contrasting patterns of morphological variation with...

Journal of Fish Biology (2013) 82, 1641–1658

doi:10.1111/jfb.12103, available online at wileyonlinelibrary.com

Contrasting patterns of morphological variation withdietary preferences in Micropogonias furnieri : insights

from stable-isotope and digestive-trait analyses

A. D’Anatro*, D. E. Naya, E. P. Lessa and O. Defeo

Departamento de Ecología y Evolucion, Facultad de Ciencias, Universidad de la Republica,Montevideo 11400, Uruguay

(Received 14 November 2012, Accepted 14 February 2013)

The dietary preferences of populations of whitemouth croaker Micropogonias furnieri , which com-monly inhabit estuarine and oceanic environments of the south-western Atlantic Ocean, wereinvestigated using stable-isotope analysis and digestive traits, and compared with previous geneticand morphometric surveys of this species. Isotopic and C:N-derived data suggested that indi-viduals from coastal lagoons are the most differentiated from the remaining localities surveyed.In contrast, the analysis of the digestive traits did not show the same differentiation pattern.The overall correlation between isotopic, molecular and morphological variations suggests thatgenetic and phenotypic differences among populations are accompanied by differential resourceuse, supporting the idea that selective forces could be playing an important role in populationdifferentiation. © 2013 The Authors

Journal of Fish Biology © 2013 The Fisheries Society of the British Isles

Key words: coastal lagoons; digestive traits; ecological differentiation; geographic variation; Río dela Plata; whitemouth croaker.

INTRODUCTION

The importance of ecologically mediated divergence in population differentiation hasbeen highlighted by several authors (Simpson, 1944; Lack, 1947; Dobzhansky, 1951;Schluter, 2000). Recent empirical evidence suggests that ecological factors can cer-tainly drive speciation processes (Barluenga et al ., 2006; Savolainen et al ., 2006),supporting previous theoretical models (Dieckmann & Doebeli, 1999). Marine envi-ronments provide excellent case studies to test the role of ecological factors in pro-moting population differentiation, as they typically allow broad dispersal in mobiletaxa and offer low travel costs compared to terrestrial habitats (Wolf et al ., 2008).Among them, coastal marine ecosystems, including estuaries, are attractive systems tostudy population differentiation as there is (1) an occurrence of species that use bothestuarine and oceanic environments, (2) ecological distinctiveness of both ecosystemsand (3) absence of geographic barriers between these environments. One of the mainrequirements of ecologically mediated population divergences is the association of

*Author to whom correspondence should be addressed. Tel.: +598 25258618 ext. 7/143; email: [email protected]

1641© 2013 The AuthorsJournal of Fish Biology © 2013 The Fisheries Society of the British Isles

1642 A . D ’ A NAT RO E T A L .

such population divergence with differential resource use, e.g . adaptation to differ-ent local conditions (Schluter & Conte, 2009). In this sense, the analysis of trophicdifferences among genetically or phenotypically differentiated populations providesinsights into the possible associations between these various classes of divergence.

Individuals from populations differing in trophic habits are also expected to differin their digestive traits. The gut represents the functional link between food intakeand metabolizable energy (Karasov, 1990; Secor, 2001), and, thus, it is expected thatanimals adjust their digestive attributes to changes in food availability and quality,in order to maximize overall energy returns and minimize maintenance costs (Sibly,1981; Naya et al ., 2008). Studies comparing digestive morphology of populationswith different trophic habits indicate a positive correlation between the size of thedigestive organs and the amount of refractory material in the diet (Hansson, 1985;Hansson & Jaarola, 1989; Corp et al ., 1997; Sassi et al ., 2007; Naya et al ., 2009;Bozinovic et al ., 2010). In addition, stable-isotope ratios of 13C and 15N have beenextensively used to disentangle the primary carbon source and to assess trophic posi-tion in complex trophic webs (Post, 2002). These same isotopes have also proven tobe very useful for differentiating among ecotypes of the same species (Harrod et al .,2005; Lowther & Goldsworthy, 2011) and for establishing short-term migratory pat-terns (Ciancio et al ., 2008). Furthermore, the use of 13C and 15N analysis to identifydifferential use of salinity habitats relies on the existence of predictable differences instable-isotope values along salinity gradients (Harrod et al ., 2005). For example, fishfeeding in different locations along a salinity gradient should have contrasting 13Cvalues as a result of relative depletion of 13C in fresh water (Fry & Sherr, 1984).The δ15N value might also provide information about food source, as unpollutedfresh waters are depleted relative to marine waters, but with less resolution than thatoffered by δ13C (Harrod et al ., 2005).

