Comparison of the population of Traplus agilis in Sistan with other populations of this species in some parts of Iran
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Fariba miranisargazi ,1,*
- Introduction: The genus Trapelus has 13 species, of which Trapelus agilis is one of
its members, which is distributed in Iran, Afghanistan, Pakistan,
India and Central Asian countries. Sixteen specimens of T. agilis were
collected from the Sistan Basin and the Central Plateau of Iran. 18
morphological traits by SPSS software. v16 was analyzed. For molecular
studies, the 16SiRNA mitochondrial gene was amplified by a polymerase
chain reaction and sent to Macrogene, South Korea, for sequencing. To
investigate phylogenetic relationships between populations, unmodified
genetic distance was taken in 6.MIEGA software. Plotype networks were
plotted to study genetic divergence in Popalt software. Genealogical
trees with maximum probability, maximum parsimony and Bayesian
inference were plotted. Morphological results show the morphological
diversity among the populations of Sistan. Molecular results also
indicate that genetic diversity among populations in T. agilis is high.
- Methods: After determining the sampling stations in Sistan, including
Miankangi, Posht Ab, Shahraki and Naravi water slopes and Zabol, as
well as determining these stations in the range of distribution of
this species in the central plateau of Iran, sampling was done.
Sampled areas are provided along with the exact address of the sample
collection site. Sampling was done entirely by hand, but this requires
tools and equipment such as suitable gloves, a bag for storing live
specimens, proper storage containers, a geolocation device (GPS), and
a camera. The collected samples are transferred to the laboratory.
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SPSS 16 software was used for statistical analysis of morphological
data. Multivariate statistical analysis was used to compare
populations. Principal component analysis or PCA is one of the most
popular multivariate analysis methods.
DNA extraction is the first step in any molecular method. After
collecting samples from the environment and transferring them
Tissues from the tongue and thigh muscles were prepared in the
laboratory and used for molecular studies. DNA extraction was
performed by ammonium acetate salt method, the steps of which are as
follows:
Transfer the finely chopped tissues to 2ml tubes. The tube is then
placed in a laboratory temperature for about 2 hours or in an
incubator for about 30 minutes at a temperature of about 40 ° C until
the alcohol in the tissue is completely evaporated.
۲. Then inside the tube 700 μl buffer (B) or Lysis buffer + 40 μl SDS
Add 20% +10 μl of proteinase K and then combine them thoroughly by
vortexing. The tubes are then transferred to an incubator at 55 ° C
for 18-20 hours.
٣. After 18-20 hours, the lysed samples are taken out of the incubator
and 340 microliters of four molar ammonium are added to each tube.
4. To mix the ingredients thoroughly, gently lower and lower the tubes
several times, then turn it to
Place at laboratory temperature for 40 to 60 minutes, then centrifuge
the tubes for 4 minutes at 4 ° C at 13,000 rpm.
5. Gently remove the upper liquid content of the precipitated area and
transfer to a new 2 ml tube, then add 1000 μl (1 ml) of cold
isopropanol (-20) or 100% alcohol to each, then add the tubes to
Gently stir several times to mix thoroughly, then centrifuge the tubes
for 30 minutes at 4 ° C at 14,000 rpm.
Discard the top 6 DNA sediment liquids and allow the remaining alcohol
in the tube to evaporate at laboratory temperature for a period of
time. Once the alcohol is completely dry, add 100 μl of double
distilled water to each tube, then refrigerate the samples at 4 ° C
for at least 24 hours to dissolve the DNA in water.
7. To evaluate the quantity and quality of the extracted DNA, a tattoo
therapist was used (Figure 2-3). For this purpose, first the device is
calibrated with distilled water, then we put 2 microliters of DNA on
the device and its quality and quantity are measured. In addition to
the light absorption ratios of 260/260 and 260/230, this device also
calculates the net concentration of soluble DNA. The calculated ratio
must be between
1. 8 to 2. 2. The closer this number is to 2, the higher the quality
of the extracted DNA, and if it is less than 108, it indicates the
presence of different proteins in the extracted DNA. On the other
hand, if this number is more than 2, it indicates the presence of
organic matter in the extracted DNA.
- Results: Morphological results The analysis of principal components analysis on
the morphological traits of I. agilis in Sistan Basin was able to
determine the morphological diversity among Sistan samples. The first
component of this analysis includes 69. 81% of the total variance and
the second component includes 19. It is 46% of the total variance.
Molecular results
In order to study and evaluate the taxonomic status of T. agilis, data
from the mitochondrial DNA sequence of gene 165 with a length of 470
nucleotides were analyzed for 16 samples. I ruderatus was considered
as an external group.
