Zoosyst. Evol. 99 (2) 2023, 473-487 | DOI 10.3897/zse.99.108048 eee BERLIN A new freshwater amphipod (Amphipoda, Gammaridae) from the Fakilli Cave, Diizce Tiirkitye: Gammarus kunti sp. nov. Murat Ozbek!, Hazel Baytaso$lu?, Ismail Aksu? 1 Department of Hydrobiology, Faculty of Fisheries, Ege University, TR-35100 Bornova - Izmir, Tiirkiyve 2 Recep Tayyip Erdogan University, Faculty of Fisheries and Aquatic Sciences, 53100 Rize, Tiirkiye https://zoobank. org/E F4B3 FAB-3E50-48 1E-98DE-8& F4B6E07F 382 Corresponding author: Murat Ozbek (murat.ozbek@ege.edu.tr) Academic editor: Luiz F. Andrade # Received 15 June 2023 # Accepted 8 August 2023 Published 4 October 2023 Abstract Aquatic species (such as fish, amphipods, isopods, hirudineans etc.) adapted to environmental conditions can live in caves connected to groundwater. The species of Niphargus and Gammarus are the most commonly encountered amphipods in caves. Turkiye is very rich in terms of karst areas and is home to more than 2000 known caves. Fakilli Cave, located in Diizce Province in the Western Anatolian Region, has a length of 1071 m. A new amphipod species belonging to the Gammarus genus has been identified from the cave and named as Gammarus kunti sp. nov. Some of the characteristic features of the newly-identified species can be listed as “Medium-large size; smooth body, well-developed and reniform eyes; non-prolonged extremities; antennal gland cone is straight and long; second antenna with setose peduncular and flagellar segments; medial palmar spine present; posterior margin of pereopod 3 densely setose; anterior margins of pereopods 6 and 7 armed with spines only; epimeral plates not pointed”. Although the mentioned features are generally seen in epigean species, the members of this species were sampled from the dark zone of the Fakilli1 Cave. The partial sequences of the COI (573 bp) and 28S (914 bp) genes of the newly-described species, Gammarus kunti sp. nov., were gen- erated. The pairwise genetic distances between the new species, Gammarus kunti sp. nov. and other species ranged from a minimum of 16.23% (G. tumaf) to a maximum of 28.27% (G. roeselii) for the COI gene and a minimum of 0.88% (G. tumaf) to a maximum of 6.81% (G. balcanicus) for the 28S gene. Phylogenies generated by the NJ and ML methods, based on the combined data, assigned the new species as an independent lineage with high support values. In addition, the ASAP method identified the new species as a single MOTU independent of other species. G. tumaf and G. baysali are the sister taxa of G. kunti sp. nov. Detailed descriptions and drawings of the extremities of the male holotype and the female allotype are given and the morphology of the newly-identified Species is compared with its relatives. Key Words benthos, cave, identification key, invertebrate, molecular identification, new species Introduction be niche-based and related to the presence of various mi- cro-habitats (Trontelj et al. 2012). Caves, mid-ocean islands, deep seas, remote lakes and extremely cold and/or hot habitats are typical examples of extreme environments. Extreme conditions can lead to more effective functioning of organisms’ adaptation and evolution mechanisms resulting in morphological changes that can be associated not only with the absence of light in caves, but also with the presence of different microhabitats. In addition, morphological changes may Gammarus, the most widely distributed epigean fresh- water genus of the Amphipoda order, has spread from the Western Palearctic to China and North America (Vainola et al. 2008). The representatives of the genus generally live in epigean habitats, but are also distributed in hy- pogean habitats, such as caves and wells (Karaman and Pinkster 1977). Reduced or vestigial eyes, elongated antennae and extremities and a non-pigmented body are Copyright Ozbek, M. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A74 some of the morphological features frequently encoun- tered in Gammarus species adapted to living in hypogean habitats (Pinkster and Karaman 1978; Fi8er 2009; Ozbek et al. 2013). Turkiye is located between the Eurasian, African and Arabian plates and is situated on the Alpine-Himalayan Mountain Belt. As a result, it 1s a karst-rich country with more than 2000 known caves (Nazik et al. 2019; Yama¢ et al. 2021). Studies on the amphipod fauna of Turkiye’s inland waters, which started with the identification of Gammarus argaeus from Mount Erciyes (Vavra 1905), have increased over time and with a total of 51 Gam- marus species reported. A total of 20 amphipod species belonging to the genus Niphargus Schiddte, 1849; Gam- marus Fabricius, 1775; Parhadzia Vigna Taglianti, 1988 and Bogidiella Hertzog, 1933 have been reported from the caves and wells of Turkiye (Ipek and Ozbek 2022). In a recent study, this number increased by one more and Gammarus tumaf was identified from Géokg6l Cave, Zonguldak Province (Ozbek et al. 2023). The study aims to examine the individuals collected from Fakilli Cave, Diizce