Description of Male, Redescription of Female, Host Record, and Phylogenetic Position of Haemaphysalis danieli

Haemaphysalis ticks are globally distributed with the greatest diversity in the Oriental region. This study aimed to primarily provide information on the morphology, host record, and preliminary phylogenetic position of a poorly known tick Haemaphysalis danieli. Herds comprised of goats and sheep were examined for this tick species in Upper Dir, Khyber Pakhtunkhwa, Pakistan. A total of 127 ticks, including males (n = 15, 11.8%) and females (n = 112, 88.2%), were collected, and morphologically identified as H. danieli. The morphological identification was confirmed through the 16S rDNA and cytochrome c oxidase (cox1) sequences. Phylogenetic analysis inferred based on 16S rDNA and cox1 showed a close evolutionary relationship of H. danieli with a conspecific from China and an undetermined Haemaphysalis sp. from China and Anatolia. A total of 32/223 (14.3%) goats in two different herds were the only host infested by H. danieli. The earliest study provided the morphological description of H. danieli male, host record, and phylogenetic position. The information provided herein could assist in minimizing the knowledge gap regarding the systematic and taxonomy of Haemaphysalis species.


Introduction
Haemaphysalis species are found in highly variable climatic conditions in different zoogeographical regions, varying from humid, mild, cold winter, well-vegetated habitats to dry, hot summers [1,2]. The species of this genus are distributed throughout the world, with the most significant number recorded in the Oriental region. Some known species of the genus Haemaphysalis are also reported from the Palearctic and Ethiopian regions [1,2].
The immature (nymphal and larval) stages and a female tick of H. danieli were collected in 1970 from different northern regions of Pakistan: Swat (currently Chitral), Gilgit Baltistan (Phandar, Babusar, and Rama Valley), Hazara district (Gitidas and Khagan Valley), and Afghanistan: Wakhan area of Badakhshan province [3]. Different wild animals of the order Rodentia such as Phodopus sungorus Pallas, 1973, Apodemus gurkha Thomas, 1924, Apodemus flavicollis Melchior, 1834, Hyperacrius wynnei Blanford, 1881, Marmota himalayana Hodgson, 1841, Cricetulus migratorius Pallas, 1773, Alticola roylei Gray, 1842, Marmota caudata Geoffroy, 1844, and Ochotona roylei Ogilby, 1839, were suggested to be the hosts for the immature stages of H. danieli, while domestic and/or wild caprine and ovine were considered the probable hosts for the adults of this tick [3]. Most of the Haemaphysalis species are associated with wild animals, but changes in their feeding behavior have been noticed. These species switched from wild hosts to domestic animals like cattle, sheep, and goats [4,9,10].
Morpho-molecular and phylogenetic analyses are among the best methods adopted for understanding the identification and systematics of known and rare and/or poorly known tick species [4,11]. To our knowledge, there is no comprehensive study on the morphology of male, host records, and phylogenetic analyses of H. danieli ticks. Therefore, the current study aimed to describe the H. danieli male ticks, host record, and preliminary phylogenetic analysis.

Ethical Statement
Ethical approval was obtained from the Advanced Studies and Research Board (Dir/A&R/AWKUM/2022/9396) of the Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan, KP, Pakistan. Oral permissions were taken from the owners of animals or herds.

Study Area
The province of Khyber Pakhtunkhwa (KP) shares its border through the international boundary with Afghanistan in the northwest and the north, and both countries are connected by the Hindu Kush range. This study was conducted in the district Upper Dir ( District Upper Dir is part of the subtropical dry and to some moist temperate regions, representing four seasons: long, cold, and severe winter, and short summer, spring, and autumn. The study district is located in a hilly, mountainous, and highly elevated area (~3000 m); average temperature (−7 • C to 35 • C), high relative humidity above 70%, and precipitation (770 mm). The Global Positioning System (GPS) was used to find the geo-coordinates of the collection points. The elevation-based map was designed through ArcGIS 10.3.1 (ESRI, Redlands, CA, USA) ( Figure 1).

Tick Collection and Preservation
Small ruminants, including 223 goats and 168 sheep kept individually and/or transhumant/nomadic herds in the tick collection sites, were examined for ticks. Ticks were directly collected from the skin by fine-tipped tweezers during the summer in July 2022. Tick specimens were stored in 1.5 mL tubes and shifted to the Department of Zoology, Abdul Wali Khan University, Mardan. The collected tick specimens were rinsed with distilled water, followed by 70% ethanol to remove any tissue debris, and preserved in 100% ethanol in well-labeled plastic tubes for further molecular analyses.

