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Original article

Lefteright asymmetries and shape analysis on Ceroglossus chilensis(Coleoptera Carabidae)

Raffaella Bravi a1 Hugo A Beniacutetez bc

a Environmental Biology Department University Roma Tre V le G Marconi 446 00146 Rome Italyb Faculty of Life Sciences University of Manchester Michael Smith Building Oxford Road Manchester M13 9PT UK c Instituto de Alta Investigacioacuten Universidad de Tarapacaacute Casilla 7-D Arica Chile

a r t i c l e i n f o

Article history

Received 9 April 2013

Accepted 31 July 2013

Available online

Keywords

Ceroglossus

Geometric morphometrics

Fluctuating asymmetry

Directional asymmetry

a b s t r a c t

Bilateral symmetry is widespread in animal kingdom however most animal can deviate from expected

symmetry and manifest some kind of asymmetries Fluctuating asymmetry is considered as a tool for

valuating developmental instability whereas directional asymmetry is inherited and could be used for

evaluating evolutionary development We use the method of geometric morphometrics to analyze left

right asymmetries in the whole body in two sites and totally six populations of Ceroglossus chilensis with

the aim to infer and explain morphological disparities between populations and sexes in this species In

all individuals analyzed we found both 1047298uctuating asymmetry and directional asymmetry for size and

shape variation components and a high sexual dimorphism Moreover a high morphological variability

between the two sites emerged as well Differences in diet could in1047298uence the expression of morpho-

logical variation and simultaneously affect body sides and therefore contribute to the symmetric

component of variation Moreover differences emerged between two sites could be a consequence of

isolation and fragmentation rather than a response to local environmental differences between sampling

sites

2013 Elsevier Masson SAS All rights reserved

1 Introduction

Most animals have a body plan that is symmetric at least

externally Some morphological symmetries result from the repe-

tition of parts in different positions or orientations (Klingenberg

et al 2002 Savriama and Klingenberg 2011) In bilaterally sym-

metric organisms bilateral symmetry can manifest itself in two

ways as object symmetry or matching symmetry (Mardia et al

2000 Klingenberg et al 2002) The 1047297rst case happens whenever

a structure is symmetric in itself and the plane of symmetry can

divide the structure into two halves that are mirror images of each

other On the other hand matching symmetry happens every time astructure is present in one left and one right copy that can be

matched as they are mirror images of each other ( Klingenberg

et al 2002 Savriama and Klingenberg 2011)

It is very hard to 1047297nd a real organism perfectly symmetric and

small asymmetries can re1047298ect phenotypic adaptation to the

environment Floate and Fox (2000) and Piscart et al (2005) sug-

gested that the degrees of phenotypic disturbancesre1047298ect the ability

of an individual to overcome the effects of stress thus more sym-

metrical individuals would have greater survival possibilities than

those with low levels of symmetry On the other hand environ-

mental pressures and geographic distances affect microenviron-

mentslocally and therefore their associated 1047298ora and fauna that are

the result of adaptation over time to a speci1047297c environment (Alibert

et al 2001 Cepeda-Pizarro et al 2003 Beniacutetez et al 2008) Or-

ganisms can deviate from the expected symmetric con1047297guration

and develop some kind of asymmetries among which are 1047298uctu-

ating asymmetry (FA) and directional asymmetry (DA) The latteroccurs whenever there is a greater development of a character on

one side of the plane of symmetry than the other Additionally a

proportion of DA has genetic bases (Van Valen 1962 Palmer and

Strobeck 1986 Pelabon and Hansen 2008 Carter et al 2009) and

its presence might be associated with the presence of develop-

mental instability (Graham et al 1993) Moreover FA is de1047297ned as

the non-directional deviation from bilateral symmetry also

expressed as individual differences between left and right sides

Those deviations are normally distributed and have the mean of

zero Because of its characteristic FAis generallyconsidered suitable

tool to infer developmental instability (Palmer and Strobeck1986)

Corresponding author Faculty of Life Sciences University of Manchester

Michael Smith Building Oxford Road Manchester M13 9PT UK Tel thorn44 (0)161

306 7102 fax thorn44 (0)161 275 5657

E-mail address hugobenitezpostgradmanchesteracuk (HA Beniacutetez)1 Tel thorn39 0657338050 fax thorn39 0657338052

