Int. J. Aquat. Biol. (2021) 9(4): 258-263
ISSN: 2322-5270; P-ISSN: 2383-0956
Journal homepage: www.ij-aquaticbiology.com
© 2021 Iranian Society of Ichthyology

Original Article
Body shape variation of Garra rufa (Teleostei, Cyprinidae) populations in the Tigris basin

in Iran using geometric morphometric analysis

Maryam Saemi-Komsari1, Hamed Mousavi-Sabet*1, Masoud Sattari1, Soheil Eagderi2, Saber Vatandoust3, Ignacio Doadrio4

1Department of Fisheries, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Guilan, Iran.
2Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran.

3Department of Fisheries, Babol Branch, Islamic Azad University, Babol, Iran.
4Biodiversity and Evolutionary Group, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain.

Article history:
Received 2 June 2020
Accepted 21 January 2021
Available online 2 5 August 2021

Keywords:
Shape variation
Phenotypic plasticity
Geometric morphometric
Iran

Abstract: Geometric morphometric method was used to examine the body shape variations among
the six populations of Garra rufa, in Iranian part of Tigris basin. A total of 15 landmark-points was
used for 170 specimens to hypothesize population differentiation of G. rufa in the six rivers and
reservoir. In discriminant function analysis, 85.9% of original grouped cases correctly classified.
Principal component analysis (PCA) and canonical variates analysis (CVA) confirmed the significant
difference between the populations. The results revealed that the studied populations are divided into
three clades based on differences in body depth, caudal peduncle length, backward moving of anal
fin. Caudal peduncle showed shortening trend in five populations. Narrower body shape was
dominated among specimens of four regions. Studies on body shape provide supporting data on
fisheries, stock management, and conservative programs.

Introduction
Geographical isolation and interbreeding are resulted
in morphometric variations among populations of a
single species (Bookstein, 1991; Torres-Dowdall et
al., 2012; Heidari et al., 2013, 2014; Kohestan-
Eskandari et al., 2014). Body shape plays an important
role in fish locomotion, feeding behavior, and
predation reflecting evolutionary adaptation in
response to environmental pressures (Webb, 1982;
Guill et al., 2003; Mousavi-Sabet and Anvarifar, 2013;
Mousavi-Sabet et al., 2018). Therefore,
morphological variations of different populations as
part of adaptation to their habitats can guarantee the
survival of the population (Nacua et al., 2010;
Paknejad et al., 2014; Vatandoust et al., 2015). A main
purpose of morphometric study is to test hypotheses
about the factors affecting body shape. Geometric
morphometrics is an approach to study shape using
landmark points (Webster et al., 2010). The landmark-
based analysis uses various statistical techniques to
exclude size, position, and orientation, therefore only

*Correspondence: Hamed Mousavi-Sabet DOI: https://doi.org/10.22034/ijab.v9i4.611
E-mail: mousavi-sabet@guilan.ac.ir

shape variables can be extracted (Webster et al., 2010;
Adams et al., 2004).

The family of Cyprinidae includes the most diverse
taxa distributing in all basins of Iran (Esmaeili et al.,
2018). One of the most phylogeographically
interesting cyprinid genus is Garra distributed in
southern, southwestern and northwestern Asia
(Esmaeili et al., 2016, 2018; Mousavi-Sabet et al.,
2019). The genus Garra Hamilton, 1822 with 151
valid species, is one of the most diverse genera of the
Labeoninae, and has a widespread distribution ranging
from East Asia to Africa (Sayyadzadeh et al., 2015;
Mousavi-Sabet and Eagderi, 2016; Froese and Pauly,
2021). In Iran, the genus is found in Tigris, Persis,
Hormuz, Makran, Mashkid, and Sistan, Jazmurian,
Kerman and Lut basins (Esmaeili et al., 2016).

Garra rufa inhabits harsh ecological conditions in
different environment having high ability to tolerance
a wide range of environmental factors that results its
wide distribution and variation in body shape (Coad,
2018). Therefore, study of population of G. rufa could

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Int. J. Aquat. Biol. (2021) 9(4): 258-263

well describe its morphometric variation and
phenotypic plasticity. Hence, in the present study, we
compare the body shape of different populations of G.
rufa from the Tigris basin using geometric
morphometric method.

