Initial steps of speciation by geographic isolation and host switch in salmonid pathogen Gyrodactylus salaris (Monogenea: Gyrodactylidae)

International Journal for Parasitology 34 (2004) 515–526 www.parasitology-online.com

Initial steps of speciation by geographic isolation and host switch in salmonid pathogen Gyrodactylus salaris (Monogenea: Gyrodactylidae)q Maria Meinila¨a, Jussi Kuuselaa, Marek S. Zie˛tarab, Jaakko Lummea,* b

a Department of Biology, University of Oulu, FIN-90014 Oulu, Finland Biological Station, Gdan´sk University, PL-80-680 Gdan´sk-Go´rki Wsch., Poland

Received 25 September 2003; received in revised form 24 November 2003; accepted 2 December 2003

Abstract To test the hypothesis that host-switching can be an important step in the speciation of gyrodactylid monogenean flatworms, we inferred the phylogeny within a cluster of parasites morphologically close to Gyrodactylus salaris Malmberg 1957, collected from Atlantic, Baltic and White Sea salmon (Salmo salar), farmed rainbow trout (Oncorhynchus mykiss), and grayling (Thymallus thymallus) from Northern Europe. The internal transcribed spacer region of the nuclear ribosomal gene was sequenced for taxonomic identification. Parasites on grayling from the White Sea Basin differed from the others by one nucleotide (0.08%), the remainder were identical to the sequence published earlier from Norway (G. salaris on salmon), England (Gyrodactylus thymalli on grayling), and the Czech Republic (unidentified salaris/thymalli on trout). For increased resolution, 813 nucleotides of the mitochondrial COI gene of 88 parasites were sequenced and compared with 76 published sequences using phylogenetic analysis. For all tree building algorithms (NJ, MP), the parasites formed a star-like phylogeny of six definite sister clades, indicating nearly simultaneous radiation. Average K2P distances between clades were 1.8– 2.6%, and internal mean distances 0.2– 1.1%. The genetic distance to the nearest known relative, Gyrodactylus lavareti Malmberg, was 24%. A variable salmon-specific mitochondrial Clade I was observed both in the Baltic Basin and in pathogenic populations introduced to the Atlantic and White Sea coasts. An invariable Clade II was common in rainbow trout farms in Sweden, Denmark and Finland; the same haplotype was also infecting salmon in a landlocked population in Russian Karelia, and in Oslo fjord and Sognefjord in Norway. Four geographically vicariant sister clades were observed on graylings: Clade III in the Baltic Sea Basin; Clade IV in Karelian rivers draining to the White Sea; Clade V in Norwegian river draining to Swedish lake Va¨nern; and Clade VI in rivers draining to Oslo fjord. The pattern fitted perfectly with the postglacial history of grayling distribution. Widely sampled clades from salmon and Baltic grayling had basal haplotypes in populations, which were isolated early during the postglacial recolonisation. The divergence between the six clades was clear and linked with their hosts, but not wide enough to support a species status for them. Parasites from the Slovakian type population of G. thymalli were not available, so this result does not mean that G. salaris and G. thymalli are synonyms. It is suggested that the plesiomorphic host of the parasite cluster was grayling, and the switch to salmon occurred at least once when the continental ice isolated Baltic salmon in an eastern freshwater refugium, 130,000 years ago. At the same time, parasites on grayling were split geographically and isolated into several allopatric refugia. The divergence among the parasite clades allowed a tentative calibration of the evolutionary rate, leading to an estimate of the divergence of 13.7 –20.3% per million years for COI coding mtDNA. The results supported the hypothesis that parallel to the allopatric mode, host switch and instant isolation by host specificity can be operated as a speciation mechanism. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Mitochondrial phylogeny; Speciation; Taxonomy

1. Introduction q

The sequences of Gyrodactylus from this study are deposited in GenBank under accession numbers AF479750 (rainbow trout type); AF540890-906 and AY472084-5 (parasites from salmon and grayling, all under name Gyrodactylus salaris); AY225306 (G. lavareti); and AY225307-8 (G. salaris, putative nuclear copies of mtDNA). * Corresponding author. Tel.: þ 358-8-553-1783/40-736-1876; fax: þ 358-8-553-1061. E-mail address: [email protected] (J. Lumme).

Ectoparasites of the genus Gyrodactylus Nordmann (Gyrodactylidae: Monogenea: Platyhelminthes) probably infect most of the world’s fish species. Only 400 species have been described, because few taxonomists have concentrated on this difficult genus. The true number of species in the world is estimated to be 20,000 (Bakke et al.,

0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2003.12.002

516

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

2002). If this diversity is borne out in future work then this genus offers a challenge for the study of evolution: why are there so many species in this narrow ecological niche? It has been suggested that the life history of these monogenean flatworms promotes speciation, via recurrent host switching (Brooks and McLennan, 1993; Cribb et al., 2002; Poulin, 2002). Switching is thought to lead to instant isolation and very rapid speciation after a host switch, and is a consequence of the characteristic life cycle of the gyrodactylids (reviewed by Cable and Harris, 2002). A test of this hypothesis, i.e. speciation following host switching and subsequent radiation, was conducted by using a phylogenetic survey of 23 closely related sympatric freshwater species of subgenus Gyrodactylus (Limnonephrotus) (Zie˛tara and Lumme, 2002). Based on comparison of internal transcribed spacer (ITS) sequences of ribosomal genes, Zie˛tara and Lumme (2002) suggested that hostswitching to distantly related fish families had repeatedly occurred in the wageneri species group, which is the ‘difficult’ species cluster of the subgenus G. (Limnonephrotus). It was predicted that with a more sensitive molecular marker than ITS, one could identify initial events of the speciation process, well before there is any morphological basis to identify the new lineages as new species (Zie˛tara and Lumme, 2002). Here we explicitly test this prediction by focusing on the problematic and economically important parasites of Atlantic salmon. An imported parasite identified as Gyrodactylus salaris is a serious pest in wild populations of Norwegian Atlantic salmon (Salmo salar L.), and its future control is important (Johnsen and Jensen, 1991). One obstacle has been that the pathogenic species (or strains) are difficult to differentiate, on morphological grounds, from other species which may not be deleterious (or less so) on the susceptible host, i.e. Atlantic salmon. Our aim was to analyse DNA sequences of Gyrodactylus from three host species: European grayling (Thymallus thymallus L.); Atlantic salmon (S. salar L.); and farmed rainbow trout (Oncorhynchus mykiss Walbaum). Tentatively, we identified them morphologically only ‘to be very close’ to the species G. salaris Malmberg, the salmon parasite described from the Baltic Sea basin, or to Gyrodactylus thymalli Zˇitnˇan, which has been described from grayling in Danubian tributaries in Slovakia. Several attempts to reveal a morphological distinction between G. salaris and G. thymalli have been based on an a priori decision about the species name as well as limited sampling, and subsequent unwarranted typological generalisation (McHugh et al., 2000). Our molecular analysis was based on the nuclear ribosomal ITS segments, allowing the systematic placement of the studied specimens (Zie˛tara and Lumme, 2002). Further we also utilised the more sensitive marker mitochondrial cytochrome c oxidase subunit I (COI, Meinila¨ et al., 2002). Our results contribute to the understanding of the a-taxonomy of this interesting parasite genus and also suggest that switching hosts can indeed lead

