639
The incongruence of nuclear and mitochondrial DNA
variation supports conspecificity of the monogenean
parasites Gyrodactylus salaris and G. thymalli
H. HANSEN*, L. MARTINSEN, T. A. BAKKE and L. BACHMANN
Natural History Museum, Department for Zoology, University of Oslo, PO Box 1172 Blindern, N-0318 Oslo, Norway
(Received 7 April 2006; revised 13 May 2006; accepted 13 May 2006; first published online 27 July 2006)
SUMMARY
The monogenean Gyrodactylus salaris Malmberg, 1957 is an economically important parasite on Atlantic salmon whereas
the morphologically very similar G. thymalli Žitňan, 1960 on grayling is considered harmless. Even molecular markers
cannot unambiguously discriminate both species. The nuclear internal transcribed spacer (ITS) sequences are identical in
both species, and although mitochondrial cytochrome oxidase I (COI) sequences show substantial variation, no support for
monophyly of either species is found. Analysis of nucleotide sequences of the intergenic spacer (IGS) have, however, been
interpreted as support for 2 species. Here, IGS and COI sequences from 81 G. salaris and G. thymalli specimens of 39
populations across the species’ distribution range were determined. Mitochondrial diversity was not reflected in the nuclear
marker. Since various 23 bp IGS repeat types usually differ by just one nucleotide and sequences primarily differ in the
number and order of repeat types, alignments may be biased and arbitrary, impeding meaningful phylogenetic analyses.
The hypothesis that parasites on rainbow trout represent hybrids of both species is rejected. The presence or absence of
particular repeat types is not considered informative. We interpret the IGS data as support for G. salaris and G. thymalli
being a single species.
Key words: concerted evolution, genetic diversity, intergenic spacer, molecular taxonomy, ribosomal DNA.
INTRODUCTION
The monogenean flatworm Gyrodactylus salaris
Malmberg, 1957 is an ectoparasite on Atlantic
salmon (Salmo salar L.) and rainbow trout
(Oncorhynchus mykiss Walbaum). G. salaris is found
both in the Baltic Sea and the Atlantic Ocean, but is
considered endemic to the Baltic area. The parasite
was introduced to Norway on several occasions
(Johnsen and Jensen, 1991 ; Johnsen et al. 1999 ;
Hansen et al. 2003), and has since its first record in
1975 been found in more than 40 Atlantic salmon
populations. G. salaris also occurs on Atlantic salmon
in at least 11 rivers of the Swedish west coast and in
several rainbow trout farms and salmon hatcheries in
Fennoscandia (Malmberg and Malmberg, 1993 ;
Koski and Malmberg, 1995 ; Buchmann et al. 2000).
The damage caused by G. salaris has initiated
many studies aimed at unambiguously identifying
the parasite. G. salaris can readily be differentiated
from most closely related species by morphology
(Ergens, 1983 ; Shinn et al. 2000, 2001) and
molecular markers (Cunningham et al. 2001 ;
Matejusová et al. 2001). However, the internal
transcribed spacers ITS1 and ITS2 of the ribosomal
* Corresponding author. Tel : +47 22851823. Fax :
+47 22851837. E-mail : haakon.hansen@nhm.uio.no
gene cluster are identical between G. salaris and
G. thymalli Žitňan, 1960, a harmless parasite
described from grayling (Thymallus thymallus L.), in
a wide geographical range (Zie( tara and Lumme,
2002). ITS1 and 2 have proven useful to delimit the
majority of Gyrodactylus species (Matejusová et al.
2001 ; Zie( tara et al. 2002 ; Zie( tara and Lumme, 2002,
2004). Morphologically the 2 species are also very
similar, but in some instances statistical classification
methods on morphometric measurements could
discriminate them (McHugh et al. 2000 ; Shinn et al.
2004). These studies were, however, based on either
a limited number of samples (McHugh et al. 2000) or
on aquarium-reared specimens (Shinn et al. 2004).
Sterud et al. (2002) and Cunningham et al. (2003)
considered some repeat types and repeat configurations in the intergenic spacers (IGS) of the ribosomal
gene cluster specific for G. thymalli and G. salaris and
suggested the IGS useful for distinguishing them.
Cloned IGS sequences from G. salaris specimens
recovered from rainbow trout, however, contained
typical repeat types of both G. salaris and G. thymalli
(Sterud et al. 2002 ; Cunningham et al. 2003).
The authors therefore suggested that the rainbow
trout parasites may be considered either hybrids
or ancestors of both G. salaris and G. thymalli.
Alternatively, the IGS arrangements of rainbow
trout parasites may just reflect the dynamics of a
Parasitology (2006), 133, 639–650. f 2006 Cambridge University Press
doi:10.1017/S0031182006000655 Printed in the United Kingdom
H. Hansen and others
repetitive region. The lack of IGS sequence variation
in parasites from rivers with recently introduced
parasites led the authors to conclude that IGS is not
suitable for discriminating recently separated populations of G. salaris.
In comparison to the very low intra- and interspecific variability of ribosomal DNA, Hansen et al.
(2003) and Meinilä et al. (2004) detected surprisingly
high sequence diversity of the mitochondrial cytochrome oxidase I (COI) gene. In a comprehensive
study including Gyrodactylus parasites from 32
Norwegian, Swedish and Latvian localities, Hansen
et al. (2003) detected 12 mitochondrial haplotypes
that grouped into 5 well-supported clades. Three
clades consisted of haplotypes found in G. salaris and
2 clades consisted of haplotypes found in G. thymalli.
