The use of geometric morphometrics in understanding ...

Parasitology International 59 (2010) 183–191

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The use of geometric morphometrics in understanding shape variability of sclerotized haptoral structures of monogeneans (Platyhelminthes) with insights into biogeographic variability Matthias Vignon ⁎, Pierre Sasal UMR 5244 CNRS EPHE UPVD, Biologie et Écologie Tropicale et Méditerranéenne, Université de Perpignan Via Domitia, 66860 Perpignan cedex, France USR 3278 CNRS EPHE, Centre de Recherches Insulaires et Observatoire de l'Environnement (CRIOBE), BP 1013, Papetoai Moorea, French Polynesia

a r t i c l e

i n f o

Article history: Received 1 July 2009 Received in revised form 18 January 2010 Accepted 24 January 2010 Available online 1 February 2010 Keywords: Monogenea Biogeography Pacific Ocean Geometric morphometrics Haptor

a b s t r a c t The sclerotized attachment organ of monogeneans has been widely used to address fundamental questions in ecology and evolution. However, traditional morphometric techniques appear to be partially inadequate and non-optimal. Traditional linear measurements mainly provide information on the size of sclerites but provide very little information, if any, on their shape. The shape of sclerites is indeed virtually unexplored and its implication for ecological and evolutionary processes remains to be analyzed. This study aims to both introduce and illustrate the use of geometric morphometrics in order to study sclerites of monogeneans in a biogeographic context. To do this, we investigated morphological variation patterns among four populations from the Pacific Ocean and six monogenean species through traditional and geometric morphometric techniques. Unlike the traditional method, the geometric morphometric method yielded a high percentage of individuals correctly classified to the four populations, providing strong evidence for phenotypic variability, divergence and local adaptation among islands without evolutionary constraint. Moreover, the traditional method also resulted in inconsistent interpretations of shape variations. This study highlighted the limitations that may arise when using traditional morphometric techniques and emphasizes that considerable information about the shape of sclerotized haptoral parts is added by using geometric morphometrics. Given the prominent taxonomic, ecological and evolutionary role of the haptor for characterizing monogeneans, we ultimately discuss the potential broad use of geometric morphometrics in a wide variety of ecological and evolutionary contexts. This powerful approach might allow a more robust estimation of the extent to which traditional evolutionary theories based on size of sclerites are congruent with their shape. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction At a broad scale, current distribution of parasites throughout large biogeographic regions may have been influenced by either radiation subsequent to colonization, or by secondary radiation in contemporary host taxa. At a narrower scale, investigating the phylogeographic patterns of widely distributed marine parasites with low dispersal ability is an important question [1,2]. Numerous parasites associated with coastal hosts have highly passive dispersal of their larval stage, limited vagility as adults and are unable to parasitize pelagic hosts from island to island. However, the broad distribution of several parasite species throughout wide biogeographic range shows their ability to travel large distances. Underlying ecological and evolutionary processes of colonization across large expanses of ocean are still unclear but it is suspected that specialist parasites may have dispersed with their hosts ⁎ Corresponding author. UMR 5244 CNRS EPHE UPVD, Biologie et Écologie Tropicale et Méditerranéenne, Université de Perpignan Via Dominitia, 52 Avenue Paul Alduy, 66860 Perpignan cedex, France. Tel.: +33 4 68 66 20 55; fax: +33 4 68 50 36 86. E-mail address: [email protected] (M. Vignon). 1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.01.006

[3–5]. Such colonization might have evolutionarily constrained parasites in several ways, including their morphological features. The monogeneans provide an excellent model system for addressing fundamental ecological and evolutionary questions on parasites regarding species richness, host specificity, community structure and host–parasite coevolutionary interaction [3,6,7]. Monogenea are known for their soft, flexible body structure. However, they possess hard structures including copulatory organs and haptoral sclerites often used to distinguish between species. Besides giving important taxonomic information on species, the haptor (sometimes along with accessory structures) has as its main function attachment of the parasite onto its host and must be adapted, as much as possible, to the microenvironment within the hosts [8–11]. Thus, morphological evolution of attachment organs is of broad interest in an evolutionary context as it may influence the specificity, the specialization as well as the reproductive segregation among conspecifics through distinct niches [7]. Among monogeneans, ancyrocephalids and diplectanids have rich species diversity and are common parasites of freshwater and marine fish occurring in tropical and subtropical waters worldwide. They are known to be highly host specific and can have a wide geographic