to rapid host-specific adaptation, isolation and genetic divergence. The process of divergence is fully comparable in mode and tempo to that caused by geographical isolation. It was also demonstrated that the rate of molecular and adaptive evolution among Gyrodactylus species is extraordinarily fast, helping to explain the high species number.

2. Materials and methods 2.1. Parasite collection and DNA analysis Specimens of G. salaris hosted by rainbow trout (O. mykiss) were obtained from several fish farms in three countries: northern Finland, southern Sweden and Denmark. For proprietary reasons the exact locations remain confidential; but the infrastructure of the rainbow trout industry makes all the farms in all these countries an essentially continuous habitat for the relatively benign parasite. The parasites were morphologically (and molecularly) separated from the Gyrodactylus lavareti Malmberg 1957 and Gyrodactylus derjavini Mikailov 1975 in the same samples. (See Bakke et al., 2002 for a complete name list of gyrodactylids infecting salmonids.) Swedish West Coast (Kattegat strait and the North Sea), Finnish (the Baltic) and Norwegian (the North Sea) parasite samples hosted by wild Atlantic salmon (S. salar) were kindly provided by Drs Go¨ran Malmberg, Perttu Koski and Bjo¨rn Ove Johnsen, respectively. In addition, we collected Gyrodactylus from juvenile salmon in the Russian rivers the Keret (anadromous salmon which feed in the White Sea) and the Pistojoki (landlocked salmon which feed in lake Kuitozero). The majority of the gyrodactylids found on grayling (T. thymallus) were obtained by fly-fishing wild grayling from various locations in the Baltic and White Sea Basins, in rivers and streams. Gyrodactylus specimens from grayling hosts in the brackish water of the Gulf of Bothnia of the Baltic Sea were kindly provided by Dr Perttu Koski. The locations and dates of sampling, along with the sample sizes are listed in Table 1. The morphological identification of parasites was based on the drawings in the original descriptions and later redescriptions (Malmberg, 1957,1970, 1993; Zˇitnˇan, 1960). All the parasites found on salmon or grayling are included in this study; i.e. they never carried any other species of gyrodactylids. Mitochondrial COI of G. lavareti Malmberg 1957 from rainbow trout farm (Koski and Malmberg, 1995) was sequenced and used as an outgroup because it is the closest known relative of G. salaris (Zie˛tara and Lumme, 2002). 2.2. DNA extraction, PCR amplification, and sequencing For molecular species identification, the internal transcribed spacers (ITS1 and ITS2) of the ribosomal RNA gene cassette were amplified and sequenced from one or more

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

517

Table 1 Sampling localities and hosts of the Gyrodactylus salaris specimens sequenced Sea Basin

Locality, country

Host species

Lat. N, Long. E

[Baltic þ ] Atlantic

Fish farms FI, S, DKa Vefsna, N Lagan/Smedjea˚n, S ¨ tran/Fageredsa˚n, S A Susea˚n, S

O. mykiss S. salar S. salar S. salar S. salar

668N, 138E 568N, 138 E 578N, 128 E 578N, 138 E

1997 1999 1997

Keret, RU

S. salar

668160 , 338330

July 2000

Pistojoki, RU Penninki, FI Oulanka PK, FI Oulanka, MN, FI Oulanka AJ, FI Olanga, RU

S. salar T. thymallus T. thymallus T. thymallus T. thymallus T. thymallus T. thymallus

658210 , 308330 658210 , 308330 658450 , 308150 668210 , 298190 668220 , 298200 668230 , 298050 668140 , 308200

July 2000 July 2000 July 2002 Aug 2000 Aug 2000 June 2001 Aug 2000

Lake Kitka, FI

T. thymallus

668160 ,288580

Sept 2001

White Sea

Baltic

0

0

Date of collection

Gyrodatylus haplotype name

N

GenBank accession #

1998–2001

Onc FI-S-DK Sal Vefsna Sal Lagan Sal Lagan Sal Lagan

22 6 2 2 1

AF479750 AF540906 AF540904 AF540904 AF540904

Sal Kereta Sal Keretb Onc FI-S-DK Thy Oulankab Thy Penninki Thy Oulankab Thy Oulankaa ThyOulankab ThyOlanga A ThyOlanga B Thy Kitka

5 5 2 2 2 5 2 2 1 1 1

AF540891 AF540892 AF479750 AF540899 AY472085 AF540899 AF540897 AF540899 AF540898 AF540900 AF540893

Thy Tornioa Thy Torniob Sal Tornio Thy Krunnita Thy Krunnitb Thy Livo ThySoivio Thy Onega