There was no support for the monophyly of either
G. salaris nor G. thymalli haplotypes. Hansen et al.
(2003) suggested that G. salaris and G. thymalli could
either be 1, 2, or several species, but did not favour
any of these alternatives. Later, Meinilä et al. (2004)
identified additional haplotypes and clades when
studying G. salaris and G. thymalli parasites from
Sweden, Finland, and Russia, but also the extended
data set did not support monophyly of either
species. Thus, Meinilä et al. (2004) were in favour of
G. salaris and G. thymalli being 1 species.
In the current study the repetitive region of the
IGS and the mitochondrial COI from parasites
from Norway, Sweden, Latvia, and one of the type
localities of G. thymalli in Slovakia (Žitňan, 1960)
were sequenced. It was of particular interest to know
whether the mitochondrial DNA variation of G.
salaris and G. thymalli is mirrored in the nuclear
genomes, i.e. whether particular IGS repeats and/or
arrangements correlate to mitochondrial DNA clades.
MATERIALS AND METHODS
Sampling of parasites and species identification
Gyrodactylus specimens were collected from Atlantic
salmon (Salmo salar L.), grayling (Thymallus
thymallus L.) and rainbow trout (Oncorhynchus
mykiss Walbaum) from 35 localities in Norway and
Sweden that drain into the North Sea/Atlantic
Ocean. Gyrodactylus specimens from Baltic salmon
were collected in 3 localities in Sweden and Latvia
that drain into the Baltic Sea (Table 1). In addition,
Gyrodactylus specimens were collected from grayling
from River Hnilec, Slovakia, a tributary to the
Danube. This is one of the type localities of
G. thymalli (Žitňan, 1960).
Classification of parasites was based on the host
species, i.e. Gyrodactylus parasites on salmon and
rainbow trout were considered as G. salaris and
individuals on grayling as G. thymalli. Thus, within
this manuscript G. salaris and G. thymalli refer to
operational taxonomic units rather than to biological
species if not stated otherwise.
640
DNA amplification, cloning and sequencing
DNA was extracted as described by Cunningham
et al. (2001). Approximately 750 bp of the mitochondrial cytochrome oxidase I gene (COI) and
600–700 bp of the intergenic spacer (IGS) of the
nuclear ribosomal DNA cassette were amplified
from the same individuals. The COI was amplified
as 2 overlapping y400 bp segments employing the
primers ZMO1, ZMO2, ZMO3 and ZMO4 (Hansen
et al. 2003). The primer pairs IGSV3 and IGSV4
(Sterud et al. 2002) were used to amplify the IGS
repeat regions of the same individuals. The PCR
reaction was performed in a GeneAmp PCR System
9700 (Applied Biosystems) and contained 1 ml of
DNA template, PCR buffer (Roche), 200 mM
dNTPs, 1 mM of each primer and 1 U Taq polymerase (Roche) in a total volume of 25 ml. Cycling
conditions were identical to those described by
Sterud et al. (2002).
PCR products were purified with QIAquick PCR
Purification Kit (Qiagen) and subsequently sequenced using BigDye chemistry on a ABI 3100
capillary sequencer (Applied Biosystems) using the
same primers as for PCR. In some instances, the IGS
amplicons yielded ambiguous sequences due to
intra-individual heterogeneity of the IGS repeat
regions (Table 1). If so, the purified PCR products
were cloned in the pCR ‘ 4Blunt-TOPO ’ plasmid
vector by means of the Zero Blunt TOPO PCR
Cloning Kit (Invitrogen) and individual clones were
sequenced. In these instances, sampling of clones
was not aimed towards exhaustive assessment of
sequence variation but biased towards clones of
different length. Sequences were proof-read and
edited in Sequencher (Gene Codes Corporation) and
labelled according to the codes used in previous
studies (Collins and Cunningham, 2000 ; Sterud et al.
2002 ; Cunningham et al. 2003). In total, IGS from
81 parasite specimens were sequenced. All sequences
were deposited in GenBank and the respective
Accession numbers are listed in Table 1.
Nucleotide sequence alignment and tree construction
Nucleotide sequences were aligned using ClustalX
(Thompson et al. 1997). Phylogenetic trees of COI
sequences were inferred by neighbour-joining (NJ,
Kimura’s 2-parameter) and maximum parsimony
(MP, unweighted, close-neighbour-interchange,
random addition of trees : 10 replications) analyses
implemented in MEGA 3.0 (Kumar et al. 2004).
Genetic distances were calculated according to
Kimura’s (1980) 2-parameter method. All mitochondrial haplotypes determined in this study as well
as those obtained by Hansen et al. (2003) were
included in the data set, i.e. the mitochondrial
haplotypes G, H, I, and J were included in the tree
construction although they were not detected in this
Nuclear and mitochondrial DNA variation in G. salaris and G. thymalli
study. The COI sequences from Gyrodactylus sp.
from alpine bullhead (AY258375) and G. lavareti
(AY225306) were used as outgroups. Bootstrap
estimates were obtained by running 1000 replicates.
The minimum spanning tree of the 23 bp IGS
repeats was constructed using the program Arlequin
(ver. 2000) (Schneider et al. 2000).
RESULTS
Cytochrome oxidase I
The mitochondrial cytochrome oxidase I (COI)
sequences were determined for 81 specimens of
G. salaris and G. thymalli from 39 localities (listed in
Table 1), and the nomenclature of the COI sequences
follows that introduced by Hansen et al. (2003).
For G. thymalli 5 new haplotypes were observed, but
none for G. salaris.
