International Journal of Environment, Agriculture and Biotechnology

Vol-10, Issue-4; Jul-Aug, 2025

Peer-Reviewed International Journal
Journal Home Page Available: https://ijeab.com/
Journal DOI: 10.22161/ijeab

Cox-1 Gene-Based Identification and Molecular Phyloge-

netics of Horseflies
Farman Ullah1*, Suleman2, Ali Ahmad3, Atta Ullah1 , Syed Azmat Shah4, Bilal Ahmad5
1Department of Zoology, University of Swabi, Swabi, Khyber Pakhtunkhwa, Pakistan
2Lecturer,Department of Zoology, Government Post Graduate College (GPGC), Swabi, 23431, Swabi, Khyber Pakhtunkhwa, Pakistan
3 School of animal sciences and technology, Shihezi University, Xinjiang, China
4 Department of Chemical and Life Science, Qurtaba University of Science and Information Technology, Peshawar, Khyber Pakhtunkhwa,
Pakistan.
5 Department of Zoology, Hazara University Mansehara, Khyber Pakhtunkhwa, Pakistan
*Corresponding Author

Received: 23 May 2025; Received in revised form: 25 Jun 2025; Accepted: 30 Jun 2025; Available online: 04 Jul 2025
©2025 The Author(s). Published by Infogain Publication. This is an open-access article under the CC BY license (https://creativecom-
mons.org/licenses/by/4.0/).

Abstract— The hematophagous flies of the family Tabanidae are involved in the transmission of various
disease-causative agents such as protozoans, helminths, bacteria, and viruses. However, molecular research
on this family is not conducted in Pakistan. This study seeks to investigate the molecular characterization of
the horseflies, feeding on buffaloes of District Swat, Khyber Pakhtunkhwa, by targeting the mitochondrial
partial Cox1 gene for the nucleotide identity/diversity and phylogenetic analysis. The Cox1 gene sequences
were compared with NCBI databases using BLASTn. The analyses revealed two Tabanus species (Tabanus
sp. 1 and Tabanus sp. 2) and one Atylotus species. Genetic comparisons found a close relationship: Tabanus
sp. 1 (93.95%) to T. superjumentarius, Tabanus sp. 2 (94.55%) belong to T. bromius, and Atylotus sp.
(93.97%) to A. agrestis. Species were identified at the genus level due to limited data availability. The nu-
cleotide identity among the collected species was Atylotus sp. vs. Tabanus sp. 1 (90.7%), Atylotus sp. vs.
Tabanus sp. 2 (89.4%), and Tabanus sp. 1 vs. Tabanus sp. 2 (90.1%). As expected, Tabanus sp. 2, clustered
together with its congener, T. bromius forming a basal clade of all other Tabanus spp included in our phy-
logenetic analysis. This means that Tabanus sp. 2 and T. bromius have some similarities and both of them
are from the Palaearctic region. However, Tabanus sp.1, though showing comparatively higher sequence
identity with T. superjumentarius (94.55%) placed on a separate branch as a sister clade of the clade con-
taining T. fontinalis and T. rubidus. T. fontinalis and T. rubidus are native to North America and their se-
quence identity with Tabanus sp.1 were 93.8% and 93.3%, respectively. Similarly, the phylogenetic tree
placed our sequenced Atylotus sp. on a separate branch inside the clade uniting all Atylotus spp. included in
our analysis containing A. agrestis as well. From our study, we concluded that the genus Atylotus is mono-
phyletic while the Tabanus genus is may be polyphyletic or paraphyletic because as it does not include all
descendants of a common ancestor. Molecular characterization of additional species of horseflies from Pa-
kistani hosts will provide a clear image of their genetic inter-relationship and their phylogenetic affinities
with other species recorded across the world. Such molecular study will provide a base for determining the
prevalence of respective species and the development of their effective management strategies.
Keywords— horseflies, PCR, phylogenetic tree, nucleotide identity and Cox-1 gene.

