Morphometric variation of Pipa pipa (Linnaeus

Herpetology Notes, volume 7: 347-353 (2014) (published online on 26 May 2014)
Morphometric variation of Pipa pipa (Linnaeus, 1758)
(Anura: Pipidae) with notes on diet and gonad development
Helena Nery Alves-Pinto1, Vanessa Kruth Verdade2* and Miguel Trefaut Rodrigues1,3
Abstract. Pipa pipa is an emblematic and endemic aquatic frog from the Amazonian Basin, but much of its biology remains
poorly known. In this paper we focused on morphometric variation, diet and gonad examination of P. pipa. We measured
23 morphometric variables in 56 individuals of P. pipa and tested for sexual dimorphism and for geographic variation using
Principal Component Analysis. Data on diet and gonad development were studied in one population. The morphometric
study showed that forearm diameter, finger and snout length are sexually dimorphic characteristics, all larger in males. The
most abundant item found in the species’ diet was Cyclopoida, whereas Heteroptera represented the group with the highest
occurrence in stomachs.
Keywords: Pipidae; sexual dimorphism; diet; morphology; reproduction
Introduction
The frog family Pipidae occurs in Africa and America
and includes peculiar aquatic species (Canatella and
Trueb, 1988; Duellman and Trueb, 1994; Frost et al.,
2006). It is monophyletic and one of the most basal
anuran groups (Canatella and Trueb, 1988; Frost et al.,
2006, Irisarri et al. 2011). Among the four genera in the
family (Hymenochirus, Pipa, Pseudhymenochirus and
Xenopus), Pipa is the only genus occurring in Central
and South America (Dunn, 1948; Canatella and Trueb,
1988; Frost, 2011), from which four out of seven
species occur in Brazil - P. arrabali Izecksohn 1976,
P. snethlageae Müller 1914, P. carvalhoi (MirandaRibeiro, 1937), and P. pipa (Trueb and Cannatella,
1986).
Pipa pipa, the largest species in the genus (Dunn,
1948; Rabb, 1961), has distinct external, osteological
Universidade de São Paulo, Instituto de Biociências,
Departamento de Zoologia, Rua do Matão, travessa 14, nº
10, Butantan 05422-970, São Paulo, SP, Brazil, e-mail:
helenanap@gmail.com
2
Universidade Federal do ABC, Centro de Ciências Naturais e
Humanas, Avenida dos Estados 5001, Bairro Bangu 09210580, Santo André, SP, Brazil
3
mturodri@usp.br
* Corresponding Author: vanessa.verdade@ufabc.edu.br
1
and behavioural characteristics (Trueb and Massemin
2000). The reproductive behaviour involves a turnover
action, during which the couple swims in vertical circles
in the water together while the eggs are released and laid
on the female back (Rabb and Rabb, 1960; Rabb and
Snedigar, 1960; Rabb, 1961). Males are smaller than
females and have thicker forearms (Zippel, 2006). The
female’s vent is rounder and more turgid, especially
during reproductive periods (Rabb and Snedigar,
1960; Rabb, 1961; Zippel, 2006). However, no other
characteristics have been investigated regarding sexual
dimorphism and those listed are either subjective or
observed only during reproductive periods.
Aspects of the biology of Pipa pipa other than
amplexus behaviour have also been neglected and
most information available comes mainly from captive
individuals (Zippel, 2006). Literature on diet of the
species recorded fish as well as anurans of genus
Leptodactylus (Deckert, 1917; Duellman, 1978). During
foraging, the species uses a combination of inertial
suction and forelimb movements, and sometimes prey
can be grasped between digits (Carreno and Nishikawa,
2010).
Reproductive biology and diet are relevant traits
of natural history that affect survival, define habitats
and behaviours (Cuello et al., 2006). The lack of
information on the ecology and biology of species is
usually the largest obstacle to management projects and
conservation of anuran fauna (Greene, 2005; Silvano
and Segalla, 2005; Verdade et al., 2012). The aim of
348
this study was to identify sexual dimorphism from
morphometric characteristics in the species Pipa pipa
and geographical variation on morphometric variables
between populations. We also provide notes on its diet
and gonad development.