The topology of the neighbour-joining tree of
the COI sequences is very similar to that published
by Hansen et al. (2003), except for the new clade
VI formed by the new haplotype N from specimens
of one of the type localities of G. thymalli (Fig. 1).
The new haplotype N is characterized by 9 diagnostic nucleotides and differs from the haplotypes of
clades I–V by 2.33–3.61 %. The new haplotypes from
Trysil and Glomma river systems grouped together
with the previously described haplotypes from the
same water systems. The 6 clades, 3 relating to
G. salaris sequences and 3 relating to G. thymalli
sequences, are well supported, but the internal
nodes remain unresolved. Figure 1 also presents
the bootstrap consensus tree of 64 equally parsimonious trees obtained in the maximum parsimony
analysis. For the majority of the obtained MP
trees the clades found in the NJ-analysis were also
recovered.
The ribosomal intergenic spacer (IGS)
IGS sequences. In total 145 IGS sequences were
obtained. Of these, 114 sequences (56 directly sequenced PCR products and 58 clones) were obtained
from 71 G. salaris individuals of 32 localities, and
31 sequences (5 directly sequenced PCR products
and 26 clones) were obtained from 10 G. thymalli
individuals of 7 localities (Table 1). All IGS sequences had the typical structure described earlier
(Collins and Cunningham, 2000 ; Sterud et al. 2002 ;
Cunningham et al. 2003). The 81 bp intervening
sequence was almost invariant ; in only 5 sequences,
3 clones and 2 PCR products, nucleotide exchanges
were detected and in one a 1 bp deletion was detected
(see Table 1). A GpA transition at position 26 and a
CpA transversion at position 33 of the intervening
sequence are shared by both G. salaris individuals
from River Göta älv, Sweden, and are therefore
considered informative.
641
The repeat regions 1 and 2. The repeats of repeat region 1 differ from each other at 1–4 positions, and the
repeats of repeat region 2 differ at 1–7 positions. The
variants A, B, D, E, and F in repeat region 1 and P, Q,
S, R, T, U and V in repeat region 2 are most abundant, and this corresponds well to previously described data (Collins and Cunningham, 2000 ; Sterud
et al. 2002 ; Cunningham et al. 2003). Twenty new
variants, 6 in region 1 and 14 in region 2, were discovered in this study. Some repeat types are more
common in either G. salaris (e.g. repeat type F) or
G. thymalli (e.g. repeat types C and V2), but no repeat
type is found exclusively in all sequences from either
species.
Evolution of the IGS
There are typical IGS arrangements for parasites
from salmon, grayling, and rainbow trout (shaded
in Table 2). IGS arrangements were considered
typical if they can be found in parasites of different
geographical origin and/or can be sequenced directly
as homogeneous PCR products. Typical IGS
arrangements of G. salaris assigned according to
different geographical origin do not result from
biased sampling. IGS arrangements 1 and 2 that are
typical for G. salaris from Norwegian rivers where
the parasite has been recently introduced are also
found in the specimens from Latvia. Typical IGS
arrangements assigned according to direct sequencing of PCR products reflect IGS sequences homogenized within a genome according to the concept
of concerted evolution (Dover, 1982). Although
intragenomic variation cannot be excluded, one
can expect one particular IGS arrangement (PCR
template) that dominates in number. In contrast,
PCR products that could not be sequenced directly
indicate substantial intragenomic IGS variation in
the genomes with no particular IGS arrangement
dominating in number. The IGS sequences of
G. thymalli are intra-individually less homogenized
than those of G. salaris. Direct sequencing of PCR
products was only possible in 5 instances and
standard IGS arrangements are more difficult to
define. However, the most common IGS arrangements of G. salaris (1, 2, 3, and 25 in Table 2) are
longer than those of G. thymalli (30, 31, and 32 in
Table 2).
The cloned IGS sequences from G. salaris from
rainbow trout show some G. thymalli-like features in
repeat region 2 while the arrangements of region 1 are
very similar to those of G. salaris from salmon. This
is consistent with the results of Sterud et al. (2002)
and Cunningham et al. (2003). Interestingly, the
IGS sequences from G. salaris on Atlantic salmon
from River Göta älv, Sweden, also have unusual
repeat arrangements in regions 1 and 2. Repeat region 1 is longer than average and includes repeat type
D that is otherwise characteristic for G. thymalli.
H. Hansen and others
Table 1. List of Gyrodactylus salaris and G. thymalli samples and the respective IGS and mtDNA haplotype characteristics and GenBank1 Accession numbers
Drainage
mtDNA cladehaplotype
Norway – Salmo salar
G. salaris
2002 Røssåga (Nordland)
G. salaris
2002 Vefsna (Nordland)
G. salaris
2002 Byaelva (Nord-Trøndelag)
Atlantic
Atlantic
Atlantic
I–A
I–A
I–A
G. salaris
G. salaris
1992
2002
Atlantic
Atlantic
I–A
I–A
G. salaris
G. salaris
2001
2001
Atlantic
Atlantic
G. salaris
G. salaris
G. salaris
2001
2001
2001
G. salaris
2001
G. salaris
G. salaris
G. salaris
2001
2001
2000
Species
Year
Locality (county)
Ogna (Nord-Trøndelag)
Batnfjordselva
(Møre og Romsdal)
Driva (Møre og Romsdal)
Litledalselva
(Møre og Romsdal)
Usma (Møre og Romsdal)
Henselva (Møre og Romsdal)
Innfjordselva
(Møre og Romsdal)
Rauma (Møre og Romsdal)1
IGS
sequencing
IGS haplotype
(see Table 2)
IGS Acc. nos.
