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Molecular circumscription of new species of Gyrocotyle
Diesing, 1850 (Cestoda) from deep-sea chimaeriform
holocephalans in the North Atlantic
Rodney A. Bray .Andrea Waeschenbach .D. Timothy J. Littlewood .
Odd Halvorsen .Peter D. Olson
Received: 14 November 2019 / Accepted: 1 March 2020 / Published online: 23 April 2020
ÓThe Author(s) 2020
Abstract Chimaeras, or ratfishes, are the only extant
group of holocephalan fishes and are the sole host
group of gyrocotylidean cestodes, which represent a
sister group of the true tapeworms (Eucestoda). These
unique, non-segmented cestodes have been known
since the 1850s and multiple species and genera have
been erected despite a general agreement that the
delineation of species on the basis of morphology is
effectively impossible. Thus, in the absence of
molecular studies, the validity of gyrocotylid taxa
and their specific host associations has remained
highly speculative. Here we report the presence of
Gyrocotyle spp. from rarely-caught deep-sea chi-
maeras collected in the North-East Atlantic, and
describe two new species: G. haffii n. sp. from the
bent-nose chimaera, Harriota raleighana Goode &
Bean, and G. discoveryi n. sp. from the large-eyed
rabbit fish, Hydrolagus mirabilis (Collett). Nuclear
ribosomal sequence data were generated for individual
parasites taken from different host species collected on
different dates and from different localities and were
combined with previously published sequences. Phy-
logenetic analyses supported the recognition of inde-
pendent lineages and clusters, indicative of species,
but were indecisive in recovering the root of the tree in
analyses that included non-gyrocotylid outgroup taxa.
The molecular data reveal variation not reflected in
morphology and point to a complex picture of genetic
divergence shaped by both isolation and migration in
the deep-sea environment.
This article was registered in the Official Register of Zoological
Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:86FB
E97B-A44F-48E5-8600-294119A7F304. This article was
published as an Online First article on the online publication
date shown on this page. The article should be cited by using
the doi number. This is the Version of Record.
This article is part of the Topical Collection Cestoda.
Electronic supplementary material The online version of
this article (doi:https://doi.org/10.1007/s11230-020-09912-w)
contains supplementary material, which is available to autho-
rized users.
R. A. Bray A. Waeschenbach D. T. J. Littlewood
P. D. Olson (&)
Division of Parasites and Vectors, Department of Life
Sciences, Natural History Museum, London SW7 5BD,
UK
e-mail: P.Olson@nhm.ac.uk
O. Halvorsen
Natural History Museum, University of Oslo,
P.O. Box 1172, Blindern, 0318 Oslo, Norway
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Introduction
Holocephalans are deep-sea, cartilaginous fish of the
chondrichthyan subclass Holocephali and its only
order, the Chimaeriformes, commonly known as rat
fishes or ghost sharks. Although highly successful in
the Palaeozoic, resulting in a rich palaeontological
fauna, the group is now represented by only 39 species
in five genera (Inoue et al., 2010). Molecular studies
confirm that the group is the sister taxon of the
Elasmobranchii and three families are recognised, the
Callorhinchidae, Chimaeridae and Rhinochimaeridae,
where the former is the sister group to the latter two
(Inoue et al., 2010). According to a relaxed molecular
clock method employed by Inoue et al. (2010), the
Holocephali arose in the Silurian Period (c.410–447
Ma), the Callorhinchidae diverged from its sister
group in the Jurassic Period (c.161–190 Ma) and the
other families diverged in the mid-Cretaceous Period
(c.98–146 Ma). Licht et al. (2012) expanded the
representation of holocephalans and reported similar
results, with the group diverging between the late
Silurian and the early Devonian. This ancient and
distinctive host group harbours similarly distinctive
parasites, and in this study, we report on those of the
cestode order Gyrocotylidea.
Gyrocotylideans are non-segmented tapeworms
known together with the Amphilinidea as cestodarians
and are the putative sister group of the true tapeworms,
or eucestodes (Waeschenbach et al., 2012). Within the
order, species of the only accepted genus Gyrocotyle
Diesing, 1850 are common, well-reported parasites of
holocephalans. Their systematics have been reviewed
previously (e.g. Colin et al., 1986; Bandoni & Brooks,
1987; Williams et al., 1987; Gibson, 1994) and the
morphological characters for distinguishing species
have been thoroughly discussed and, according to
Williams et al. (1987), been found wanting. Moreover,
in addition to the lack of reliable morphological
characters for species identification, the method of
preservation has been shown to have a significant
effect on their morphology, making it difficult to
provide reliable species identifications post-preserva-
tion (Colin et al., 1986). In the absence of such
characters, it appears that many specimens have been
identified historically on the basis of their host species.