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distribution. Several species indeed occur throughout the entire IndoWest Pacific Ocean and may have dispersed with their hosts [3–5]. Such monogeneans provide interesting models to study morphological variation of attachment organs over a large biogeographic range and to test for the evolutionary constraint of haptor shape. Attachment by the haptor generally involves the use of hooks, suckers, glue(s) and/or clamps. Only sclerotized haptoral structures are considered for morphometric analysis as their morphology remains unchanged after fixation. For ancyrocephalid species the haptor contains two pairs of hooks (i.e. dorsal and ventral anchors), two connective bars (one dorsal and one ventral) and seven pairs of marginal hooks [12]. Additional haptoral accessory sclerites/structures may also be present. Traditionally, morphometric approaches are based on the application of standard multivariate analyses of arbitrary collections of linear distance measurements (‘trusses’), ratios, and angles. For example, the traditional analyses of ancyrocephalids involve five, four and one landmarks for anchors, bars and marginal hooks respectively [13] (Fig. 1a). Those points correspond to a total of 17 linear measurements. Similarly, only a few landmarks are commonly used for other monogenean families (see Diplectanidae in Fig. 1b). However, such a traditional approach while providing information on the overall size of sclerotized haptoral parts, provides very little information, if any, on their shape (Fig. 2). This is especially important in a biogeographic context because subtle intra-specific shape variation may not be revealed using a traditional approach. Specifically, the analysis of a limited set of linear distances, ratios or angles frequently fails to capture the complete spatial arrangement of the anatomical points (landmarks) on which the measurements are based [14]. Due to the lack of discrimination of traditional methods, several authors added arbitrary additional landmarks to take into account the maximum shape information [15,16] or find an informative combination of variables [17–19], leading to the construction of analyses on a case-by-case basis [20,21]. As more landmarks are included in an analysis, the minimum number of distances needed to fix the relative landmark positions increases to the point of being unrealistic [14]. Thus, traditional method does not consider the spatial relationships among the variables measured. This complete retention of geometric information from data collection through analysis and visualization is the reason for using alternative methods when studying sclerites of monogeneans. Coordi-

nates of these same landmarks, however, concisely encode all the information in any subset of distances between them. Intuitively, one expects methods that are able to take such additional information into account to have greater statistical power. Traditional methods also only allow one to visualize statistical relationships either numerically or as scatter plots, not as estimates of the shapes themselves [22]. So far, no attempt has been made to study and quantify the shape of sclerotized haptoral parts and its implication for ecological and evolutionary processes remains virtually unexplored. To date, several techniques such as geometric morphometric (GM) methods and elliptic Fourier analysis are commonly being used to describe and measure morphological variability [14,23]. While the two methods provide valuable alternative to the traditional one, only the former method is considered in this manuscript. This method analyzes the geometry among the locations of all landmarks simultaneously rather than the linear distances between pairs of landmarks (‘trusses’). Unlike traditional approach, GM shape variables preserve the geometry of anatomical structure throughout the analysis and therefore it is possible not only to perform statistical comparisons of shape, but also to generate graphical representations of mean forms to study trends in shape variation. Over the last decades, GM has been successfully used in analyzing shape of numerous organisms within and among samples [24]. Despite its advantage, the geometric morphometrics approach has never been applied to monogeneans (except [25]). This study aims to investigate morphological variation patterns among several populations of monogeneans in the Pacific Ocean in a broad biogeographic context. To do this, we analyzed six monogenean species (five Ancyrocephalidae and one Diplectanidae) infecting the same lutjanid host (Perciformes, Lutjanidae) from four populations in the Southwest Pacific Ocean (Fiji, New Caledonia, Society archipelago and Marquesas Islands). More specifically, we tested for the evolutionary constraint of haptor shape due to the colonization (i.e. correspondence between geographical and shape proximity across islands). In this context, the study also aims to both introduce and illustrate the use of geometric morphometrics (landmarks and semi-landmarks) in order to study sclerotized haptoral parts of monogeneans. Results are compared with the traditional morphometric technique (trusses) and we ultimately discuss the potential use of GM in a wide variety of ecological and evolutionary contexts when studying monogeneans.

Fig. 1. Landmark definition and morphometric measurements of haptor sclerotized parts for a) Ancyrocephalidae (according to Gussev [34]) and b) Diplectanidae (according to Amine and Euzet [35]). Ancyrocephalidae: Total length of anchor (a), length of base (b), length of inner root (c), length of outer root (d), length of point (e); total length of marginal hook (f); total length of the bar (g), total width (h); median width (i). Diplectanidae: Total length of anchor (a), point length (b), hilt length (c), grid length (d), blade opening (e); distance between grip and hilt (f); total length of dorsal and lateral bar (g), total width (h).

