Evolutionary view on sexual dimorphism

and shape variation in Iranian

populations of Hypera postica (Coleoptera:
Curculionidae)

Ehsan Sanaei, Marjan Seiedy & Farzaneh

Momtazi

Zoomorphology
Evolutionary, Comparative and
Functional Morphology

ISSN 0720-213X
Volume 134
Number 4

Zoomorphology (2015) 134:541-552

DOI 10.1007/s00435-015-0279-2

1 23
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 Author's personal copy
Zoomorphology (2015) 134:541–552
DOI 10.1007/s00435-015-0279-2
ORIGINAL PAPER
Evolutionary view on sexual dimorphism and shape variation
in Iranian populations of Hypera postica (Coleoptera:
Curculionidae)
Ehsan Sanaei1 • Marjan Seiedy1 • Farzaneh Momtazi2
Received: 21 April 2015 / Revised: 31 July 2015 / Accepted: 1 September 2015 / Published online: 1 October 2015
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract The alfalfa weevil (Hypera postica) is a
worldwide variable pest that is established in almost all
Iranian alfalfa fields. As a result of the large distribution
range and the climatic diversity in Iran, population differ-
entiation had been observed, which was largely unstudied.
Previous morphological studies have suggested a local
adaptation of Hypera postica populations; yet, traditional
morphological markers are not powerful enough to reveal
subtle changes in morphological characters among popu-
lations or even gender within populations. The robust sta-
tistical geometric morphometric analysis promised to be
able to detect small changes in shape. In this study, we used
Fourier outline analysis to compare the shape of pronotum,
elytra and rostrum of five Iranian populations of the alfalfa
weevil from three well-separated geographic areas (Karaj,
Jovein and Tuyserkan). Besides population divergence, the
sexual shape dimorphism was investigated. The results
show a significant shape difference between males and
females, at inter- and intrapopulation levels for H. postica.
These results suggest the influence of a local natural
selection on the studied characters.
Communicated by A. Schmidt-Rhaesa.
& Marjan Seiedy
mseyyedi@ut.ac.ir
Ehsan Sanaei
ehsansanai@ut.ac.ir
Farzaneh Momtazi
Momtazi.f@inio.ac.ir
1 even emphasized on the variation among populations of
School of Biology and Center of Excellence in Phylogeny of
Living Organisms, College of Science, University of Tehran,
Tehran 14155-6455, Iran
2
Iranian National Institute for Oceanography and Atmospheric
Science (INIOAS), Tehran, Iran
Keywords Elytra shape  Fourier outline analysis 
Pronotum shape  Rostrum shape  Sexual shape
dimorphism
Introduction
Due to its high adaptability to a wide range of ecological
conditions, efficiency in nitrogen fixation and excellent
nutritional value as animal feed, alfalfa (Medicago sativa
L.) has become an essential crop (Deshpande et al. 2002).
In Iran, more than 600,000 hectares of alfalfa are planted
yearly across large parts of Iran (Tohidfar et al. 2013). The
alfalfa weevil, Hypera postica (Gyllenhal, 1813), is one of
the most important insect pests of alfalfa crop in the world
(Summers 1998). Many studies have been performed in
order to understand the biology of H. postica (Khanjani and
Pourmirza 2004; Moradi-Vajargah et al. 2011; Zahiri et al.
2010a, b, 2014). Except some recent studies (Sanaei et al.
2014, 2015a) there is still a lack of detailed information
about Iranian populations of this species.
The alfalfa weevil is originally a Palaearctic species
(Kuhar et al. 2000) that is widely distributed in Europe,
Asia and northern Africa (Coskuncu and Gencer 2010;
Skuhrovec 2013). Further it has been introduced to North
America in 1904 (Titus 1909). The species shows a high
intrapopulation variation in body shape and color that has
caused many systematic problems including several
nomenclatural changes (Michelbacher 1940; Skuhrovec
2013; Warner 1962). The former name, Hypera variabilis,
this species (Michelbacher 1940; Srivastava 1959). In
addition, the huge morphological variation in Hypera
species group has led to a variety of taxonomical problems.
At the moment, we have more than 18 synonyms of H.
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542
postica that need deep taxonomic revision (Skuhrovec
2013). After the first introduction to America, three strains
were identified (Bundy et al. 2005; Hsiao 1993) which
show slightly morphological differences (Bland 1984;
Pienkowski et al. 1969), but high ecological differences
(Bundy et al. 2005; Goosey 2009; Hsaio 1996; Hsiao and
Hsiao 1985). Further, molecular analysis suggested sig-
nificant differences between H. postica populations (Bött-
ger et al. 2013; Erney et al. 1996; Kuwata et al. 2005). This
prior knowledge on population variability of the species in
the USA and Japan suggests that it is important to inves-
tigate additional populations of H. postica, especially in its
native range. Such interesting results may prove the use-
