Geographic pattern of shell morphology in the endemic freshwater musselUnio ravoisieri (Bivalvia: Unionidae) from northern Tunisia

Chiheb Fassatoui, Amel Ben Rejeb Jenhani and Mohamed Salah Romdhane

Unite de Recherche Ecosystemes et Ressources Aquatiques (UR13AGRO1), Institut National Agronomique de Tunisie, Universite de Carthage,43, Avenue Charles Nicolle, Cite Mahrajene 1082, Tunis, Tunisia

Correspondence: C. Fassatoui; e-mail: fassatouichiheb@yahoo.fr

(Received 6 January 2014; accepted 19 August 2014)

ABSTRACT

Unio ravoisieri is an endemic freshwater mussel inhabiting North Africa rivers. Morphological diversityin U. ravoisieri was examined at eight sites covering the three main drainage systems in northernTunisia. Variation was assessed using 10 shell measurements in 193 specimens. Univariate and multi-variate analyses were applied to test the effects of sex and sites on shell traits. Simple and partial Mantelcorrelations on external and internal shell traits and geographic locations of populations were per-formed to test for spatial effects. Our results showed significant differences in shell morphometric char-acters among locations, but no evidence of sexual size dimorphism. Mantel correlations betweengeographic and all shell morphometric distances were significant, indicating an isolation-by-distancepattern. However, partial Mantel correlation did not detect a significant correlation between internaland external shell morphometric distances after controlling for geographic distance. The nonparametricSpearman correlations between geographical parameters (latitude, longitude and altitude) and exter-nal shell traits, as well as growth index were significant, but no significant relationships were demon-strated for internal traits or for condition index. Assessment of the underlying processes involved inthis differentiation is not straightforward, because of the likely complex interaction between genetic andenvironmental factors.

INTRODUCTION

Freshwater mussels of the family Unionidae are the most diversegroup of freshwater bivalve molluscs worldwide (Bogan, 2008).They have a broad distribution that includes all continentsexcept Antarctica (Haas, 1969; Bogan, 2008). Unio ravoisieri(Deshayes, 1847) is an endemic species with limited distributionin North Africa, including Algeria and Tunisia (Graf &Cummings, 2011; Khalloufi et al., 2011). This mussel is oftenfound in shallow rivers on gravel and silt bottoms. As with allunionid mussels, U. ravoisieri has a complex life cycle with asedentary-benthic juvenile and adult stage, and a larval stage,the glochidium, starting with a short planktonic dispersivephase followed by a parasitic phase. The host of the parasitic glo-chidium is commonly a specific teleostean fish (Barnhart, Haag& Roston, 2008). Unio ravoisieri is gonochoristic; fertilization isinternal and occurs in the gill chambers of females, after an ex-ternal liberation of sperm into the water column and their inhal-ation through inhalant siphons of females.

Nowadays, the rapid loss of freshwater biodiversity hasbecome a central policy question and a major concern world-wide (Ricciardi & Rasmussen, 1999; Lydeard et al., 2004).Freshwater molluscs are a critically endangered group of organ-isms and show high estimated rates of decline (Regnier,Fontaine & Bouchet, 2009). There are a variety of causes

that contribute to the vulnerability of freshwater mussels, bothin terms of abundance and diversity (Bogan, 1993; Strayeret al., 2004; Dudgeon et al., 2006; Downing, Van Meter &Woolnough, 2010). The first major contributor is the anthropo-genic pressure through over-exploitation of biota, habitat dis-turbance, water quality alteration by pollution and flowmodifications by dredging, canalization and weir construction.Among the main demographic reasons behind the decline infreshwater mussels are sedentary lifestyle, long life span, slowgrowth, low reproductive rates, predation and decline in fishhost populations. Climate change in recent decades, followed bythe problems of invasion by exotic species and migration ofnative species, also contribute to the extinction risk.

Another major reason for extinction of freshwater mussels isthe high degree of endemism. This aspect is typical for fresh-water habitats where species often have a small effective popula-tion size, narrow ecological amplitude and a limitedbiogeographic range (Strayer & Dudgeon, 2010). Consequently,the threat situation can be aggravated when habitats arechanged, degraded or heavily modified, especially in specieswith low dispersal ability, such as unionid mussels (Burlakovaet al., 2011).

