Vol.
32 No.
November 2025 1577-1583 DOI:10.
4308/hjb.
ISSN: 1978-3019
EISSN: 2086-4094
H A Y AT I
Journal of Biosciences Research Article Different Carapace Asymmetry Among Sexes in Vulnerable Tortoise:
Chelonoidis carbonarius Spix 1824 (Testudines: Testudinida.
Arcesio Salamanca-Carreyo1*.
Pere M.
Parys-Casanova2.
Brando E.
Pinzyn-Guzmyn1.
Daniel A.
Dyaz-Caviedes1 Facultad de Medicina Veterinaria y Zootecnia.
Universidad Cooperativa de Colombia, 50001 Villavicencio.
Colombia Department de Bromatologya.
Universitat Oberta de Catalunya, 08018 Barcelona.
Spain
ARTICLE INFO
Article history:
Received February 11, 2025 Received in revised form April 27, 2025 Accepted June 8, 2025 Available Online August 27, 2025 KEYWORDS:
biotic stress, development stability, environmental stressors, fluctuating asymmetry.
Copyright .
2025@ author.
ABSTRACT
Developmental stability (DS) is characterized by an individual's ability to resist random environmental and/or genetic perturbations during their development.
Fluctuating Asymmetry (FA) is the most common means of assessing developmental stability (DS) in bilateral traits.
To date, little attention has been paid to sexual differences in FA among tortoises.
The aim was to assess the levels of carapace scutation symmetries in a sample of 45 wild adult red-footed tortoises.
Chelonoidis carbonarius .
males and 32 female.
, from the Arauca plains (Colombi.
using geometric morphometric techniques.
The landmark configuration, based on the dorsal scute sutures of the carapace, consisted of 7 symmetric pairs with three landmarks along the axial plane, and was tested.
Procrustes ANOVA reflected sex-related FA .
<0.
, with males tending to exhibit a more pronounced asymmetry.
Although we cannot identify the potential sources responsible for the detected developmental instability, our results suggest a high degree of stress and highlight that human intervention in Arauca is affecting wildlife.
Similar studies in the future, correlated with an estimate of human impact, could provide irrefutable proof of causality between FA and environmental stressors.
The results of this study may represent an estimate of the physical status of the Chelonoidis carbonarius (Spix 1.
population currently being studied.
Introduction Bilaterally symmetrical organisms can exhibit different types of asymmetry, including Fluctuating Asymmetry (FA).
Directional Asymmetry (DA), and Antisymmetry (AS) (Graham et al.
Graham et al.
FA is defined as the deviation of the symmetry of an individual from its ideal symmetry.
is considered a negative indicator of the individualAos ability to resist random and small developmental accidents (Palmer 1.
These developmental accidents are commonly the result of genetic or * Corresponding Author E-mail Address: asaca_65@yahoo.
environmental stress (Auffray et al.
Carter et al.
Inbreeding has been linked to reduced health in populations and, consequently, to body symmetry.
DA occurs whenever there is a greater development of a character on one side than the other (Kharlamova et Lotto and Byguelin 2.
DA typically has a genetic basis (Leuniak 2.
Finally.
AS is a bimodal asymmetry that is random concerning side, where the lack of symmetry is distinguished by a departure from a Gaussian distribution of side differences (Mancini et For the quantification of these asymmetries, size and shape must be assessed separately (Sforza et Geometric morphometric approaches have been used for this purpose.
Carreyo AS et al.
Chelonoidis carbonarius was described as Testudo carbonaria by Spix in 1824 (Echeverry-Alcendra It is a tortoise species from South America, distributed in open areas (Barros et al.
Rhodin et al.
from northern Paraguay and Argentina through the cis-Andean region to Panama (GallegoGarcya et al.
Cacciali et al.
Rhodin et They are medium-sized tortoises that can reach over 40 cm (Barros et al.
, characterized by dark-colored, loaf-shaped carapaces with a lighter patch in the middle of each scute.
However, they exhibit considerable variation in morphological characteristics (Barros et al.
Gallego-Garcya et al.
No size or sex dimorphism has been described (Barros et al.
, but morphological population differences are recognized within the species between areas, primarily in plastron scutes, carapace width, and head length (Barros et al.
carbonarius is frequently kept as a pet, and overcollection, as well as habitat destruction, have caused it to be close to extinction (Gallego-Garcya et al.