fulness of understanding the biogeographical patterns,
population dynamics and evolutionary trajectories of this
globally distributed pest [e.g., for designing integrated pest
management strategies (Böttger et al. 2013)].
Even though classic morphological analyses are still
regarded a powerful tool to distinguish taxa and some-
times even intraspecific populations, traditional morpho-
logical analyses have failed in the past to distinguish
American populations of the alfalfa weevil (Bland 1984;
Erney et al. 1996; Kuwata et al. 2005; Pienkowski et al.
1969). Classic morphological analyses are often limited to
few measurements; however, the development of geo-
metric morphometrics, along the advances in statistical
methods, provides a powerful tool to study the shape
variation of animal populations (Adams et al. 2004;
Viscosi and Cardini 2011).
Morphological characters might employ for revealing
significant differences between sexes. Investigation of
sexual dimorphism was always an interesting field of
research for scientists and especially for entomologists that
usually deals with non-obvious dedicated characters for
sexes (Benı́tez 2013b). Thus, finding such a character
allows more convenient sex determination. On the other
hand, the evolutionary explanation of sexual dimorphism in
different taxa and the effect of sexual selection in the
evolutionary progress are still ambiguous (Ridley 2003;
Stillwell and Davidowitz 2010). Understanding the mor-
phological variation of homologous characters between
sexes would reveal the direction of selective pressures and
let create hypotheses about it (Tatsuta et al. 2004).
According to high pressure of fecundity selection, insects
usually are observed with larger females than males (Still-
well et al. 2014). Sexual size dimorphisms in most of the
external anatomic parts were limited in length and width
measurements. Therefore, traditional morphology is not
powerful enough to describe the minor shape differences of
current anatomic structures. Calculating the sexual shape
dimorphism of H. postica by powerful geometric morpho-
metric analysis was the first goal of this research. Here,
possible evolutionary reasons for sexual shape dimorphism
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Zoomorphology (2015) 134:541–552
of studied anatomic structure in alfalfa weevil will be
discussed.
Despite its importance as an agricultural pest in Iran
(Sanaei et al. 2015b), little is known about Iranian H.
postica populations. To fill this gap, a Fourier outline
analysis was performed. Alfalfa weevils were collected at
six locations in three major cities in the Northern Provinces
of Iran (Karaj, Jovein and Tuyserkan). The climatic con-
ditions of these three localities are different. In addition,
there is a geographic isolation between Karaj 1 and Karaj 2
stations and also Tuyserkan 1 and Tuyserkan 2 stations as
well. We quantified pronotum, elytra and rostrum shape as
variables for our analysis. The studied traits have been
suggested to be either of high taxonomic value or ecolog-
ically meaningful. The goal of this part was determining
the amount of shape differences between studied popula-
tions and using this information to estimate the population
divergence of H. postica.
Materials and methods
Sampling and image preparation
Three major localities including Karaj (Alborz Province),
Tuyserkan (Hamadan Province) and Jovein (Razavi Kho-
rasan Province) were selected (Fig. 1). In each locality, two
alfalfa farms were chosen as sampling locations. The two
chosen farms from Jovein were very close to each other
(\10 km); hence, they were considered as one station. A
total of 200 specimens was collected in spring and summer
2013 using sweep nets. The dates of collection, coordinates
of each location and the total number of specimens are
given in Table 1. Images were captured in dorsal view
using a digital camera connected to a binocular stereoscope
(Hund Stereo-Zoom-Microscope, Model: Wiloskope).
Several pictures from each specimen with different focus
were taken and were combined by Combine-ZM program
(Hadley 2008), to get a sharper board picture.
Geometric morphometric and statistical analysis
During preparation, some specimens showed deformations
of the pronotum, elytra or rostrum parts and were therefore
omitted from geometric morphometric analysis. The final
sample sizes for pronotum, elytra and rostrum were 193,
167 and 189 specimens, respectively (Table 2). As the
studied morphological features only provided limited true
landmarks, a closed curve on these using tpsDig 2.17 was
recorded (Rohlf 2013) by outline method. In tpsDig soft-
ware, 150 equally spaced points for pronotum and rostrum
and 200 points for elytra were sampled along the outline
(Fig. 2). Elliptical Fourier analysis (EFA) has been done
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Zoomorphology (2015) 134:541–552 543

