Evolution, 58(5), 2004, pp.

1001–1018

MOLECULAR PHYLOGENY OF CONGENERIC MONOGENEAN PARASITES

(DACTYLOGYRUS): A CASE OF INTRAHOST SPECIATION
ANDREA ŠIMKOVÁ,1,2,3 SERGE MORAND,4,5 EDOUARD JOBET,6,7 MILAN GELNAR,1,8 AND OLIVIER VERNEAU2,9
1 Department of Zoology and Ecology, Faculty of Science, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic
2 Parasitologie
Fonctionnelle et Evolutive, UMR 5555 CNRS-UP, Université, 52 Avenue Paul Alduy,
66860 Perpignan Cedex, France
3 E-mail: [email protected]
4 Centre de Biologie et de Gestion des Populations, Campus international de Baillarguet, CS 30016, Montferrier sur Lez cedex,

34988 Montpellier, France

5 E-mail: [email protected]
6 Laboratoire Génome et Développement des Plantes, UMR 5096 CNRS-UP, Université, 52 Avenue Paul Alduy,

66860 Perpignan Cedex, France

7 E-mail: [email protected]
8 E-mail: [email protected]
9 E-mail: [email protected]

Abstract. Dactylogyrus species (Dactylogyridae: Monogenea) are a group of monogenean gill parasites that are highly
specific to freshwater fish of the family Cyprinidae. Dactylogyrus species were sampled from 19 cyprinids and one
percid collected in Europe. Using partial 18S rDNA and ITS1 sequences, a phylogeny of 51 Dactylogyrus species
was reconstructed to investigate the patterns of parasite speciation and diversification. Three main Dactylogyrus lineages
were recognized from all phylogenetic trees, that is, analysis of 18S rDNA alone and combined 18SrDNA and ITS1.
The first lineage associates the Dactylogyrus species of Cyprinus carpio and Carassius auratus of the Cyprininae; the
second associates Dactylogyrus species of the Gobioninae, Pseudorasbora parva of the Rasborinae, and Ctenophar-
yngodon idella of the Cyprininae; and the third associates Dactylogyrus species of the Leuciscinae and Alburninae
and Barbus barbus of the Cyprininae. Our results suggest that the genus Dactylogyrus is of quite recent origin and
that these three lineages separated from each other in a very short period of time. Host subfamily mapping onto the
parasite tree inferred from analysis of the combined dataset showed that the Cyprininae could be plesiomorphic hosts
for Dactylogyrus. Dactylogyrus parasites would have secondarily colonized the Percidae and representatives of the
Leuciscinae, Alburninae, Gobioninae, and Rasborinae. Comparison of host and parasite phylogenetic relationships
indicated that a very high number of parasite duplications occurred within two of the three Dactylogyrus lineages.
Dactylogyrus diversification can be mainly explained by sympatric intrahost speciation events that seem to be correlated
to strict host specificity. Moreover, the present study shows that the congeneric parasites speciating within one host
tend to occupy niches within hosts differing at least in one niche parameter.

Key words. Cyprinidae, Dactylogyrus, intrahost speciation, molecular phylogeny, Monogenea, niche preference.

Received October 20, 2003. Accepted January 8, 2004.

The search for patterns and processes of evolution to un- 1991), thus facilitating host switches. In that case, phylo-
derstand speciation and diversification of parasites has been genetically closely related parasite species infesting different
one of the main subjects in evolutionary biology (Brooks and host species are exemplified by incongruent host and parasite
McLennan 1993). As suggested by Brooks and MacLennan phylogenies. However, sympatric speciation may also be con-
(1993), one important point is the investigation of host spec- sidered when a single host species is infested by a mono-
ificity that can be the basis of parasite speciation. Parasite phyletic parasite lineage. In that case, it represents intrahost
speciation may occur in allopatry, that is, on two distinct speciation or parasite duplication, that is, the parasite spe-
geographically isolated host species, or sympatry, that is, ciates without a corresponding host cospeciation event and
either on distinct host species by a host shift or in the same this leads to two or more lineages of parasites being present
host species by duplication. on single host species (Paterson and Gray 1997). Vickery and
Cospeciation in host-parasite assemblages was first re- Poulin (1998) suggested that this could explain the occur-
ported for pocket gophers and their chewing lice (Hafner and rence of congeneric parasite species in the same host species.
Nadler 1988; Hafner et al. 1994). In that case, allopatric Finally, it is sometimes difficult to distinguish between sym-
parasite speciation was involved, following geographical iso- patric and allopatric speciation when parasite extinction has
lation of their hosts. Thus, when host lineages speciate, par- occurred during host evolution (Page et al. 1998; Paterson
asites on the descendant host species may also speciate, which and Banks 2001). In that case, the phylogenetic patterns of
is illustrated by congruent host and parasite phylogenies. The parasites cannot be easily explained, even in the light of host
presence of sister species in small areas (e.g., isolated islands relationships.
or lakes) might imply sympatric speciation (Coyne and Price The Monogenea, as a group of ectoparasites with a direct
2000), which can present an outcome of competition for re- life cycle that predominantly live on the gills and skin of
sources (Dieckmann and Doebeli 1999). When considering fish, seem to be an appropriate model for studying the process
only parasites, sympatric speciation might be the result of of parasite diversification, mainly because of their high spe-
association of the host with particular ecological conditions cies richness and morphological and ecological diversity
(Klassen and Beverley-Burton 1987; Guégan and Agnèse (Poulin 2002). Moreover, monogeneans are highly host (re-
1001
q 2004 The Society for the Study of Evolution. All rights reserved.
1002 ANDREA ŠIMKOVÁ ET AL.