Morphological Identification of Ticks
Tick specimens were morphologically identified and confirmed up to the species level using a stereomicroscope (StereoBlue-euromex SB.1302-1, Arnhem, The Netherlands) with 3.5-135×, using a standard taxonomic identification key based on different morphological

Tick Collection and Preservation
Small ruminants, including 223 goats and 168 sheep kept individually and/or transhumant/nomadic herds in the tick collection sites, were examined for ticks. Ticks were directly collected from the skin by fine-tipped tweezers during the summer in July 2022. Tick specimens were stored in 1.5 mL tubes and shifted to the Department of Zoology, Abdul Wali Khan University, Mardan. The collected tick specimens were rinsed with distilled water, followed by 70% ethanol to remove any tissue debris, and preserved in 100% ethanol in well-labeled plastic tubes for further molecular analyses.

DNA Extraction and PCR
A total of 15 tick specimens (two males and thirteen females) were subjected to genomic DNA extraction for molecular identification. The tick specimens were crushed with sterilized pestles in 1.5 mL Eppendorf tubes. The genomic DNA was extracted from each tick specimen using a phenol-chloroform protocol [12] per the standard guidelines. The DNA pellet was hydrated by adding 30 µL of PCR water "nuclease-free". The extracted genomic DNA was quantified using NanoDrop (Nano-Q, Optizen, Daejeon, Korea).

DNA Sequencing and Phylogenetic Analyses
The PCR amplified amplicons were purified using the GeneClean II Kit (Qbiogene, Il-lkirch, France) following the manufacturer's protocol. A total of 30 amplified or purified products (one for 16S rDNA and one for cox1 from each tick specimen) were sequenced bidirectionally in a commercial company (Macrogen, Inc., Seoul, Republic of Korea) by the Sanger sequencing method. The obtained sequences were trimmed and assembled in SeqMan V. 5.0 (DNASTAR, Inc., Madison, WI, USA) to remove low-quality nucleotide sequences and primer regions. The purified sequences were subjected to BLAST (Basic Local Alignment Search Tool: https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 4 September 2022) [15] using the searching tool NCBI (National Center for Biotechnology Information). The homologous sequences were downloaded from NCBI in FASTA format for phylogenetic analysis. These sequences were aligned in BioEdit Sequence Alignment Editor V. 7.0.5 (Raleigh, NC, USA) [16] using ClustalW Multiple alignments [17] with the obtained and an outgroup sequence (Haemaphysalis punctata). The phylogenetic trees for 16S rDNA and cox1 sequences were constructed according to the Maximum Likelihood method in MEGA-X (Molecular Evolutionary Genetics Analysis) with a 1000 bootstrapping value and Kimura 2-parameter model [18]. The coding sequences (cox1) were aligned by MUSCLE [19]. The obtained sequences formed the dataset's final positions.

Collected Ticks and Host Record
A total of 127 ticks were collected and all were morphologically identified as H. danieli (Figures 2 and 3), comprised of males (n = 15, 11.8%), females (n = 112, 88.2%), and no immature stages of the tick were collected. All H. danieli ticks were found only on goats in two different herds containing goats and sheep. These two herds were found at two different locations (Dare Khwar and Samasu Khwar) of Upper Dir, separated by a distance of approximately 4-5 km (km). Nearly four ticks/host, a total of 32/223 (14.3%) goats, were infested by H. danieli ticks.

Description of Haemaphysalis danieli 3.2.1. Description of Male
Idiosoma: Ornamentation is indistinct on the conscutum, with reddish brown to dark reddish color; body is oval shape with length (excluding capitulum) 2.1 mm, greatest width 1.6 mm, (Figure 2A (a,b). Conscutum is pear-shaped, about twice as long as wide, and is widest at the level of the fourth leg, Figure 2A (a,b); punctuations are shallow, mostly small, and of medium size, distributed slightly dense and not very uniform, Figure 2C (c). Scapulae are short and blunt; the margins are narrow and shallow; cervical grooves are deep, linear anteriorly and converging posteriorly; lateral grooves are narrow and long, enclosing the first two anterior festoons; eyes absent; 11 festoons ranging from 0.13 to 0.17 mm in width and 0.17 to 0.2 mm in length, Figure 2A (a,b). Stigmas are oval-elongated with a narrower part dorsally and rounded macula located on the antero-inferior side, Figure 2F (b); anus with "Y" anal groove, "Y" tail reaching the festoon and the lateral arms exceed the anus anteriorly ( Figure 2B). The genital apron is located between the coxa II, Figure 2D (b).