PCR products
PCR products
PCR products
1
1
1
AY490445
AY490517, AY490518
AY490439–AY490442
PCR products
PCR products
1
1
AY490438
AY490514–AY490516
I–A
I–A
AY486494
AY486529, AY486530
AY258346, AY486489–
AY486491
AY486488
AY258355, AY486527,
AY486528
AY486538, AY258342
AY486533, AY486534
PCR products
PCR products
1
1
AY490533, AY490534
AY490526, AY490527
Atlantic
Atlantic
Atlantic
I–A
I–A
I–A
AY486495, AY486496
AY486535, AY486536
AY486492, AY486493
PCR products
PCR products
PCR products
1
1
1
AY490446, AY490447
AY490528, AY490529
AY490443, AY490444
Atlantic
I–A
PCR products
1
AY490530–AY490532,
AY490538, AY490539
Atlantic
Atlantic
Atlantic
I–B
I–B
III – F
Clones
PCR products
PCR products
2, 16, 22
1, 2
1
AY490448–AY490454
AY490512, AY490513
AY490455–AY490457
Atlantic
Atlantic
III – F
III – F
AY258338, AY258339
AY486537,
AY486542–AY486543
AY486497
AY486525, AY486526
AY258371–AY258372,
AY486498
AY486519, AY486520
AY486539–AY486541
PCR products
PCR products
1
1
AY490502, AY490503
AY490535–AY490537
AtlanticU
AtlanticU
AtlanticU
V–K
V–L
V–M
AY486549
AY486552, AY486553
AY486547, AY486548
Clones
Clones
PCR products
41–45
30, 34
32
AY490411–AY490417
AY490429–AY490436
AY490409, AY490410
AtlanticU
V–O
AY486550
Clones
33, 36, 37, 40
AY490418–AY490424
642
Signaldalselva (Troms)
Skibotnelva (Troms)
Lærdalselva
(Sogn og Fjordane)
G. salaris
2002 Drammenselva (Buskerud)
G. salaris
Lierelva (Buskerud)
Norway – Thymallus thymallus
G. thymalli 2002 Åsta (Oppland)
G. thymalli
Rena (Hedmark)1
G. thymalli 1997 Gudbrandsdalslågen
(Oppland)
G. thymalli 2003 Måsåbekken, Lake Mjøsa
(Oppland)
CO1 Acc. nos.
1997
Valåe, Lake Lesjaskogsvatnet
(Oppland)
G. thymalli 2002 Trysilelva (Hedmark)
Sweden – Oncorhynchus mykiss
G. salaris
2002 Lake Bullaren – fish farm
(Göteborg and Bohus)
Sweden – S. salar
G. salaris
2001 Göta älv (Västra Götaland)
G. salaris
2002 Surtan (Halland)
G. salaris
2001 Ätran (Halland)
G. salaris
2001
G. salaris
2001
G. salaris
G. salaris
AtlanticU
V–Q
AY486545, AY486546
PCR products
30
AY490407, AY490408
Atlantic2
IV – P
AY486544
PCR products
31
AY490437
Atlantic
III – F
AY146590, AY486503
Clones
1, 8, 9, 26, 27, 28, 29
AY490395–AY490406
Atlantic2
Atlantic
Atlantic
II – E
I–A
I–A
PCR products
Clones
Clones
25
2, 6, 10, 12, 13, 15, 21
1, 4, 14, 18
AY490491, AY490492
AY490478–AY490485
AY490461–AY490467
Atlantic
I–A
AY258374, AY486512
AY486508, AY486509
AY258348, AY258349,
AY486500
AY486521, AY486522
Clones
1, 3, 4, 7
AY490504–AY490509
Atlantic
I–A
AY486517, AY486518
PCR products
1
AY490500, AY490501
2001
2001
Ätran (Högvadsån/Kogstorp)
(Halland)
Ätran (Høgvadsån/Ullared)
(Halland)
Susenån (Halland)
Nissan (Halland)
Atlantic
Atlantic
2001
2001
Fylleån (Halland)
Genevadsån (Halland)
Atlantic
Atlantic
G. salaris
2001
Stensån (Halland)
Atlantic
I–C
Clones
Clones
PCR products
PCR products
Clones
PCR products
PCR products
1, 3, 7, 11, 20, 23
4, 5
1, 3
2
6, 19, 24
1
1
G. salaris
2001
Stensån (Brostorp) (Halland)
Atlantic
I–C
I–B
I–B
I–C
Clones
PCR products
PCR products
PCR products
PCR products
17
1
2
2
1
AY490510, AY490511
Baltic
Baltic
Atlantic
AY486531, AY486532
AY486511
AY486510
AY486515, AY486516
AY258364,
AY486513, AY486514
AY258361, AY486501,
AY486502
AY486524
AY486523
AY258370, AY486499
AY258368
AY486504–AY486506
AY490519–AY490525
AY490486–AY490490
G. salaris
G. salaris
I–C
I–A
I–C
I–C
I–C
AY490458, AY490459
AY490460
AY490471–AY490473
Baltic
I–D
AY486507
Clones
1, 2
AY490474–AY490477
Danube
VI – N
AY486551
Clones
30, 35, 38, 39
AY490425–AY490428
G. salaris
2002 Torneälven (Norrbotten)
G. salaris
2002 Vindelälven (Västerbotten)
G. salaris
2002 Tvååkersån (Halland)
Latvia – S. salar
G. salaris
2002 Gauja – fish farm
Slovakia – Thymallus thymallus
G. thymalli 2003 Hnilec
1
Samples partly or in total from laboratory strains ; 2 connected to the Baltic Sea via the Göta canal.