Materials and Methods
A total of 170 specimens of G. rufa were collected
from six rivers of Iranian part of the Tigris basin,
including Godarkhosh (33°30′16″N, 45°54′3″E),
Sirvan (35°05'12.3"N 46°05'19.2"E), Chardavol
(33°45′N 46°34′E), Leileh (35°03'30.9"N 45°57'
27.7"E), Baneh rivers (36°00'31.1"N 45°54'15.6"E),
and Siah-Gav twin lakes (32°52'03.1"N 47°42'03.7"E)
using electrofishing device during September 2013 -
September 2015. The collected fish were preserved in
10% buffered formalin after anesthesia. Sexual
dimorphism does not appear in Genus Garra;
therefore, sex determination has not been carried out.
The left side of each individual (with dorsal and anal
fins were fixed by pins) was photographed, then 15
homologous landmark-points were defined and
digitized on 2D images using tpsDig2 software
version 2.16 to extract body shape data (Rohlf 2004)
(Fig. 1).

Generalized Procrustes analysis (GPA) carried out
on data to remove non-shape data. Principal
Component Analysis (PCA) was used to explain the
variance-covariance structure to summarize the
variation among the specimens. The Multivariate
analysis of variance (MANOVA) and canonical
variates analysis (CVA) were used to investigate
power of distinction among groups. Mahalonobis
distance also calculate to reveal the distance between
the studied populations in terms of morphology. All

statistical analyzes were done using PAST software
(Hammer, 2012) at the 95% confidence limit.
Clustering analysis was performed as the Euclidean
square distance clustered algorithm (Sneath and
Sokal, 1973).

Results
PCA showed 52.81% of shape variations of the first
two components derived from the variance-covariance
matrix. However, the screen plot in PCA showed, four
component situated above the Jullife broken line
(Julliffe cut-off=5.996e-05) include 74.67% of
variance with significant level (Table 1). The CVA/
MANOVA identified significant differences in body
shape among the studied populations of G. rufa
(P=2.305e-60 Wilkils lambda=2.67). The pairwise

Figure 1. Defined landmark points to extract the body shape data. 1. Anterior tip of the premaxilla; 2. Center of the eye; 3. Dorsal edge of the
head vertical to the eye center; 4. End of operculum; 5. Nape at the beginning of the scale; 6 and 7. Origin and insertion of the dorsal fin; 7. Upper
margin of caudal peduncle; 9. Center of caudal peduncle; 10 Lower margin of caudal peduncle; 11 & 12. Insertion and origin insertion of the anal
fin; 13. Origin of the pectoral fin; 14. The lower beginning of gill slit; 15. Ventral edge of the head vertical to the eye center.

Figure 2. The results of Canonical discrimination analysis (CVA)
of the six studied population of the Garra rufa in Tigris basin with
respect to the first two canonical variables based on body shape
extracted from landmarks.

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Saemi-Komsari et al./ Body shape variation of Garra rufa populations in the Tigris basin

Hotelling's test of the groups showed significant
differences in all groups (P<0.0001). The CVA plot
depicted based on the first two CVs clustered G. rufa
population into three distinct groups and all
population, showed in some extends overlapping (Fig.
2). Mahalonobis distance showed the most distances
of the chardavol population from others in terms of the
body shape (Table 2).

The body shape changes were latero-dorsal shift of
the pectoral fin, shorter caudal peduncle, and smaller
head with longer snout in Chardavol population. In
Sirvan population, lower body depth and shorter
caudal peduncle became highlighted. Population of
Baneh can be identified by having lower body depth

and longer caudal peduncle, whereas that of Leilerizan
showed lower body depth, and shorter caudal
peduncle. However, the Godarkhosh population
showed ventral position of the snout, shorter caudal
peduncle, and deeper body depth. Population of Siah-
Gav spring can be identified by the deepest body
depth, ventral position of the snout and short caudal
peduncle.

Discriminant analysis (DA) revealed 85.9% of
original grouped cases correctly classified. 67.6% of
cross-validated grouped cases correctly classified
(Table 3). The dendrogram derived from cluster
analysis of Euclidean square distances showed that six
populations of G. rufa segregated from each other into

Table 1. Eigenvalue and variance of the first four principle component of six studied populations of the Garra rufa in Tigris basin of Iran.

PC Eigenvalue % Variance
1 0.000896986 35.16
2 0.000450501 17.659
3 0.000299303 11.732
4 0.000198041 7.7628

total 72.31%

Table 2. Mahalanobis distance analysis for the six studied population of the Garra rufa in Tigris basin of Iran.