Province, Turkiye, in terms of morphological and molecular features. Detailed descrip- tions and drawings of the extremities of the male holotype and female allotype are given and the morphology of the newly-identified species is compared with its relatives. Materials and methods Sampling area Fakillt Cave is located in Fakilli Village, 8 km south- east of Akcakoca Town, Duizce Province, NW Turkiye. The total length of the cave is 1071 m and 350 m from the cave entrance is open for visitors. The entrance of the cave, which is 100 ms above sea level, has a width of 5-10 m and a ceiling height of 5-6 m. From the en- trance of the cave, the sections are passed through long narrow corridors. There are many natural features includ- ing galleries, stalactites and stalagmites going in vari- ous directions inside the cave, which was registered as a first-degree protected area by the Ministry of Culture and Tourism’s Regional Board of Protection of Cultural and Natural Assets (Zengin and Eker 2020). Morphological identification Individuals were collected with a hand aspirator from the dark zone of the cave, fixed in 70% ethanol in the field and transported to the laboratory for taxonomic identification. Specimens were dissected under a stereomicroscope, straightened with forceps and body length was measured from the base of the first antennae to the base of the tel- son. Permanent slides of the male holotype individual were prepared using the high-viscosity mount, CMCP- zse.pensoft.net Ozbek, M. et al.: Gammarus kunti sp. nov. from the Fakill: Cave, Turkiye 10. Photographs of the extremities were taken with a dig- ital camera connected to an Olympus CX41. A digitiser board (Wacom PTH-451) and a standard pen connected to a PC were used for detailed drawings of the extremi- ties. Scaled drawings of the extremities were made on the photographs (Coleman 2003). The geographical location of the cave is shown in Fig. 1. The collected samples are kept in the Museum of the Faculty of Fisheries, Ege Uni- versity (ESFM). Molecular identification DNA extraction, PCR amplification and Sequencing Total DNA was extracted on the Automated DNA iso- lation device (QIAcube Qiagen, Germany) according to the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Ger- many) protocol. Mitochondrial cytochrome c oxidase subunit I gene (COI) and the nuclear large subunit ribo- somal RNA gene (28S) were amplified from the extracted DNA. Amplification of the COI marker was performed with the primers UCOIF (5’- TAWACTTCDGGRT- GRCCRAAAAAYCA-3’) and UCOIR (5’- ACWAAY- CAYAAAGAYATYGG-3’) according to the PCR proto- col of Costa et al. (2009). Amplification of the 28S marker was performed with the primers 28F (5’- TTAGTAGGG- GCGACCGAACAGGGAT-3’) and 28R (5’- GTCTTC- GCCCCTATGCCCAACTGA-3’) according to the PCR protocol of Hou et al. (2007). PCR products of the COI and 28S genes were purified by using the QIAquick PCR Purification Kit (Qiagen). Bidirectional sequencing of both PCR products was per- formed with an ABI PRISM 3730x1 Genetic Analyser using a BigDye Terminator 3.1 cycle sequencing ready reaction kit (Applied Biosystem) according to the Sanger method at Macrogen Europe. Molecular data analyses We sequenced the partial sequences of the COI and 28S genes from one individual to perform molecular analy- ses and generate the genetic record of the new species. In addition, we downloaded a total of 27 reference se- quences (COI and 28S sequences for each species) from the GenBank (NCBI: National Centre for Biotechnology Information) for use in molecular analyses. Detailed in- formation on the sequences used in molecular analyses is given in Table 1. The raw COI and 28S sequences of the new species were corrected by checking their chromatograms in Bioedit 7.2.5 programme (Hall 1999). All sequences were then aligned with the Clustal W method (Thompson et al. 1994), trimmed at the ends and converted to a FASTA format file. The pairwise genetic distances were calculat- ed separately for both genes according to the uncorrected p-distance in MEGA X software (Kumar et al. 2018). Zoosyst. Evol. 99 (2) 2023, 473-487 4, Fakilh Cave Ankara Antalya ‘MEDITERRANEAN SEA : a 28° 30° 32° 4° 475 Trabzon Erzurum 4 * \ 2 3 0 8 een ae Diyarbakir Figure 1. The habitus of the male holotype (up) and the type locality of Gammarus kunti sp. nov. (down). To perform the phylogenetic analyses, the COI and 28S sequences, both newly-generated and download- ed from GenBank, were added end-to-end to obtain a concatenated dataset (28S+COI) for each species. Phy- logeny of Gammarus species was estimated by using Neighbour-Joining (NJ) and Maximum Likelihood (ML) methods in MEGA_X software. The NJ tree was generat- ed according to the p-distance parameter. The ML tree was generated according to the General Reversible Time (GTR) with gamma-distributed invariant sites (G+I) model (Tavaré 1986) and the best-fit substitution model was selected with the lowest Akaike Information Crite- rion (AIC) score in jModelTest 0.1.1 (Posada 