Research Article Descriptive Study of Some Osteological Parts of Rosy Stone Lapper ( Garra rossica ) from Mashkid Basin of Iran

Since osteological structures of ﬁshes provide important biological and ecological information, studying these structures is valuable. On the other hand, due to lack of data on the osteology of stone lappers, the present study was conducted to provide detailed descriptive osteology of Garra rossica from Mashkid basin, southeastern Iran, comparing it with those of G. typhlops from the Bagh-e Loveh cave, Iran, G. rossica from Nahang River, Mashkid Basin, and G. persica from Zahak River, Sistan basin. For this purpose, 15 specimens of G. rossica were captured from Ladiz River, Mashkid Basin, using an electroﬁshing device, and ﬁxed in 10% buﬀered formalin. Then, the specimens were cleared and stained with alcian blue and alizarin red for osteological in-vestigations. A detailed description of the osteological features of G. rossica was provided. Based on the results, several diﬀerences were observed between the four species. G. rossica can be distinguished from G. typhlops , G. persica , and G. rossica based on the shape of suspensorium and opercular series, pharyngobranchial bones, ventral and pectoral girdles, caudal, dorsal, and anal ﬁns skeleton, and shape and number of infraorbital elements.


Introduction
e cyprinid genus Garra Hamilton 1822 has a wide geographic distribution from Borneo, China, and southern Asia, through the Middle East, Arabian Peninsula, and East Africa to West Africa [1]. ere are about 73 species in Garra [2], among which 14 species are recognized from Iran. Esmaeili et al. [3] listed four species G. persica, G. rufa, G. variabilis, and G. rossica [3]; recently, Sayyadzadeh et al. [4] reviewed the members of the genus in the Persian Gulf and Oman Sea basins and recognized six epigean species, including G. barreimiae, G. longipinnis, G. persica, G. rossica, G. rufa, and G. variabilis, and two subterranean species, including G. typhlops and G. widdowsoni, and described a new species from the region as G. mondica. Mousavi-Sabet and Eagderi [5] described a new species of G. lorestanensis. Esmaeili et al. [6] reviewed Garra genus species and introduced a new species, G. amirhosseini. Mousavi-Sabet et al. [7] described a new species of G. roseae. Moreover, Zamani-Faradonbe et al. [8] described G. meymehensis and G. tiam. Species of the genus Garra inhabit a wide range of substrates (muddy, sandy, and rocky bottoms) in streams, rivers, pools, and lakes [9]. ey are primarily freshwater species but are also reported from brackish waters [1]. is genus is characterized by small-to-moderate body size and elongate and almost cylindrical body shape having a rounded snout with an inferior and crescent-shaped mouth, a horny lower jaw, usually fringed upper lip continuous with the snout, and a suctorial disc with a free posterior margin [2].
Despite the introduction of modern techniques such as DNA sequencing and barcoding, osteology, due to its reliability, still plays an important role in the systematic studies of fishes and comprises a major percent of today's works. Since osteological characteristics can provide valuable information in taxonomy and phylogenetic relationships of fishes [10], the present study was conducted to provide detailed descriptive osteology of G. rossica from the Ladiz River, Mashkid Basin, Iran.

Material and Method
Fifteen specimens of G. rossica were collected from Ladiz River, Mashkid Basin (total length: 60.77 ± 5.53 mm, mean ± SD) using the gill net and electrofishing device and anesthetizing in 1% clove oil, then fixed in 10% buffered formalin, and transferred to the laboratory for further examinations. e specimens were cleared and stained with alizarin red S and alcian blue according to the protocol of Taylor and van Dyke [11] and Sone and Parenti [12] with some modifications for osteological examination. e cleared and stained specimens were studied using a stereomicroscope (Leybold Didactic GmbH model) and their skeletal elements were dissected and scanned by a scanner (HP Scanjet 3770) equipped with a glycerol bath. Drawing of the specimens was performed using CorelDraw X7 software. e terminology of skeletal elements follows Howes [13] and Rojo [14]. e detailed osteological features of Garra typhlops from the Tigris basin were provided by Jalili and Eagderi [15], G. rossica from Nahang River, Mashkid Basin, by Saemi-Komsari et al. [16], and G. persica from Zahak River, Sistan Basin, by Zamani-Faradonbe and Keivany [17].

