Parasitol Res (2007) 100:1389
–
1394
DOI 10.1007/s00436-006-0423-5
Haakon Hansen & Tor A. Bakke & Lutz Bachmann
Received: 5 September 2006 / Accepted: 21 November 2006 / Published online: 10 January 2007
#
Springer-Verlag 2007
Abstract In recent years, the mitochondrial haplotype diversity of the monogenean ectoparasites Gyrodactylus salaris Malmberg, 1957 on Atlantic salmon and G. thymalli
Ž it ň an, 1960 on grayling has been studied intensively to understand the taxonomy and phylogeography of the two species. According to these studies, neither species can be considered monophyletic, but unfortunately, the geographic sampling has mostly been restricted to Fennoscandia. Only few samples from continental Europe have been analysed, and samples from the United Kingdom have not been included at all. Gyrodactylosis is a notifiable disease in
Europe and is in the UK considered the most important exotic disease threat to wild Atlantic salmon populations. In this study, we report six new mitochondrial haplotypes of
G. thymalli from England, Poland, and Norway detected by sequencing 745 bp of the cytochrome oxidase I gene. The six new haplotypes add five new clades to a neighborjoining dendrogram deduced on the basis of the currently known 44 mitochondrial haplotypes for G. thymalli and G.
salaris.
We conclude that G. thymalli established in the UK along with the immigration of grayling. There is currently no reason to suspect that this parasite is a threat to Atlantic salmon in the UK, although its infectivity to salmon stocks in the UK has not been tested.
Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/
10.1007/s00436-006-0423-5 and is accessible for authorized users.
H. Hansen (
*
)
:
T. A. Bakke
:
L. Bachmann
Department of Zoology, Natural History Museum,
University of Oslo,
P.O. Box 1172 Blindern, N
–
0318 Oslo, Norway e-mail: haakon.hansen@nhm.uio.no
Introduction
The monogenean parasite Gyrodactylus thymalli
Ž it ň an
1960 was first described on grayling ( Thymallus thymallus
L.) from Danubian tributaries in Slovakia (
Ž it ň an
1960
). In recent years, the species has attracted particular attention due to its striking morphological and genetic similarity to
G. salaris Malmberg 1957, a parasite with devastating effect on Atlantic salmon ( Salmo salar L.) populations in
Norway and Russia (see e.g. Johnsen et al.
1999 ; Hansen et
al.
2003 ). The economic importance of salmon has initiated
several comprehensive studies on the taxonomy and diagnostics of G. thymalli and G. salaris .
The morphological differentiation of the two species has been possible to some extent (McHugh et al.
2000
; Shinn et al.
2004 ), but only few populations were included in these
studies. As the morphology of the opisthaptor of Gyrodactylus is also known to be affected by abiotic factors in the macroenvironment such as e.g. temperature (Mo
1991
;
Dávidová et al.
2005
), morphological discrimination of the two species may not be straightforward. The internal transcribed spacers (ITS 1 and 2) of the nuclear ribosomal gene cluster are practically identical in the two species
(Zi ę tara and Lumme
2002 ), although a few populations
with slightly different sequences have been reported
(Lindenstrøm et al.
2003
; Jørgensen et al.
2006
; Robertsen et al.
2007
). Further, it has been suggested that the repeat regions of the intergenic spacer of the nuclear ribosomal gene cluster may be useful for differentiating the two species (Sterud et al.
2002 ; Cunningham et al.
2003 ), but
this has recently been rejected (Hansen et al.
2006 ).
Analyses of the mitochondrial cytochrome oxidase I
(COI) gene have detected high genetic divergence between geographically isolated populations of G. thymalli and G.
salaris (Hansen et al.
2003 ; Meinilä et al.
2004 ) Several

1390 well-supported haplogroups have been described, but the analyses of COI sequences did not reveal evidence for the monophyly of either species. In contrast, the data strongly suggest conspecificity of G. thymalli and G. salaris
(Hansen et al.
2003
; Meinilä et al.
2004 ; Hansen et al.
2006
). Currently, there is no morphological or molecular marker available that can unambiguously discriminate the two species, and differences in host preference (Soleng and
Bakke
2001
; Bakke et al.
2002 ; Sterud et al.