Collected Ticks and Host Record
A total of 127 ticks were collected and all were morphologically identified as H. danieli (Figures 2 and 3), comprised of males (n = 15, 11.8%), females (n = 112, 88.2%), and no immature stages of the tick were collected. All H. danieli ticks were found only on goats in two different herds containing goats and sheep. These two herds were found at two different locations (Dare Khwar and Samasu Khwar) of Upper Dir, separated by a distance of approximately 4-5 km (km). Nearly four ticks/host, a total of 32/223 (14.3%) goats, were infested by H. danieli ticks.

Description of Male
Idiosoma: Ornamentation is indistinct on the conscutum, with reddish brown to dark reddish color; body is oval shape with length (excluding capitulum) 2.1 mm, greatest width 1.6 mm, (Figure 2A (a,b). Conscutum is pear-shaped, about twice as long Gnathosoma: Length from apices to posterior margin of the basis 0.37 mm; the basis capituli is 1.8 times wider than long (including the cornua), with the posterior margin being straight and having thick, short, and blunt cornua, Figure 2C (a). Palpi are elongated and clavate, with a length of four articles as follows: article 1, 0.07 mm; article 2, 0.17 mm. The distal end of article 2 is noticeably wider, being 1.5 times longer than the third article. Article 3 was 0.12 mm, without external spur; article 4 is in the apical pit, visible ventrally; hypostome is slightly shorter than the palpi, having length 0.24 mm and width at base 0.12 mm; dental formula is 5/5, distally a rosette is visible, Figure 2D (a).
Legs were short and robust (Figure 2A,B). All coxae have a well-developed inner spur, similar in size and shape, Figure 2E (a,b), slightly longer than the width of its base, narrow and blunt or firmly pointed at the end, and slightly curved to the outside, Figure 2E (a,b) and Figure 2F (a). Tarsus I tapers distally, measuring a length of 0.29 mm, with a clear, oval area on the dorsum of tarsi I, Haller's organ.
Chaetotaxy: Small and tiny hairs can be observed on the palps and legs.

Redescription of Female
Idiosoma: Scutum lacks ornamentation, having a dark brown color. This tick possesses an elliptical-shaped body with a maximum length (excluding capitulum) of 2.86 mm and a maximum width of 2.09 mm ( Figure 3A (a-d). Scutum is pear-shaped, slightly longer than width, is widest between the second and third legs, narrows backward, with a blunt end, Figure 3 (b); punctuations are thick and deep, distributed densely, but not uniform, Figure 3 Figure 3A (a,b). Stigma is pear-shaped, tapering towards the outside, Figure 3E (a); anus with "Y" shaped anal groove, "Y" tail extending towards the central festoon and the lateral arms diverging anteriorly and exceeding the anus, ( Figure 3B). The genital aperture is located between the coxae II and III with a U-shaped, Figure 3D (b).
Gnathosoma: Length from the anterior top to the posterior bottom 0.66 mm ( Figure 3C); basis capituli is about 3 times wider than long; cornua absent, Figure 3C (a); the porose areas are moderate size, oval, and distantly spaced, with a shallow depression between them, Figure 3C (a). Ventrally, basis capituli is wide and short, with the posterior edge arced, Figure 3F (a). Palpi elongated and clavate, with a length of four articles as follows: article 1, 0.13 mm, article 2, 0.29 mm, narrowing from front to back, the distal end of article 2 is wider, 1.8-1.9 times longer as the third article 3, 0.16 mm, the posterior margin is straight, article 4, 0.1 mm (located in the third article pit) and only visible ventrally, Figure 3C (a). Hypostome is slightly shorter than the palpi with a length of 0.54 mm and width at base 0.25 mm, dental formula is 5/5, end with a crone, Figure 3F (a). For more detailed information on female H. danieli, see the standard key proposed by the expert [3].
Legs: The main features of each leg are the same as those of male ticks, Figure 3A-F. Chaetotaxy: Fine hairs can be seen on the palps and legs.

Molecular Analyses
DNA was extracted from all 15 H. danieli ticks. The nucleotide sequences of the expected size, one 16S rDNA and one cox1 per tick, were obtained. The sequences belonging to the same partial fragments were found to be identical. Two consensus sequences were obtained, including 16S rDNA (390 bp) and cox1 (675 bp), which were considered for further analyses. By BLAST analysis, the 16S rDNA showed 98. The consensus DNA sequences of H. danieli were submitted to GenBank under accession numbers OP435750 (16S rDNA) and OP435801 (cox1).