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Fig. 2. Hypothetical sclerites with identical landmarks but distinct shapes. The overall superimposition according to landmarks is provided on the right.

2. Materials and methods 2.1. Sites, host and parasite collection One host (Lutjanus kasmira, Lutjanidae) was collected in four localities in the Southwest Pacific Ocean between 2006 and 2007: Moorea Island (17°30′S, 149°50′W, Society archipelago, French Polynesia), Ua Huka Island (8°57′S, 139°35′W, Marquesas Islands, French Polynesia), Viti Levu Island (18°10′S, 178°24′E) and New Caledonia Island (22°29′S, 166°25′E) (Fig. 3). Fish were collected using a spear gun from both the inner and outer reefs at depths ranging from 5 to 40 m. All snappers sampled were sexually mature and their length overlapped throughout all localities. Fish were examined for parasites under a dissecting microscope. Monogeneans were prepared and mounted using either ammonium picrate-glycerine [26] or Berlese fluid. All monogeneans were identified using sclerotized parts of the haptor and reproductive organs (male copulatory organs and vaginal armaments) according to [27–30]. All individuals examined for morphological purpose were collected on the same specific part of the gills and were approximately of the same size throughout Islands. Monogeneans were observed using a light microscope equipped with a digital camera and then drawn with Adobe Illustrator using a computerised digitizing system. All individuals for which the complete outline of the entire sclerites was not available (slides of poor quality, up to 80% of individuals examined) were excluded from subsequent analysis. Measurements were made from the numeric drawings with the help of a custom-made rule previously calibrated, following the advices of Roff and Hopcroft [31]. Only anchors and bars were analyzed as they are not subject to large variation from contraction or flattening on fixation. Marginal hooks were not considered because they were smaller than other sclerites and it was difficult to discern their overall outline accurately with the light microscope. Fluctuating asymmetry between the right and left sides of each pair of ventral and dorsal anchors was not assessed and relative measurements were made equally on both sides. Morphological features of the attachment organ

were measured on a total of 480 individuals belonging to six species (20 individuals per species and per locality, five ancyrocephalid and one diplectanid species in four localities). 2.2. Genetic analysis All monogeneans studied were already described and recorded from hosts in the westernmost part of the Pacific Ocean (i.e. Australia and China Sea) and their presence in New Caledonia, Fiji, Society archipelago and Marquesas islands constitute an important range expansion. To confirm the identity of the monogenean species throughout their wide biogeographic range, we used D1–D2 domain of LSU rDNA from specimens collected in French Polynesia and compared sequences with those previously available in GenBank from individuals collected in the westernmost part of the Pacific Ocean. D1–D2 domain of LSU rDNA generally allows for specific identification of ancyrocephalid and diplectanid monogeneans [27,28,32]. Total genomic DNA was extracted using the micro-genomic DNA purification kit by EZNA based on the protocol of DNA purification from one entire individual. The D1–D2 domain of LSU rDNA was PCR amplified using primers C1 (forward; 5′ACCCGCTGAATTTAAGCAT-3′) and D2 (reverse; 5′-TGGTCCGTGTTTCAAGAC-3′) [33]. PCR reactions were performed in a 25 µL reaction volume containing: 2.5 µL of 10× Buffer (1.5 mM Mg2+), 200 µM of each dNTP, 0.5 mM MgCl2, 0.5 µM of each primer, 1 U Taq DNA Polymerase and 1.2 µL genomic DNA template using the following conditions: an initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. Quality and quantity of PCR products were determined on a 2% TBE (0.5×) agarose gel with 2% EtBr. 2.3. Traditional morphometric method Sixteen and twelve haptoral linear measurements were taken to the nearest micrometer for Ancyrocephalidae [according to 34] and Diplectanidae [according to 35], respectively (Fig. 1). For each monogenean species, a forward stepwise linear discriminant analysis was performed on overall morphological variables in order to discriminate between the four island populations. 2.4. Geometric morphometrics

Fig. 3. Sampling locations in the Southwest Pacific Ocean.