Meanwhile, sequence data are available for only three
putative species.
In this paper we use partial large nuclear ribosomal
subunit (lsrDNA; domains D1–D3) and complete
small nuclear ribosomal subunit (ssrDNA) sequences
to reconstruct a phylogenetic network of Gyrocotyle
spp. from five chimaeras: Chimaera monstrosa L.,
Hydrolagus mirabilis (Collett), Hydrolagus colliei
(Lay & Bennett) (Chimaeridae), Harriotta raleighana
Goode & Bean (Rhinochimaeridae) and Cal-
lorhinchus milii Bory de Saint-Vincent (Callorhinchi-
dae). Based on these data, we recognise and name two
new species of Gyrocotyle from the bent-nose chi-
maera, Ha. raleighana, and the large-eyed rabbit fish,
Hy. mirabilis. These deep-sea host species are not
commonly seen and, although probably not rare at
depths greater than 1,000 metres, are difficult to
capture, requiring specialised equipment and consid-
erable effort.
Materials and methods
Specimen collection, preservation and morphological
study
Chimaeras were collected by RAB using a semi-
balloon otter trawl during three research cruises in the
North-East Atlantic aboard the National Environmen-
tal Research Council research vessel RRS Discovery
(April 2001 and September/October 2002). The fishes
were immediately dissected and worms extracted and
fixed briefly in Berland0s fluid and preserved in 80%
ethanol for morphological examination, and in 100%
ethanol for molecular analysis. In some cases, worms
were cut into separate parts before fixation. Whole-
mounts were stained with Mayer0s paracarmine,
cleared in beechwood creosote and mounted in
Canada balsam. Measurements were made through a
drawing tube on an Olympus BH-2 microscope using a
Digicad Plus digitising tablet and Carl Zeiss KS100
software adapted by Imaging Associates, and are
quoted in micrometres. Where two-dimensions are
given length precedes width. Type- and voucher
material has been submitted to the Natural History
Museum, London, UK (NHMUK). Additional speci-
mens for molecular analysis were obtained from
waters off Norway (Tromsø, Finnmark and Bergen)
and Tasmania, Australia, and published sequences of
three Gyrocotyle species were included in the analy-
ses. A list of taxa including collection information and
sequence accession numbers is given in Table 1.
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286 Syst Parasitol (2020) 97:285–296
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Table 1 Gyrocotyle species collection information and GenBank sequence accessions
Species Label Host Locality Coordinates Depth (m) Collection
date
lsrDNA ssrDNA
Gyrocotyle haffii n. sp. Gcot6 Hariotta raleighana Goode &
Bean
North Atlantic, Goban Spur
a
49.7666°N, -2.3500°W 1,631–1,653 15.iv.2001 MN657006 MN655880
Gyrocotyle discoveryi n. sp. Gcot Hydrolagus mirabilis (Collett) North Atlantic, Goban Spur
a
49.8166°N, -11.7333°W 1,175–1,250 15.iv.2001 MN657011 –
Gcot2 North Atlantic, Goban Spur
a
49.8166°N, -11.7333°W 1,175–1,250 15.iv.2001 MN657003 MN655879
Gcot3 North Atlantic, Goban Spur
a
49.8166°N, -11.7333°W 1,175–1,250 15.iv.2001 MN657004 –
Gcot4 North Atlantic, Goban Spur
a
49.8166°N, -11.7333°W 1,175–1,250 15.iv.2001 MN657005 –
Gcot7 North Atlantic, Porcupine Bight
a
51.2500°N, -11.9166°W 1,200 30.ix.2002 MN657007 MN655881
Gcot8 North Atlantic, Porcupine Bight
a
51.2500°N, -11.9166°W 1,200 30.ix.2002 MN657008 –
Gcot9 North Atlantic, Goban Spur
a
49.8333°N, -12.0833°W 1,360–1,240 19.x.2002 MN657009 –
Gyrocotyle nybelini
(Furhmann, 1931)
Gyrol Chimaera monstrosa L. Coast of Finnmark, Norway
b
na na 2.xi.2001 MN657016 MN655885
Gyrocotyle confusa van der
Land & Dienske, 1968
Gycon Chimaera monstrosa L. Coast of Finnmark, Norway
b
na na 2.xi.2002 MN657014 –
Gyrocotyle urna (Grube &
Wagener in Wagener,
1852)
Gcot11 Chimaera monstrosa L. North Atlantic, Goban Spur
a
49.8333°N, -12.0833°W 1,360–1,240 19.x.2002 MN657010 MN655882
Gurna Coast of Troms, Norway
b
na na 11.xi.2002 MN657013 MN655884
Gurna2 Coast of Finnmark, Norway
b
na na 2.xi.2001 MN657012 –
