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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(2): 601-614, April-June 2021 (Published May 14, 2021)

Sexual dimorphism in the turtle Kinosternon scorpioides (Testudines:

Kinosternidae) from Marajó Island, Brazilian Amazon

Joilson Silva da Silva

Brenda Stefany dos Santos Braga

Juliane da Silva Costa

Leandro Schlemmer Brasil

Verônica Regina Lobato de Oliveira-Bahia

Relionan Pimentel Leal

José Ribamar Felipe Marques

Diva Anélie de Araújo Guimarães

1. Institute of Biological Sciences, Universidade Federal do Pará, Belém, Pará, Brazil; jss26joilson@gmail.com,

brenda.braga@icb.ufpa.br, jsc.zootecnista@gmail.com (*Correspondence), leandrobrasilecologia@gmail.com,

veronicaoliveirabahia@gmail.com, diva@ufpa.br

2. Empresa Brasileira de Pesquisa Agropecuária, Embrapa Amazônia Oriental, Belém, Pará, Brazil;

relionan.leal@gmail.com, jrrffm@me.com

Received 08-VII-2020. Corrected 06-II-2021. Accepted 22-IV-2021.

ABSTRACT

Introduction: Morphometrics analysis is an efficient and low-cost technique used in studies of sexual dimor-

phism in turtles. Kinosternon scorpioides scorpioides, scorpion mud turtle, has a wide phenotypic variation,

depending on the area of its occurrence. Objective: The objective of this work was to identify the anatomical

sexual difference of K. s. scorpioides, adults and hatchlings, through morphometric analysis; and relate the

weights of adult animals to environmental factors (temperature and rainfall) in Marajó Island, Brazil. Methods:

The sample collection was carried out from March to September 2018, covering both the rainy season (January

to July) and the dry season (August to December). For the biometric analysis, 95 adults and 21 hatchlings were

used, in which the length and width of the carapace and plastron, height of the shell, and weight were measured

(adults only). For the geometric morphometry analysis, 21 adults and 13 hatchlings were used, in which 27 coor-

dinates of anatomical landmarks were inserted in each image of the carapace and 11 in the plastron. Hatchlings

were sexed by histology which was enabled by the identification of the ovaries and testicles. Results: The results

showed the existence of dimorphism in adults. The plastron and height were higher in females, which had a

more rounded carapace than males. This characteristic may be related to the species’ sexual strategy, where

males impose copulation. Histologically, it was possible to identify the ovaries and testicles in the hatchlings, but

there was no anatomical sexual difference, despite the tendency to differentiate in the analysis of carapace PCA.

Conclusions: Sexual dimorphism in K. s. scorpioides may play an important role on its reproductive behavior,

which is synchronized with environmental events. This fact suggests that the reproductive strategies of this spe-

cies would be severely affected by changes in the ecosystem.

Key words: biometrics; geometry morphometrics; scorpion mud turtle; sexual difference; turtles.

Silva da Silva, J., dos Santos Braga, B.S., da Silva Costa, J.,

Schlemmer Brasil, L., Lobato de Oliveira-Bahia, V.R.,

Pimentel Leal, R., Felipe Marques, J.R., & de Araújo

Guimarães, D.A. (2021). Sexual dimorphism in the turtle

Kinosternon scorpioides (Testudines: Kinosternidae) from

Marajó Island, Brazilian Amazon. Revista de Biología

Tropical, 69(2), 601-614. https://doi.org/10.15517/rbt.

v69i2.42834

https://doi.org/10.15517/rbt.v69i2.42834

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Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(2): 601-614, April-June 2021 (Published May 14, 2021)

The turtles or Testudines are represented

by 356 species, and considering the subspecies,

they total 478 modern taxa, divided into 13

families (Rhodin et al., 2017). Of this total, 20

% are found in South America (Souza, 2004;

Ferreira-Júnior, 2009). Thirty-six species are

known in Brazil (Costa & Bérnils, 2018), out

of which 17 terrestrial species have been iden-

tified in the Brazilian Amazon, 15 aquatic and

two terrestrial species.