stricted to one or a few host species) and niche specific (re- et al. 1996), although, occasionally reported on the Percidae
stricted to several microhabitats within the same host spe- (Gusev 1985; Valtonen et al. 1990; Cone et al. 1994; Hayward
cies), and it has been shown that congeneric species could 1997). In this genus, a high number of congeneric species
coexist on the same host because of a low level of interspe- coexisting on the same host species has been reported (Rohde
cific competition (Rohde 1977, 1979, 1991; Šimková et al. 1989; Kennedy and Bush 1992; Šimková et al. 2000, 2002),
2000). To date, several models of congeneric monogenean and several studies have been conducted on host specificity
species have been investigated to study the process of parasite within the Cyprinidae (Guégan and Agnèse 1991; Guégan
diversification using phylogenies (Littlewood et al. 1997; and Lambert 1991; El Gharbi et al. 1994; Lambert and El
Desdevises et al. 2002; Huyse and Volckaert 2002). Gharbi 1995; Šimková et al. 2001). The coexistence of Dac-
Congeneric gill parasites belonging to Lamellodiscus tylogyrus species on the same host was suggested to be fa-
(Monopisthocotylea), a group of parasites specific to marine cilitated by niche distances and differing morphology of re-
fish of the Sparidae, were used as a model by Desdevises et productive apparatus (Šimková et al. 2002; Jarkovský et al.
al. (2002). These parasites are either generalists, infesting 2004).
different host species, or specialists that may infest one host Similarly, the Cyprinidae is the most diverse family among
species. Desdevises et al. (2002) suggested that the genus freshwater fish with about 2000 species (Helfman et al. 1997).
was old and that intraspecific morphological variability could Seven subfamilies are recognized, among them six with rep-
explain their ability to colonize different host species. They resentatives occurring in Europe (Acheilognathinae, Albur-
proposed that the rapid speciation of Lamellodiscus that infest ninae, Cyprininae, Gobioninae, Leuciscinae, and Rasbori-
hosts living in sympatry happened without any cospeciation nae), the most diverse being the Leuciscinae (Winfield and
and intrahost speciation. Similarly, no intrahost speciation Nelson 1991). It has been believed for a long time that cyp-
was reported for Gyrodactylus (Monopisthocotylea), a genus rinids originated in Asia and later dispersed to Europe, Africa,
occurring in multiple parasite lineages on single host species and North America (Winfield and Nelson 1991). Neverthe-
of freshwater and marine fish (Huyse and Volckaert 2002). less, it is still questioned whether eastern Asia was an im-
Zietara and Lumme (2002) proposed the host switch as the portant interchange for cyprinids or a center of speciation
model of Gyrodactylus speciation facilitated by their partic- (Durand et al. 2002).
ular reproductive system. The present study carried out phylogenetic analyses, based
Polystomes (Polyopisthocotylea) are geographically wide- on partial 18S, or small subunit, ribosomal RNA gene (rDNA)
spread endoparasitic monogeneans infecting mainly amphib- and ribosomal RNA gene internal transcribed spacer 1 (ITS1)
ians and freshwater turtles. These parasites are morpholog- sequences obtained from 51 Dactylogyrus species sampled
ically very similar, are highly host specific, and congeneric from cyprinids and one percid species of central Europe. The
species can be found on the same host species within turtles, process of parasite speciation and diversification, especially
but infecting different niches (i.e., oral cavities, urinary blad- whether congeneric parasites coexisting on the same host
ders, and conjunctival sacs). Littlewood et al. (1997), by species have speciated via intrahost speciation and whether
investigating phylogenetic relationships of six polystome phylogenetically related parasites occupied similar or differ-
species of turtles, showed no intrahost speciation and con- ent microhabitats, was investigated.
cluded that the occurrence of congeneric species within the
same host species could be the result of either host switching MATERIALS AND METHODS
or cospeciation.
Parasite Sampling
Therefore, one may ask whether parasite speciation may
occur within a single host. Moreover, within one host species, A total of 51 Dactylogyrus species, including specialist and
different congeneric species can occupy different niches, that generalist species, were collected from freshwater fish of the
is, microhabitats within the host (Rohde 1989). Therefore, Morava River basin in the Czech Republic (central Europe).
one could also ask whether phylogenetically closely related Among them, 49 species parasitizing fish species belonging
congeneric species parasitize similar or different microhab- to the family Cyprinidae (19 cyprinid species) and two spe-
itats within hosts. In the case of site-specific polystome cies parasitizing Gymnocephalus cernuus belonging to the
monogeneans, congeneric species infecting the same niche family Percidae, were recognized. A total of 44 Dactylogyrus
within the different host species (even if geographically iso- species were collected only on one host species, and seven
lated host species) are more closely related than congeners Dactylogyrus species were collected on more than one host
infecting different sites of the same host species (Littlewood species. For generalist species, several individuals of the dif-
et al. 1997). Following the hypotheses of Rohde (1991), niche ferent host species were collected. Dactylogyrus species with
segregation or reproductive isolation between congeneric their hosts are given in Table 1. This sample is representative
species parasitizing the same host species has been predicted, of the fauna living in central Europe (71 species of Dactyl-
to prevent competition and to increase intraspecies mating ogyrus and 34 host species are known).
contacts (Šimková et al. 2000; Morand et al. 2002). Fish sampling was performed as described in Ergens and
The model of congeneric monogeneans of the genus Dac- Lom (1970). Parasites were removed from the gills, placed
tylogyrus (Dactylogyrinae: Dactylogyridae: Monopisthoco- on slides, covered by a coverslip, and identified using a light
tylea) was investigated in this study for the following reasons: microscope equipped with phase contrast, differential inter-
Dactylogyrus is a highly diversified group within the Mono- ference contrast, and digital image analysis (MicroImage 4.0
genea (Gusev 1985) with more than 900 nominal species for Windows, Olympus Optical Co., Hamburg, Germany).
mainly restricted to freshwater fish of the Cyprinidae (Gibson The sclerotized parts of the parasite attachment organ (the
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1003

TABLE 1. Dactylogyrus sequences obtained in this study with their host species and GenBank accession numbers for partial 18SrDNA
and ITS1. Numbers 1, 2, and 3 on the right column refer to the Dactylogyrus lineages deduced from the parasite neighbor-joining tree.
Dactylogyrus species and their hosts investigated for niche preference are indicated by asterisks.

Fish species Dactylogyrus species Accession number Dactylogyrus lineage

Abramis ballerus auriculatus AJ564112 3
chranilowi AJ564117 3
Abramis brama* falcatus AJ564130 3
wunderi* AJ564164 3
zandti* AJ564165 3
Abramis sapa propinquus AJ564147 3
Alburnus alburnus* alatus* AJ564109 3
fraternus* AJ564136 3
minor* AJ564143 3
parvus* AJ564146 3
Aspius aspius ramulosus AJ564149 3
tuba AJ564158 3
Barbus barbus* carpathicus* AJ564115 3
dyki AJ564127 3
malleus* AJ564142 3
Abramis bjoerkna* cornoides* AJ564118 3
cornu* AJ564119 3
distinguendus* AJ564125 3
sphyrna* AJ564155 3
Carassius auratus* anchoratus* AJ564111 1
dulkeiti* AJ564126 1
formosus* AJ564135 1
inexpectatus* AJ564138 1
intermedius* AJ564139 1
vastator* AJ564159 1
Chondrostoma nasus chondrostomi AJ564116 3
ergensi AJ564128 3
vistulae AJ564160 3
Ctenopharyngodon idella lamellatus AJ564141 2
Cyprinus carpio achmerovi AJ564108 1
anchoratus AJ490161 1
extensus AJ564129 1
Gobio albipinatus finitimus AJ564133 2
Gobio gobio cryptomeres AJ564123 2
Gymnocephalus cernuus amphibothrium AJ564110 3
hemiamphibothrium AJ564137 3
Leuciscus cephalus* fallax AJ564132 3
folkmanovae* AJ564134 3
nanoides* AJ564144 3
prostae* AJ564148 3
vistulae* AJ564161 3
vranoviensis* AJ564163 3
Leuciscus idus crucifer AJ564122 3
ramulosus AJ564150 3
tuba AJ564157 3
vistulae AJ564162 3
Phoxinus phoxinus borealis AJ564113 3
Pseudorasbora parva squameus AJ564156 2
Rutilus rutilus* caballeroi* AJ564114 3
crucifer* AJ564120 3
fallax* AJ564131 3
nanus* AJ564145 3
rarissimus* AJ564151 3
rutili* AJ564152 3
similis* AJ564153 3
sphyrna AJ564154 3
Scardinius erythrophthalmus* crucifer AJ564121 3
difformis* AJ490160 3
difformoides* AJ564124 3
izjumovae* AJ564140 3

opisthaptor; i.e., the central hooks called anchors, seven pairs for parasite determination according to Gusev (1985). Some
of marginal hooks, one connective bar [dorsal in this case] parasite specimens were fixed in a mixture of glycerine and
or two connective bars [dorsal and ventral]) and reproductive ammonium picrate and deposited in the collection of the De-
organs (vaginal armaments and copulatory organs) were used partment of Zoology and Ecology, Masaryk University (Brno,
1004 ANDREA ŠIMKOVÁ ET AL.