After grouping the populations, the genetic distance between them was
calculated. The results show that the highest genetic distance between
Kerman cloud is with other clouds and the lowest genetic distance is
between Gonabad and Ghaen clouds. Sistan cloud has the highest genetic
distance with Mazandaran, Kohgiluyeh and Boyer Ahmad clouds.
After drawing the genealogical tree by BI method, the ML genealogy
tree and the MP parsimony tree, which is considered as an outgroup in
all three trees of Irapelus ruderatus, have a similar pattern and
generality in all three trees for species I. agilis is shown as
follows: Clouds of Mazandaran, Kohgiluyeh and Boyer Ahmad, Cloud T.
Sanguinolenfus and a specimen of Sistan are in a tributary, and the
clouds of Qaen, Gonabad, and the cloud of Sistan are in a tributary.
Cloud Sitan is scattered among the branches. Kerman cloud is separated
from other clouds
- Conclusion: One of the most controversial groups of the Agamidae family is the
Irapelus agilis species collection, which has been the subject of many
reptile journals and magazines since 1804, when Olivier collected it
from the outskirts of Baghdad in eastern Iraq. This species species
has a very wide distribution range within the territory of Iran,
Pakistan, Afghanistan and Central Asian countries. The populations of
the southern regions (southern Iran, southern Pakistan and
Afghanistan) show great diversity in morphological traits, which
indicates a long history of evolution. These populations are
traditionally divided into three subspecies, the populations of
eastern Iraq if any, the populations of southwestern Iran and central
Iran as subspecies of T. a. are considered agilis. While the
populations of eastern and southeastern Iran, southern Afghanistan,
southern and western Pakistan and northern India are considered as the
second traditional subspecies of Ta, isolepis. In contrast, the
populations of the northern part (Northeast of Iran and Central Asia)
are a homogeneous group of
T. a. sanguinolentus, which has low phenotypic changes and indicates
the possibility of their recent invasion of the southern part of the
post-Tuscan police icy period.
In 1961, Leviton and Anderson demonstrated the statistical
significance of the diversity of scales in different populations.
Wettstein in 1951 stated that there are three subspecies
agilis, isolepis, Sanguinolentus are not acceptable because these
parts are not based on morphology. A collection of 51 samples
collected by Anderson from southwestern Iran in 1963 has 76-95 scales
around his body. 18 samples by
Tuck collected from northeastern Iran in 1979. He has 74-63 scales all
over his body. Six examples from Afghanistan show that there are 76-72
scales. 9 examples from Kandahar in southeastern Afghanistan have
74-60 scales. The specimens of Kandahar (southeast Afghanistan) are
somewhat smaller than the specimens of the lowlands of Afghanistan and
Iran. Finally, Rastegar Pouyani in 1999, two subspecies of T. , a,
pakistanesis and Ta
khuzistanesis for species T. introduced agilis. Among these
subspecies, there are three subspecies in Iran
These are:
T. , a, agilis, T. a. sanguinolentus, T. a. khuzistanesis
Is
In this study, different populations (T. agilis, Olivier (1804) in the
Sistan Basin and some areas of the Central Plateau of Iran were
studied based on morphological and genetic data. The existence of
different ecological conditions in the habitats occupied by this taxon
is attributed to the fact that it lives in different habitats and the
habitats are at different levels in terms of height.
Genetic results show that the clouds of Mazandaran, Kohgiluyeh and
Boyer Ahmad, the cloud of T. sanguinolents and a specimen of Sistan
are in a fork, and the clays of Qaen, Gonabad, and a cloud of Sistan
are in a fork.
Kerman cloud is separated from other clouds. On the other hand, Kerman
cloud has the greatest genetic distance with other clouds. According
to the haplotype network, Ghaen and Gonabad clouds are shared with
each other and Mazandaran, Kohgiluyeh and Boyer-Ahmad clouds also
share their haplotypes, which is probably due to the short
geographical distance or the same living conditions between these
clouds. There are several different haplotypes of Cloud Sistan between
haplotypes, indicating genetic diversity. Kerman haplotype with high
genetic diversity is placed separately and needs comprehensive and
extensive studies. On the other hand, the existence of a very large
area of the Central Plateau has caused the rapid distribution of
this species on the plateau and the existence of habitat diversity and
geographical barriers in a very diverse
The Central Plateau of Iran and the Sistan Basin have probably led to
the localization of this species and the occurrence of genetic and
morphological variations.
- Keywords: Genetic distance, phylogeny, morphology