Fig. 1 Locality of four

populations: 1 Karaj 1, 2 Karaj
2, 3 Jovein, 4 Tuyserkan 1, 5
Tuyserkan 2

Table 1 Information about coordinates, altitude, number of specimens and distribution of sexes at each station and in total; the two Jovein
stations were merged and considered as one station in the statistical analysis
Station Location Coordinates Altitude Date of collecting Number of males Number of females Total number

1 Karaj 35°480 04.60 N 050°570 39.60 E 1315 m June 3, 2013 17 31 48

2 Karaj 35°410 36.50 N 051°090 29.30 E 1193 m June 3, 2013 21 36 57
3 Jovein 35°380 10.30 N 057°240 14.20 E 1140 m July 3, 2013 7 8 15
4 Jovein 36°410 30.40 N 057°200 56.50 E 1103 m July 3, 2013 4 13 17
5 Tuyserkan 36°260 02.80 N 048°400 31.70 E 1712 m July 14, 2013 29 21 50
6 Tuyserkan 34°310 16.20 N 048°180 43.40 E 1657 m July 14, 2013 6 7 13
Total 84 115 200

Table 2 Number of specimens and distribution of sexes in each station that was used in the analyses: A. pronotum analysis, B. elytra analysis
(*due to few number of specimens in Tuyserkan 2 station, this station was removed from statistical analysis of elytra), C. rostrum analysis
Karaj 1 Karaj 2 Jovein Tuyserkan 1 Tuyserkan 2
Male Female Male Female Male Female Male Female Male Female

Pronotum 17 28 21 34 11 18 27 22 7 6
Elytra 15 29 19 31 7 17 26 17 4* 2*
Rostrum 17 30 21 34 11 20 28 16 6 6

(Giardina and Kuhl 1977; Kuhl and Giardina 1982) by
EFAWin program (Isaev and Denisova 1995). The pro-
gram GMTP (Taravati 2010) was used to convert the
tpsDig output file format to the EFAWin format.
The first ten harmonics were used to describe the shape
variation of the pronotum and rostrum. For elytra, the first
nine harmonics were used to study the shape variations in
elytra. Each harmonic corresponds to four coefficients, An,
Bn for x, and Cn, Dn for y, and defines an ellipse on the XY-
plane (Hautier et al. 2009). Generally the first coefficient
describes size, the second shape (analogous to length–width
ratio), and the subsequent coefficients finer details of shape
(EFAWin). These are independent variables and therefore
can be used in statistical programs. Shape is mathematically
defined as all the geometric features of an object except its
size, position and orientation (Dryden and Mardia 1998).
Due to the goal of this study that was focused mainly on
shape variation rather than size variation, first harmonic was
removed from analysis in order to eliminate the size effect
in statistical analysis. Position and orientation effects were
removed automatically by EFAWin program.
The output file of EFAWin that includes harmonic
coefficients could be employed as raw data for statistical
analysis. PAST version 1.89 (Hammer et al. 2001) was
utilized to transfer the EFAWin outputs into SPSS. Har-
monic coefficients were used as statistical variables in

