2
and 28S sequences from Pacific H. pambanensis
showed no variation. However, the ITS2 region of
H. postorchis showed a relatively high level of
variation between the samples from Pacific and
Atlantic oceans. A total of six bases (2.3%) differed
between sequences from the Pacific Ocean (west, off
Queensland; east, off Hawaii) and the North West
Atlantic Ocean/Caribbean, resulting in the formation
of two clades representing these two oceanic regions
(Fig. 2). Stacy (2008) also found evidence of two
distinct ITS2 and mitochondrial cytochrome c
oxidase I (COI) genotypes within H. postorchis,
123
76
Syst Parasitol (2015) 90:67–79
Fig. 2 Maximum likelihood analysis of the relationships of species of Hapalotrema and Learedius learedi based on the ITS2 region.
Sequences from off Florida are sourced from Stacy (2008). Sequences for L. learedi from off Florida/Caribbean and Hawaii/Florida
correspond with two genotypes, Variants A and B (as identified by Stacy, 2008), respectively. Numbers in parentheses denote the
number of replicates where applicable. Scale-bar indicates the number of nucleotide substitutions per site. Abbreviations: QLD,
Queensland; HI, Hawaii; Fl, Florida; CAR, Caribbean
Fig. 3 Maximum likelihood analysis of the relationships of species of Hapalotrema and Learedius learedi based on the 28S rDNA
region. Sequences from Florida sourced from Stacy (2008). Numbers in parentheses denote the number of replicates where applicable.
Scale-bar indicates the number of nucleotide substitutions per site. Abbreviations: QLD, Queensland; HI, Hawaii; Fl, Florida
representing samples from off Hawaii and the North
West Atlantic/Caribbean region. The results shown
here support these findings and extend the data to
include sequences from the Western Pacific.
123
Although L. learedi also demonstrated some variation in the ITS2 between the two oceanic regions, it
was at a lower level than observed for H. postorchis (2
bases, 0.77%). Comparable levels of variation were
Syst Parasitol (2015) 90:67–79
seen between sequences for H. synorchis sampled
from off Queensland and Florida (QU937894.1).
Stacy (2008) observed two genetic variants of L.
learedi. However while variant A was restricted to
the Atlantic region, variant B was found in both the
Pacific and Atlantic Oceans, suggesting that a clear
geographical boundary between genotypes does not
exist in this species.
The variation discussed above may be indicative
of the presence of multiple cryptic species (particularly in H. postorchis), or alternatively may be
interpreted as intraspecific geographical variation.
Nolan & Cribb (2005) discussed the use of the ITS2
rDNA region in taxonomic analyses and noted that
intraspecific variation in this region is often low or
absent. Given that the level of intraspecific molecular variation is likely to be variable between taxa, a
comparison with levels of genetic variation among
similar species may be a useful yardstick when
determining whether separate species should be
recognised (Bray & Cribb, 2014). The level of
variation observed in H. postorchis between geographical regions is greater than that observed in
other species of the same genus in this study (e.g. H.
pambanensis) and may therefore suggest the presence of two cryptic species or subspecies.
The geographical distribution of host species plays
an important role in the exchange of genetic information among parasites (Hoberg & Adams, 2000; Hoberg
& Klassen, 2002) and subsequent potential for speciation. Although the species of marine turtles studied
here generally have a cosmopolitan distribution and
are known to range over thousands of kilometres
(Limpus, 2008a, b, c), the geographical barrier of the
Americas is likely to prevent significant mixing of
Pacific and Atlantic turtle populations (Bowen et al.,
1992; Bowen & Karl, 2007). As a result, the potential
exists for divergence among parasites of turtles from
these two regions. In many cases, genetically identified cryptic species may be separated a posteriori by
detailed morphological re-examination (Bray &
Cribb, 2014). To date, no detailed morphological
description of Atlantic specimens of H. postorchis has
been published, so it is not yet possible to assess the
morphological variation in specimens from the two
regions. One description of L. learedi from the
Atlantic Ocean is available (Werneck et al., 2014).
This study from off Brazil reported no notable
differences between the Atlantic specimens and
77
descriptions published from the east Pacific (off
Mexico), excepting a slightly longer cirrus-sac. Further fine scale morphological studies will be required
in order to properly investigate the possibility of the
presence of cryptic species assemblages.
Phylogeny
An unexpected finding of the phylogenetic analysis
was the nesting of L. learedi among species of
Hapalotrema. Learedius learedi bears similarity in
overall form to species of Hapalotrema, being of
comparable overall shape and size. Previous studies
(Price, 1934; Byrd, 1939; Platt, 2002) separate Learedius from Hapalotrema primarily on the presence
(Hapalotrema) or absence (Learedius) of testes posterior to the ovary. Beyond this, morphological differences appear minor. The eggs of the members of the
two genera are reportedly morphologically indistinguishable (Wolke et al., 1982) and species of both
genera are found primarily in the heart and associated
major vessels. All reports in recent years identify
species of Learedius as L. learedi rather than any of the
four other named Learedius spp. (Aguirre et al., 1998;
Cordero-Tapia A et al. 2004; Santoro et al., 2006, 2007;
Werneck et al., 2006; Stacy et al., 2010; Chen et al.,
2012), none of which have been reported since 1981
(Gupta & Mehrotra, 1981). Poor sample quality and
quantity is a common problem when working with
spirorchiids as samples are often collected opportunistically from dead turtles. As a result, morphological
subtleties can be difficult to recognise. Learedius
learedi may prove to be the only valid species in this
genus, but it is possible that multiple species will be
recognised eventually. Regardless, as L. learedi represents the type-species of the genus, the phylogenetic
information provided here suggests that the genus is
potentially synonymous with Hapalotrema.
Acknowledgements We acknowledge the assistance of staff
at turtle rehabilitation facilities including Underwater World
(Mooloolaba, QLD) and Australia Zoo (Beerwah, QLD) for
providing deceased turtles for parasitological examination.
Additionally, we appreciate the assistance of the United States
Geological Survey National Wildlife Health Centre in providing
access to Hawaiian samples. Thanks also due to Paul Eden for
works with primers and parasite collection, and Brian Stacy for
his comments on this manuscript. This project has been fully
funded by the Australia Research Council Linkage Project Grant
LP110100569.
123
78
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