Gyro2 Unknown fjord, Norway
c
na na 10.xi.2003 MN657015 MN655883
Gyro Unknown fjord, Bergen, Norway
a
na na 6.vi.1996 AF286924.2 AJ228782
Gyrocotyle rugosa Diesing,
1850
Grg Hydrolagus colliei (Lay &
Bennett)
Gulf of Alaska, Torch Bay, Alaska
d
58.3142°N, -136.8036°W na 1997 AF286925.2 AF124455
Gyrocotyle sp. Gyc Callorhinchus milii Bory de
Saint-Vincent
Tasman Sea, off Hobart, Australia
e
na na 1.x.1997 EU343735 EU343741
a
Collector: R. A. Bray;
b
Collector: C. Vollelv;
c
Collector: K. MacKenzie;
d
Collector: G Tyler;
e
Collector: K. Rohde
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Molecular analysis
Ethanol was removed from tissue samples by soaking
in tris-EDTA buffer overnight or by evaporation at
room temperature. Total genomic DNA was extracted
using the DNeasy blood and tissue kit (Qiagen). Partial
lsrDNA (domains D1-D3; c.1,400 bp) was amplified
using LSU5 or ZX-1 ?1200R or 1500R primers; in
the case of Gyrocotyle confusa van der Land &
Dienske, 1968, only a short fragment of 512 bp could
be sequenced from a fragment amplified using primers
900F ?1500R. Complete ssrDNA (c.2,000 bp) was
amplified using WormA and WormB primers for a
subset of the taxa (see Table 1). PCRs were carried out
in 25 ll reaction volumes using puRe Taq Ready-to-go
PCR beads (Amersham Biosciences, Little Chalfont,
UK) and 1 llofa10lM solution of each primer.
Cycling conditions included an initial denaturation for
5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C
denaturation, 30 s at 55 °C(lsrDNA)or54°C(ssrDNA)
and 2 min at 72 °C, followed by a final hold of 7 min at
72 °C. Amplicons were purified using a QIAquick Gel
Extraction Kit or a QIAquick PCR Purification Kit
(Qiagen, Hilden, Germany). Sequencing of both
strands was carried out on an Applied Biosystems
3730 DNA Analyser, using Big Dye version 1.1. PCR
and internal sequencing primers for lsrDNA are given
in Littlewood et al. (2000), except for ZX-1 which was
modified from van der Auwera et al. (1994) as shown
in bold: ACC CGC TGA ATT TAA GCA TAT.
Primers for ssrDNA are given in Littlewood & Olson
(2001). Contigs were assembled using Sequencher 4.5
(GeneCodes Corporation, Ann Arbor, USA) and
manually checked for ambiguous and incorrect base
calls. Sequence identity was verified using the Basic
Local Alignment Search Tool (BLAST) (www.ncbi.
nih.gov/BLAST/).
Phylogenetic analysis
Gene-specific alignments were made for all available
gyrocotylidean sequences along with multiple repre-
sentatives of either caryophyllidean or spathebothri-
idean species (see Olson et al., 2008) used as
outgroups. In addition, a gyrocotylidean-only
sequence alignment was made. Sequences were
aligned with MAFFT version 7.149b (Katoh, 2008)
using 1,000 cycles of iterative refinement and the
genafpair algorithm. Alignment masks for ambigu-
ously aligned positions were generated using
GBLOCKS (Castresana, 2000; Talavera &
Castresana, 2007) using less stringent settings, and
were further refined by eye in Mesquite version 3.5
(Maddison & Maddison, 2018). Alignments with
indicated exclusion sets are available from the NHM
Data Portal at https://doi.org/10.5519/0003327.
MrModeltest2 (Nylander, 2004) was used to select a
model of nucleotide substitution using Akaike&s
information criterion. Data were partitioned into three
character sets: (i) partial lsrDNA; (ii) complete
ssrDNA; and (iii) partial lsrDNA ?complete ssrDNA.
Phylogenetic trees were constructed using Bayesian
inference with MrBayes, version 3.2 (Ronquist &
Huelsenbeck, 2003). Likelihood settings were set to
nst = 6, rates = invgamma, ngammacat = 4 (equivalent
to the GTR?I?G model of evolution). In the com-
bined analysis, parameters were estimated separately
for each gene. Four chains (temp = 0.2) were run for
15,000,000 generations and sampled every 1,000th
generation; 10,000,000 generations were discarded as
‘burn-in&. The ‘burn-in&period was determined as the
point when the average standard deviation of split
frequency values were \0.01.