The lack of numerous biological (i.e. traditional) landmarks is a problem when considering sclerotized haptoral structures of monogeneans because the entire mathematical framework of geometric morphometrics was initially developed on the comparison of homologous landmarks from specimen to specimen. To analyze outlines in the same analytical framework as landmarks, outlines must be digitized as a series of discrete points (semi-landmarks distributed along a homologous outline) that can be treated as though they were landmarks [36]. However, it is important to recognize the compromises resulting from

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using landmarks of doubtful homology [36,37]. In order to study simultaneously all points as we did in this study, several adjustments are possible when computing generalized least square Procrustes superimposition of landmarks configuration and semi-landmarks. One of them was developed by Green [38] and Bookstein [36] and consists in sliding additional points along a tangential direction so as to remove tangential variation, because contours should be homologous from subject to subject whereas their individual points need not. This variation can be removed by minimizing either bending energy or Procrustes distance with respect to a mean reference form [36]. The justification for this sliding technique is that differences in relative positions of semilandmarks along the curve cannot be informative because this spacing was defined arbitrarily. For the geometric morphometrics approach, each haptoral part was analyzed separately. Each sclerite shape was digitized using traditional landmarks plus additional semi-landmarks. Semi-landmarks were distributed at equal intervals along the outline of sclerites between traditional landmarks. The total number of points (landmarks and semi-landmarks) used in this study (100 for anchors and 80 for bars) results in a compromise between curve precision and the degree of freedom for subsequent statistical analysis (see Discussion). Consequently, the sub-number of points between each pair of consecutive landmarks was adjusted in order to obtain equal intervals between all consecutive points along the overall outline of each sclerite. The coordinates of each landmark and semi-landmark were digitized using the software tpsDig version 2.10 [from the TPS package 39]. Generalized least square Procrustes superimposition of landmark and semi-landmark coordinates was then computed with tpsRelw version 1.45, minimizing bending energy with respect to a mean reference form [36,38] to determine the criteria for sliding semi-landmarks along outlines (see Discussion for details). The geometric size of each specimen was estimated by the centroid size, defined as the square root of the sum of squared distances from all landmarks to the centroid of the configuration [40]. We performed several statistical and graphical analyses to explore sclerite shape variation among populations. Shape differences can be quantified among groups by estimating the minimal shape parameters (referred to as partial warps) needed to deform the form of the consensus configuration to each specimen. Partial warp contains shape information that can be analyzed using conventional multivariate statistical methods [41]. First, the partial warp scores plus the uniform components, derived using thin-plate spline decomposition of the bending energy matrix from the partial Procrustes aligned landmark and semi-landmark coordinates, were used to perform a principal component analysis (PCA) of the specimens using tpsRelw. The shape changes modelled by the thin-plate splines technique can be decomposed into two parts, the uniform and non-uniform components [42,43]. The uniform components (u1 and u2) express global (affine) variation in shape, whereas the non-uniform components describe local (non-affine) shape changes at different geometric scales. The first uniform component (u1) corresponds to the stretching of a landmark configuration along the x-axis (width of sclerites), whereas the second uniform component (u2) refers to a shearing along the y-axis (width of sclerites). The uniform components were tested for significant differences among populations by multivariate analysis of variance (MANOVA). Moreover, the regression of partial warps and uniform components onto each axis allowed the estimation of the relative contribution of uniform components. This was given by the difference between unexplained variances with and without uniform components appended. In addition, we performed a canonical variates analysis (CVA) using the software CVAGen version 6o [from the IMP package 44] with method of dimensionality reduction based on PCA [45] to determine if the four populations differed in sclerites shape. Statistical significance of the CVA was tested using pairwise multiple comparisons among populations by transforming generalized distances into Hotelling's T2 values. The utility of the canonical variates (CVs) for discriminating among populations was also evaluated using the Mahalanobis distances of specimens from the group mean. The means are computed

