601

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

602

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).

603

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.

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.

604

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.

605

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.