Results
e ethmoid region of the neurocranium is flat and contains the kinethmoid, preethmoid-I, lateral ethmoid, supraethmoid, vomer, and nasal bones ( Figure 1). e supraethmoid is broad and has two vertical and horizontal sections; the vertical section of this bone is located on the dorsal part of the vomer and the anterior part of the parasphenoid. e horizontal section bears two small anterior processes and a shallow middle depression with cartilaginous lateral margins ( Figure 1). e posterior part of the vomer is pointed and overlapped with the anterior part of the parasphenoid and its anterior part has a V-shaped notch. e anterior part of the vomer is thicker than its posterior part. e ventral face of the vomer is smooth. e preethmoid-I bones are small and semicircular in shape and are present in the anterior-lateral edge of the vomer (Figure 1). e lateral ethmoid is located as a wall between the ethmoid and orbital regions; the lateral part of the lateral-ethmoid bears two anterior and posterior processes. A cylindrical kinethmoid exists between the maxillary bones ( Figure 1). e orbital region comprises the frontals, parasphenoid, ptersphenoids, orbitosphenoids, and circumorbital series. e frontal is a large bony element of the skull roof with a serrated anterior edge and bent ventrally; it bears a midlateral pointed process. e supraorbital canal is enclosed by the lateral margin of the frontal. e two orbitosphenoids are fused via their ventral process that is also connected to the parasphenoid (Figure 1). e ptersphenoid is concaved in shape with two ventral processes for connecting to the parasphenoid and bears two pores. e ptersphenoid is dorsally attached to the frontal, anteriorly to the orbitosphenoid, and posteriorly to the sphenotic. e anterior half of the parasphenoid is wider and is of typical shape with a serrated anterior rim and its posterior half has a triangular structure that its posterior margin bifurcates via a deep groove and has some pores in its middle part ( Figure 1). e otic region includes the parietal, epiotic, sphenotic, pterotic, and prootic bones. e parietal bone is almost square-shaped, and its posterior-lateral margin overlapped asymmetrically and laterally the dorsal part of the pterotic and epiotic bones. e epiotic is oval-shaped with a posterior process ( Figure 1); it is situated between the pterotic and supraoccipital bones. e pterotic is a quarter-circle in shape ( Figure 1) and its posterior-lateral part is well-developed and probably fused to the dermopterotic. e sphenotic bears a lateral process that connects the middle process of the frontal; this bone is ventrally attached to the prootic and posteriodorsally to the pterotic ( Figure 1). e two prootics are connected to each other ventrally and to the parasphenoid dorsally by a descending process. is bone has two pores on its anterior part. e occipital region is composed of the supraoccipital, exoccipital, and basioccipital. e supraoccipital is pentagon in shape and has a blade-shaped crest; its lateral part is connected to the epiotic. e exoccipital bears a large ventral foramen on its middle concave part. In the dorsal part of the basioccipital, there are a pointed pharyngeal process and a concaved masticatory plate pointing laterally. e pharyngeal process has a dorsal fossa and a ventromedial ridge ( Figure 1). e upper jaw is made up of the paired maxilla, premaxilla, and unpaired kinethmoid. e two maxillae are located at the dorsal side of the premaxilla; the anterior part of the maxillae is wider than the posterior part. A notch on the lateral edge of the maxillae is marked. e premaxilla is wide, L-shaped, and articulated with the maxillae and dentary and the kinethmoid bone is situated between the two maxillae ( Figure 2(a)). e lower jaw is composed of three bones, including the dentary, articular and retroarticular. e dentary is L-shaped, and its anterior edge is flat. e coronoid process situated on the dorsal middle part of the dentary is oriented posteriorly. e middle part of the articular is wide and its posterior part bears an articulatory facet. e triangularshaped retroarticular is a small bone situated under the posterior part of the articular; the posterior part of the retroarticular is connected to the interopercle via the retroarticular-interopercle ligament. A small and elongated coronomeckelian is observed on the medial face of the articular (Figure 2(a)). e suspensorium series is formed of the hyomandibular, ectopterygoid, endopterygoid, metapterygoid, symplectic, quadrate, and palatine (Figure 2(a)). e hyomandibular bone is almost triangular in shape: its dorsal part is wider and posterior part bears two protuberances; the ventral part of the hyomandibular is