2008). The nodal support of the NJ and ML analyses was comput- ed with the bootstrap test (Felsenstein 1985) using 1000 pseudoreplicates. To root the Gammarus phylogeny, Pontogammarus robustoides (also see Table 1) was used as an outgroup in the analyses. The species delimitation analysis was carried out using the ASAP (Assemble Species by Automatic Partitioning) method, based on COI data. To implement the ASAP method, we used the Kimura 2-parameter (K2P) distanc- es and transition/transversion ratio (R:1.4) settings at the web address https://bioinfo.mnhn. fr/abi/public/asap/. The transition/transversion ratio (R) for the COI data was calculated in MEGA X software. zse.pensoft.net 476 Table 1. Information of sequences used in molecular analyses. Secymin, Poland Targu Bujor, Romania Simferopol, Crimea, Ukraine Kolasin, Montenegro Sarajevo, Bosnia and Herzegovina Vistula, Poland Bela Palanka, Serbia Lazaropole, Macedonia . varsoviensis (T) . kischineffensis (T) . spelaeus (T) . balcanicus (T) . bosniacus (T) . leopoliensis (T) . Stojicevici (T) G. halilicae (T) Species Locality Gammarus kunti sp. nov. (T) Fakilli Cave, Turkiye G. tumaf (T) Gokgol Cave, Turkiye G. baysali (T) Cumayani Cave, Turkiye G. kesslerianus (T) Simferopol, Crimea, Ukraine G. komareki (T) Ca. 200.km SE Sofia, Bulgaria G. komareki Mazandaran, lran G. rambouseki (T) Bitola, Macedonia G. roeselii Netherlands G. fossarum (T) Regensburg, Germany G. plaitisi Tinos, Komi, Greece G. uludagi Evia, Greece G. monspeliensis (T) Montpellier, France G. ibericus Lascaux, France G. pulex (T) Slovenia G. lacustris Bled, Slovenia G. italicus Rieti, Lazio, Italy G G G G G G G G. pljakici Galicica planina, Macedonia G. stankokaramani (T) Ohrid, Macedonia G. salemaai Gradiste, Macedonia Pontogammarus Delta Volgi, Russia robustoides Note: (T) Topotype samples of nominal taxa. Results Gammarus kunti sp. nov. https://zoobank. org/25D58B26-4577-460C-A F00-0D20B 1D04397 Figs 1-7 Type material. Holotype. Male, 11.5 mm (ESFM-MA- LI/20-15), Akcakoca District, Diizce Province, Turkiye (41°3'7.01"N, 31°10'38.70"E), 16-xitt.2020; collected by M. Elverici. Paratypes. 3 males and 5 females, (ESFM-MA- LI/20—16), same data as holotype. Diagnosis. A medium-large species. Body smooth, pigmentation weak; eyes well-developed, ovoid; extrem- ities not prolonged; second antenna with setose pedun- cular and flagellar segments; antennal gland cone long; medial palmar spine present; posterior margin of pereo- pod 3 densely setose; anterior margins of pereopods 5 to 7 armed with spines and a few short setae; epimeral plates not pointed; inner ramus of uropod 3 longer than 0.75 of the outer one; telson weakly armed. Description of male holotype. Head: Rostrum ab- sent, inferior antennal sinus deep, rounded. Eyes kid- ney-shaped; shorter than the diameter of the first pedun- cular segment of antenna 1 (Figs 1, 5G). zse.pensoft.net Ozbek, M. et al.: Gammarus kunti sp. nov. from the Fakill: Cave, Turkiye 28S COl References OP650556 OP642558 This study ON751931 ON749780 Ozbek et al. (2023) ON751932 ON749781 Ozbek et al. (2023) JF965721 JF965909 Hou et al. (2011) JF965725 JF965913 Hou et al. (2011) JF965723 JF965911 Hou et al. (2011) JF965770 JF965946 Hou et al. (2011) JF965771 JF965947 Hou et al. (2011) JF965696 JF965886 Hou et al. (2011) MT999102 MT999049 Hupato et al. (2020) JF965817 JF965986 Hou et al. (2011) JF965738 JF965923 Hou et al. (2011) JF965713 JF965901 Hou et al. (2011) JF965767 JF965943 Hou et al. (2011) JF965728 JF965915 Hou et al. (2011) JF965716 JF965904 Hou et al. (2011) JF965818 JF965987 Hou et al. (2011) MG987529 MG987571 Copilas-Ciocianu et al. (2018) JF965801 JF965971 Hou et al. (2011) JF965640 JF965834 Hou et al. (2011) JF965680 JF965872 Hou et al. (2011) JF965734 JF965919 Hou et al. (2011) JF965808 JF965978 Hou et al. (2011) JF965711 JF965900 Hou et al. (2011) JF965758 JF965936 Hou et al. (2011) JF965806 JF965976 Hou et al. (2011) JF965780 JF965955 Hou et al. (2011) JF965822 JF965990 Hou et al. (2011) Antennae: Antenna | is as long as half of the body length; the length ratio of the peduncular segments is 1:0.67:0.4; peduncle segments bear a few groups of min- ute setae; the length of the setae is much shorter than the segment where they are implanted; the main flagellum with 32 segments; each segment bears a few short setae in distal side; aesthetasc absent; accessory flagellum 6 segmented (Fig. 3A). Antenna 2 is shorter than antenna 1 (ratio 1:0.67); the antennal gland cone is straight, reaches to the distal end of the third peduncular segment; setation is rich both on peduncular and flagellar segments; pedun- cular segments 4 and 5 bear many groups of setae; the setae on the ventral part of the peduncle segments are lon- ger than the dorsal ones and can be up to 1.5 times longer than the diameter of the segment; flagellum consists of 15 segments; flagellar segments are setose and swollen; each segment bears many long setae on both dorsal and ventral sides; calceoli absent (Fig. 3B). Mouthparts: Left