Studies on the biological processes related

to the growth, change in the size, shape and

body pattern of turtles, can be carried out

through morphometric analysis of the carapace

and plastron. This technique has been applied

to distinguish morphological variations by ana-

tomical landmarks (Monteiro & Reis, 1999),

proving to be accurate for phylogenetic stud-

ies and sexual differentiation of these animals

(Valenzuela, Adams, Bowden, & Gauger, 2004;

Depecker, Berge, Penin, & Renous, 2006;

Ferreira-Júnior, Treichel, Scaramussa, & Scal-

foni, 2011; Sönmez, Bağda, Candan, & Yilmaz,

2019). Morphological analyses allow research-

ers to precisely identify the shape and size of

biological organisms, allowing the observa-

tion of their morphological changes (Van Der

Molen, Martínez-Abadía, & González, 2007).

It has as an advantage of increasing the feasi-

bility of collecting samples, as only an analysis

of the photograph of the animal is necessary

(Lyra, Hatadani, Azeredo-Espin, & Klaczko,

2010). It is important to understand that most

of the techniques used in sexing chelonian

hatchlings are largely impractical and invasive,

even requiring to slaughter the animal (Valen-

zuela et al., 2004). Therefore, it is necessary to

apply non-invasive techniques, to differentiate

the sex of the hatchlings. Sexing is a useful tool

in management practices in the wild or captiv-

ity, enabling the correct intervention for the

reintroduction and release of animals, or in the

formation of groups.

The scorpion mud turtle (Kinosternon

scorpioides scorpioides) is geographically dis-

tributed from Panama to Argentina and inhab-

its permanent, semi-permanent and temporary

aquatic environments (Iverson, 2010; Berry

& Iverson, 2011). This species is one of the

smallest turtles of the Amazon forest, measur-

ing from 18 to 27 cm in length (Vogt, 2008).

Its conservation status in the Brazilian terri-

tory is categorized as least concern (Vogt et

al., 2015), despite it being the only species of

the Kinosternidae family occurring in Brazil

(Viana, Santos, & Antunes, 2015). It is severely

exploited by illegal consumption and trade in

many places (Ferrara et al., 2016), especially

in Marajó island, State of Pará, Brazil (Cristo,

Baía-Júnior, Silva, Marques, & Guimarães,

2017). In addition, there are few conservation

and management programs for this species.

This animal is well adapted to captive condi-

tions; however, it is necessary to expand the

knowledge about its nutritional requirements

and management (Costa et al., 2017). In this

instance, there is little information of the

aspects regarding the reproductive biology

of the free-living species. Nevertheless, it is

known that they copulate on land and in shal-

low waters. Their nests are found at the base

of the roots of shrubs, away from water bodies

(Ferrara et al., 2016). Sex is determined by the

temperature of incubation during embryonic

development (Ewert & Nelson, 1991; Rueda-

Almonacid et al., 2007), and sexual maturity is

attained when the animal reaches 2.8 to 5 years

old (Vogt, 2008).

The phenotypic difference between adult

males and females, raised in captivity, is related

to body size, plastron shape, weight, tail size

and pigmentation in the male’s head (Castro,

2006). The adult female measures an aver-

age of 15.26 cm in length and 430.08 g in

weight, and the male 14.79 cm and 314.05

g, respectively (Castro, 2006). Generally, the

first nesting occurs, when the female reaches

10 cm in carapace length (Barreto, Lima, &

Barbosa, 2009). In males, sexual maturity is

reached at 10 cm (Barreto et al., 2009) to 13.2

cm in carapace length (Vogt, 2008). However,

a variation in the body size has been found in

the wild, where males may be larger or smaller

in relation to females (Berry & Iverson, 2011).

Hatchling turtles in captivity are on average 2.6

cm long and weigh 2.8 g (Costa et al., 2017).

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They do not show any apparent sexual dimor-

phism until they reach 20 months of age (9.4

cm in length), when there is an increase in the

tail and the presence of pigmentation on the

head of the males (Castro, 2006).

Thus, the main objective of this research

was to identify the sexual morphological differ-

ence of K. s. scorpioides through the morpho-

metric analysis of adult animals and hatchlings

turtles, in addition to relating the weight mea-

surements of adult animals to environmental

factors (temperature and rainfall) in Marajó

island, Pará, Brazil.

