Zoosyst. Evol. 100 (3) 2024, 989-1004 | DO! 10.3897/zse.100.121692 Gpgtusrue ror BERLIN Gammarus sezgini sp. nov. (Arthropoda, Amphipoda, Gammaridae), anew amphipod species from the Eastern Black Sea region of Ttirkiye Hazel Baytasoélu', Ismail Aksu!, Murat Ozbek? 1 Recep Tayyip Erdogan University, Faculty of Fisheries and Aquatic Sciences, 53100 Rize, Turkiye 2 Ege University, Faculty of Fisheries, 35100 Izmir, Turkiye https://zoobank. org/8CF9597B-97B7-4E42-AE6A-55AD07C25878 Corresponding author: Hazel Baytasoglu (gokbuluthazel@gmail.com) Academic editor: Luiz F. Andrade # Received 25 March 2024 Accepted 17 June 2024 Published 11 July 2024 Abstract A new amphipod species belonging to the genus Gammarus was identified in the rivers of the Eastern Black Sea Region of Turkiye: G. sezgini sp. nov. The authors described the new species using a taxonomic approach that combines morphological and molecular data. The newly identified species belongs to the G. komareki species complex because of the setation of antenna 2, pereopods 3 and 4, and the uropod 3. Some of its characteristic features are as follows: A medium-large species (holotype male, 9.8 mm). The body is yellowish; no dorsal keel or hump; eyes well developed, kidney-shaped; extremities not elongated; the second antenna bears numerous groups of long setae on the peduncle and flagellar segments; antennal gland cone long, not curved; the posterior margin of pereopod 3 is densely setose; the setae on the posterior edge of pereopod 4 are shorter and fewer in number; the anterior margins of pereopods 5 to 7 bear spines in the male; epimeral plates are not pointed. The newly identified species looks similar to G. komareki but differs from it by having a longer antennal gland cone, having fewer D-setae (33) in the third segment of the mandible palp, having shorter setae on the ventral part of the peduncular segment of the antenna 2, and having longer antenna 1, having fewer setae along the posterior margins of pereopods 3 and 4, and the absence of setae along the anterior margins of merus and carpus of pereopod 7. The new species is distinct from its relatives by high genetic distance (COI: 17.10% and 28S: 0.88%) and was resolved from them as an independent lineage with high support (ML: 78%, NJ: 70%, and BI: 1.0) in all phylogenetic results, based on the concatenated dataset (28S+COI). Additionally, species delimitation analyses (ASAP and PTP) based on the COI gene supported the conclusion that the new species constitutes an independent lineage. Detailed descriptions and drawings of the male holotype and the female allotype are given, and the morphology of the newly identified species is compared with that of its relatives. Key Words Eastern Black Sea, freshwater, identification, molecular analysis, taxonomy Introduction The order Amphipoda Latreille, 1816, is comprised of six suborders represented by approximately 11,000 species. The suborder Senticaudata Lowry & Myers, 2013, which also includes the family Gammaridae Latreille, 1802, en- compasses around 6,000 species, hosting nearly all fresh- water species and numerous marine benthic species. The genus Gammarus Fabricius, 1775, with approximately 200 described species, exhibits a wide distribution in the Holarctic region. Previous studies suggest that Gammarus originated from ancient Tethys and then diversified due to plate tectonic activities between Eurasia and A frica/India (Hou et al. 2011; Horton et al. 2024). Studies on Gammarus species in Turkiye began in the early 20" century and have continued until the present day (Vavra 1905; Coifman 1938; Bacescu 1954; Ozbek and Ustaoglu 1998, 2001, 2005a, 2005b; Sari et al. 2001; Balik et al. 2004; Ozbek et al. 2004, 2007; Akbulut et al. 2009; Albayrak and Ozulu% 2016; Ozbek and Ozkan 2017; BaytasoSlu and Goézler 2018). Especially in recent years, newly recorded species from the inland waters Copyright Baytasoglu, H. 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. 