To comply with the regulations set out in article 8.5
of the amended 2012 version of the International Code
of Zoological Nomenclature (ICZN, 2012), details of
all new taxa have been submitted to ZooBank. For
each new taxon, the Life Science Identifier (LSID) is
reported in the taxonomic summary.
Results
Molecular analyses
Bayesian inference analysis of the combined ssr/
lsrDNA data is shown in Fig. 1, and the results of
analyses of the individual gene partitions are given in
Supplementary Figures S1 and S2. Nodes supported by
\0.95 posterior probabilities were collapsed. Table 2
gives the corrected (GTR?I?G) pairwise distances
estimated for each gene. Analyses including either
caryophyllidean or spathebothriidean outgroup taxa
failed to robustly resolve relationships among the
gyrocotylidean samples, as a consequence of the need
to exclude large numbers of sites that lacked clear
positional homology between ingroup and outgroup
sequences. For example, the lsrDNA alignment
including caryophyllidean outgroup taxa required
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60% of the sites to be excluded whereas an alignment
including only gyrocotylidean sequences required
only 25% and thus included a greater number of
informative characters among the ingroup sequences.
We therefore chose to maximise the number of
informative sites by aligning the gyrocotylidean
sequences to themselves and consequently present
our results as un-rooted networks.
All data partitions showed that the most divergent
taxon by an order of magnitude was G. nybelini
(Fuhrmann, 1931) Bandoni & Brooks, 1987 from C.
monstrosa collected off Norway (Table 2); inset boxes
in Fig. 1and Supplementary Figures S1 and S2 were
required to depict its full branch length relative to the
other taxa. This was followed by Gyrocotyle sp. from
C. milii off Australia. Among the samples collected
from the North-East Atlantic, those from Hy. mirabilis
formed a tight cluster with good separation from the
other branches of the network and are described below
as Gyrocotyle discoveryi n. sp. A specimen from Ha.
raleighana similarly formed a distinct lineage in the
network and has been described below as Gyrocotyle
haffii n. sp. This lineage was connected in an
unresolved node with G. confusa and G. nybelini, also
from C. monstrosa off Norway. Samples identified as
G. urna (Grube & Wagener in Wagener, 1852)
Wagener, 1858 from C. monstrosa showed consider-
able divergence, both among Norwegian fjords and
between these and the North Atlantic, whereas the
sample identified as G. rugosa Diesing, 1850 from Hy.
colliei from the Gulf of Alaska was closer to the G.
urna samples from Norway than they are to the G.
urna sample from the North-East Atlantic. The
possibility that G. urna/G. rugosa represents a single,
variable species is discussed below.
Class Cestoda
Order Gyrocotylidea Poche, 1926
Family Gyrocotylidae Benham, 1901
Genus Gyrocotyle Diesing, 1850
Fig. 1 Unrooted, consensus network of Gyrocotyle species based on combined complete ssr ?partial lsrDNA. Nodes supported by\
0.95 posterior probabilities have been collapsed. Boxed inset shows the topology including the full branch subtending G. nybelini.
Sample labels are given in Table 1
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Table 2 Corrected (GTR?I?G) pairwise distances between samples (above the diagonal: ssrDNA; below the diagonal: lsrDNA)
12345678 91011121314151617
Gyrol Gcot2 Gcot3 Gcot4 Gcot7 Gcot8 Gcot9 Gcot11 Gcot Gurna2 Gurna Gycon Gyro2 Gyro Gcot6 Grg Gyc
1 Gyrol 0.099 0.099 0.104 0.104 0.101 0.101 0.099 0.097 0.12
2 Gcot2 0.253 0 0.01 0.01 0.011 0.011 0.015 0.014 0.027
3 Gcot3 0.252 0.001
4 Gcot4 0.177 0 0.001
5 Gcot7 0.185 0 0.028 0 0.01 0.01 0.011 0.011 0.015 0.014 0.027
6 Gcot8 0.185 0 0.028 0 0
7 Gcot9 0.185 0 0.028 0 0 0
8 Gcot11 0.157 0.029 0.066 0.022 0.024 0.024 0.024 0.005 0.005 0.006 0.013 0.008 0.029
9 Gcot 0.256 0 0.002 0 0.001 0.001 0.001 0.03
10 Gurna2 0.127 0.03 0.076 0.019 0.025 0.025 0.025 0.007 0.031
11 Gurna 0.12 0.03 0.075 0.019 0.025 0.025 0.025 0.007 0.031 0 0 0 0.014 0.003 0.029
12 Gycon 0.273 0.115 0.111 0.08 0.071 0.071 0.071 0.033 0.116 0.043 0.043
13 Gyro2 0.165 0.03 0.067 0.02 0.025 0.025 0.025 0.01 0.031 0.005 0.005 0.043 0 0.014 0.004 0.03
14 Gyro 0.215 0.03 0.03 0.021 0.03 0.03 0.03 0.012 0.031 0.006 0.006 0.063 0.001 0.014 0.004 0.034
15 Gcot6 0.194 0.045 0.045 0.032 0.045 0.045 0.045 0.024 0.045 0.023 0.024 0.045 0.025 0.026 0.016 0.032
16 Grg 0.228 0.038 0.04 0.022 0.039 0.039 0.039 0.017 0.038 0.013 0.013 0.071 0.014 0.014 0.027 0.03
17 Gyc 0.189 0.039 0.047 0.036 0.041 0.041 0.041 0.039 0.04 0.04 0.04 0.092 0.037 0.036 0.05 0.04
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Gyrocotyle haffii n. sp.