Materials and Methods
We studied 61 individuals of Pipa pipa from three
different sources: individuals (n=32) collected in
January 2007 near the left bank of Abacaxis River, São
Sebastião village, Borba municipality, Amazonas state,
Brazil (4º18’32” S, 58º 38’11” W) (MZUSP 142744–
775); individuals (n=4) collected in April 2010 at the
right bank of Madeira river, Abunã village, Porto Velho
municipality, Rondônia state, Brazil (9º37’45” S, 65º
27’19” W) (MZUSP 143285–286, 144395–396); and
individuals (n=25) from different localities, deposited at
the Museu de Zoologia da Universidade de São Paulo
(MZUSP) collection (MZUSP 1006, 2036, 15903,
16066, 16068, 23062, 23119, 23215, 23374–76, 23795,
37519, 54799–800, 58534–35, 80450, 80454–55,
80474–78).
The sample from São Sebastião village (Abacaxis
River) was assembled from a pond approximately six
meters long by six meters wide and 30 centimetres deep,
in an area susceptible to seasonal flooding. The water
was muddy, and leaf litter and mud covered the bottom.
The stream where individuals from Abunã village
were found was approximately one meter deep and
three meters wide, and had similar aspects as the São
Sebastião pond. All individuals were euthanized using
a lethal dose of anaesthetic , preserved in 10% formalin
and stored in 70% ethanol.
Morphometric studies
We tested sexual dimorphism in 32 individuals from
the São Sebastião sample, in which sex was determined
by gonad examination. The other samples were not
included in this analysis since gonad examination was
not possible. Sexual maturity was determined based on
the snout-vent length (SVL) and gonad development.
According to Schuette and Ehrl (1987), only individuals
larger than 60 mm are considered adults. Although
sexual maturity may vary from one population to
the other, we still considered this size limit since all
individuals were larger than 60 mm with the exception
of one, a 36 mm juvenile.
Thirteen measurements were obtained and analysed
in this study: snout-vent length (SVL); body height
(BOH) taken from dorsal to the ventral side of the body
Helena Nery Alves-Pinto et al.
considering an imaginary mid-line between head and
urostile; tibia length (TIL); eye diameter (ED); hand
length (HL); fingers 1 to 4 length (F1 – 4 L); foot length
(FOL) (Verdade and Rodrigues 2007); mouth (Mt);
forearm length (FL) (Márquez-Garcia et al. 2009);
forearm diameter (FD) from dorsal to the ventral side
in an imaginary mid-line between elbow and hand; toes
1 to 5 length (T1 – 5 L) from base to the tip of each toe;
interdigital membranes 1 to 4 length (IM1 – 4 L) from
the base to the tip considering an imaginary mid-line
between toes; and snout length (SL) from the tip of the
mid-line of the jaw to the tip of the snout. We measured
variables on the left side of all individuals, with a digital
calliper.
To test sexual dimorphism we used variables’ residuals
from a regression analysis in order to avoid biases in
morphometric comparisons. Levene’s and Shapiro Wilk
tests were performed to test normality and residual’s
variance. A One-way ANOVA was made with normal
and homogeneous data, whereas a Mann-Whitney U
test was performed with the nonparametric residuals.
We studied morphological geographic variation using
all three samples: from São Sebastião, Abunã and
previously specimen deposited at MZUSP, totalizing
61 individuals (one young excluded from the original
sample) of Pipa pipa from 12 different localities. We
performed Principal Component Analysis to explore
any group formation based on morphometric variation.
All variables listed earlier in this study were included
in the analysis. Statistics was performed in SPSS 20.0
for Windows.
Gonad development
Gonadal studies were restricted to the sample of São
Sebastião, since we could not examine internal anatomy
from the other populations. Testis length, diameter and
volume were measured and compared to male’s weight
and size using correlation analysis. Diameter, weight
and number of developed ova were measured.
Diet
Dietary studies were also restricted to the sample of
São Sebastião, since we could not remove stomach
contents from the other populations. The approximated
time between collection of animals and fixation was of
one day. All individuals had their gastric and intestinal
contents analysed under a stereoscope microscope. Prey
length and diameter were measured, and prey category
identified to the level of Order. Degraded items were
not considered because they could not be identified.
Morphometric variation of Pipa pipa with notes on diet and gonad development
349
Figure 1. Geographic distribution of Pipa pipa based on literature data (Vaz-Silva and Andrade 2009) and on our data.
The frequency of appearance of each prey, as well as
the occurrence of stomachs that contained each prey
was determined. Prey volume was approximated to
the ellipsoid volume, calculated based on the formula
of a prolate spheroid V = 4/3π * L/2 * (W/2)2, where
V=volume, L=length, and W=width.