Effective conservation measures require an understanding oftaxonomy and the recognition of the different populations

#The Author 2014. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved

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within species. It also requires information about fitness and lifehistory of populations such as age, growth and longevity. Thesedata are especially critical in assessing the risk of extinction ofrare and endangered species (Dennis, Munholland & Scott,1991).

Morphometric methods remain useful approach to differenti-ate species, lineages or populations within species. This methodprovides a complement to more direct methods of genetic andenvironmental stock identification (Cadrin, 2000). Some geo-graphic variations in morphology, growth or reproductive pat-terns may not be consistent with genetic variation. In somesituations such variation is related to phenotypic plasticity or toadaptive phenomena related to selection pressures under differ-ent environmental conditions (Thompson, 1991; Pigliucci, 2005;Garland & Kelly, 2006). Plasticity is well documented in nature(Fordyce, 2006) and has been reported in Unionidae (Zieritzet al., 2010; Inoue et al., 2013).

Traditional morphometrics is still a widely used technique,able to discriminate populations, but geometric morphometricsprovides a more powerful statistical approach for analysing mor-phological variation (Zelditch et al., 2004). During the lastcentury, intraspecific variation in shell morphology has widelybeen investigated in the family Unionidae. However, few studieshave combined morphological variability, fitness (survival andreproduction) and life-history data (age, growth, etc.) with bio-geographic data (Zieritz & Aldridge, 2009, 2011).

The current work focuses on the morphological characteriza-tion of the endemic freshwater musselU. ravoisieri in Tunisia. Forthis purpose, eight samples, covering most of their geographicaldistribution range in northern Tunisian rivers, were examinedthrough external and internal shell morphometric data andsome indices related to growth and condition. The specific goalsof this study were: (1) to examine the patterns of morphological

variation in U. ravoisieri; (2) to determine their geographical dis-tribution and to consider the historical processes responsible and(3) to evaluate the biogeographical correlates of morphologicalplasticity, growth and condition. The findings should provideuseful information for the implementation of future conservationstrategies for this species.

MATERIAL AND METHODS

Study sites and sample collection

A total of 193 Unio ravoisieri specimens were collected from eightrivers in northern Tunisia between April and July 2012 (Fig. 1,Table 1). The rivers belong to the three major watersheds repre-senting the main drainage systems in northern Tunisia. Most ofthe rivers in which we found freshwater mussels in are not inter-connected; only three of them have a geographical continuitythroughout the year: namely Mejerda River, Maleh River andRaghay River (Fig. 1). All these rivers lie above the Tunisianmountains and coincide with two different bioclimatic zones:humid stage for Kebir, Barbra, Maaden, Douimiss and Sejnanerivers and subhumid stage for the rest of the rivers (Gounot &Le Houerou, 1988).Sampling was carried out by visual inspection and by hand.

Identification as U. ravoisieri was based on the external shellmorphology and colour, by comparison with data reported inthe literature, as recently described by Khalloufi et al. (2011).The geographic coordinates of each sample were measured usinga Garmin 60 GPS. Samples were transported alive to the labora-tory and placed in numbered aquaria containing water fromtheir original river until dissection and measurements. Samplecollection details of each river are presented in Table 1.

Figure 1. Map of northern Tunisian rivers and collection sites for Unio ravoisieri. Abbreviations: BR, Barbra River; DM, Douimiss River; KB, KebirRiver; MA, Maaden River; MJ, Mejerda River; ML, Maleh River; RG, Raghay River; SE, Sejnane River. Samples coded as in Table 1.

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Sex and age determination

The sex of each individual was determined by examination ofgonadal fluid under a light microscope. Mussels showing oogonia,oocytes or larvae (glochidia) of any developmental stage wereassigned as females. The occurrence of spermatozoa and/orsperm morulae indicated male sex (Heard, 1975).

Shell growth bands in bivalves result from seasonal oscillationsin growth and changes in food availability, but also depend onthe physiological state of individuals (maturation, spawningevent, etc.) and environmental factors (habitat change, tem-perature, etc.). Identification and interpretation of annualgrowth bands in U. ravoisieri followed Aldridge (1999). Wintergrowth cessations were indicated by thick rings displayed on thesurface of the shell and were distinguished from other disturb-ance rings by their continuation around the entire valve. Theage of each specimen was estimated by counting the number ofannual winter rings, and hence the maximum age attained byeach sample was determined. These counts were made by asingle operator and rechecked by another.

