Descriptive osteology of Paracobitis persa Freyhof , Esmaeili , Sayyadzadeh & Geiger , 2014 , an endemic species from Kor River basin of Iran

This study was carried out to provide a detailed osteological structure of the newly described crested loach, Paracobitis persa. For this purpose, ten specimens were collected from the Malsosjan spring, in the Kor River basin and fixed in 5% buffered formalin. After clearing and staining, their skeletal structure was examined. The results were compared to the other members of the family Nemacheilidae particularly P. hyrcanica and P. iranica. Based on the results, P. persa can be distinguished from other members of this family by a combination of characters, including having lacking sesamoid ossifications, having six hypurals, a triangular-shaped lateral ethmoid with pointed ends, alveolar bony capsule, no contact between retroarticular and dental, lack of the pre-ethmoid-I and basibrancial-4.

Paracobitis persa, described from the Kor river drainage of Iran (Maloosjsn Spring and from Sivand River), is distinguished by having a prominent, irregularly shaped mid-lateral stripe, a shallow adipose crest, and the tube of the anterior nostril not fully overlapping the posterior nostril when folded back (Freyhof et al. 2014).There is little information is available about biological features of this recently described and threatened species (Esmaeili and Valavi 2016); therefore, the present study was conducted to provide a detailed osteological characteristics of this species.The results of this study can be used as a reference for its comparison with the other members of the genus Paracobitis.The results will also provide a basis for further phylogenetic study of Iranian members of the Paracobitis group using osteological data.

Materials and Methods
Ten specimens of P. persa (Fig. 1) with mean standard length of 62±10.5 mm were collected by electrofishing device from the Malsosjan Spring, in the Kor River basin (Fars Province, Sothern Iran).
The collected specimens were anesthetized in 1% clove solution and then fixed in 5% buffered formaldehyde.For osteological examination, the specimens were cleared and stained with alizarin red S and alcian blue based on Taylor and Van Dyke (1985).The left sides of the skeletal structures photographed using a scanner (Epson V600) equipped with a glycerol bath.The skeletal structure of specimens was observed and studied by a Leica MS5 stereomicroscope.The obtained images were drawn using CorelDrawX6 software.Nomenclature and abbreviation of the skeletal elements were done according to Prokofiev (2009Prokofiev ( , 2010)), Azimi et al. (2015aAzimi et al. ( , 2015bAzimi et al. ( , 2015c) ) and Nikmehr andEagderi (2016a, 2016b).

Results
The posterior part of the neurocranium is oval in shape and wider.The ethmoid region consists of the lateral ethmoid, supraethmoid-ethmoid, pre-ethmoid-II, kinethmoid and pre-vomer (Figs.2a, b, c, 4).The supraethmoid-ethmoid is narrow with serrated rim posteriorly.This bone bears a groove on its dorsal face and is ventrally fused to the pre-vomer (Fig. 2a, b).The posterior edge of the lateral ethmoid is concaved.The unpaired cylindrical kinethmoid possesses two mid-lateral processes (Fig. 3a).The pre-ethmoid-II is short and cylindrical in shape; its middle part is slightly curved.This bone is connected to the pre-palatine laterally, to the anterior part of the pre-vomer posteriorly and to the maxilla anteriorly.
The orbital region comprises the frontals, parasphenoid, pterosphenoids, and orbitosphenoids.The frontal is the largest bony element of the skull roof; it is connected to the orbitosphenoid anterolaterally.The parietals, frontals, and supraoccipital enclose the fontanel.The parasphenoid is the longest bone in the ventral face of the neurocranium; its anterior and posterior ends are bifurcated.The pterosphenoid is connected to the frontal dorsally and to the sphenoid postero-laterally; its posterior margin is curved, forming a cavity along with the prootic and parasphenoid (Fig. 2c).The orbitosphenoid is connected to the lateral-ethmoid ventrally, to the parasphenoid laterally, and to the pterosphenoid posteriorly.ethmoid is narrow with serrated rim posteriorly.This bone bears a groove on its dorsal face and is ventrally fused to the pre-vomer (Fig. 2a, b).The posterior edge of the lateral ethmoid is concaved.The unpaired cylindrical kinethmoid possesses two mid-lateral processes (Fig. 3a).The pre-ethmoid-II is short and cylindrical in shape; its middle part is slightly curved.This bone is connected to the pre-palatine laterally, to the anterior part of the pre-vomer posteriorly and to the maxilla anteriorly.
