Morphological and Molecular Evolution Are Not Linked in Lamellodiscus (Plathyhelminthes, Monogenea)
PLoS ONE (2011)
- ISSN: 19326203
- DOI: 10.1371/journal.pone.0026252
- PubMed: 22022582
Available from
Tim Poisot's profile on Mendeley.
or
Abstract
Lamellodiscus Johnston & Tiegs 1922 (Monogenea, Diplectanidae) is a genus of common parasites on the gills of sparid fishes. Here we show that this genus is probably undergoing a fast molecular diversification, as reflected by the important genetic variability observed within three molecular markers (partial nuclear 18S rDNA, Internal Transcribed Spacer 1, and mitonchondrial Cytochrome Oxidase I). Using an updated phylogeny of this genus, we show that molecular and morphological evolution are weakly correlated, and that most of the morphologically defined taxonomical units are not consistent with the molecular data. We suggest that Lamellodiscus morphology is probably constrained by strong environmental (host-induced) pressure, and discuss why this result can apply to other taxa. Genetic variability within nuclear 18S and mitochondrial COI genes are compared for several monogenean genera, as this measure may reflect the level of diversification within a genus. Overall our results suggest that cryptic speciation events may occur within Lamellodiscus, and discuss the links between morphological and molecular evolution.
Available from
Tim Poisot's profile on Mendeley.
Page 1
Morphological and Molecular Evolution Are Not Linked in Lamellodiscus (Plathyhelminthes, Monogenea)
Morphological and Molecular Evolution Are Not Linked
in Lamellodiscus (Plathyhelminthes, Monogenea)
Timothe´e Poisot1,2*, Olivier Verneau3, Yves Desdevises1,2
1UPMC Univ Paris 06, UMR 7232, Biologie Inte´grative des Organismes Marins, Observatoire Oce´anologique, Banyuls-sur-Mer, France, 2CNRS, UMR 7232, Biologie
Inte´grative des Organismes Marins, Observatoire Oce´anologique, Banyuls-sur-Mer, France, 3UMR 5110 CNRS-UPVD, Centre de Formation et de Recherche sur les
Environnements Me´diterrane´ens, Universite´ de Perpignan Via Domitia, Perpignan, France
Abstract
Lamellodiscus Johnston & Tiegs 1922 (Monogenea, Diplectanidae) is a genus of common parasites on the gills of sparid
fishes. Here we show that this genus is probably undergoing a fast molecular diversification, as reflected by the important
genetic variability observed within three molecular markers (partial nuclear 18S rDNA, Internal Transcribed Spacer 1, and
mitonchondrial Cytochrome Oxidase I). Using an updated phylogeny of this genus, we show that molecular and
morphological evolution are weakly correlated, and that most of the morphologically defined taxonomical units are not
consistent with the molecular data. We suggest that Lamellodiscus morphology is probably constrained by strong
environmental (host-induced) pressure, and discuss why this result can apply to other taxa. Genetic variability within nuclear
18S and mitochondrial COI genes are compared for several monogenean genera, as this measure may reflect the level of
diversification within a genus. Overall our results suggest that cryptic speciation events may occur within Lamellodiscus, and
discuss the links between morphological and molecular evolution.
Citation: Poisot T, Verneau O, Desdevises Y (2011) Morphological and Molecular Evolution Are Not Linked in Lamellodiscus (Plathyhelminthes, Monogenea). PLoS
ONE 6(10): e26252. doi:10.1371/journal.pone.0026252
Editor: Dirk Steinke, Biodiversity Insitute of Ontario-University of Guelph, Canada
Received March 14, 2011; Accepted September 23, 2011; Published October 12, 2011
Copyright: 2011 Poisot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tpoisot@um2.fr
Introduction
Describing new species solely on the basis of their morphology is
often not straightforward, and especially so for small-bodied
organisms that display few morphological features on which to
rely. A good illustration is highlighted in monogenean parasitic
flatworms, where the main morphological structures used for
species identification, namely the hard parts of the host attachment
apparatus (haptor) and male copulatory organ, often require
expert advice to discriminate closely related species, and although
displaying phylogenetic conservatism in some genera [1], may
display variations with environmental conditions [2–4] and host
species [5,6], eventually leading to speciation [7]. It is not clear
whether morphological variation within a species should be linked
to an ongoing speciation process, if it emerges as a combination of
inter-individual variation and (potentially host-induced) polymor-
phism in the population, or involves any combination of these
factors. In the specific case of monogeneans, the haptoral parts,
because they are used by the parasite to attach to its host, are likely
to be more strongly affected by phenotypic plasticity in generalist
species (i.e. using several host species with varying gill character-
istics [8]), even if this process appears to be limited [9]. It is now
well described that species can engage in phenotype switching to
cope with complex (in the case of parasites, multi-hosts)
environments [10], which result in the coexistence of potentially
different forms of the same species [11]. In this case, the existence
of different morphotypes would not correspond to different
species, rendering molecular evaluation of the taxonomic situation
necessary. This problem is obviously more difficult to tackle when
there are few characters on which identification can be conducted,
and when these characters are directly under environmental
control (as is the case for the hard haptoral parts of the
monogeneans).
Lamellodiscus (Monogenea, Diplectanidae) are gill parasites of
sparid fish throughout the world [12]. In the past ten years, over
twelve new species have been described within this genus in
Mediterranean and African fishes [13–19]. These species were
described solely on the basis of very few morphological variations
in comparison to previously known Lamellodiscus species. The
morphological differences between recently described species and
their already described counterparts are often tedious to observe in
light microscopy, making them highly questionable. The difficul-
ties in species assignment in Lamellodiscus were highlighted in
previous molecular analyses, which showed that some species like
Lamellodiscus virgula and L. obeliae, because of their high similarities
in sequences coding for 18S and ITS1 (% differences are
respectively 0 and 0.27), could be synonymous species [20]. A
more striking example is Furnestinia echeneis, that belongs to the
genus Lamellodiscus [21], despite its blaring morphological diver-
gence from previously known Lamellodiscus species. Most notably,
F. echeneis attachment apparatus only harbors one lamellodisc,
instead of two for all other Lamellodiscus species. The above
examples stress that morphology should not be viewed as a
consistently reliable tool in systematic investigation, and recent
studies showed how this conclusion applies for other monogeneans
[22] and free-living animals [23].
Beyond the use of molecular data for species-assignment
purposes, a recent study by Hansen and colleagues [24], looking
PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26252
in Lamellodiscus (Plathyhelminthes, Monogenea)
Timothe´e Poisot1,2*, Olivier Verneau3, Yves Desdevises1,2
1UPMC Univ Paris 06, UMR 7232, Biologie Inte´grative des Organismes Marins, Observatoire Oce´anologique, Banyuls-sur-Mer, France, 2CNRS, UMR 7232, Biologie
Inte´grative des Organismes Marins, Observatoire Oce´anologique, Banyuls-sur-Mer, France, 3UMR 5110 CNRS-UPVD, Centre de Formation et de Recherche sur les
Environnements Me´diterrane´ens, Universite´ de Perpignan Via Domitia, Perpignan, France
Abstract
Lamellodiscus Johnston & Tiegs 1922 (Monogenea, Diplectanidae) is a genus of common parasites on the gills of sparid
fishes. Here we show that this genus is probably undergoing a fast molecular diversification, as reflected by the important
genetic variability observed within three molecular markers (partial nuclear 18S rDNA, Internal Transcribed Spacer 1, and
mitonchondrial Cytochrome Oxidase I). Using an updated phylogeny of this genus, we show that molecular and
morphological evolution are weakly correlated, and that most of the morphologically defined taxonomical units are not
consistent with the molecular data. We suggest that Lamellodiscus morphology is probably constrained by strong
environmental (host-induced) pressure, and discuss why this result can apply to other taxa. Genetic variability within nuclear
18S and mitochondrial COI genes are compared for several monogenean genera, as this measure may reflect the level of
diversification within a genus. Overall our results suggest that cryptic speciation events may occur within Lamellodiscus, and
discuss the links between morphological and molecular evolution.
Citation: Poisot T, Verneau O, Desdevises Y (2011) Morphological and Molecular Evolution Are Not Linked in Lamellodiscus (Plathyhelminthes, Monogenea). PLoS
ONE 6(10): e26252. doi:10.1371/journal.pone.0026252
Editor: Dirk Steinke, Biodiversity Insitute of Ontario-University of Guelph, Canada
Received March 14, 2011; Accepted September 23, 2011; Published October 12, 2011
Copyright: 2011 Poisot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tpoisot@um2.fr
Introduction
Describing new species solely on the basis of their morphology is
often not straightforward, and especially so for small-bodied
organisms that display few morphological features on which to
rely. A good illustration is highlighted in monogenean parasitic
flatworms, where the main morphological structures used for
species identification, namely the hard parts of the host attachment
apparatus (haptor) and male copulatory organ, often require
expert advice to discriminate closely related species, and although
displaying phylogenetic conservatism in some genera [1], may
display variations with environmental conditions [2–4] and host
species [5,6], eventually leading to speciation [7]. It is not clear
whether morphological variation within a species should be linked
to an ongoing speciation process, if it emerges as a combination of
inter-individual variation and (potentially host-induced) polymor-
phism in the population, or involves any combination of these
factors. In the specific case of monogeneans, the haptoral parts,
because they are used by the parasite to attach to its host, are likely
to be more strongly affected by phenotypic plasticity in generalist
species (i.e. using several host species with varying gill character-
istics [8]), even if this process appears to be limited [9]. It is now
well described that species can engage in phenotype switching to
cope with complex (in the case of parasites, multi-hosts)
environments [10], which result in the coexistence of potentially
different forms of the same species [11]. In this case, the existence
of different morphotypes would not correspond to different
species, rendering molecular evaluation of the taxonomic situation
necessary. This problem is obviously more difficult to tackle when
there are few characters on which identification can be conducted,
and when these characters are directly under environmental
control (as is the case for the hard haptoral parts of the
monogeneans).