mandible (Fig. 2A) with 5-toothed incisor, lacinia mobilis with 3 dentitions, molar tritura- tive. The first article of palp without setae, the second one bears 12 setae; the setae become shorter from dis- tal to proximal. The third segment has 28 D-setae, 4-5 E-setae, one group of A-setae and one group of B-setae. C-setae absent. Zoosyst. Evol. 99 (2) 2023, 473-487 477 H ) nel aa it Figure 2. Gammarus kunti sp. nov., (male holotype). A. Left mandible; B. Right mandible; C. Maxilla 2; D. Lower lip 1; E. Max- illiped; F. Left maxilla 1; G. Right maxilla 1; H. Upper lip. zse.pensoft.net 478 Ozbek, M. et al.: Gammarus kunti sp. nov. from the Fakill: Cave, Turkiye — —s sce, — iN ey Bip, a —* a = —* —ji_), oS =" ———— a — i Figure 3. Gammarus kunti sp. nov., (male holotype). A. Antenna 1; B. Antenna 2; C. Gnathopod 1; C’. Palm of gnathopod 1; D. Gnathopod 2; D’. Palm of gnathopod 2. zse.pensoft.net Zoosyst. Evol. 99 (2) 2023, 473-487 Right mandible (Fig. 2B) has a 4-toothed incisor and bifurcate lacinia mobilis. Right maxilla I (Fig. 2G) is asymmetric to the left, it has 14 plumose setae along the inner margin of the inner lobe. The outer lobe bears 11 distal stout serrate spines and some tiny setules on the inner margin. Palp of the outer lobe with no setae in the first segment and six stout spines and two simple setae on the distal part of the sec- ond segment, in addition to two marginal setae along the outer margin. The second article of left palp elongated and bears 8 spines and 3 simple setae on its distal part and no setae along the outer margin (Fig. 2F). Lower lip (Fig. 2D) has no inner lobe and bears nu- merous small simple setae along the distal margins of both lobes. Upper lip (Fig. 2H) with numerous minute setules in the distal part. Maxilla IT (Fig. 2C) has 20-25 simple setae in the distal part of the outer lobe and a few tiny hairs along the outer margin. The inner lobe also has 8—10 simple setae in the distal part in addition to 15 plumose setae located in a diag- onal row along the inner margin. There are also a few tiny hairs in the proximal part of the inner margin of the lobe. Maxilliped (Fig. 2E) inner plate has 3 tooth-like spines and a spine in the distal part and the distal corner, re- spectively. Additionally, there are 7 plumose setae along the inner margin of the lobe. Outer plate armed with 5—6 serrate stout setae in the distal part and 12 spines along its inner margin. Coxal plates: Coxal plate 1 is rectangular, the distal part slightly widened, the ventral margin slightly convex and bears 4 antero-distal setae and one postero-distal seta in addition to some tiny setules along the ventral margin (Fig. 3C). Coxal plate 2 is in the shape of an elongat- ed rectangle, distal part narrower than the proximal, the ventral margin is highly convex, antero-distal part with 5 setae and postero-distal part with one seta (Fig. 3D). Coxal plate 3 is similar to coxal plate 2 in shape, with 3 and | setae in the antero- and postero-distal ends, respec- tively (Fig. 4A). The ventral edge of the fourth coxal plate is slightly convex and bears 3 and 6 setae along the an- teroventral and posterior margins, respectively (Fig. 4B). Coxal plate 5 (Fig. 5A) and Coxal plate 6 exhibit a bilo- bate structure (Fig. 5B), each having one seta in the an- terior lobes and four and one setae in the posterior lobes, respectively. Coxal plate 7 is characterised by the pres- ence of five setae on the postero-ventral margin (Fig. 5C). Gnathopods: Basal segment of gnathopod 1 bears many long setae along both margins, the length of the setae can be as long as twice the diameter of the segment. Ischium bears a group of setae in the postero-ventral corner. Carpus is triangular and bears four groups of se- tae along the anterior margin in addition to many setae groups on both ventral and posterior sides. Propodus pyr- iform, the length/width ratio is 1: 0.57, anterior margin with two groups of setae, medial palmar spine is pres- ent, postero-distal corner armed with two strong spines in addition to some small spines, posterior margin bears 479 4—5S groups of setae. Dactylus reaches the postero-distal corner and bears a simple seta along the outer margin in addition to a small setule around the distal part of the in- ner margin (Fig. 3C, C’). Basis and ischium of gnathopod 2 have a similar seta- tion to that of gnathopod 1. Merus and carpus are more se- tose than those of gnathopod 1. Carpus triangular, densely setose along the posterior margin in addition to two groups of setae along the anterior margin. Propodus is densely se- tose and has a sub-rectangular shape, the length/width ra- tio is 1: 0.53, anterior margin bears 6 groups of setae, pos- terior margin with many groups of setae, medial palmar Spine is present, the postero-distal corner 1s armed with two strong spines in addition to some small spines. Dac- tylus reaches the postero-distal corner and bears a simple seta along the outer margin in addition to a small setule around the distal part of the inner margin (Fig. 