2002
) remain the main argument in favour of considering G. thymalli and
G. salaris valid species (Hansen et al.
2006 ).
The European grayling is widely distributed in northern and continental Europe from the Ural mountains to the
British Isles (Northcote
1995 ), and its phylogeography has
been studied intensively analysing both mitochondrial data and microsatellites (Koskinen et al.
2000
,
2002
; Gum et al.
2005 ). Koskinen et al. ( 2000
) found three main assemblages of mitochondrial haplotypes that diverged before the late
Pleistocene, and it was concluded that Northern Europe was colonized from at least two ancient refugia. Grayling populations in the UK were shown to be closely related to haplotypes from the River Rhine, Germany (Gum et al.
2005
).
Comparative studies on G. thymalli have, so far, mostly relied on grayling from northeastern and nortwestern/ central European populations. Unfortunately, no samples from the UK have so far been included. In a survey of
Gyrodactylus parasites in the UK, Denham and Long
( 1999
) found G. thymalli in six grayling populations in
England. Although the morphology of G. thymalli from the
UK has been studied (McHugh et al.
2000
; Shinn et al.
2004
), no molecular data are available. The UK is officially considered free of G. salaris , but UK authorities are afraid of G. salaris being introduced into the country. In risk assessment studies,
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G. salaris has been considered the most important exotic disease threat to wild Atlantic salmon populations in the UK (Peeler and Thrush
2004 ; Peeler et
al.
2004
,
2006
). A possible conspecificity of G. thymalli and G. salaris would formally mean that G. salaris is already present in the UK, although G. thymalli is considered harmless to Atlantic salmon. The molecular characterization of Gyrodactylus sp. on grayling in the UK is thus of importance to both management authorities and the scientific community.
In this paper, we report the molecular characterization of
G. thymalli from the River Test, Hampshire, England.
Along with other new and previously published sequences from G. thymalli and G. salaris populations in continental
Europe and Fennoscandia, we address the mitochondrial haplotype diversity of G. thymalli and G. salaris with particular focus on the origin of G. thymalli in the UK and on the taxonomic status of G. thymalli and G. salaris .
Materials and methods
The Gyrodactylus specimens reported on in the current study were collected from six localities of grayling in
United Kingdom, Poland, and Norway. In the subsequent analyses, we have also included the cytochrome oxidase I
(CO1) sequences from previous publications (Hansen et al.
2003
,
2006
; Meinilä et al.
2004
), new sequences from populations analysed earlier, and four unpublished sequences from GenBank (AY840222, AY840223,
AY840224, DQ180333). All details about the new samples are listed in Table
1
, and a complete list of all sequences included in this study is provided as supplementary material .
Table 1 Details on the G. thymalli haplotypes presented in this study
Locality River system Drains into
Latitude/ longitude
Sampling date
River Test, United
Kingdom
Solerudbekken,
Lake Mjøsa,
Norway
Lake Murusjøen,
Norway
Fjellfrøselva,
Norway
River Brda near
Rytel, Poland
River Gwda near
Lêdyczek, Poland
Test
Glomma
Ångermanälv Baltic
Målselva
Sea
Nordic
Vistula
Odra/Oder
Sea
Baltic
Sea
Baltic
Sea
English
– channel
Oslofjord 60°45
′
32.76
″
N,
11°4
′
41.52
″
E
64°28
′
27.28
″
N,
14°2
′
17.22
″
E
69°2
′
26.56
″
N,
19°21
′
30.83
″
E
53°45
′
3.25
″
N,
17°46
′
17.23
″
E
53°31
′
57.71
″
N,
16°57
′
2.75
″
E
–
18.05.2003
08.07.2003
2
1
1
17
–
19.07.2003
1
01.11.2003
02.11.2003
1
1
Number of specimen
Mitochondrial haplotype
GenBank accession nos.
ITS CO1
V
R
W
S
T
U
DQ916138
DQ916137
DQ919059
Not sequenced
Not sequenced
Not sequenced
DQ159924
DQ159925
DQ159913
DQ159928
DQ159921
DQ159922
DQ159923

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DNA extraction, PCR amplification of the ITS1, 5.8S,
ITS2 and COI, and DNA sequencing followed protocols described elsewhere (Cunningham et al.