Contents lists available at ScienceDirect

Acta Oecologica

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e a c t o e c

1146-609X$ e see front matter 2013 Elsevier Masson SAS All rights reserved

httpdxdoiorg101016jactao201307007

Acta Oecologica 52 (2013) 57e62



Finally it is postulated that the degree of phenotypic symmetry

is a mate-choice criterion and as consequence anomalies in sym-

metry can be frequent enough to affect the evolution of sexual

behavior recurrence of asymmetrical sexual selected characters

could happen if they are important under selection (West-

Eberhard 2005) The processes of sexual selection are believed to

modify normally maintained symmetry in traits under stabilized

selection and consequently an increase in FA is expected (Moslashller

and Pomiankowski 1993 Tomkins and Simmons 2003)

Ceroglossus chilensis (Eschscholtz 1829) (Coleoptera Carabidae)

has 26 subspecies distributed from the Maule Region and the

extreme south of the Ayseacuten Region (Chile) it is also present in

Argentina and is the southernmost species and the one with the

widest distribution in Chile This species prefers xeric habitats and

it is more tolerant to arid conditions than its congeners It is still

matter of debate if its size which is relatively large for a carabid is

related to its ability to resist the aridity of the environment ( Jiroux

2006) Some evidences indicate that the development and envi-

ronmental instability of C chilensis which is a species with high

activity density can be affected by modi1047297ed environmental con-

ditions (Briones and Jerez 2007 Beniacutetez et al 2008 2011) Previ-

ous studies in C chilensis have demonstrated that the similarities of

males and females are directly associated with the sex ratio andvery lowly with sexual dimorphism the latter detectable only un-

der a microscopy inspection (Beniacutetez et al 2010b 2011)

In the present study we analyze leftright asymmetries with the

method of geometric morphometric in the whole body of several

populations of Ceroglossus chilensis to provide an explanation of

the morphological disparities between populations and sexes in

this species

2 Materials and methods

21 Data acquisition

Three populations in mature forest (F1 F2 and F3) and three

populations in Second-growth forest (S1 S2 and S3) ( Fig 1) werecollected in 1047297eld during January 2007 12 pitfall traps per site were

installed for 3 days and 3 nights and separated from each other by

approximately 5 m The sex of each specimen was determined

under an optical microscope based on the presence of antennal

careens (Beniacutetez et al 2010a)

22 Shape analyses samples and measurement error

A total of 477 specimens of C chilensis were used for the

morphometric analyses For each individual we took a picture of

the ventral side with an Olympus X-715 digital camera we then

digitized 28 landmarks (LMs anatomical homologous points) on

every picture by TpsDig 210 (Rohlf 2006) (Fig 2) All analyses

were then run using MorphoJ software version 105d (Klingenberg2011)

Once obtained the Cartesian xe y coordinates for all landmarks

the shape information wasextracted with a full Procrustes1047297t (Rohlf

and Slice 1990 Dryden and Mardia 1998) taking into account the

object symmetry of the structure Procrustes superimposition is a

procedure that removes the information of rotation position and

orientation and standardizes each specimen to unit centroid size

(that is the he square root of the summed squared Euclidean dis-

tances from each landmark to the specimen centroid and provides

an estimation of the size of the studied structure (Dryden and

Mardia 1998)) Because of the symmetry of the structure re1047298ec-

tion is removed by including the original and mirror image of all

con1047297gurations in the analysis and simultaneously superimposing

all of them (Klingenberg et al 2002)

We also provided informations on the studied structure sym-

metry for observing eventual phenomena of directional asymmetry

or 1047298uctuating asymmetry and to better understand the observed

differences between the investigated populations DA occurs

whenever one characterdeveloped more in one side of the plane orplanes of symmetry than in the other (Van Valen1962) while FA is

de1047297ned as those random differences which occur between the left

and right sides in symmetrical organisms Fluctuating asymmetry

has been used as an indicator of the level of developmental stability

and in de1047297ning the in1047298uence of both environmental and genetic

stress on development (Van Valen 1962) Measurement error (ME)

is of critical importance when analysing FA (eg Palmer 1994) To

assess the signi1047297cance of FA relative to ME 50 individual beetles

were digitized twice We then applied a conventional analysis of

variance (ANOVA) on centroid size for size and a Procrustes ANOVA

for shape taking into account the values of MS of the ANOVA

23 Statistical analyses

To examine the amount of symmetric variation and asymmetry

we used Procrustes ANOVA as assessed for studies on object sym-

metry (Klingenberg and McIntyre 1998 Klingenberg et al 2002

Klingenberg and Monteiro 2005) To avoid the assumption of

having an equal and independent variation at all points we per-

formed a MANOVA test for symmetric component and asymmetry

This test is used to compare the individualere1047298ection interactions

to measurement error the latter estimated from the total variation

of the entire landmark con1047297guration (Klingenberg et al 2002)