3D, D’). Pereopods: Anterior and posterior margins of the basal segment of pereopod 3 bear long setae, the setae along the posterior margin are much longer than those in the anterior margin, posterior margins of the merus, carpus and propo- dus bear long setae, the setae can be more than three times the diameter of the segment where they are implanted. Dactylus slim, a minute plumose seta occurs on the outer margin; the inner margin with two small setules (Fig. 4A). The basal segment of pereopod 4 has a similar setation to that of pereopod 3. Ischium, merus, carpus and propo- dus have groups of setae along their posterior margins, but they are much shorter and less than those in pereopod 3, the length of the setae can be as long as (or slightly longer) than the diameter of the segment where they are implanted. Dactylus slim, a minute plumose seta occurs on the outer margin; the inner margin with two small setules (Fig. 4B). Posterior margins of the basal segments of pereopods 5 to 7 are more or less convex and bear many short setae, anterior margins with 5—7 small spines and no setae pres- ent on the inner surfaces of the basal segments; no spine exists in the postero-ventral corner of the basal segment of pereopod 7. Pereopods 6 and 7 bear no setae along the anterior margins of ischium, merus and carpus, while pereopod 5 has a few setae longer than the accompanying spines along with the mentioned segments. Propodus of pereopods 5 to 7 with 2-3 groups of long setae groups along their outer margins in addition to 5—6 groups of small spines along their inner margins. Dactylus slim, a minute plumose seta occurs on the outer margin; the inner margin with two small setules (Fig. SA—C). Epimeral plates: They are neither curved nor sharply pointed. Epimeral plate 1 bears 2 long setae in addition to 4—5 setules along the anterior margin, the postero-ven- tral corner is angular (Fig. 5D). Epimeral plate 2 bears 5—6 setae in the antero-ventral corner, the ventral margin is armed with 1 spine and two short setae, the posterior margin with 4—5 setules, the postero-ventral corner is an- gular (Fig. SE). Epimeral plate 3 is slightly pointed; the antero-ventral corner bears 3—4 setae; the ventral margin is armed with 3 spines; the posterior margin bears 6—7 setules (Fig. 5F). zse.pensoft.net 480 Ozbek, M. et al.: Gammarus kunti sp. nov. from the Fakill: Cave, Turkiye C-E F Figure 4. Gammarus kunti sp. nov., (male holotype). A. Pereopod 3; B. Pereopod 4; C. Uropod 1, D. Uropod 2; E. Uropod 3; F. Telson. zse.pensoft.net Zoosyst. Evol. 99 (2) 2023, 473-487 481 1mm D-F 1mm A-C, G,H Figure 5. Gammarus kunti sp. nov., (male holotype). A. Pereopod 5; B. Pereopod 6; C. Pereopod 7; D. Pleopod 1; E. Pleopod 2; F. Pleopod 3; G. Head; H. Urosomites. zse.pensoft.net 482 Urosomites: Not elevated. Each segment bears a me- dian and two dorsolateral groups of armaments; each of them consists of 1—2 spines and 3-4 accompanying setae (Fig. 5H). Uropods: Uropod 1 has a spine in the disto-ventral cor- ner of the base; the peduncle is longer than the rami; the length ratio is about 1:0.7. Peduncle with a spine in the outer margin of the proximal part in addition to 3 spines along the inner margin and 3 spines in the distal part. The inner ramus is slightly longer than the outer ramus and bears 3-4 spines along their inferior margin in addition to 4—5 distal spines. The outer ramus with 2 spines along the inferior margin 1n addition to 4—5 distal spines (Fig. 4C). Uropod 2 is smaller than the first one; the length ra- tio is about 1:0.6; the peduncle segment is slightly longer than the rami and bears 2+2 spines along the inner mar- gin and the distal part, respectively. The outer margin is bare. The length and armaments of both rami are similar to each other, bearing 2—3 spines along their inner and outer margins in addition to 4—5 longer spines on their distal tips (Fig. 4D). Uropod 3 is setose and bears simple and plumose setae. The peduncle segment is much shorter than the outer ramus and the length ratio is about 1:0.41. The outer ramus is two articulated and densely setose along both margins; the outer margin bears 2 groups of spines accompanied by groups of long simple setae; the inner margin with plumose setae; the second arti- cle is well developed and longer than the surrounding distal spines. The inner ramus is about 0.77 the length of the outer ramus. It bears two spines along the outer margin in addition to groups of simple and plumose setae; the inner margin bears both simple and plumose setae (Fig. 4E). Telson: Telson lobes cleft, each lobe bears 2 spines and 2—3 simple setae in their distal parts. The setae are longer than the spines. There are 2—3 groups of short se- tae on the dorsal surface of the lobes in addition to two plumose setules. The length/width ratio of each lobe is about 1:0.5 (Fig. 4F). Etymology. The species epithet is derived from the name of scientist Dr. Kadir Bo&a¢ Kunt, who has valu- able contributions to the Arachnida species of Turkiye and sent