2001
; Matejusová et al.
2001 ; Hansen et al.
2003
). Nucleotide CO1 sequences were aligned using ClustalX (Thompson et al.
1997 ), and
neighbor-joining dendrograms based on Kimura ’ s twoparameter (K2P) method (Kimura
1980
) were constructed using MEGA version 3.0 (Kumar et al.
2004
).
Results and discussion
In this study, we present six new mitochondrial haplotypes that have been identified by sequencing 745 bp of the cytochrome oxidase I (COI) gene from seven Gyrodactylus specimens from six localities (Table
1
). Based on the sequencing of the ITS1 and ITS2, the specimens from River
Test, UK and from Lake Mjøsa (Solerudbekken) and Lake
Murusjøen, Norway were first identified as G. salaris / G.
thymalli . Compared to the
“ standard
”
ITS1 and ITS2 sequence of G. salaris / G. thymalli (GenBank accession number AF328871, Zi ę tara and Lumme
2002 ), the speci-
mens from the River Test and Lake Mjøsa had single nucleotide substitutions in the ITS. The specimen from Test had a substitution at position 177 of ITS2, and the specimen from Lake Mjøsa had a substitution at position 268 of ITS1, respectively. ITS sequences that differ by one or a few nucleotide substitutions from the standard ITS sequence have been reported earlier for some G. salaris / G. thymalli populations (Lindenstrøm et al.
2003
; Jørgensen et al.
2006 ;
Robertsen et al.
2007
).
A compilation of all available COI sequences of G.
salaris / G. thymalli yields a dataset consisting of 44 haplotypes (see supplementary material ). The vast majority of samples in this compilation have been collected in
Fennoscandia; only few specimens originate from Central and East Europe. The alignment of 745 bp of the 44 haplotypes that have been used in this study can be retrieved from ftp://ftp.ebi.ac.uk/pub/databases/embl/align/
ALIGN_001070.dat.
The CO1 haplotype V found in the two specimens from the River Test, Hampshire, UK was unique and found in a separate clade in the neighbor-joining tree presented in
Fig.
1
(average K2P distance to all other known haplotypes,
0.0263). Based on the current dataset, we can therefore exclude that G. thymalli was recently introduced into the
United Kingdom from any other known European G.
thymalli populations. It is more likely that G. thymalli established in the UK along with the immigration of grayling. The mitochondrial haplotypes of grayling in the
UK show a close relationship to grayling from the Rhine
(Gum et al.
2005
), and grayling seem to have followed the same migration pattern that has been suggested for other
1391 freshwater fish species (see e.g. Nesbø et al.
1999 ; Hänfling
et al.
2002 ; Gum et al.
2005 ).
Gyrodactylus salaris / G.
thymalli have, so far, not been recorded on salmon in the
UK. If the assumption is correct that G. thymalli on grayling in the River Test is well established and not the result of a recent introduction, the same argument also supports the assumption that G. thymalli on grayling is currently not spreading to salmon. It is noteworthy that the mitochondrial haplotype observed in G. thymalli on grayling in the River Test is not related to any haplotype detected so far in one of the pathogenic strains of G.
salaris . Accordingly, there is currently no reason to believe that G. thymalli on grayling in the UK is an immediate threat for UK salmon stocks, although its infectivity to salmon stocks in the UK has not yet been tested.
The G. thymalli specimens from the Polish rivers Brda and Gwda were sampled and analysed to increase the knowledge about the mitochondrial DNA variation in samples from Central and Eastern Europe. Both haplotypes (T and U) differ substantially from any other known G. salaris / G. thymalli haplotypes (average K2P distances, 0.0214 and 0.0246, respectively) and can, therefore, be considered representatives of two further clades. At first glance, it seems that the haplotype from
Brda groups together with haplotypes found in G. salaris on salmon, but there is no bootstrap support for such a haplogroup.
Three further G. thymalli specimens from the Norwegian river Målselva and the lakes Mjøsa and Murusjøen were also analysed. The haplotype found in the specimen from
Mjøsa (R) groups together (average K2P distance within the clade, 0.0048) with other haplotypes found in G.
thymalli specimens from the Glomma drainage system in
Norway (clade V in Hansen et al.