Type-host:Harriotta raleighana Goode & Bean
(Chimaeriformes: Rhinochimaeridae), bent-nosed
chimaera.
Type-locality: Goban Spur (49°460N, 12°210W, depth
1,631–1,653 m, 22-23.iv.2001; RRS Discovery Cruise
252, No. 13951/14), North-East Atlantic.
Type-material: Holotype (NHMUK.2019.11.21.1),
paratype (NHMUK.2019.11.21.2).
Site in host: Spiral intestine.
Representative DNA sequences: MN655880
(ssrDNA); MN657006 (lsrDNA, domains D1-D3).
ZooBank registration: The Life Science Identifier
(LSID) for Gyrocotyle haffii n. sp. is urn:lsid:-
zoobank.org:act:7717F9D6-4C9D-4C59-8D0A-
4C5E306B4671.
Etymology: The species is named in honour of our late
colleague and friend Professor Harford ‘Haffi0Wil-
liams in recognition of his contribution to the under-
standing of the Gyrocotylidea.
Description
[Based on a single intact, immature whole worm and
second immature worm from which the central portion
had been excised for molecular analysis; Figs. 2,3].
With characters of the order. Body elongate with
minute annular ridges; no large lateral flap. Length
23,504; greatest width near anterior extremity, 3,131.
Rosette relatively small with few crenulations, 1,943
long. Anterior sucker large, oval, 1,750 91,223.
Reproductive system immature; anlagen commences
2,852 from anterior extremity, 8,191 long; consisting
of a long, narrow patch of stained tissue reaching, and
a branched section passing, towards lateral margin of
worm; apparently opening at c.268 from anterior
extremity. Only other evidence of reproductive organs
is putative vitelline glands scattered around posterior
extremity of anlagen.
Diagnosis
Gyrocotyle haffii n. sp. can be diagnosed from other
congeners on the basis of unique nucleotide characters
in our rDNA alignments (listed as alignment position-
nucleotide): ssrDNA: 218-T, 723-A, 746-C, 747-A,
748-G, 1,158-T, 1,654-G, 1,673-C, 2,115-C; lsrDNA:
612-T, 837-A, 875-T, 1,306-A, 1,395-C, 1,402-A,
1,501-G.
Figs. 2–6 Images and drawings of the new Gyrocotyle species. 2, Photomicrograph of Gyrocotyle haffii n. sp. holotype (NB: the
specimen is immature) ex Harriotta raleighana, Goban Spur (13951/14); 3, Line-drawing of Gyrocotyle haffii n. sp., holotype; 4,
Photomicrograph of Gyrocotyle discoveryi n. sp. holotype ex Hydrolagus mirabilis (Goban Spur; 15063/103a); 5. Line-drawing of
Gyrocotyle discoveryi n. sp., holotype; 6, Gyrocotyle discoveryi n. sp. paratype ex Hydrolagus mirabilis (Goban Spur; 15066/124a).
Scale-bars:10mm
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Remarks
As far as we are aware there is only one previous report
of a gyrocotylidean from Ha. raleighana, the bent-
nose chimaera. Parukhin (1966) reported ‘‘Gyrocoty-
loides nybelini Fuhrmann, 1931’’ in this host from the
South Atlantic Ocean. Parukhin (1968) repeated this
report saying (in translation) ‘‘Found in Callorhynchus
capensis. Two adult parasites were found in two fish.