In addiction the shape variation in the entire data set were

assessed using principal component analysis (PCA) based on the

covariance matrix of symmetric and asymmetry components of

shape variation The 1047297rst one is the average of left and right sides

and represents the shape variation component whereas the

Fig1 Map of Chile and the Ayseacuten Region indicating the study area and the sampling

sites Circles Second-Growth Stand Squares Forest

R Bravi HA Beniacutetez Acta Oecologica 52 (2013) 57 e6258



asymmetry component represents the individual left-right differ-

ences (Klingenberg et al 2002)

Finally differences between populations and sexes were

assessed performing a canonical variate analysis (CVA) a multi-

variate statistical method used to 1047297nd the shape characters that

best distinguish among multiple groups of specimens The results

were reported as Mahalanobis distance that is a multivariate

measure of distance relative to the within sample variation All the

analyses are computed as permutation test with 10000 permuta-

tion runs

3 Results

The estimate of both size and shape were accurate as the mea-

surement error was smaller than the true FA(MS values for

individual side 00003 in comparison with the individual values

02 for size and 31e-5 to 65e-5 for shape respectively) Procrustes

ANOVA indicates that variation in size and symmetric shape be-

tween populations and sexes is highly signi1047297cant Moreover DA

and FA result highly signi1047297cant for the symmetric component of

shape variation (Table 1) MANOVA test were used for calculating

the non-isotropic variation at each landmark and con1047297rmed the

presence of highly signi1047297cant DA in the sample (Pillai tr frac14 078

P lt 00001) PCA then was used to identify and describe the pattern

of individual variation (DA) and 1047298uctuating asymmetry As for

the symmetric component of the variation (DA) the 1047297rsts four

PCs accounted for 5429 of the total shape variation

(PC1 thorn PC2 thorn PC3 thorn PC4 frac141962 thorn 1458 thorn 1068 thorn 942) and

the whole set of landmarks contribute quite equally to the variation

among individuals (Fig 3a) Differently the variation for the 1047298uc-

tuating asymmetry (asymmetric component of shape variation)

was due for the majority to the PC1 and only for a reduced part to

the PC2 with the 1047297rsts two PCs that complexly accounted for

5739 of the total shape variation (PC1 thorn PC2 frac14 4904 thorn 835)

(Fig 3b)The high morphological variability among the whole sample

was analyzed and displayed with CVA analyses for assessing dif-

ferences among populations and sexual dimorphism Due to the

fact that differences between the F and S sites are statistically

signi1047297cative (Mahalanobis distance frac14 182 P lt 00001) we per-

formed all the subsequent analyses separately

As for the S site concerns CVA analysis showed a high

morphological variability between the three populations (S1 S2

S3) in which a strong sexual dimorphism emerged (Fig 4a and

Table 2) As the F site concerns CVA analysis also showed high

morphological variability between the three populations (F1 F2

F3) and a strong sexual dimorphism visible as a separation be-

tween males and females of the same population (Fig 4b and

Table 3) Moreover in the F site CVA analysis also showed that thethree female populations are more similar to each other and

resulted as clustered together whereas male populations emerged

as more different to each other and resulted as separate groups (see

Fig 4b) On the whole the majority of differences were due tothe F3

population that differed substantially to the others and showed the

higher differences between males and females

4 Discussion

The use of landmarks to study bilaterallysymmetric animals has

been frequently questioned (Mitteroecker 20 09) since they in-

crease the degrees of freedom without adding much additional

information (Dryden and Mardia 1998) leading to high values in

the variance-covariance matrix (Alibert et al 2001) Here we usedthe entire organism in accordance with the methodology proposed