MATERIALS AND METHODS

Data collection: Samples from 95 wild

adults K. s. scorpioides, for traditional mor-

phometrics (biometrics) and weight analy-

sis, were collected in Cachoeira do Arari,

Mesoregion of Marajó island, Pará, Brazil,

specifically in Guajarás farm (0°37’7.50” S &

48°58’59.68” W) (Fig. 1). The animals were

captured manually in the wild, followed by

release. No anesthetic was used, with a short

physical containment time. In addition, photo-

identification technique of the animal’s head

was used for records, as it has different marks,

avoiding recapture. The meteorological data

were obtained from the National Meteorologi-

cal Institute of Brazil. The annual temperature,

rainfall and humidity, for 2018, were: 26.16 ±

4.15 ºC, 987.8 ± 9.66 mm, and 78.59 ± 13.00

% for the rainy season (January to July), and

27.61 ± 3.33 ºC, 56.1 ± 4.35 mm, and 72.58 ±

10.02 % for the dry season (August to Decem-

ber), respectively (Moraes, Costa, Costa, &

Costa, 2005). The research was carried out

between March to September 2018, 48 females

and 25 males were captured in the rainy season,

and 13 females and nine males in the dry sea-

son. The reason that few animals were captured

during the dry season, is due to the behavior of

this species, which remains in estivation buried

in the soil, and it is hardly observed in the fields

(Berry & Iverson, 2011; Cristo et al., 2017).

Samples from 21 adults and 13 hatchling

turtles, for geometric morphometry study, were

collected from the scientific research breed-

ing facility of Empresa Brasileira de Pesquisa

Agropecuária - Embrapa Amazônia Oriental

(License number 7310/2014-SEMAS-PA),

located in Salvaterra, Marajó island, Pará, Bra-

zil, (0°42’26.90” S & 48°33’34.70” W) (Fig.

1). In addition to the 13 samples, more eight

hatchlings were also obtained for biometry, a

total of 21 animals. Hatchlings were sexed by

histological analysis of the gonads. The testi-

cles and ovaries were fixed in Bouin’s solution

and preserved in 70 % alcohol. The samples

were dehydrated in an increasing ethanolic

solution (70, 80, 90 and 100 %), diaphanized in

xylol and included in paraffin. Posteriorly, his-

tology sections 5 µm were stained with Hema-

toxylin and Eosin, and the slides were analyzed

with optical microscopy.

The research performed were approved

by the Chico Mendes Institute for Biodiver-

sity Conservation (ICMBio-SISBIO 59314-1),

the Ethics Committee for Animals Research

- Universidade Federal do Pará (Authorization

number CEUA 7749240817), and the Ethics

Committee for Use of Animals - Embrapa

(Authorization number 001-2016).

Morphometrics: Ninety-five adults scor-

pion mud turtle (61 females and 34 males)

and 21 hatchlings (17 females and four males)

were used for biometric analysis. Ten anatomi-

cal points were identified on the animals, and

the following morphometric characters were

analyzed (Castro, 2006; Costa et al., 2017):

1- Rectilinear carapace length (CL); 2- Rec-

tilinear carapace width (CW); 3- Rectilinear

plastron length (PL); 4- Rectilinear plastron

width (PW); 5- Shell height (HCP), as shown

in Fig. 2.

Adult animals were measured with a 300

mm pachymeter, 0.02 mm scale (MTX®), and

the hatchlings with a 150 mm pachymeter,

0.02 mm scale (Starfer). A digital scale (FWB

Model 41375), range from 1 g to 7 kg, was used

to weigh adults. Weights of the hatchlings was

not obtained, because the samples were ana-

lyzed after the removal of the animal organs.

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Twenty-one adults (11 females and 10

males) and 13 hatchlings (nine females and

four males) scorpion mud turtles were selected,

by the more visible intersections of the shields,

for geometric morphometry analysis. The ref-

erence points were measured according to

the intersections of the vertebral and internal

shields of the carapace (Valenzuela et al., 2004;

Mendes, 2017). Twenty-seven coordinates of

anatomical landmarks (Ferreira-Júnior et al.,

2011; Mendes, 2017) were inserted in each

image of the carapace, and 11 in the plastron

(Fig. 3). This study used type-1 anatomical

landmarks (juxtapositions of different tissues),

and the reference points were marked with a

TPSDIg2 software, and analyzed in the Mor-

phoJ software (Klingenberg, 2011).