Czech Republic). Other parasites were preserved in 95% eth- at positions 274–294 of D. anchoratus accession no.
anol before DNA extraction. AJ490161. Sequencing was carried out using ABI Prism Big
Dye Terminator Cycle Sequencing kit (Applied Biosystems,
DNA Extraction and Polymerase Chain Reaction Foster City, CA) and electrophoresis was performed on an
automated sequencer (ABI373 DNA Sequencer). Sequences
Individual parasites were removed from ethanol, dried, and
were read and corrected using Sequencher software (Gene
DNA was extracted using the standard phenol-chloroform
Codes Corp., Ann Arbor, MI). New sequences were deposited
method (Sambrook et al. 1989). For generalist species, in-
in EMBL (see Table 1 for their accession numbers).
dividuals from different host species were investigated. Total
DNA obtained from individual parasites was resuspended in
25 ml distilled water. Partial 18S rDNA and entire ITS1 re- Phylogenetic Analyses of Dactylogyrus
gion were amplified in one round using primer S1 (59- DNA sequences were aligned using the ED program in the
ATTCCGATAACGAACGAGACT-39) that anneals in the MUST package (Philippe 1993). Gaps and ambiguously
terminal region of the 18S gene (Sinnappah et al. 2001) and aligned regions were removed. First, phylogenetic analysis
H7 (59-GCTGCGTTCTTCATCGATACTCG-39; Sinnappah using partial 18S rDNA alone was performed to polarize
et al. 2001) or IR8 primers (59-GCTAGCTGCGTTCTTCA- Dactylogyrus evolution as the variability of this marker al-
TCGA-39; Šimková et al. 2003) that anneal in the 5.8S rDNA. lows for unambiguous alignment with the outgroup. For this
Each amplification reaction was performed in a final volume analysis seven other monogenean species belonging to dif-
of 25 ml containing 1.5 units of Taq polymerase, 1X buffer, ferent subfamilies of the Dactylogyridae were included, fol-
1.5 mM MgCl2, 200 mM of each dNTP, 0.8 mM of each lowing the phylogenetic relationships previously inferred for
polymerase chain reaction (PCR) primer and 3 ml DNA. PCR Dactylogyridae (Šimková et al. 2003). Among them two spe-
was carried out with the following steps: 4 min at 958C fol- cies, Thaparocleidus vistulensis (accession no. AJ490165) of
lowed by 35 cycles of 1 min at 958C, 1 min at 558C, and 1 the Ancylodiscoidinae and Cleidodiscus pricei (AJ490168)
min 30 sec at 728C, and 10 min of final elongation at 728C. of the Ancyrocephalinae, were used for rooting the tree. Five
Electrophoresis was performed on a 1% agarose gel stained other species previously found to form a group including
with ethidium bromide for DNA visualization. PCR products Dactylogyrus species were also used (i.e., Pseudodactylogy-
were cut from the gel and purified using siliconized glass- roides apogonis, Pseudodactylogyrus anguillae, and P. haze
wool. DNA was precipitated and resuspended in sterile water. of the Pseudodactylogyrinae and Thylacicleidus sp. and
Some of the PCR products were sequenced directly. Others Pseudohaliotrema sphincteropus of the Ancyrocephalinae;
were ligated into the pGEM-T vector and cloned in Esche- AB065115, AJ490162, AB0651141, AJ490169, AJ287568,
richia coli JM109 competent cells when the amount of pu- respectively). For generalist species, only the sequence of the
rified DNA after PCR was too low for direct sequencing. individual parasitizing the most commonly infested host was
Recombinant colonies were checked following Sekar’s pro- retained, so 58 species were analyzed. Distance trees were
cedure (1987) and plasmids were purified with the Wizard generated with a neighbor-joining (NJ) algorithm based on
Plus miniprep kit (Promega, Madison, WI). At least three Kimura two-parameter distances (Kimura 1980) and per-
clones were sequenced per species. formed with PAUP* 4b10 (Swofford 2002). Support values
for internal nodes were estimated using a bootstrap resam-
DNA Sequencing pling procedure with 1000 replicates (Felsenstein 1985).
The direct sequencing of purified PCR products as well as Maximum likelihood (ML) and NJ analyses based on ML
recombinant plasmids was performed using the same primers distances were also conducted using PAUP from the best
as for PCR or universal primers (T7 and SP6) supplied by appropriate model (TrNf 1 G 1 I in this case) selected by
Promega. Moreover, for sequencing species included in the the ModelTest program (Posada and Crandall 1998). The
D. minor group (group 3, see Results), two forward primers search of the best ML tree was done using a branch-swapping
IF8 (59-AACTGTTCAATCATCGTCGTG-39; Šimková et al. algorithm (TBR, tree bisection reconnection). One hundred
2003) and newly designed IF9 (59-ATCCGCCGACTCT- replicates for ML and 1000 replicates for NJ were calculated
GACTGGA-39) and two reverse primers IR5 (59-TA- using the same model as above, using the TBR branch-swap-
CGGAAACCTTGTTACGAC-39; Sinnappah et al. 2001) and ping algorithm for ML.
newly designed IR9 (59-RRGACTCACCCGAAGGGAG-39) Maximum parsimony (MP) analysis was also performed
were used. For sequencing of species included in the D. an- using heuristic search with stepwise random addition se-
choratus group (groups 1 and 2, see Results), the same in- quence running on unweighted informative characters. Sup-
ternal primers were used, with the exception of IF9, which port values for internal nodes were estimated after 1000 rep-
was replaced by IF10 (59-YMTTCTCCCTTCGGGTGAGT- licates using the TBR branch-swapping algorithm.
39) also designed for this study. Further analyses were performed using combined sequenc-
Sequences obtained, including partial 18S rDNA, complete es from partial 18S rDNA and ITS1 without including other
ITS1, and partial 5.8S rDNA ranged from 962 to 1130 bp. species belonging to the Dactylogyridae as ITS1 sequences
Primers IF8 and IR5 anneal in the 18S rDNA at positions of Dactylogyrus species could not be aligned with ITS1 se-
234–254 and 471–490, respectively, of D. difformis sequence quences of the seven species of Dactylogyridae used in the
accession no. AJ490160. The primers IF9 and IR9 anneal in analysis of 18S rDNA. The analyses of combined 18S rDNA
the ITS1 region at positions 223–243 and 348–367, respec- and ITS1 data were done to obtain a better phylogenetic
tively, of D. difformis, and IF10 anneals in the ITS1 region resolution within the Dactylogyrus genus. NJ analysis was
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1005

TABLE 2. Fish species used for phylogenetic analysis, with the exception of Gobio albipinatus, for which the cytochrome b gene has
not yet been sequenced. Asterisks indicate species for which Dactylogyrus species were recorded. The taxonomic position and GenBank
accession numbers for cytochrome b sequences are indicated for each species.

Order Family Subfamily Species Accession number

Cypriniformes Balitoridae Crossostoma lacustre M91245
Characidae Astyanax mexicanus AF045997
Cyprinidae Cyprininae Barbus barbus* Y10450
Carassius auratus* AF051858
Cyprinus carpio* X61010
Ctenopharyngodon idella* AF051860
Gobioninae Gobio albipinatus* no sequence available
Gobio gobio* Y10452
Rasborinae Pseudorasbora parva* Y10453
Leuciscinae Abramis ballerus* AY026409
Abramis bjoerkna* Y10442
Abramis brama* Y10441
Abramis sapa* AY026408
Aspius aspius* AY026398
Chondrostoma nasus* AY026402
Leuciscus cephalus* Y10446
Leuciscus idus* AY026397
Phoxinus phoxinus* Y10448
Rutilus rutilus* Y10440
Scardinius erythrophthalmus* Y10444
Alburninae Alburnus alburnus* Y10443
Perciformes Percidae Ammocrypta clara AF045350
Etheostoma kennicotti AF045341
Gymnocephalus cernuus* AF045356
Perca flavencens AJ001521
Perca fluviatilis AF045358
Zingel streber AF045360
Salmoniformes Salmonidae Oncorhynchus mykiss L29771