2 3 3 1 1 2 2 5

AF540902 AF540903 AF540905 AF540894 AF540895 AF540896 AY472084 AF540901

Tornio, Poroeno, FI

T. thymallus

69805 , 21851

Aug 2001

Tornio, Va¨ha¨kurkkio, FI Krunnit (sea), FI

S. salar T. thymallus

688350 , 228180 658250 , 258000

2001 1999

Livojoki, FI Soivio, FI Pjalma (Onega), RU

T. thymallus T. thymallus T. thymallus

658560 , 278340 658430 , 298210 628240 , 358550

Oct 2000 July 2002 Aug 2001

Only the number of complete 813 bp of mtDNA COI sequences is given. FI, Finland; S, Sweden; DK, Denmark; RU, Russia; N, Norway. Parasites from rainbow trout (Oncorhynchus mykiss) farms in Finland, Sweden and Denmark were pooled, because they invariably had the same Gyrodactylus haplotype. b Rainbow trout farm A was infected by this haplotype only once (1998 sample), next year it again had standard Onc FI-S-DK haplotype. a

specimens of the majority of the populations mentioned in Table 1 by methods described in Zie˛tara and Lumme (2002). Molecular methods for DNA extraction, PCR amplification, sequencing development of primers for partial G. salaris COI gene are described elsewhere (Meinila¨ et al., 2002). We used Invitrogen’s TA-cloning kit to clone PCR products for separating mitochondrial genes from putative nuclear copies (pseudogenes), which were observed in few parasite strains. 2.3. Alignment of the sequences and phylogenetic analysis The L- and H-strand sequences covering approximately the 820 bp region of the COI gene of various G. salaris lineages and outgroup were aligned and checked with Sequencher 4.0.5 (GeneCodes). The length of the final alignment was 813 bp, but several of the GenBank sequences were shorter. To investigate the phylogenetic relationships of the different mitochondrial lineages, neighbour joining (NJ, Kimura’s two parameter distance), and maximum parsimony (MP) trees were produced, with 1000 bootstrap replicates. The calculations were performed using Mega version 2.1 program package (Kumar et al., 2001). The topology of trees produced by both methods was identical.

The sequences of Gyrodactylus from this study are deposited in GenBank under accession numbers AF479750 (rainbow trout type); AF540890-906 and AY472084-5 (parasites from salmon and grayling, all under name G. salaris); AY225306 (G. lavareti); and AY225307-8 (G. salaris, putative nuclear copies of mtDNA). Host fish species, collection dates and localities, haplotype names and numbers, and accession numbers are listed in Table 1. The large published data of G. salaris and G. thymalli by Norwegian authors (Hansen et al., 2003) were retrieved from GenBank and included in the phylogenetic analysis; expanding significantly the geographic coverage and generality of the conclusions.

3. Results 3.1. The nuclear ribosomal ITS region is almost invariable The 1232 bp of the ITS regions of a total of ten specimens of Gyrodactylus, from S. salar, O. mykiss and T. thymallus and representing the main Clades I –IV (Figs. 1 and 2) were subjected to PCR and sequencing. With the exception of parasites from the River Oulanka (GenBank accession number AF484544), all the ITS sequences

518

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

Fig. 1. Location of the sampling sites of Gyrodactylus on Atlantic salmon (Salmo salar) and grayling (Thymallus thymallus). The shading covers the Baltic Sea drainage area. The mtDNA clades of parasites on both hosts are indicated by symbols, and different haplotypes on salmon are marked by letters referring to the phylogenetic trees in Figs. 2 and 3. The hypothetical directions of postglacial arrival of the four grayling-specific parasite clades are indicated by arrows, based on grayling phylogeography of Koskinen et al. (2002b) and Weiss et al. (2002). The cross indicates approximate position of rivers Hron and Hnilec, type locality of Gyrodactylus thymalli in Slovakia. The squared area is redrawn as enlarged in Fig. 4.

examined were identical (AF328871), which is in accordance with the previous studies in Norway, Britain and Czech Republic (Cunningham, 1997; Cunningham et al., 2001; Mate˘jusova´ et al., 2001). The ITS region of

Gyrodactylus found on grayling from the River Oulanka (Clade IV, the White Sea drainage) differed from the rest of the sequences by a single nucleotide substitution along 1232 bp, i.e. by 0.08%.

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

519

Fig. 2. Unrooted Neighbour Joining tree of Gyrodactylus salaris haplotypes. The bootstrap support of the nodes (%) was based on 1000 replicates of Neighbour Joining, and Maximum Parsimony models, respectively. The haplotypes marked with asterisk were published by Hansen et al. (2003), and the capital letters after host abbreviation refer to their haplotype designations. The host species are abbreviated as follows: Sal, Salmo salar; Thy, Thymallus thymallus; and Onc, Oncorhynchus mykiss.

3.2. Nucleotide and amino acid variation in the mitochondrial COI gene A total of 83 variable nucleotide sites were revealed within 826 bp long alignment of mitochondrial COI gene sequences from 164 available specimens of Gyrodactylus. Of the substitutions, 87.5% were transitions. There were 11 amino-acid replacements in a total of 275 codons. Twentynine different haplotypes were observed. The mean K2P distance between the 29 haplotypes was 2.04 ^ 0.26%, with a maximum of 3.42%. To obtain better comprehension of the scale of the variation at different taxonomic levels, we sequenced the same mtDNA region of a closely related species, G. lavareti which frequently infects Finnish rainbow trout farms, together with G. salaris (Koski and Malmberg, 1995). Comparison of the 813 bp region of the COI of Gyrodactylus haplotype Onc FI-S-DK (AF479750) with the G. lavareti haplotype (AY225306) revealed 166 nucleotide substitutions between the species (K2P distance 24%). The number of fixed nucleotide differences between the species was 154, including six amino acid replacements and 147 synonymous

substitutions; i.e. only 4.5% of the changes resulted in nonsynonymous substitutions. The approximately 17:1 bias towards synonymous substitutions indicated that the sequences represented a functional gene region. The low transition/transversion ratio of 1.1 suggested saturation at the third codon position. Differences were observed also in codon usage, as G. lavareti had a GC in 32% of its third codon positions, whereas for G. salaris this measure was 46%. Thus, the ‘nearest’ relative was quite distant, in comparison with variation among the strains in focus. For drawing the phylogenetic tree in Fig. 3, we also included the sequence of an unnamed parasite of the Alpine bullhead, Cottus poecilopus (AY258375, Hansen et al., 2003) to make the outgroup more balanced by randomisation. 3.3. Construction of the mtDNA phylogeny of divergent strains We constructed phylogenies based on distance methods (neighbour joining tree, NJ, Fig. 2, and minimum evolution, ME) or MP. The grand topology of the tree is very robust. The bootstrap values given along the unrooted NJ tree in

520

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

Fig. 3. Neighbour Joining tree of the Gyrodactylus salaris haplotypes rooted with the sequence of Gyrodactylus lavareti and Gyrodactylus sp. from Cottus poecilopus (Hansen et al., 2003). The bootstrap values are percentages over 1000 replicates. The host species are abbreviated as follows: Sal, Salmo salar; Thy, Thymallus thymallus; and Onc, Oncorhynchus mykiss.