connected to the interhyal. is bone is articulated to the posterior part of the neurocranium with two hyomandibular articulatory condyles and to the opercle by opercular articulatory condyle. e ectopterygoid, endopterygoid, metapterygoid, symplectic, and quadrate form a bony complex connecting to the anterior part of the neurocranium via the palatine. A long symplectic is enclosed by the metapterygoid posteriorly, by the quadrate anteriorly and by the preopercle posteriorly. e anterior part of the palatine bears three processes and slightly deep depression for connecting to the vomer and preethmoid-I. e opercular series consists of four bones including the opercle, preopercle, subopercle, and interopercle bones. e opercle bone that is the largest element of this series has an opercular process in anterodorsal part and this bone has a socket joint that is connected to the hyomandibular. An L-shaped preopercle presents in the anterior side of the opercle and ventral side of the hyomandibular; its vertical part is longer than that of its wider horizontal part. e ventral part of the preopercle overlaps the dorsal part of the interopercle. e subopercle is long, and its anterior part is broad (Figure 2(a)). e hyoid arch consists of the unpaired basihyal and urohyal and the paired epihyals, hypohyals, and ceratohyals and three pairs of branchiostegal rays. e urohyal is the largest element that has two vertical and horizontal sections. e posterior margin of its horizontal section is pointed and its middle part is wider. In addition, the anterior part of its horizontal section is branched. e posterior part of the ceratohyal is wider than its anterior part; its anterior part is branched and connected to the dorsal and ventral hypohyals. e dorsal and ventral hypohyals are fused. e basihyal is T-shaped, situated between the hypoyals (Figure 2(a)).
In the circumorbital series, the number of the infraorbital bones was the same in the studied specimens. ere are four infraorbital and one supraorbital elements. e first circumorbital, i.e., lacryamal, is the largest element of this series. e supraorbital is oval-shaped and located at the lateral side of the frontal. e suborbital canal is enclosed by the infraorbital bony elements (Figure 2(b)). e branchial apparatus includes five types of bones, including five pairs of the ceratobranchials, four pairs of the epibranchials, three pairs of the hypobranchial and two pairs of the pharyngobranchial, and three unpaired basibranchial. e fifth ceratobranchial is crescent in shape with a dental. ere are three rows of pharyngeal teeth with a dental formula of 2.4.5-5.4.2 (Figure 3). e dorsal fin bears one unbranched ray and nine branched rays, eight pterygiophores and one stay bone (Figure 4(a)). e first pterygiophore is the largest, U-shaped (bifurcates), and supports unbranched ray and first branched ray. e first pterygiophore is next to the 22th and 23th preural. A tetrangular stay bone supports the last branched ray. e anal fine originates at 12th preural. is fin has two unbranched and seven branched rays that were supported by six pterygiophores and one small stay bone (Figure 4(b)). e caudal skeleton consists of the last centrum with the epural, parhypural, pleurostyle, uroneural, and six hypurals bones ( Figure 5(c)). e pleurostyle is fused to the last centrum (urostyle). e neural arch of the second centrum is consumptive in some specimens. e first hypural is the largest one and attached to the parhyporal. e epural is a long bone positioning at the dorsal part of the neural arch of the first vertebra. ere are 31-35 vertebra, including 15-17 cranial and 16-18 caudal centra. e pectoral girdle consists of the cleithrum, coracoid, mesocoracoid, scapula, posttemporal, and radials ( Figure 6(a)). e ventral part of the cleithrum is wider with a lateral process. is bone bears two wide horizontal and vertical portions; its ventral part bears an anteromedial downward process connecting to the anterior part of the coracoid. e posterior part of the coracoid is wider than its anterior part and bears an ascending process for connecting to the mesocoracoid. e posttemporal is a small bone that connects the pectoral girdle to the pterotic (Figure 1(b)). e medial part of the coracoid is bent ventrally and has a small pore. A semicircular scapula is located between cleithrum and coracoid bones; this bone bears a large foramen and is articulated to the first unbranched ray. e ventral part of the mesocoracoid is V-shaped and attached the coracoid to the cleithrum; the dorsal part of the mesocoracoid is broadened and attached to the medial surface of the cleithrum. e pectoral fin bears four radials; the lateral radial is the thickest and three others are long and flat.