performed as described above from Tamura Nei distances ing the same model. MP analysis was performed as described
including a proportion of invariable characters and gamma for previous analyses.
distribution (TrN 1 I 1 G). One thousand bootstrap replicates
were conducted following the same evolutionary model. ML Mapping of Host Subfamilies onto the Parasite Tree
analyses were also performed on the TrN 1 I 1 G model
The host subfamily for each Dactylogyrus species was
selected by ModelTest. One hundred replicates were calcu-
mapped onto the NJ tree inferred from analysis of combined
lated following the same model and using the same procedure
data on 51 Dactylogyrus species, using MacClade version
described for the analyses of 18S rDNA alone. MP analysis
4.0.1 with Farris optimization (Maddison and Maddison
was performed as described in the case of 18S rDNA.
1992). Host species were divided into subfamilies according
to Nelson (1994) and are given in Table 2. For parasite spe-
Host Phylogeny
cies that have been recorded on different host species (gen-
The phylogeny of cyprinids has been previously investi- eralists) of different host subfamilies, mapping was per-
gated using mitochondrial markers (Briolay et al. 1998; Gilles formed with the host subfamily from which parasites were
et al. 1998, 2001; Zardoya and Doadrio 1999; Zardoya et al. examined and analyzed. It should be noted that this host
1999; Durand et al. 2002), but not all species used in our subfamily corresponds to the most infested host species.
study were included. Thus, we reconstructed the host phy-
logeny from cytochrome b sequences retrieved from Gen- Comparison of Host and Parasite Phylogenies
Bank, including 18 representatives of the Cyprinidae, one of
TreeMap 1.0 (Page 1994) was used to represent host-par-
the Balitoridae, one of the Characidae, and six of the Per-
asite associations using the Dactylogyrus tree inferred from
cidae. The tree was rooted with Oncorhynchus mykiss from
analysis of combined data and the cyprinid tree after ex-
the Salmonidae. All species with their accession numbers are
cluding fish species not infested by Dactylogyrus. Gobio al-
given in Table 2.
bipinatus was added to the fish topology as a sister species
The ModelTest program was used for selecting the best
of G. gobio considered as a monophyletic group (Baruš and
evolutionary model for ML analysis (TVM 1 I 1 G). Support
Oliva 1995).
values for internal nodes were estimated using a bootstrap
resampling procedure with 100 replicates following a branch-
Mapping of Niche Preference
swapping algorithm (TBR) on the same model. NJ analysis
was performed using evolutionary parameters selected by The mapping of niche preference was performed using the
ModelTest. One thousand replicates were calculated follow- same methodology as for mapping of host subfamilies. For
1006 ANDREA ŠIMKOVÁ ET AL.

niche preference, data concerning niche position (i.e., the with bootstrap proportions (BP) supported the monophyly of
position on the gills within the hosts) were recorded for 33 Dactylogyrus (BP 5 84; see Fig. 1).
Dactylogyrus species obtained from eight different cyprinid ML analysis was performed on the TrNf 1 G 1 I model,
species (asterisks in Table 1). We could not obtain niche data with the following parameters: substitution rate matrix: A-C
for all Dactylogyrus species from all host species investigated 5 1.000, A-G 5 4.1211, A-T 5 1.000, C-G 5 1.000, C-T
because of either low host sample or low parasite sample. 5 5.4533, G-T 5 1.000; proportion of invariable sites 5
Some fish species were found only rarely, and even when 0.4342; and rate heterogeneity approximated by a gamma
using a relatively high number of host individuals we ob- distribution (four rate categories), a 5 0.5912. Surprisingly,
tained only a few individual specimens of several Dactylo- the best ML tree (not shown) shows nonmonophyly of Dac-
gyrus species. tylogyrus. Indeed, the group including D. cryptomeres, D.
The parasite niche was investigated as previously described finitimus, D. squameus, and D. lamellatus was positioned as
in Šimková et al. (2000). For a description of the gill arch, the sister group of the Pseudodactylogyrinae. Following NJ
see Figure 6A. The niche position of each parasite individual analysis using TrNf 1 G 1 I, species of Pseudodactylogyr-
was recorded (i.e., its position on the gill arch, gill segment, inae are not nested within Dactylogyrus species. Neverthe-
gill area), and the position where the maximum number of less, the monophyly of Dactylogyrus was again not supported.
parasite individuals was found was considered as the pre- MP analysis was also performed. In MP analyses, 100 equally
ferred niche. The preferred niche (gill arch, segment, and parsimonious trees with 393 steps were retained (consistency
area) was mapped onto the phylogenetic tree obtained from index, CI 5 0.550, retention index, RI 5 0.639). However,
the NJ analysis using combined 18S rDNA and ITS1 se- BP values for MP show a very weak phylogenetic resolution.
quences. No statistically significant differences among NJ on Kimura
two-parameter, ML tree, NJ based on ML distance (TrNf 1
RESULTS G 1 I), and the equally most parsimonious 100 trees were
found (Shimodaira-Hasegawa test implemented in PAUP*
Molecular Divergence within Generalist Species 4b10, P . 0.05).
This incongruence (monophyly vs. nonmonophyly of Dac-
For each of the seven generalist species investigated, we tylogyrus) may be due to different evolutionary rates between
sequenced at least two individuals recovered from each dif- Dactylogyrus species and the other species used as outgroup.
ferent host species. Whereas no differences were detected Li (1997) stated that ML methods may become inconsistent
between individuals collected from the same host species, if the rate of evolution is assumed to be uniform when in
differences were observed between individuals collected fact it is not. We observed highly variable substitution rates
from different host species. Pairwise comparisons showed among the branch lengths in the tree inferred from the ML
0.4% molecular divergence between D. anchoratus specimens approach, suggesting that this assumption may be violated.
from Cyprinus carpio and Carassius auratus; no difference Furthermore, we noted that rate heterogeneity was different
between D. crucifer specimens from Rutilus rutilus, Scardi- between Dactylogyrus species and other dactylogyrids. Fi-
nius erythrophthalmus, and Leuciscus idus; 0.3% molecular nally, the high number of parameters for the TrNf 1 G 1 I
divergence between D. fallax specimens from R. rutilus and model involves a higher variance of branch-length estimation
Leuciscus cephalus; 1% molecular divergence between D. and therefore may lead to incorrect reconstruction of internal
ramulosus specimens from Aspius aspius and L. idus; 0.6% short branches (Nei and Kumar 2000). For those reasons, we
molecular divergence between D. sphyrna specimens of retained the NJ tree based on the Kimura two-parameter dis-
Abramis bjoerkna and R. rutilus; and no difference between tances that shows the monophyly of Dactylogyrus.
D. tuba specimens of Aspius aspius and L. idus. Finally, 0.2% Within Dactylogyrus we observe three monophyletic lin-
divergence was calculated between D. vistulae specimens eages, which are moderately supported (BP values). The first
from Chondrostoma nasus and L. idus; the same order of (further referenced as clade 1) includes the Dactylogyrus spe-
divergence (1.2%) was found between D. vistulae specimens cies from two species of the Cyprininae; the second (clade
from C. nasus and L. idus with specimens from L. cephalus. 2) associates one Dactylogyrus species from the Cyprininae,
The genetic divergence between the two most closely related one from the Rasborinae, and two from the Gobioninae; and
species was 1.4% (D. caballeroi and D. crucifer), and the the third (clade 3) includes mainly Dactylogyrus species par-
genetic divergence between other pairs was more than 2.6%. asitizing species of the subfamilies Leuciscinae and Albur-
ninae, three Dactylogyrus species of Barbus barbus (subfam-
Phylogenetic Analysis Using Partial 18S rDNA ily Cyprininae), and two of the family Percidae. While no
clear phylogenetic pattern is observed among the three clades,
For the phylogenetic analyses based on partial 18S rDNA
relationships between species within clades 1 and 2 are better
alone we used all Dactylogyrus species sequenced for this resolved than relationships between species of clade 3 (see
study and seven other dactylogyrids. The sequence alignment Fig. 1).
comprised 440 unambiguously alignable positions after re-
moving gaps and ambiguous aligned regions, of which 150
Phylogenetic Analysis Using Combined Data
were variable with 106 parsimony informative. The first anal-
(18S rDNA and ITS1)
ysis was performed using NJ with Kimura two-parameter
because of equal nucleotide base frequencies (about 25%). The partition homogeneity test as implemented in PAUP*
The tree obtained from the Kimura two-parameter distances 4b10 (Swofford 2002) was used to test the congruence of the
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1007