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Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

I. INTRODUCTION of amino acids or DNA — to explain the evolutionary rela-

Horseflies are blood-feeding insects that belong to the Tab- tionships among a set of organisms. The approach has
anidae family. Both humans and livestock suffer greatly gained prominence due to the technological explosion of
from their itchy and painful bites. A range of infectious DNA sequencing and the accessing of sequence information
agents, including protozoa, helminths, bacteria and viruses, for genes and proteins through publicly available internet
can be carried by and spread by these insects. Both veteri- dbs. The selection of a certain molecule for a phylogenetic
nary and medical important must comprehend their part in analysis is not arbitrary; molecules evolve in various ways
the spread of disease [1]. and rates [11].

The adult Tabanidae bug has to scrape and lick its mouth- They are an ecologically, and medically significant group,
parts. It is a robust, muscular flyer. There are more than 20 and evolutionary relationships within the Tabanidae family
families within Brachycera, with Tabanidae being the larg- are still poorly understood[12]. The family Scionini in-
est with about 4455 species spread across 144 genera. Some cludes a total of seven genera and is primarily distributed
1300 Tabanidae species are known to exist in the genus through the Southern Hemisphere, most notably in Austral-
Tabanus. Horsefly females feed on blood by biting people asia and South America. Both Bayesian and maximum like-
and animals; this can cause anaemia and atopic dermatitis, lihood approaches show strong monophyly of the Scionini
among other health problems [2]. This family is found but not of the genus Goniops, which occurs in the northern
worldwide, with 415 species in Africa, 244 species recorded hemisphere [13].
in India, 335 species in the Americas, 120 species in Malay- Expanding the Characterisation of tabanid mitogenomes is
sia, and over 100 species in Thailand [3]. Without a doubt, essential for species identification, phylogenetic studies,
horse fly morphological traits aid in identification and clas- and epidemiological research. Utilising existing mitochon-
sification. Nevertheless, depending solely on their morpho- drial genome databases, we used Illumina sequencing for
logical traits has some drawbacks [4]. Identifying dipteran decode of the mitochondrial genomes of six horsefly spe-
species accurately can be challenging and potentially risky cies[14]. Subsequently, we analyzed their evolutionary re-
due to inadequate physical characteristics and taxonomic lationships with other species in the Tabanomorpha infraor-
expertise. Consequently, molecular techniques such as der. The newly sequenced mitogenomes exhibited a con-
DNA barcoding are commonly employed to facilitate spe- sistent 37-gene circular topology typical of Tabanomorpha.
cies identification in this group[5]. The DNA barcoding of Phylogenetic analysis unveiled non-monophyletic relation-
the mitochondrial COX-1 gene sequence enables the iden- ships within the genus Tabanus of horseflies [15].
tification of rang arthropods, including tabanid flies [6]. The Tabanidae family has not been studied in Pakistan, par-
This is more effective than morphological identification as ticularly at the molecular level. To bridge this gap, we col-
this method allows genus/species level identification at any lected tabanid samples from the Swat district. The objec-
developmental stage, immatures or adults. DNA barcoding tives were: 1) to identify horseflies using COX-1 gene se-
methods are widely adopted in bio-diversity studies owing quences, 2) to compare nucleotide identity between the
to their ability for fast and accurate species identification identified horseflies and their congeners. and 3) to investi-
[7]. DNA barcoding method PCR amplification of a target gate the phylogenetic relationship among the identified
DNA of species. With DNA barcoding, the target species is horseflies and their congeners.
identified by performing PCR amplification of their
DNA[8]. The mitochondrial gene COX-1 is the most widely
used target for this purpose, with the amplified region typi- II. MATERIALS AND METHODS
cally ~658 base pairs in size. This technique enables accu- 2.1 Sample collection and study area
rate species identification [9].
A total of 250 tabanid samples were collected from various
The horsefly family (Tabanidae) contains 78 species di- districts in Swat KPK, Pakistan, including Barikot, Kabal,
vided into 2 subfamilies, and 10 genera in Croatia (species Matta Khwazakhela, Babuzai, and Bahrain. These tabanid
identification is mainly based on morphology). The most di- samples were physically collected while they were actively
verse genera are Tabanus (19 species), Hybomitra (7 spe- biting livestock to obtain blood. This method proved effec-
cies) and other genera. Morphological identifications were tive as it capitalized on the insects' preoccupation with
confirmed through DNA barcoding using the COX-1 gene, bloodsucking, making them easier to collect. The samples
with 16 new Barcode Index Numbers (BINs) added to the were washed with saline solution four times and were iden-
Barcode of Life Database (BOLD) [10]. tified using a standard taxonomic key outlined by Oldroyd
Phylogenetics is a common method in molecular biology in 1954 and further elaborated upon by Cameron in 2014a.
and evolutionary biology. It uses molecular data sequences

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 Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

Fig.2.1. Map of study area (Swat).