The carapace and plastron of the scorpion

mud turtle were recorded with an f/2.8 aperture

digital camera (Canon Eos Rebel T5) equipped

with a 60 mm macro lens, mounted on a level

tripod, at a focal distance of 15 cm in height

from the object (Domingues, 2015; Mendes,

2017). A 30 cm ruler was used for scale refer-

ence. Posteriorly, files were imported into the

tpsUtil software, the distortion generated by

unwanted curves was removed statistically, and

converted the JPEG-format photographs into

TPS (Haas, 2011).

Statistical analysis: First, one permuta-

tional multivariate analysis of variance test

(PERMANOVA) was generated, and later one

t-test for each variable, both using a Bonfer-

roni alpha of 0.025 to avoid type I error. The

PERMANOVA was generated with a biometric

data matrix using Euclidean distance (Ander-

son, 2005), separately, differentiated the males

from the females. The homogeneity of variance

for the t-test was tested with the Levene test

(Vieira, 1997). Pearson’s correlation coefficient

was applied to check the degree of association

Fig. 1. Arari microregion in Marajó island, Pará, Brazil. Grey area for the municipality of Cachoeira do Arari and Salvaterra,

places of samples collection of K. scorpioides scorpioides.

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between the weight and the biometric measure-

ments of adult animals. The normality of the

variables for Pearson’s Correlation was tested

with the Shapiro Wilk test (Vieira, 1997). The

correlation analyses between environmental

variables and weight were performed using

the GAM test (generalized additive model),

due to the appearance of non-linear patterns in

the data (Sothe, Camargo, Gerente, Rennó, &

Monteiro, 2017). All analyses were performed

using the R software (R Core Team, 2018).

PERMANOVA was made with the adonis

function of the Vegan package (Oksanen et al.,

2010). The t-test was performed with the t.test

function, the Levene test with the levene.test

function, the Shapiro test with the shapiro.test

function, all from the basic R

package. The

GAM model was made with the gam function

of the gam package (Hastie, 2015) using a

Bonferroni alpha of 0.025 to avoid type I error.

Analysis of geometric morphometry was

performed on the MorphoJ software. Males

and females were graphically ordered through

the analysis of the principal components (PCA)

(Klingenberg, 2011). Analysis of the canoni-

cal variation (CVA) and discriminant function

was performed to test the differences. Thus,

it was possible to visualize the variations by

deformation grades in vector displacement

(Valenzuela et al., 2004).

Fig. 2. Corporal biometrics in Kinosternon scorpioides scorpioides (2 cm scale). CL: Rectilinear carapace length, straight

line from the nuchal to the supracaudal shield. CW: Rectilinear carapace width, distance between the edges of the marginal

shields. PL: Rectilinear plastron length, straight line from the gular shields to the anal junction. PW: Rectilinear plastron

width, distance between the ends of the intersection line of the pectoral and abdominal shields. HCP: Shell height, distance

between the vertebral shields from the carapace to the plastron.

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RESULTS

No significant difference was observed

among the biometric measurements observed

between adult males and females, consid-

ering all variables together (PERMANOVA,

pseudo-F = 3.488, R² = 0.036, P = 0.082).

No significant difference between adult males

and females was observed by Student-t on the

length of the carapace (T = -0.559, df = 69.737,

P = 0.577), width of the carapace (T = 1.286, df

= 92.843, P = 0.201), width of the plastron (T

= 1.950, df = 90.031, P = 0.054) and the weight

(T = 2.110, df = 900.350, P = 0.037). However,

a difference was observed between males and

females on the length of the plastron (T =

2.286, df = 92.252, P = 0.024) and the height of

the shell (T = 3.767, df = 92.384, P = 0.0001).

Thus, adult females have longer plastrons and

taller shells than males, despite the similar

carapace measurements (Table 1).

Strong correlations were observed

between weight and body measurements of

adult females, which were greater than 90 %

for carapace and plastron length, and 80 % for

carapace and plastron width and shell height.

Fig. 3. Anatomical landmarks of Kinosternon scorpioides scorpioides (2 cm scale). A. Carapace of adult turtle; B. Adult

plastron; C. Carapace of hatchlings turtle; D. Hatchlings plastron.

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Males also showed strong correlations between

weight and body measurements, with carapace

width and plastron length greater than 70 %,

followed by carapace length greater than 60

%, plastron width greater than 50 % and shell

height at 40 % (Table 2).