Morphometric measurements

Shell morphometrics were obtained by measuring the rightvalves of 193 individuals from the 8 sample sites for 11 shelltraits (Fig. 2). Total shell width (TSW) alone was measuredusing both valves. Measurements were taken to the nearest0.01 mm with a digital calliper (RUPAC m). The measurementsfall into two groups: four external traits of the shell including thelengths of the cardinal and lateral teeth (DTC and DTL respect-ively), and six internal traits related to the growth of the adduct-or muscles and visceral mass (Fig. 2).

Prior to analysis, morphological measurements were correctedfor size-dependent variation using an allometric approach asdescribed by Reist (1985). We used the total shell length (TSL)to standardize the remaining 10 measurements according to thefollowing formula of Reist (1985): M¼ logMo�b�ðlogTSLo�logTSLsÞ, whereM denotes the predicted measurement for a givenindividual, Mo is the observed measurement for a given individual,TSLo is the observed TSL value for a given individual, TSLs is theoverall mean value of TSL across all individuals and b is the slope ofthe regression of logMo on log TSLo for each sample.

Measurement of condition and growth indices

During dissection, the shell was opened, the soft body removedand the total weight of wet tissue (TWT) to the nearest 0.001 gwas determined immediately. The condition index (CI) wascalculated by the following equation adopted from Kanget al. (2007): CI ¼ ðTWT=Shell volumeÞ � 100. Shell volume(V) was estimated by applying the formula for an ellipsoid:V ¼ 4=3� p� TSL=2� TSH=2� TSW=2, where TSH and

TSW are the total shell height and the total shell width, respect-ively. This index estimates the proportion of the available shellcavity capacity utilized by the bivalve’s soft tissues, and thusreflects tissue accrual or loss between samples. Besides, it is usedas an ecophysiological variable to characterize apparent healthof bivalves, and to summarize the physiological activity of theanimals (growth, reproduction, secretion, etc.) under given en-vironmental conditions (Lucas & Beninger, 1985).

The growth index (GI) was determined as the ratio betweenTWT in grams and the age. This index reflects longevity andgrowth rates of freshwater mussels and therefore allows insightsinto lifetime reproductive success and population structure. Inaddition, we constructed the graphics of TSL against age inorder to compare the approximate growth curves between sites.

Statistical analysis

Sexual size dimorphism in shell morphometric traits was exam-ined within each sample and for each measurement. The differencebetween both sexes was evaluated by the Aspin-Welch’s t-test forunequal variances (Snedecor & Cochran, 1989) using MicrosoftOffice Excel 2007.

Table 1. Sampling locations in northern Tunisia, geographical coordinates, altitude above sea level, date of collection, number of Unio ravoisieri persample (n, number of females/males in brackets) and average total shell length (TSL, in mm) per sample.

Watersheds River names Code Coordinates Altitude (m) Collection date n (C/F) TSL (mean+SD)

Ichkeul watershed Douimiss River DM N 37812′03.26′′ E 09837′26.50′′ 7 25 April 2012 10 (4/6) 52.96+5.91

Sejnane River SE N 37811′33.88′′ E 09834′46.50′′ 4 6 May 2012 15 (5/10) 52.75+3.05

North-west watershed Barbra River BR N 36846′29.70′′ E 08833′45.30′′ 117 21 May 2012 23 (15/8) 75.62+10.54

Kebir River KB N 36855′09.11′′ E 08845′14.66′′ 8 25 April 2012 34 (22/12) 48.04+4.39

Maaden River MA N 36854′21.63′′ E 09806′23.60′′ 86 7 July 2012 19 (9/10) 56.51+11.18

Mejerda watershed Mejerda River MJ N 36828′09.70′′ E 08832′32.00′′ 170 21 May 2012 27 (13/14) 77.25+10.93

Maleh River ML N 36830′01.70′′ E 08834′32.20′′ 173 21 May 2012 33 (18/15) 55.78+6.72

Raghay River RG N 36829′04.40′′ E 08832′14.50′′ 164 21 May 2012 32 (13/19) 50.45+5.83

SD, standard deviation.