990 Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye of Turkiye have been evaluated not only based on their morphological characteristics but also through molecular analysis (Rewicz et al. 2016; Ozbek et al. 2023a, 2023b). So far, 55 species belonging to the genus Gammarus have been identified in Turkish inland waters (Ipek and Ozbek 2022; Ozbek et al. 2023a, 2023b). The Eastern Black Sea Basin covers Artvin, Rize, Tra- bzon, Gumtshane, Giresun, and Ordu provinces. The riv- ers of the area are mainly fed by precipitation and have a regular regime. The flow rate is normal during the summer months, while the flow rate increases with melting snow. Some of the rivers flow directly into the Black Sea after a short flow, and some of them originate in central Anatolia and reach the Black Sea by crossing the North Anatolian Mountains (Selim 2011). In previous studies conducted in this basin, six species [G. balcanicus Schaferna 1922, G. birsteini Karaman & Pinkster, 1977, G. kischineffensis Schellenberg 1937, G. komareki Schaferna 1923, G. pu- lex pulex (Linnaeus 1758), and G. fossarum Koch 1835] were reported (Karaman 2003). However, no detailed dis- tribution data for these species has been presented to date. This study aimed to investigate the amphipod sam- ples collected from streams (Balat-Yesildere-Tasl1) 1n the Eastern Black Sea Basin (Rize) of Turkiye both morpho- logically and genetically. As a result of the study, a new amphipod species was described, Gammarus sezgini sp. nov., detailed descriptions and drawings of the extremi- ties of the male holotype and female allotype were given, and the morphology of the newly described species was compared with its relatives. Materials and methods Study area Samplings were conducted in Balat Stream, Tasl1 Stream, and Yesildere Stream within the borders of Rize province, the northeastern part of Turkiye. Balat Stream 1s a tribu- Figure 1. Map of the sampling area and the localities. zse.pensoft.net tary of the Buyuk Stream, which flows from Rize/Cayeli district to the Black Sea. There are trout farming facili- ties on the stream. Yesildere Stream is a tributary of the Tashi Stream, flowing from Rize/Andon Hot Springs to the Black Sea. In these locations, there are trout farming facilities and tea collection centers. The map of the sta- tions where the species was identified is given in Fig. 1. A map was created using the QGIS v.3.8.3-Zanzibar soft- ware available at http://diva-gis.org (Fig. 1). Data collection and analysis Samplings were carried out at three stations in October 2019 and September 2020. A 30x30 cm sized hand net (D-Frame net) with a 250 u mesh size was used to collect the specimens. The collected samples were placed in plas- tic sample containers, and the labels on which the date, the name of the station, the coordinate, the altitude, and the name of the city where they are located are written both inside the container and on the outside. The first fix- ation of the samples was made with 96% alcohol in the field. The samples brought to the laboratory were cleared of their sludge under tap water with the help of sieves with a mesh size of 4 mm-—63 um. Each individual was exam- ined under a Leica MC 170 HD brand stereomicroscope. Morphological identification One adult male and one female individual from the sam- ples were selected as holotype and allotype individuals, respectively. Both selected individuals were kept in a lac- tic acid and 10% NaOH solution for 2 hours. The holotype male individual was photographed under a stereomicro- scope before being dissected. After holotype and allotype individuals were dissected in a glycerin alcohol solution, permanent slides were prepared with a CMCP- 10 high-vis- cosity mount. Detailed photographs of the extremities 41.000 42.000 Zoosyst. Evol. 100 (3) 2024, 989-1004 were taken with a 5-megapixel resolution digital camera mounted on an Olympus CK X-41 model