using the a priori group assignments [37]. The predicted group membership of each specimen is determined by assigning each specimen to the group whose mean is closest to the specimen (under the Mahalanobis distance based on effective CVs). To visualize localized sclerite shape differences among populations–individuals, we generated thin-plate spline deformation grids [40] with scaling option α = 0 [41] along the first canonical or principal axis using tpsRelw. In order to test for the evolutionary constraint of haptor shape due to stepping-stone colonization across islands, we computed a regression of the partial warps onto two kinds of variables using tpsReg program version 1.31. First, dummy variables corresponding to islands ordered from west to east were used, (i.e. 0, 1, 2 and 3 for New Caledonia, Fiji, Moorea and Ua Huka, respectively). Second, geographical distances from the westernmost island were used (i.e. 0, 1300, 3350 and 4650 km for New Caledonia, Fiji, Moorea and Ua Huka, respectively). Both regressions allow assessment of the extent to which there is a continuity in shape variation across the four populations. Regressions were tested for significance using a permutation test (based on 1000 random permutations). This corresponds to traditional Mantel permutation procedure to assess the increase of shape differences over geographical distances. Centroid size was tested for differences among populations by single classification analysis of variance (ANOVA). The major axes of variation in partial warps (i.e. first relative warps) were regressed on centroid size for the assessment of allometric localized shape variation among population within each species. The uniform components were also regressed on centroid size for the assessment of ontogenetic uniform shape variation. Regressions between the partial warps, centroid size and canonical variates were computed with the TPSRegr program, version 1.31. 3. Results The identical rDNA sequences obtained from specimens collected in French Polynesia compared to that from the south-western most part of the Pacific Ocean (accession numbers: Euryhaliotrema chrysotaeniae AF026115; Haliotrema anguiformis DQ537375; Haliotrema spirotubiforum DQ157656) for three species confirmed that the present study focuses on the same species which have indeed a wide geographic range (thousands of kilometers). For the remaining species, the examination of fine morphological features of the body and genitalia also allows an assumption that the specific identity of monogeneans does not vary over the Pacific Ocean. The traditional approach only allowed discrimination of H. anguiformis among the four populations (100% correct identification, Wilks' lambda=0.001, pb 0.001) and Haliotrematoides patellacirrus to a lesser degree (80% correct identification, Wilks' lambda=0.005, pb 0.001) (Table 1). The other four species remain undiscriminated (pN 0.1) on the basis of all morphometrics made on sclerites (both anchors and bars). The forward stepwise linear discriminant analysis revealed for H. anguiformis that only six measurements allowed complete discrimination between the four populations and only nine were selected by the analysis, including predominantly morphometrics from both dorsal and ventral anchors (Fig. 4). Using GM, the uniform components and the relative warps are not significantly related to the centroid size (R2 b 0.2, p N 0.1). This reveals no allometric shape variation in our dataset and allows considering shape and size as independent factors. ANOVA showed non-significant difference in centroid size among populations within each species (p N 0.1). Relative warp analysis showed high variability among overall individuals for all species and several groups were formed on the ordination of first relative warps, especially when considering anchors (Fig. 5). However, PCA did not allow discrimination among populations. CVA revealed significant morphological differences among the population for five species out of six when considering dorsal or ventral anchors (Hotelling's T2 test, p b 0.01) (Table 1). Discriminations were not

M. Vignon, P. Sasal / Parasitology International 59 (2010) 183–191 Table 1 Summary of linear discriminant analysis for the four populations according to traditional linear measurements and geometric morphometrics. Monogenean species

Percentage of correct identification Linear measurements F

NC

M

UH

Geometric morphometrics Total

Diplectanum fusiforme

20

50

60

45

44

Euryhaliotrema chrysotaeniae

35

30

50

50

41

Haliotrema anguiformis

100 100 100 100 100

Haliotrema spirotubiforum

45

5

65

Haliotrematoides longitubocirrus

60

60 100 100

80

Haliotrematoides patellacirrus

50

25

40

55

50

30

41

da va db vb da va db vb da va db vb da va db vb da va db vb da va db vb

F

NC

M

UH

Total

100 100 50 40 100 100 50 55 100 100 70 65 45 70 45 35 85 90 55 35 80 90 50 65

100 100 35 65 100 100 60 35 100 100 75 55 50 35 15 65 90 95 65 75 85 85 60 50

100 100 60 55 100 100 45 40 100 100 55 40 35 50 65 75 100 100 40 45 100 100 55 35

100 100 40 25 100 100 40 55 100 100 50 70 75 60 45 45 100 100 35 40 100 100 45 40

100* 100* 46 46 100* 100* 49 46 100* 100* 62 56 51 53 42 55 93* 96* 49 48 91* 93* 52 47

The percentage of correct identification is provided for each locality (20 individuals) as well as for overall localities (80 individuals; F: Fiji; NC: New Caledonia; M: Moorea; UH: Ua Huka). For geometric morphometrics, discriminant analysis were performed separately on each sclerite (da: dorsal anchor; va: ventral anchor; db: dorsal bar; vb: ventral bar) and statistical significance of pairwise multiple comparisons was estimated using Hotelling's T2 (*, p b 0.01).

significant when considering bars for any species as well as for H. spirotubiforum when considering either anchors or bars (Hotelling's T2, p N 0.05). In all CVA performed, the first two discriminant axis contributed for only 20–40% of total non-uniform shape differentiation among populations. In fact at least the first ten relative warps were generally needed to explain 90% of total non-uniform shape variations, emphasizing numerous complex and localized morphological differences (on the base, inner root, outer root and the point). For all species except H. spirotubiforum, both uniform components showed a significant difference among populations (MANOVA, p b 0.01), revealing that geographical differences were partly due to global (affine) variation in shape (Fig. 6). However, the uniform components were generally responsible for no more than 20% of the total shape variation (most of

Fig. 4. Summary of the stepwise linear discriminant analysis for the four populations of H. anguiformis. The graph shows the contribution of each morphological feature to the overall separation among populations.