Sequences with variation in the intervening sequence.
U
AY490498, AY490499
AY490493–AY490497
AY490468–AY490470
Nuclear and mitochondrial DNA variation in G. salaris and G. thymalli
G. thymalli
Samples are from different localities within the Glomma drainage system.
643
H. Hansen and others
644
A
A
100
C
72
59
87
75
26 29 L
32 J
60
I
83
O
51
III
F
67
L
65
J
V
I
56
83
O
M
Q
97
Q
G
N
62
89
E
K
M
99 P
91 H
C
D
II
F
K
99
I
D
E
99
B
B
G
IV
P
H
89
87
N
VI
G. lavareti
G. sp.
99
0·05
Fig. 1. Neighbour-joining dendrogram (Kimura’s two parameter) and maximum parsimony bootstrap consensus tree
(unweighted close-neighbour-interchange, random addition of trees) of mitochondrial haplotypes of Gyrodactylus salaris
and G. thymalli (see Table 1). Haplotypes from this study and from Hansen et al. (2003) are included. The depicted
haplotypes and clades (Roman capitals) are listed in Table 1. Bootstrap support is indicated as percentages of 1000
replicates. Scale bar refers to a genetic distance of 0.05.
In repeat region 2 the SU repeat motif is also typical
for G. thymalli.
are also not specific for any particular clade of
mitochondrial haplotypes.
A hypothetical evolutionary history of the ribosomal
intergenic spacer repeats
DISCUSSION
The nucleotide sequences of IGS repeats from
regions 1 and 2 show high levels of sequence
similarity that indicate common ancestry. Maximum
similarity is observed between repeat types B4, E, E1,
E3, H, J, and I from region 1 which all share
17 identical positions with repeat type V2 in repeat
region 2. A minimum spanning tree (Fig. 2) illustrates that the IGS repeat types are more similar
within each repeat region than between repeat
regions. Furthermore, there is no indication of
sequence homogenization between the 2 repeat
regions.
Nuclear versus mitochondrial variation in G. salaris
and G. thymalli
The observed IGS arrangements were compared
to the clades of mitochondrial COI haplotypes (see
Table 1). The mitochondrial haplotype E that has so
far only been observed in G. salaris specimens from
River Göta älv, Sweden, occurs together with the
typical IGS repeat arrangements 25. Otherwise,
there is no match of specific mitochondrial haplotypes with specific IGS arrangements. The typical
repeat arrangements 1, 2, 3 found in G. salaris occur
in specimens that bear mitochondrial haplotypes of
both clades I and III. The typical repeat arrangements 30, 31 and 32 found in G. thymalli specimens
In this study sequence data of the ribosomal intergenic spacer (IGS) regions and the mitochondrial
cytochrome oxidase I (COI) genes of G. salaris
and G. thymalli that complement earlier IGS
(Collins and Cunningham, 2000 ; Sterud et al.
2002 Cunningham et al. 2003) and COI data
sets (Hansen et al. 2003 ; Meinilä et al. 2004) are
presented.
Sequence variation in the CO1 gene
The COI data largely confirm the clades and tree
topologies reported by Hansen et al. (2003) and
Meinilä et al. (2004). The new COI haplotype N
detected in the G. thymalli specimens from one of the
type localities in Slovakia adds an additional wellsupported clade. In total, there are now 8 mtDNA
clades (6 included in this study and 2 described by
Meinilä et al. (2004)). In the present data set, clades I
and II are specific for Gyrodactylus parasites on
salmon and clade III for Atlantic salmon and rainbow trout. Clades IV–VI are specific for grayling as
are two further clades described by Meinilä et al.
(2004). The data are still most consistent with considering G. salaris and G. thymalli to be either
one polytypic species or several different species,
each relating to one of the distinct mitochondrial
clades described here, by Hansen et al. (2003) or by
Meinilä et al. (2004).
Nuclear and mitochondrial DNA variation in G. salaris and G. thymalli
645
Table 2. Alignment of IGS haplotypes of Gyrodactylus salaris and G. thymalli
(The sequences and codes of the 23 bp repeat types are given in Table 3. The most common/characteristic haplotypes are
shaded. This alignment was constructed manually and reflects the authors’ understanding of similarity of the repeats. Host :
S=Salmo salar, T=Thymallus thymallus, O=Oncorhynchus mykiss.)