binocular micro- scope. For detailed drawings of the extremities, a digitizer board (Wacom PTH-451) and a standard pen connected to the computer were used. Image processing programs were used in the drawings of the extremities, and the drawing techniques specified by Coleman (2003) were followed. Molecular identification Total DNA isolation, PCR amplification, and sequencing The DNA of Gammarus specimens was extracted on the Qiacube Automated DNA Isolation Device (Qiagen, Va- lencia, CA) according to the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) protocol. The mitochondrial cytochrome c oxidase subunit I gene (COI) was ampli- fied with the primers UCOIF (5’-TAWACTTCDGGRT- GRCCRAAAAAYCA-3’) and UCOIR (5’-ACWAAY- CAYAAAGAYATYGG-3’) as described by Costa et al. (2009), and cycle conditions were as follows: 3 min. first denaturation at 95 °C, followed by 35 cycles of denaturing for 30 sec. at 95 °C, annealing for 30 sec. at 47 °C, exten- sion for 45 sec. at 72 °C, and final extension for 7 min. at 72 °C. The nuclear large subunit ribosomal RNA gene (28S) was amplified with the primers 28F (5’-TTAGTAGGGG- Table 1. Information on sequences used in molecular analyses. Species Locality 991 CGACCGAACAGGGAT-3’) and 28R (5’-GTCTTCGC- CCCTATGCCCAACTGA-3’) as described by Hou et al. (2007), and cycle conditions were as follows: 3 min. first denaturation at 95 °C, followed by 35 cycles of denaturing for 35 sec. at 95 °C, annealing for 30 sec. at 62 °C, exten- sion for 1.15 min. at 72 °C, and final extension for 7 min. at 72 °C. The QIAquick PCR Purification Kit (Qiagen) was utilized to purify the amplified PCR products. Two-direc- tional sequencing of PCR products was performed with an ABI PRISM 3730x1 Genetic Analyser using a BigDye Terminator 3.1 cycle sequencing ready reaction kit (Ap- plied Biosystems) at Macrogen Europe. Molecular data analysis We carried out analyses to genetically compare the potential new species with its congeners and to generate its first mo- lecular records. We sequenced the COI and 28S genes of a total of five specimens from three populations (Balat, Yesil- dere, and Tasli streams) of the new species (see “Genetic material” section). In addition, we downloaded the COI and 28S sequences of valid Gammarus species from GenBank. Detailed information on these species is available in Table 1. The raw COI and 28S sequences of the new species were corrected by checking their chromatograms in the Bioedit 7.2.5 program (Hall 1999). All sequences were 2 G. G. sezgini sp. nov. (T) sezgini sp. nov. (T) sezgini sp. nov. ( . ( .( <= — no sezgini sp. no sezgini sp. no kunti (T) tumaf (T) baysali (T) kesslerianus (T) komareki (T) komareki rambouseki (T) roeselii fossarum (T) plaitisi uludagi monspeliensis (T) ibericus pulex (T) lacustris italicus varsoviensis (T) kischineffensis (T) spelaeus (T) balcanicus (T) bosniacus (T) leopoliensis (T) stojicevici (T) halilicae (T) pljakici stankokaramani (T) salemaai + V ) T) ) Balat stream, Rize, Turkiye Balat stream, Rize, Turkiye Yesildere stream, Rize, Turkiye Yesildere stream, Rize, Turkiye Tasli stream, Rize, Turkiye Fakilli Cave, Turkiye Gokgol Cave, Turkiye Cumayani Cave, Turkiye Simferopol, Crimea, Ukraine ca 200 km SE Sofia, Bulgaria Mazandaran, Iran Bitola, Macedonia Netherlands Regensburg, Germany Tinos, Komi, Greece Evia, Greece Montpellier, France Lascaux, France Slovenia Bled, Slovenia Rieti, Lazio, Italy Secymin, Poland Targu Bujor, Romania Simferopol, Crimea, Ukraine Kolasin, Montenegro Sarajevo, Bosnia and Herzegovina Vistula, Poland Bela Palanka, Serbia Lazaropole, Macedonia Galicica planina, Macedonia Ohrid, Macedonia Gradiste, Macedonia Pontogammarus robustoides Delta Volgi, Russia Note: (T) Topotype samples of nominal taxa. 28S Col References PP456724 PP457381 This study PP456725 PP457382 This study PP456726 PP457383 This study PP456727 PP457384 This study PP456728 PP457385 This study OP650556 OP642558 Ozbek et al. (2023a) ON751931 