by Alibert et al (2001) which considers the variation in form ho-

listically This approach includes the differentiation of sexes and

allows a more realistic view of the variation in shape in a defor-

mation network without an obvious sexual differentiation like

horns and mandibles in beetles

The use of geometric morphometrics plus multivariate statisti-

cal techniques provides a graphic visualization of the morpholog-

ical variation of the individuals In this study we used geometric

morphometric approach to investigate the variation of body shape

in an austral population of Ceroglossus chilensis ANOVA and CVA

analyses showed a high morphological differentiation among sites

and populations These variations among sampling sites are mainly

due to differences in shape more than in size

Table 1

Procrustes ANOVA for both centroid size (CS) and shape (SH) of Ceroglossus chilensis

characterized by object symmetry

SS MS df F P

CS BP 0000002 0 5 409 00012

SD 0000001 0000001 1 1364 00003

FA 0000034 0 415

SH BP 00353 0000271 130 1075 lt00001

SD 00223 0000858 26 34 lt00001FA 0272 00000252 10790 17 lt00001

DA 000998 0000384 26 2587 lt00001

Characterized by object symmetry Sums of squares (SS) and mean squares (MS) are

in units of Procrustes distances (dimensionless)

BP frac14 variation between populations (S1 S2 S3 F1 F2 F3) FA frac14 1047298uctuating

asymmetry DA frac14 directional asymmetry

Fig 2 Ceroglossus chilensis Selection of 28 landmarks in the ventral view The bar line

represent 1 cm

R Bravi HA Beniacutetez Acta Oecologica 52 (2013) 57 e62 59



Although these variations are catchable only at a microscopical

scale they may be suf 1047297cient to assume a sexual selection on theinsects The morphological variation in the abdomen we observed

on the whole was greater in females than in males this is an

essential morphological character that allows a female to produce a

huge quantity of eggs and therefore have a high fecundity and

greater 1047297tness than other females (Andersson 1994 Cepeda-

Pizarro et al 1996 Beniacutetez et al 2010a 2010b)

It is frequently suggested that morphological variation of in-

dividuals may be strongly dependent upon unfavorable environ-

mental conditions (Adams and Funk 1997 Tatsuta et al 2001) In

fact individuals under environmental noise could develop any kind

of asymmetries (Van Valen 1962)

Directional asymmetry occurs throughout the animal kingdom

(Palmer 1996 Klingenberg et al 1998) and a lefteright axis has

now been demonstrated for most major phyla including all the

principal model organisms in developmental biology The statistical

power of a Procustes ANOVA analysis allowing sharper detection of differences in directions with smaller variability enabled us to

determine that the body shape of C chilensis but not the size

showed a directional asymmetry statistically signi1047297cant although

not very sharp (for details see Table 1) Over half of the symmetric

component of shape variation is associated with the 1047297rst four PCs

with no huge variation between them indicating that there is no

external pressure determining shape change (high sexual dimor-

phism or allometry) Nevertheless the high variation found on F

sites particularly the distinction of a female group in F3 could be

explained by the scarce anthropic intervention on these pop-

ulations generating a better environmental balance and hence

higher phenotypic heterogeneity coming out as morphological

differences between sites On the contrary it is observed that sec-

ondary forests of S populations in contrast to F are subject to

Fig 3 Symmetric and asymmetry components of shape variation a) the 1047297rsts four PCs of the symmetric component used to describe the pattern of symmetric part of shape

variation that is the variation in the averages of the original and re1047298ected landmark con1047297gurations constrained to symmetry (the starting shape) b) the 1047297rsts four PCs of the

asymmetry component used to describe the variation of individual asymmetries (FA) from the symmetric consensus con1047297guration (the starting shape) The signs and the scores for

PCs are arbitrary and they are of thorn006 unit of Procrustes distance In each 1047297gure the starting shapes are in grey and the target shapes are in black




deeper changes and therefore there is higher pressure on the

phenotype This observation could be assumed to be an adaptive

response coming out as low morphological changes or as 1047298

uctu-ating asymmetry which is present in both sites but in different

degrees (see Tables 1 and 2)