IGS
haplo
type Host Repeat region 1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
S, O
S
S
S
S
S
S
O
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
O
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
A
A
A
A
A
A
A
A5
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
A
B
A
B
B
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B I
B B
B
A
B
B
B
B B
B
B
B
B
B
B
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
D
B
B
B
B
B
A
B
B
B
B
A
B
A
A
A
A
A
B
B
B
B
B
B
B
B
B
J
B
A
A
A
B
C
D
D
B
B
D
D
B
B
D
D
D
D
D
D
Repeat region 2
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
D
D
D
D
D
D
D
D
B
B
B
B
B
B
B
B
B
B
B
B
B
B
F
F
D
B
B
F
F
F
F
B
B
B
B
F
B
B
B
B
B
B
B
B
B
B
F
F
F
F
F
B
B
B
B
B
B
B
B
B
B
B
B
K
B
B
B
B
B
B
B
B
D
F
B
B
B
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
E
E
E
E
E
E
E
E
E E
E
E
E
E
E
E
B
E
E
E5
E
E
E
E
E4
E
B B
B
B B
E
E E
E
E
E
E
E
E
E
E
E
E
E
E
D D
Meinilä et al. (2004) suggested that the mitochondrial clades specific for grayling parasites
reflected ice-age refugia due to congruence with 4
postulated refugia for grayling (Koskinen et al. 2000 ;
Weiss et al. 2002). Accordingly, one may assume
a further ice-age refugium for grayling bearing
parasites with the new haplotype N. Alternatively,
grayling from any particular ice-age refugium is
infected with parasites bearing mitochondrial
haplotypes from more than one clade. For us it seems
likely that any further geographically isolated
P
P
P
P
P
P
P
P
P
P
P8
P
P
P
P
P
P
P
P
P
P
P
P
P
P5
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
D D P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P7
P
P
P
P
P
P
P
P
P
P
P
P
P6
P
P
P
O
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P9
P
P
P
P
P
P
P
P
P
P
S
P
P
P
P
P
P
P
P
P
P
P
Q
P Q
Q
Q
Q
P Q
Q
Q
Q
P Q9
Q
P Q
P Q
Q
Q
P Q
P Q
Q
Q
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
N W R V
R T R V
Q
Q
P Q
Q
Q
Q
P
R
R
R
R
S
S
S
S
S
S
S
S
S
S
S
Q
Q
Q
Q
R4
Q
Q8
Q
Q
Q
Q
R
R
Q
Q
P
S
S
S
S
S
S
S
S
T
T
W
W
W
T
W
T
T
T
W
T
T
T
W
T
T
T
W
T
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
T
U
U
U
U
U
R
R
R
R
R
V
V
V
V
V
Q
Q
Q
Q
Q
R
R
R
R
R
R
R
R
R
R
V
V
V
V
V
U3
V
V
V
V
Q
Q
Q
Q
Q
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
V4
V
V
V
U
V
V
V
V
V
V
V2
M
V
V
R
R
R
R
R
R
R
V
M
V2
V5
V2
V2
V2
R
V Q
Q
Q
Q
Q
V2 R
V2 R
Q
R
R
R
R
R
R
population of G. thymalli being sampled will provide
haplotypes belonging to a new mitochondrial clade.
We favour the assumption of COI clades mirroring
geographically isolated parasite strains that evolved
independently after a bottleneck associated with the
spreading into new water systems.
IGS repeat types
Twenty new IGS repeat types were found in this
study, most of which are rare and expected to result
H. Hansen and others
646
A2
A4
A5
J
B
A
EI
E3
E4
E5
E
Repeat region 1
B3
B5
B6
B7
C
I
H
DI
D
F
A3
K
AI
B4
V5
M
N
V3
V
V4
R4
R2
R3
Q8
Q9
Q2
Q3
Q5
Q6
Q7
V2
Repeat region 2
Q
R
Z
U2
U3
U
S
W
P8
T
P9
P
P6
O
P7
P3
P4
P5
X
Fig. 2. Minimum spanning tree of individual IGS repeats found in Gyrodactylus salaris and G. thymalli. The tree was
constructed in Arlequin (Ver. 2000) (Schneider et al. 2000). The corresponding sequences of the individual repeats
can be found in Table 3. Labelling follows earlier studies (Collins and Cunningham, 2000 ; Sterud et al. 2002 ;
Cunningham et al. 2003).
Repeat region 1
Repeat region 2
Code
Sequence
Reference
Code
Sequence
Reference
A
AI
A2
A3
A4
A5
B
B3
B4
B5
B6
B7
C
D
DI
E
EI
E3
E4
E5
F
H
I
J
K
GTCCTTCAGTGTAGAACCGTACA
GTCCTTCAGTGTAGAACCGTACG
GTTCTTCAGTGTAGAACCGTACA
GTCCTTCAGTGTAAAACCGTACA
GTCCTTCAGTGGAGAACCGTACA
GTCCTCCAGTGTAGAACCGTACA
GTCCTTCAGTGTAGAGCCGTACA
GTCCTTCAGTGTAGAGCCGTGCA
GTCCTTCAGTGTAGAGCCGTACG
GTCCTTCGGTGTAGAGCCGTACA
GTCCTTCAGTGAAGAGCCGTACA
GTCCTTCAGTGTAGAGCCGCACA
GTCCCTCAGTGTAGAGCCGTACA
GTCATTCAGTGTAGAGCCGTACA
GGCATTCAGTGTAGAGCCGTACA
GTCCTTTAGTGTAGAGCCGTACA
CTCCTTTAGTGTAGAGCCGTACA
GCCCTTTAGTGTAGAGCCGTACA
GTCCTTTAGTGTAAAGCCGTACA
GTCCTTTAGTGTAGGGCCGTACA
GTCATTCAGGGTAGAGCCGTACA
GTCCTTTAGAGTAGAGCCGTACA
GTCTTTCAGTGTAGAGCCGTACA
GACCTTCAGTGTAGAGCCGTACA
GTCCTTCAGTGTAAAGCCGTACA
Collins and Cunningham 2000
Sterud et al. 2002
Sterud et al. 2002
Cunningham et al. 2003
Cunningham et al. 2003
this study
Collins and Cunningham 2000
Cunningham et al. 2003
Cunningham et al. 2003
Cunningham et al. 2003
Cunningham et al. 2003
Cunningham et al. 2003
Sterud et al. 2002
Sterud et al. 2002
Cunningham et al. 2003
Collins and Cunningham 2000
Cunningham et al. 2003
Sterud et al. 2002
this study
this study
Collins and Cunningham 2000
Cunningham et al. 2003
this study
