Is Garra rezai (Teleostei, Cyprinidae) a species known only from two widely disjunct areas in the Tigris drainage?

Garra rezai was recently described from two geographically distant areas in the Tigris drainage: upper Yanarsu River (eastern Türkiye) and Bouein-Sofla Creek (Iran). In the scope of this study, we aimed to ascertain the distribution ranges of G. rezai and its morphologically most similar congener G. rufa in Türkiye by examining 1165 specimens from 73 lots, which were collected between 1957 and 2023 and currently curated in two broad fish collections. To achieve this, we focused on two important diagnostic morphological characters which distinguish these two species: scales on predorsal mid-line between dorsal-fin origin and nape, and branched dorsal-fin rays. The results revealed that G. rufa is a widely distributed species in the Tigris-Euphrates catchment, while G. rezai is regionally widespread, with populations identified in at least six different regions within the Tigris catchment. Addition - ally, G. rezai is documented for the first time in the upper Euphrates. Furthermore, we identified the drainage areas where G. rezai co-exists with G. rufa .


Introduction
Fish taxonomy, which provides a foundation for scientific research, involves the classification and naming of fish species.Taxonomy helps identify and document different species; it is also essential tasks for assessing biodiversity, tracking changes in populations and implementing effective conservation strategies (Mace 2004).Different species of Garra Hamilton, 1822 have gained popularity for their use in spa treatments.These species are also known as doctor fish or nibble fish.They are small freshwater fish belonging to the family Cyprinidae (Ruane et al. 2013;Aydın and Akhan 2020).Although they are not considered threatened or endangered on a global scale (Freyhof 2014), some threats such as habitat degradation and pollution might impact their populations.
Garra is one of the largest genera of the family Cyprinidae, containing approximately 150 species (Majeed et al. 2019).The species belonging to this genus are small to medium-sized fish which usually live in the bottom of fast-flowing rivers and mountain streams.However, some small-sized species of the genus Garra -which were previously considered under the genus Hemigrammocapoeta Pellegrin, 1927 -prefer vegetated and relatively more stagnant habitats.Approximately a decade ago, Hemigrammocapoeta was considered as a synonym of the genus Garra by Behrens-Chapuis et al. (2015).Garra is widely distributed from east, southeast, south and southwest Asia to tropical Africa (Zhang and Chen 2002;Kottelat 2020).
Another interesting point about Garra rezai is its similarity to its two congeneric species (Garra variabilis (Heckel, 1843) and G. rufa) distributed in the Euphrates-Tigris catchment.Although G. rezai is morphologically closer to G. rufa, it is genetically closer to G. variabilis, which has one pair of barbels, a small mental disc and a comparatively different body shape.
The description of Garra rezai raised some questions: Is this species a threatened species with a very limited range?Is G. rezai restricted to these two localities, or is it more widespread?Are G. rezai and G. rufa sympatric/ syntopic?
Overall, the aims of this study are: i) to investigate the morphologically-based diagnostic characters of G. rezai and G. rufa, ii) to reveal the distribution of these species in Türkiye and iii) to answer the above-mentioned questions by analysing samples from two broad fish collections.

Materials and methods
In order to determine the distribution ranges of Garra rufa and G. rezai in Türkiye, 1165 specimens from 73 lots (Suppl.material 1) were examined from the Recep Tayyip Erdogan University Zoology Collection of the Faculty of Fisheries (FFR) and Collection of Ege University Faculty of Fisheries (ESFM).Material was examined in ESFM collected by electro-fishing and hand scoops between 1957 and 1997 and those in FFR collected with DC electro-fishing equipment between 2005 and 2023.
Since both collection samples were fixed in formaldehyde, it was not possible to perform a molecular study.However, considering the critical diagnostic characters (scales on predorsal mid-line between dorsal-fin origin and nape [PreDs]; branched dorsal fin rays [DFR]; total gill rakers on first branchial arch [GR]) determined by Mousavi-Sabet et al. (2022), many samples preserved in FFR and ESFM were examined.
In this sense, DFR and PreDs were counted for all specimens found in FFR and ESFM.In cases where the species could not be identified with these two critical diagnostic characters, individuals were identified by counting GR, the third critical diagnostic character.The reason why GR could not be counted in all specimens is that the gill covers are less open and the gill arches are smaller in Garra spp.compared to other species, so that the gill lamella has to be removed and dissection from the upper and lower parts of the operculum opening has to be performed in order to count the gill spines.However, the collection authorities did not consent to the partial dissection of over a thousand Garra specimens in the FFR and ESFM that were part of the study.The counting methods were followed as described by Armbruster (2012).