The whitemouth croaker Micropogonias furnieri (Desmarest 1823) is widely dis-tributed along the western Atlantic Ocean coasts from Mexico (20◦ N) to the Gulf ofSan Matías, Argentina (41◦ S) (Issac, 1988; Vazzoler, 1991). This species, similar tomost species of the family Sciaenidae, uses estuaries and river mouths as reproductiveand nursery areas. In Uruguay, the main spawning season of M . furnieri is duringspring and summer (Acuna et al ., 1992), mainly using the Río de la Plata and Lagunade Rocha (Fig. 1; Vizziano et al ., 2002). Several studies suggest that the completelife cycle of this fish may take place in these localities (Vizziano et al ., 2002; Jau-reguizar et al ., 2008). In Laguna de Rocha, individuals reach maturity at total lengths(LT) 11–12 cm less than specimens from the Atlantic Ocean, which attain maturityat c. 30 cm (Vizziano et al ., 2002). This phenomenon has also been reported for thisspecies in Lagoa dos Patos, southern Brazil (Castello, 1986; Haimovici & Gatto,1996). Genetic (D’Anatro et al ., 2011) and geometric morphometric (D’Anatro &Lessa, 2011) analyses showed a moderate population differentiation of M . furnierifrom Laguna de Rocha relative to other localities surveyed along the Uruguayanand Argentinean coasts. In addition, mitochondrial DNA (Pereira et al ., 2009),microsatellite (D’Anatro et al ., 2011) and geometric morphometric (D’Anatro &Lessa, 2011) studies also suggest that M . furnieri from the Río de la Plata mayrepresent a partially differentiated population. Previous morphological and meristicanalyses have also proposed the existence of a differentiated population in the Río dela Plata compared with individuals sampled in the Atlantic Ocean waters of Brazil(Vazzoler, 1971; Haimovici & Gatto, 1996), Uruguay (Galli, 2001) and Argentina

© 2013 The AuthorsJournal of Fish Biology © 2013 The Fisheries Society of the British Isles, Journal of Fish Biology 2013, 82, 1641–1658

M O R P H O L O G I C A L VA R I AT I O N I N M I C RO P O G O N I A S F U R N I E R I 1643

Uruguay

Argentina

Laguna de Castillos

Laguna de Rocha

La Paloma

Piriápolis

Atlantic Ocean

Río de la Plata

Montevideo

Villa Gesell25 km

N

Fig. 1. Sampling localities of Micropogonias furnieri analysed. Approximate geographic co-ordinates: Lagunade Castillos 34◦ 18′ 00′′ S; 53◦ 55′ 00′′ W; Laguna de Rocha 34◦ 37′ 00′′ S; 54◦ 15′ 00′′ W; La Paloma34◦ 39′ 30′′ S; 54◦ 08′ 30′′ W; Piriapolis 34◦ 52′0 10′′ S; 55◦16′ 40′′ W; Montevideo 34◦ 38′ 00′′ S; 56◦

16′ 00′′ W; Villa Gesell 37◦ 14′ 00′′ S, 56◦ 44′ 00′′ W. , the limit of the Río de la Plata. Modifiedfrom D’Anatro & Lessa (2011).

(Cotrina, 1986; Figueroa & Díaz de Astarloa, 1991). In contrast, allozyme variationstudies failed to confirm this pattern (Maggioni et al ., 1994). Thus, several indepen-dent lines of evidence showed a complex population structure along south-westernAtlantic Ocean coasts. Because of the apparent absence of geographical barriersbetween localities, it was suspected that adaptation to local environmental condi-tions could be playing an important role in population differentiation (D’Anatro &Lessa, 2011). In this sense, it is hypothesized that, if divergent selection is promoting


1644 A . D ’ A NAT RO E T A L .

population differentiation via adaptation to local trophic regimes, differences in thedigestive traits and δ13C and δ15N values among individuals from different environ-ments should occur in line with the genetic and phenotypic differences mentionedabove. Alternatively, individuals may not differ significantly in their trophic profilesin relation to environmental heterogeneity and simply exhibit a neutral isolation bydistance pattern. The primary objectives of this work were (1) to characterize the13C and 15N values of several putative populations of M . furnieri , (2) to make infer-ences about the possible short-term individual movement patterns between differentlocalities using the same stable isotopes as markers and (3) to analyse the geographicvariation in digestive traits in the same populations. In particular, whether variationin isotopic and digestive tract attributes is associated with documented phenotypicand genetic differences between M . furnieri in the Uruguayan Atlantic front, theLaguna de Rocha and the Río de la Plata was investigated.

MATERIALS AND METHODS

S T U DY A R E A A N D S A M P L E C O L L E C T I O NMicropogonias furnieri from Uruguay were obtained during November of 2009 (Laguna

de Rocha, n = 10; La Paloma, n = 7; Piriapolis, n =7; Montevideo, n = 10; Laguna de Castil-los, n = 8; Fig. 1) and March of 2010 (Piriapolis, n =5; Montevideo, n = 8) from artisanalfisheries. Individuals from Villa Gesell (Argentina, n = 20; Fig. 1) were sampled duringNovember 2009 by the R.V. Aldebaran (DINARA-MGAP). Only 10 individuals from VillaGesell were employed for the stable-isotope analyses. Laguna de Rocha is a coastal lagoonconnected to the Atlantic Ocean via a sandy bar; Laguna de Castillos is also a coastal lagoon,but it is connected to the Atlantic Ocean via the Valizas creek. Villa Gesell and La Palomaconstitute oceanic environments; Piriapolis is located in a transitional zone between the Ríode la Plata and the Atlantic Ocean. Geographical co-ordinates of the localities sampled aregiven in Fig. 1. All the 75 specimens of M . furnieri analysed were classified as adults bygonad inspection and LT [following Issac (1988) and Vizziano et al . (2002)].