FIG. 1. Neighbor-joining tree based on Kimura two-parameter distances inferred from analysis of partial 18S rDNA sequences of 51
Dactylogyrus species including seven representatives of the Dactylogyridae (Thaparocleidus vistulensis, Cleidodiscus pricei, Pseudohal-
iotrema shincteropus, Thylacicleidus sp., Pseudodactylogyroides apogonis, Pseudodactylogyrus anguillae, P. haze), with T. vistulensis and
C. pricei taken as outgroup. Numbers along branches indicate bootstrap percentages resulting from the different analyses in the order:
neighbor joining/maximum parsimony/maximum likelihood. Values lower than 50 are indicated with dashes.
1008 ANDREA ŠIMKOVÁ ET AL.

two datasets: 18S and ITS1. As no significant difference was Leuciscinae and Alburninae (((D. similis, D. alatus) D. sphyr-
found (P 5 0.100), further analyses were performed with the na) D. vistulae). In all cases, parasites of the three host sub-
51 Dactylogyrus species examined in this study using com- families are monophyletic. Conversely, the Dactylogyrus spe-
bined sequences from partial 18S rDNA and ITS1. The align- cies of the two species that are the most common hosts for
ment of the partial 18S rDNA and ITS1 sequences comprised Dactylogyrus, L. cephalus and R. rutilus, do not form mono-
712 unambiguously alignable positions after gaps were re- phyletic groups.
moved, of which 246 were variable with 201 parsimony in-
formative. The ML analysis was performed on the model TrN Phylogeny of Cyprinid Species Using Cytochrome b
1 I 1 G with the following parameters: substitution rate The alignment of cytochrome b sequences comprised 1140
matrix: A-C 5 1.0000, A-G 5 3.0453, A-T 5 1.0000, C-G alignable positions, of which 545 were variable with 487
5 1.0000, C-T 5 3.7850, G-T 5 1.0000; the proportion of parsimony informative. The selected model for ML analysis
invariable sites 5 0.5341; and rate heterogeneity approxi- was TVM 1 I 1 G with the following parameters: A-C 5
mated by a gamma distribution (four rate categories), a 5 0.6912, A-G 5 8.1493, A-T 5 0.4777, C-G 5 0.3696, C-T
0.7423. The topology of the best ML tree was similar to the 5 8.1493, G-T 5 1.0000; the proportion of invariable sites
topology inferred from NJ analysis on the Tamura Nei dis- 5 0.4887; and rate heterogeneity approximated by a gamma
tances including one proportion of invariable characters and distribution (four rate categories), a 5 0.8008. The best ML
gamma distribution (TrN 1 I 1 G). MP analysis was also tree is reported in Figure 3. Whereas the monophyly of the
performed and 100 equally parsimonious trees with 951 steps Cyprinidae is well supported, the monophyly of the different
were retained (CI 5 0.428, RI 5 0.621). The strict consensus host subfamilies is moderately supported. The only repre-
displays similar topology to the NJ and ML trees, that is, it sentative of Alburninae is nested within Leuciscinae. The
supports the clustering of species found from the NJ and ML Gobioninae and Rasborinae are sister subfamilies, but only
analyses with BP of more than 50%. No statistically signif- one species from these two subfamilies was examined. Fi-
icant differences among NJ, ML, and trees obtained by MP nally, monophyly of the Cyprininae was not found, but the
were found (Shimodaira-Hasegawa test implemented in divergence of this subfamily appears to be the most basal
PAUP* 4b10, P . 0.05), therefore we only present the NJ event within the Cyprinidae. NJ using the parameters selected
tree giving BP values obtained from all three analyses (Fig. by ModelTest was also performed. Three equally parsimo-
2). nious trees with 2909 steps were retained (CI 5 0.331, R 5
The phylogenetic relationships inferred from 18S rDNA 0.429), but no statistically significant differences were found
analysis (Fig. 1) do not allow us to clearly specify which of among the topologies of the trees obtained from ML, NJ, and
the three reported lineages diverged first. Therefore, the tree MP analyses (Shimodaira-Hasegawa test implemented in
obtained from combined 18S rDNA and ITS1 data was drawn PAUP* 4b10, P . 0.05), and BP values obtained from ML,
to reflect the phylogenetic relationships described in the anal- NJ, and MP analyses are reported in Figure 3.
ysis of 18S rDNA sequences, that is, three Dactylogyrus
groups. Each of the three Dactylogyrus groups is well sup- Mapping Host Subfamilies onto the Parasite
ported (BP values more than 90). Moreover, the midpoint Phylogenetic Tree
rooting technique was applied as implemented in PAUP*
4b10 and highlights the first Dactylogyrus group as the most The fish subfamilies were mapped onto the parasite phy-
basal lineage. logeny inferred from the analysis of the combined dataset
Several general trends are inferred from all analyses. The (18S rDNA and ITS1). This mapping was done with the
first lineage (group 1, Fig. 2) is divided into two sister sub- assumption that the first Dactylogyrus group has the most
groups. The first associates two specialists found on C. carpio basal divergence, following the results obtained from the
that are related to the two specialists found on C. auratus. analysis of combined 18S 1 ITS1 using a midpoint rooting
The second one associates four species found on C. auratus. technique.
However, one of them, D. anchoratus, is also found on C. However, following the results using only partial 18S
carpio. rDNA, where lineages 1 and 2 are not well supported, three
The second lineage (group 2, Fig. 2) is composed of four hypotheses should be mentioned when considering which lin-
species. Two Dactylogyrus species found on Gobio species eage is basal. When groups 1 (Fig. 4) or 2 (not shown) are
are related to D. squameus from P. parva. The fourth species, considered as the most basal taxa, the Cyprininae is hypoth-
D. lamellatus from Ctenopharyngodon idella, is the most bas- esized to be a plesiomorphic host subfamily for Dactylogyrus.
al. All associations within the first and second lineages are In that case, the Percidae would have been secondarily col-
well supported by BP in all analyses. onized by Dactylogyrus species. However, when group 3 (not
The third lineage (group 3, Fig. 2) can be divided in two shown) is considered as the most basal taxon, the plesiom-
groups: the first one associates two Dactylogyrus species of orphic host subfamily is equivocal. It could be representatives
the Percidae (Gymnocephalus cernuus), and the second as- of the Cyprininae, Leuciscinae, or Percidae. In all cases, par-
sociates all remaining Dactylogyrus species, that is, parasites asites of B. barbus (Cyprininae) and of A. alburnus (Albur-
of the Leuciscinae, A. alburnus of the Alburninae, and B. ninae) are derived from host fish of the Leuciscinae.
barbus of the Cyprininae. Phylogenetic interrelationships
Comparison of Host and Parasite Phylogenies
among species of this group are in general moderately or
poorly supported or unresolved with the exception of one Although phylogenetic relationships are not fully resolved
subgroup of generalist parasites that associates species of the for the parasites, several cases of parasite duplication within
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1009

FIG. 2. Neighbor-joining tree based on Tamura-Nei distances, including one proportion of invariable characters and gamma distribution,
inferred from analysis of combined partial 18S rDNA and ITS1 sequences of 51 Dactylogyrus species. Numbers along branches indicate
bootstrap percentages resulting from different analyses in the order: neighbor joining/maximum parsimony/maximum likelihood. Values
lower than 50 are indicated with dashes.
1010 ANDREA ŠIMKOVÁ ET AL.