Pelvic girdle includes the paired basipterygium, metapterygium, lateral pterygium, and radials ( Figure 6(b)). e anterior part of the basipterygium bone is bifurcate; this bone has a posterior long and a midlateral processes. e two L-shaped lateral pterygiums are located at the posterolateral side of the pelvic bone. ere are two paired radial and one paired metapterygium in the pelvic girdle.

Discussion
e present study provides a detailed skeletal description of rosy stone lapper (G. rossica) from Ladiz River, Mashkid Basin. Rosy stone lapper showed differences compared to G. rossica from Nahang River, Mashkid Basin, G. persica from Zahak River, Sistan Basin, and G. typhlops, a closely related species resulting from an evolutionary adaptation to a subterranean system. Most members of Garra genus are found in mountain streams and flowing waters [2], whereas G. typhlops inhabits subterranean habitats with stagnant waters [2]. ere are differences in the suspensorium and opercular series, pharyngobranchial bones, ventral and pectoral girdles, caudal, dorsal, and anal fins skeleton, infraorbital series elements, and branchial apparatus between G. rossica of this study and G. typhlops provided by Jalili and Eagderi, [15], G. rossica from Nahang River provided by Saemi-Komsari et al. [16], G. persica from Zahak River, Sistan Basin, studied by Zamani-Faradonbe and Keivany [17]. All bones in suspensorium and opercular series in G. rossica are narrower and longer than G. typhlops. e pharyngobranchial bones in G. rossica are wide, while these bones are small in G. typhlops. Garra rossica has four infraorbital and one supraorbital elements, whereas these bones in G. typhlops had six infraorbital elements and in G. rossica from Nahang River had five. e basipterygium in G. rossica is bifurcate and its posterior process is rounded, whereas in G. typhlops, the basipterygium is trifurcate and its posterior process is pointed. Garra rossica bears narrow and long cleithrum and coracoid bones in the pectoral girdle, whereas G. typhlops bears broad and wide bones. e caudal skeleton in G. rossica has 6 hypural plates, long epural, and narrow and a long neural spin, whereas G. typhlops bears 7 hypural plates, a short epural, and neural spin. Furthermore, differences were observed between the osteological structures of the median unpaired fins of G. rossica and G. typhlops. e dorsal fin in G. rossica has one unbranched ray and nine branched rays and eight pterygiophores, whereas G. typhlops bears three unbranched and seven branched rays and nine pterygiophores; G. rossica from Nahang River had three unbranched ray and nine branched rays and eight pterygiophores and G. persica had two to three unbranched rays and eight to ten branched rays and nine pterygiophores. e anal fin in G. rossica has two unbranched and seven branched rays and is supported by six pterygiophores and one small stay bone, while this fin in G. typhlops had two unbranched and six branched rays, seven pterygiophores and one stay bone, G. rossica from Nahang River had two unbranched and six branched rays and is supported by six pterygiophores and one small stay bone and one unbranched and one branched rays that were supported by six pterygiophores and one small stay bone in G. persica. e branchial apparatus in G. rossica has five pairs of the ceratobranchials while G. rossica from Nahang River and G. persica had four pairs; G. persica had three pairs of pharyngobranchial, while G. rossica in this study has two pairs; there are three rows of the pharyngeal teeth with a formula of 2.4.5-5.4.2 in G. rossica, whereas the dental formula was 3.5.6-6.5.3 in G. rossica from Nahang River. e osteological features of G. rossica can reflect changes in its composing structures with a new adaptation to mountain streams that can be described as evolutionary novelties that gradually accumulated as modified anatomical 4 International Journal of Zoology structures as phenotypic plasticity during about 5 million years since it is divergent from their common ancestor with the other species in Garra genus.

Data Availability
All the data used for this study were obtained in the "Department of Natural Resources (Fisheries Division), Isfahan University of Technology" and are available for everyone.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  International Journal of Zoology