ON749780 Ozbek et al. (2023b) ON751932 ON749781 Ozbek et al. (2023b) 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) zse.pensoft.net 992 Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye then aligned with the Clustal W method (Thompson et al. 1994). The pairwise genetic distances were calculated for the COI and 28S genes according to the uncorrected p-distance in the MEGA X software (Kumar et al. 2018). To reconstruct the phylogeny of the genus Gammarus, the COI and 28S sequences of all species were added end-to-end, resulting in a concatenated data set (28S+- COI) for each species. Phylogeny was estimated by using Neighbour-Joining (NJ; Saitou and Nei 1987), Maximum Likelihood (ML; Felsenstein 1981) methods in MEGA X software, and Bayesian inference (BI) in MrBayes v3.2.1 (Ronquist et al. 2012). The appropriate nucleotide substi- tution models were selected according to the Akaike Infor- mation Criterion (AIC) and Bayesian Information Criteri- on (BIC) in jModeltest 0.1.1 (Posada 2008). The NJ tree was constructed using the p-distance node support, which was calculated (Felsenstein 1985) using 1000 bootstrap pseudo-replicates. The ML tree was constructed using the Tamura-Nei model (TN93; Tamura and Nei 1993) with gamma-correction and invariant sites (G+I), and node support was calculated with 500 bootstrap pseudo-repli- cates. The BI tree was constructed using the TN93+G+I model. The analysis was run for 5 million generations with Metropolis-coupled Monte Carlo Markov Chains (MCMC) sampled every 1000 generations. As burn-in, the first 25% of generations were discarded. The convergence of the runs was confirmed using Tracer v1.7.1 (Rambaut et al. 2018). The iTOL (Interactive Tree of Life; https://itol. embl.de/), a web-based program, was used to visualize the BI tree. In all phylogenies, Pontogammarus robustoides (Sars, 1894) (Table 1) was chosen as the outgroup. We applied one distance-based method, Assemble Species by Automatic Partitioning (ASAP; Puillandre et al. 2020), and one tree-based method, Poisson Tree Processes (PTP; Zhang et al. 2013), to identify the Molecular Operational Taxonomic Units (MOTUs) based on the COI dataset. To implement the ASAP method, we used the Kimura 2-param- eter (K2P) distances and transition/transversion ratio (R:1.4) settings at the web address https://bioinfo.mnhn.fr/abi/pub- lic/asap/. The transition/transversion ratio (R) for the COI data was calculated in MEGA X software. PTP with a maxi- mum likelihood solution was implemented via a web server (http://mptp.h-its.org/#/tree) (accessed on February 2, 2024). Results Gammarus sezgini sp. nov. https://zoobank.org/BE51BA07-80D8-4832-96F3-594D0CD087FF Figs 2-7 Material examined. Holotype: Turxty « Male; 9.8 mm; Rize Province, Yesildere stream/Balat stream/ Tasli Stream; coordinates: 40.9493°N, 40.5394°E / 41.0227°N, 40.7130°E / 40.8701°N, 40.5859°E. Spec- imens collected by Hazel BAYTASOGLU; 16 October 2019 and 1 September 2020. Holotypes with paratypes are stored under catalog number RTEU-FFR200001; zse.pensoft.net (GenBank accession numbers: PP457383, PP457384 for COI, and PP456726, PP456727 for 28S; PP457381, PP457382 for COI, and PP456724, PP456725 for 28S; PP457385 for COI and PP456728 for 28S). Paratypes: 38 males and 34 females, same data as the holotype. Genetic material. RTEU-FFR-DNA K2, K4, Yesildere stream, Rize Province, Turkiye, 40.9493°N, 40.5394°E (GenBank accession numbers: PP457383, PP457384 for COI, and PP456726, PP456727 for 28S) - RTEU-FFR- DNA K5, K8, Balat stream, Rize Province, Turkiye, 41.0227°N, 40.7130°E (GenBank accession numbers: PP457381, PP457382 for COI, and PP456724, PP456725 for 28S) - RTEU-FFR-DNA K%9, Tasli stream, Rize Prov- ince, Turkiye, 40.8701°N, 40.5859°E (GenBank acces- sion numbers: PP457385 for COI and PP456728 for 28S). Diagnosis. A medium-large species. The body ts yel- lowish; no dorsal keel or hump; the eyes are well de- veloped; kidney-shaped; the