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the variance being explained by local variations). While CVA only allowed statistical test of the null hypothesis of no morphological differences among at least two populations, the assignment based on Mahalanobis distances revealed complete discrimination among the four populations when using either dorsal and ventral anchors for Diplectanum fusiforme, E. chrysotaeniae, H. anguiformis, Haliotrematoides longitubocirrus and H. patellacirrus. Table 1 summarizes results of discriminant analysis (traditional linear measurements) and CVA (GM). Interestingly, when considering both traditional and GM methods, bars remain poorly informative in the present biogeographic context (not selected in the results of the forward stepwise linear discriminant analysis, and low percentage of correct identification, respectively). The permutation test showed non-significant correspondence (p N 0.1) between shapes and geographic distance among the four islands for any sclerite of any species examined. 4. Discussion 4.1. Advantages of GM over traditional methods Seven basic limitations are associated with the traditional method. First, linear truss lengths are generally strongly positively related to the size of haptoral sclerites and removing the effect of size variation from shape variation is a difficult task. Second, variation among samples becomes more difficult to assess when homologous landmarks do not define complexes trusses. Third, different shapes can yield identical sets of truss lengths because the locations of trusses relative to each other are not quantified. Fourth, the exclusion of potentially important geometric shape information suggests that there can be reduced statistical power to distinguish variation among samples. Fifth, analysis of shape using traditional method lacks interpretable representation. There was indeed no way to reconstitute and represent the complete shape assessed using many component trusses. Sixth, the traditional approach does not allow proper measurement of the amount of difference between shapes, leading to invalid morphological interpretations. Lastly, most multivariate tests (such as discriminant analysis) traditionally used with linear measurements bypass statistical assumption. As already emphasized by several authors [18,46], while most morphological features of the sclerites are highly correlated and thus redundant, most studies bypass the fact that tests require mutually independent factors. However, such bypass results in overestimating the efficacy of discriminant functions and may lead to misunderstanding morphological results. Actually, only a few studies select variables before performing multivariate tests, avoiding collinearity and redundancy, while this last limitation is not intrinsic to the traditional method, but results from a widespread improper use of statistics. Considering those restrictions, there are several theoretical and practical reasons for favouring GM methods over traditional ones. All points previously identified were widely discussed in the literature [see for example 47,48]. Only considerations specific to monogeneans are discussed below. When considering traditional methods, it is not obvious how to visually interpret results, especially when discriminant analyses combine measurements from haptoral sclerites as well as from copulatory organs [see for example 49,50]. The best way to interpret which parts of the sclerites vary among groups consists in using forward stepwise linear discriminant analysis on overall morphological descriptors [see for example 17]. This is illustrated in Fig. 4 for H. anguiformis (the only one species for which traditional method allows complete reclassification among the four islands populations). In this case, the length of point and total length of dorsal anchor are the two most discriminant measurements. However, such method does not encourage intuitive understanding of accurate shape differences. When GM is applied on the same species among the four populations, the interpolating function thin-plate spline provides visual as well as quantitative interpretation (Figs. 4 and 6) and also revealed morphological

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Fig. 5. Scatter plot of the two first relative warps from the four populations with deformation grids (thin-plate) depicting shape differences among them (at 3× magnification). a) Dorsal anchor and b) ventral anchor from H. anguiformis.