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544
Fig. 2 Locations of defined points for each anatomic structure with
determining the start point. a 150 points were digitized on each
pronotum in clockwise direction. Starting point was in the right upper
edge of pronotum. b. 200 points were digitized on elytra in clockwise
direction. In the dorsal view, the posterior margin of elytra was the
place of start point. c 150 points were digitized on each rostrum. The
SPSS version 19 (Brosius 2011) in order to do discriminant
function analysis (DFA), multivariate analysis of variance
(MANOVA), hierarchical cluster analysis (HCA) and
principle component analysis (PCA) for pronotum, elytra
and rostrum separately. DFA, MANOVA and PCA were
carried out to assess intraspecific differences between sta-
tions and sexes. In order to find similarities and differences
of material from different stations in a dendrogram, HCA
has been done using the linkage between group methods.
Squared Euclidean distances were used as the interval
measurement for calculating distances between clusters.
Most effective harmonics that contributed to the separation
of stations for each analysis were determined by investi-
gating the structure matrix of canonical discriminant
functions (CDF) extracted from DFA. There was always no
important variable for separating grouping variables in
DFA. By detecting substantial harmonic, the important
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pictures captured in frontal view and start point was in the lowest
point of left eye in the front area. The points follow left eye’s border
clockwise and pass the apex of rostrum that contained mouthparts.
Then the points came down to reach inner border of right eyes in
frontal view. The lowest line of the eye distances was the final curve
of points
possible change in certain anatomic structure among pop-
ulations was described in EFA.
Result
The results of DFA and MANOVA for all three anatomic
structures among gender demonstrate that the shape of
pronotum, elytra and rostrum differ between males and
females. Thus the DFA, MANOVA and HCA between
stations were done for male and female specimens sepa-
rately. Among all multivariate analyses, a distinctive
separation of sexes or stations was never observed in PCA
analysis for none of the three external anatomic parts.
Here, six dendrograms according to the shape of prono-
tum, elytra and rostrum for male and female specimens
were presented in Fig. 5a–f.
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Zoomorphology (2015) 134:541–552 545

Table 3 Value of significance differences (P value) based on four independent assumption tests; Pillai’s Trace, Wilks’ Lambda, Hotelling’s Trace and Roy’s Largest Root in MANOVA for
Pronotum analysis

The certain significance differences (P \ 0.005) were marked by ’*’. Due to significant differences via sexes in the shape of all three anatomic parts, the MANOVA was run for male and female
Roy’s largest

P \ 0.005*
P \ 0.005*
P \ 0.005*
The DFA could correctly separate 73.1 % of males and

root
females. No effective variable was observed that was
mostly responsible for gender separation in DFA. Further,

Among populations for female specimens

P \ 0.005*

P \ 0.005*
the MANOVA revealed significant differences between

Hotelling’s
sexes (P \ 0.05). (The amount of significant differences

0.001*
trace
between gender and stations in MANOVA for each ana-
tomic part is given in Table 3.) Four functions were cal-

P \ 0.005*

P \ 0.005*
culated in DFA from pronotum coefficients with respect to
stations as grouping variable for males and females inde-

lambda

0.004*
Wilks’
pendently. The result of DFA indicated 94 % stations’
separation of male specimens and 91.7 % stations’ sepa-

P \ 0.005*

P \ 0.005*
ration in female specimens. For male and female speci-

Pillai’s
mens, the first two functions with the highest significance

0.011*
trace
value (P \ 0.05 for both male and female specimens) were
chosen for drawing scatter plots in order to document the

Roy’s largest
separation of the five populations in pronotum’s shape

P \ 0.005*
(Fig. 3a, b). Also this analysis showed that harmonic 4.3

0.001*
0.180
(third coefficients of fourth harmonic) in male specimens

root
and harmonic 5.2 (second coefficients of fifth harmonic)
played an important role in separation of stations. The

Among populations for male specimens

P \ 0.005*
Hotelling’s
changes of pronotum by adding each harmonic in EFAWin
program are shown in Fig. 4a. Most effective harmonics

0.697
0.125
trace
(fourth and fifth harmonics) demonstrate important changes
in pronotum shape. The important change in shape of male

P \ 0.005*
specimens via stations is observed when the basal part of

lambda
pronotum has a tendency for central inward curving to Wilks’

0.641
0.208
scutellum rather than being more flat. The more flatted base
of pronotum is observed in Jovein populations and seems
P \ 0.005*
this is responsible for assumed population divergence in
Pillai’s

DFA of pronotum shape. The changes in harmonic fifth

0.585
0.306
trace

that were responsible mostly for female specimens have

not being detectable and describable. The MANOVA
Roy’s largest

P \ 0.005*
P \ 0.005*

among stations had confirmed the results of DFA by

revealed significant differences between stations in male
0.031*

and female specimens separately (Table 3).