Lamellodiscus (Monogenea, Diplectanidae) are gill parasites of
sparid fish throughout the world [12]. In the past ten years, over
twelve new species have been described within this genus in
Mediterranean and African fishes [13–19]. These species were
described solely on the basis of very few morphological variations
in comparison to previously known Lamellodiscus species. The
morphological differences between recently described species and
their already described counterparts are often tedious to observe in
light microscopy, making them highly questionable. The difficul-
ties in species assignment in Lamellodiscus were highlighted in
previous molecular analyses, which showed that some species like
Lamellodiscus virgula and L. obeliae, because of their high similarities
in sequences coding for 18S and ITS1 (% differences are
respectively 0 and 0.27), could be synonymous species [20]. A
more striking example is Furnestinia echeneis, that belongs to the
genus Lamellodiscus [21], despite its blaring morphological diver-
gence from previously known Lamellodiscus species. Most notably,
F. echeneis attachment apparatus only harbors one lamellodisc,
instead of two for all other Lamellodiscus species. The above
examples stress that morphology should not be viewed as a
consistently reliable tool in systematic investigation, and recent
studies showed how this conclusion applies for other monogeneans
[22] and free-living animals [23].
Beyond the use of molecular data for species-assignment
purposes, a recent study by Hansen and colleagues [24], looking
PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26252
Page 2
for differences between the two monogeneans Gyrodactylus thymalli
and Gyrodactylus salaris, revealed the existence of several lineages,
unveiling a higher than expected diversity. Bakke and colleagues
[25] reported a similar result, proposing that there could be as
many as 20000 Gyrodactylus species, due to their fast ability to
diverge both on molecular and morphological characters. Due to
the fact that gyrodactylids pose severe economic problems in
aquaculture, they have been more extensively studied than any
other monogenean genera, which explain that few data are
available for other monogenean genera.
In this study, we used three genetic markers, the 39 extremity of
the 18S rRNA gene, the Internal Transcribed Spacer 1, and
approximately 300 base pairs within the first subunit of the
mitochondrial Cytochrome C Oxidase I, COI, to estimate the level
of divergence at the intra- and interspecific levels in Lamellodiscus.
We focused on recently described species from the morphological
group ignoratus [14] that are characterized by simple lateral dorsal
bars in the haptor and an en lyre (made of two loosely bound hard
parts resembling the shape of a lyre) male copulatory organ.
Several features of L. ignoratus s.l. (sensu lato, i.e. the group
comprised of L. ignoratus s.s., L. falcus, L. neifari, L. confusus and L.
diplodi) make it a suitable group for such a study: its four taxa are
discriminated by discrete morphological differences. Finally, these
species occur on a limited number of sparid hosts: Diplodus sargus,
D. vulgaris, D. annularis, D. puntazzo, Lithognathus mormyrus and Salpa
salpa.
Our goals were (i) to assess the taxonomic status of these
recently described Lamellodiscus species: L. neifari, L. falcus, L.
confusus, L. diplodi; (ii) to check whether or not these species are
closely related to L. ignoratus (sensu stricto, henceforth referred to as
L. ignoratus s.s.), thus comparing the relative merits of morpholog-
ical and molecular investigation of species status in this genus; and
(iii) to evaluate the level of molecular diversity in Lamellodiscus,
within and between species and discuss how it can assist in species
assignment problems.
Materials and Methods
1 Fish and parasite sampling
Fishes were sampled near Banyuls-sur-Mer (42u28947N,
3u08910E), by free diving. Two hosts species were collected,
Diplodus sargus and D. vulgaris, as they are known to harbor several
Lamellodiscus species belonging to the L. ignoratus s.l. subgroup [12].
Immediately after capture, fish were killed by a sharp blow on the
top of the head, and dissected. Gills were removed, and examined
at most 30 minutes after removal, under a light stereomicroscope
(Olympus SZ61), to check for the presence of Lamellodiscus.
Parasites were isolated from the gills, and placed on a slide to be
examined under light microscope (Olympus CX41, 400 times
magnification). Species identification was carried out based on the
shape of the opistohaptor and male copulatory organ [15].
Parasites were the preserved and stored individually in 96%
ethanol before DNA extraction.
2 DNA extraction and amplification
DNAs were extracted from dried samples in a mixture of 70 ml
of Chelex (100 mg/ml) and 15 ml of Proteinase K (10 mg/ml) at
55 uC for one hour. Reactions were then stopped at 100 uC for 15
minutes and kept at 4 uC until used.
Three markers were used in our analysis: the 39 terminal
fragment of the 18S rDNA (18S), the Internal Transcribed Spacer
1 (ITS1) and partial mitochondrial gene Cytochrome Oxidase I
(COI). Until now, only the 18S had been used for phylogenetic
analysis in Lamellodiscus [20,26,21].
The 18S-ITS1 fragment was amplified in one round with
primers L7 (forward, 59-TGATTTGTCTGGTTTATTCCGAT-
39) and IR8 (reverse, 59-GCTAGCTGCGTTCTTCATCGA-39)
as designed by Verneau and colleagues [27] and Sˇimkova´ and
colleagues [28] while COI was amplified with primers LCO1P
(forward, 59-TTTTTTGGGCATCCTGAGGTTTAT-39) and
HCox1P2 (reverse, 59-TAAAGAAAGAACATAATGAAAATG-
39), after Littlewood and colleagues [29]. PCR were performed
using the following cycles: 6 minutes at 95uC, then 35 cycles as
follows: 1 minute at 95uC, 1 minute at 48uC, and 2 minutes at
72uC. A final elongation was conducted for 10 minutes at 72uC.
PCR fragments were run in 1% agarose gels and purified using
Nucleospin Extract II Gel extraction kit (Macherey-Nagel). They
were sent to Macrogen Inc. (Korea) for sequencing. Sequences for
this study were deposited in GenBank with numbers EU259028 to
EU259032 and JF427625 to JF427661.
3 Distance computation and phylogenetic analysis
Due to the difficulty to align ITS1 even within a single genus, the
following analyses were done on COI and 18S only. GenBank [30] was
first queried to retrieve 18S and COI sequences from monogeneans
(species for which at least 3 sequences were available were included).
ClustalW2 [31] was used to align all sequences for each marker with
default settings, using the alignment of Lamellodiscus species as a
reference (for both 18S and COI). The ambiguously aligned parts were
removed using Gblocks [32,33], which retained 473 unambiguous
positions out of 537 in the original 18S sequences. Uncorrected
pairwise distances (excluding indels) were computed using the dist.dna
function of the APE package [34] for R 2.9.0 [35]. Numbers of
sequences by genus and species are listed in Tables 1 and 2.
Due to the difficulty of obtaining enough sequences of COI in
Lamellodiscus, the phylogenetic reconstruction was computed on the
18S fragment only. Evolutionary models were tested using
ModelTest [36] and selected with regard to their AIC score, using
PAUP* 4.0b10 [37]. Trees were inferred using two probabilistic
approaches: maximum likelihood with a non-parametric bootstrap
validation using PhyML [38] (using a GTR model with 49% of
invariant sites and a Gamma shape parameter of 0.46), and
Bayesian inference (using MrBayes 3.1.2 [39,40], using 2 runs of 4
chains during 2.106 generations, a burnin value of 25% of the saved
trees, sampled every 100 generations; convergence was assessed
using Tracer v. 1.5 [41], the average standard deviation of split
frequencies was checked to be less than 0.01, and the potential scale
reduction factors at the end of the runs were less than 1.01 for both
the model parameters and the bipartitions in the consensus trees).
The trees were rooted using Diplectanum aequans [42]. Each time the
phylogenetic pattern obtained casted doubt upon the taxonomic
status of a group of individuals, ITS1 sequences were manually
aligned to help in determining whether they belong to the same
species, as it has been previously suggested that ITS1 could be
aligned within but not between Lamellodiscus species [20,8].
However, given that this criterion deserves a more formal
investigation, ITS1 is used along with other criteria such as genetic
distance and phylogenetic pattern to assess species status.
Results
1 Phylogeny of Lamellodiscus
Our updated Lamellodiscus phylogeny (Figure 1–the ML version is
presented, as both reconstruction methods gave congruent
topologies), using the 39 end of 18S ribosomal DNA carries new
information regarding the previous phylogeny obtained by
Desdevises and colleagues [42], using the same portion of the 18S
ribosomal DNA (but based on fewer species and using only one
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26252
and Gyrodactylus salaris, revealed the existence of several lineages,
unveiling a higher than expected diversity. Bakke and colleagues
[25] reported a similar result, proposing that there could be as
many as 20000 Gyrodactylus species, due to their fast ability to
diverge both on molecular and morphological characters. Due to
the fact that gyrodactylids pose severe economic problems in
aquaculture, they have been more extensively studied than any
other monogenean genera, which explain that few data are
available for other monogenean genera.
In this study, we used three genetic markers, the 39 extremity of
the 18S rRNA gene, the Internal Transcribed Spacer 1, and
approximately 300 base pairs within the first subunit of the
mitochondrial Cytochrome C Oxidase I, COI, to estimate the level
of divergence at the intra- and interspecific levels in Lamellodiscus.
We focused on recently described species from the morphological
group ignoratus [14] that are characterized by simple lateral dorsal
bars in the haptor and an en lyre (made of two loosely bound hard
parts resembling the shape of a lyre) male copulatory organ.
Several features of L. ignoratus s.l. (sensu lato, i.e. the group
comprised of L. ignoratus s.s., L. falcus, L. neifari, L. confusus and L.
diplodi) make it a suitable group for such a study: its four taxa are
discriminated by discrete morphological differences. Finally, these
species occur on a limited number of sparid hosts: Diplodus sargus,
D. vulgaris, D. annularis, D. puntazzo, Lithognathus mormyrus and Salpa
salpa.