, appearing as AuVulnerableAy in the 2021 TFTSG Draft Red List (Rhodin et al.
The anthropogenic disturbance in the habitat, resulting from the continuous human exploitation of the Arauca plains, contributes to environmental deterioration, subjecting C.
carbonarius to ecological stress and thereby promoting developmental instability in the species.
Therefore, we hypothesize that human intervention in the environment where these turtles live may be related to the presence of possible Hybridization and consanguinity are excluded, as we have worked with an open wild To date, not much attention has been paid to asymmetric studies or sexual differences in FA in In the present study, the scutation asymmetries between sexes in the shell of the wild red-footed tortoise (C.
from the Arauca plains were analyzed and compared using geometric morphometric techniques.
Our data aims to compare possible differences in sexual homeostasis among these animals.
Materials and Methods Sampling Area The present study was carried out in the department of Arauca (Colombi.
located on the Orinoco Basin .
he AuLlanos OrientalesA.
in the extreme East, bordering Venezuela, situated between the Arauca and Casanare rivers, and covering a huge area of 23.
818 km2 .
07A05A25 N, longitude: 70A45A42 W, altitude: 128 .
(Figure .
Seventy-five per cent of the land is flat.
The soil is covered by native grass.
The temperature varies from 28AC to 35AC (Arauca 2.
This area experiences minimal tourist impact, with waste from multinational crop companies posing its primary threat.
Data Collection Data were obtained from 45 wild adult individuals .
males, 34.
7 cm total carapace length.
32 females, 20.
2 cm total carapace lengt.
of C.
carbonarius, during the dry season (February to March 2.
(Salamanca-Carreyo et al.
This tortoise is Figure 1.
Map of Colombia.
Arauca Department is located in the AuLlanos OrientalesAy of Eastern Colombia.
Red circle: sampling Source: authors HAYATI J Biosci Vol.
32 No.
November 2025 diurnal and typically inhabits tropical dry forests.
It is an omnivorous animal.
In different areas, digital pictures were obtained in the field, with the camera held approximately 40-50 cm above each tortoise, focused on the center of their central dorsal aspect.
We only used the carapacial scores because the carapacial photographs are less susceptible to tilting during the photographing process .
he animal can rest flat and inside its shel.
A single digital picture of each animal was obtained.
To prevent distortion and optical aberrations, no zoom was used, and the tortoises were maintained in a neutral position, using props to minimize parallax errors.
All the field-collected specimens displayed no injuries from predators or additional scutes.
Photographs were taken with a Nikon P530 camera featuring a 42y optical zoom.
Identification of sex was based on the presence or absence of a pronounced plastron concavity.
An average straight carapace length 9 cm is reported, although records of up to 51 cm are also available (Echeverry-Alcendra 2.
A ruler was inserted previously on each image.
After their measurement and photography, the animals were released at the same site where they were captured.
In no case were animals sacrificed, or traumatic manipulation or samples obtained that could cause pain.
Geometric Morphometrics A landmark configuration based on the dorsal scute sutures of the carapace, comprising seven symmetric pairs with three landmarks along the axial plane, was tested (Figure .
Three types of landmarks can be recognized.
Our landmarks were of type I, e.
, localized at the intersection of sutures and therefore easy to identify The captured images were transformed using TpsUtil v.
40 software (Rohlf 2015.
, and landmarks were recorded using TpsDig v.
26 software (Rohlf Scale was eliminated by setting the centroid size .
he square root of the sum of squared distances between each landmark and the carapace centroi.
the same in all specimens (Bookstein 1.
Because measurement error may either hide or bias fundamental differences between the sides (Palmer 1.
, the first author repeated the measurements for all traits in all samples twice to minimize inconsistencies or errors in plotting landmark points.
The set of original configurations and mirrored copies .
ncluding the replicated measurement.
was then superimposed .
caled, translated, and rotate.
following the Procrustes method of generalized least squares superimposition.
Shape spaces are curved, non-Euclidean spaces.
apply the usual methods of statistics, it is necessary to project shape space onto a linear Euclidean space, so that the distribution of points in the tangent space can be used as a good approximation of their distribution in shape space (Klingenberg and McIntyre 1998.