Similarly a prominent role of phenotypic plasticity may also

explain the discrepancy in the patterns of covariation between the

symmetric shape variation among individuals and 1047298uctuating

asymmetry

Our results show that shape differences we obtained fromdifferent populations could reasonably be a consequence of isola-

tion and fragmentation that happened in our sample sites rather

than simply a response to local environmental differences between

sampling sites

Fig 4 Scatter plot by the canonical variate analysis (CVA) a) for the S populations b) for the N populations Both graphics show the presence of sexual dimorphism and differences

between populations

Table 2

Pairwise comparisons by the canonical variate analysis performed between the three sites of the S population Results are reported as Mahalanobis distance (and p-values)

after 10000 permutation runs

S1 F S1 M S2 F S2 M S3 F

S1 M 377 (lt00001)

S2 F 254 (lt00001) 387 (lt00001)

S2 M 390 (lt00001) 311 (lt00001) 327 (lt00001)

S3 F 277 (lt00001) 390 (lt00001) 336 (lt00001) 461 (lt00001)

S3 M 448 (lt00001) 271 (lt00001) 435 (lt00001) 312 (lt00001) 426 (lt00001)




Acknowledgments

The authors thank to Dra Viviane Jerez and Dr Raul Briones for

the collaboration in 1047297eld and ideas of this research and to Mrs

Maria Raquel Lazo de la Vega for help in the English of the manu-

script This work was supported by grant N 20611372-3 from the

Direccioacuten de Investigacioacuten Universidad de Concepcioacuten

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dimorphism in Neochlamisus bebbianae leaf beetles multivariate applicationsof the thin-plate spline Syst Biol 46 (1) 180e194Alibert P Moureau B Dommergues JL David B 2001 Differentiation at a

microgeographical scale within two species of ground beetle Carabus auro-nitens and C nemoralis (Coleoptera Carabidae) a geometrical morphometricapproach Zool Scr 30 299e311

Andersson M 1994 Sexual Selection Princeton University Press Princeton Beniacutetez H Briones R Jerez V 2008 Asimetriacutea Fluctuante en dos poblaciones de

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Beniacutetez H Vidal M Briones R Jerez V 2010a Sexual dimorphism andmorphological variation in populations of Ceroglossus chilensis (Eschscholtz1829) (Coleoptera Carabidae) J Entomol Res Soc 12 87e95

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Dryden IL Mardia KV 1998 Statistical Shape Analysis Wiley ChichesterFloate KD Fox AS 2000 Flies under stress a test of 1047298uctuating asymmetry as a

biomonitor of environmental quality Ecol Appl 10 (5) 1541e1550

Graham JH Freeman DC Emlen JM 1993 Antisymmetry directional asym-metry and dynamic morphogenesis Genetica 89 121e187

Jiroux E 2006 In Magellanes Collection (Ed) Le Genre Ceroglossus vol 14Klingenberg CP 2011 MORPHOJ an integrated sof tware package for geome tric

morphometrics Mol Ecol Res 11 353e357Klingenberg CP Barluenga M Meyer A 2002 Shape analysis of symmetric

structures quantifying variation among individuals and asymmetry Evolution56 1909e1920

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morphometric methods for any type of symmetry BMC Evol Biol 11 280 Tatsuta H Mizota K Akimoto SI 2001 Allometric patterns of heads and genitalia

in the stag beetle Lucanus maculifemoratus (Coleoptera Lucanidae) AnnEntomol Soc Am 94 462e466

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Table 3

Pairwise comparisons by the canonical variate analysis performed between the three sites of the F population Results are reported as Mahalanobis distance (and p-values)


F1 F F1 M F2 F F2 M F3 F

F1 M 385 (lt00001)

F2 F 165 (00001) 402 (lt00001)

F2 M 405 (lt00001) 187 (lt00001) 381 (lt00001)

F3 F 182 (lt00001) 339 (lt00001) 168 (lt00001) 326 (lt00001)

F3 M 676 (lt00001) 537 (lt00001) 619 (lt00001) 424 (lt00001) 563 (lt00001)




Finally it is postulated that the degree of phenotypic symmetry

is a mate-choice criterion and as consequence anomalies in sym-

metry can be frequent enough to affect the evolution of sexual

behavior recurrence of asymmetrical sexual selected characters

could happen if they are important under selection (West-

Eberhard 2005) The processes of sexual selection are believed to

modify normally maintained symmetry in traits under stabilized

selection and consequently an increase in FA is expected (Moslashller

and Pomiankowski 1993 Tomkins and Simmons 2003)