In addition, six larvae were found in one of them. In
addition to C. capensis, specimens were found in two
Hariota [sic] raleighana. In both cases there were two
specimens. Previously, this species was observed in
the Atlantic in Chimaera monstrosa’’. In addition, it
seems likely that the records of ‘cestode adults0from
Ha. raleighana,Hy. mirabilis and C. monstrosa from
the Rockall Trough off NW Scotland by Mauchline &
Gordon (1984) refer to Gyrocotyle spp.
There is no reliable morphological character to
differentiate this species or indeed any of the gyro-
cotylidean species circumscribed by molecular means.
Therefore, the species is diagnosed by its relatively
marked sequence divergence from those of recognised
species.
Gyrocotyle discoveryi n. sp.
Type-host:Hydrolagus mirabilis (Collett) (Chimaer-
iformes: Chimaeridae), large-eyed rabbitfish.
Type-locality: Goban Spur, North-East Atlantic.
Other localities: Porcupine Seabight (51°090N,
11°550W, depth 1,200 m, 30.xi.2002, RRS Discovery
Cruise, No. 15048-14, 15); Goban Spur (49°490N,
11°440W, depth 1,175–1,250 m, 27.iv.2001, RRS
Discovery Cruise 252, No. 13963/17, 20, 24, 72;
49°410N, 11°530W, depth 1,053–1,077 m, 23.iv.2001,
RRS Discovery Cruise 252, No. 13962/4; 49°470N,
11°580W, depth 1,240–1,360 m, 19.x.2002, RRS
Discovery Cruise D266, No. 15066-124, 125;
51°090N, 11°550W, depth 1200 m, 30.ix.2002, RRS
Discovery Cruise D266, No. 15063-103), North-East
Atlantic.
Type-material: Holotype (NHMUK 2019.11.21.3),
paratypes (NHMUK.2019.11.21.4-13 from Goban
Spur; NHMUK.2019.11.21.14-19 from Porcupine
Sea Bight).).
Site in host: Spiral intestine.
Representative DNA sequences: MN655879 and
MN655881 (ssrDNA); MN657003-MN657005,
MN657007- MN657009, MN657011 (lsrDNA,
domains D1-D3).
ZooBank registration: The Life Science Identifier
(LSID) for Gyrocotyle discoveryi n. sp. is urn:lsid:-
zoobank.org:act:7B028A0B-B8EB-495E-A9F1-
F29DDB60B89A.
Etymology: The species is named after the RRS
Discovery, the NERC research vessel on which the
specimens were collected.
Description
[Based on 17 specimens; Figs. 4–6.] With characters
of the order. Body relatively squat, with deeply
crenulated margins, 8,634–17,586 94,996–8,439
(12,071 96,676), width 36–98 (60)% of length.
Anterior sucker distinct, 1,149–1,621 (1,347) long,
596–1,022 (771) wide. Uterus large, in central part of
body, 1,546–2,996 (2,232) from anterior extremity,
2,453–6,226 (4,533) long, 35–43 (38)% of body
length. Rosette distinct, fairly complex, 1,797–3,092
(2,575) long, junction with soma not clear. Eggs
tanned, operculate, 85–97 939–56 (89 949).
Diagnosis
Gyrocotyle discoveryi n. sp. can be diagnosed from
other congeners on the basis of unique nucleotide
characters in our rDNA alignments (listed as align-
ment position-nucleotide): ssrDNA: 176-G, 782-G,
862-G, 973-C; lsrDNA: 573-A, 800-T, 1,245-C, 1,246-
C, 1,247-G, 1,360-T, 1,369-T, 1,375-C, 1,379-G,
1,382-G, 1,391-A, 1,449-T, 1,468-T, 1,477-T.