Fig. 2. were derived from 1000 repeats of the NJ and MP, respectively (MEGA). A tree rooted with outgroups was essentially the same (Fig. 3). The ingroup parasites formed a star-like phylogeny. Six main clades emerged, two with low bootstrap support. Mean genetic distances between clades were 1.8– 2.6%. Four of them were specific to grayling and geographically separated (allopatric); one was specific for Atlantic salmon irrespective of the sea area; and one clade was found in many rainbow trout cultures, but also observed to be able to infect S. salar, in three rivers.

The mitochondrial clade I was found in parasites hosted by Atlantic salmon in the White Sea, the Baltic and the North Sea populations. The basal branches Sal E Go¨te a¨lv and Sal Keret1 haplotype have diverged from the others very early, and the bootstrap support for including Sal E Go¨te a¨lv into this clade is critically low (bootstrap percentage for 1000 replicates NJ/MP 53/20). However, the host species Salmo salar is an important synapomorphy supporting this placement. Mean genetic distance between haplotypes within this clade, basal branches included, was 0.9 ^ 0.2% (K2P).

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

The mitochondrial Clade II was also a separate branch (NJ/MP 100/99), containing a single invariable haplotype in spite of wide sampling. This haplotype was the most numerous among our samples, because it was found in several rainbow trout farms (Finland, Sweden, and Denmark). Exactly the same haplotype was also observed in the landlocked salmon population in the River Pistojoki, draining to Kuitozero in Russian Karelia; as well as in rivers Lierelva and Drammen in Oslo fjord and Laerdalselven in Sognefjord, Norway (Hansen et al., 2003). The Clade III (Fig. 2) was specific for grayling in the Baltic Sea basin, which was sampled widely. The basal branch found was Thy Onega in the River Pjalma, draining to Lake Onega, which (we presume) had diverged at quite an early stage from the common lineage (NJ/MP 71/62). Also the haplotype Thy Kitka diverged early from the Baltic ancestor. It lives in a lake which was connected to Baltic Sea up until 8400 BP , but then tilted to drain eastwards to White Sea. Mean distance within the clade is large, 1.1 ^ 0.2%, indicating that this clade is the most variable of all. Grayling parasites from the River Oulanka-Olanga (Kovdozero river system), and Penninki-Pistojoki (Kuitozero and river Kemi system), draining eastwards to the White Sea, were included in a mitochondrial Clade IV, with a high bootstrap support (NJ/MP 98/97). Mean genetic distance within clade was 0.2 ^ 0.1%. Grayling parasites of clades V and VI were from geographically close locations in Norway (Hansen et al., 2003). However, the river systems are historically separated: Trysilelva is a tributary of Klara¨lven, which maintains a landlocked Baltic salmon population of Lake Va¨nern in Sweden and belongs to geographically defined Baltic Basin (Nilsson et al., 2001). It had two haplotypes, belonging ˚ sta together (NJ/MP 100/99). Glomma and its tributaries A and Rena drain directly into the Oslo fjord (NJ/MP 99/99). The haplotypes in clades among grayling differed as much from each other as from clades I and II, occurring in salmon. Mean distances between grayling specific clades were 2.0– 2.5%, while mean distance between clades on different host varied from 1.8 to 2.6%.

4. Discussion 4.1. Taxonomic problem setting: what did we study? For the parasites studied in this paper, two species names were available: G. salaris Malmberg, and G. thymalli Zˇitnˇan. Current molecular ITS and mitochondrial DNA data and the associated geographical and host specificity observations could be used to support two opposite taxonomic solutions to explain the phylogenetic tree in Fig. 2. We stress that these hypotheses only concern the geographical area and the lineages studied, and are not meant to, or cannot challenge the species status of

521

the original Slovakian G. thymalli, or the British observations on this species. Hansen et al. (2003) suggested three alternatives. Their two-species scenario (i) was that there is a polytypic G. salaris, including clades I and II, and a polytypic G. thymalli, including clades V and VI in their data. This now obviously includes also clades III and IV. This suggestion implies that the host species is a basis for species definition, which can be rejected by several available species concepts (Wheeler and Meier, 2000). None of the nodes below the level of six clades of the phylogenetic tree had significant bootstrap support, i.e. there is no molecular sequence evidence for connecting grayling parasites in one group and salmon parasites in another, or for any other grouping. All the six clades are genuinely sister clades. It is also important to note that in spite of forming separate clades, the two widely sampled ones (I and III) are molecularly not strictly delineated and coherent. That is, the most basal branches resist statistical classification. Our taxonomic hypothesis 1, that all the six clades represent one species only, corresponds to suggestion (ii) of Hansen et al. (2003). The similarity of all nuclear ITS sequences, irrespective of the host and sampling location, support naming a single ‘polytypic’ species, which by rule of priority is G. salaris. Currently a consensus has not been reached within the taxonomic community of species definitions via sequence data (e.g. Mallet and Willmott, 2003; Seberg et al., 2003; Tautz et al., 2003). Even if DNA is accepted as a useful tool, there are no explicit rules to tell, how much divergent the species should be, in ITS or in any DNA segment. Zie˛tara and Lumme (2002, 2003) suggested that among Gyrodactylus, while the species are so difficult to demarcate morphologically, a divergence of 1% in ITS could be a practical limit to separate species, but this proposal is open for debate. Taxonomic hypothesis 2, that there are several species was suggestion (iii) of Hansen et al. (2003). The mitochondrial sequence data could be interpreted as showing that there are six different parasite species, assigned as clades in Figs. 2 and 3, with limited intraspecific variation. The host specificity, deduced from the structure of the tree and observed also in two intimately sympatric situations in the field, supports the idea of separate species. Of course, the host delineates the parasites into two groups only and for consistency, the clades with a similar degree of genetic divergence should be treated as separate as well. According to hypothesis 2, there are four species on grayling and one species restricted to salmon. The sixth species is a moderate generalist in artificial environment, alternating between salmon and rainbow trout. As a counterargument against the multiple species hypothesis (2), the molecular discontinuity between the clades perhaps is not clear enough to support it. Most widely sampled Clade I in salmon and Clade III in grayling contained very basal branches which were not highly supported members of their respective clades, in spite of