Ceroglossus chilensis (Eschscholtz 1829) (Coleoptera Carabidae)

has 26 subspecies distributed from the Maule Region and the

extreme south of the Ayseacuten Region (Chile) it is also present in

Argentina and is the southernmost species and the one with the

widest distribution in Chile This species prefers xeric habitats and

it is more tolerant to arid conditions than its congeners It is still

matter of debate if its size which is relatively large for a carabid is

related to its ability to resist the aridity of the environment ( Jiroux

2006) Some evidences indicate that the development and envi-

ronmental instability of C chilensis which is a species with high

activity density can be affected by modi1047297ed environmental con-

ditions (Briones and Jerez 2007 Beniacutetez et al 2008 2011) Previ-

ous studies in C chilensis have demonstrated that the similarities of

males and females are directly associated with the sex ratio andvery lowly with sexual dimorphism the latter detectable only un-

der a microscopy inspection (Beniacutetez et al 2010b 2011)

In the present study we analyze leftright asymmetries with the

method of geometric morphometric in the whole body of several

populations of Ceroglossus chilensis to provide an explanation of

the morphological disparities between populations and sexes in

this species

2 Materials and methods

21 Data acquisition

Three populations in mature forest (F1 F2 and F3) and three

populations in Second-growth forest (S1 S2 and S3) ( Fig 1) werecollected in 1047297eld during January 2007 12 pitfall traps per site were

installed for 3 days and 3 nights and separated from each other by

approximately 5 m The sex of each specimen was determined

under an optical microscope based on the presence of antennal

careens (Beniacutetez et al 2010a)

22 Shape analyses samples and measurement error

A total of 477 specimens of C chilensis were used for the

morphometric analyses For each individual we took a picture of

the ventral side with an Olympus X-715 digital camera we then

digitized 28 landmarks (LMs anatomical homologous points) on

every picture by TpsDig 210 (Rohlf 2006) (Fig 2) All analyses

were then run using MorphoJ software version 105d (Klingenberg2011)

Once obtained the Cartesian xe y coordinates for all landmarks

the shape information wasextracted with a full Procrustes1047297t (Rohlf

and Slice 1990 Dryden and Mardia 1998) taking into account the

object symmetry of the structure Procrustes superimposition is a

procedure that removes the information of rotation position and

orientation and standardizes each specimen to unit centroid size

(that is the he square root of the summed squared Euclidean dis-

tances from each landmark to the specimen centroid and provides

an estimation of the size of the studied structure (Dryden and

Mardia 1998)) Because of the symmetry of the structure re1047298ec-

tion is removed by including the original and mirror image of all

con1047297gurations in the analysis and simultaneously superimposing

all of them (Klingenberg et al 2002)

We also provided informations on the studied structure sym-

metry for observing eventual phenomena of directional asymmetry

or 1047298uctuating asymmetry and to better understand the observed

differences between the investigated populations DA occurs

whenever one characterdeveloped more in one side of the plane orplanes of symmetry than in the other (Van Valen1962) while FA is

de1047297ned as those random differences which occur between the left

and right sides in symmetrical organisms Fluctuating asymmetry

has been used as an indicator of the level of developmental stability

and in de1047297ning the in1047298uence of both environmental and genetic

stress on development (Van Valen 1962) Measurement error (ME)

is of critical importance when analysing FA (eg Palmer 1994) To

assess the signi1047297cance of FA relative to ME 50 individual beetles

were digitized twice We then applied a conventional analysis of

variance (ANOVA) on centroid size for size and a Procrustes ANOVA

for shape taking into account the values of MS of the ANOVA

23 Statistical analyses

To examine the amount of symmetric variation and asymmetry

we used Procrustes ANOVA as assessed for studies on object sym-

metry (Klingenberg and McIntyre 1998 Klingenberg et al 2002

Klingenberg and Monteiro 2005) To avoid the assumption of

having an equal and independent variation at all points we per-

formed a MANOVA test for symmetric component and asymmetry

This test is used to compare the individualere1047298ection interactions

to measurement error the latter estimated from the total variation

of the entire landmark con1047297guration (Klingenberg et al 2002)