2003
), which makes geographic sense. The particular haplotype S found in the
River Målselva (average K2P distance to all other known haplotypes, 0.0226), groups, although with low bootstrap support, within a clade of haplotypes specific for grayling in drainages in the Northern Baltic and the Lake Onega
(clade III of Meinilä et al.
2004 ). River Målselva drains into
the Norwegian Sea, but is located geographically very close to and connected to the river Torneälv that drains into the
Baltic Sea. The topology of the neighbor-joining tree may, therefore, reasonably reflect the colonization of river
Målselva with G. thymalli of Baltic origin. The haplotype
W of the sample from Murusjøen represents a separate clade. This lake drains into the Baltic Sea through the
Swedish river Ångermanälv, which is far south of the River
Torneälv and belongs to a water system that has until now not been screened for G. thymalli .
The new mitochondrial haplotypes of G. thymalli presented in this study did not improve the grouping of clades in the neighbor-joining tree. There is still no support

1392
0.05
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2004
Hansen et al., 2003 Meinilä et al.,
86
A
80
Sal Vefsna
B
C
D
Sal Lizhma
Sal Keret b
Sal Tornio
T
Sal Kumsha
Sal Keret a
F
96
E
Thy Oulanka a
Thy Olanga b
Thy Penninki
Thy Olanga A
Thy Oulanka b
W
Thy Onega
86
93
S
Thy Kitka
Thy Soivio
Thy Livo
Thy Tornio4 S b
85
82
Q
R
Thy Krunnit
Thy Tornio3
Thy Tornio a a Thy Krunnit
Thy Tornio b
98
86
M
I
O
V
I
I
98
91
G
L
P
H
N
V
U
J
K V
IV
IV
G. lavareti , AY225306
I
I
IV
III
III
VI
VI
V
V
G . sp., AY258375
Hansen et al., 2006
I
I
V
V
IV
VI

ƒ
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Fig. 1 Neighbor-joining dendrogram of the 44 mitochondrial haplotypes described for G. salaris and G. thymalli . Only bootstrap values exceeding 80 are shown. Clades of haplotypes as identified by Hansen et al.
2003
, Meinilä et al.
2004 , and Hansen et al.
2006
are indicated.
Haplotypes recovered from parasites on salmon are in grey ; those recovered from parasites on grayling are in black . Haplotype F has, so far, been recovered from parasites on salmon, rainbow trout, and
Arctic charr.
Black arrows point to new haplotypes presented in this study.
Interrupted lines have been shortened by a length identical to the scale bar for basal nodes nor for the monophyly of either G. salaris or G. thymalli . Haplotypes found in particular drainage systems usually group together, whereas haplotypes from parasites of geographically separated drainage systems constitute different clades. This result is in line with previous analyses of the phylogenetic relationships of mitochondrial haplotypes of G. salaris and G. thymalli
(Hansen et al.
2003
,
2006
; Meinilä et al.
2004
). For example, eight different haplotypes that have now been recovered from geographically distant populations of grayling within the Norwegian river Glomma all fall within one well-supported clade (clade V in Hansen et al.
2003 ;
Fig.
1 ).
We conclude that the occurrence of G. thymalli on grayling in the UK is the result of a postglacial introduction together with its grayling hosts and that there is currently no reason to suspect that this parasite is pathogenic to
Atlantic salmon in the UK. However, its infectivity to salmon stocks in the UK remains to be tested. Furthermore, we consider it unlikely that extended geographic sampling of G. salaris and G. thymalli will reveal mitochondrial haplotypes that will allow for a better understanding of the phylogenetic relationships of the mitochondrial haplogroups or of the postglacial phylogeographic pattern of G. salaris and G. thymalli .
Acknowledgement We thank Andy Shinn for providing samples from UK, Stanislaw Cios for collecting material in Poland, and Siw
Ellinor Aagaard for assisting the sampling of the Norwegian
Murusjøen population. Kjetil Olstad helped in collecting the Trysil,
Murusjøen and Målselva samples, and Erik Brenna helped with the parasitological examination of the fish. The project was supported by the Norwegian Research Council
’ s Wild Salmon Programme (project no. 145861/720) and the National Centre for Biosystematics (project no. 146515/420), co-funded by the NRC and the NHM, University of
Oslo, Norway.
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