Results
As a result, we determined that Garra rufa is still a widespread species in the Tigris-Euphrates catchment and G. rezai is regionally widespread, with populations in at least six different regions in the Tigris.Furthermore, G. rezai is recorded for the first time in the upper Euphrates.Our study indicates that G. rufa is considerably more widespread compared to G. rezai.Based on the collections examined in this study, G. rufa was observed in almost the entire Euphrates, except in the trout zones and throughout the Tigris, except in the Great Zap, Hezil and the eastern part of the Botan.The presence of G. rufa in the Menfez Stream near Hezil, as well as in the drainage areas flowing into Botan from the north (Destumi and Bitlis streams), suggests the possibility of G. rufa inhabiting the eastern side of Botan and Hezil.However, none of the specimens examined in the Great Zap in this study was identified as G. rufa.These assumptions, of course, require confirmation, especially through molecular studies.
On the other hand, it was observed that G. rezai is more dominant in all regions of the Tigris where these two species have sympatric distribution.However, in Kaynarca Stream, the only sympatric area in the Euphrates, G. rufa was dominant (Fig. 1).This may be attributed to G. rezai's preference for clean and shallow streams rather than large rivers.Kaynarca Stream, dominated by G. rufa, is larger and more turbid compared to streams dominated by G. rezai in the Tigris.In summary, based on the data obtained from this study, we can assume that, in areas where these two species co-exist, G. rezai is dominant in clear, shallow and small streams, whereas G. rufa is dominant in turbid, large and relatively deep streams.Sympatric comparison of G. rufa and G. rezai in Kaynarca, Menfez, Botan and Yanarsu drainage areas are presented in Fig. 2.
Diagnosis.Garra rufa is distinguished from all the species of Garra in adjacent waters in having a combination of the following characters: Breast and belly covered by scales, scales embedded in skin, rarely ab- Common names.Tigris garra.
Diagnosis.Garra rezai is distinguished from all the species of Garra in adjacent waters in having a combination of the following characters: Breast with embedded scales, predorsal mid-line covered by (12)13-18(19-21) scales, gular disc short and wide, 35-40 total lateral-line scales, 5½(rarely 4½ and 6½) transverse scale rows between lateral line and dorsal origin, 3½-4½ transverse scale rows between lateral line and pelvic origin, 15-18 circumpeduncular scales, axillary scale at pelvic origin large, 11-16 total gill rakers, usually 7½ branched dorsal rays, eye fully developed.
Distribution in Türkiye.Known only from Kaynarca Stream (Murat drainage) in Euphrates.In Tigris; Yanarsu, Botan, Menfez, Hezil and Batman drainages, as well as from Aktoprak Stream, an uppermost drainage of Tigris River.
IUCN Status.Not Evaluated.The results of this study revealed that G. rezai is distributed in at least seven different drainage areas in Türkiye.Although G. rezai is known from only one locality in Iran outside Türkiye, we expected it to inhabit also other localities in Iran, Iraq and possibly Syria.In summary, this species occurs in various drainage areas and, in general, its populations appear to be in good condition; its IUCN status is suggested to be Least Concern.
Here, we selected the most diagnostic and easily distinguishable two characters: PreDs and DFR.
Based on the two critical diagnostic characters focused on in this study, Mousavi-Sabet et al. (2022)  Our morphological examination demonstrated that 1016 of these specimens belong to G. rufa, while 149 belong to G. rezai (Table 1 and Suppl   Our previous in-situ observations and examination of the collection specimens in FFR and ESFM revealed that there is an important relationship between Garra rezai, the main species of this study and the recently described Turcinoemacheilus ekmekciae Kaya, Yoğurtçuoğlu, Aksu, Bayçelebi & Turan, 2023, particularly concerning their distribution ranges.As mentioned in the Results section, G. rezai individuals were found from Kaynarca Stream (upper Murat River drainage, Euphrates), as well as from Yanarsu, Botan, Menfez, Hezil and Batman drainage areas (tributaries of the Tigris River).T. ekmekciae was described from Kaynarca Stream (upper Murat River drainage), as well as recorded from Yanarsu, Botan, Nerduş and Batman drainage areas (tributaries of the Tigris River) (Kaya et al. 2024).It is obvious that both species have a very similar distribution pattern.In fact, the most interesting point in the distribution of these two species is that they were not expected to be distributed in different rivers and inhabit limited areas.Despite this, the fact that these two species -in addition to their expected distribution outside the Tigris River -also occur in the Murat River, the most important tributary of the Euphrates River, shows that there may be a strong relationship between the Murat and Tigris Rivers.In particular, the distribution of Turcinoemacheilus ekmekciae, which is not known to inhabit more than one different river and belongs to a genus with a generally limited distribution range and G. rezai, which is thought to have a regional distribution in certain areas (Fig. 1), in both the Yanarsu and Murat Rivers indicates that the connection of these two rivers naturally points to the elevation of at least part of the Muş South Mountains not being very ancient.
This study has shown that the distribution of G. rezai is not restricted and that many previous records of G. rufa (e.g.Hashemzadeh et al. (2015); Kaya et al. (2016)) may actually belong to G. rezai.Therefore, this study will shed light on studies to determine the distribution of this species in Iran, Iraq and Syria.We strongly encourage morphological and molecular studies to reveal the distribution of G. rezai and G. rufa in Iran, Iraq and Syria.

Figure 1 .
Figure 1.Distribution of the Garra rufa and G. rezai in Türkiye.

Figure 5 .
Figure 5. Frequency distributions of G. rufa and G. rezai for PreDs and DFR.

Table 1 .
Frequency distributions of G. rufa and G. rezai for PreDs and DFR.