522

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

the obvious synapomorphic salmon host in Clade I. One suggested definition of phylogenetic species concept is based on the ‘three-times rule’ (Palumbi et al., 2001). It states that when the branch length leading to the mtDNA sequences of a tentative species is three times longer than the average mtDNA sequence diversity observed within that species, then also nuclear genes are predicted to be monophyletic, which could be used as a kind of definition of species (Palumbi et al., 2001). This ‘rule’ has been criticised (Hudson and Turelli, 2003), but it was not nearly fulfilled among our widely sampled Baltic Gyrodactylus strains in both salmon and grayling. However, this rule strongly supports the monophyly of all six clades together, when compared with G. lavareti. The data available now cover wider range than in any earlier molecular or morphological studies of the G. salaris complex. However, there are a number of parasite populations which were not available for this study, especially among grayling races and species in the eastern part of the range (Koskinen et al., 2002c); the Danubian drainage (including the type locality of G. thymalli); in the Adriatic area, in England, and in continental Europe (Weiss et al., 2002). It would be interesting to place them into the phylogenetic framework presented in Fig. 2. We suggest that the taxonomic hypothesis 1 for the populations studied here is the most parsimonious and consequently, we use only one species name, G. salaris Malmberg, for the whole complex. However, and importantly, we do not discount that even the six lineages here may have taken the first essential steps leading to speciation. In order to remind the wide content of this species name, we may use it in a form G. salaris cluster, or G. salaris sensu lato. 4.2. How does the phylogeny test the hypothesis of speciation by host switch? From the molecular systematic position of G. salaris s.l. in the subgenus G. (Limnonephrotus) (Zie˛tara and Lumme, 2002), it is clear that G. lavareti Malmberg is the closest known sister taxon; much closer than the common salmonid farm parasites Gyrodactylus truttae Gla¨ser; Gyrodactylus teuchis Lautraite, Blanc, Thiery, Danie and Vigneulle; and G. derjavini: all members of wageneri species group (Zie˛tara and Lumme, 2002). G. lavareti is probably a parasite of the whitefish Coregonus, but the specimens sequenced were collected from a rainbow trout farm. The most probable ancestor of all wageneri group species was a parasite of the minnow Phoxinus phoxinus. Host switch over host fish family borders had evidently occurred at least five times in this group but in the minimum scenario, only once to salmonids (Zie˛tara and Lumme, 2002). The mitochondrial data in this paper suggest that the G. salaris cluster is a monophyletic evolutionary clade, distinctly separated from the nearest relative G. lavareti. The phylogenetic tree based on mtDNA variation was like a star (Fig. 2), expanding from a single ancestral form to six clades.

The rainbow trout was imported to Europe some 150 years ago from North America, and it has since acquired G. salaris Clade II from an unknown European source. The parasites of this clade show affinities to salmon and repulsion against grayling in cross infection experiments (Bakke et al., 2002), suggesting salmon as the natural host for it. The most speculative hypothesis derived from this suggestion is that G. salaris Clade II is endemic in the Oslo fjord salmon population, and the susceptibility of the Drammen and Lierelva salmon is due to gene flow from Atlantic populations. However, the lack of genetic variation in Clade II indicates a bottleneck, probably caused by a single introductory infection event to rainbow trout and consequent fugitive life history. Among the non-domesticated G. salaris s.l., one of the clades is hosted by salmon, and four by grayling. The geographically widely distributed reports of G. thymalli indicate that the grayling may be globally the most common host of this complex (see e.g. Denham and Long, 1999), and is therefore expected to be the plesiomorphic host for ancestor of G. salaris s.l. Thus, the minimum natural hostswitching event has supposedly been a single step from grayling to Atlantic salmon. If the natural host of Clade II also is salmon, then two switches are needed, or a rapid geographical split and isolation of the salmon clade, which is consistent with the pattern observed among grayling parasites. On the basis of the star-like phylogenetic tree, it can be concluded that the switch to Atlantic salmon coincides temporally with the separation of allopatric geographical lineages still hosted by grayling. A possible time for this event is thus the latest Weichselian glaciation of Northern Europe, which begun some 130,000 years ago. We suggest that the continental ice separated the ancestor of modern G. salaris s.l. into several freshwater refugia surrounding the Scandinavian Ice Sheet in the east and south (Kvasov, 1979; Mangerud et al., 2001). In one of those refugia, Atlantic salmon also was isolated from seawater, into a lake suitable for wide dispersal of the parasite (e.g. Nilsson et al., 2001). We suggest that this ecologically new situation opened up the opportunity for Gyrodactylus parasitising grayling to switch to salmon, which being now nonanadromous was demographically optimal for developing a large parasite population. The freshwater salmon population postglacially re-colonised the Baltic Basin (Nilsson et al., 2001), and was now perfectly co-adapted with its new parasite. Another possible salmon refugium (for Clade II) was North Sea Ice lake and the headwaters of present day river Elbe, an area well know as a refugium for southern Scandinavian populations of bullhead (Kontula and Va¨ino¨la¨, 2001) or grayling (Koskinen et al., 2002b) and possibly also for salmon (Nilsson et al., 2001). Locating the North Sea salmon refugium is uncertain because the populations of continental Europe went extinct and the remaining descendants have been mixed by postglacial gene flow.