Figure 2. Eleven shell traits measured for each individual of Unioravoisieri sampled from northern Tunisia. Abbreviations: TSL, total shelllength; TSH, total shell height; TSW, total shell width of two valves;HAR, height of anterior adductor muscle scar; WAR, width of anterioradductor muscle scar; HPR, height of posterior adductor muscle scar;WPR, width of posterior adductor muscle scar; DAP, distance betweenventral margin of anterior adductor muscle scar and ventral margin ofposterior adductor muscle scar; DPM, distance between pallial line andventral shell margin; DTC, distance between tip of beak and anteriorend of cardinal teeth; DTL, distance between tip of beak and posteriorend of lateral tooth. Internal shell traits related to adductor muscles andvisceral mass are italicized, while remaining measurements representexternal shell traits and lengths of cardinal and lateral teeth.

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After having verified normal distribution and homogeneity ofvariances of standardized shell sizes with a Shapiro-Wilk’s test(Shapiro & Wilk, 1965) and a Levene’s test (Levene, 1960) re-spectively, the differences between samples were examined by aone-way analysis of variance (ANOVA) for each trait independ-ently. Tukey’s honestly significant difference (HSD) tests wereused for post hoc comparisons. When there is a significant devi-ation from normality and/or the data proved to be nonhomoge-neous, the nonparametric Kruskal-Wallis test (Kruskal &Wallis, 1952) followed by Mann-Whitney U-test pairwise com-parisons was conducted. Unless otherwise stated, statistical ana-lyses were calculated with XLSTAT v.2013.3 for Windows(Addinsoft). The alpha level was set at 0.05 for all statistical testsand a Bonferroni correction was applied when necessary.

Discriminant function analysis (DFA) was performed to identifypopulation groups in the pooled sample and to clarify the varia-tions for shell traits between samples. DFA was carried out for ex-ternal, internal and all shell measurements separately. To facilitateinterpretation, the graphical presentations of the DFA were builtwith 95% confidence interval ellipses around centroids of eachsampling locations (von Zuben et al., 1998). Discriminant analysiswas also used to estimate the percentage of individuals that wereclassified correctly, and Wilks’ lambda (l) was calculated as anoverall test statistic for each and all shell measurements.

Isolation-by-distance (IBD) between samples was tested usingboth simple and partial Mantel tests as implemented in the soft-ware PASSaGE v. 2 (Rosenberg & Anderson, 2011). Significanceof correlations between distance matrices was assessed using9999 permutations to estimate P-values. Simple Mantel test(Mantel, 1967) was used to compare two distance matrices be-tween pairwise samples based on geographic and squaredMahalanobis distances (Mahalanobis, 1936) using all shell mea-surements. Geographic distances were calculated in kilometresas straight-line distances between localities using Google Earthsoftware. These distances range from 1.8 to 126 km betweensites. The Mahalanobis distance measures the distance betweentwo samples on the basis of multiple variables. This is similar tothe Euclidean distance between coordinates but accounts for co-variance among variables and variation within variables. Apartial Mantel test (Fortin & Gurevitch, 2001) was performed totest for the correlation between three distance matrices in order toestimate their influence. It investigates the correlation betweentwo matrices while controlling the effect of a third one, and thustries to remove spurious correlations. We used a matrix of geo-graphic distances and two matrices of squared Mahalanobis dis-tances corresponding to external and internal shell measurements.We computed (1) the correlation between external shell trait andgeographic distance matrices while controlling for the effect of in-ternal shell traits, (2) the correlation between internal shell traitsand geographic distance matrices while controlling for the effect ofexternal shell traits and (3) correlation between external and in-ternal shell trait distance matrices while controlling for the effect ofgeographic distances. Concordance represents a significant posi-tive relationship, discordance a significant negative relationshipand independence a nonsignificant relationship between matrices.

The nonparametric Spearman’s rank correlation test (rs) wasapplied in order to evaluate the relation of each morphologicalcharacter and index measurements (CI and GI) with ecogeo-graphic parameters of the sampling locations (latitude, longi-tude and altitude).