2.2 DNA Extraction amplification. PCR conditions, initial denaturation at 94°C

Total genomic DNA was extracted from horse fly samples for 5 min, followed by denaturation at 94°C for 30 sec, an-
for molecular analysis using the Wizprep DNA extraction nealing at 40°C for 1 min and extension at 72°C , continued
kit. After the material has been cleaned, extract genomic for 40 cycles. The amplification was extended for 10
DNA using the Wizprep DNA extraction kit. To ensure a minutes at 72°C, after which the PCR product was observed
successful DNA extraction, strictly adhere to the kit instruc- in 1% agarose gel with UV light under the Gel-Doc system.
tions. Reagent addition, temperature incubation, and DNA Table 2.1 PCR Reagents and their volumes
purification are the steps. Make sure that the prescribed se- Reagents Concentration
quence and times are followed. The DNA was kept in −20 ◦
C for further analyses. Obtaining pure genomic DNA that ddH2O 10.0 µL
is appropriate for later uses is the aim. Achieved DNA qual- Master Mix 10.0 µL
ity facilitates many uses, including sequencing and PCR. Templet DNA 2.5 µL
2.3 Polymerase Chain Reaction (PCR) and Gel Electro-
Forward primer 0.50 µL
phoresis
Reverse primer 0.50 µL
After DNA extraction the samples were subjected to a PCR
machine, the LCO1490-HC02198 Primers (Votýpka et al., Total volume 25.0 µL
2019), and the master mix used for the Cox-1 gene in PCR
Table 2.2 Condition for gel electrophoresis
Requirements Current Time Volume Voltage
Eluted DNA Adjust 25 minutes 3.5 µL 120 V

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Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

2.4 Bioinformatics analysis

With the use of the BLAST (Basic Local Alignment Search
Tool) program, the query sequence of the COX-1 gene un-
dergoes comparison with established sequences of the
COX-1 gene within the NCBI. This software identifies re-
semblances between the new sequence and an archive of
recognized sequences. Through these resemblances,
BLAST can infer the probable species of the organism from
which the sample originated.
2.5 Nucleotides identity
This alignment phase allowed meaningful and reliable com- Fig.3.1. Entire specimen of Atylotus spp.
parisons to be made by ensuring that the sequences were
placed correctly. To improve the quality of the analysis, the
trimmed aligned sequences were trimmed using BioEdit.
Trimming involved focusing on retained regions of the
COX-1 gene and excluding only sequences that were not
much related or comparable. Nucleotide identities were fi-
nally computed based on pairwise alignments among the se-
quences.
2.6 Phylogenetic tree
The identified horseflies and their relatives were examined
for evolutionary adjacency by constructing a phylogenetic
tree using MEGA 11.0 software (www.megasoft-ware. Fig.3.2 Head view of Atylotus spp.
net/dload_win_beta). Sequences were initially aligned us-
ing ClustalW, which reliably matched homologous sites.
The general-time reversible substitution model (GTR) was
chosen for data as it is a correct and accurate representation
of the rates of evolutionary change in sequences. Subse-
quently, MEGA 11.0 software was used to remove poorly
aligned positions for better accuracy and more reliable re-
sults of phylogenetic trees. A phylogenetic tree was then
generated based on the BI method to illuminate the evolu-
tionary relationship among horseflies and from other spe-
cies. The tree was visualised with Figtree v. 42
(http://tree.bio.ed.ac.uk/software/figtree/) as a graphical
tree format for easier and clearer visualization. This paved
the way for a greater understanding of the genetic diversity Fig.3.3 Photograph of the entire specimen of Tabanus sp.
and evolutionary connections between horseflies and close
relatives. These shocking findings were revealed after ex-
amining the combined capabilities of Figtree and MEGA
11.0, which demonstrated it is possible to better understand
the phylogenetic history of the horseflies and their phyloge-
netic relationships within their genera.