differences already supported by previous traditional method. In fact, GM allows graphical representations of shape variation and the generation of deformation grids yielded clear and visually interpretable figures (allowing precise location of morphological changes). Because GM imposes to analyze each haptoral part separately, distinct comparison of anchors and bars is likely to carry complementary meaningful information from a biological point of view. However, patterns of covariation between sets of shapes can be easily explored [51] as well as the search for common causal factors among haptoral parts [52]. Despite its advantages, Procrustes based geometric morphometrics is not suitable to find diagnostic features within a structure which maximally contribute to differences among groups and can be readily used on the field. In this context, results from discriminant analysis from traditional methods are much more effective. Other fundamental advances of GM over traditional approaches include the way one can measure the amount of difference between shapes (using Procruste distance). When considering sclerites, Mahalanobis distances were traditionally calculated using discriminant analysis and used as a measure of morphometric distances between groups [50,53]. However such distances only consider discriminant axis and do not provide a suitable and absolute measure of the overall amount of shape difference between groups. This is a prominent point as shape comparison is used as a key feature in several ecological and evolutionary contexts. For example, infracommunities of congeneric species living on the same habitat allow us to address questions about reproductive isolation, niche restriction and niche specialization [7,53]

and xenocommunities raise the question of host specificity [11,50,54]. In those examples, morphometric distances of the attachment organ are traditionally compared to niche overlapping, male copulatory dissimilarity or host specificity based on Mahalanobis distances. Because all previous analyses were based on size (morphometric measurements) of sclerites, it is pertinent to examine to what extent size and shape (morphology) covary, as illustrated in Fig. 7. Taking into account morphology may allow adjustment or revision of the proposed hypothesis. In fact the GM method provides many advantages over traditional methods. While differences in linear measurements necessarily involve differences in shapes (illustrated by H. anguiformis and H. longitubocirrus, Table 1) except for strict homothety, the corollary is not true as differences in shape do not necessarily involve distinct linear measurements (illustrated by D. fusiforme, E. chrysotaeniae and H. patellacirrus, Table 1). As a consequence, studying the overall shape of sclerotized haptoral structures with GM method is more prone to detect differences among groups rather than traditional one. However, GM should be applied cautiously when using semi-landmarks. The use of semilandmarks as we have done in this study indeed imposes methodological constraints which must be carefully examined (see below). 4.2. The use of semi-landmarks There are a number of ways to identify semi-landmarks on a segment of the curve under analysis (including increments along the length of the curve, increments along the length of a chord connecting the ends of the

Fig. 6. Morphometric differences of ventral anchor from H. anguiformis between Moorea and Fiji. a) Complete deformation of major discriminant warps, b) uniform (affine) component and c) localized (non-affine) component (at 5× magnification).

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Fig. 7. Comparison of geographical differences among the four populations of H. anguiformis when considering Procrustes distances (mean of dorsal and ventral anchors) and Mahalanobis distances using discriminant analysis. Rooted with another outgroup species.

curve and increments of an angle subtended by the curve). Ultimately, regardless of the methods of identifying and superimposing semilandmarks, a curve will be represented by a large number of semilandmarks better than it will be by a smaller number. If curvature is simple, a small subset of semi-landmarks may be enough to characterize the curve, which implies that most of the semi-landmarks do not contribute to additional information. On the other hand, if the curvature has a high but consistent complexity the information provided by landmarks and some semi-landmarks might be nearly equivalent. However, increasing the number of semi-landmarks on a curve means it will play a larger role in superimposition and comparison [37]. This raises the question to what extent curvature between two landmarks is an important feature of variation. Thus, determining the most appropriate number of semi-landmarks to digitize is a compromise and depends on the geometry/variability of the curve and its relationship to other features represented by landmarks as well as on the sample size and criteria for rejecting the null hypothesis using conventional statistical tests. Of particular concern is the possibility that the different methods of handling semi-landmarks might influence results [55]. Thus, the use of semi-landmarks needs to have special consideration when applying GM to biological structures with few or no landmarks. Unfortunately, it is not clear what criteria might a priori be used to appropriately analyze outlines in the same analytical framework as landmarks. Nonetheless, it is always possible to perform a sensitivity analysis on a case-by-case basis [see for example 45,55]. 4.3. Biogeographic shape variations Results provide strong evidence that individuals collected from several localities in the Southwest Pacific Islands show significant and consistent geographical variability for five species out of six examined. However, previous studies have already emphasized the importance of several confounding effects which can affect the morphological features of sclerotized haptoral structures. Those geographical differences for the five ancyrocephalids investigated, were one or two orders of magnitude below those differences found among species. Previous studies already examined geographical variation of sclerites in monogeneans of the genera Kuhnia and Pseudokuhnia from various regions in the Atlantic and Indo-Pacific Oceans, and found considerable geographical variation [56–59]. In addition, such variation remained unrevealed when using the traditional approach, except for H. anguiformis and H. longitubocirrus to a lesser degree. This may be explained because geographical differences were predominantly due to non-uniform components which remain mostly unassessed by traditional trusses. Interestingly, only anchors (both ventral and dorsal) correlate with geographical patterns whereas bars are poorly informative in a biogeographic context. Procrustes superposition reveals that intra-populational variability of bars is indeed important compared with anchors (as illustrated in Fig. 8 for example). The higher intra-populational morphological variability for the bars has already been reported for several ancyrocephalid