RESULTS

Sexual size dimorphism

The Aspin-Welch’s t-test showed no evidence of sexual size di-morphism in external shell traits, except for the cardinal teethlength (DTC) in the Kebir sample (P ¼ 0.048). However,

internal shell traits demonstrated a significant differencebetween males and females for the anterior adductor musclescar height in the Douimiss (P ¼ 0.003) and Kebir (P ¼ 0.008)samples, for the posterior adductor muscle scar width (WPR) inthe Raghay sample (P ¼ 0.033) and for the distance betweenpallial line and ventral shell margin (DPM) in the Sejnanesample (P ¼ 0.042). It seems that the sexual size dimorphism inUnio ravoisieri is extremely slight, and significant t values couldbe explained by the sampling effect. In the light of these results,the specimens of both sexes could be pooled for further analyses.

Differences between samples in shell size means

A normal distribution was verified for all transformed morpho-metric shell characters, with the exception of DPM (P ¼ 0.019).However, Levene’s test indicated that variances were not homo-geneous for 3 out of 10 measurements (DPM, P ¼ 0.027; DTC,P ¼ 0.005; WPR, P ¼ 0.040). Both ANOVA and Kruskal-Wallis test revealed the existence of highly significant differencesamong samples for variation in all morphometric traits (all P ,0.0001). The post hoc Tukey’s HSD and Mann-Whitney U-testcomparisons (see Supplementary material, Table S1) indicatedthat Sejnane and Maaden samples differs statistically only bythe distance between ventral margin of anterior and posterioradductor muscle scars (DAP), and Douimiss sample differs stat-istically from Maaden and Sejnane samples only by TSH. Itshowed also that Barbra and Mejerda samples are characterizedby a large DPM, whereas the Kebir sample is characterized by areduced TSW and showed a significant difference from all theother samples, except for the Douimiss sample. Regarding teethlength, the Raghay sample has a reduced DTL and showed asignificant difference from all the other samples, except for theBarbra sample. However, the Sejnane sample has a reducedDTC and differs statistically from the other samples, with theexception of the Douimiss andMaaden samples.

Discriminant function analyses

No multicolinearity was registered between variables (Pearson’scorrelation coefficients were all ,0.4, tolerance .0.68 andVIF , 1.47) and therefore all transformed morphometric shellcharacters could be used in the DFA. The Wilk’s lambda testsindicated a strong difference between samples when the morp-hometric characters were compared by means of discriminantanalysis (Table 2). Overall Wilks’ lambda performed for all size-correctedmorphometric characters (l ¼ 0.04505)was also highlysignificant (approximate F70, 1033 ¼ 10.36; P , 0.0001).Details of DFA results for external, internal and all shell meas-

urement traits in U. ravoisieri are provided in Table 3. Plots of

Table 2. Summary of discriminant function analysis for morphometrictraits measured inUnio ravoisieri samples from northern Tunisia.

Wilks’ Lambda F-remove P-value Tolerance

TSH 0.059 8.020 0.000000 0.723

TSW 0.062 9.321 0.000000 0.779

DTC 0.055 5.729 0.000006 0.884

DTL 0.054 5.198 0.000021 0.927

HAR 0.057 6.934 0.000000 0.753

WAR 0.067 12.123 0.000000 0.842

HPR 0.061 8.957 0.000000 0.682

WPR 0.055 5.344 0.000015 0.759

DPM 0.077 17.930 0.000000 0.898

DAP 0.060 8.137 0.000000 0.786

Abbreviations of shell traits are shown in Figure 2.

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samples along the first two canonical variables for the threemeasure types are shown in Figure 3. The first two discriminantfunctions gave a cumulative percentage of the variance of morethan 85% for external shell traits, 75% for internal shell traitsand 60% for all measurements. External shell traits illustratedseveral overlapping grouping with a unique separation alongaxis 1 of the Kebir sample from the rest. Internal shell traitsshowed less overlapping with a positive trend of separation alongaxis 1 for the Raghay, Barbra and Mejerda samples. However,the use of both measurements together gives the best and themost informative separation with the highest Eigenvalues(Table 3). It revealed that samples can be grouped in three clus-ters. The first group contains the sample of Kebir River, thesecond includes samples from Barbra, Mejerda and RaghayRivers and the third group puts together the remaining samples.

When considering all shell measurements, the morphometricdiscriminant analysis correctly classified, on average, 74.09% ofthe individuals into their original population (Table 4). Thehighest classification success rate was observed for samples fromSejnane River with 93.33%. The percentage of individuals cor-rectly classified was lowest for the Maaden sample with 52.63%.