To confirm whether the possible differences in the isotopic values of M . furnieri sampledat different time periods, November 2009 and March 2010, correspond with differences introphic sources, temporal estimates of baseline variation of δ13C and δ15N were obtained bysampling two species of bivalves, depending on their availability (November 2009: Piriapolis,Mytilus edulis platensis , n = 5; Montevideo, Brachidontes rodriguezii , n = 5; March 2010:Piriapolis, M . e. platensis , n = 5; Montevideo, B . rodriguezii , n = 5). These species wereselected because marine bivalves are mainly phytoplankton filter feeders (Rouillon & Navarro,2003) and they are the most abundant taxa in the respective areas from where they werecollected.

S TA B L E - I S OT O P E A NA LY S E S A N D C : NIn the laboratory, white muscle tissues of M . furnieri were excised from the right or

left flank of each fish. Samples were oven-dried at 55◦ C for 48 h and then pulverizedand weighed. Lipids were not removed because the low lipid content of fish muscle tissueprobably does not affect the 13C proportion (Penchaszadeh et al ., 2005; Corbisier et al .,2006; Garcia et al ., 2007) and also because the protocols commonly used to remove lipidsmay adversely affect nitrogen isotope integrity (Grey et al ., 2002). In addition, Post et al .(2007) proposed that it is not necessary to account for lipids in tissues of aquatic animalswith a C:N ratio of <3·5, and the present results support this decision not to extract lipidsfrom muscle tissue samples. For the analyses of stable isotopes of 13C and 15N, samples wereweighed (0·5–0·8 mg) and encapsulated into tin cups prior to combustion in an elementalanalyser (Flash EA 112) coupled to a mass spectrometer (Finnigan MAT DELTAplus XL;



www.thermoscientific.com). Based on the values produced by a laboratory standard (leucine),the precision of estimations (s.d.) were c. 0·1‰ for 13C and 0·3‰ for 15 N. The stable-isotoperatios were expressed as δ values in ‰ as follows: δX = 1000 [(Rsample Rstandard

−1) −1],where X is 13C or 15 N and R is the corresponding ratio, 13C:12C or 15 N:14 N. Standardsused for the analyses were Vienna Peedee belemnite for C and atmospheric N2 for N. TheC:N values were calculated from the percentage of carbon and nitrogen in each sampledetermined from an exact mass of material analysed in the elemental analyser (Harrod et al .,2005). These analyses were carried out at Centro de Aplicaciones de Tecnología Nuclearen Agricultura Sostenible (CATNAS), Facultad de Agronomía, Universidad de la Republica.Muscle tissues obtained from bivalves were analysed following the same procedures.

S TAT I S T I C A L A NA LY S E S O F F I S H I S OT O P I CA N D C : N D E R I V E D DATA

All variables derived from stable-isotope analyses were ln transformed in order to nor-malize data and stabilize variance. To analyse the spatial variability in the isotopic values ofM . furnieri , a discriminant function analysis (DFA) was carried out on the ln-transformeddata matrix derived from isotopic analysis of M . furnieri sampled during 2009 (Harrod et al .,2005). Pearson’s correlation analyses were used to study the relationships between standardlength (LS) of fish sampled during 2009 and their isotopic and C:N values.

In order to analyse the possible existence of an isolation by distance pattern in the isotopicdifferentiation among populations, a Procrustean fit between Mahalanobis distances derivedfrom the DFA and linear geographic distances among the localities sampled were explored,using the ProTest computer programme with 9999 randomizations (Jackson, 1995; Peres-Neto & Jackson, 2001). Linear geographic distances between population pairs were obtainedwith Google Earth software (www.google.com/earth). ProTest was also used to explore thecorrelation between Mahalanobis distances of the DFA and pair-wise estimates of geneticand morphological distances reported by D’Anatro & Lessa (2011). That work analysedM . furnieri sampled in the San Luis locality instead of Piriapolis for the genetic analyses.Because of the geographical proximity of these two localities (c. 27 km apart), it was assumedthat individuals from these two coastal points constitute the same putative population. Inorder to reduce dimensionality prior to ProTest analyses, multidimensional scaling (MDS)analyses were performed on all the distance matrices to be evaluated. As a subsequent step,the Procrustean fit was implemented between the two dimensions obtained in each MDSanalysis. To fix the possible effect of geographic distances on the correlation between genetic,morphological and Mahalanobis distances, partial ProTest analyses were also carried out. Thepartial ProTest was carried out on the residuals obtained from the multiple linear regressions ofeach dimension obtained in the MDS analyses of the morphological, genetic and Mahalanobisdistances, and the first two dimensions of the MDS performed on the geographic distancematrix (Peres-Neto & Jackson, 2001).

In order to evaluate the temporal variation in the isotopic values of M . furnieri , statisticaldifferences in δ13C and δ15N of M . furnieri between Piriapolis and Montevideo in differentmonths (November 2009 to March 2010) were assessed using a factorial ANOVA. The sameanalysis was also used to evaluate differences in baseline isotopic values between Piriapolisand Montevideo in these months, derived from the analysis of mussel tissue samples. In bothcases, post hoc comparisons were evaluated by Tukey HSD tests.