root
each anatomic part among gender and stations are given

Elytra analysis
P \ 0.005*
P \ 0.005*
Hotelling’s

0.031*

The DFA among sexes stated that 84.5 % of specimens

trace
Among sexes in total specimens

could be correctly separated by gender. The structure

matrix of DFA also revealed coefficient 3.4 (third har-
P \ 0.005*
P \ 0.005*

specimens separately among stations

monic) was the most important variable for separating

lambda

0.031*
Wilks’

gender by DFA. The third harmonic is responsible for a

more rounded shape of the apex of the elytra compared to a
more slender apex. In precise observation, the more roun-
P \ 0.005*
P \ 0.005*

ded apex was dedicated to females and more slender one to

Pillai’s

0.031*
trace

males, although the differences were too weak and here no

sexual shape dimorphism to all specimens was assigned.
Pronotum

The MANOVA among gender illustrated significant dif-

Rostrum
Elytra

ferences between sexes (Table 3). Due to a lack of speci-

mens from Tuyserkan 2 station, this station was omitted

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Zoomorphology (2015) 134:541–552
b Fig. 3 Scatter plot of DFA among stations based on two first
components. a DFA of pronotum shape in male specimens, b DFA of
pronotum shape in female specimens, c DFA of elytra shape in male
specimens, d DFA of elytra shape in female specimens, e DFA of
rostrum shape in male specimens, f DFA of rostrum shape in female
specimens. Note in the elytra analysis (c and d), the Tuyserkan 2
station was removed from the statistical analysis
from elytra analysis. Three functions were calculated in
DFA from elytra coefficients with respect to stations as
grouping variables for males and females independently.
The result of DFA indicated 74.6 % stations’ separation in
male and 77.7 % in female specimens. For male analysis,
the first two functions by 0.487 and 0.730 P value and for
female analysis, first two functions by 0.001 and 0.221
P value were employed for drawing scatter plot (Fig. 3c,
d). There were no effective variables with respect to station
separation, either in male specimens, or in female. The
results of MANOVA for elytra shape in male and female
specimens among stations clarified that despite lack of
significant differences in stations for male specimens, the
female showed significant differences between stations
(Table 3).
Rostrum analysis
The results of DFA, while taking sexes as grouping vari-
ables, demonstrated 82.4 % separation in gender. The
fourth coefficients of the second harmonic (harmonic 2.4)
had an important role in sex separation in DFA. This sec-
ond harmonic is responsible for early changes of rostrum
shape in EFA, and therefore it was not applicable to
changes in males or females (Fig. 4c). The MANOVA for
sexes indicated significant differences between genders in
rostrum shape (Table 3). The DFA correctly separated
78 % of male specimens and 78.3 % of female specimens
into five stations. From four functions that were calculated
from DFA with respect to stations as grouping variable,
first two functions by P value 0.130 and 0.808 were applied
in scatter plot for male specimens (Fig. 3e). Also for
female specimens, first two functions by P value \0.005
and 0.001 were employed in drawing scatter plot (Fig. 3f).
The structure matrix reveals no certain variable that was a
key factor in distinguishing stations in male specimens of
DFA. In DFA for females, harmonic 8.1 seems to be the
important variables in separations. The eighth harmonic
was one of the last harmonics in EFA that showed unde-
tectable dedicate changes in rostrum shape (Fig. 5c). Even
though there was no significant difference between stations
in MANOVA for male specimens, there was a significant
difference between stations for female specimens in
MANOVA.
547
Discussion
Within the species border, there are several markets that
have been employed by taxonomists in order to track the
evolutionary progress (Mayr and Ashlock 1991). In sys-
tematic studies, morphologic variables are trusted, they are
target of selection, detectable, applicable, and they usually
were shaped under polygenic control (Garnier et al. 2005).
Traditional morphological characters have been limited to
measurements of few traits, whereas geometric morpho-
metrics allows quantifying the shape of a particular struc-
ture (Mutanen and Pretorius 2007). By considering the
complete shape of particular structure, the quantity and
quality of variables for statistical analysis greatly increase,
which is why geometric morphometric analysis has
become a favored tool for statistics multidimensional
comparison of morphological characters (Bichain et al.
2007). In our study, the shape of the pronotum, elytra and
rostrum of five different populations of H. postica from
Iran was analyzed and investigated for sex differences. The
results showed the sexual shape dimorphism and popula-
tion divergence in alfalfa weevil.
Sexual shape dimorphism
Even though the sexual size dimorphism is a commonly
observed phenomenon in insects that usually advocated
larger females than males (Fairbairn 1997; Stillwell et al.
2014), studies of sexual shape dimorphism are relatively
modern (Benı́tez 2013a; Benı́tez et al. 2011a, b; Tatsuta
et al. 2004). Investigation for sexual shape dimorphism is a
crucial guide in determining the evolutionary pressures of
this phenomenon in each taxon (Benı́tez 2013b; Bertin
et al. 2002). Studies of sexual size dimorphism in Iranian
populations of alfalfa weevil have revealed a larger
pronotum, elytra and rostrum in females related to strong
fecundity selection rather than sexual selection (Sanaei
et al. in print). The results of MANOVA strongly con-
firmed the significance of sexual size dimorphism in shape
of pronotum, elytra and rostrum. However, the rate of
dimorphism was varying in each character. The differen-
tiation and direction of selective pressure in sexual shape
dimorphism of pronotum is non-significant or vague and
non-trackable. DFA for rostrum illustrated more sexual
shape dimorphism separation by 82.4 % correctly classi-
fied specimens into gender, but again failed to reveal exact
differences between male and female. Females have a
somewhat longer rostrum which is used for making holes
in alfalfa stems in order of laying eggs there. Also there is
no evidence for sexual dimorphism in mouthparts of alfalfa
weevil (Bland 1984). It is possible that the difference in
rostrum shape between genders is related to egg-laying
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548 Zoomorphology (2015) 134:541–552