Our goals were (i) to assess the taxonomic status of these
recently described Lamellodiscus species: L. neifari, L. falcus, L.
confusus, L. diplodi; (ii) to check whether or not these species are
closely related to L. ignoratus (sensu stricto, henceforth referred to as
L. ignoratus s.s.), thus comparing the relative merits of morpholog-
ical and molecular investigation of species status in this genus; and
(iii) to evaluate the level of molecular diversity in Lamellodiscus,
within and between species and discuss how it can assist in species
assignment problems.
Materials and Methods
1 Fish and parasite sampling
Fishes were sampled near Banyuls-sur-Mer (42u28947N,
3u08910E), by free diving. Two hosts species were collected,
Diplodus sargus and D. vulgaris, as they are known to harbor several
Lamellodiscus species belonging to the L. ignoratus s.l. subgroup [12].
Immediately after capture, fish were killed by a sharp blow on the
top of the head, and dissected. Gills were removed, and examined
at most 30 minutes after removal, under a light stereomicroscope
(Olympus SZ61), to check for the presence of Lamellodiscus.
Parasites were isolated from the gills, and placed on a slide to be
examined under light microscope (Olympus CX41, 400 times
magnification). Species identification was carried out based on the
shape of the opistohaptor and male copulatory organ [15].
Parasites were the preserved and stored individually in 96%
ethanol before DNA extraction.
2 DNA extraction and amplification
DNAs were extracted from dried samples in a mixture of 70 ml
of Chelex (100 mg/ml) and 15 ml of Proteinase K (10 mg/ml) at
55 uC for one hour. Reactions were then stopped at 100 uC for 15
minutes and kept at 4 uC until used.
Three markers were used in our analysis: the 39 terminal
fragment of the 18S rDNA (18S), the Internal Transcribed Spacer
1 (ITS1) and partial mitochondrial gene Cytochrome Oxidase I
(COI). Until now, only the 18S had been used for phylogenetic
analysis in Lamellodiscus [20,26,21].
The 18S-ITS1 fragment was amplified in one round with
primers L7 (forward, 59-TGATTTGTCTGGTTTATTCCGAT-
39) and IR8 (reverse, 59-GCTAGCTGCGTTCTTCATCGA-39)
as designed by Verneau and colleagues [27] and Sˇimkova´ and
colleagues [28] while COI was amplified with primers LCO1P
(forward, 59-TTTTTTGGGCATCCTGAGGTTTAT-39) and
HCox1P2 (reverse, 59-TAAAGAAAGAACATAATGAAAATG-
39), after Littlewood and colleagues [29]. PCR were performed
using the following cycles: 6 minutes at 95uC, then 35 cycles as
follows: 1 minute at 95uC, 1 minute at 48uC, and 2 minutes at
72uC. A final elongation was conducted for 10 minutes at 72uC.
PCR fragments were run in 1% agarose gels and purified using
Nucleospin Extract II Gel extraction kit (Macherey-Nagel). They
were sent to Macrogen Inc. (Korea) for sequencing. Sequences for
this study were deposited in GenBank with numbers EU259028 to
EU259032 and JF427625 to JF427661.
3 Distance computation and phylogenetic analysis
Due to the difficulty to align ITS1 even within a single genus, the
following analyses were done on COI and 18S only. GenBank [30] was
first queried to retrieve 18S and COI sequences from monogeneans
(species for which at least 3 sequences were available were included).
ClustalW2 [31] was used to align all sequences for each marker with
default settings, using the alignment of Lamellodiscus species as a
reference (for both 18S and COI). The ambiguously aligned parts were
removed using Gblocks [32,33], which retained 473 unambiguous
positions out of 537 in the original 18S sequences. Uncorrected
pairwise distances (excluding indels) were computed using the dist.dna
function of the APE package [34] for R 2.9.0 [35]. Numbers of
sequences by genus and species are listed in Tables 1 and 2.
Due to the difficulty of obtaining enough sequences of COI in
Lamellodiscus, the phylogenetic reconstruction was computed on the
18S fragment only. Evolutionary models were tested using
ModelTest [36] and selected with regard to their AIC score, using
PAUP* 4.0b10 [37]. Trees were inferred using two probabilistic
approaches: maximum likelihood with a non-parametric bootstrap
validation using PhyML [38] (using a GTR model with 49% of
invariant sites and a Gamma shape parameter of 0.46), and
Bayesian inference (using MrBayes 3.1.2 [39,40], using 2 runs of 4
chains during 2.106 generations, a burnin value of 25% of the saved
trees, sampled every 100 generations; convergence was assessed
using Tracer v. 1.5 [41], the average standard deviation of split
frequencies was checked to be less than 0.01, and the potential scale
reduction factors at the end of the runs were less than 1.01 for both
the model parameters and the bipartitions in the consensus trees).
The trees were rooted using Diplectanum aequans [42]. Each time the
phylogenetic pattern obtained casted doubt upon the taxonomic
status of a group of individuals, ITS1 sequences were manually
aligned to help in determining whether they belong to the same
species, as it has been previously suggested that ITS1 could be
aligned within but not between Lamellodiscus species [20,8].
However, given that this criterion deserves a more formal
investigation, ITS1 is used along with other criteria such as genetic
distance and phylogenetic pattern to assess species status.
Results
1 Phylogeny of Lamellodiscus
Our updated Lamellodiscus phylogeny (Figure 1–the ML version is
presented, as both reconstruction methods gave congruent
topologies), using the 39 end of 18S ribosomal DNA carries new
information regarding the previous phylogeny obtained by
Desdevises and colleagues [42], using the same portion of the 18S
ribosomal DNA (but based on fewer species and using only one
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26252
Page 3
individual per species). The L. ignoratus s.l. group is not supported,
with a bootstrap value of 17% for its most basal node (PP,0.5).
Within this group, individuals from several putative species (both
previously known and recently described from morphology) cluster
together. Moreover, individuals from the same species are not
clustered in this tree (for example, the sequence obtained for L.
coronatus clusters in between the sequences obtained for L. ignoratus),
which can be due to the fact that this part of the tree is overall poorly
supported. However, so as to gain further insight on the species
status of such groups, we checked that the ITS1 sequences could be
aligned. The clusters of individuals for which this alignment was
possible are outlined in grey boxes in Fig. 1, suggesting that these
groups might have taxonomical relevance, but were incorrectly
attributed to the various species of the ignoratus group. COI was not
used in phylogenetic analyses due to the difficulties of getting a large
number of sequences, as no standard amplification protocol for this
marker exists.
2 Intraspecific and interspecific pairwise distances
From the partial 18S, the mean of uncorrected pairwise distances
between all sequences available for Lamellodiscus is 5.7%. The
distance between L. elegans (AF294956) and L. parisi (AY038198), the
most divergent sequences, is 9.2%. For the least divergent
sequences, L. fraternus (AY038191) and L. ergensi (AY038190), the
distance is 0.6%. Within L. ignoratus s.l. individuals (n = 17), we were
able to align all ITS1 sequences in two groups (containing 11 and 6
individuals), suggesting that all specimens within these groups
belong to the same species (named L. ignoratus and L. neifari on Fig. 1),
thus highlighting incongruences between morphological and
molecular identifications. The mean distance for 18S of L. ignoratus
s.l. considered as a single taxonomic entity is 2.46%.
Pairwise distances for the markers COI and 18S are listed in
Tables 1 and 2, respectively. Variability within the genus
Lamellodiscus is the highest observed in our sample for the COI
gene. Concerning the 18S, the genus Lamellodiscus is more variable
than any other monogenean genera, except Gyrodactylus, as indicated
by the higher genetic distances. The amount of variability correlates
with the taxonomic level for COI (i.e. isolates are less variable than
species, and species less than genera), but not for 18S. It should be
noted that this result is likely to be influenced by the fact that
sampling effort was stronger on some monogenean genera, and that
should be kept in mind when interpreting these observations.
Discussion
1 Cryptic speciation in Lamellodiscus?
Because of their strong potential for diversification, monogene-
ans are a promising model to study biodiversity issues [43]. This
assumption is supported by the estimation of 25000 monogenean
species by Rohde [44]. Note that Bakke and colleagues [25]
estimated 20000 species in the genus Gyrodactylus only, making it
one of the most speciose animal genera known. Gyrodactylus is one
of the most studied monogenean genera, because of the impact of
some species in aquaculture [45,46], but data for other
monogeneans are lacking. Here we suggest that Lamellodiscus,
compared to other monogeneans, is characterized by a high
molecular diversity at both intraspecific and intrageneric level.
Among the two main morphologically defined groups, ergensi was
poorly supported by the molecular phylogeny, while ignoratus forms
an unsupported cluster of individuals. In addition, the situation
within each putative group is complex: phylogenetic support is
weak within each group, where distinct morphs are found, among
which some are close to each other from molecular data (grey
boxes in Fig. 1). Within the ergensi group, the small sample size
precludes any meaningful observation. Within the ignoratus group,
while supported nodes exist, they are not compatible with groups
that could be delineated using morphological characters. For
example, individuals from L. ignoratus s.s. are interspersed in the L.
Table 1. Mean pairwise distances (m.p.d.) for the 245 bp long
fragment of the COI gene in several monogenean taxa.
Clade Order Rank Sample size m.p.d.
Euryhaliotrema grandisM isolate grap 5
E. grandis M isolate gram 8
E. grandis M isolate grah 4 0.0032
E. grandis M isolate gral 9 0.0067
Haliotrema aurigae M species 16 0.0070
Gyrodactylus lavareti M species 4 0.0081
G. arcuatus M species 7 0.0085
Wetapolystoma almae P species 3 0.0122
G. salaris M species 16 0.0153
Lamellodiscus furcosus M species 3 0.0327
E. grandis M species 27 0.0510
G. lucii M species 9 0.0514
Protopolystoma
xenopodi
P species 5 0.0688
L. neifari M unclear 2 0.0696
Protopolystoma
simplicis
M species 3 0.0860
Protopolystoma spp. P genus 10 0.1312
Gyrodactylus spp. M genus 38 0.1596
Polystomatidae P family 28 0.1766
Lamellodiscus spp. M genus 11 0.1988
Legend for column Order: P is for Polyopisthocotylea, M for Monopisthocotylea.
m.p.d: mean of pairwise distances. Species for which at least 3 sequences were
available were included in the analysis.
doi:10.1371/journal.pone.0026252.t001
Table 2. Mean pairwise distances for the 641 bp long
fragment of the 18S ribosomal DNA in several monogenean
taxa.