Although there are visual trends, there was

no relationship in adult female’s weight con-

sidering rainfall increased (Explained deviance

= 12.5 %, R²(adj) = 0.08, F = 1.922, edf = 3,

Ref.df = 3.37, P = 0.123, Fig. 4A), as weight

and temperature in males (Explained deviance

= 17.7 %, R²(adj) = 0.128, F = 2.609, edf = 2,

Ref.df = 2.161, P = 0.077, Fig. 4B).

Geometric morphometry results showed

an overlap of the anatomical landmarks in the

principal component analysis (PCA) of the

carapace of adult K. s. scorpioides, despite

the trend in the differentiation between the

sexes. Males were more distributed within

the PC2 negative values, while females were

distributed between PC1 negative values and

PC2 positive values (Fig. 5A). PC1 explained

38.88 % (Eigenvalues = 0.001) of the variation

and the PC2 explained 28.13 % (Eigenval-

ues = 0.0007). The greatest variation within

the sample occurred at landmarks 17 and 18,

these points are left-posterior and right to

the fifth vertebral shield, respectively, and

landmark 19 located caudally to the fifth ver-

tebral shield (Fig. 3A). The canonical varia-

tion analysis (CVA) showed the significant

TABLE 1

Biometric measurements (t-test) and weight of wild Kinosternon scorpioides scorpioides, adult male (N = 34)

and female (N = 61), in Marajó Island, Pará, Brazil

Variables

Average ± SD

Female

Min - Max

Average ± SD Male

Min - Max

T df P

CL (mm) 144.37 ± 14.37 104.2-171.8 146.07 ± 14.03 90.22-168.84 0.56 69.74 0.577

CW (mm) 92.50 ± 9.77 62.08-106.8 90.52 ± 5.18 80.08-101.98 1.29 92.84 0.201

HCP (mm) 55.71 ± 6.91 35.6-68.9 51.68 ± 3.50 43.64-58.8 3.77 92.38 0.0001*

PL (mm) 131.91 ± 14.23 90.98-154.4 126.54 ± 8.64 101.68-149.34 2.29 92.25 0.024*

PW (mm) 65.37 ± 7.0 42.62-77.08 63.03 ± 4.66 50.28-69.28 1.95 90.03 0.054

Weight (g) 401.07 ± 106.36 133-590 362.77 ± 69.92 236-587 2.11 90.35 0.037

CL: carapace length; CW: carapace width; HCP: carapace height; PL: plastron length; PW: plastron width; SD: standard

deviation; Min-Max F: minimum and maximum female values; Min-Max M: minimum and maximum male values; T:

standardized T-score; df: degrees of freedom; P: the attained level of significance; *: P values that showed significant results

of sexual dimorphism (t-test) using alpha P < 0.025.

TABLE 2

Pearson’s correlation coefficient between weight and biometric measurements of wild Kinosternon scorpioides

scorpioides, adult females (N = 61) and males (N = 34)

CL CW HCP PL PW Weight (g)

F M F M F M F M F M F M

CL 1.000 1.000

CW 0.859 0.567 1.000 1.000

HCP 0.838 0.383 0.787 0.314 1.000 1.000

PL 0.937 0.528 0.863 0.726 0.845 0.441 1.000 1.000

PW 0.874 0.390 0.840 0.717 0.817 0.379 0.905 0.660 1.000 1.000

WEIGHT 0.932 0.667 0.852 0.773 0.846 0.458 0.931 0.778 0.849 0.555 1.000 1.000

CL: carapace length, CW: carapace width, HCP: carapace height, PL: plastron length, PW: plastron width.

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difference (Mahalonobis P < 0.001, Procrustes

P = 0.0002) between the carapaces of adult

males and females (Fig. 5B). The discriminant

function analysis also showed a significant dif-

ference between the sexes of adults (P < 0.001).

The graphical reconstruction of the PCA indi-

cated a difference between the shields of the

carapace of the females, which are larger than

the males. This aspect makes the carapace of

females rounded, while the males had an oval

shape (Fig. 5C).