L A B O R AT O RY A NA LY S E S O F D I G E S T I V E T R A I T S

The LS of each fish was measured (±0·1 cm) and then stomach, intestine, liver and visceralfat were removed. Stomach and intestines were dissected and their lengths (LSt and LI) weremeasured (±0·05 cm). Stomach and intestine were washed with running water, dried withpaper towels and then weighed (M St and M I) (±0·01 g); the number of pyloric caeca (N PC)was then determined. Total mass of liver (M L) (±0·01 g) and the eviscerated body mass(M EB) (±0·1 g) were also measured.


1646 A . D ’ A NAT RO E T A L .

S TAT I S T I C A L A NA LY S I S O F DATA O N D I G E S T I V E T R A I T S

L I commonly varies with M EB, and for that reason data must be standardized for sizeeffects before making comparisons among individuals (Kramer & Bryant, 1995). For thispurpose, the effect of M EB on LI was removed employing the method described by Wag-ner et al . (2009), a modification of those described by Kramer & Bryant (1995). Allometricgrowth creates a power function relationship between LI and M EB, and the intercept of thelinearized equation, lnLI and lnM EB, represents the coefficient of the power function, whichsummarizes the size-independent component of the growth trajectory of gut length. Conse-quently, if homogeneity of slopes of the linearized functions among groups was met, theintercept serves as an index of size-standardized intestinal length (I LI) that can be comparedamong individuals or groups (Wagner et al ., 2009). Correlation between I LI and trophicposition was estimated using δ15N as a proxy of trophic level, instead of converting δ15Ninto other estimates of trophic position (Wagner et al ., 2009). Because LSt also varies withM EB, a similar index (I LSt) was constructed to evaluate the size-free differences in LStamong groups. Differences in number of N PC, I LI and I LSt among groups were evaluatedusing ANOVA.

After removing outliers revealed by initial exploratory analyses, between-mass differencesof the different organs were evaluated by a size-free discriminant function analysis (SFDFA).The SFDFA was performed on the ln-transformed residuals of M L, M I and M St, obtainedfrom a MANCOVA using M EB as a covariate. Prior to these statistical analyses, data wereexamined for assumptions of normality and homoscedasticity, using Kolmogorov–Smirnovand Levene tests (P < 0·05).

A Procrustean fit was also implemented with ProTest between Mahalanobis distancesderived from the isotopic and C:N analyses and the Mahalanobis distances from the SFDFA,following the approach described above. The same analysis was used to explore the cor-relation between isotopic and Mahalanobis distances mentioned above and the geographicdistances between paired populations, as well as with the genetic and morphological dis-tances reported by D’Anatro & Lessa (2011). Again, partial ProTest analyses were carriedout between Mahalanobis distances from the SFDFA and all the distances mentioned above,i .e. isotopic and C:N, genetic and morphological, in order to assess the effect of geographicdistances on these correlations.

RESULTS

I S OT O P I C A N D C : N D E R I V E D DATA

Descriptive statistics of δ13C and δ15N values of M . furnieri per site are outlinedin Fig. 2. The C:N values for M . furnieri sampled during 2009 were (mean ± s.d.):Laguna de Rocha = 3·217 ± 0·173, La Paloma = 3·274 ± 0·149, Montevideo =3·264 ± 0·216, Laguna de Castillos = 3·296 ± 0·079, Piriapolis = 3·291 ± 0·166and Villa Gesell = 3·312 ± 0·072 2010, while C:N values for individuals sampledduring 2010 were: Montevideo = 3·193 ± 0·080 and Piriapolis = 3·100 ± 0·082.Positive correlations between δ15N and LS and between δ13C and LS were found(r = 0·818 and 0·645, P < 0·001 in both cases). No significant correlation wasfound between C:N and LS (r = −0·064, P > 0·05).

The DFA performed on the ln-transformed data derived from the isotopic andC:N analyses of M . furnieri did not show a clear segregation among all groupssurveyed (Fig. 3). Nevertheless, individuals from coastal lagoons, especially thosefrom Laguna de Castillos, were clearly discriminated from the other groups evaluatedalong factor 1. δ15N was the most influential variable in factor 1, and explained 96%of the variance. The remaining two factors account for the rest of the varianceand did not contribute to the segregation of samples. The percentage of correctly



20

18

16

14

12

10

8–24 –22 –20 –18 –16 –14 –12

δ13C

δ15N

Fig. 2. Mean ± s.d. of δ13C and δ15N of Micropogonias furnieri populations: Laguna de Rocha ( ), La Paloma( ), Montevideo ( ), Laguna de Castillos ( ), Piriapolis ( ) and Villa Gesell ( ).

classified individuals was above 42·86% in all cases (Table I), with individuals fromLaguna de Castillos and Laguna de Rocha showing the highest values. As expected,the largest Mahalanobis distances squared were between individuals from Lagunade Castillos and the other localities surveyed, followed by comparisons betweenLaguna de Rocha and the remaining localities (Table I). A DFA carried out withδ13C values arithmetically corrected for lipid content, based on C:N ratios (Postet al ., 2007), yielded comparable results. The ProTest analysis did not reveal acorrelation between distances obtained from isotopic and C:N analyses and lineargeographic distances between paired localities (m2 = 0·837, P > 0·05). In contrast,the same analysis found a statistically significant correlation between isotopic andgenetic distances (m2 = 0·364, P < 0·05) and also with the morphological distances,but with a marginal probability value in this case (m2 = 0·353, P > 0·05). PartialProTest results showed a marked statistical correlation between isotopic and geneticdistances (m2 = 0·173, P < 0·05) as well as between isotopic and morphologicaldistances (m2 = 0·298, P < 0·05).