The otic region includes the parietal, epiotic, sphenotic, pterotic and prootic.The parietal is approximately square-shaped and its posterior margin covers the anterior part of the epiotic (Fig. 2a).The pterotic is quarter-circle in shape connecting to the epiotic and sphenotic dorso-laterally and to the prootic and exoccipital ventro-laterally (Fig. 2a, c).The epiotic is oval-shaped and bears a posterior process (Fig. 2a), situating between the pterotic and supraoccipital.The sphenotic forms the lateral wall of the neurocranium (Fig. 2b) and is connected to the prootic ventrally and to the parietal postero-dorsally.The two prootics are connected to each other ventrally and to the parasphenoid dorsally by a descending process.The prootic bears two large pores on its anterior part.
The occipital region is composed of the supraoccipital, exoccipitals and basioccipital.The supraoccipital is pentagon in shape in dorsal view and its anterior margin is involved in the formation of the fontanel.In the ventral part of the basioccipital, a pharyngeal process is present (Fig. 2c).The anterior hyomandibular facet is formed by the pterosphenoid, sphenotic and prootic; and the posterior one by the sphenotic and pterotic.The fontanel is covered by connective tissue and extended longitudinally.
The upper jaw is made up of the maxilla and premaxilla.The maxillary possesses a midlateral ascending and an anterior descending processes; it has also a distal process on its dorsal border.The premaxilla is a thin L-shaped bone and its vertical portion is narrower; the horizontal part has a process on its middle-anterior margin.The lower jaw is composed of the dentary, articular, retroarticular and coronomeckelian (Fig. 3b).The coronomeckelian is present on the medial face of the lower jaw situated in the dorsal part of the articular.The dentary is the largest element of the lower jaw possessing the narrow ramus dentalis and a wide coronoid process.The suspensorium is formed by the hyomandibular, ectopterygoid, endopterygoid, metapterygoid, autopalatine, symplectic and quadrate (Fig. 4).The hyomandibular is almost triangular in shape and its dorsal part is wider.It bears two dorsal condyles which are articulated to the neurocranium.The ventral part of the hyomandibular is connected to the interhyal.The ectopterygoid, endopterygoid, metapterygoid, symplectic and quadrate form a bony complex connecting to the anterior part of the neurocranium via the auto-palatine.The ventral edge of the ectopterygoid is jagged.The endoptrygoid has a mid-dorsal protuberance that is connected to the metaptrygoid and ectoptrygoid ventrally; this bone articulated to the auto-palatine by an anterior condyle.The metapterygoid is roughly rectangular in shape; its lateral part is caved and its ventral part has a pointed process.The quadrate bears a pointed and stretched abdominal process bending posteriorly.The symplectic is a triangular bone located between the quadrate and hyomandibular.
The opercular series includes the opercle, preopercle, subopercle, and interopercle (Fig. 4).The opercle is a large and triangular bone, and bears a rodshaped process antero-dorsally for connection to the levator operculi muscle.The ventral margin of the opercle is connected to the subopercle that is narrow and strip-like.The interopercle is a narrow bone; it is pointed at its two ends.The posterior part of the preopercle bears two processes.The branchial apparatus includes five pairs of the ceratobranchials, four pairs of the epibranchials, three pairs of the hypobranchials and basibranchials and two paired infrapharyngobranchial (Fig. 5a).The ceratobranchials are the largest element and fifth one is modified as the pharyngeal teeth.The third basibranchial is longer than the two others.The epibranchial-4 has a ventral plate-like protrusion.The second hypobranchial is attached to the first basibranchial anteriorly.The hyoid arch consists of the epihyals, hypohyals, ceratohyals, urohyal, basihyal and three pairs of the branchiostegal rays.The urohyal is narrow and bears two ventral and dorsal parts; its dorsal part is blade-like and perpendicular and the ventral part is bifurcated.The basihyal is a T-shaped bone with a small anterior fossa (Fig. 5b).The paired ventral and dorsal hypohyal are tightly connected together.The ceratohyal is the largest bone of this arch situating between the hypohyal and epihyal.The first and second branchiostegal are positioned, respectively, in the middle of the ceratohyal and at the junction of the ceratohyal and epihyal.The third one is positioned in the middle of the epihyal.