The plastron analysis of PCA of the adults

showed that there is no separation between

the sexes of scorpion mud turtle, as both are

distributed in the two main axes (Fig. 5D). PC1

explained 34.94 % (Eigenvalues = 0.0003) of

the variation and the PC2 explained 20.95 %

(Eigenvalues = 0.0002). The greatest varia-

tion within the sample occurred in landmarks

7, 8 and 9. Located on the left lateral portion

of the plastron (Fig. 3B), on the left side of

the intersection of the pectoral and abdominal

shields (landmark 9), abdominal and femoral

shields (landmark 8), and femoral and anal

(landmarks 7). However, analyses of CVA (Fig.

5E) (Mahalonobis P < 0.0001, Procrustes P =

0.0002) and discriminant function were signifi-

cant (P < 0.001). The graphical reconstruction

of the PCA showed the distinction between the

sexes (Fig. 5F).

PCA analysis of the carapace of hatchlings

K. s. scorpioides turtles showed an overlap

of anatomical landmarks, despite the trend in

differentiating between the sexes. Males were

more distributed within the PC1 and PC2

positive values; while females were distrib-

uted between positive and negative PC1 and

negative PC2 values (Fig. 5G). PC1 explained

30.08 % (Eigenvalues = 0.001) of the variation

and the PC2 explained 24.53 % (Eigenvalues

= 0.0008). The greatest variation within the

sample occurred in landmarks 3 and 4, located

caudally to the first vertebral shield (Fig. 3C).

However, in the canonical variation analysis

(CVA), it was not possible to find a significant

difference (Mahalonobis P = 0.022, Procrustes

P = 0.074) between the carapaces of male and

female hatchlings (Fig. 5H). Furthermore, the

discriminant function analysis did not show

a separation between the sexes (P > 0.001).

Graphical reconstruction of the PCA showed

the similarity between the carapace of males

and females (Fig. 5I).

The plastron analysis of PCA of the hatch-

lings showed that the dispersion of male and

female was homogeneous between values of

PC1 and PC2. PC1 explained 36.51 % (Eigen-

values = 0.0005) of the variation and PC2

explained 17.52 % (Eigenvalues = 0.0003). The

greatest variation within the sample occurred

Fig. 4. Analysis of the correlation between the weight of adult Kinosternon scorpioides scorpioides, A. Females and B.

Males and the environmental variables (rainfall and temperature) in Marajó island, Brazil.

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Fig. 5. Analysis of geometric morphometry of the Kinosternon scorpioides scorpioides. Adult individuals are represented

from A to F. A. Dispersion of the anatomical landmarks of the carapace; B. Analysis of CVA, 10 000-times amplified of the

carapace of adult. C. Graphical reconstruction of the carapace; D. Dispersion of the anatomical landmarks of the plastron;

E. Analysis of CVA, amplified 10 000 times, of the plastron; F. Graphical reconstruction of the plastron. The same analyzes

are observed on the hatchlings individuals from G to L, respectively. * indicates overlapping between females and males,

black lines: shape of the carapace, gray lines: consensus configurations; gray dots or bars: female, black dots or bars: male;

1: females and 2: males.

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in landmarks 3 and 9, located on the right

and left laterolateral portion of the plastron,

respectively (Fig. 3D). It was not possible to

observe a separation of the sexes of scorpion

mud turtle (Fig. 5J); neither the CVA analysis

(Fig. 5K) (Mahalonobis P = 0.006, Procrustes P

= 0.511) nor the discriminant function were not

significant (P > 0.001). The similarity between

the plastron of males and females (Fig. 5L) was

possible to observe in the graphical reconstruc-

tion of the PCA.

Histological sections of the gonads of the

hatchlings K. s. scorpioides showed the cortical

region of the ovaries with primordial follicles,

well delimited by the connective tissue stroma.

The testicles were covered by the tunica albu-

ginea, and interstitial cells were observed in

the stroma between the seminiferous tubules,

which contained the spermatogonia.

There was no significant difference in bio-

metric values observed with male and female

hatchlings, considering all variables together

(PERMANOVA, pseudo-f = 1.116, R² = 0.055,

P = 0.369) and in any measure tested with the

t-test (Table 3).

DISCUSSION

The identification of dimorphism between

the sexes of turtles, through corporal mor-

phometry, gives valuable tools for application

in taxonomy and management of species. This

study was the first to use morphometric analy-

sis in a natural population of K. s. scorpioides

on Marajó Island, Pará, Brazil.

The results indicated that there is sexual

dimorphism in wild adult scorpion mud turtles.