The factorial ANOVA did not show statistically significant differences in δ13C orδ15N values of M . furnieri , either between localities (P > 0·05) or sampling times(P > 0·05). In contrast, the same analysis revealed statistically significant differencesin baseline estimates of δ13C among localities (P < 0·001), but not for δ15N values(P > 0·05). When baseline estimate samples taken at different times were evaluated,both δ13C and δ15N values showed significant differences (P < 0·05 and <0·01).There was no statistically significant effect on the locality × sampling time interac-tion in both cases (P < 0·05 for both δ13C and δ15N values). Results obtained afterTukey HSD tests are shown in Fig. 4.


1648 A . D ’ A NAT RO E T A L .

3·0

2·5

2·0

1·5

1·0

0·5

–0·5

–1·0

–1·5

–2·0

–2·5

–3·0

–3·5–6 –4 –2 0

Factor 1 (99·19% of variance explained)

Fact

or 2

(0·

70%

of

vari

ance

exp

lain

ed)

2 4 6 8 10

0

Fig. 3. Scatterplot of the factors 1 and 2 of the discriminant function analysis performed on the data matrixof δ13C, δ15N and C:N values obtained from the analysis of muscle samples of Micropogonias furnierifrom: Laguna de Rocha ( ), La Paloma ( ), Montevideo ( ), Laguna de Castillos ( ), Piriapolis ( )and Villa Gesell ( ) (Wilks’ λ = 0·085, P < 0·001).

D I G E S T I V E T R A I T S

Descriptive statistics of digestive traits analysed are shown in Table II. The I LIwas obtained as: I LI = lnLI − lnM EB

0·357 and I LSt as: I LSt = lnLSt − lnM EB0·403.

The lower trophic levels were occupied by some individuals of Laguna de Castillosand Laguna de Rocha, but I LI did not differ among groups (ANOVA: P > 0·05).The I LI showed a negative correlation with δ15N values, used as a proxy of trophic

Table I. Percentage of individuals of Micropogonias furnieri correctly assigned in the classi-fication matrix of the discriminant function analysis and Mahalanobis distances between pairedpopulations. Values in bold denote statistical significant differences between population pairs

after Bonferroni correction, at the P < 0·05 level

Squared Mahalanobis distances between paired populations% of correctly

classifiedindividuals

Laguna deRocha

LaPaloma Montevideo

Laguna deCastillos Piriapolis

Laguna de Rocha 90·000 –La Paloma 42·857 14·234 –Montevideo 50·000 6·866 1·401 –Laguna de Castillos 100·000 27·848 81·187 61·510 –Piriapolis <0·001 9·133 0·641 0·192 67·743 –Villa Gesell 50·000 7·681 1·518 0·177 62·695 0·248



–16·5

–17·0

–17·5

–18·0

δ13C –18·5

Piriápolis Montevideo

–19·0

–19·5

–20·0

–20·5c

b

a.b

a

(a)

(b)

δ15N

Piriápolis Montevideo

11·0

10·5

10·0

a.b

a

a

b

9·5

9·0

8·5

8·0

Fig. 4. Isotopic values (mean ± s.d.) of (a) δ13C and (b) δ15N of mussels sampled between localities in 2009( ) and 2010 ( ). Different lower-case letters denote statistically significant differences at the P < 0·05level, after Tukey HSD tests.

position (Fig. 5). The ANOVA also did not find differences in N PC or I LSt amonggroups (P > 0·05 in both cases).

Contrary to the DFA performed on the data derived from the isotopic and C:Nanalyses, the SFDFA did not discriminate among localities (Fig. 6). The percentage ofcorrectly classified individuals of the SFDFA was very low in almost all cases, withthe exception of M . furnieri from Villa Gesell (Table III). The highest Mahalanobisdistances were between Villa Gesell and Laguna de Rocha, and between the formerlocality and Montevideo, followed by the comparison involving Laguna de Rochaand Piriapolis and Laguna de Castillos with Villa Gesell and Piriapolis, respectively(Table III). None of these distances were statistically significant (P > 0·05 in allcases). Results obtained with the ProTest and partial ProTest analyses did not showsignificant correlations of the Mahalanobis distances of the SFDFA with any of theother distance matrices analysed (P > 0·05 in all cases).


1650 A . D ’ A NAT RO E T A L .

Tab

leII

.D

escr

iptiv

est

atis

tics

ofth

edi

gest

ive

trai

tsof

Mic

ropo

goni

asfu

rnie

ri

Loc

ality

sam

pled

Lag

una

deR

ocha

La

Palo

ma

Mon

tevi

deo

Lag

una

deC

astil

los

Piri

apol

isV

illa

Ges

ell

Var

iabl

en

Mea

n±

s.d.

nM

ean

±s.

d.n

Mea

n±

s.d.

nM

ean

±s.

d.n

Mea

n±

s.d.

nM

ean

±s.

d.