III. RESULTS
3.1 Morphological Identification of Horseflies and Pho-
tography
By using a taxonomic key, it is possible to identify the
Atylotus spp (Genus Atylotus) and Tabanus (Genus Taba-
nus) specimens (as described in the material method). Fig.3.4 Head view of Tabanus spp.

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Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

3.2 Identification of Horseflies through the COX-1 gene 93.6% of Atylotus sp. for A. agrestis, A. fuscipes, and A.
sequence nigromaculatus, respectively.
In the present study, COX-1 gene partial sequencing was Table 3.1. The % nucleotide identity of identified spe-
accomplished for three species; two of them from the genus cies/and some congers.
Tabanus (designated as Tabanus sp. 1 and Tabanus sp. 2) S.No. Names of Species Nucleo-
and one belonging to the genus Atylotus (Atylotus sp.). tide
Comparison of the gene sequences to databases of genetic identity
information in the NCBI was carried out by using the Basic in %
Local Alignment Search Tool (BLAST) program. However,
due to limited sequence data, only the genera of these spec- 1 Tabanus sp. 1& Tabanus Sp2. 90.1
imens could be identified, not the individual species. Nota- 2 Tabanus sp. 1& Tabanus fontinalis 93.8
bly, Tabanus sp. 1 exhibited a significant 93.95% similarity 3 Atylotus sp. & Atylotus nigromacu- 91.2
with Tabanus superjumentarius (Accession No. latus
KM2433535). Notably, the genome of T. superjumentarius
was sequenced in the United States for North Carolina State 4 Tabanus sp. 1& Tabanus rubidus 90.3
University before July 15, 2014 (Morita et al., 2014). Addi- 5 Tabanus sp. 1& Tabanus bromius 94.9
tionally, Tabanus sp. 2 demonstrated a significant genetic
6 Atylotus sp. & Atylotus fuscipes 88.8
similarity of 94.55% with Tabanus bromius (Accession No.
MK941824), which was previously sequenced on May 16, 7 Atylotus sp. & Atylotus agrestis 94.0
2019, at Eskisehir Technical University in Turkey (Sanal et 8 Atylotus fuscipes & Atylotus nigro- 98.9
al., 2021). Moreover, Atylotus sp. showed a 93.97% simi- maculatus
larity with Atylotus agrestis (Accession No. OL534387),
9 Atylotus agrestis & Atylotus nigro- 98.9
which was sequenced earlier on November 17, 2021, in
maculatus
South Africa (Williams et al., 2022).
10 Atylotus agrestis & Atylotus fusci- 97.9
3.3 Nucleotide identity
pes
The nucleotide names in among sequenced horseflies and
11 Tabanus bromius & Atylotus nigro- 90.5
their congeners are below. It is evident from table 3.1 that
maculatus
Atylotus sp. is 90.7% identical in nucleotide sequence to
Tabanus sp. 1, whereas with Tabanus sp. and 2, the identity 12 Tabanus bromius & Atylotus fusci- 89.0
is a little bit lower- 89.4%. Nucleotide identity between pes
Tabanus sp. 1 and Tabanus sp. 2 is 90.1%. These differ- 13 Tabanus bromius & Atylotus agres- 90.3
ences are evident in the distinct clades observed in the phy- tis
logenetic tree depicted in Figure 3.5 Upon thorough exami-
14 Atylotus sp. & Tabanus bromius 90.7
nation of Table 3.1, distinct patterns of nucleotide identity
among the listed species emerge. For instance, Tabanus sp.1 15 Tabanus rubidus & Atylotus nigro- 90.2
exhibits a nucleotide identity of 93.8% with T. fontinalis, maculatus
while with T. rubidus, the identity slightly decreases to 16 Tabanus rubidus & Atylotus fusci- 89.5
93.3%. In contrast, T. fontinalis and T. rubidus share a pes
higher nucleotide identity of 94.0%. With a 94% nucleotide
17 Tabanus rubidus & Atylotus agres- 90.5
identity between T. fontinalis and T. rubidus, it suggests a
tis
shared ancestry between these two species, as indicated by
their arrangement in the evolutionary tree diagram. Con- 18 Atylotus sp. & Tabanus Sp1. 90.7
versely, the nucleotide identities of Tabanus sp.1 with T. 19 Atylotus sp. & Tabanus Sp2. 89.4
fontinalis and T. rubidus, at 93.8% and 93.3% respectively,
20 Atylotus sp. & Tabanus fontinalis 89.8
slightly fall short of the 94% threshold. This positioning
suggests that in the phylogenetic tree, Tabanus sp.1 is 21 Atylotus sp. & Tabanus rubidus 89.8
closely related to T. fontinalis and T. rubidus. 22 Tabanus sp. 1& Atylotus agrestis 91.4
When we examined the data for Tabanus sp. 2 and T. bro- 23 Tabanus sp. 1& Atylotus fuscipes 90.0
mius we obtained a high nucleotide identity of 95%. genetic
24 Tabanus sp. 1& Atylotus nigro- 91.2
identity of Atylotus species, just as 94.0%, 92.6% and
maculatus