Clade Order Rank Sample size m.p.d
Dactylogyrus crucifer M species 3
D. vistulae M species 3
Pseudodactylogyrus spp. M genus 17 0.0017
Lamellodiscus neifari M unclear 4 0.0027
Dactylogyrus spp. M genus 61 0.0238
L. ignoratus s.l. M LITU 17 0.0246
Polystomoides spp. P genus 8 0.0251
Lamellodiscus spp. M genus 46 0.0476
Gyrodactylus salaris M species 156 0.0514
G. thymalli M species 31 0.0832
Gyrodactylus spp. M genus 341 0.1175
Polystomatidae P family 86 0.3371
See Table 1 for legend. The group formed by L. ignoratus s.l. (Figure 1) was
given the status of litu sensu Pleijel and Rouse [62].
doi:10.1371/journal.pone.0026252.t002
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26252
with a bootstrap value of 17% for its most basal node (PP,0.5).
Within this group, individuals from several putative species (both
previously known and recently described from morphology) cluster
together. Moreover, individuals from the same species are not
clustered in this tree (for example, the sequence obtained for L.
coronatus clusters in between the sequences obtained for L. ignoratus),
which can be due to the fact that this part of the tree is overall poorly
supported. However, so as to gain further insight on the species
status of such groups, we checked that the ITS1 sequences could be
aligned. The clusters of individuals for which this alignment was
possible are outlined in grey boxes in Fig. 1, suggesting that these
groups might have taxonomical relevance, but were incorrectly
attributed to the various species of the ignoratus group. COI was not
used in phylogenetic analyses due to the difficulties of getting a large
number of sequences, as no standard amplification protocol for this
marker exists.
2 Intraspecific and interspecific pairwise distances
From the partial 18S, the mean of uncorrected pairwise distances
between all sequences available for Lamellodiscus is 5.7%. The
distance between L. elegans (AF294956) and L. parisi (AY038198), the
most divergent sequences, is 9.2%. For the least divergent
sequences, L. fraternus (AY038191) and L. ergensi (AY038190), the
distance is 0.6%. Within L. ignoratus s.l. individuals (n = 17), we were
able to align all ITS1 sequences in two groups (containing 11 and 6
individuals), suggesting that all specimens within these groups
belong to the same species (named L. ignoratus and L. neifari on Fig. 1),
thus highlighting incongruences between morphological and
molecular identifications. The mean distance for 18S of L. ignoratus
s.l. considered as a single taxonomic entity is 2.46%.
Pairwise distances for the markers COI and 18S are listed in
Tables 1 and 2, respectively. Variability within the genus
Lamellodiscus is the highest observed in our sample for the COI
gene. Concerning the 18S, the genus Lamellodiscus is more variable
than any other monogenean genera, except Gyrodactylus, as indicated
by the higher genetic distances. The amount of variability correlates
with the taxonomic level for COI (i.e. isolates are less variable than
species, and species less than genera), but not for 18S. It should be
noted that this result is likely to be influenced by the fact that
sampling effort was stronger on some monogenean genera, and that
should be kept in mind when interpreting these observations.
Discussion
1 Cryptic speciation in Lamellodiscus?
Because of their strong potential for diversification, monogene-
ans are a promising model to study biodiversity issues [43]. This
assumption is supported by the estimation of 25000 monogenean
species by Rohde [44]. Note that Bakke and colleagues [25]
estimated 20000 species in the genus Gyrodactylus only, making it
one of the most speciose animal genera known. Gyrodactylus is one
of the most studied monogenean genera, because of the impact of
some species in aquaculture [45,46], but data for other
monogeneans are lacking. Here we suggest that Lamellodiscus,
compared to other monogeneans, is characterized by a high
molecular diversity at both intraspecific and intrageneric level.
Among the two main morphologically defined groups, ergensi was
poorly supported by the molecular phylogeny, while ignoratus forms
an unsupported cluster of individuals. In addition, the situation
within each putative group is complex: phylogenetic support is
weak within each group, where distinct morphs are found, among
which some are close to each other from molecular data (grey
boxes in Fig. 1). Within the ergensi group, the small sample size
precludes any meaningful observation. Within the ignoratus group,
while supported nodes exist, they are not compatible with groups
that could be delineated using morphological characters. For
example, individuals from L. ignoratus s.s. are interspersed in the L.
Table 1. Mean pairwise distances (m.p.d.) for the 245 bp long
fragment of the COI gene in several monogenean taxa.
Clade Order Rank Sample size m.p.d.
Euryhaliotrema grandisM isolate grap 5
E. grandis M isolate gram 8
E. grandis M isolate grah 4 0.0032
E. grandis M isolate gral 9 0.0067
Haliotrema aurigae M species 16 0.0070
Gyrodactylus lavareti M species 4 0.0081
G. arcuatus M species 7 0.0085
Wetapolystoma almae P species 3 0.0122
G. salaris M species 16 0.0153
Lamellodiscus furcosus M species 3 0.0327
E. grandis M species 27 0.0510
G. lucii M species 9 0.0514
Protopolystoma
xenopodi
P species 5 0.0688
L. neifari M unclear 2 0.0696
Protopolystoma
simplicis
M species 3 0.0860
Protopolystoma spp. P genus 10 0.1312
Gyrodactylus spp. M genus 38 0.1596
Polystomatidae P family 28 0.1766
Lamellodiscus spp. M genus 11 0.1988
Legend for column Order: P is for Polyopisthocotylea, M for Monopisthocotylea.
m.p.d: mean of pairwise distances. Species for which at least 3 sequences were
available were included in the analysis.
doi:10.1371/journal.pone.0026252.t001
Table 2. Mean pairwise distances for the 641 bp long
fragment of the 18S ribosomal DNA in several monogenean
taxa.
Clade Order Rank Sample size m.p.d
Dactylogyrus crucifer M species 3
D. vistulae M species 3
Pseudodactylogyrus spp. M genus 17 0.0017
Lamellodiscus neifari M unclear 4 0.0027
Dactylogyrus spp. M genus 61 0.0238
L. ignoratus s.l. M LITU 17 0.0246
Polystomoides spp. P genus 8 0.0251
Lamellodiscus spp. M genus 46 0.0476
Gyrodactylus salaris M species 156 0.0514
G. thymalli M species 31 0.0832
Gyrodactylus spp. M genus 341 0.1175
Polystomatidae P family 86 0.3371
See Table 1 for legend. The group formed by L. ignoratus s.l. (Figure 1) was
given the status of litu sensu Pleijel and Rouse [62].
doi:10.1371/journal.pone.0026252.t002
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26252
Page 4
Figure 1. Phylogeny of several Lamellodiscus species obtained by maximum likelihood and Bayesian inference. As topologies obtained
with both reconstruction methods gave congruent topologies and similar branch lengths, the most resolved tree, obtained by maximum likelihood,
was retained and is presented here. Bootstrap values (1000 replicates) and posterior probabilities (.0.5. Dashes correspond to values ,0.5) are
indicated at each node. The clusters of individuals for which the alignment of ITS1 was possible are outlined in grey boxes. Thick black lines indicate
ergensi and ignoratus groups.
doi:10.1371/journal.pone.0026252.g001
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26252
with both reconstruction methods gave congruent topologies and similar branch lengths, the most resolved tree, obtained by maximum likelihood,
was retained and is presented here. Bootstrap values (1000 replicates) and posterior probabilities (.0.5. Dashes correspond to values ,0.5) are
indicated at each node. The clusters of individuals for which the alignment of ITS1 was possible are outlined in grey boxes. Thick black lines indicate
ergensi and ignoratus groups.
doi:10.1371/journal.pone.0026252.g001
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26252
Page 5
ignoratus s.l. group, and one of the L. abbreviatus individuals is
outside a strongly supported clade containing the other L.
abbreviatus isolates. Based on partial 18S rRNA gene sequences,
several species appear to be either not monophyletic (e.g. L.
ignoratus, L. ergensi) or invalid (such as L. coronatus which clusters
within L. furcosus individuals), which suggests that Lamellodiscus
could be either more diversified than expected, or that there is a
gap between the morphological characterization of species and
their evolutionary relatedness (these two propositions not being
mutually exclusive). This claim is supported by the magnitude of
pairwise distances observed, particularly at the lower taxonomic
levels, between Lamellodiscus individuals.
Three main factors can be invoked to explain the putative high
ability for diversification in monogeneans, reflected in the high
molecular and morphological diversity observed in Lamellodiscus.
First, habitat heterogeneity is likely to be greater for small-bodied
organisms (such as Lamellodiscus), thus favoring their diversification
[47–49]. Poulin [50] observed this pattern in monogenean
ectoparasites : there are far more small-bodied fish monogeneans
than large-bodied ones. Second, monogeneans have a direct life
cycle. Life-cycle complexity has been suggested to affect speciation
rates in parasites [43]. Within Platyhelminthes, which share a
common origin of the parasitic lifestyle, the Monogenea is the only
group in which an adaptive evolutionary radiation has occurred
[51]. Because of their small body size and direct life cycle,
monogeneans have an important potential for diversification [28].
Finally, the genus Lamellodiscus appears to be composed of more
species with a wide host range than any other monogenean
genera, which has been suggested to add some molecular
variability [8] and potential for speciation [6,52–54].
Relying on morphology alone led previous researchers to
consider as belonging to the same species some morphs that were
found in different clades in our molecular phylogeny. This is
emphasized by the situation of L. ignoratus s.l., where none of the
described species receives support based on the molecular data.