LS

(cm

)10

32·30

±6·2

17

44·76

±3·9

610

46·70

±2·9

58

22·44

±2·0

46

39·08

±1·8

420

41·73

±5·4

3L

St(c

m)

106·5

4±

1·23

78·5

7±

2·52

108·3

5±

1·84

82·8

9±

0·75

66·5

7±

1·47

207·3

7±

2·60

LI

(cm

)10

42·49

±5·7

47

66·43

±12

·6010

58·90

±13

·978

22·99

±5·9

26

62·83

±9·8

320

56·38

±22

·70N

PC10

9·40

±0·9

77

8·43

±1·1

310

8·80

±0·9

28

9·25

±0·7

16

8·83

±0·7

520

8·90

±1·0

7M

St(g

)10

3·82

±2·9

37

8·09

±4·5

810

6·39

±3·8

38

0·85

±0·4

66

4·45

±1·2

820

6·71

±4·1

3M

I(g

)10

10·73

±9·1

17

24·73

±5·4

110

29·70

±11

·598

2·64

±1·1

76

14·00

±3·3

320

20·40

±10

·89M

L(g

)10

7·92

±4·7

97

15·40

±7·1

310

26·38

±7·9

98

1·90

±0·7

86

12·25

±3·0

720

18·83

±18

·81M

EB

(g)

1060

3·86

±36

2·93

713

70·09

±19

9·99

1015

77·48

±30

8·50

814

8·02

±56

·166

938·4

5±

130·4

920

1094

·09±

404·6

6

n,

num

ber

ofin

divi

dual

ssu

rvey

edpe

rlo

calit

y;L

S,st

anda

rdle

ngth

;L

St,

stom

ach

leng

th;

LI,

inte

stin

ele

ngth

;N

PC,

num

ber

ofpy

lori

cca

eca;

MSt

,st

omac

hm

ass;

MI,

inte

stin

em

ass;

ML

liver

mas

s;M

EB

,ev

isce

rate

dbo

dym

ass.



80·8

1·0

1·2

1·4

1·6

1·8

2·0

2·2IL

I

10 12 14 16 18 20

δ15N

Fig. 5. Linear regression between intestinal length index (I LI) and trophic position of Micropogonias furnieri ,expressed as δ15N for : Laguna de Rocha ( ), La Paloma ( ), Montevideo ( ), Laguna de Castillos ( ),Piriapolis ( ) and Villa Gesell ( ). The curve was fitted by y = 1·008 + 0·035x (r = 0·363, P<0·01).

DISCUSSION

Results obtained with isotopic and C:N derived data suggested that individualsfrom Laguna de Castillos and Laguna de Rocha are the most differentiated amongall the localities surveyed. On the other hand, M . furnieri from Río de la Plataand the Atlantic Ocean did not differ in their δ13C, δ15N or C:N values, suggestingthat M . furnieri feeds both in estuarine and oceanic environments. Contrary to theisotopic and C:N results, digestive traits did not show a clear differentiation amonglocalities.

P O P U L AT I O N S T RU C T U R E O F M . F U R N I E R I I N F E R R E DF RO M I S OT O P I C A N D C : N VA L U E S

Results obtained with the DFA did not suggest the same differentiation patternas in previous genetic (Pereira et al ., 2009; D’Anatro & Lessa, 2011; D’Anatroet al ., 2011) or morphological (D’Anatro & Lessa, 2011) studies. Besides, ProTestanalyses suggested an overall correlation between the isotopic and C:N distances andthe morphological and genetic distances reported by D’Anatro & Lessa (2011). Thisconcordance is more evident in individuals from coastal lagoons, especially thosefrom Laguna de Rocha.

The δ13C values obtained suggested that individuals from coastal lagoons feedon different carbon sources from M . furnieri that inhabit the Río de la Plata or theAtlantic Ocean. The DFA also separated the M . furnieri from the coastal lagoonsfrom those of the other localities surveyed. Considering M . furnieri from Lagunade Rocha, the segregation proposed by the DFA is in line with the genetic and


1652 A . D ’ A NAT RO E T A L .

5

4

3

2

1

0

–1

–2

–3

–4–4 –3 –2 –1

Factor 1 (72·97% of variance explained)

Fact

or 2

(20

·20%

of

vari

ance

exp

lain

ed)

0 1 2 3 4

Fig. 6. Scatterplot of the factors 1 and 2 of the size-free discriminant function analysis performed over thedata matrix derived from the analysis of the digestive traits of Micropogonias furnieri from: Laguna deRocha ( ), La Paloma ( ), Montevideo ( ), Laguna de Castillos ( ), Piriapolis ( ) and Villa Gesell( ) (Wilks’ λ = 0·913, P > 0·05).

morphological results obtained by D’Anatro & Lessa (2011) and D’Anatro et al .(2011). In contrast, individuals from Laguna de Castillos were not discriminated bythe genetic or morphological analyses mentioned above, but were the most differ-entiated considering the isotopic and C:N analyses. Micropogonias furnieri fromthis lagoon also showed the lowest relative trophic position and were δ13C-depleted(Fig. 2). Additionally, M . furnieri from Laguna de Castillos showed the greatest s.d.