Spatial patterns of morphological variation

The regression analysis between pairwise Mahalanobis distanceperformed for all shell traits and geographic distance matricesyielded a highly significant positive correlation (r ¼ 0.560; P ¼0.007), suggesting an IBD pattern between samples. The rela-tionship between external shell traits (Mahalanobis distance)and geographical proximity was also significant (r ¼ 0.553; P ¼0.003) when controlled for internal shell traits. Moreover, thepartial Mantel test showed that, even when external shell traitswas taken into account, geographical proximity still had animpact on internal shell traits (r ¼ 0.464; P ¼ 0.022). However,internal shell traits did not carry significant additional informa-tion about external shell traits (r ¼ 20.430; P ¼ 0.051) whengeographical proximity was set as a constant.

Growth, age and condition

The collected shells ranged in age from 5 to 18 years, with amedian age of 9 (quartiles ¼ 7 and 11). Mussel maximum ages

were variable among samples, suggesting a different longevitybetween sites (Fig. 4). At Mejerda and Barbra rivers, withthe highest growth in shell length, longevity exceed 16 years,while at Kebir River, with the lowest growth, the maxi-mum age found was 10 years. At Maleh and Maaden rivers,samples showed an intermediate maximum age (13 years) andgrowth. However, in the rivers of Douimiss, Sejnane andRaghay, the maximum age detected was 9 years, although thegrowth of their shell length was greater than in the Kebirsample.

The differences in means of growth and condition indicesbetween each sample and all samples combined are presentedin Figure 5. The mean values of the CI were relatively closebetween sites. However, variations between samples in the GIwere significant. The Kruskal-Wallis analyses show highly sig-nificant differences, especially in the Mejerda sample for CI andin Mejerda, Barbra and Kebir samples for GI. The post hocMann-Whitney U-test classified the samples into three homoge-neous groups based on condition and growth indices, but classi-fications were dissimilar. For the CI, Douimiss showed nodifference from all samples. However, based on the GI, theBarbra and Mejerda samples were the most differentiated fromthe others with the highest indices.

Geographic scale of condition, growth and shell size variation

Spearman’s rank correlation results between geographic vari-ables of the sampling locations, CI, GI and shell measurementsare presented in Table 5. In general, CI was unrelated to alti-tude, latitude or longitude. Correlations between GI as well asthe majority of the external shell traits and elevation were posi-tive and significant, while correlations with latitude and longi-tude were negative and significant. The only exception wasDTL which demonstrated a significant inverse trend with thegeographic parameters compared with the other external shelltraits. The relationships of internal shell traits with geographicparameters were similar to those of the external shell traits, butwere not all significant. Consequently, the geographic scale ofvariation in growth and shell size in U. ravoisieri is characterizedby increasing values with elevation, from north to south andfrom east to west.

Table 3. Summary of canonical discriminant functions generated on the basis of external, internal and all shell measurements in Unio ravoisieri samplesfrom northern Tunisia.

Function

1 2 3 4 5 6 7

External shell traits

Eigenvalue 0.705 0.423 0.138 0.058

Variance (%) 53.246 31.955 10.388 4.412

Cumulative (%) 53.246 85.201 95.588 100

Canonical correlation 0.643 0.545 0.348 0.235

Internal shell traits

Eigenvalue 1.455 0.730 0.481 0.187 0.022 0.014

Variance (%) 50.370 25.255 16.660 6.463 0.765 0.487

Cumulative (%) 50.370 75.625 92.285 98.747 99.513 100

Canonical correlation 0.770 0.650 0.570 0.397 0.147 0.118

All shell traits

Eigenvalue 1.684 0.994 0.758 0.451 0.259 0.155 0.118

Variance (%) 38.107 22.487 17.164 10.197 5.859 3.518 2.669

Cumulative (%) 38.107 60.594 77.758 87.954 93.813 97.331 100

Canonical correlation 0.792 0.706 0.657 0.557 0.453 0.367 0.325

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DISCUSSION

We investigated shell morphological variation, growth and con-dition among eight samples of Unio ravoisieri covering most of itsgeographical distribution range in northern Tunisia. Our resultsrevealed a strong morphological structure, a considerable growthvariation and a relative low differentiation in CI between thepopulations. These trends were strongly influenced by biogeo-graphic parameters, except for CI.The main result of the study was the high level of variation in

shell characters between locations. This variation was observedamong the river basins even though the geographic scale was rela-tively small. Similar results were reported in a recent geometric

Figure 3. Discriminant function analysis based on external (A),internal (B) and all shell traits (C) standardized according to Reist(1985) for all samples of Unio ravoisieri from northern Tunisia. Ellipsesrepresent 95% confidence interval around the centroid for eachsample. Abbreviations of samples and shell traits as in Table 1 andFigure 2, respectively.