Fig. 8. Procrustes superimposition of sclerites from H. anguiformis collected in Moorea after sliding semi-landmarks to minimize bending energy (consensus and variability estimated using 20 specimens).

species [60]. On the other hand, low variability in shape of anchors may be due to mechanical constraint [61,62] as anchors allow for specific attachment of monogeneans to a specific host, in a precise micro-habitat. Moreover, differences between bars and anchors may reveal their distinct ontogenetic origin [63,64]. Alternatively, bars which are thicker than anchors and have distinct chemical composition [65–68], might be more difficult to observe flat and also more prone to distortion during fixation and mounting. Further investigations using either digestive or sonication techniques and SEM [69,70], minimizing differences due to fixation and preparation, may reveal to what extent bars are actually informative. The discrimination of populations over a large biogeographic range prompts an examination of the underlying ecological or evolutionary mechanisms. How these parasites have dispersed so widely in the Pacific Ocean is a significant question in marine monogenean biogeography [1,2]. Monogeneans are characterized by highly passive dispersal of larval stages (oncomiracidium) and limited vagility as adults. Moreover, they are not taken with pelagic fish larvae from island to island because larvae are not parasitized [71]. The processes of colonization across large expanses of ocean are still unclear. However, the broad distribution of several parasite species throughout the Pacific Ocean clearly shows their ability to travel large distances [72]. All ancyrocephalid and diplectanid species studied are known to be very host specific, parasitizing only a few lutjanid hosts with little host switching [27–30,73]. Thus, parasites may have dispersed with their hosts [3–5] which are suspected to have dispersed from the Indonesia–Philippines region [74–76]. Such colonization might have evolutionarily constrained the haptor shape. However, without an obvious longitudinal continuum in shape, the precise shape of sclerotized haptoral structures at each island is more likely the result of local adaptation to specific environmental conditions as well as to the morphological features of the gills of their hosts, rather than phylogenetic constraint. Alternatively, in the absence of gene flow across the Pacific Ocean, the shape of sclerotized haptoral structures may result from a genetic drift. However, considering the prominent role of the haptor, it is

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highly unlikely. Thus, phenotypic changes among localities are likely due to phenotypic plasticity between distinct host/environment factors [7,57–59,77–80]. This is in accordance with general considerations about the size of sclerites which is suspected to be adaptive, and possibly linked to host specificity [review by 7]. For example, it has been suggested that it was necessary to develop large attachment organs in large hosts, as large fish may impose stronger mechanical constraints on ectoparasites [11,81]. It remains to be determined to what extent the shape of sclerites affect host or micro-habitat specificity and to what extent shape and size covary? Shape variation is widespread in nature and embodies both a response to, and a source for, evolution and natural selection. To detect patterns of shape evolution, one must assess the quantitative genetic underpinnings of shape variation as well as the selective environment that the organisms have experienced. Future population genetic studies on both hosts and parasites throughout their ranges may reveal the origin of dispersal, the patterns of colonization–speciation–diversification throughout the Pacific Ocean, and the congruence within this host– parasite complex [3,5,72]. In particular, this may allow insight into the influence of host diversity on parasite speciation/diversification [4,82]. Considering the prominent taxonomic and ecological role of the haptor among monogeneans, it is amazing that the intrinsic shape components of sclerites has remained virtually unexplored via modern, accurate, and quantitative methods prior to this study. Because all previous analyses were mainly based on the size of sclerites, it is of interest to examine to what extent the size and shape covary. Over the last few decades, GM have been successfully used in analyzing the shape of numerous organisms, not only analytically but biologically [24], and its use represents a powerful novel analytical approach in understanding shape variability of sclerotized haptoral structures for monogeneans.

Acknowledgements We are grateful to Pascal Ung for field assistance in Polynesia and Jean-Lou Justine for his support in New Caledonia (facilities and expertise). The authors would like to thank Louis H. Du Preez and Robert Adlard for revising this manuscript as well as Bruno Frédérich for his look at morphological analysis as well as anonymous reviewers who made highly valuable suggestions to improve the manuscript. This study was supported by CNRS (UMS 2978 and UMR 5244).

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