In addiction the shape variation in the entire data set were

assessed using principal component analysis (PCA) based on the

covariance matrix of symmetric and asymmetry components of

shape variation The 1047297rst one is the average of left and right sides

and represents the shape variation component whereas the

Fig1 Map of Chile and the Ayseacuten Region indicating the study area and the sampling

sites Circles Second-Growth Stand Squares Forest













tion runs

3 Results









































4 Discussion



















Table 1



SS MS df F P

CS BP 0000002 0 5 409 00012

SD 0000001 0000001 1 1364 00003

FA 0000034 0 415

SH BP 00353 0000271 130 1075 lt00001

SD 00223 0000858 26 34 lt00001FA 0272 00000252 10790 17 lt00001

DA 000998 0000384 26 2587 lt00001






represent 1 cm

















































asymmetry




sampling sites


between populations

Table 2




S1 M 377 (lt00001)

S2 F 254 (lt00001) 387 (lt00001)

S2 M 390 (lt00001) 311 (lt00001) 327 (lt00001)

S3 F 277 (lt00001) 390 (lt00001) 336 (lt00001) 461 (lt00001)

S3 M 448 (lt00001) 271 (lt00001) 435 (lt00001) 312 (lt00001) 426 (lt00001)




Acknowledgments






References






































Table 3




F1 M 385 (lt00001)

F2 F 165 (00001) 402 (lt00001)

F2 M 405 (lt00001) 187 (lt00001) 381 (lt00001)

F3 F 182 (lt00001) 339 (lt00001) 168 (lt00001) 326 (lt00001)

F3 M 676 (lt00001) 537 (lt00001) 619 (lt00001) 424 (lt00001) 563 (lt00001)













tion runs

3 Results









































4 Discussion



















Table 1



SS MS df F P

CS BP 0000002 0 5 409 00012

SD 0000001 0000001 1 1364 00003

FA 0000034 0 415

SH BP 00353 0000271 130 1075 lt00001

SD 00223 0000858 26 34 lt00001FA 0272 00000252 10790 17 lt00001

DA 000998 0000384 26 2587 lt00001






represent 1 cm

















































asymmetry




sampling sites


between populations

Table 2




S1 M 377 (lt00001)

S2 F 254 (lt00001) 387 (lt00001)

S2 M 390 (lt00001) 311 (lt00001) 327 (lt00001)

S3 F 277 (lt00001) 390 (lt00001) 336 (lt00001) 461 (lt00001)

S3 M 448 (lt00001) 271 (lt00001) 435 (lt00001) 312 (lt00001) 426 (lt00001)




Acknowledgments






References






































Table 3




F1 M 385 (lt00001)

F2 F 165 (00001) 402 (lt00001)

F2 M 405 (lt00001) 187 (lt00001) 381 (lt00001)

F3 F 182 (lt00001) 339 (lt00001) 168 (lt00001) 326 (lt00001)

F3 M 676 (lt00001) 537 (lt00001) 619 (lt00001) 424 (lt00001) 563 (lt00001)

















































asymmetry




sampling sites


between populations

Table 2




S1 M 377 (lt00001)

S2 F 254 (lt00001) 387 (lt00001)

S2 M 390 (lt00001) 311 (lt00001) 327 (lt00001)

S3 F 277 (lt00001) 390 (lt00001) 336 (lt00001) 461 (lt00001)

S3 M 448 (lt00001) 271 (lt00001) 435 (lt00001) 312 (lt00001) 426 (lt00001)




Acknowledgments






References






































Table 3




F1 M 385 (lt00001)

F2 F 165 (00001) 402 (lt00001)

F2 M 405 (lt00001) 187 (lt00001) 381 (lt00001)

F3 F 182 (lt00001) 339 (lt00001) 168 (lt00001) 326 (lt00001)

F3 M 676 (lt00001) 537 (lt00001) 619 (lt00001) 424 (lt00001) 563 (lt00001)




Acknowledgments






References






































Table 3




F1 M 385 (lt00001)

F2 F 165 (00001) 402 (lt00001)

F2 M 405 (lt00001) 187 (lt00001) 381 (lt00001)

F3 F 182 (lt00001) 339 (lt00001) 168 (lt00001) 326 (lt00001)

F3 M 676 (lt00001) 537 (lt00001) 619 (lt00001) 424 (lt00001) 563 (lt00001)


Bravi & Benitez 2013

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