As the process of extracting tangent landmark coordinates from curved objects can introduce errors due to non-coplanarity Figure 2.
Pairs of landmarks .
situated on sutures of carapace scutes .
ight: anal regio.
As the carapace is a symmetric structure with object symmetry, characterized by an axis of symmetry that passes through the landmark configuration, the symmetry axis can partially align with the three remaining landmarks on the mid-sagittal line Pertiwi MP Carreyo AS et al.
(Klingenberg and McIntyre 1998a.
Webster and Sheets 2.
, we tested whether the observed shape variation was sufficiently slight.
This analysis was conducted using TpsSmall v.
33 software (Rohlf 2015.
The asymmetrical component was quantified as the difference between the original and the ideally symmetrical configuration, based on the displacement of unpaired landmarks along the mid-sagittal axis and the average displacement of paired landmarks moving in any direction.
For the quantification of asymmetry, a displacement of unpaired landmarks is allowed in the direction perpendicular to the mid-sagittal axis while paired landmarks move freely.
The analysis done was specifically for object symmetry, with the symmetry axis passing through the structure.
Statistical Analysis A multivariate regression of the asymmetric component against centroid size was also performed to detect allometry for each sex.
There was no change in asymmetry according to size, shape asymmetric variables, and centroid size, which were found to be largely uncorrelated in the sample .
= 0.
10,000
randomization round.
To examine the amount of asymmetric variation, we used a Procrustes two-way mixed model ANOVA (Klingenberg et al.
Klingenberg & Monteiro 2.
for each sex and shape separately.
The AuindividualsAy effect refers to the variation in size and shape among The main effect is Ausides,Ay which indicates the variation between sides.
The Auindividual x side interactionAy is a mixed effect, representing the failure of individuals to be the same between sides .
eft and Lastly, measurement error, estimated from the total variation of the entire landmark configuration, was The AuindividualsAy effect refers to the variation among individual samples and can be interpreted as variation in symmetry.
The effect of Auindividual x side interactionAy is the measure of FA, and the effect of AusideAy is the measure of DA (Klingenberg 2.
To verify the data for AS, we visually examined scatter plots of vectors representing left-right differences for each landmark after superimposition using the Procrustes This vector plot depicts the displacement of all landmarks between the target form and those on the reference form.
Analytical procedures that incorporate geometric morphometrics in testing for FA are available from numerous seminal publications (Klingenberg et Klingenberg 2002.
Klingenberg & Monteiro Finally, a Canonical Variate Analysis (CVA) was performed to compare the asymmetric components between sexes.
Mahalanobis distances were used for this test.
All analyses were performed using MorphoJ v.
(Klingenberg 2.
, with permutation tests conducted across 10,000 permutation runs.
For all analyses, =0.
Results Although the carapace is markedly convex, when we compared the 2D Procrustes distances to the tangent space distances .
Euclidean distances in tangent spac.
, the relation was very close to linear for all the data, e.
, it appeared an excellent correlation between the tangent and the shape space .
orrelation between the tangent space onto Procrustes distance=0.
This result proved the acceptability of the dataset for further statistical analysis in the Euclidean space.
Initially, intra-observer error, which is associated with the placement of landmarks, was evaluated.
The results showed that the variation among samples (MS = 0.
was higher than the variation between double distributions of landmarks (MS = 0.
hence, high intra-observer agreement was ascertained.
The absence of clustering of vectors .
s the equivalent of bimodal distributions of left-right difference.
provided no support for the presence of AS (Figure .
Procrustes ANOVA shows the mean squares for the mixed-model ANOVA and its corresponding effects (Table .
The significant interaction of Auindividual x sideAy confirmed the presence of FA for both sexes, but the side factor .
ndicating DA) was not detected to be statistically Canonical Variate Analysis (CVA) revealed statistically significant differences between sexes.
is, a significant factor analysis (FA) was found for both sexes .
<0.
10,000 permutation round.
There are more degrees of freedom in Procrustes ANOVA than in conventional ANOVA because the squared deviations are summed over all the landmark coordinates .
nstead of a single sum of squares in conventional ANOVA).
Therefore, the number of degrees of freedom is that for ordinary ANOVA multiplied by the shape dimension, which is, for two-dimensional coordinate data, twice the number of landmarks minus four .
he number of coordinates minus two dimensions for translation and one each for scaling and rotatio.