Measures related to plastron and height were

more evident in females. Although the carapace

has biometric similarity between the sexes, the

geometric shape observed in the PCA graphical

representation demonstrated that this structure

is more elongated in males, when compared

to the rounded aspect of the female. Varia-

tions in body size according to the geographi-

cal location was observed, specifically in K.

scorpioides natural populations, where males

may have greater or smaller carapace lengths

than females (Acuña-Mesén & Arturo, 1994;

Márquez, 1995; Iverson, 2010; Berry & Iver-

son, 2011; Moura et al., 2015). Furthermore,

females of this species have a more curved

body shape, and a larger plastron than males

(Berry & Iverson, 2011). Such body character-

istics in Kinosternidae, according to Berry and

Shine (1980) would be related to sexual behav-

ior strategies, where males of the same size or

larger than females impose copulation, instead

of being chosen through courtship. In addition,

it is possible that population genetic variability

influences morphological pattern of this group.

It is known that the K. s. scorpioides has

a seasonal reproductive period, which starts

in late December, with the mating peak in late

March and early April, and laying between

TABLE 3

Biometric measurements of hatchlings in captivity of Kinosternon scorpioides scorpioides,

and significance test between males (N = 4) and females (N = 17)

Variables

Average ± SD

Min-Max

Average ± SD M

Min-Max

T df

CL (mm) 29.11 ± 2.25 24.72-33.30 27.15 ± 5.55 19.500-32.600 0.69 3.24 0.533

CW (mm) 19.68 ± 3.78 11.00-28.20 21.27 ± 4.67 17.880-28.100 -0.63 3.97 0.561

HCP (mm) 12.18 ± 2.10 9.40-17.20 10.87 ± 2.02 8.500-13.120 1.16 4.66 0.301

PL (mm) 23.33 ± 3.86 10.00-27.96 25.32 ± 1.92 23.500-27.660 -1.48 9.74 0.170

PW (mm) 14.02 ± 0.90 13.00-15.90 15.81 ± 3.15 13.740-20.500 -1.8 9.74 0.169

F: female; M: male; CL: carapace length; CW: carapace width; HCP: carapace height; PL: plastron length; PW: plastron

width; SD: standard deviation; Min-Max F: minimum and maximum female values; Min-Max M: minimum and maximum

male values; T: standardized T-score; df: degrees of freedom; P: the attained level of significance.

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late June and early August, concentrated in the

rainiest season on Marajó Island, where there

is more food supply (Costa et al., 2017). This

period is followed by the dormancy behavior

of the animals during the dry season (Cristo

et al., 2017). In the present study, weight gain

in females was not statistically significant cor-

related with the reduction in rainfall, and in

males with the increase in temperature. How-

ever, it is known that any minimal change in

environmental factors could affect the growth

and reproduction of the turtles (Lovich, Ennen,

Agha, & Gibbons, 2018). Furthermore, the

result of this work demonstrated a biological

trend that, in the wild, adult females and males

showed weight gain at least until the rainy

period, thus storing energy reserves for both

reproduction and the dormancy season. As

observed in the literature, Homopus signatus

signatus (Loehr, Hofmeyr, & Henen, 2007) and

Chelonia mydas (Stubbs, Marn, Vanderklift,

Fossette, & Mitchell, 2020), the environmental

changes (e.g., temperature and rainfall) have a

strong impact on the growth and reproduction

of turtles, being associated with the availability

of food. Based on this mechanism, reproduc-

tion in wild animals occurs in the period of the

year most propitious for the survival of parent

and young, and in reptiles the environmental

factors may directly trigger physiological of

this process (Kuchling, 1999).

Concerning hatchlings, ovarian histology

was characterized by the presence of primordial

follicles, and the testicles by the observation of

the seminiferous tubules. Hatchling biometry

was similar to the results observed by Costa et

al. (2017). However, there was no sexual differ-

ence in relation to body morphometry, despite

the tendency to differentiate in the PCA analy-

sis of the carapace. Possibly, the small sample

number of males (N = 5) used in this work

could have contributed to the non-morphomet-

ric differentiation, which was also a negative

factor in the analyses of C. mydas (Sönmez

et al., 2019). Even though, the sexual identi-

fication of Caretta caretta hatchlings was also

only possible through the histological observa-

tion of the gonads, there was no difference in

geometric morphometry (N = 174, Mendes,

2017). In contrast, in hatchlings of Podocnemis

expansa (N = 230) and Chrysemys picta (N =

87), sexed by radioimmunoassay or by gonadal

macroscopic observation, it was possible to dif-

ferentiate sexual dimorphism using geometric

morphometry (Valenzuela et al., 2004).