The pectoral girdle consists of the cleithrum, supracleithrum, coracoid, mesocoracoid, scapula, posttemporal, supratemporal and radials (Fig. 6a).The cleithrum bears two wide descending and ascending portions; its ventral part bears an anteromedial downward process connecting to the anterior part of the coracoids.Also, it has a dorsal process on its ascending part for connecting to the supracleithrum.The supratemporal is a small bone locating on the anterior margin of the posttemporal.The posttemporal is thin and long, and located between the supracleithrum and supratemporal.The anterior part of the coracoid is narrower.The anterior parts of the cleithrum and coracoid are connected; the posterior part of the coracoid bears an ascending process for connecting to the mesocoracoid.The dorsal part of the mesocoracoid is broadened and attached to the medial side of the cleithrum.A semicircular scapula is located between cleithrum and coracoid bones; this bone bears a large foramen.
The pelvic girdle includes the paired pelvic bone, pelvic splint and radials (Fig. 6b).The anterior part is narrower.There is a U-shaped hollow on the anterior margin of the pelvic bone.The two L-shaped pelvic splints are located at the postero-lateral side of the pelvic bone.There are three radial bones positioning along the postero-lateral side of the ventral bone.
The Weberian apparatus is formed by four anterior centra with their ossicles, including the tripus, intercalarium, scaphium and claustrum (Fig. 7a).The claustrum is circular in shape and situated at the dorsal side of the scaphium.The intercalarium is small and positioned between the scaphium and Y-shaped tripus.The first centrum has two lateral processes which are ligamentously connected to the pectoral girdle.The second, third, and fourth centra participate in the formation of bony capsule.The second and third centra are fused together.The parapophysis of the forth centrum is modified and bears processes to forms the posterior part of the bony capsule.In the lateral part of the bony capsule, two openings are observed that anterior one is larger.The surface of the bony capsule is vascular.The right and left lobes of the bony capsule are symmetrical and divided by the manubrium (Fig. 7a).
The dorsal fin bears 4 unbranched, 7½ branched rays, 8 pterygiophores and one stay bone.The first pterygiophore is bifurcated and the largest elements, situating in the front of the 14 th centrum (Fig. 7b).A triangular stay bone supports the last branched ray.
The Anal fin consists of 3 unbranched, 5½ branched rays, 6 pterygiophores and one stay bone (Fig. 7c).The first pterygiophore is bifurcated situating in the front of the 26 th vertebra.The caudal skeleton consists of four centra with the epural, parahypural, pleurostyle, uroneural and six hypurals bones (Fig. 7d).The pleurstyle is fused to the last centrum.The hypural-1 reaches to the ventral edge of the terminal centrum and not connected to the parahypural.The epural is a long, narrow and free bone, connected to the pleurostyle ventrally by connective tissue.The neural process of the 4 th centrum become wide and bifurcated.
In P. iranica, the frontal is wider (Nikmehr and Eagderi 2016a) than that of P. hircanca (Azimi et al. 2014) and P. persa.Unlike the members of the genera Metaschistura, Paraschistura, Oxynoemacheilus and Iskandaria (Prokofiev 2010), there is a bony bridge between the parietal and pterotic in P. persa.In P. hircanica, the posterior part of the maxilla is wider (Azimi et al. 2014) than that of P. persa.
The number of unbranched and branched rays of the anal fin is 3 and 5½, respectively in P. persa, and P. hyrcanica; whereas they are 3½ and 5 in P. iranica (Azimi et al. 2014), respectively.Furthermore, the number of unbranched and branched rays of the dorsal fin is 3 and 7 in P. iranica (Nikmehr and Eagderi 2016a), but they are 4 and 7½ in P. hyrcanica and P. persa, respectively.
The neural processes of the 3 rd centrum has three processes in P. hircanica (Azimi et al. 2014), whereas that of P. iranica (Nikmehr and Eagderi 2016a) and P. persa has no dorsal process.According to Prokofiev (2009), there are 6 hypurals in the genus Paracobitis as observed in P. persa and P. iranica (Nikmehr and Eagderi 2016a), whereas there is five hypurals present in P. hircanica (Azimi et al. 2014).However, six hypurals are present in O. bergianus (Jalili and Eagderi 2014).
As a result, P. persa is distinguished from other members of the family Nemacheilidae, particularly P. hyrcanica and P. iranica by a combination of characters, including having lacking sesamoid ossifications, having six hypurals, a triangular-shaped lateral ethmoid with pointed ends, alveolar bony capsule, no contact between retroarticular and dental, lack of the pre-ethmoid-I and basibranchial-4.