Table 4. Results of the morphometric discriminant analysisclassification showing the numbers and percentage of specimens of Unioravoisieri correctly classified into their original population using all shellmeasurements.

Sample code Number % correct

BR 14/23 60.87

DM 8/10 80.00

KB 27/34 79.41

MA 10/19 52.63

MJ 22/27 81.42

ML 26/33 78.79

RG 22/32 68.75

SE 14/15 93.33

Total 143/193 74.09

Samples coded as in Table 1.

Figure 4. TSL values vs estimated age in years showing estimates ofgrowth curves and maximum ages (represented by dashed lines)determined in Unio ravoisieri samples from northern Tunisia. Samplescoded as in Table 1.

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morphometric analysis in U. delphinus collected from three differ-ent habitats (estuary, river and intermittent streams) of thePortuguese Guadiana (Morais et al., 2014).

Morphometric variation is a product of the interactionbetween genotype and environment. Several hypotheses of evo-lutionary forces, or their interaction, can explain this phenotypicdivergence among U. ravoisieri populations. Those most usuallyreported in the literature are geographic isolation, genetic drift,divergent natural or sexual selection and phenotypic plasticity(Chris Funk &Murphy, 2010).

The hypothesis of isolation by geographical distance predictsthat increasing distance between populations, reflecting decreas-ing gene flow (or ‘migration’), should result in increasing pheno-typic divergence (Slatkin, 1987; Hendry, Day & Taylor, 2001).In our results, the correlation of pairwise morphological andpairwise geographical distance across all shell characters showed

a clear IBD pattern. Structural connectivity among habitatsand dispersal rates are two key factors affecting the migrationlevel. Thus, the existence of physical and/or biological barrierssuch as decrease in water currents, predation pressure, long sed-entary adult phase, low reproductive rate, short larval dispersalstage and the need for an intermediate host to complete the lifecycle limit the connectivity between populations. The strongmorphological differentiation between locations associated withIBD, in addition to the sedentary habit of the species, suggestthat long-distance dispersal is likely to be limited inU. ravoisieri.

Genetic drift is a random phenomenon producing fluctuationin allele frequencies, and it affects small populations more strongly,potentially leading to loss of genetic diversity. Although theeffect of genetic drift is well established for neutral molecularmarkers, its effect on phenotypic divergence is less clear. Wecannot conclude anything about the effect of genetic drift onmorphological variation in U. ravoisieri. Nevertheless, the in-creasing rarity of the species in Tunisia (Khalloufi et al., 2011),with our evidence of IBD, suggest that genetic drift could playa role.

Another plausible explanation of the observed morphologicalvariation in U. ravoisieri populations is selection, under the effectof anthropogenic, environmental or demographic pressures. Againwe have no direct evidence of this. Molecular genetic studies arerequired to test hypotheses of drift and selection.

Morphological variation in U. ravoisieri was found in both ex-ternal and internal shell traits, but their relative magnitude wasnot the same. According to the DFA results, internal shell traitsare more informative in distinguishing populations than externalshell traits. Partial Mantel correlation analyses showed that geo-graphical variation of external shell traits was independent ofvariation in internal shell traits. This is probably related to thefact that these sets of traits are likely subject to different environ-mental factors, selective agents and genetic control.

Our analyses revealed significant correlations between geo-graphical parameters (altitude, latitude and longitude), andboth GI and external shell traits. On another hand, significantcorrelations were not observed for all internal shell traits or forthe CI. The geographic gradients in GI and external shell traitssuggest possible phenotypic plasticity in U. ravoisieri. This is inagreement with general ecogeographical rules that describeempirical trends between morphological and environmental

Figure 5. Box-plots of condition index (A) and growth index (B) for theeight samples of Unio ravoisieri from northern Tunisia. The box portion ofthe plot represents the interquartile range from first to third quartile andthe line drawn through the box represents the median. The verticalwhiskers represent the highest and lowest values in the dataset, excludingoutliers, which are marked as open circles. Dotted lines represent themean value for all samples combined. Asterisks indicate significantdifferences between mean values of each sample and of all sites combined(Kruskal-Wallis test: *P, 0.05; **P, 0.01 and ***P, 0.001). Solid linesabove boxes connect sites that were not significantly different (P . 0.05).Samples coded as in Table 1.