the materials for this study. Description of females. Smaller than males. Except for the sexual dimorphism indicated for the genus Gammarus, females do not show obvious differences from males. At first glance, the morphological differences between the female allotype and the male holotype can be listed as follows: less setose antenna 2, not swollen flagellar segments of antenna 2, less setose gnathopod 2 and more setose pereopods 4—7 (Figs 6, 7). Variability: Some of the paratypes are immature. The eyes are kidney-shaped or slightly oval. The num- ber of flagella segments in antenna | varies between 26 and 29. Similarly, there are 10—11 flagellar segments in antenna 2. zse.pensoft.net Ozbek, M. et al.: Gammarus kunti sp. nov. from the Fakill: Cave, Turkiye Molecular data analyses We generated the partial sequences of the COI (573 bp) and 28S (914 bp) genes of the newly-described species, Gam- marus kunti. After all sequences were aligned, the total length is 1489 bp including gaps. While no stop codon, in- sertion, deletion and a gap was detected in the protein-cod- ing mtDNA COI gene, there were insertions and deletions in the nuclear 28S gene. Additionally, newly-generated sequences are deposited in GenBank accession numbers, for COI; OP642558 and 28S; OP650556. Thus, the first genetic record of the newly-described species was created. We performed the genetic comparison of the new species with the reference sequences of the topotype samples of the nominal taxa in GenBank. In the absence of sequences of topotype samples, correct sequences considered repre- sentative of the species were preferred (Table 1). For the COI gene, the pairwise genetic distance amongst the species ranged from a minimum of 5.24% (G. stankokaramani - G. salemaai) to a maximum of 28.62% (G. kesslerianus - G. plaitisi). The pairwise ge- netic distances between the new species Gammarus kunti sp. nov. and the other species ranged from a minimum of 16.23% (G. tumaf) to a maximum of 28.27% (G. roeselii). For the 28S gene, the pairwise genetic distance amongst the species ranged from a minimum of 0.11% (G. halili- cae - G. pljakici) to a maximum of 7.84% (G. rambouseki - G. stojicevici). The pairwise genetic distances between the new spe- cies Gammarus kunti sp. nov. and the other species ranged from a minimum of 0.88% (G. tumaf) to a maximum of 6.81% (G. balcanicus). The genetic distance of the new species to the nearest species 1s approximately three times greater for the COI gene and eight times greater for the 28S gene than the minimum genetic distance between valid Gammarus species. This indicates that the new spe- cies 1s well differentiated genetically. All pairwise genetic distance values amongst Gammarus species are given in Suppl. material 1. Phylogenies generated by the NJ and ML methods, based on the concatenated data, yielded fully compatible trees. Except for a few branches (ML:16—67%; NJ:25— 69%), the other branches (ML: 82—100%; NJ: 83—100%) in the phylogenies were generally resolved and supported with high bootstrap values. G. tumaf Ozbek et al., 2023 and G. baysali Ozbek et al., 2013 are the sister taxa of G. kunti sp. nov. The phylogenetic position of the new species, Gammarus kunti sp. nov., indicates an indepen- dent lineage supported by high bootstrapping values (for NJ: 95%, for ML: 91%; Fig. 8). The species delimitation analysis we implemented ac- cording to the ASAP method, based on COI data, identi- fied 26 MOTUs (molecular operational taxonomic units) for 27 Gammarus species. The best ASAP score had 1.5 (p=0.01) at a threshold distance of 0.079053. The anal- ysis identified species G. stankokaramani and G. sale- madi as a single MOTU. The new species formed a single MOTU independent of other species. Zoosyst. Evol. 99 (2) 2023, 473-487 483 A-G Figure 6. Gammarus kunti sp. nov., (female allotype). A. Antenna 1; B. Antenna 2; C. Gnathopod 1; D. Gnathopod 2; E. Pereopod 3; F. Pereopod 4; G. Uropod 3; H. Telson. zse.pensoft.net 484 Ozbek, M. et al.: Gammarus kunti sp. nov. from the Fakill: Cave, Turkiye LAA = —— = ae Figure 7. Gammarus kunti sp. nov., (female allotype). A. Pereopod 5; B. Pereopod 6; C. Pereopod 7. Discussion Gammarus kunti sp. nov. 1s a species belonging to the Gam- marus pulex-group due to the setation of the posterior part of pereopods 3 and 4 and the setation of uropod 3 (Kara- man and Pinkster 1977). Gammarus kunti sp. nov. shows close proximity to G. baysali and G. tumaf, considering the genetic analysis results (Fig. 8). In addition, the newly-de- scribed species show some similarities with G. kessleri- anus and G. komareki (Schaferna, 1922). G. kesslerianus has not been recorded from Turkiye, while G. komareki has been reported from the entire Black Sea Region of Turkey, including the Thrace Region (Ipek and Ozbek 2022). Although Gammarus kunti sp. nov. 1s genetically and morphologically close to G. baysali, it differs from G. baysali in having several morphological features. The newly-identified species is smaller than