Table III. Individuals of Micropogonias furnieri correctly assigned (%) in the classificationmatrix of the size-free discriminant function analysis and Mahalanobis distances betweenpaired populations. None of these distances were statistically significant at the P < 0·05 level

Squared Mahalanobis distances between paired populationsIndividuals correctlyclassified

individuals(%)

Laguna deRocha

LaPaloma Montevideo

Laguna deCastillos Piriapolis

Laguna de Rocha 30·00 –La Paloma <0·001 0·119 –Montevideo 12·50 0·142 0·169 –Laguna de Castillos <0·001 0·076 0·090 0·183 –Piriapolis <0·001 0·292 0·058 0·179 0·234 –Villa Gesell 94·44 0·411 0·101 0·356 0·230 0·064



in δ13C values, suggesting that individuals from this lagoon may feed over a widerrange of carbon sources with respect to individuals from other localities (Fig. 2). Toa lesser extent, M . furnieri sampled from Laguna de Rocha also showed a lowertrophic position and were δ13C-depleted. Considering M . furnieri from Laguna deRocha, the isotopic differentiation is accompanied by moderate genetic differentia-tion and also by variation in the external morphology, partially driven by divergentselection (D’Anatro & Lessa, 2011). Thus, the idea that population differentiationin M . furnieri is mediated by natural selection seems plausible. Although baselineestimates of δ13C or δ15N were not obtained from coastal lagoons, it is reasonableto assume that the isotopic differences between M . furnieri from these lagoons andthose from the Río de la Plata and Atlantic Ocean indeed reflect differential resourceuse by M . furnieri , because of the pronounced ecological differences in theseenvironments. For example, the values of δ13C or δ15N obtained for M . furnierifrom Laguna de Rocha agree with those reported by Rodríguez-Grana et al . (2008)for the same species in this coastal lagoon (c. −18 and 14‰ for δ13C and δ15N,respectively). The congruency between the results provided by Rodríguez-Granaet al . (2008) and those obtained in this work, indicate that differences in the isotopicsignals of M . furnieri from coastal lagoons and the other localities analysed heretruly reflect ecological differences, as these isotopic signals values were apparentlysustained through time. Additional temporal samplings of the remaining localitiessurveyed in this work are needed to completely address this question.

Lacustrine trophic webs can be divided into pelagic and littoral, the latter beingmore influenced by allochthonous carbon sources [e.g . 13C of terrestrial origin; Post(2002)]. Perhaps, M . furnieri that inhabit coastal lagoons participate in both thetrophic webs and therefore exhibit a high variance in the δ13C values. In addition,D’Anatro & Lessa (2011) suggested a moderate to high level of gene flow betweenLaguna de Castillos and the other localities [number of migrants per generation (N m)c. 4·12, estimated as N m = 0·25 (F ST

−1 − 1)], which could also explain the highvariance in δ13C values of M . furnieri sampled in this lagoon. These estimates ofgene flow reflect historical demographic processes, and the current movement patternof the individuals could be quite different. Considering Laguna de Rocha, the caseis somewhat different, as M . furnieri from this lagoon are also genetically andmorphologically differentiated from the other sites. This lagoon is connected to theAtlantic Ocean via a sandy bar that creates two well-delimited zones: the southernzone, with a clear oceanic influence, and the northern zone, mainly driven by afreshwater regime. Regardless of this evident zoning, Rodríguez-Grana et al . (2008)proposed that biological assemblages and trophic webs were essentially the same insouthern and northern regions of Laguna de Rocha, suggesting that species inhabitingthis lagoon make extensive use of the entire area. Consequently, the northern area ofLaguna de Rocha could provide M . furnieri with prey of terrestrial origin, which,when combined with prey of oceanic origin, could explain the high variance in theδ13C values of M . furnieri from this lagoon. This detailed information is not availablefor Laguna de Castillo but it can be inferred that similar processes may be takingplace in this lagoon where there is a high variance of δ13C values in M . furnieri .It is important to bear in mind that the oceanic influence in Laguna de Castillosis less than in Laguna de Rocha, as the former is connected to the Atlantic Oceanvia the Valizas Creek, a less direct connection than a sandy bar. Finally, isotopicvalues showed by M . furnieri from coastal lagoons could also be influenced by body


1654 A . D ’ A NAT RO E T A L .

size, as individuals sampled in these lagoons are often smaller than their oceanic orestuarine counterparts (Vizziano et al ., 2002), thus influencing the trophic positionof M . furnieri (Penchaszadeh et al ., 2005).