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Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

25 Tabanus sp. 2 & Tabanus fontinalis 93.8 amount of genetic alteration, evident from the extended
branch length observed in the phylogenetic tree. The sepa-
26 Tabanus sp. 2 & Tabanus rubidus 90.3
rate position of Tabanus sp. 1 indicates that it diverged evo-
27 Tabanus sp. 2 & Tabanus bromius 94.9 lutionarily for possible adaptation to the unique extreme
28 Tabanus sp. 2 & Atylotus agrestis 89.8 environmental conditions of KPK Swat
29 Tabanus sp. 2 & Atylotus fuscipes 88.8 The another subclade represents a distinct subgroup formed
by Tabanus sp. 2 and T. bromius (MK941824), distinguish-
30 Tabanus sp. 2 & Atylotus nigro- 89.4
ing them from other species. This topology clearly indicates
maculatus
that Tabanus sp. 2 deviates significantly from other mem-
31 Tabanus fontinalis & Tabanus ru- 94.0 bers of its group. However, despite this distinction, both
bidus Tabanus sp. 2 and T. bromius (MK941824) are species en-
32 Tabanus fontinalis & Tabanus bro- 92.6 demic to the Palaearctic region, specifically located in the
mius Swat district of KP province. Consequently, they share the
same clade and exhibit a bootstrap score of 90, indicating a
33 Tabanus fontinalis & Atylotus 89.8
common ancestor. The sequenced Tabanus sp. 2 and T. bro-
agrestis
mius (MK941824) demonstrate considerable genetic varia-
34 Atylotus fuscipes & Tabanus fontin- 88.6 tion from other members within the formed monophyletic
alis group.
35 Tabanus rubidus & Tabanus bro- 92.1 Atylotus represents another prominent clade comprising six
mius species categorized into two subclades. The sequenced
36 Tabanus fontinalis & Atylotus ni- 89.7 Atylotus sp. was grouped alongside A. fuscipes MW01386,
gromaculatus A. nigromaculatus MW337211, and A. agrestis OL534387.
A. agrestis and the Atylotus sp. series exhibit close linkage.
A. agrestis has a wide distribution spanning the Palaearctic
3.3 Phylogenetic relationship and Afrotropical regions, as well as China, Egypt, India,
The evolutionary tree depicted in Figure 3.5 contains nine- Saudi Arabia, and Sri Lanka. Similarly, Atylotus sp. is found
teen (19) species and a single outgroup, Sarcophaga rufi- in the Palaearctic region, aligning closely with A. agrestis.
cornis, that represents the infraorder Muscomorpha, order They cluster together in the phylogenetic tree, suggesting a
Diptera. The primary clades observed in the phylogenetic strong evolutionary connection due to their shared geo-
tree are the Tabanus and Atylotus clades. The Tabanus graphic regions. The evolutionary tree constructed in this
clade includes numerous subclades and sister clades. Taba- study highlights that although Tabanus sp. 1 and Tabanus
nus sp. 1 forms a robust clade together with the sister to the sp. 2 belong to the same genus, they form distinct clades.
penultimate common ancestor of T. fontinalis (MZ769395) Conversely, Atylotus sp. is grouped with Atylotus species
and T. rubidus (MT132391). T. fontinalis, and T. rubidus, within its own clade. Hence, from our study, we concluded
both native to North America, are closely related if the evo- that the genus Atylotus is monophyletic while the Tabanus
lutionary tree is any indication due to their proximal posi- genus is may be polyphyletic or paraphyletic because as it
tion in the same clade. Our sequenced Tabanus sp.1 dis- does not include all descendants of a common ancestor
plays a separate positioning from the T. rubidus based on the provided phylogenetic tree.
(MT132391) species, likely attributed to prolonged geolog-
ical isolation. This divergence signifies a substantial