Remarks
Mauchline & Gordon (1984) reported a ‘‘cestode’’ in
Hy. mirabilis from the Rockall Trough off NW
Scotland, which is, as far as we are aware, the only
possible record of a Gyrocotyle from this host. Two
species of Gyrocotyle,G. major van der Land &
Templeman, 1968 and G. abyssicola van der Land &
Templeman, 1968, have been reported from its
congener, the small-eyed rabbit fish Hydrolagus
affinis (de Brito Capello) on the edges of the
continental shelf off the eastern coast of Newfound-
land (van der Land & Templeman, 1968). These two
species are illustrated as much more elongate than our
specimens, with less complex lateral wrinkling and
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292 Syst Parasitol (2020) 97:285–296
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small rosettes. The worms were recovered from frozen
hosts, so the gross morphology may well not be of
significance in differentiating these species. Subse-
quently, these two taxa have been reported from the
same host species off south-western Greenland by
Karlsbakk et al. (2002) and, puzzlingly, from a
rhinochimaerine species, the straight-nosed rabbit fish
Rhinochimaera atlantica Holt & Byrne, off the
Scotian Shelf by Hogans & Hurlbut (1984). In the
North-East Atlantic, R. atlantica has not been found
harbouring Gyrocotyle, but it does harbour the stro-
bilate tapeworm Chimaerocestos prudhoei Williams
& Bray, 1984 and a congeneric host, the Pacific
spookfish R. pacifica (Mitsukuri, 1895) also harbours a
species of Chimaerocestos Williams & Bray, 1984
(see Caira et al., 1999,2014). Other records of
Gyrocotyle spp. from Hydrolagus spp. are from the
Pacific Ocean (see Bandoni & Brooks, 1987).
There are no reliable morphological characters to
differentiate this species or indeed any of the gyro-
cotylidean species circumscribed by molecular means.
Therefore, the species is diagnosed by its relatively
marked sequence divergence from those of recognised
species.
Discussion
Colin et al. (1986) made a careful study, based on
1,361 specimens, of the morphological characters used
for distinguishing species of Gyrocotyle and con-
cluded that, due to the great contractibility of the
worms, their reactions to different fixation techniques
and the state of the worms at fixation (e.g. alive, dead,
from frozen hosts), some characters were of limited or
no value, i.e. total length and breadth, the degree of
lateral crenulation, the complexity of the rosette, the
distribution of body spines and the morphology of the
eggs. In effect, they came to the conclusion that
Gyrocotyle spp. could not be reliably identified using
morphological characters. Indeed, these authors con-
sidered Gyrocotyle confusa and Gyrocotyloides nybe-
lini as synonyms of G. urna, and the genus
Gyrocotyloides Fuhrmann, 1930 as synonymous with
Gyrocotyle. When an unidentified ‘‘chimaera cesto-
darian’’ was reported in the Caribbean chimaera
Chimaera cubana Howell Rivero by Bunkley-Wil-
liams & Williams (2004), they reckoned that ‘‘most
authors agree that only one morphologically highly
variable species of cestodarian is found in chimaeras,
but some confusion exists about calling it Gyrocotyle
rugosa Diesing, 1850 or G. urna (Grube & Wagener,
1852)’’.
Despite the difficulties in identifying Gyrocotyle
spp. on the basis of morphology and the controversies
in the literature relative to the specific and generic
status of various morphological forms (e.g. Colin
et al., 1986; Bandoni & Brooks, 1987; Williams et al.,
1987), there have been few investigations utilising
molecular data. Simmons et al. (1972) utilised DNA
hybridisation to confirm the distinctness of four
species of Gyrocotyle from the Pacific Ocean. Bristow
& Berland (1988), Berland et al. (1990) and Bristow
(1992), using electrophoresis, fatty acid chemistry and
biological characteristics, retained three species as
distinct, but did not recognise the genus Gyrocoty-
loides. Olson & Caira (1999) generated an ssrDNA
sequence of Gyrocotyle rugosa (Grg, Table 1), Olson
et al. (2001) added partial lsrDNA data for this species
and generated ssrDNA and lsrDNA data for G. urna
(Gyro, Table 1), and Olson et al. (2008) generated ssr/
lsrDNA sequences for Gyrocotyle sp. (Gyc, Table 1).
In 2007, Waeschenbach et al. completed the lsrDNA
sequence of G. urna (i.e. Gyro) and in 2012 comple-
mented this with large fragments of mitochondrial
genome data (Waeschenbach et al., 2012).
Each of the latter studies were aimed at resolving
higher-level interrelationships of eucestodes and did
not attempt to address the interrelationships or validity
of named species and genera within the order. In this
paper we make a first attempt at this, using ribosomal
sequences from a variety of gyrocotylids, including
some identified by other workers. The inability to root
the resulting trees negated the ability to define clades,
but the results still provide a picture of the relative
genetic distances between samples and how they are
interconnected within the network. The species G.
nybelini,G. confusa and G. haffii n. sp. are part of an
unresolved trichotomy, but are separated by long
branches from the other samples and from each other.
This indicates that G. haffii n. sp. is not conspecific
with G. nybelini, suggesting in turn that Parukhin
(1966,1968) may have been incorrect in reporting G.
nybelini from the host Ha. raleighana.