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

The phylogenetic structure of the clades I and III gives circumstantial support to the scenario of late and quick divergence. Among the Baltic basin grayling populations represented in Clade III, Lake Onega was the first large freshwater reservoir to recover under the Scandinavian Ice cap about 11,000 years ago, and it was isolated from other parts of Baltic basin from the beginning. The haplotype, Thy Onega, is basal in the clade. Also Lake Kitka contains a parasite Thy Kitka that diverged at an early stage from the Baltic clade. Lake Kitka was a separate lake, connected to the Baltic Basin since 9000 BP by a short river, but the connection to Baltic was cut off 8400 years ago, by the postglacial land uplift (Heikkinen and Kurimo, 1977, Fig. 4). Since then, fish fauna in lake Kitka and connected rivers have been isolated from other natural sources, because the eastwards draining River Kitka has a 9 m waterfall, Jyra¨va¨, above the uppermost level of the White Sea Ice Lake (Koutaniemi, 1999), which supposedly acts as a barrier. The nearest populations of Clade IV in river Oulanka-Olanga and Pistojoki are geographically very close to the Baltic neighbours, as shown in map in Fig. 4, but still unmixed, due to sedentary habits of grayling (Koskinen et al., 2002a). The headwater populations are

523

relicts of the high water level phase of the White Sea Ice lake, of 10,000– 8000 years ago and Ancylus lake, 9000– 8000 BP (Koutaniemi, 1999). The structure and internal variation of the salmonspecific clade corresponds the structure of the Baltic grayling clade, even if the samples represent the North Sea, the Baltic and the White Sea. It is generally assumed that the infections in Norway and the White Sea are caused by imported parasites. The monophyly of salmon Clade I supports this hypothesis. The source of the White Sea infections is, for logistical reasons, believed to be in the area of the Onega lake. The salmon parasite from Onega has not yet been molecularly analysed, but the basal position of Sal Keret1 resembles the position of Thy Onega in its own clade. The origin of infection in Keret has been traced to a helicopter-carried canvas bag used to transport salmon parr in lake Onega area and, in the same day, from another hatchery (Vyg) to river Keret (Professor Evgeny Ieshko, personal communication). The Swedish haplotype Sal E Go¨te a¨lv (Hansen et al., 2003) is aberrant among the Swedish west coast populations. It remains to be investigated if the landlocked salmon populations in the rivers draining to Lake Va¨nern

Fig. 4. Details of the sampling sites of grayling parasites in the Baltic Sea and the White Sea watershed area. Symbols are the same than in Fig. 1 (O, Clade III; P, Clade IV; p , Clade II). Darkly shaded Lake Kitka drainage area (B) was flowing to the Baltic via Livo River until 8400 BP , and after that eastwards, to the White Sea. Drainage areas B and C were connected during the highest level of White Sea Ice Lake about 9000 BP , but presently there are no natural connections available for grayling migration between B and C.

524

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

carry Gyrodactylus of this haplotype. The lake drains now westwards to Kattegat, but the landlocked salmon was observed to belong to the Baltic maternal lineage, as a relict from Na¨rke strait, which was the first Atlantic connection of recovering Baltic Sea, about 10,000 years ago (Nilsson et al., 2001). The relatedness of haplotypes observed on salmon from Southern Baltic and Swedish west coast (Hansen et al., 2003) are interesting. Haplotype Sal D Gauja from Latvia is only one nucleotide different from type Sal C Susea˚nStensa˚n, which occupies several of the Swedish west coast rivers. It is tempting to suggest that the Swedish west coast parasite population is mostly natural, due to parasites’ dispersal together with the slowly emigrating Baltic host (Nilsson et al., 2001). Malmberg and Malmberg (1993) characterised the gyrodactyliasis at this secondary contact zone of salmon as ‘balanced’, suggesting a degree of coadaptation of host and parasite. The epidemic history of the serious gyrodactyliasis of Atlantic salmon in Norway was resolved by Hansen et al. (2003). Three separate origins of Norwegian salmon infection are clear. In the north, Skibotnselva and Signaldalen populations are infected by the parasite type Sal B found also in Vindela¨lven and Tornio/Torne river in Gulf of Bothnia. The headwaters of Torne river and Signaldalelven are very close to each other along the Norwegian – Swedish watershed. The second infection (Sal A) is the main epidemic, which reportedly started in the middle Norway and has subsequently spread in rivers between Røssa˚ga and ¨ tran and Rauma. This specific parasite type also exists in A Surtan in Swedish west coast, where the history of Baltic salmon stocking is well documented. Due to scarcity of Baltic observations, the haplotype Sal A has not yet been found there. The third Norwegian infection in Oslo fjord and Laerdalselven is obviously transmitted via rainbow trout, if not endemic in Oslo fjord, which is less probable. In the field, sympatric co-occurrence of clades has been observed in two cases. The Gyrodactylus specimens obtained from salmon and grayling in the Rivers Tornio (Finland) and Pistojoki (Karelia, Russia) turned out to represent different mitochondrial clades. In River Tornio, the salmon parasites (n ¼ 66; haplotypes Sal B and Sal T, distribution data under preparation) were typical for Baltic salmon. Those on grayling (haplotypes Thy Tornio1 and 2, n ¼ 5) belonged to the Clade III of Baltic graylings. In the Karelian Pistojoki, the salmon parasites were from the rainbow trout specific Clade II, and the graylings had parasites from the Clade IV of the White Sea Basin graylings. In river Keret, all the parasites from salmon (n ¼ 135, distribution data not under preparation) were Clade I haplotypes, while also graylings of Keret have been reported to carry G. thymalli. Thus, the parasites of different clades seem to be host-specific even in closely sympatric situations, but this has yet to be confirmed by a large field study.