FIG. 3. Maximum-likelihood tree inferred from analysis of cytochrome b sequences of 18 representatives of the Cyprinidae, one of the
Balitoridae, one of the Characidae, six of the Percidae, and one of the Salmonidae. Selected model was TVM 1 I 1 G. Numbers along
branches indicate bootstrap percentages resulting from different analyses in the order: maximum likelihood/maximum parsimony/neighbor
joining. Values lower than 50 are indicated with dashes.

host species (i.e., intrahost speciation) can be recognized by two specialist parasites. Those duplications were sup-
(Fig. 5). Dactylogyrus duplications were found within the ported by BP of more than 50 except those found within S.
host species A. alburnus, which is infested by three specialist erythrophthalmus, A. brama, and B. barbus (see Figs. 2, 5).
parasites; within S. erythrophthalmus, which is also infested Two duplication events can be recognized within R. rutilus.
by three specialist parasites; within C. nasus, which is in- The first one is characterized by the two specialist parasites
fested by two specialists; within A. bjoerkna for the three D. rutili and D. nanus (BP . 70 from all analyses) and the
specialist parasites D. cornoides, D. cornu, and D. distin- generalist parasite D. fallax (BP , 50), and the second one
guendus; within L. cephalus for the two specialists D. na- by the specialist D. caballeroi and the generalist D. crucifer
noides and D. folkmanovae; within A. brama infested by two (BP . 90 from all analyses), the two generalist species in-
specialists D. zandti and D. wunderi; within B. barbus infested festing predominantly R. rutilus. All those duplication events
by two specialists; and within G. cernuus, which is infested occurred within lineage 3. Similarly, some duplication events
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1011

FIG. 4. Mapping of host subfamilies onto the parasite neighbor-joining tree inferred from analysis of combined data. The first Dactylogyrus
group is considered as the most basal lineage within Dactylogyrus following the most likely scenario of Dactylogyrus evolution.

were also reported within lineage 1. Dactylogyrus duplica- festing S. erythrophthalmus from the Leuciscinae (lineage 3,
tions were found within the host species C. carpio (BP . c in Fig. 5), but this cospeciation is not supported by bootstrap
60), which is infested by two specialist parasites, and within (Fig. 2). Even if the Alburninae are recognized to be poly-
C. auratus for which two duplication events were reported. phyletic and nested within Leuciscinae, A. alburnus is a sister
The first gave rise to two specialist species (D. vastator and species of S. erythrophthalmus (Figs. 3, 5). Thus, it is likely
D. intermedius, BP 5 100) and the second (BP . 95) to three that these two groups of parasites cospeciated with their host
specialist parasites and one generalist, that is, D. anchoratus species and later diversified within hosts. Dactylogyrus pros-
also infests C. carpio. No duplication event was reported tae would have switched to L. cephalus before intrahost spe-
within lineage 2. ciation occurred on S. erythrophthalmus.
Besides intrahost duplication events, several cospeciation Finally, several host switches must be invoked to explain
events (numbers in Fig. 5) can also be recognized from the discordant host and parasite relationships. That is the case,
comparison of host and parasite phylogenies. In lineage 2, for instance, for Dactylogyrus species parasitizing B. barbus
the two sister species D. finitimus and D. cryptomeres para- that are nested within the species of the Leuciscinae.
sitize two congeneric host species, G. albipinatus and G. go-
bio respectively (a in Fig. 5). Moreover, Dactylogyrus of Mapping of Niche Preference onto the Phylogenetic Tree
Gobio species are a sister group to D. squameus of P. parva
(b in Fig. 5). Because Gobio species and P. parva form a The preferred niche of each Dactylogyrus species was
monophyletic group, cospeciation events can be suggested mapped onto their phylogeny. For parasite species found on
to explain similarities in host and parasite relationships. Sim- more than one host species (D. sphyrna and D. crucifer), the
ilarly, the parasite species infesting A. alburnus from the position on the fish species with the highest parasite abun-
Alburninae are closely related to Dactylogyrus species in- dance was chosen. This position, however, was similar to
1012 ANDREA ŠIMKOVÁ ET AL.

FIG. 5. Tanglegram of Dactylogyrus and cyprinid species deduced from comparison of the parasite neighbor-joining tree inferred from
analysis of combined data (18SrDNA and ITS1 sequences) with the topology of a fish phylogeny that includes only species infested by
Dactylogyrus species. Gobio albipinatus was added to the fish phylogeny as a sister species of Gobio gobio considering that they are
monophyletic. Intrahost duplications are depicted on the trees. Dactylogyrus speciation by intrahost duplication supported by bootstrap
percentage greater than 50 are given in bold. Potential coevolution events are indicated by a, b, and c.
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1013

that observed when considering individuals of a given Dac- lutionary patterns (e.g., case study of marine monogenean
tylogyrus species obtained from more than one host species. parasites by Desdevises et al. 2002). Therefore, we studied
The first mapping shows the preferred position on the gill highly specific monogenean parasites of the genus Dactylo-
arch (Fig. 6B). The position on the second gill arch seems gyrus that infest freshwater fish of the Cyprinidae. This study
to be ancestral and several changes toward the first, third, represents a large parasite sampling from central European
and fourth arches were noted. The evidence for strong chang- fish.
es in the arch position was found within the groups of par-
asites on C. auratus. The clade including the specialist Dac- Dactylogyrus Origin
tylogyrus species parasitizing B. barbus, L. cephalus, S. er-
On the basis of all phylogenetic analyses using partial 18S
ythrophthalmus, and A. alburnus shows a shift from the po-
rDNA separately or combined 18S rDNA and ITS1 data, three
sition on the second to the third gill arch with changes toward
main lineages of Dactylogyrus species were recognized: (1)
first and second gill arches for parasites of S. erythrophthal-
the clade of parasites of C. carpio and C. auratus, both rep-
mus.
resentatives of the Cyprininae; (2) the lineage that includes
The preferred segment position on the gill arches was
parasite species of C. idella (Cyprininae), P. parva of the
mapped onto the phylogeny (Fig. 6C). The position on the
Rasborinae (this species is considered as Gobioninae by Chen
dorsal segment is found to be the ancestral state with several
et al. 1984) and Gobio of the Gobioninae; and (3) the lineage
changes toward the medial and ventral segments for the spe-
that includes parasite species of B. barbus (Cyprininae), Leu-
cies in terminal positions. Changes in preferred segment po-
ciscinae, Alburninae, and Percidae, all of European origin.
sition were found between the branches within monophyletic
Phylogenetic relationships between species within each of
groups of the lineages of Dactylogyrus species parasitizing
the three main lineages were better resolved when analyzing
the same host species.
combined data. The 18S rDNA represents a well-conserved
The preferred area position on the gill arches was mapped
gene that evolves relatively slowly (Hillis and Dixon 1991).
onto the phylogeny (Fig. 6D). The position on the distal area
This explains why it has been widely used for the study of
is found to be the ancestral state with several changes toward
plathyhelminth relationships (Littlewood and Olson 2001;
the central and proximal areas. The same observation as in
Olson and Littlewood 2002). However, fast-evolving se-
the case of preferred segment position was noted, that is, the
quences, for instance the ITS1 region, could provide useful
changes in preferred areas are found between the branches
phylogenetic information for resolving relationships within
within the group of Dactylogyrus species parasitizing the
groups of recent origin (Booton et al. 1999). Desdevises et
same host species. However, when Dactylogyrus species of
al. (2002) showed in the case of monogenean species be-
one clade and parasitizing the same host (i.e., recognized as
longing to Lamellodiscus that ITS1 sequences could not be
a case of intrahost duplication) were separated by their arch
aligned among species, which suggested an older age of this
position, then the same segment or area positions are often
group of parasites than hypothesized from a morphological
found in the phylogenetic tree. Looking at changes for all
point of view. It may also be the result of an increased sub-
positions, we can note that no sister groups have identical
stitution rate in this genus. Our results inferred from analysis
niches and at least one of the niche parameters (arch, segment,
of ITS1 sequences suggest that Dactylogyrus is of quite recent
or area) has changed.
origin and that the three lineages separated from each other
in a very short period of time. This is confirmed by our
DISCUSSION
alignment of ITS1 sequences derived from all Dactylogyrus
The Dactylogyrus-Cyprinidae Model species and by the lack of basal resolution in the genus.
In general, parasites with direct life cycle and free-living
Plesiomorphic Host Subfamily
infectious stages attaching to the external surface of their
hosts are believed to have a narrow host range (i.e., they are Results inferred from mapping host subfamily onto the
specialists) and to have coevolved with their hosts, in contrast parasite phylogenetic tree could suggest three scenarios con-
to parasites with indirect life cycles (Littlewood et al. 1997; cerning plesiomorphic hosts for Dactylogyrus. Evolutionary
Paterson and Poulin 1999). Therefore, highly specific mono- scenarios considering either lineage 1 or 2 as displaying the
geneans could be considered an appropriate model for study- most basal divergence, both support the hypothesis that Asi-
ing the evolution of host-parasite associations to investigate atic Cyprininae could be the ancestral hosts. However, the
patterns and processes of parasite speciation. Nevertheless, scenario involving basal divergence of lineage 3 proposes an
different levels of correspondence between patterns of par- equivocal solution where either representatives of the Cy-
asite speciation and host specificity have been shown across prininae, Leuciscinae, or Percidae would be plesiomorphic
different models of congeneric monogenean parasites (Lit- hosts. In the latter case, if we consider that Dactylogyrus first
tlewood et al. 1997; Bentz et al. 2001; Desdevises et al. 2002; evolved within percids, then it would imply that Dactylogyrus
Huyse and Volckaert 2002), suggesting importance of the species became extinct within the Percidae, except in one
host species in the process of parasite diversification. species, G. cernuus, and at least in the Characidae and Bal-
When considering that evolution of freshwater fish is more itoridae, which are the most basal taxa within Cypriniformes.
constrained than that of marine fish (i.e., it is correlated to Indeed, the majority of Dactylogyrus species infest represen-
the history of freshwater movements and dispersals; Tsigen- tatives of the Cyprinidae. For these reasons, we consider this
opoulos and Berrebi 2000), then freshwater fish may repre- possibility very unlikely. The second solution is to consider
sent more suitable models for studying host-parasite coevo- the Leuciscinae as the primitive host subfamily for Dactyl-
1014 ANDREA ŠIMKOVÁ ET AL.