Fig. 4 Effect of each harmonic on the shape of specific anatomic structure in EFA. The changes of first 8 harmonics for five random specimens
are given. a The pronotum analysis, b the elytra analysis and c the rostrum analysis

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Zoomorphology (2015) 134:541–552
behavior (Oberprieler et al. 2007). According to DFA, the
highest sexual shape dimorphism was observed in elytra by
84.5 % separation. The EFA demonstrated two different
tendencies in apex of elytra. More rounded apex in females
and more slender apex in males were indicated. Larger
female elytra in length and width seem to be related to
fecundity selection (Sanaei et al. in print). Sexual shape
dimorphism in the apex of elytra might be related to
amount of egg deposition or mounting behavior, but more
functional studies are needed for testing this hypothesis.
Although the results of geometric morphometric study
clearly showed sexual shape dimorphism in pronotum,
elytra and rostrum, these changes are too small to be
employed as sex-discriminative characters. Only the geo-
metric morphometric study was able to indicate these
subtle changes for us.
Iranian populations
Population divergence is a first step in the speciation pro-
cess (Foster et al. 1998; Husemann et al. 2014), and it is
affected by economic issues when we are considering
population divergence of important and variable pests such
as alfalfa weevil. Each population of H. postica is faced
with unique ecological conditions and is affected by natural
selection independently (Salt and van den Bosch 1967).
However, gene flow can decrease the intrapopulation dif-
ferences and increase the total diversity.
The limited and preliminary molecular study of Iranian
populations illustrated the high mitochondrial genetic
diversity in Iranian specimens without any clear population
divergence pattern (Sanaei et al. 2014). Our geometric
morphometric analysis was able to detect subtle changes in
shape of anatomic structures among populations. With
respect to diversity of weather condition, Iran is large with
diversity of weather conditions in different parts of the
country (Zehzad et al. 2002). The Jovein population is
located in east and the Karaj and Tuyserkan populations are
located in west of Iran, and they also differ from each
others by annual perceptions (Ngdir 2015). Karaj is under
effect of Alborze Mountains, and Tuyserkan is under effect
of Sahand Mountains (Zehzad et al. 2002). In addition, this
geographic isolation seems to appear between Karaj 1 and
Karaj 2 and also Tuyserkan 1 and Tuyserkan 2. The Karaj
city is a barrier between Karaj 1 and Karaj 2 populations,
and also the climatic conditions of these two areas are
different. The same theory is assumed for Tuyserkan 1 and
Tuyserkan 2 populations.
Because of sexual shape dimorphism that is observed in
pronotum, elytra and rostrum of alfalfa weevil, a multi-
variate analysis of stations was run for male and female
specimens separately. Even though the length and width of
pronotum showed no significant differences between
549
populations (Sanaei et al. 2015a), there is a high level of
population variation in shape of pronotum. The shape of
the pronotum is a useful variable for explaining inter-
specific variation in beetles (Benı́tez et al. 2011a; Garnier
et al. 2005; Taravati 2010). For genus Hypera, most keys
discriminate species based on pronotum shape (Balalaikins
2012; Skuhrovec 2009) with inexact description. The
geometric morphometric study of pronota has introduced
shape of pronotum as important taxonomic variables for
the study of H. postica populations, and probably it would
be helpful in taxonomic studies of other Hypera species.
The most important detectable change in pronotum may
occur at the base of pronotum, whereas it is tended to be
flat or not.
Elytra represent another character exhibiting substantial
variation between species or even populations (Faccoli
2006; Nakamura 1983; Rankin and Arnqvist 2008). In the
case of rostrum study in weevils, the length and shape of
rostrum shows strong ecological signals and has evolved in
response to different host plants (Oberprieler et al. 2007).
The shape of elytra and rostrum showed a less degree of
divergence in populations. MANOVA for elytra and ros-
trum shape among stations had no significant differences
between male specimens, but this was significant for
females. The amount of separation in DFA of elytra and
rostrum was, respectively, 77.7 and 78.3 %. The scatter
plot of DFA confirmed low isolation of stations for elytra
and rostrum (Fig. 5d, e).
High degree of differences in shape of the pronotum and
low rate of divergence of the elytra and the rostrum in the
studied populations may be due at least in part to the iso-
lation of the habitat. According to this study, the population
divergence is low and also the PCA failed to confirm dis-
tinct separation. Alfalfa weevil is a flight-capable species,
and after harvesting, large numbers of them fly to avoid
unfavorable conditions (Prokopy and Gyrisco 1965).
However, like several pest species, the flight ability is not
the key factor for long distribution range of alfalfa weevil
(Summers 1998). The trade of alfalfa products between
localities might have resulted in the admixture of popula-
tions in some parts and had prevented stronger pressure
effects in evolutionary processes. For example, Hamadan
Province, where Tuyserkan 1 and Tuyserkan 2 populations
are located, is one of the main culture centers of alfalfa
crop (Mobtaker et al. 2012), from which alfalfa hay is
transported across all over Iran. H. postica only feeds on
fresh alfalfa, but the pupae and eggs can survive the long
journey on alfalfa hay; the individuals can be introduced to
new localities by this way. For determining how popula-
tions are similar or different to each other, the HCA was
used for each sex in each character (totally six dendro-
grams suggested). As discussed below, only the shape of
the pronotum shows distinct population differences, and
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550 Zoomorphology (2015) 134:541–552