4.3. Estimate of the molecular divergence rate of mtDNA The phylogeographic pattern of the six separate parasite clades on grayling and salmon suggests that they have been isolated from a common ancestor simultaneously, into four refugia situated around the Continental Ice Cap. The last major glaciation cycle started 130,000 years ago. Using this to calibrate the divergence rate, we estimate a nucleotide substitution rate from 13.7 to 20.3% per million years, from the mean divergence between the clades. Most divergence rate estimates rely on calibrations based on paleontological evidence from higher animals, the generation times of which are considerably longer than in flatworm Platyhelminthes, and specifically, in Gyrodactylus. Their generation time may be as short as 4 days in suitable conditions, when the females reproduce through automictic parthenogenesis (Cable and Harris, 2002). It has been demonstrated, with mutation accumulation lines of Caenorhabditis elegans (Nematoda), that the mitochondrial base substitution rate in these lineages was 890% per million years, which was more than two orders of magnitude higher than what was observed in pedigree analyses (Denver et al., 2000). For both Bradybaenid and Acatinellinid land snails isolated on an oceanic island, the divergence rates have been estimated to be 10 – 13% per million years for 16S and 12S rRNA genes; regions that are considered to be more conservative than COI (Chiba, 1999; Watanabe and Chiba, 2001). This indicates that the divergence rate estimates in G. salaris are not extraordinary. Our results suggest two possible lines of speciation for the parasite studied here. The lineages which were isolated by geographical barriers rapidly accumulated molecular differences. This process perhaps is reversible, and as long as the lineages parasitise the original host, there is no need to question their conspecificity. After a rare successful host switch, Gyrodactylus parasites are able to evolve rapidly to a host specific lineage, as suggested by Brooks and McLennan (1993), and confirmed among wageneri species group by Zie˛tara and Lumme (2002). A new lineage is perhaps instantly isolated from the ancestral population, due to predominantly sex-free propagation and intimate contact on hosts in all life stages (Cable and Harris, 2002). The rate of evolution, both molecular (mitochondrial) and adaptive (host specificity), seems to be very rapid. Our results have a pertinence to fish disease prevention. At least one of the G. salaris lineages is spreading through the rainbow trout vector, and it is able to infect wild Atlantic salmon. On the other hand, contemporary parasites on grayling seem to be specific enough to stay on grayling most of the time. However, what is clear is that the phylogeny of the salmon specific lineage indicates that it switched from grayling to salmon at least once; suddenly and successfully.

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

Acknowledgements We would like to thank the people who supplied us with parasites: Perttu Koski, Kurt Buchmann, Go¨ran Malmberg, and Bjo¨rn Ove Johnsen. Even more people participated in the expeditions to catch grayling, and other fish: Alexei Veselov, Igor Bakhmet, Dimitry Stepanov, Ahti Karusalmi, Ari-Pekka Huhta, Kalevi Kuusela, Ari Pesonen, Leo Koutaniemi, and Igor Shurov. Laboratory help from Hannele Parkkinen is gratefully acknowledged. David Hughes and several anonymous referees helped to sharpen the message. The work was supported by the Finnish Academy (grant no. 63797), Emil Aaltonen Foundation (to M.M.), Suomen Kulttuurirahasto (to M.M.), and Naturpolis Kuusamo (to J.K.). The Finnish Ministry of Agriculture and Forestry supported the Karelian expeditions.

References Bakke, T.A., Harris, P.D., Cable, J., 2002. Host specificity dynamics: observations on gyrodactylid monogeneans. Int. J. Parasitol. 32, 281–308. Brooks, D.R., McLennan, D.A., 1993. Parascript: Parasites and the Language of Evolution. Smithsonian Institution Press, Washington, DC. Cable, J., Harris, P.D., 2002. Gyrodactylid developmental biology: historical review, current status and future trends. Int. J. Parasitol. 32, 255–280. Chiba, S., 1999. Accelerated evolution of land snails Mandarina in the Oceanic Islands: evidence from mitochondrial DNA sequences. Evolution 53, 460 –471. Cribb, T.H., Chisholm, L.A., Bray, R.A., 2002. Diversity in the Monogenea and Digenea: does lifestyle matter? Int. J. Parasitol. 32, 321–328. Cunningham, C.O., 1997. Species variation within the internal transcribed spacer (ITS) region of Gyrodactylus (Monogenea: Gyrodactylidae) ribosomal RNA genes. J. Parasitol. 83, 215 –219. Cunningham, C.O., Mo, T.A., Collins, C.M., Buchmann, K., Thiery, R., Blanc, G., Lautraite, A., 1999. Redescription of Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel and Vigneulle, 1999 (Monogenea: Gyrodactylidae); a species identified by ribosomal RNA sequence. Syst. Parasitol. 48, 141–150. Denham, K.L., Long, J., 1960. Occurrence of Gyrodactylus thymalli Zˇitnˇan, 1960 on grayling, Thymallus thymallus (L.), in England. J. Fish Dis. 22, 247–252. Denver, D.R., Morris, K., Lynch, M., Vassilieva, L.L., Thomas, W.K., 2000. High direct estimate of the mutation rate in the mitochondrial genome of Caenorhabditis elegans. Science 289, 2342– 2344. Hansen, H., Bachmann, L., Bakke, T.A., 2003. Mitochondrial DNA variation of Gyrodactylus spp. (Monogenea, Gyrodactylidae) populations infecting Atlantic salmon, grayling, and rainbow trout in Norway and Sweden. Int. J. Parasitol. 33, 1471– 1478. Heikkinen, O., Kurimo, H., 1977. The post-glacial history of Kitkaja¨rvi, North-Eastern Finland, as indicated by trend-surface analysis and radiocarbon dating. Fennia 153, 1–32. Hudson, R.R., Turelli, M., 2003. Stochasticity overrules the three-times rule: genetic drift, genetic draft, and coalescence times for nuclear loci versus mitochondrial DNA. Evolution 57, 182–190. Johnsen, B.O., Jensen, A., 1991. The Gyrodactylus story in Norway. Aquaculture 98, 289–302.