This situation could be interpreted in two ways. First, most species
in the ignoratus group might be paraphyletic. By paraphyletic, we
mean that within a cluster of related individuals belonging to the
same species, one or several individuals from another species
branch out. The existence of paraphyletic species has already been
observed both for free-living [55] and symbiotic [56–58] taxa. In
our sample, some pairwise genetic distances between individuals
from different species (e.g. 0.6% between L. fraternus and L. ergensi
based on partial 18S rDNA) were found to be lower than some
intra-specific distances. This situation strongly suggests that two or
more species are not a monophylum [59]. Second, Lamellodiscus
may contain more species that our current estimation. Several
studies aimed to characterize new species in this genus in the last
few years [13,15,17], based on very small morphological
variations. However, these recently described species are not
easily differentiated from others (neither from morphological nor
molecular analyses), and according to the molecular evidence
presented here, it might be more conservative to consider that they
are species inquirenda (i.e. species of doubtful identity).
The fact that the recently described species are not necessarily
valid must not lead us to lump all L. ignoratus s.l. individuals into a
single species. Indeed, pairwise distances within L. ignoratus s.l. are
higher than for any other species pairs (Table 1), and comparable
to those observed in other genera (the partial 18S rDNA diversity
of L. ignoratus s.l. is comparable to what was observed between
Dactylogyrus spp. and between Polystomoides spp., see Table 2), and
while they do not correspond to what could be delineated based on
morphological character, there are some supported clades in the
ignoratus group. This result suggests that several species could exist
in the L. ignoratus s.l. group, but due to a high diversity and putative
cryptic speciation, an intensive sampling is still needed to gather
enough data to detect them.
The precise knowledge of which taxa are species is crucial, because
species, contrary to higher level taxa, are not only an outcome of
evolution, but are also directly involved in evolutionary processes
[60,61]. We face the same problem in Lamellodiscus: our current view
of the evolution of this genus [42] was inferred according to what we
thought to be species; if what we called species was rather an
assemblage of dissociated taxonomical units, some of the mechanisms
thought to act in Lamellodiscus evolution (such as radiation by host
switch followed by speciation) need to be re-evaluated in the light of
revised species delineation and an updated phylogeny. Before
assessing intraspecific and intrageneric genetic diversity, it is
important to be sure which taxa are given the species status. In
such a situation, it could be useful to use the Least-inclusive
taxonomic unit (LITU) concept [62], that is considering several
individuals as forming a clade, without making further assumptions
about the taxonomic position of this clade. Our results suggest that
the ignoratus group is highly diversified, and is likely to be formed by
several OTUs; we suggest to give this group the status of LITU, and
to wait for further investigation to determine its exact taxonomic
status. According to these results, our view of the taxonomy and,
consequently, of the evolution of Lamellodiscus needs to be reassessed.
2 Phylogeny and morphology seem to be unlinked in
Lamellodiscus
The molecular variability (as approximated by the pairwise
distance at several taxonomical depths) in Lamellodiscus was
compared to what was found in other monogenean genera. For
COI, Lamellodiscus shows the most important interspecific distances;
for 18S, Lamellodiscus is nearly twice as variable as Dactylogyrus, but
less than half as variable as Gyrodactylus, thought to be the most
variable monogenean genus [63]. Despite this important molecular
variability, however, there are very little clearly distinguishable
morphologies within the genus Lamellodiscus. Amine and Euzet [14]
defined two morphological groups in this genus, named ignoratus
(formed by L. ignoratus s.l., L. fraternus, L. knoeppfleri and L. erythrini) and
ergensi (formed by L. ergensi, L. sanfilippoi, L. kechemirae and L. baeri).
Our results (Figure 1) are congruent with this classification, with two
notable exceptions: L. knoeppfleri, L. fraternus and L. erythrini were
found to belong to the ergensi group. According to our phylogeny,
the ignoratus group is only formed of the taxa belonging to L.
ignoratus s.l. Within each group, however, there is no link between
morphological features and phylogenetic position, mostly because
none of the individuals of a single putative species cluster together. A
similar situation was reported by Hay and colleagues [64]. They
observed that the tuatara, Sphenodon punctatus, while being a living
fossil (its morphology is strikingly similar to the fossil specimens), and
having a slow metabolism, a long generation time, and a slow rate of
reproduction, is the species having the highest rate of molecular
evolution observed amongst vertebrates. Hence, a high molecular
divergence is not necessarily linked to important morphological
changes, and the assumption that rates of molecular and
morphological evolution are inherently correlated [65] is likely to
be untrue in certain genera, which could be the case in Lamellodiscus.
Given that we were able to align the ITS1 of several individuals,
we are able to make suggestions as to the species status of some
morphotypes. We found molecular evidences that, despite some
morphological divergences on the shape of the hard parts and
copulatory organs, L. furcosus and L. coronatus form a single species
(that we call L. furcosus). The ITS1 of L. ergensi, L. oliveri, L. fraternus
and L. gussevi can be aligned, suggesting that all of these
morphotypes should be considered as a single species, that we call
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26252
outside a strongly supported clade containing the other L.
abbreviatus isolates. Based on partial 18S rRNA gene sequences,
several species appear to be either not monophyletic (e.g. L.
ignoratus, L. ergensi) or invalid (such as L. coronatus which clusters
within L. furcosus individuals), which suggests that Lamellodiscus
could be either more diversified than expected, or that there is a
gap between the morphological characterization of species and
their evolutionary relatedness (these two propositions not being
mutually exclusive). This claim is supported by the magnitude of
pairwise distances observed, particularly at the lower taxonomic
levels, between Lamellodiscus individuals.
Three main factors can be invoked to explain the putative high
ability for diversification in monogeneans, reflected in the high
molecular and morphological diversity observed in Lamellodiscus.
First, habitat heterogeneity is likely to be greater for small-bodied
organisms (such as Lamellodiscus), thus favoring their diversification
[47–49]. Poulin [50] observed this pattern in monogenean
ectoparasites : there are far more small-bodied fish monogeneans
than large-bodied ones. Second, monogeneans have a direct life
cycle. Life-cycle complexity has been suggested to affect speciation
rates in parasites [43]. Within Platyhelminthes, which share a
common origin of the parasitic lifestyle, the Monogenea is the only
group in which an adaptive evolutionary radiation has occurred
[51]. Because of their small body size and direct life cycle,
monogeneans have an important potential for diversification [28].
Finally, the genus Lamellodiscus appears to be composed of more
species with a wide host range than any other monogenean
genera, which has been suggested to add some molecular
variability [8] and potential for speciation [6,52–54].
Relying on morphology alone led previous researchers to
consider as belonging to the same species some morphs that were
found in different clades in our molecular phylogeny. This is
emphasized by the situation of L. ignoratus s.l., where none of the
described species receives support based on the molecular data.
This situation could be interpreted in two ways. First, most species
in the ignoratus group might be paraphyletic. By paraphyletic, we
mean that within a cluster of related individuals belonging to the
same species, one or several individuals from another species
branch out. The existence of paraphyletic species has already been
observed both for free-living [55] and symbiotic [56–58] taxa. In
our sample, some pairwise genetic distances between individuals
from different species (e.g. 0.6% between L. fraternus and L. ergensi
based on partial 18S rDNA) were found to be lower than some
intra-specific distances. This situation strongly suggests that two or
more species are not a monophylum [59]. Second, Lamellodiscus
may contain more species that our current estimation. Several
studies aimed to characterize new species in this genus in the last
few years [13,15,17], based on very small morphological
variations. However, these recently described species are not
easily differentiated from others (neither from morphological nor
molecular analyses), and according to the molecular evidence
presented here, it might be more conservative to consider that they
are species inquirenda (i.e. species of doubtful identity).
The fact that the recently described species are not necessarily
valid must not lead us to lump all L. ignoratus s.l. individuals into a
single species. Indeed, pairwise distances within L. ignoratus s.l. are
higher than for any other species pairs (Table 1), and comparable
to those observed in other genera (the partial 18S rDNA diversity
of L. ignoratus s.l. is comparable to what was observed between
Dactylogyrus spp. and between Polystomoides spp., see Table 2), and
while they do not correspond to what could be delineated based on
morphological character, there are some supported clades in the
ignoratus group. This result suggests that several species could exist
in the L. ignoratus s.l. group, but due to a high diversity and putative
cryptic speciation, an intensive sampling is still needed to gather
enough data to detect them.
The precise knowledge of which taxa are species is crucial, because
species, contrary to higher level taxa, are not only an outcome of
evolution, but are also directly involved in evolutionary processes
[60,61]. We face the same problem in Lamellodiscus: our current view
of the evolution of this genus [42] was inferred according to what we
thought to be species; if what we called species was rather an
assemblage of dissociated taxonomical units, some of the mechanisms
thought to act in Lamellodiscus evolution (such as radiation by host
switch followed by speciation) need to be re-evaluated in the light of
revised species delineation and an updated phylogeny. Before
assessing intraspecific and intrageneric genetic diversity, it is
important to be sure which taxa are given the species status. In
such a situation, it could be useful to use the Least-inclusive
taxonomic unit (LITU) concept [62], that is considering several
individuals as forming a clade, without making further assumptions
about the taxonomic position of this clade. Our results suggest that
the ignoratus group is highly diversified, and is likely to be formed by
several OTUs; we suggest to give this group the status of LITU, and
to wait for further investigation to determine its exact taxonomic
status. According to these results, our view of the taxonomy and,
consequently, of the evolution of Lamellodiscus needs to be reassessed.
2 Phylogeny and morphology seem to be unlinked in
Lamellodiscus
The molecular variability (as approximated by the pairwise
distance at several taxonomical depths) in Lamellodiscus was
compared to what was found in other monogenean genera. For
COI, Lamellodiscus shows the most important interspecific distances;
for 18S, Lamellodiscus is nearly twice as variable as Dactylogyrus, but
less than half as variable as Gyrodactylus, thought to be the most
variable monogenean genus [63]. Despite this important molecular
variability, however, there are very little clearly distinguishable
morphologies within the genus Lamellodiscus. Amine and Euzet [14]
defined two morphological groups in this genus, named ignoratus
(formed by L. ignoratus s.l., L. fraternus, L. knoeppfleri and L. erythrini) and
ergensi (formed by L. ergensi, L. sanfilippoi, L. kechemirae and L. baeri).