Considering M . furnieri from Río de la Plata, the variance in δ13C values islower than that obtained for individuals from coastal lagoons (Fig. 2). A recentwork using δ13C and δ15N (Botto et al ., 2011) showed that the Río de la Platareceives a significant amount of nutrients of foreign origin, in addition to theprimary production, which could explain the variance in the δ13C values observedin estuarine individuals of M . furnieri . The similar δ13C and δ15N values ofM . furnieri from the Río de la Plata and Atlantic Ocean suggest that eitherallochthonous carbon sources are used to a lesser extent by individuals that spawnwithin the Río de la Plata or this carbon source is used evenly by individuals thatreproduce in the Río de la Plata or oceanic waters (see Fig. 2).

T E M P O R A L VA R I AT I O N I N δ1 3 C A N D δ1 5 N VA L U E S

Stable isotope analysis has been used extensively to establish short-term move-ment patterns in different animals (Hobson, 1999; Rubenstein & Hobson, 2004), butthis technique has some limitations. For instance, individuals inhabiting differentenvironments may exhibit a great variability in their isotopic values (Wunder et al .,2005) or these values may vary depending on the age, sex or body size of individuals(Lott et al ., 2003). Micropogonias furnieri sampled in estuarine or oceanic Atlanticwaters did not differ in their δ13C or δ15N values, both between localities or samplingtimes (November to March). Baseline estimations of δ13C and δ15N showed statisti-cally significant differences between sites (e.g . Montevideo and Piriapolis) and alsowithin sites at the beginning and at the end of reproductive periods (November 2009and March 2010). It is important to note that two different species of mussels wereused to obtain baseline estimates, and this fact could introduce bias in these estima-tions (Xu et al ., 2011). Homogeneity in δ15N values between both species presentedin Fig. 4 suggests, however, that this may not be the case, at least for baselineestimations. In addition to the limitations of the isotopic techniques for tracing indi-vidual migration mentioned above, the isotopic turnover rate for 13C and 15N inwhite muscle of M . furnieri is unknown. Nevertheless, assuming a muscle turnoverrate of the order of weeks for the isotopes used in the analyses (Hobson & Clark,1993) and considering baseline estimation of δ13C and δ15N to be reliable, the resultssuggest that M . furnieri moves freely between estuarine and oceanic waters duringits reproductive season. This suggestion must, however, be interpreted with caution.

Analysing otolith morphology, Norbis & Verocai (2005) proposed the existenceof two different stocks of M . furnieri in the Río de la Plata, with older and largerindividuals spawning earlier during the reproductive season and younger and smallerones reproducing at the end of the spawning season. As Norbis & Verocai (2005)mentioned, this strategy could be considered as an adaptive advantage in relation tothe environmental variability of the Río de la Plata during the spawning season. Thepresent results suggest that individuals sampled at the beginning or at the end ofthe reproductive period share the same feeding grounds, supporting the hypothesisthat this is a size-specific spawning behaviour that is not related to a differentialuse of feeding resources.



D I G E S T I V E T R A I T S A N D T RO P H I C P O S I T I O N O FM I C RO P O G O N I A S F U R N I E R I

Optimal digestibility theory suggests a direct link between digestive systemattributes and food quality (Sibly, 1981). If individuals specialize on differentfood types, variation in the morphology of digestive organs is also conceivable(Olsson et al ., 2008). The SFDFA performed on the digestive system traits didnot segregate individuals from coastal lagoons, in spite of the clear isotopic andC:N differentiation observed among individuals from different localities. Likewise,stomach and intestinal lengths, expressed as I LSt and I LI, did not show statisticallysignificant differences among individuals from different localities. Despite this, LIshowed a negative relationship with trophic position, expressed as δ15N (Fig. 5).Thus, it could be hypothesized that individuals with a lower trophic position feedon some easily digestible prey, and for that reason they tend to have a shorterintestine, but at present not enough dietary data are available to confirm this.

Rodríguez-Grana et al . (2008) proposed that the trophic position of M . furnierifrom Laguna de Rocha varies among seasons, but not among sites. Stomach contentanalysis of M . furnieri from this lagoon revealed the existence of bivalves, benthicinvertebrates, small fishes and vegetable debris as main items (Rodríguez-Granaet al ., 2008). Mendoza-Carranza & Vieira (2008) analysed the diet of M . furnieri inseveral localities along the Brazilian coast, including the estuary of Lagoa dos Patos,and proposed that this species is a generalist-opportunist, which allows M . furnieri tooccupy a wide diversity of environments. These results agree with those obtained inthis work, which showed a marked difference in trophic position and a high variancein the carbon sources used by M . furnieri , according to the localities analysed.

In summary, D’Anatro & Lessa (2011) proposed that a moderate amount of thegenetic differentiation among populations of M . furnieri is accompanied by morpho-logical displacements that could be influenced by divergent selection, e.g . in Lagunade Rocha and Río de la Plata relative to oceanic waters. The differential resourceuse of individuals from coastal lagoons revealed by the isotopic and C:N analysesappears to be in line with these results, supporting the idea that selective pressures,i .e. differential resource use, could be playing an important function in populationdifferentiation of this species, although other factors cannot be ruled out.

We thank C. Rivera and M. Feijoo for assistance during the fieldwork and DINARA(MGAP-Uruguay) for providing samples from Villa Gesell, Argentina. We also want toacknowledge three anonymous reviewers for constructive comments on an earlier versionof this manuscript. Financial support was provided by CSIC (Uruguay) and ANII (Uruguay).

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