525

Kontula, T., Va¨ino¨la¨, R., 2001. Postglacial colonization of Northern Europe by distinct phylogeographic lineages of the bullhead, Cottus gobio. Mol. Ecol. 10, 1983– 2002. Koski, P., Malmberg, G., 1995. Occurrence of Gyrodactylus (Monogenea) on salmon and rainbow trout in fish farms in Northern Finland. Bull. Scand. Soc. Parasitol. 5, 76–88. Koskinen, M.T., Sundell, P., Piironen, J., Primmer, C.R., 2002a. Genetic assessment of spatiotemporal evolutionary relationships and stocking effects in grayling (Thymallus thymallus, Salmonidae). Ecol. Lett. 5, 193– 205. Koskinen, M.T., Nilsson, J., Veselov, A.Je., Potutkin, A.G., Ranta, E., Primmer, C.R., 2002b. Microsatellite data resolve phylogeographic patterns in European grayling, Thymallus thymallus, Salmonidae. Heredity 88, 391–401. Koskinen, M.T., Knizhin, I., Primmer, C.R., Schlo¨tterer, C., Weiss, S., 2002c. Mitochondrial and nuclear DNA phylogeography of Thymallus spp. (grayling) provides evidence of ice-age mediated environmental perturbations in the world’s oldest body of fresh water, Lake Baikal. Mol. Ecol. 11, 2599– 2611. Koutaniemi, L., 1999. Physical characteristics and palaeogeography of the Oulanka-Paanaja¨rvi region of the Finnish–Karelian border. Fennia 177, 3–9. Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: Molecular Evolutionary Genetics Analysis Software. Arizona State University, Tempe, AZ. Kvasov, D.D., 1979. The Late-Quaternary history of large lakes and inland seas of Eastern Europe. Ann. Acad. Sci. Fenn. A 127, 1–71. Mallet, J., Willmott, K., 2003. Taxonomy: renaissance or Tower of Babel? Trends Ecol. Evol. 18, 57– 59. Malmberg, G., 1957. Om fo¨rekomsten av Gyrodactylus pa˚ svenska fiskar. ˚ rskrift, 1956. Skrifter Utgivna av So¨dra Sveriges Fiskerifo¨rening, A Malmberg, G., 1970. The excretory systems and the marginal hooks as a basis for the systematics of Gyrodactylus (Trematoda, Monogenea). Ark. Zool. 23, 1– 235. Malmberg, G., 1993. Gyrodactylidae and gyrodactylosis of salmonidae. Bull. Fr. Peche Piscic. 328, 5–46. Malmberg, G., Malmberg, M., 1993. Species of Gyrodactylus (Platyhelminthes, Monogenea) on salmonids in Sweden. Fish. Res. 17, 59 –68. Mangerud, J., Astakhov, V., Jakobsson, M., Svendsen, J.I., 2001. Huge iceage lakes in Russia. J. Quaternary Sci. 16, 773–777. McHugh, E.S., Shinn, A.P., Kay, J.W., 2000. Discrimination of the notifiable pathogen Gyrodactylus salaris from G. thymalli (Monogenea) using statistical classifiers applied to morphometric data. Parasitology 121, 315– 323. Mate˘jusova´, I., Gelnar, M., McBeath, A.J.A., Collins, C.M., Cunningham, C.O., 2001. Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei). Int. J. Parasitol. 31, 738– 745. Meinila¨, M., Kuusela, J., Zie˛tara, M.S., Lumme, J., 2002. Primers for amplifying ,820 bp of highly polymorphic mitochondrial COI gene of Gyrodactylus salaris. Hereditas 137, 72–74. Nilsson, J., Gross, R., Asplund, T., Dove, O., Jansson, H., Kelloniemi, J., Kohlman, K., Lo¨ytynoja, A., Nielsen, E.E., Paaver, T., Primmer, C.R., ¨ st, T., Lumme, J., 2001. Titov, S., Vasema¨gi, A., Veselov, A., O Matrilinear phylogeography of Atlantic salmon (Salmo salar L.) in Europe and postglacial colonization of the Baltic Sea area. Mol. Ecol. 10, 89– 102. Palumbi, S.R., Cipriano, F., Hare, M.P., 2001. Predicting nuclear gene coalescence from mitochondrial data: the three-times rule. Evolution 55, 859–868. Poulin, R., 2002. The evolution of monogenean diversity. Int. J. Parasitol. 32, 245–254. Seberg, O., Humphries, C.J., Knapp, S., Stevenson, D.W., Petersen, G., Scharff, N., Andersen, N.M., 2003. Shortcuts in systematics? A commentary on DNA-based taxonomy. Trends Ecol. Evol. 18, 63– 65. Tautz, D., Arxtander, P., Minelli, A., Thomas, R.H., Vogler, A., 2003. A plea for DNA taxonomy. Trends Ecol. Evol. 18, 70–74.

526

M. Meinila¨ et al. / International Journal for Parasitology 34 (2004) 515–526

Watanabe, Y., Chiba, S., 2001. High within-population mitochondrial DNA variation due to microvicariance and population mixing in the land snail Euhadra quaesita (Pulmonata: Bradybaenidae). Mol. Ecol. 10, 2635–2645. Weiss, S., Persat, H., Eppe, R., Schlo¨tterer, C., Uiblens, F., 2002. Complex patterns of colonization and refugia revealed for European grayling Thymallus thymallus, based on complete sequencing of the mitochondrial DNA control region. Mol. Ecol. 11, 1393–1407. Wheeler, Q.D., Meier, R., 2000. Species Concepts and Phylogenetic Theory. A Debate. Columbia University Press, New York.

Zie˛tara, M.S., Lumme, J., 2002. Speciation by host switch and adaptive radiation in a fish parasite genus Gyrodactylus (Monogenea: Gyrodactylidae). Evolution 56, 2445–2458. Zie˛tara, M.S., Lumme, J., 2003. The crossroads of molecular, typological and biological species concepts: two new species of Gyrodactylus Nordmann, 1832 (Monogenea: Gyrodactylidae). Syst. Parasitol. 55, 39 –52. ¨ sche Zˇitnˇan, R., 1960. Gyrodactylus thymalli sp. nov. aus den Flossen der A (Thymallus thymallus L.). Helminthologia 2, 266–269.