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