Our results (Figure 1) are congruent with this classification, with two
notable exceptions: L. knoeppfleri, L. fraternus and L. erythrini were
found to belong to the ergensi group. According to our phylogeny,
the ignoratus group is only formed of the taxa belonging to L.
ignoratus s.l. Within each group, however, there is no link between
morphological features and phylogenetic position, mostly because
none of the individuals of a single putative species cluster together. A
similar situation was reported by Hay and colleagues [64]. They
observed that the tuatara, Sphenodon punctatus, while being a living
fossil (its morphology is strikingly similar to the fossil specimens), and
having a slow metabolism, a long generation time, and a slow rate of
reproduction, is the species having the highest rate of molecular
evolution observed amongst vertebrates. Hence, a high molecular
divergence is not necessarily linked to important morphological
changes, and the assumption that rates of molecular and
morphological evolution are inherently correlated [65] is likely to
be untrue in certain genera, which could be the case in Lamellodiscus.
Given that we were able to align the ITS1 of several individuals,
we are able to make suggestions as to the species status of some
morphotypes. We found molecular evidences that, despite some
morphological divergences on the shape of the hard parts and
copulatory organs, L. furcosus and L. coronatus form a single species
(that we call L. furcosus). The ITS1 of L. ergensi, L. oliveri, L. fraternus
and L. gussevi can be aligned, suggesting that all of these
morphotypes should be considered as a single species, that we call
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26252
Page 6
L. ergensi. The individuals belonging to L. neifari and L. diplodi, as well
as some L. ignoratus individuals, display enough ITS1 similarity to
allow their sequences to be aligned. As for other species, we suggest
that these species are invalid, and that only L. neifari should be
retained. The situation is similar for L. falcus, L. ignoratus, L.
abbreviatus and L. confusus. We suggest that these morphotypes belong
to the L. ignoratus species, and that the others are invalid. More data
(e.g. other genes) are needed to confirm this pattern, as shown by the
lack of support from 18S sequences in this part of the tree. However,
we did not rule out the possibility that diversification is acting within
these species, which is likely given the important genetic divergence
observed within Lamellodiscus. Owing to the relatively low support of
some nodes in the phylogeny (Fig. 1), we suggest that Lamellodiscus
may be highly diverse, and our understanding of their taxonomical
status will benefit from an increased genetic sampling.
The apparent discrepancies between morphological features and
molecular phylogeny could be explained by the strong selective
pressures parasites have to cope with. Morphology in Lamellodiscus
(and in most monogeneans) is mainly studied by looking at the
sclerotized parts of the haptor and the male copulatory organ, which
is a putative factor in the initiation of intra-host speciation events
amongst monogeneans [66,28]. In Lamellodiscus, as in most (if not all)
Monogenan genera, no information is available about the degree of
morphological dissimilarity that must be achieved between two
shapes of male copulatory organs to trigger a reproductive isolation
event leading to speciation. However, all species in the ignoratus and
ergensi groups do harbor an en lyre male copulatory organ, with some
variation between species [14]; this supports the hypothesis that these
groups share a direct common ancestor. The monophyly of both
ignoratus and ergensi groups is strongly supported by morphological
characters, such as the split of lateral dorsal bars, and this result is
supported by our phylogeny (modulo the position of L. knoeppfleri, L.
fraternus and L. erythrini, which may be cases of convergent evolution,
or merely reflect the difficulty to determine what constitutes a
‘‘character’’ [67]). It seems that, while the strong (e.g. number of
lamellodiscs, number of pieces in the lateral bars) differences
between organ shapes are linked to the taxonomic position of species,
the small differences (e.g. shape or width of some parts of dorsal and
ventral hooks) are not indicative of a speciation process [68].
However, molecular data suggest that some features (non-split bars
in some species within the ergensi group) may display an
evolutionary convergence, under environmental (that is, host-
induced) pressure.
3 Genetic diversity in Lamellodiscus and other
monogeneans
During this study, we assessed mean uncorrected genetic
pairwise distances based on two molecular markers (the 59 end
of the 18S gene, and about 300 base pairs within COI) on several
monogenean genera. For the COI gene (Table 1), it seems that the
mean pairwise distance is an appropriate reflection of the
taxonomic position: intra-specific uncorrected pairwise genetic
distances range from 0% (in Euryhaliotrematoides grandis individuals
from the same isolate) to 8% (between individuals of Protopolystoma
simplicis); intra-generic distances range from 13% (for Protopolystoma
spp.) to 19% (for Lamellodiscus). The notable exception to this
pattern is the Polystomatidae family, with an intra-family distance
of 17%. However, the latter result may be due to the fact that few
sequences are available to cover a whole family [69], thus
potentially decreasing the mean distance, and emphasizing the
need to gather more genetic data at broad taxonomical scales.
Another explanation is that larger bodied monogeneans might be
less speciose than smaller bodied organisms. Another explanation
is that chelonian polystomes arose very early, in the Lower
Triassic, namely 200 Million years ago [70]. This may explain
large divergences observed between species of different genera.
For the 18S gene (Table 1), however, the pattern of correspon-
dence between taxonomic position and mean pairwise distance is
lost. While some species display very few variations (the sequences
we retrieved for Dactylogyrus crucifer and D. vistulae showed no
differences), others (such as G. salaris and G. thymalli thought to be a
single species [71]) harbor a level of intra-specific divergence
comparable or superior to the one found in the Lamellodiscus and
Gyrodactylus genera. Altogether, these results indicate that analyses
of pairwise genetic distances to assess taxonomic status, although
used in diverse biological systems [72–74], should be conducted
cautiously as not all markers display the same behavior of
congruence between genetic distance and taxonomical rank.
It seems that the evolutionary rate of some markers, such as 18S
rDNA, is lineage specific (e.g. Gyrodactylus seems to evolve faster than
Lamellodiscus, itself evolving faster than Dactylogyrus), whereas in other
markers, such as COI, mean distance correlates with taxonomic
position. These results can be due to different evolutionary rate in
these markers (COI is mitochondrial and coding, 18S is nuclear and
structural), but may also be linked to sampling effort: where some
genera have undergone an important sampling effort (e.g.
Gyrodactylus), few molecular data are yet available for others or,
when they are, they often come from a single study, often limited to
a single geographic area despite the broad geographical distribution
of Lamellodiscus [75]. The question of whether the current amount of
available data allows us to capture the majority of the genetic
diversity in monogeneans remains pending. Moreover, it is likely
that the lifestyle of the various taxa will matter in determining the
genetic diversity; for example, how viviparous and egg laying
monogeneans differ in this extent is yet to be investigated.
4 Concluding remarks
This study suggests that the degree of variability displayed by the
different markers used here is impacted by the taxonomic position of
the group investigated. Here, this variability is linked to the taxonomic
position for COI, but not for 18S. In comparison to the important
genetic variability displayed by Lamellodiscus, there is a relative
morphological conservatism, suggesting the action of environmental
(host-induced) selection pressures on the shape of several haptoral parts.
Acknowledgments
We are grateful to Pascal Romans for invaluable help in fish sampling,
Gwenael Piganeau for help with computing and interpretation of genetic
distances, Fre´de´ric Delsuc for pointing out GBlocks, and two anonymous
referees who greatly helped improving a previous version of this
manuscript. We also thank Louis Euzet for sharing his extensive knowledge
on Lamellodiscus.
Author Contributions
Conceived and designed the experiments: TP OV YD. Performed the
experiments: TP OV. Analyzed the data: TP OV YD. Wrote the paper:
TP OV YD.
References
1. Vignon M, Pariselle A, Vanhove MPM (2011) Modularity in attachment organs
of African Cichlidogyrus (Platyhelminthes, Monogenea, Ancyrocephalidae ) reflects
phylogeny rather than host specificity or geographic distribution. Biological
Journal of the Linnean Society 102: 694–706.
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26252
as some L. ignoratus individuals, display enough ITS1 similarity to
allow their sequences to be aligned. As for other species, we suggest
that these species are invalid, and that only L. neifari should be
retained. The situation is similar for L. falcus, L. ignoratus, L.
abbreviatus and L. confusus. We suggest that these morphotypes belong
to the L. ignoratus species, and that the others are invalid. More data
(e.g. other genes) are needed to confirm this pattern, as shown by the
lack of support from 18S sequences in this part of the tree. However,
we did not rule out the possibility that diversification is acting within
these species, which is likely given the important genetic divergence
observed within Lamellodiscus. Owing to the relatively low support of
some nodes in the phylogeny (Fig. 1), we suggest that Lamellodiscus
may be highly diverse, and our understanding of their taxonomical
status will benefit from an increased genetic sampling.
The apparent discrepancies between morphological features and
molecular phylogeny could be explained by the strong selective
pressures parasites have to cope with. Morphology in Lamellodiscus
(and in most monogeneans) is mainly studied by looking at the
sclerotized parts of the haptor and the male copulatory organ, which
is a putative factor in the initiation of intra-host speciation events
amongst monogeneans [66,28]. In Lamellodiscus, as in most (if not all)
Monogenan genera, no information is available about the degree of
morphological dissimilarity that must be achieved between two
shapes of male copulatory organs to trigger a reproductive isolation
event leading to speciation. However, all species in the ignoratus and
ergensi groups do harbor an en lyre male copulatory organ, with some
variation between species [14]; this supports the hypothesis that these
groups share a direct common ancestor. The monophyly of both
ignoratus and ergensi groups is strongly supported by morphological
characters, such as the split of lateral dorsal bars, and this result is
supported by our phylogeny (modulo the position of L. knoeppfleri, L.
fraternus and L. erythrini, which may be cases of convergent evolution,
or merely reflect the difficulty to determine what constitutes a
‘‘character’’ [67]). It seems that, while the strong (e.g. number of
lamellodiscs, number of pieces in the lateral bars) differences
between organ shapes are linked to the taxonomic position of species,
the small differences (e.g. shape or width of some parts of dorsal and
ventral hooks) are not indicative of a speciation process [68].