this study
this study
M
N
O
P
P3
P4
P5
P6
P7
P8
P9
Q
Q2
Q3
Q5
Q6
Q7
Q8
Q9
R
R2
R3
R4
S
T
U
U2
U3
V
V2
V3
V4
V5
W
X
Z
TACTATTACCGTGGAGCCGTACG
TACTATTACCGTGGAGCCGTAGG
TACTAATACCGTGCAGCCGTAGG
TACTAATACCGTGTAGCCGTAGG
TACTAACACCGTGTAGCCGTAGG
TGCTAATACCGTGTAGCCGTAGG
TACTAATAACGTGTAGCCGTAGG
TACTAATGCCGTGTAGCCGTAGG
TACTAATACCGTATAGCCGTAGG
TACTAATACCGTGTAGCCGTATG
TACTAATACCGTGTAGCCGTACG
TATTATTACCGTAGAGCCGTACG
TATTACTACCGTAGAGCCGTACG
TATTATTACCGTAGAGCCGTACA
TATTATTACCGTAGAGCCGTAGG
TGTTATTACCGTAGAGCCGTACG
TATTATTACCGTAGAGCCGCACG
TATTATTACAGTAGAGCCGTACG
TATTATTATCGTAGAGCCGTACG
CACTATTACCGTGGAGCCGTAGG
CACTATTACCGTGGAGCCGTATG
CACTATTACCGTGGAGCAGTAGG
CACTATTACCGTGGAGCCGTACG
TACTTATACTGTAGAGCCGTAGG
TACTTATACCGTGGAGCCGTACG
TACTTATACCGTAGAGCCGTACG
TACTTATACCGTAGAGCCGTGCG
TACTTATACCGTAGAGCCGCACG
TACTTTTACCGTGAAGCCGTAGG
TACTTTTACCGTAGAGCCGTACG
TACTTTTACCGTGGAGCCGTAGG
TACTTTTACCCTGAAGCCGTAGG
TACTTTTACCGTAGAGCTGTACG
TACTTATATCGTGGAGCCGTACG
TACTAATACCGTGTAGCCGAAGG
TATTATTACCGTAGAGCCGTCCG
this study
this study
this study
Collins and Cunningham 2000
Cunningham et al. 2003
Cunningham et al. 2003
this study
this study
this study
this study
this study
Collins and Cunningham 2000
Sterud et al. 2002
Cunningham et al. 2003
Cunningham et al. 2003
Sterud et al. 2002
Cunningham et al. 2003
this study
this study
Collins and Cunningham 2000
Cunningham et al. 2003
Sterud et al. 2002
this study
Sterud et al. 2002
Collins and Cunningham 20001
Sterud et al. 2002
Cunningham et al. 2003
this study
Sterud et al. 2002
Sterud et al. 2002
Cunningham et al. 2003
this study
this study
Cunningham et al. 2003
Cunningham et al. 2003
Cunningham et al. 2003
1
Nuclear and mitochondrial DNA variation in G. salaris and G. thymalli
Table 3. Sequence and corresponding codes of the repeat types of the intergenic spacer region of Gyrodactylus salaris and G. thymalli
Repeat type originally described by Collins and Cunningham 2000, but later found to contain errors. Corrected by Sterud et al. 2002.
647
H. Hansen and others
from point mutations affecting individual rDNA
repeats. Not surprisingly, most new repeat types
were detected in cloned sequences. Accordingly,
some rare IGS repeat types that have been described
earlier (Collins and Cunningham, 2000 ; Sterud et al.
2002 ; Cunningham et al. 2003) were not found in the
present study. However, some new repeat types were
obtained from direct sequencing of PCR products
and are therefore considered characteristic for the
analysed Gyrodactylus specimens ; E4, J, P5 and R4
were detected in G. salaris from River Göta älv,
Sweden, and contribute to IGS arrangement 25,
and ; O was detected in G. thymalli from River
Gudbrandsdalslågen, Norway, carrying the mitochondrial haplotype M.
According to Sterud et al. (2002) and Cunningham
et al. (2003) some IGS repeat types may be specific
and suitable for discriminating G. salaris and
G. thymalli. Sterud et al. (2002) found repeat types C
and D only in G. thymalli, with C only occurring in
specimens from Trysil, Norway. Repeat types F and
T were only found in G. salaris from salmon while
S and U were absent. Later, Cunningham et al.
(2003) found D also in one clone from G. salaris from
rainbow trout in Berlin, Germany. They also found
repeat type F in G. salaris from rainbow trout in
western Sweden. Here repeat type T was also found
in one clone from G. thymalli from Måsåbekken,
Norway, and repeat types S and U in G. salaris from
River Göta älv, Sweden. If rare and clone-specific
repeat types are not considered, currently only repeat
type F is diagnostic but not found in all G. salaris
populations. This is in line with Cunningham et al.
(2003) who suggested dynamic IGS patterns ; all
repeat types can accordingly be found in all populations but with different frequencies. Presence or
absence of certain repeat types is thus not considered
informative.
Evolution of the repetitive region of the IGS
The sequence similarity of the 23 bp IGS repeats
suggests common ancestry and it can be assumed that
repeat regions 1 and 2 result from a duplication of just
one ancestral repeat region. Following the duplication both repeat regions evolved independently.
In the lack of sequence data from closely related
species it is impossible to reconstruct the sequence
and repeat arrangement of the ancestral IGS repeat
region. According to the concept of concerted
evolution (Dover, 1982) both repeat regions will be
homogenized in individuals, populations and species
by recombination processes such as, for example,
unequal crossing-over and replication slippage.