G. baysali. Addi- tionally, having well-developed eyes, shorter antenna 1, more setose antenna 2, not elongated extremities and not setose anterior margins of pereopods 5—7 are some of the discriminant characteristics of G. kunti (Table 2). G. kunti sp. nov. also resembles G. tumaf which is re- ported from the Gokgol Cave, Zonguldak Province by zse.pensoft.net the same authors of the present study in 2023. The new- ly-identified species differs from it by having reniform eyes, while eyes are minute in G. tumaf. Inner lobe of right maxilla 1 bears 14 and 20 plumose setae in G. kunti and G. tumaf, respectively. G. kunti has six stout spines in the palp of right maxilla 1, while the number of the stout spines is five in G. tumaf. In addition, the newly-iden- tified species has 15 plumose setae in the inner lobe of maxilla 2, while G. tumafhas 20 plumose setae (Table 2). The new species is also similar to Gammarus obruki Ozbek, 2012 by having kidney-shaped eyes, setose antenna 2 and armaments and setation of pereopods 5-6, but differs from it by being smaller and having much shorter antenna 1 and shorter inner/outer lobe ratio of uropod 3. In addition, G. kunti has 14 plumose setae in the inner lobe of right maxilla 1, while it has 18 in G. obruki. Similarly, the new species has two setae along the outer margin of the palp of the right maxilla 1, while G. obruki has three setae in the mentioned part of maxilla 1. Inner lobe of maxilla 2 bears 15 plumose setae in the newly-identified species and the number is 21 in G. obruki. Similarly, the number of D-setae in the palp of the mandible in G. kunti and G. obruki differs from each other (28 vs. 37, respectively) (Table 2). Zoosyst. Evol. 99 (2) 2023, 473-487 98/99 95/91 83/384 94/96 100/100 485 ON751931+ON749780 Gammarus tumaf (Gokgol Cave Turkiye) ON751932+ON749781 Gammarus baysali (Cumayani Cave Turkiye) OP650556+0P642558 Gammarus kunti sp. nov. (Fakilli Cave Turkiye) JF965721+JF965909 Gammarus kesslerianus(Simferopol Crimea Ukraine) JF965725+JF965913 Gammarus komareki (SE Sofia Bulgaria) 88/92 100/100 50/46 84/82 33/39 100/100 JF965723+JF965911 Gammarus komareki (Mazandaran Iran) JF965770+JF965946 Gammarus rambouseki (Bitola Macedonia) JF965771+JF965947 Gammarus roeselii (Netherlands) JF965696+JF965886 Gammarus fossarum (Regensburg Germany) MT999102+MT999049 Gammarus plaitisi (Tinos Komi Greece) JF965817+JF965986 Gammarus uludagi (Evia Greece) JF965738+JF965923 Gammarus monspeliensis (Montpellier France) 84/81 96/97 100/100 100/100 JF965713+JF965901 Gammarus ibericus (Lascaux France) JF965767+JF965943 Gammarus pulex (Slovenia) JF965728+JF965915 Gammarus lacustris (Bled Slovenia) JF965716+JF965904 Gammarus italicus (Rieti Lazio Italy) JF965818+JF965987 Gammarus varsoviensis (Secymin Poland) 62/52 69/67 , 34/29 99/96 f MG987529+MG987571 Gammarus kischineffensis (Targu Bujor Romania) JF965801+JF965971 Gammarus spelaeus (Simferopol Crimea Ukraine) JF965640+JF965834 Gammarus balcanicus (Kolasin Montenegro) JF965680+JF965872 Gammarus bosniacus (Sarajevo Bosnia and Herzegovina) 18/22 25/16 40/36 100/100 91/94 100/99 JF965734+JF965919 Gammarus leopoliensis (Vistula Poland) JF965808+JF965978 Gammarus stojicevici (Bela Palanka Serbia) JF965711+JF965900 Gammarus halilicae (Lazaropole Macedonia) JF965758+JF965936 Gammarus pijakici (Galicica planina Macedonia) JF965806+JF965976 Gammarus stankokaramani (Ohrid Macedonia) JF965780+JF965955 Gammarus salemaai (Gradi te Macedonia) JF965822+JF965990 Pontogammarus robustoides (Delta Volgi Russia) a | 0.050 Figure 8. Maximum Likelihood (ML) phylogenetic tree generated, based on the concatenated dataset (28S+COI). ML and NJ methods yielded the same topologies and, therefore, only the ML tree is shown. The bootstrap values of NJ and ML are shown on nodes (NJ/ML). Table 2. Some of the morphological features of Gammarus kunti sp. nov., G. baysali, G. tumaf and G. obruki. Characters Gammarus kunti sp. nov. G. baysali G. tumaf G. obruki Body length 11.5 mm 18.1 mm 12.6 mm 21.0 mm Eyes Kidney-shaped eyeless minute Kidney-shaped Body colour whitish colourless, whitish whitish yellowish Antenna 1 32+6 flagellar segments 41+6 flagellar segments 30+5 flagellar segments 52+6 flagellar segments Antenna 2 peduncular and flagellar peduncular and flagellar peduncular and flagellar fifth peduncular and flagellar segments densely setose straight, reaches to the distal end of the third peduncular segment with 14 plumose setae segments setose straight, reaches to the distal end of the third peduncular segment with 19 plumose setae Antennal gland cone Inner lobe of right maxilla 1 Palp of right maxilla 6 stout spines, 2 setae 1 along the anterior margin 6 stout spines, 4 setae along the anterior margin segments densely setose straight, reaches to the distal end of the third peduncular segment with 20 plumose setae 5 stout spines, 2 setae along the anterior margin segments densely setose straight, not reaching to the distal end of the third peduncular segment with 18 plumose setae 6 stout spines, 3 setae along the