Results
Morphometric studies
We found 17 males and 15 females in the São
Sebastião sample. The One-way ANOVA showed that
males and females do not differ on snout-vent length
(p= 0.650, F=0.210), but forearm diameter (FD) (p=0.00
F=26.833), finger 2 length (p=0.00; F=12.522) and
snout length (SL) (p=0.03, F=10.614) were significantly
different between males and females. SL represented
between 3.4 to 4.8% of SVL in females, while in males
it ranged from 3.8 to 5.4% of SVL.
To investigate the geographic variation in
morphometric variables of Pipa pipa, data were taken
from 12 localities (Figure 1), nine of which were not
registered by Vaz-Silva and Andrade (2009), who
summarized the distribution of the species. Differences
between groups did not clearly split populations.
According to the Principal Component Analysis, the
350
Helena Nery Alves-Pinto et al.
Figure 2. Plot of scores of the first and second components for the Principal Component Analysis, showing the geographic
variation of different populations of Pipa pipa, based on morphological characters.
first component explained 47.98% of the variation, and
the second explained 26.93%. SVL (0.846), mouth, (Mt)
(0.838) and foot length (FOL) (0.834) were the variables
that most contributed to the separation of groups in the
score 1, whereas finger 2 length (F2L), finger 1 length
(F1L) and finger 4 length (F4L) (0.721; 0.715; 0.712)
contributed the most in score 2 (Figure 2).
Gonad development
Testis volume increased positively and proportionally
with weight (r2 = 0.909, p=0.035) and SVL (r2 = 0.807,
p=0.001). The two mature females (with developed
ova, ready to reproduce; MZUSP 142751, 142768)
had large, yellow ova, in which the vegetative and
the animal poles had the same colour (Figure 3, Table
1). Inside the ovary, smaller ova were found, and the
oviduct occupied a great portion of the visceral cavity.
In immature females the ovary was light yellow and
long, parallel to the kidneys.
Diet
Only 15 out of the 32 digestive tubes examined
contained identifiable items (Table 2). Diet contained
mainly arthropods, of which Cyclopoida was more
abundant than other items and had the highest frequency
(67%), followed by Diptera larvae (7.3%) and
Heteroptera (6.3%). Heteroptera showed the highest
occurrence in stomachs. Even though decapods were not
abundant (3.1%), they represented the higher volume
(61.9 mm3), whereas Cyclopoida represented only 8.5
mm3 (Table 2). Skin fragments were found in three
stomachs, and their appearance could either indicate
these have been from an anuran of the genus Rhinella
Fitzinger 1926 or from its own individual of Pipa pipa,
since many amphibians are known to eat their own skin
(dermatophagy) (Weldon et al. 1993).
Discussion
Morphometric studies
Forearm diameter and snout length were the only
sexually dimorphic characteristics in Pipa pipa, both
larger in males. Forearm diameter was previously
reported in the literature as sexual dimorphic in Pipa
pipa (Zippel, 2006), but the longer snout is a novelty that
may help to externally distinguish sex of individuals.
According to Zippel (2006), literature shows that SVL
is larger in females, whereas we found no differences
between sexes. These contradictory results may be due
Morphometric variation of Pipa pipa with notes on diet and gonad development
Figure 3. Mature female of Pipa pipa with developed ova.
SVL = 110 mm.
Table 1. Data from the mature females from São Sebastião
sample.
Table 1.
Field
Number
(MZUSP)
SVL
(mm)
Weight
(g)
Number of
eggs
Weight of all eggs
(g)
Average
diameter of eggs
(mm)
142751
110
85
74
3.856
4.26
142768
107
110
101
4.614
4.02
351
to the age of the individuals in the samples studied: the
size of the ovule found in females from São Sebastião
were smaller than the fully developed ones described
in literature (Rabb and Snedigar, 1960), and therefore
probably under development (Monnet and Cherry,
2002).
In some anurans males are significantly larger than
females, especially when males are territorial and fight
each other (Shine, 1979; Woolbright, 1983). In Pipa
pipa males are extremely territorial and fight frequently.
Rabb and Rabb (1963) described the vocalization and
physical contact during a fight, in which mainly the
head and forearm were used. This might explain the
larger forearm diameter in males from São Sebastião.