The biological significance of the mor-

phological difference in adult animals, could

be related to the reproductive behavior of the

species. In addition, the results of this work

reinforce the idea that environmental events

are the triggers that support the reproductive

strategy of these animals. As the degradation

of the environment and large scale hunting

are the greatest threats to the species (Berry &

Iverson, 2011; Cristo et al., 2017), its repro-

ductive strategy suggests that it would not be

adapted to severe anthropogenic changes in the

ecosystem. Thus, the data obtained in this work

provide information for a better understanding

in situ and ex situ management of the species.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

Our thanks to Embrapa Amazônia Orien-

tal - Campus Experimental Emerson Salimos,

and the Guajarás farm for the logistical sup-

port. Joilson Silva da Silva and Brenda Stefany

dos Santos Braga thank the Coordenação de

Aperfeiçoamento de Pessoal de Nível Supe-

rior (CAPES) and Postgraduate Program in

Biodiversity and Conservation of the Universi-

dade Federal do Pará for master’s scholarship.

Juliane da Silva Costa and Brenda Stefany dos

Santos Braga thank CAPES and Post-Graduate

Program in Animal Science of the Universi-

dade Federal do Pará for doctorate scholarship.

612

Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(2): 601-614, April-June 2021 (Published May 14, 2021)

The authors thank Michele Singh (Caribbean

Agricultural Research and Development Insti-

tute), for assistance with the comments, and

Martín Roberto Del Valle Alvarez for translat-

ing the summary into Spanish.

RESUMEN

Dimorfismo sexual de la tortuga Kinosternon

scorpioides (Testudines: Kinosternidae) en

la isla de Marajó, Amazonia brasileña

Introducción: La morfometría es una técnica efi-

ciente y de bajo costo, utilizada en estudios de dimorfis-

mo sexual en tortugas, mediante el marcado de puntos

anatómicos. Este grupo incluye Kinosternon scorpioides

scorpioides, con una amplia variedad fenotípica, depen-

diendo del área en la que se encuentra presente. Objetivo:

Este estudio tuvo como objetivo identificar la diferencia

sexual entre K. s. scorpioides, adultos y juveniles, a través

del análisis morfométrico; y verificar la relación entre las

medidas de peso para adultos y los factores ambientales

(temperatura y precipitación) en la isla de Marajó, Brasil.

Métodos: La recolecta se realizó de marzo a septiembre

de 2018, cubriendo tanto la temporada de lluvias (enero a

julio) como la estación seca (agosto a diciembre). Para el

análisis biométrico, se utilizaron 95 animales adultos y 21

crías, en los que se midió la longitud y el ancho del capa-

razón y el plastrón, la altura del casco y el peso en adultos.

Para la morfometría geométrica, se utilizaron 21 adultos y

13 juveniles, en los que se identificaron 27 coordenadas

de puntos de referencia anatómicos en el caparazón y 11

en el plastrón. Los individuos recién eclosionados fueron

sexados por histología de rutina, que permite identificar

ovarios y testículos. Resultados: Los datos analizados

mostraron que existe dimorfismo sexual en adultos. El

plastrón y la altura fueron mayores en las hembras, que

tenían un caparazón más redondeado que los machos. Esta

característica puede estar relacionada con la estrategia

sexual de la especie, donde los machos imponen la cópula.

En los juveniles recién eclosionados fue posible identificar

histológicamente los ovarios y los testículos, pero no hubo

diferencias sexuales en relación con la morfometría corpo-

ral, a pesar de la tendencia a diferenciarse en el análisis de

PCA de caparazón. Conclusiones: El dimorfismo sexual en

K. s. scorpioides puede desempeñar un papel importante en

su comportamiento reproductivo, que ocurre en sincronía

con los eventos ambientales. Este hecho sugiere que las

estrategias reproductivas de esta especie se verían grave-

mente afectadas por los cambios en el ecosistema.

Palabras clave: biometría; diferencia sexual; morfometría

geométrica; tortuga casquito; quelonio.

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