123
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Zoomorphology (2015) 134:541–552
b Fig. 5 Dendrograms (based on between group linkage method) from
HCA for showing similarity between stations for each anatomic part.
This analysis was carried out separately for males and females:
a Pronotum-male, b pronotum-female, c elytra-male, d elytra-female,
e rostrum-male, f rostrum-female. The X axis is the distance
measurement that calculated by squared Euclidean distance method.
The Y axis demonstrated the locations of populations: K1 Karaj 1, K2
Karaj 2, J Jovein, T1 Tuyserkan 1 and T2 Tuyserkan 2. The
Tuyserkan 2 station was omitted from elytra analysis (c and d)
the HCA was considered only based on pronotum shape.
The results of EFA and our direct observation revealed that
the base of the pronotum is more flatted in Jovein popu-
lation. Hence, the Jovein population should be separated
from other eastern populations. The male dendrogram and
female dendrogram were different to each other. When all
specimens were assumed in the analysis, the dendrogram
from HCA was similar to dendrogram of females (Fig. 5b).
The shape difference in pronotum of the Jovein population
and also the longest distance between Jovein and other
populations convinced us to designate Jovein station as
eastern population of Iran, well separated from the western
populations. Long distances lead to differences in envi-
ronment and ecological factors and reduce the chance of
accidental interbreeding.
The significant difference in pronotum shape between
western and eastern populations is consequence of a pop-
ulation divergence which can influence on ecological
aspects of each population. Such differences should be first
detected and then are applied for pest management strate-
gies. For instance, like American strains, it is possible that
western and eastern populations have different response to
each parasitoid species. Therefore, the results of this study
can follow by other researchers to achieve an efficient pest
management strategy against alfalfa weevil.
Acknowledgments The authors are indebted to Prof. Alireza Sari
for editing an early version of the manuscript and Dr. Hassan Rahi-
mian for his valuable help. The research was supported by the
University of Tehran, which is greatly appreciated.
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