Gyrocotyle sp. from Callorhynchus milii off
Hobart, Australia, forms another long branch in the
network and, on this basis, is likely to represent an
undescribed species. The hosts of this lineage of
123
Syst Parasitol (2020) 97:285–296 293
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Gyrocotyle are unusual chimaeras known commonly
as ghost sharks or elephant fish and are restricted to the
temperate coasts of Australia and New Zealand. Not
deep-sea dwelling, they constitute part of the fisheries
in both countries and are commonly taken, suggesting
that the collection and study of their gyrocotylid
parasites should make the circumscription of this
putatively novel species easier than for species whose
hosts are rarely obtained.
Gyrocotyle urna, with six samples from C. mon-
strosa clustering with ‘G. rugosa’ from Hydrolagus
colliei off Alaska, may well represent a complex of
similar species, or is a single widespread species in
northern waters with contacts via the deep Arctic
Ocean. There is also distinct divergence between G.
urna specimens from different Norwegian fjords.
These may be as deep as 1,300 m and, like most
fjords, are deeper than the adjacent sea and generally
have a sill at their mouth formed by the glacier0s
terminal moraine. This topology may explain the
apparent isolation of Gyrocotyle populations from
different fjords as indicated by their genetics.
Gyrocotyle discoveryi n. sp., represented by seven
samples from Hy. mirabilis in the North-East Atlantic,
is almost genetically homogeneous. The regions of the
two sites of collection are adjacent, with the Goban
Spur forming the relatively shallow bank at the
southern margin of the Porcupine Seabight. The
samples formed a tight cluster that most likely
represents a clade specific to the large-eyed rabbitfish.
As far as we are aware, the only gyrocotylids
previously reported from the North-East Atlantic are
the three species known from C. monstrosa, the
commonly found holocephalan in the region. These
are G. urna, the most commonly reported, and two
rarer forms, G. confusa and G. nybelini. As stated
previously, the latter species has been housed by
various authors in the genus Gyrocotyloides, but this
has been more commonly accepted as a synonym of
Gyrocotyle (see Gibson, 1994). In contrast, our data
lend some support to the recognition of Gyrocoty-
loides as a distinct genus, given its far greater genetic
divergence in comparison to the other samples,
including those obtained from far reaching parts of
the globe.
Conclusions
The Gyrocotylidea is a small, but common group of
cestodes of holocephalans with a widespread distri-
bution characteristic of a relictual parasite group
restricted to a relictual host group. The mostly deep-
sea habitat of their hosts represents an unusually
stable environment in which this host-parasite system
evolved and likely explains their long-term persis-
tence. Other features of the deep-sea, including fjords,
are likely to have structured these systems in ways that
are not immediately obvious until topography and
mechanisms of isolation are considered, and may
account for why genetic divergences do not strongly
correlate with the degree of geographical separation
among samples. Their systematics has been hitherto
reliant on morphology and host-associations which in
most cases have failed to satisfactorily distinguish
species. It is therefore imperative that molecular
investigations be employed to guide the circumscrip-
tion of natural groups. Our results indicate that
Gyrocotyle comprises not one cosmopolitan, non-
Figs. 7, 8 Host images. 7, Harriota raleighana (longnose
chimaera) suspended in tank for photography. 8, Some of the
Hydrolagus mirabilis (large-eyed rabbitfish) specimens
investigated
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294 Syst Parasitol (2020) 97:285–296
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specific species, but a group of distinct, mostly host-
specific, species that cannot be distinguished by
morphology. Although the recognition of individual
lineages and sequence clusters as species is problem-
atical and probably provisional, establishing these
conceptions now is justified by the fact that the hosts of
the new species are rarely seen; the specimen of the
long-nosed chimaera Ha. raleighana (Fig. 7) is the
only one RAB has examined in over 30 years of
marine trawling, whereas Hy. mirabilis (Fig. 8)is
found in numbers, but only at particular depths and
localities.
Acknowledgements RAB is indebted to the crew and officers
of the NERC research vessel RRS Discovery, to Professor
Imants ‘Monty0Priede of the Oceanlab Aberdeen, Dr Nigel
Merrett and Ms Mary Spencer Jones of the Natural History
Museum (NHM), London, and to other colleagues for help with
the collection of specimens. We thank Roman Kuchta (Institute
of Parasitology, Czech Republic) for bringing accessioning
errors in published gyrocotylidean sequences to our attention.
We thank the NHM0s Sequencing Unit for assistance in gene
sequencing.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
Ethical approval All applicable institutional, national and
international guidelines for the care and use of animals were
followed.
Open Access This article is licensed under a Creative Com-
mons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any med-
ium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative
Commons licence, and indicate if changes were made. The
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the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.
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