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Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

Fig.3.5. Phylogenetic relationship of collected species of horseflies and their congeners through BL method as a base on
partial sequence of mitochondrial cox-1gene. Sarcophaga ruficornis is an outgroup organism from the infraorder Musco-
morpha order Diptera.

Table 3.1. The species taxonomy and GenBank accession numbers included for phylogenetic analysis in this study. Bold se-
quences indicate new sequenced species.
Name of species Systematic position Accession No.
Tabanus sp. 1 Insecta, Diptera XXXX
Tabanus sp. 2 Insecta, Diptera XXXX
Atylotus sp. Insecta, Diptera XXXX
Tabanus fontinalis Insecta, Diptera MW013786
Tabanus rubidus Insecta, Diptera MT132391
Atylotus nigromaculatus Insecta, Diptera MW337211
Atylotus fuscipes Insecta, Diptera MW013786
Atylotus agrestis Insecta, Diptera OL534387
Tabanus bromius Insecta, Diptera MK941824
Tabanus melanocerus Insecta, Diptera MW152006
Atylotus miser Insecta, Diptera OM991885
Atylotus horvathi Insecta, Diptera OM991884
Tabanus chrysurus Insecta, Diptera ON506245
Tabanus pleskei Insecta, Diptera ON556503
Tabanus moderator Insecta, Diptera MW152025
Tabanus molestus Insecta, Diptera MW152025
Tabanus autumnalis Insecta, Diptera MZ563367
Tabanus superjumentarius Insecta, Diptera KM243535
Sarcophaga ruficornis Insecta, Diptera (Muscomorpha) KM538688

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Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