anterior margin Maxilla 2 inner lobe with 15 plumose _ inner lobe with 21 plumose setae setae Number of D-setae 28 34 Pereopods not elongated elongated Pereopods 6-7 anterior margins without —_ anterior margins with setae setae Uropod 3 setose, inner/outer lobe setose, inner/outer lobe ratio: 0.77 ratio: 0.9 inner lobe with 20 plumose setae 28 not elongated anterior margins without setae setose, inner/outer lobe ratio: 0.75 inner lobe with 21 plumose setae 37 slightly elongated anterior margins without setae setose, inner/outer lobe ratio: 0.9 zse.pensoft.net 486 At first glance, the newly-identified species looks sim- ilar to G. komareki by the setation of the antennae, by the presence and the shape of the eyes, uropod 3 and telson, but the following characters are different. In G. kunti sp. nov., the antennal gland cone reaches to the distal end of the third peduncular segment, but it is shorter (roughly halfway) in G. komareki. Similarly, G. kunti sp. nov. has less D-setae in the third segment of the mandible palp (28 in G. kunti; 40 in G. komareki), less setose pereopod 4 and not setose anterior margins of pereopods 6—7. An- other important differentiation is that, in males, the seta- tion on the carpus and merus posterior margin of the 4" pereiopod is significantly shorter in G. kunti (subequal to the diameter of underlying segment) than in G. komareki (longer than the diameter of underlying segment). G. kunti sp. nov. also resembles Gammarus komareki aznavensis Ozbek & Rasouli, 2014 in terms of setation of antenna 2, pereopods 3 and 4, but the newly-identi- fied species differs from G. komareki aznavensis by its larger size, by having smaller eyes, longer antenna 1, by absence setae along the anterior margins of pereopod 6 and by the shorter setation of the telson (Ozbek and Rasouli 2014). The newly-identified species differs from G. kessleri- anus by the body length (smaller), having fewer flagellar segments in antenna 2 and shorter endopod of uropod 3. Gammarus kesslerianus werneri S. Karaman 1934 was identified from Iznik Lake, NW Anatolia. After S. Karaman’s record, the subspecies has been never re-de- scribed and collected again until G.S. Karaman’s re-de- scription (Karaman 2018). He elevated the subspecies to the specific rank as Gammarus werneri and transferred it into the Gammarus balcanicus-group. So, Gammarus kunti sp. nov. distinctly differs from G. werneri because the newly-identified species belong to the Gammarus pulex-group. Gammarus kunti sp. nov. differs from Gammarus ram- bouseki (S. Karaman, 1931) by having reniform eyes, by the absence of long setae on the peduncular segments of antenna 1, by the absence of long setae along the ante- rior margins of pereopod 5 to 7 and by the presence of plumose setae on uropod 3. Additionally, G. rambouseki has less setose antenna 2 and more setose urosomites and telson (Karaman and Pinkster 1977). Gammarus kunti sp. nov. is similar to Gammarus fossa- rum Koch, 1836 by having reniform eyes, a setose posteri- or margin of pereopod 3 and the armaments of pereopods 5 to 7. However, the newly-identified species differ from G. fossarum by having much more setose antenna 2 and by having a more elongated inner lobe of the uropod 3. Studies conducted in recent years suggest that the west- ern Black Sea Region of Turkiye is quite rich in terms of freshwater amphipods. Many new and endemic species have been identified from the caves and water bodies in the region (Andreev and Kenderov 2012; Karaman 2012: Ozbek 2012; Ozbek et al. 2013, 2023). To reveal the bio- diversity of Turkish inland waters, studies supported by molecular analyses should be increased. zse.pensoft.net Ozbek, M. et al.: Gammarus kunti sp. nov. from the Fakill: Cave, Turkiye Acknowledgements The authors would like to thank Mert Elverici and Kadir Bogac¢ Kunt (collectors and biologist experts); Baris Kay- maz, Hilmi Umut Demiriz, Ozlem Kaya, Burak Gezer (sup- port access to caves and aquatic habitats and sportive cav- ing within the Turkish Caving Federation); Gokhan Eren Cankaya, Ertugrul Kulaksizoglu (support at various stages within the scope of the project, within the body of Kasif Consulting, Reporting, Organisation Company); Mustafa Uzun (the director of the Natural Assets branch of the Turk- ish Ministry of Environment, Urbanisation and Climate Change, General Directorate of Conservation of Natural Assets). 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Springer, 1-10. https://doi.org/10.1007/978-3-030- 65501-3_ 1 Zengin B, Eker M (2020) The effects of cave tourism on Ak¢akoca tour- ism: Fakilli Cave example. Journal of Turkish Tourism Research 4(1): 220-233. https://doi.org/10.26677/TR1010.2020.309 Supplementary material | The pairwise genetic distance values amongst the Gammarus species, based on the COI dataset (below the diagonal) and 28S dataset (above the diagonal) Authors: Murat Ozbek, Hazel Baytasolu, Ismail Aksu Data type: xls Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.99.108048.suppl1 zse.pensoft.net