FIG. 6. Mapping of niche within hosts preferred by Dactylogyrus species. (A) Gill arch division: gill segments: D, dorsal; M, medial;
V, ventral; gill area: d, distal; c, central; p, proximal; gill surface: in, inner; out, outer; gill hemibranch: A, anterior; P, posterior. (B)
Gill arch, (C) gill segment, and (D) gill area were mapped onto the parasite phylogeny.

ogyrus. In that case, it would imply that Dactylogyrus orig- the distribution of the latter family, that is, its absence in
inated in the ancestor of the Leuciscinae and secondarily Africa and eastern Asia, limited diversification in Europe,
colonized the Percidae and Cyprininae. This scenario also and extensive diversity in North America, it is very likely
seems unlikely if we consider that east-central Asia is the that host transfer occurred in Europe from fish of the Cy-
center of speciation of the Cyprininae. How then can we prininae.
explain host switches from European to Asian host fish,
whereas the divergence of Asian cyprinins appears basal Pattern and Process of Parasite Diversification
within the Cyprinidae (Fig. 3)? Indeed, C. auratus and C.
carpio, the hosts for eight parasite species in our study, are The nested position of Dactylogyrus species parasitizing
considered to have originated in Asia and been recently im- B. barbus within the species of the Leuciscinae suggests host
ported in Europe. Furthermore, there is no paleontological switching from host fish of the Leuciscinae. It has been pro-
evidence for their presence in Europe during the Pleistocene posed that Western Palearctic Barbus would have originated
(Baruš and Oliva 1995). However, it should be noted that in eastern Asia and migrated to Europe and Asia during the
the origin of different subfamilies of the Cyprinidae and dis- Miocene–Upper Oligocene (Tsigenopoulos and Berrebi
persal migration routes are still debated (Durand et al. 2002). 2000). However, many species belonging to the genus Barbus
When considering the above arguments and host subfamily are of European origin (Cunha et al. 2002). It has also been
mapping, we propose that Dactylogyrus originated within the shown that Barbus divergence is of the same order as the
Cyprininae and secondarily colonized ancestral hosts of the radiation of the Leuciscinae genera and that both are con-
Leuciscinae and several species of the Percidae. Concerning sidered to have their center of speciation in Siberia (Durand
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1015

FIG. 6. Continued.

et al. 2002). The biogeography of this species could explain balleroi, D. crucifer, and D. rarissimus), D. crucifer mainly
when this capture of parasites has occurred. Whether nu- infests R. rutilus and occasionally L. idus and S. erythro-
merous host switches can be inferred from the comparison phthalmus. Within the second parasite clade of R. rutilus (D.
of host and parasite relationships (Fig. 5), their interpretations rutili, D. nanus, and D. fallax), D. fallax has been more often
would at the moment be too speculative. However, we can reported from R. rutilus than from L. cephalus. Within the
assume that parasites of B. barbus of the Cyprininae and of parasite clade of C. auratus (D. formosus and D. anchoratus),
A. alburnus of the Alburninae are derived from parasites of D. anchoratus has also been reported from C. carpio. How-
Leuciscinae host fish. ever, because of low sampling across the two latter host spe-
Although several nodes are not well supported, the mo- cies, we are currently unable to know which of these two
lecular phylogeny shows a consistent pattern of relationships host species is preferred for D. anchoratus. Nevertheless,
of Dactylogyrus species, as well as a very high number of sympatric speciation seems to be closely correlated to strict
intrahost speciation events (i.e., parasite duplications), com- host specificity as suggested by Vickery and Poulin (1998).
pared to very few cospeciation events. Eight duplication Our observations suggest that the majority of Dactylogyrus
events were recognized within lineage 3 across seven host parasites first speciated by intrahost duplication and second-
species and three within lineage 1 across two host species. arily colonized new host species that are closely related. This
These speciation events gave rise mainly to specialist species, confirms the suggestion of Gusev (1985) who defined so-
that is, parasite species that infest a single host species. How- called basic host species for many generalist species (host
ever, there are several exceptions where two duplication species when Dactylogyrus species occurs in high abundance)
events gave rise to both specialist and generalist parasite and rare host species (other phylogenetically closely related
species. Within the first parasite clade of R. rutilus (D. ca- species). Our results contradict the investigations of Guégan
1016 ANDREA ŠIMKOVÁ ET AL.