Mutations will generate new repeat types that may
be homogenized or may disappear. In addition,
recombination may alter the number and order of
repeats. Although most of the variation may be
selectively neutral, the length of the IGS may affect
648
transcription (e.g. Zentgraf et al. 1990 ; Zentgraf and
Hemleben, 1992). Footprints of concerted evolution
can be seen clearly in the IGS dataset as particular
IGS haplotypes are homogenized in the genomes and
can be unambiguously sequenced as PCR products.
The rare repeat types detected through cloning
illustrate the significance of mutations in the concept
of concerted evolution. Since the IGS repeats
are only 23 bp long, there may be a relatively high
probability that homoplasic repeat types appear
independently.
This particular mode of evolution of the ribosomal
IGS region makes it impossible to align nucleotide
sequences unambiguously. Alignments are biased
towards the researchers’ understanding of similarity
and may not reflect homologous positions. We
therefore consider phylogenetic trees based on IGS
data such as, for example, that put forward by
Cunningham et al. (2003) not informative. Although
the authors were aware of the problem and excluded
‘ difficult ’ sequences from the data set, they presented a phylogenetic tree with very low bootstrap
support depicting a ‘ grayling form ’ and a more
derived ‘ salmon form ’ of IGS which they took as
support for G. thymalli and G. salaris being 2 species.
Nuclear versus mitochondrial variation in G. salaris
and G. thymalli
Since phylogenetic analyses of IGS sequences of
G. thymalli and G. salaris are not straightforward, the
IGS features were related to the clades of mitochondrial haplotypes. The clade II-specific IGS
arrangement 25 will not be considered in this
context, since this clade/haplotype currently only
includes the G. salaris specimens from River Göta
älv, Sweden ; any peculiarity of the IGS of this strain
will by default be specific for mitochondrial clade II.
Three features within repeat region 1 can be
related to mitochondrial clades : (i) repeat type D
occurs in clades II and IV–VI, but does not occur in
clades I and III ; (ii) repeat type F is only found in
parasites bearing mitochondrial haplotypes from
clades I and III. Repeat type F differs from D by only
1 nucleotide substitution ; we therefore assume that F
replaced D in clades I and III ; (iii) repeat type B at
the first position of repeat region 1 is restricted to
clades IV–VI. Within repeat region 2, two features
can be related to mitochondrial clades : (1) the repeat
arrangement SU does not occur in clade I; (2) repeat
type V2 that is considered ancestral due to its position
in the minimum spanning network is restricted to
clade IV.
Taxonomic implications of the IGS sequences
According to our interpretation there are no diagnostic repeat types or repeat arrangements for either
G. salaris or G. thymalli. More exhaustive cloning
Nuclear and mitochondrial DNA variation in G. salaris and G. thymalli
and sequencing of IGS is expected to support this
assumption. Nevertheless, there is a trend in favour
of a ‘ grayling form ’ and a ‘ salmon form ’ of IGS as
described by Cunningham et al. (2003). However,
repeat arrangements similar to the ‘ grayling form ’ in
salmon parasites from River Göta älv, Sweden, challenges this view. Parasites from this locality have the
distinct clade II mitochondrial haplotype E (Hansen
et al. 2003). Other authors such as Meinilä et al. (2004)
do not accept haplotype E as a separate clade and
consider this haplotype as belonging to clade I.
As already noticed by Sterud et al. (2002) and
Cunningham et al. (2003), the IGS of Norwegian and
Swedish G. salaris populations from rainbow trout
share features with both the ‘ grayling form ’ and the
‘ salmon form ’. Cunningham et al. (2003) suggested
that G. salaris on rainbow trout may either be a hybrid between G. salaris from salmon and G. thymalli
from grayling or the ancestor of both forms. All
currently known COI sequences of G. salaris from
rainbow trout (Hansen et al. 2003 ; Meinilä et al.
2002, 2004) are haplotype F and if true the putative
mother must bear the same haplotype. So far, this
haplotype in the wild has only been found in G. salaris
from the Norwegian rivers Drammenselva, Lierelva,
and Lærdalselva, where the parasite was recently
introduced (Hansen et al. 2003). We consider it more
likely that the IGS of G. salaris on rainbow trout
results from recombination of 2 ancestral G. thymalli
IGS haplotypes prior to host switches to rainbow
trout and salmon. This hypothesis is favoured since
the most important step in generating an IGS
haplotype, as observed today in G. salaris on rainbow
trout, is recombination.
According to our opinion, the IGS data are most
consistent with G. salaris and G. thymalli being a
single polytypic species. This hypothesis has been
first suggested as one of three possibilities by Hansen
et al. (2003) and was also favoured later by Meinilä
et al. (2004). Different mitochondrial haplotypes may
be suitable to characterize clades or strains that are
separated geographically and/or are infecting different salmonid hosts. The major remaining argument
in favour of considering G. salaris and G. thymalli as
2 valid species is their different host preference
(Soleng and Bakke, 2001 ; Bakke et al. 2002 ; Sterud
et al. 2002). However, host switches are frequent
in Gyrodactylus (Zie( tara and Lumme, 2002) and
host preferences may depend on the particular host
(Bakke et al. 1990 ; Bakke et al. 1996) and/or parasite
strains (Lindenstrøm et al. 2003) in question. Until
now, only a very limited number of G. salaris and
G. thymalli populations have been tested for host
preference, and therefore host preference should be
used with caution in Gyrodactylus taxonomy.
We are grateful to Vladka Hanzelova for providing
G. thymalli samples from Slovakia. We thank Lars
Karlsson, Ingemar Perä, Thrond Haugen, Kjetil Olstad
and Dag Gammelsæter for collecting material in Sweden
649
and Norway. 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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