Table 3. Classification matrix showing the number and percentage of individuals that were correctly classified.

sites Godarkhosh Sirvan Siagav Chardavol Leilerizan Bane Total
Original (%) Godarkhosh 89.3 3.6 3.6 0 3.6 0 100
Sirvan 10.7 78.6 7.1 0 3.6 0 100
Siagav 3.3 6.7 80 0 3.3 6.7 100
Chardavol 0 0 4.8 90.5 4.8 0 100
Leilerizan 0 3.1 6.2 0 90.6 0 100
Baneh 0 3.2 6.5 3.2 0 87.1 100
Cross-validate (%)
Godarkhosh 67.9 14.3 3.6 0 14.3 0 100
Sirvan 25 53.6 14.3 3.6 3.6 0 100
Siagav 3.3 16.7 50 0 16.7 13.3 100
Chardavol 4.8 0 19 66.7 4.8 4.8 100
Leilerizan 3.1 3.1 6.2 0 84.4 3.1 100
Baneh 3.2 3.2 6.5 3.2 3.2 80.6 100

Cross validation is done only for those cases in the analysis. In cross validation, each case is classified by the
functions derived from all cases other than that case. 85.9% of original grouped cases correctly classified. 67.6%
of cross-validated grouped cases correctly classified.

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three distinct clusters, G. rufa from the Siah-Gav and
Baneh appeared in one cluster, along with Godarkhosh
population, Leileh and Chardavol formed the second
one, and the last one refers to the Sirvan population
(Fig. 4).

Discussions
The experimental phenotypic discrepancies among the
G. rufa populations discovered morphologically
separated stocks in the studied populations of Tigris
basin in Iran. Phenotypic plasticity can be implied by
phenotypic variations among the population
specimens. Head and mouth shape variation can be
considered as reflective of differences in selection of
food items and direction of feeding (Langerhans et al.,
2003). A fish with a mouth oriented upward usually
feeds in the water column vs from bentic feeding
behavior (Andersson et al., 2005). Morphological
adaptations in freshwater fishes according to wide
variety of physiological and environmental conditions
result in genetic divergence and/or phenotypic
plasticity (Gatz, 1979; Wainwright et al., 1994; Eklov
et al., 2006). Phenotypic plasticity responded to
environmental variations refer to niche patterns of

resource utilization, behavior, and/or habitat use
(Gatz, 1979; Wainwright et al., 1994; Eklov et al.,
2006; Langerhans, 2008). Moreover, diet pattern
could influence morphology, while particular dietary
items induce morphological change within or among
populations (Wainwright et al., 1994). However,
abiotic factors including food abundance, temperature
(Hossain et al. 2010), body shape (Beacham, 1990),
amount of food (Currens et al., 1989), and type of food
or feeding mode (Pakkasmaa, 2001; Peres-Neto and
Magnan, 2004; Proulx and Magnan, 2004) as well as
biotic factors affect phenotypic plasticity (He et al.,
2013). As mentioned before, in our study Siah-Gav
reservoir showed the deepest body depth after
Godarkhosh vs. narrower body in other riverine habit.
Similarly, Haas et al. (2010) found that deeper-bodied
Cyprinella (Cyprinidae) are indicative of reservoir
compared to riverine habitats. Our study on population
of G. rufa in Chardavol showed lately higher position
of the pectoral fin. It is supposed that lateral
positioning of the pectoral fins correlates with the
locomotory characteristics of particular species to
improve maneuvering (Webb, 1982; Bandyopadhyay
et al., 1997).

Figure 2. Dendrogram derived from cluster analyses of morphometric variables on the basis of Euclidean distance of the six studied population of
the Garra rufa in Tigris basin of Iran. The mean shape of species in relation of consensus shape of the Garra rufa are represented.

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Overall, studies on some species confirmed that
shape differences could be related to trophic ecology
(Costa and Cataudella, 2007), regarding local
adaptation and ecological radiations (Schluter and
McPhail, 1992; Langerhans et al., 2003). Overall,
geographical isolation plays key role on producing
morphological variation in fish species (Yamamoto et
al., 2006). Importance of environmental conditions
and biogeographical patterns on morphological
differentiation within communities is linked to the
zoogeographical history of a region (Hoagstrom and
Berry, 2008). Selective pressures influencing
individual mechanisms revealed how the process of
evolution moves within the population in response to
adaptation in their habitat.

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