Males with hypertrophied forearms are common among
anurans (Duellman and Trueb, 1994), and Liao et
al. (2012) showed that forelimbs tend to be stronger
in amplectant male specimens of the species Bufo
andrewsi, helping to keep the grip on the female during
amplexus.
Some dimorphic structures may be used in competition
for mates between males as a strategy during contests,
pushing or holding the contender. However, in some
cases the structures may indicate male’s size, strength
and fitness without being effectively used in combat
(Willemart and Gnaspini, 2003). Snout length is an
example of a structure that is not directly used in combat,
but that could indicate size superiority.
The morphological variation among samples studied
may be suggestive of geographic variation. Nevertheless,
considering our limited geographic covering, this could
only be confirmed with additional sampling comprising
a larger number of individuals per locality.
Gonad development
Table 2. Description of preys found as part of the diet in Pipa
pipa. (N) number of individuals; (%n) frequency; (Oc) number
of stomachs with that prey; (%Oc) number of stomachs with
that
prey
Table
2. per total of stomachs; (∑ Vol.) sum of the volumes.
Order
Amphibia
Anura (skin)
3
3.1
3
15.0
5.4
Insecta
Heteroptera
6
6.3
3
15.0
3.4
Diptera
3
3.1
3
15.0
Homoptera
2
2.1
2
10.0
Hymenoptera
4
4.2
4
20.0
Coleoptera
1
1.1
1
5.0
Orthoptera
1
1.1
1
5.0
7
7.3
2
10.0
6.3
Diptera larvae
(chironomidae)
N
%n
Oc
%Oc
volume
Class
(mm3)
6.2
Aracnida
Acari
1
1.1
1
5.0
0.0
Crustacea
Decapoda
3
3.1
3
15.0
61.9
Copepoda
Cyclopoida
64
67.0
5
25.0
8.5
Total = 95
Rabb and Snedigar (1960) found ova with an average
diameter of 6 mm in females that spawned between 96
and 273 eggs. Even though the number of ova recorded
in the literature is similar to the number we found, their
sizes suggest that females from this study were not fully
ready for reproduction. Smaller ova as well as the low
number of reproductive females in our sample may be
explained either by the seasonality (early rainy season,
when this species reproduce), or due to young age of the
population sample.
Diet
According to the results, Pipa pipa has an opportunist
diet related mainly to the aquatic environment.
Literature recorded animals captured in nature to feed
on fish - Erytrinus erytrinus (Bloch and Schneider
352
1801) (Duelman, 1978) and a frog genus Leptodactylus
Fitzinger 1826 (Deckert, 1917). In captivity it was
recorded to feed on different types of items, from
bovine meet to copepods (Rabb and Snedigar, 1960;
Rabb, 1969; Palmer, 1994).
The little amount of preys found in the sample studied
in this work may be a consequence both of the gap time
between the animals were collected and euthanized,
allowing the preys to be digested; or scarcity of prey
items due to the season, since the water level of the
ponds may influence the frequency and availability
of prey in the habitat, as well as competition among
individuals (Deckert, 1917; Duelman, 1978; Palmer,
1994). Nevertheless, composition of Pipa pipa`s diet
was similar to other species in the same genus: P.
aspera, P. arrabali, and P. carvalhoi were all recorded
to have ingested arthropods, such as nymphs and larvae
of insects, and aquatic invertebrates, as well as flying
and terrestrial prey (Trueb and Massemin 2000, Canedo
et al., 2006; Garda et al. 2006). The latter may indicate
either that these fall into the pond or that these species
go forage in land (Buchacher, 1993).
Conclusions
This study presented some new information about
the species Pipa pipa, such as new sexually dimorphic
morphometric structures, as well as its occurrence in new
geographic locations. Furthermore, it added preliminary
information about geographic morphometric variation
among populations, suggesting further studies with
larger samples should be informative. Finally, although
seasonality may have interfered in our sample, more
information was provided regarding Pipa pipa`s diet
and gonad characteristics.
Acknowledgements. We thank J. M. Guellere, J. Cassimiro, M.
Teixeira Jr, S. M. Sousa, P. Nunes and M. Sena for help in field;
C. Mello and H. Zaher for the access to MZUSP collection. We
are very thankful for S. Bueno, D. Caetano and P. Fuentes for
the identification of diet items. Teresa Ávila and an anonymous
reviewer for revising the article. FAPESP and CNPq provided
funding for this project. Authorization for collection was given
by IBAMA to MTR (Proc. 10126-1).
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Accepted by Mirco Solé