Phylogenetic Analysis
In the phylogenetic analysis based on 16S rDNA (Figure 4), the H. danieli of the present study clustered with the same species and an undetermined Haemaphysalis sp. compared to the phylogenetic tree based on cox1 (Figure 5), where H. danieli was clustered only with the same species. In both phylogenetic analyses, H. danieli appeared in a monophyletic branch with different species (H. tibetensis).

Discussion
Genus Haemaphysalis counts the second most diversified genus of existing tick fauna, containing 176 species [2,20,21]. With the existence of large diversity in ticks of the Oriental region and the lack of systematic knowledge associated with terms of precise identification and categorization, these ticks have mostly remained unidentified accurately. Herein, we described the male morphology, host record, and preliminary phylogenetic position of H. danieli.
Haemaphysalis danieli collected from goats represents the first confirmed host and male tick record in the country, while formerly, only a female tick was collected from vegetation inhabited by wild ibex and many rodents [3]. Herein, female ticks outnumbered male ticks, which could be attributed to unequal hatching and mortality rates [22-

Discussion
Genus Haemaphysalis counts the second most diversified genus of existing tick fauna, containing 176 species [2,20,21]. With the existence of large diversity in ticks of the Oriental region and the lack of systematic knowledge associated with terms of precise identification and categorization, these ticks have mostly remained unidentified accurately. Herein, we described the male morphology, host record, and preliminary phylogenetic position of H. danieli.
Haemaphysalis danieli collected from goats represents the first confirmed host and male tick record in the country, while formerly, only a female tick was collected from vegetation inhabited by wild ibex and many rodents [3]. Herein, female ticks outnumbered male ticks, which could be attributed to unequal hatching and mortality rates [22][23][24], and such tick species may have a selective advantage [25]. Furthermore, it could also depend on the season and area of collection. Comparing the collection points of current and previous studies suggests that it might be due to habitat expansion, or the migration of hosts associated with this tick [3], as currently, this tick has been collected from goats in Upper Dir about 102 km away from the previous collection done in the Tirich Mir valley. In our current study, all ticks were collected at an altitude reaching up to ∼3000 m in the western Himalayas in view of the fact that H. danieli prefers rocky biotopes at high altitudes [3], as all members of the subgenus Allophysalis were reported from high terrains (1600-4000 m) of Himalayan and Hindu Kush ranges [4]. Some life stages of species belonging to the subgenus Allophysalis are unknown because of inhabiting such harsh topography, and thus being difficult for researchers to find [2].
Descriptions of male and female H. danieli revealed that moderate-sized coxal spurs exhibit hook-like or spiny shapes to firmly attach to the host [4]. As a member of the subgenus Allophysalis, H. danieli females possess elongated palpi, pointing towards the primitive group of ticks [26]. The description of H. xinjiangensis developed confusion as a new species in China [27], confirmed by Hoogstraal as a new species of the subgenus Allophysalis [4]. Kolonin (2009) believed that H. xinjiangensis is a synonym of H. pospelovashtromae, but to support his view, he did not provide any conclusive evidence. Therefore, Guglielmone et al. [2,28] treated H. xinjiangensis as a synonym of H. danieli.
When morphology becomes unreliable, the rapid and precise identification of any life stage of a tick can be achieved through molecular approaches by using phylogenetic analysis [29][30][31][32][33][34], targeting mitochondrial genes like 16S rRNA and cox1 [33][34][35][36]. In the phylogenetic trees, the obtained partial sequences of 16S rDNA and cox1 clustered with the species from the same subgenus, e.g., H. danieli and H. tibetensis reported from China. Furthermore, H. tibetensis clustered as a sister clade to H. danieli in the phylogenetic trees, which authenticated the relation between the two species and classified them into the same subgenus Allophysalis [4]. Additionally, the morphological description has been validated by the current phylogenetic analyses.

Conclusions
This study rediscovered a poorly known H. danieli tick, found the host record, and genetically characterized it for the first time. The study confirmed that H. danieli is endemic to the mountains of the Hindu Kush-Himalayan (HKH) region located at the junction of the Oriental and Palearctic regions. Besides contributing to the genomic data of Haemaphysalis ticks enlisted in the Oriental region, this study may assist in understanding the systematic and taxonomy of Haemaphysalis species.