and Agnèse (1991) on congeneric African cyprinids and their closely related species within a host, it could be hypothesized
Dactylogyrus species. The comparison of Dactylogyrus phy- that their mode of speciation (i.e., intrahost speciation) should
logeny based on morphological grounds with a host phylog- be closely related to the evolution of attachment organs (i.e.,
eny (with fish species belonging only to a single genus) based opisthaptor) morphology and reproductive system, as sug-
on genetic markers revealed no intrahost speciation, but did gested by Rohde (1989). However, the morphology of the
suggest cospeciation and host switching events (Guégan and attachment apparatus of the Dactylogyrus genus is complex,
Agnèse 1991). including central and marginal hooks and connective bars.
Parasite duplication is a coevolutionary scenario that has The number of the morphological characters studied so far
been recently considered to explain host-parasite relation- would not allow for phylogenetic reconstruction based on
ships (Paterson and Gray 1997; Johnson et al. 2003). Paterson morphology when using a parasite sample as large as the 51
and Poulin (1999) showed that intrahost speciation could be species in the present study. The evolution of complex char-
more likely than cospeciation events within chondracanthid acters and the comparison of these with molecular evolution
copepods and their teleost hosts. They suggested that the require further studies in the future.
large geographical distances among hosts could explain this Gusev (1985) suggested that there are some morphological
pattern of speciation. However, in the case of Dactylogyrus features, mainly in the sclerotized parts of opisthaptor and
species, intrahost speciation is observed within hosts that are reproductive organs, that differ between Dactylogyrus species
not geographically isolated but living in sympatry. of Cyprininae and Leuciscinae subfamilies, and some of these
In the tree reported in Figure 3, four of the several gen- may be common for several Dactylogyrus species occurring
eralist species, D. alatus, D. similis, D. sphyrna, and D. vis- on the same or closely related species. However, there is no
tulae, form a clade of large generalists with similar morpho- evidence that morphological characters help determine the
logical features (mainly strongly developed central hooks and groups within Dactylogyrus, in contrast to the genus Gyro-
similar shape of the third pair of marginal hooks, which dif- dactylus, where the protonephridial system and opisthaptor
ferentiate this group from other Dactylogyrus species). Al- have been used (Malmberg 1970). However, the morpholog-
though two species (D. alatus and D. similis) were sequenced ical characters of the attachment apparatus and reproductive
from only one host species, their presence on other host spe- organs for Dactylogyrus species are sufficient for species dis-
cies was recorded during this study (D. alatus on L. idus and crimination, and there is no need to use the molecular markers
D. similis on L. cephalus were not sequenced due to the low for facilitating species discrimination, as suggested Zietara
number of parasite individuals collected from those hosts). and Lumme (2002) for Gyrodactylus species.
According to Gusev (1985) and Moravec (2001), those spe- Our findings on intrahost speciation are closely related to
cies may parasitize a wide host range. Like the generalists hypotheses explaining species coexistence. Coexistence of
included among a majority of specialists from the molecular Dactylogyrus species on the same host is facilitated by dif-
phylogeny (see above), those four generalist species also oc- fering morphology of copulatory organs, or niche center dis-
cur in high abundance in one host species and only rarely on tances, which prevent competition and increase intraspecific
other host species. Because those species form a clade, it mating contacts (Šimková et al. 2002). The present study
could be hypothesized that the ancestral parasite was able to shows that parasites speciating within one host (intrahost
colonize a wide range of hosts with consequent speciation of speciation) tend to occupy niches differing at least in one
new species within one host species. New parasite species parameter (gill arch, segment, or area). The same result was
preferentially choose this host species and occasionally infest obtained in a study of Dactylogyrus assemblages coexisting
closely related host species, maybe because of their large on one fish species (i.e., roach) by Šimková et al. (2000).
body size. The presence of several large species could pre- However, molecular phylogeny indicates that Dactylogyrus
clude species coexistence. species parasitizing roach do not form a monophyletic group.
Molecular divergence among generalist Dactylogyrus spe- In that case, isolation by niche segregation is important for
cies collected from the different host species was about 1%. congeneric species. Moreover, when different Dactylogyrus
This could suggest that there is low gene flow between dif- species occupy similar or closely situated niches, reproduc-
ferent populations of the same Dactylogyrus species parasit- tive barriers could also reinforce reproductive isolation be-
izing different host species, which may potentially reduce tween congeners (Rohde and Hobbs 1986; Rohde 1991; Mor-
interbreeding among the different populations. However, and et al. 2002; Jarkovský et al. 2004).
such suggestion needs further investigation in the future. Finally, the mapping of niche position within the fish gills
When comparing the mode of Dactylogyrus species spe- indicates that there is an ancestral position (possibly a site
ciation with that of Gyrodactylus (also a monogenean genus more protected against water current), from which other po-
with high number of parasite species), host switch was ac- sitions seem to be derived.
cepted for Gyrodactylus, and adaptive radiation was sug-
gested to be the consequence of host switch to a new family Conclusions
(Zietara and Lumme 2002). Those differences may be ex-
plained by different reproductive strategy (viviparous Gy- Phylogenetic relationships of Dactylogyrus species com-
rodactylus vs. oviparous Dactylogyrus). In the case of Gy- bined with host subfamily mapping suggest that the genus
rodactylus species, only one specimen may be required originated recently within fish of the Cyprininae. They would
achieve a successful switch to a new host if the host defense have secondarily evolved by colonizing different host species
system is tolerated. of the subfamilies Rasborinae, Gobioninae, Leuciscinae, and
As Dactylogyrus species form groups of phylogenetically Alburninae and the family Percidae by host switching and
 MOLECULAR PHYLOGENY OF DACTYLOGYRUS 1017

cospeciation. The main process of Dactylogyrus diversifica- Middle East inferred from cytochrome b DNA: evolutionary
tion corresponds to sympatric speciation (i.e., intrahost spe- significance of this region. Mol. Phylogenet. Evol. 22:91–100.
El Gharbi, S., E. Birgi, and A. Lambert. 1994. Monogènes Dac-
ciation), which seems correlated to strict host specificity. This tylogyridae parasites de Cyprinidae du genre Barbus d’Afrique
kind of speciation was previously very rarely recognized in Nord. Syst. Parasitol. 27:45–70.
host-parasite associations and the model Dactylogyrus-Cy- Ergens, R., and J. Lom. 1970. Causative agents of fish diseases.
prinidae presents the first case study where intrahost speci- (In Czech.) Academia Praha, Prague.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach
ation has been recorded for monogenean parasites. Moreover, using the bootstrap. Evolution 39:783–791.
the present study shows that congeneric parasites speciating Gibson, D. I., T. A. Timofeeva, and P. I. Gerasev. 1996. A catalogue
within one host tend to occupy niches differing at least in of the nominal species of the monogenean genus Dactylogyrus
one niche parameter. Diesing, 1850 and their host genera. Syst. Parasitol. 35:3–48.
Gilles, A., G. Lecointre, E. Faure, R. Chappaz, and G. Brun. 1998.
Mitochondrial phylogeny of the European cyprinids: implica-
ACKNOWLEDGMENTS tions for their systematics, reticulate evolution, and colonization
time. Mol. Phylogenet. Evol. 10:132–143.
This study was supported by Research Project of the Mas- Gilles, A., G. Lecointre, A. Miquelis, M. Loerstcher, R. Chappaz,
aryk University, Brno, project number J07/98:143100010. and G. Brun. 2001. Partial combination applied to phylogeny of
AŠ was founded by the Grant Agency of the Czech Republic, European cyprinids using the mitochondrial control region. Mol.
project number 524/03/P108. The stay of AŠ in France was Phylogenet. Evol. 19:22–33.
supported by a postdoctoral fellowship from the Ministère Guégan, J.-F., and J.-F. Agnèse. 1991. Parasite evolutionary events
inferred from host phylogeny: the case of Labeo species (Te-
de la Recherche in France. We would like to thank M. On- leostei, Cyprinidae) and their dactylogyrid parasites (Monoge-
dráčková, R. Sonek, M. Nováková, and K. Kořı́nková of Mas- nea, Dactylogyridae). Can. J. Zool. 69:595–603.
aryk University, Brno, the Czech Republic, for their help in Guégan, J.-F., and A. Lambert. 1991. Dactylogyrids (Platyhelmin-
collecting material; P. Jurajda from the Institute of Vertebrate thes: Monogenea) of Labeo (Teleostei: Cyprinidae) from West
African coastal rivers. J. Helminthol. Soc. W. 58:85–99.
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