HAYATI J Biosci Vol.
No.
6,XX,
XX XX 2025
32 No.
November Figure 3.
Clustering of vectors for 45 individuals .
males and 32 female.
of Chelonoidis carbonarius.
The vector plot depicts the displacement of all landmarks between the target form and those on the reference form.
The absence of clustering of these vectors does not suggest antisymmetry Table 1.
ANOVA Procrustes analysis of dorsal shape of 45 red-footed tortoise Chelonoidis carbonarius carapaces .
males and 32 female.
A/A/Males .
Effect Individual Side Individual* Side Error 1 63y10-05 19y10-05 64y10-05 75y10-08 77y10-05 30y10-05 85y10-07 A/Females .
Effect Individual Side Individual* Side Error 1 Discussion Developmental instability (DI) refers to an organismAos inability to buffer the perturbation of a developmental process under given genetic and/or environmental conditions (Jung et al.
Many studies have shown that DI can be caused by genetic and environmental stresses, such as nutritional deficiencies, genetic diseases, and parasitosis (Jung et al.
Fluctuating asymmetry (FA, random departures from perfect symmetr.
has been established as an indicator of developmental stress, implying a measurable degree of instability (DI) (Graham et al.
Jung et al.
The use of geometric morphometrics, combined with multivariate statistical techniques, yielded a low measurement error, providing clear information on the carapacial asymmetry of adult males and females of C.
Our results reflected a significant FA for both sexes.
To date, available studies of asymmetries among testudines are scarce and sometimes yield contradictory For instance.
Dillard .
found no shell symmetric differences between the two populations of Pseudemys species (P.
concinna and P.
Still.
Davis and Grosse .
found FA in Trachemys scripta, as did BEncilE et al.
in Testudo graeca ibera, while Buica and Cogalniceanu .
detected low levels of FA in this same species.
In a study on T.
hermanni hermanni and T.
hermanni boettgeri, fluctuating asymmetry was observed between the shape of the left and right shield patterns of the plastrons (Parys-Casanova Carreyo AS et al.
et al.
Similarly.
Goessling et al.
detected fluctuating asymmetry in Gopherus polyphemus.
The lack of a relationship between asymmetry and body size, another conclusion of our study, has been observed in studies on other species (BEncilE et al.
Buica and Cogalniceanu 2013.
Goessling et al.
Considering that FA is the product of cumulated effects from stresses over the development history, detected levels of FA demonstrate an inability to buffer developmental noise and achieve an equilibrium.
In any case, as animals were sampled from different areas in Arauca and no consanguinityAia possible developmental perturbation Aiwas expected, our results suggest a high degree of stress and highlight that human intervention in Arauca is affecting wildlife.
No FA studies have been reported in C.
therefore, it cannot be compared.
In conclusion, wild Chelonoidis carbonarius from the Arauca plains in eastern Colombia exhibit fluctuating asymmetry on their carapaces, with sex differences.
These results may suggest a high degree of stress and highlight that human intervention in Arauca is affecting the sexes of C.
carbonarius differently.
The results of this study may represent an estimate of the physical status of the C.
carbonarius Spix 1824 population currently Globally, results highlight the need for increased conservation and management of red-footed tortoises in populations experiencing reduced fitness.
Similar studies in the future, correlated with an estimate of human impact, could provide proof of causality between developmental instabilities and environmental stressors, in Araucanian and other wild populations.
Acknowledgements The authors would like to express their sincere gratitude to the Research CommitteeAiCONADI (Project ID 2.
Aiof the Universidad Cooperativa de Colombia for funding this research, and to the owners of Araucanian farms, who allowed access to their properties for tortoise collection.
Institutional Review Board Statement This study conforms to the appropriate guidelines for the welfare of reptiles.
No ethical approval was necessary, as field research consisted of gentle manipulation of animals for measuring, weighing, and photographing, which were immediately released at the same capture Neither sample collection nor pain manipulation was done.
The procedures of the National Environmental License Authority of Colombia, as outlined in Resolution No.
01877 (October 22, 2.
, were followed.
Data Availability Statement
The datasets used and/or analyzed during the current
study are available from the second author on reasonable References