IV. DISCUSSION (MW238412) was investigated by [17]. The results showed

In this study, samples were collected from Swat, and their characteristic identity patterns: the sequence of A. agrestis
DNA were amplified by PCR targeting the COX-1 gene. from Saudi Arabia proved from 92% to 99% nucleotide
Following amplification, sequencing of the COX-1 gene re- similar to tabanid isolated from other regions. The nearest
vealed that Tabanus sp.1 and Tabanus sp.2 exhibited se- tabanid isolates from varied regions such as the Central Af-
quence identities of 93.95% and 94.55% with T. super- rican Republic (CAR) (MK396308), India(KM111678),
jumentarius and T. bromius, respectively. Moreover, Atylo- Gabon(MK396326), Thailand (MG426116) and the USA
tus sp. showed 93.97% sequence identity to A. agrestis. (KM243535) ,estimated a highest nucleotide identity of
According to [6], the specimens were originally identified 91% to 92.80% with the T. par sequence of Saudi Arabia.
based on morphology as Ha. turkestanica, C. vanderwulpi, In the present study, the phylogenetic relationship of Taba-
C. dissectus, T. chrysurus, T. pleskei and Hy. sp. were nus sp. 1 clade and T. frontinalis and T. rubidus and the
known only to genus because species-specific information Tabanus sp. 2 clade and Tabanus bromius were studied.
was not available. When amplified cox1 sequences were Similarly, the Atylotus sp. clade was examined alongside A.
compared with sequences deposited in GenBank agrestis, A. fuscipes, and A. nigromaculatus. Hence, from
(MT231188, OM991886, OM991887, NC062705, our study, we concluded that the genus Atylotus is mono-
NC062705, and MT410834), identities of 93.5%, 93.8%, phyletic while the Tabanus genus is may be polyphyletic or
96.2%, 95.62%, 96.48%, and 96.15% were observed, re- paraphyletic because as it does not include all de-ascendants
spectively. of a common ancestor based on the provided phylogenetic
The findings of this study revealed distinct genetic vari- tree. In a previous cladistic study by[6] , Maximum Likeli-
ances between Tabanus sp.1 from KP Swat and the indige- hood (ML) analysis was utilized to construct a phylogenetic
nous species of North America, T. fontinalis and T. rubidus. tree. The findings from the analysis consistently supported
Although T. fontinalis and T. rubidus exhibit 94% identi- the delineation of six recognized branches. It was observed
ties at the nucleotide level, Tabanus sp. 1 has nucleotide that Hy. sp. grouped within these branches alongside other
identities of 93.8% and 93.3% to T. fontinalis and T. ru- species belonging to the genus Hybomitra, such as H. astur,
bidus. This slight divergence suggests a genetic distinction H. lurida, and H. bimaculata. Moreover, the research indi-
of Tabanus sp. 1 from the native North American species cated H. turkestanica is a member of the genus Haemato-
(T. fontinalis and T. rubidus), likely attributable to signifi- pota, and T. pleskei and T. chrysurus fall under the genus
cant geographical separation over time. According to Tabanus. Moreover, six Atylotus horseflies were virtually
[16].In contrast to species inhabiting the Nearctic and Neo- huddled on a single branch, while C. dissectus and C.
tropical regions, Tabanus species exhibit genetic unique- vanderwulpi horseflies were embedded on the branch of the
ness as they originate from the Afrotropical region. The nu- Chrysops horseflies. This study confirms the current taxo-
cleotide alignment exhibited interesting patterns as re- nomic classification and supports the idea that all genera are
ported by [17] specimens of H. lurida exhibited close monophyletic. COX-1 sequences obtained from genera in
matches with those from the Nearctic and Neotropical re- three tribes of the Tabanidae family (Atylotus, Hybomitra,
gions, whereas specimens of both T. par and A. agrestis Tabanus, Haematopota and Philoliche), and from the
from Saudi Arabia were more similar to populations in the Chrysops genera were used for ML and BI phylogenetic
Afrotropical region than the Palearctic. Notably, molecular analyses, according to . All COX-1 sequences found in tab-
research unveiled a heightened level of genetic resemblance anids are clustered with their congeners. The monophyly of
between tabanid specimens from Saudi Arabia and those the family Tabanidae was strongly supported by both to-
from other species worldwide. This study uncovered that pologies[6]. 1. Monophyly Tabanidae are monophyletic as
Tabanus sp. 2 and T. bromius share an astonishing 95% previously proposed [18, 19]. The above results coincide
similarity at the nucleotide level, which suggests a remark- with earlier studies showing that based on genetic data (nu-
able affinity between the two species especially, as alluded clear (28S) and mitochondrial (COX-1) genes) and by mor-
to above as they are both present in the Palaearctic. In con- phology (genitalia and external features) Tabanidae is a
trast, 94.0% of nucleotide identity was found between the monophyletic group[6].
sequenced Atylotus sp. and A. agrestis, indicating a close
genetic relationship of the two. The other two were A. V. CONCLUSION
agrestis species that are widely distributed; the Palaearctic
The molecular study of Tabanid from Pakistan which indi-
and Afrotropical A. agrestis was previously identified from
cates the two genera Atylotus and Tabanus with 93.95%
China, Egypt, India, Saudi Arabia, and Srilanka. Nucleotide
similarity between Tabanus sp. 1 and T superjumentarius
identity of Saudi Arabian specimens of A. agrestis
(Accession No. KM2433535), while Tabanus sp. 2 shares
(MW243943), H. lurida (MW265638) and T. par

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https://dx.doi.org/10.22161/ijeab.104.2 24
Ullah et al. Cox-1 Gene-Based Identification and Molecular Phylogenetics of Horseflies

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