However, molecular data suggest that some features (non-split bars
in some species within the ergensi group) may display an
evolutionary convergence, under environmental (that is, host-
induced) pressure.
3 Genetic diversity in Lamellodiscus and other
monogeneans
During this study, we assessed mean uncorrected genetic
pairwise distances based on two molecular markers (the 59 end
of the 18S gene, and about 300 base pairs within COI) on several
monogenean genera. For the COI gene (Table 1), it seems that the
mean pairwise distance is an appropriate reflection of the
taxonomic position: intra-specific uncorrected pairwise genetic
distances range from 0% (in Euryhaliotrematoides grandis individuals
from the same isolate) to 8% (between individuals of Protopolystoma
simplicis); intra-generic distances range from 13% (for Protopolystoma
spp.) to 19% (for Lamellodiscus). The notable exception to this
pattern is the Polystomatidae family, with an intra-family distance
of 17%. However, the latter result may be due to the fact that few
sequences are available to cover a whole family [69], thus
potentially decreasing the mean distance, and emphasizing the
need to gather more genetic data at broad taxonomical scales.
Another explanation is that larger bodied monogeneans might be
less speciose than smaller bodied organisms. Another explanation
is that chelonian polystomes arose very early, in the Lower
Triassic, namely 200 Million years ago [70]. This may explain
large divergences observed between species of different genera.
For the 18S gene (Table 1), however, the pattern of correspon-
dence between taxonomic position and mean pairwise distance is
lost. While some species display very few variations (the sequences
we retrieved for Dactylogyrus crucifer and D. vistulae showed no
differences), others (such as G. salaris and G. thymalli thought to be a
single species [71]) harbor a level of intra-specific divergence
comparable or superior to the one found in the Lamellodiscus and
Gyrodactylus genera. Altogether, these results indicate that analyses
of pairwise genetic distances to assess taxonomic status, although
used in diverse biological systems [72–74], should be conducted
cautiously as not all markers display the same behavior of
congruence between genetic distance and taxonomical rank.
It seems that the evolutionary rate of some markers, such as 18S
rDNA, is lineage specific (e.g. Gyrodactylus seems to evolve faster than
Lamellodiscus, itself evolving faster than Dactylogyrus), whereas in other
markers, such as COI, mean distance correlates with taxonomic
position. These results can be due to different evolutionary rate in
these markers (COI is mitochondrial and coding, 18S is nuclear and
structural), but may also be linked to sampling effort: where some
genera have undergone an important sampling effort (e.g.
Gyrodactylus), few molecular data are yet available for others or,
when they are, they often come from a single study, often limited to
a single geographic area despite the broad geographical distribution
of Lamellodiscus [75]. The question of whether the current amount of
available data allows us to capture the majority of the genetic
diversity in monogeneans remains pending. Moreover, it is likely
that the lifestyle of the various taxa will matter in determining the
genetic diversity; for example, how viviparous and egg laying
monogeneans differ in this extent is yet to be investigated.
4 Concluding remarks
This study suggests that the degree of variability displayed by the
different markers used here is impacted by the taxonomic position of
the group investigated. Here, this variability is linked to the taxonomic
position for COI, but not for 18S. In comparison to the important
genetic variability displayed by Lamellodiscus, there is a relative
morphological conservatism, suggesting the action of environmental
(host-induced) selection pressures on the shape of several haptoral parts.
Acknowledgments
We are grateful to Pascal Romans for invaluable help in fish sampling,
Gwenael Piganeau for help with computing and interpretation of genetic
distances, Fre´de´ric Delsuc for pointing out GBlocks, and two anonymous
referees who greatly helped improving a previous version of this
manuscript. We also thank Louis Euzet for sharing his extensive knowledge
on Lamellodiscus.
Author Contributions
Conceived and designed the experiments: TP OV YD. Performed the
experiments: TP OV. Analyzed the data: TP OV YD. Wrote the paper:
TP OV YD.
References
1. Vignon M, Pariselle A, Vanhove MPM (2011) Modularity in attachment organs
of African Cichlidogyrus (Platyhelminthes, Monogenea, Ancyrocephalidae ) reflects
phylogeny rather than host specificity or geographic distribution. Biological
Journal of the Linnean Society 102: 694–706.
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26252
Page 7
2. Ergens R, Gelnar M (1985) Experimental verification of the effect of
temperature on the size of hard parts of opisthaptor of Gyrodactylus katharineri
Malmberg, 1964 (Monogenea). Folia Parasitologica 32: 377–380.
3. Mo TA (1991) Variations of opisthaptoral hard parts of Gyrodactylus salaris
Malmberg, 1957 (Monogenea: Gyrodactylidae) on parr of Atlantic Salmon Salmo
salar L. in laboratory experiments. Systematic Parasitology 20: 11–19.
doi:10.1007/BF00009707.
4. Da´vidova´ M, Jarkovsky´ J, Matejusova´ I, Gelnar M (2005) Seasonal occurrence
and metrical variability of Gyrodactylus rhodei Zitnan 1964 (Monogenea,
Gyrodactylidae). Parasitology research 95: 398–405. doi:10.1007/s00436-005-
1311-0.
5. Mo TA (1993) Seasonal variations of the opisthaptoral hard parts of Gyrodactylus
derjavini Mikailov, 1975 (Monogenea: Gyrodactylidae) on brown trout Salmo trutta
L. parr and Atlantic salmon S. salar L. parr in the River Sandvikselva, Norway.
Systematic Parasitology 26: 225–231. doi:10.1007/BF00009730.
6. Poisot T, Desdevises Y (2010) Putative speciation events in Lamellodiscus (Monogenea:
Diplectanidae) assessed by a morphometric approach. Biological Journal of the
Linnean Society 99: 559–569. doi:10.1111/j.1095-8312.2009.01381.x.
7. Bueno-Silva M, Boeger WA, Pie MR (2011) Choice matters: incipient speciation
in Gyrodactylus corydori (Monogenoidea: Gyrodactylidae). International Journal for
Parasitology 41: 657–667. doi:10.1016/j.ijpara.2011.01.002.
8. Kaci-Chaouch T, Verneau O, Desdevises Y (2008) Host specificity is linked to
intraspecific variability in the genus Lamellodiscus (Monogenea). Parasitology 135:
607–616.
9. Matejusova´ I, Koubkova´ B, Gelnar M, Cunningham CO (2002) Paradiplozoon
homoion Bychowsky & Nagibina, 1959 versus P. gracile Reichenbach-Klinke, 1961
(Monogenea): two species or phenotypic plasticity? Systematic Parasitology 53:
39–47.
10. Poisot T, Bever JD, Nemri A, Thrall PH, Hochberg ME (2011) A conceptual
framework for the evolution of ecological specialization. Ecology Letters 14:
841–851. doi:10.1111/j.1461-0248.2011.01645.x.
11. Libby E, Rainey PB (2011) Exclusion rules, bottlenecks and the evolution of
stochastic phenotype switching. Proceedings of the Royal Society B: Biological
Sciences: rspb.2011.0146-. doi:10.1098/rspb.2011.0146.
12. Euzet L, Combes C, Caro A (1993) A checklist of Monogenea of Mediterranean
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p.
13. Neifar L, Euzet L, Oliver G (2004) Lamellodiscus (Plathelminthes, Monogenea,
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Pagrus (Teleostei, Sparidae). Zoosystema 26: 365–376.
14. Amine F, Euzet L (2005) Deux espe`ces nouvelles du genre Lamellodiscus Johnston
& Tiegs, 1922 (Monogenea: Diplectanidae) parasites de Sparidae (Teleostei) des
coˆtes de l9Alge´rie. Systematic Parasitology 1922: 187–196.
15. Amine F, Euzet L, Kechemir-Issad N (2007) Lamellodiscus theroni sp. nov.
(Monogenea, Diplectanidae), a gill parasite from Diplodus puntazzo (Teleostei,
Sparidae) from the Mediterranean Sea. Acta Parasitologica 52: 305–309.
doi:10.2478/s11686-007-0052-x.
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groupe morphologique ‘‘ignoratus’’, parasites de Diplodus sargus et D. vulgaris
(Teleostei: Sparidae). Systematic Parasitology 64: 37–45. doi:10.1007/s11230-
005-9016-4.
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Parasite 14: 281–285.
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Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26252
temperature on the size of hard parts of opisthaptor of Gyrodactylus katharineri
Malmberg, 1964 (Monogenea). Folia Parasitologica 32: 377–380.
3. Mo TA (1991) Variations of opisthaptoral hard parts of Gyrodactylus salaris
Malmberg, 1957 (Monogenea: Gyrodactylidae) on parr of Atlantic Salmon Salmo
salar L. in laboratory experiments. Systematic Parasitology 20: 11–19.
doi:10.1007/BF00009707.
4. Da´vidova´ M, Jarkovsky´ J, Matejusova´ I, Gelnar M (2005) Seasonal occurrence
and metrical variability of Gyrodactylus rhodei Zitnan 1964 (Monogenea,
Gyrodactylidae). Parasitology research 95: 398–405. doi:10.1007/s00436-005-
1311-0.
5. Mo TA (1993) Seasonal variations of the opisthaptoral hard parts of Gyrodactylus
derjavini Mikailov, 1975 (Monogenea: Gyrodactylidae) on brown trout Salmo trutta
L. parr and Atlantic salmon S. salar L. parr in the River Sandvikselva, Norway.
Systematic Parasitology 26: 225–231. doi:10.1007/BF00009730.
6. Poisot T, Desdevises Y (2010) Putative speciation events in Lamellodiscus (Monogenea:
Diplectanidae) assessed by a morphometric approach. Biological Journal of the
Linnean Society 99: 559–569. doi:10.1111/j.1095-8312.2009.01381.x.
7. Bueno-Silva M, Boeger WA, Pie MR (2011) Choice matters: incipient speciation
in Gyrodactylus corydori (Monogenoidea: Gyrodactylidae). International Journal for
Parasitology 41: 657–667. doi:10.1016/j.ijpara.2011.01.002.