Table 5. Results of the nonparametric Spearman’s rank correlation (rS)test between site geographic variables (latitude, longitude and altitude)and condition index (CI), growth index (GI) and shell measurementcharacters (see Fig. 2 for abbreviations) in Unio ravoisieri samples fromnorthern Tunisia.

Spearman’s rank correlation (rS)

Latitude Longitude Altitude

CI 0.039 20.081 0.096

GI 20.324*** 20.324*** 0.184*

TSH 20.260*** 20.231** 0.236***

TSW 20.275*** 20.229** 0.244***

DTC 20.377*** 20.431*** 0.298***

DTL 0.262*** 0.411*** 20.155*

HAR 20.053 20.090 20.156*

WAR 20.566*** 20.549*** 0.389***

HPR 20.230** 20.238*** 0.139

WPR 0.028 0.027 0.006

DPM 20.315*** 20.397*** 0.076

DAP 20.065 0.017 0.197**

Significance levels: *P , 0.05, **P , 0.01 and ***P , 0.001.

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variation (Mayr, 1956; Gaston, Chown & Evans, 2008). In fresh-water molluscs, the most reported ecogeographic rule is Ortmann’slaw of stream position (Ortmann, 1920), which attributes variationin shell form to the response of individuals to an environmental gra-dient from upstream to downstream in single river systems. Suchclinal changes in shell morphology have been observed in severalunionid species (Mackie & Topping, 1988; Graf, 1998; Hornbach,Kurth &Hove, 2010).

Growth as well as longevity differences in U. ravoisieri sampleswere mostly coincident with geographic groups generated fromthe morphometric traits. However, the sample groups based onCI showed different results that were not consistent with thespatial distribution of morphometric groups. Differences ingrowth and shell size between samples are commonly attributedto the influence of temperature (correlated with latitude and/orelevation) and food availability. However, differences in condi-tion are under the combined effect of many intrinsic and extrinsicfactors, including population density, size, age, gonad develop-ment, nature of the substratum, temperature, food availability,environmental stress such as pollution, parasitism, pathogens,etc. (reviewed in Filgueira et al., 2013). The combined effect ofthese factors may be important, even at small geographic scales,leading to differences between populations that are located inclose proximity.

Regarding sexual size dimorphism in the shell of U. ravoisieri,statistical analysis showed that the differences between sexeswere globally not significant. Kotrla & James (1987) reportedno differences in shell size between males and females in theunionids Villosa villosa and Elliptio icterina, but found significantdifferences in shape. Zieritz & Aldridge (2011) showed thatsexual dimorphism in shell width of Anodonta anatina occurred insome populations, but not others. This observation has beenexplained by an indirect effect of environmental conditions,which may affect shell morphology.

In conclusion, our study demonstrates phenotypic divergencein shell morphology among U. ravoisieri populations and high-lights the importance of internal shell characters in describingthis variation. The causes of this variation, however, requirefurther study. The significant geographical heterogeneities in shellmorphology as well as in growth and longevity have implicationsfor the conservation management of the species. Accordingly, anyfuture conservation plan should manage each watershed separately.

SUPPLEMENTARY MATERIAL

Supplementary material is available at Journal of MolluscanStudies online.

ACKNOWLEDGEMENTS

We wish to express our gratitude to the teams from ResearchUnit Ecosystems & Aquatic Resources (UR13AGRO1, INAT/University of Carthage) for their valuable support. We aregrateful to A. Bogan, A. Zieritz and D. Graf for helpful com-ments on the manuscript. We also sincerely thank the twoanonymous referees and editors for useful comments and sugges-tions on the manuscript. Special thanks to all persons whohelped in collecting specimens. This research was financiallysupported by the Institution of Agriculture Research andHigher Education, and the Ministry of Higher Education, Sci-entific Research and Information and Communication Tech-nologies of the Tunisian republic.

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