8. Kaci-Chaouch T, Verneau O, Desdevises Y (2008) Host specificity is linked to
intraspecific variability in the genus Lamellodiscus (Monogenea). Parasitology 135:
607–616.
9. Matejusova´ I, Koubkova´ B, Gelnar M, Cunningham CO (2002) Paradiplozoon
homoion Bychowsky & Nagibina, 1959 versus P. gracile Reichenbach-Klinke, 1961
(Monogenea): two species or phenotypic plasticity? Systematic Parasitology 53:
39–47.
10. Poisot T, Bever JD, Nemri A, Thrall PH, Hochberg ME (2011) A conceptual
framework for the evolution of ecological specialization. Ecology Letters 14:
841–851. doi:10.1111/j.1461-0248.2011.01645.x.
11. Libby E, Rainey PB (2011) Exclusion rules, bottlenecks and the evolution of
stochastic phenotype switching. Proceedings of the Royal Society B: Biological
Sciences: rspb.2011.0146-. doi:10.1098/rspb.2011.0146.
12. Euzet L, Combes C, Caro A (1993) A checklist of Monogenea of Mediterranean
fish. In: Second International Symposium on Monogenea. Montpellier-Se`te. 58
p.
13. Neifar L, Euzet L, Oliver G (2004) Lamellodiscus (Plathelminthes, Monogenea,
Diplectanidae) nouveaux parasites branchiaux des poissons marins du genre
Pagrus (Teleostei, Sparidae). Zoosystema 26: 365–376.
14. Amine F, Euzet L (2005) Deux espe`ces nouvelles du genre Lamellodiscus Johnston
& Tiegs, 1922 (Monogenea: Diplectanidae) parasites de Sparidae (Teleostei) des
coˆtes de l9Alge´rie. Systematic Parasitology 1922: 187–196.
15. Amine F, Euzet L, Kechemir-Issad N (2007) Lamellodiscus theroni sp. nov.
(Monogenea, Diplectanidae), a gill parasite from Diplodus puntazzo (Teleostei,
Sparidae) from the Mediterranean Sea. Acta Parasitologica 52: 305–309.
doi:10.2478/s11686-007-0052-x.
16. Amine F, Euzet L, Kechemir-Issad N (2006) Description de deux nouvelles
espe`ces de Lamellodiscus Johnston & Tiegs, 1922 (Monogenea: Diplectanidae) du
groupe morphologique ‘‘ignoratus’’, parasites de Diplodus sargus et D. vulgaris
(Teleostei: Sparidae). Systematic Parasitology 64: 37–45. doi:10.1007/s11230-
005-9016-4.
17. Amine F, Euzet L, Kechemir-Issad N (2007) Description de Lamellodiscus confusus
n. sp. (Monogenea: Diplectanidae), parasite de Sarpa salpa (Teleostei: Sparidae).
Parasite 14: 281–285.
18. Amine F, Neifar L, Euzet L (2006) Lamellodiscus sanfilippoi n. sp. (Monogenea,
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Phylogenetics and Evolution 38: 241–249. doi:10.1016/j.ympev.2005.08.013.
69. Verneau O, Palacios C, Platt T, Alday M, Billard E, et al. (2011) Invasive species
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doi:10.1017/S0031182011000333.
70. Verneau O, Bentz S, Sinnappah ND, Preez L du, Whittington I, et al. (2002) A
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Biological Sciences 269: 535–543. doi:10.1098/rspb.2001.1899.
71. Huyse T, Buchmann K, Littlewood DTJ (2008) The mitochondrial genome of
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proach for Gyrodactylus species and strain identification. Gene 417: 27–34.
doi:10.1016/j.gene.2008.03.008.
72. Bradley RD, Baker RJ (2001) A test of the genetic species concept: cytochrome-b
sequences and mammals. Journal of Mammalogy 82: 960–973.
73. So¨ller R, Warnke K, Saint-Paul U, Blohm D (2000) Sequence divergence of
mitochondrial DNA indicates cryptic biodiversity in Octopus vulgaris and supports
the taxonomic distinctiveness of Octopus mimus (Cephalopoda: Octopodidae).
Marine Biology 136: 29–35. doi:10.1007/s002270050004.
74. Beaumont M, Barratt EM, Gottelli D, Kitchener AC, Daniels MJ, et al. (2001)
Genetic diversity and introgression in the Scottish wildcat. Molecular Ecology
10: 319–336.
75. Justine JL, Briand MJ (2010) Three new species, Lamellodiscus tubulicornis n. sp., L.
magnicornis n. sp. and L. parvicornis n. sp. (Monogenea: Diplectanidae) from
Gymnocranius spp. (Lethrinidae: Monotaxinae) off New Caledonia, with the
proposal of the new morphological group ‘tubulicornis’ within Lamellodiscus
Johnston & Tiegs, 1922. Systematic Parasitology 75: 159–179. doi:10.1007/
s11230-009-9224-4.
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26252
Alaska and paraphyly within the Ursidae. Molecular Phylogenetics and
Evolution 5: 477–494. doi:10.1006/mpev.1996.0044.
56. Brown JM, Pellmyr O, Thompson JN, Harrison RG (1994) Phylogeny of Greya
(Lepidoptera: Prodoxidae ), Based on Nucleotide Sequence Variation in
Mitochondrial Cytochrome Oxidase I and II: Congruence with Morphological
Data interactions. Molecular Biology and Evolution 11: 128–141.
57. Lymbery AJ, Thompson RC (1996) Species of Echinococcus: pattern and process.
Parasitology Today 12: 486–91.
58. Ellis JT, Morrison DA, Liddell S, Jenkins MC, Mohammed OB, et al. (1999)
The genus Hammondia is paraphyletic. Parasitology 118: 357–362.
59. Harrison G (1992) Linking Evolutionary Pattern and Process. In: Howard DJ,
Berlocher SH, eds. Endless forms: Species & speciation Oxford University Press.
pp 19–31.
60. de Queiroz K, Donoghue MJ (1988) Phylogenetic systematics and the species
problem. Cladistics 4: 317–338.
61. Luckow M (1995) Species concepts: assumptions, methods, and applications.
Systematic Botany 20: 589–605.
62. Pleijel F, Rouse GW (2000) Least-inclusive taxonomic unit: a new taxonomic
concept for biology. Proceedings of the Royal Society B: Biological Sciences 267:
627–630. doi:10.1098/rspb.2000.1048.
63. Hansen H, Bakke TA, Bachmann L (2007) DNA taxonomy and barcoding of
monogenean parasites: lessons from Gyrodactylus. Trends in parasitology 23:
363–367. doi:10.1016/j.pt.2007.06.007.
64. Hay J, Subramanian S, Millar C (2008) Rapid molecular evolution in a living
fossil. Trends in Genetics 24: 103–106. doi:10.1016/j.tig.2007.12.005.
65. Omland KE (1997) Correlated Rates of Molecular and Morphological
Evolution. Evolution 51: 1381–1393.
66. Morand S, Simkova A, Matejusova I, Plaisance L, Verneau O, et al. (2002)
Investigating patterns may reveal processes: evolutionary ecology of ectoparasitic
monogeneans. International Journal for Parasitology 32: 111–119.
67. Donoghue MJ, Sanderson MJ (1994) Complexity and homology in plants. In:
Hall BK, ed. Homology: the hierarchical basis of comparative biology Academic
Press. pp 393–421.
68. Pouyaud L, Desmarais E, Deveney M, Pariselle A (2006) Phylogenetic relationships
among monogenean gill parasites (Dactylogyridea, Ancyrocephalidae) infesting
tilapiine hosts (Cichlidae): systematic and evolutionary implications. Molecular
Phylogenetics and Evolution 38: 241–249. doi:10.1016/j.ympev.2005.08.013.
69. Verneau O, Palacios C, Platt T, Alday M, Billard E, et al. (2011) Invasive species
threat: parasite phylogenetics reveals patterns and processes of host-switching
between non-native and native captive freshwater turtles. Parasitology: in press.
doi:10.1017/S0031182011000333.
70. Verneau O, Bentz S, Sinnappah ND, Preez L du, Whittington I, et al. (2002) A
view of early vertebrate evolution inferred from the phylogeny of polystome
parasites (Monogenea: Polystomatidae). Proceedings of the Royal Society B:
Biological Sciences 269: 535–543. doi:10.1098/rspb.2001.1899.
71. Huyse T, Buchmann K, Littlewood DTJ (2008) The mitochondrial genome of
Gyrodactylus derjavinoides (Platyhelminthes: Monogenea) – a mitogenomic ap-
proach for Gyrodactylus species and strain identification. Gene 417: 27–34.
doi:10.1016/j.gene.2008.03.008.
72. Bradley RD, Baker RJ (2001) A test of the genetic species concept: cytochrome-b
sequences and mammals. Journal of Mammalogy 82: 960–973.
73. So¨ller R, Warnke K, Saint-Paul U, Blohm D (2000) Sequence divergence of
mitochondrial DNA indicates cryptic biodiversity in Octopus vulgaris and supports
the taxonomic distinctiveness of Octopus mimus (Cephalopoda: Octopodidae).
Marine Biology 136: 29–35. doi:10.1007/s002270050004.
74. Beaumont M, Barratt EM, Gottelli D, Kitchener AC, Daniels MJ, et al. (2001)
Genetic diversity and introgression in the Scottish wildcat. Molecular Ecology
10: 319–336.
75. Justine JL, Briand MJ (2010) Three new species, Lamellodiscus tubulicornis n. sp., L.
magnicornis n. sp. and L. parvicornis n. sp. (Monogenea: Diplectanidae) from
Gymnocranius spp. (Lethrinidae: Monotaxinae) off New Caledonia, with the
proposal of the new morphological group ‘tubulicornis’ within Lamellodiscus
Johnston & Tiegs, 1922. Systematic Parasitology 75: 159–179. doi:10.1007/
s11230-009-9224-4.
Morphology and Phylogeny in Lamellodiscus
PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26252
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