extremities are not elon- gated; the second antenna bears numerous groups of long setae on the peduncle and flagellar segments; the antennal gland cone is straight and reaches to the distal end of the third peduncular segment; posterior margin of pereopod 3 densely setose; the setae on the posterior edge of pereopod 4 are shorter and fewer in number; the anterior margins of pereopods 5 to 7 bear spines in the male, while they bear long setae along with the spines in females; epimeral plates are pointed; the inner ramus of uropod 3 is slightly longer than 0.8 of the outer one; each telson lobe bears a pair of spines distally and setae longer than the spines. Description of male holotype. Head: Rostrum ab- sent, inferior antennal sinus deep, rounded. Eyes kid- ney-shaped; length is slightly shorter than the diameter of the first peduncular segment of antenna | (Fig. 2). Antennae: Antenna | (Fig. 4A) is half as long as the body length; the length ratio of the peduncular segments is 1:0.75:0.38; peduncle segments bear a few groups of minute setae; the length of the setae is much shorter than the segment where they are implanted; the main flagel- lum with 32 segments; each segment bears a few short setae in distal side; aesthetasc absent; accessory flagel- Figure 2. Habitus of the holotype male of Gammarus sezgini Sp. nov. Zoosyst. Evol. 100 (3) 2024, 989-1004 g93 E. Right mandible; F. Left mandible; G. Left maxilla 1; H. Right maxilla 1; G’, H’. Detail of the left and right maxilla 1. lum with five segments. Antenna 2 (Fig. 4B) is shorter than antenna 1 (ratio 1:0.7); the antennal gland cone is straight and reaches the distal end of the third pedun- cular segment; setation is rich both on peduncular and flagellar segments; peduncular segments 4 and 5 bear many groups of setae; the setae on the ventral part of the peduncle segments are shorter than the diameter of the segment but longer than those on the dorsal part; fla- gellum consists of 12 segments; flagellar segments are setose and swollen; each segment bears many long setae groups on both dorsal and ventral sides; calceoli absent. Mouthparts: Upper lip (Fig. 3B) with numerous min- ute setules in the distal part. Left mandible (Fig. 3F) with 4-toothed incisor, lacin- ia mobilis with 4 dentitions, molar triturative. The first article of palp without setae; the second one bears 14 se- tae; the setae become shorter from distal to proximal. The third segment has 33 D-setae, 4-5 E-setae, one group of A-setae, and one group of B-setae. C-setae absent. Right mandible (Fig. 3E) has a 4-toothed incisor and bifurcate lacinia mobilis. Right maxilla 1 (Fig. 3H, H’) is asymmetric to the left; it has 16 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 (one of them lost) and three setae (two of them robust) on the distal part of the second segment, in addition to a marginal seta along the outer margin. The second article of left palp is elongated and bears 10 spines, five simple setae on its distal part, and no setae along the outer margin (Fig. 3G, G’). Lower lip (Fig. 3A) has no inner lobe and bears nu- merous small simple setae along the distal margins of both lobes. zse.pensoft.net 994 Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye Figure 4. Extremities of the holotype male of Gammarus sezgini sp. nov. A. Antenna 1; B. Antenna 2; C. Gnathopod 1; C’. Palm of Gnathopod 1; D. Gnathopod 2; D’. Gnathopod 2. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 989-1004 O95 Figure 5. Pereopods of the holotype male of Gammarus sezgini sp. nov. A. Pereopod 3; B. Pereopod 4; C. Pereopod 5; D. Pereopod 6; E. Pereopod 7. zse.pensoft.net 996 Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye a —— a” - LY \ LY 1 1 1 1 ' Figure 6. Extremities of the holotype male of Gammarus sezgini sp. nov. A. Pleopod 3 and Epimeral plate 3; B. Pleopod 2 and Epimeral plate 2; C. Pleopod 1 and Epimeral plate 1; D. Uropod 2; E. Uropod 1; F. Uropod 3; G. Telson. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 989-1004 O97 Figure 7. Appendages of the allotype female of Gammarus sezgini sp. nov. A. Antenna 1; B. Antenna 2; C. Gnathopod 1; D. Gna- thopod 2; E. Pereopod 3; F. Pereopod 4. zse.pensoft.net 998 Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye Maxilla 2 (Fig. 3C) has 20-30 simple setae in the dis- tal part of the outer lobe and a few tiny hairs along the outer margin. The inner lobe also has 10—15 simple setae in the distal part in addition to 14—15 (two of them lost) plumose setae located in a diagonal row along the inner margin. There are also a few tiny hairs on the proximal part of the inner margin of the lobe. Maxilliped (Fig. 3D) inner plate has three tooth-like Spines and a spine in the distal part and the distal cor- ner, respectively. Additionally, there are 10 plumose se- tae 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 (Fig. 4C) is rectangular, the distal part slightly widened, the ventral margin slightly convex, and bears four antero-distal setae and two poste- ro-distal setae. Coxal plate 2 (Fig. 4D) 1s in the shape of an elongated rectangle; distal part narrower than the prox- imal; the ventral margin is highly convex; anterodistal part with five setae; and postero-distal part with one seta. Coxal plate 3 (Fig. 5A) is similar to coxal plate 2 in shape, with four and two setae in the antero- and postero-distal ends, respectively. The ventral edge of the coxal plate 4 (Fig. 5B) is slightly convex and bears three and eight se- tae along the anteroventral and posterior margins, respec- tively. Coxal plate 5 (Fig. 5C) bilobate and has one and five setae in the anterior and posterior lobes, respectively. Coxal plate 6 (Fig. 5D) bilobate and has one seta in the posterior lobe. Coxal plate 7 (Fig. SE) is characterized by the presence of four setae on the postero-ventral margin. Gnathopods: Basal segment of gnathopod 1 (Fig. 4C, C’) bears many long setae along both margins; the length of the setae can be longer than twice the diameter of the seg- ment. Ischium bears a group of setae in postero-ventral cor- ner. Merus is in diamond shape and bears some setae along its disto-posterior margin. Carpus is triangular and bears two groups of setae along the anterior margin, in addition to many setae groups on both ventral and posterior sides. Propodus pyriform, the length/width ratio is 1:0.60; anterior margin with four groups of setae; medial palmar spine is present; posterodistal corner armed with two strong spines in addition to some small spines; posterior margin bears 4—5 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 inner margin. The basis and ischium of gnathopod 2 (Fig. 4D, D’) are similar to those of gnathopod 1 but have denser se- tae. Merus and carpus are more setose than those of gna- thopod 1. Carpus is triangular, densely setose along the posterior margin, in addition to two groups of setae along the anterior margin. Propodus is densely setose and has a sub-rectangular shape; the length/width ratio is 1: 0.6; anterior margin bears six groups of setae; posterior mar- gin with many groups of setae; medial palmar spine is present; the postero-distal corner is armed with six strong spines. 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 inner margin. zse.pensoft.net Pereopods: Anterior and posterior margins of the basal segment of pereopod 3 bear long setae; the setae along the posterior margin are longer than those in the anterior mar- gin; posterior margins of the merus, carpus, and propodus 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. 5A). The basal segment of pereopod 4 (Fig. 5B) has a similar setation to that of pereopod 3. Ischium, merus, carpus, and propodus have groups of setae along their posterior mar- gins, but they are much shorter and less than those tn pereo- pod 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. Posterior margins of the basal segments of pereopods 5 to 7 (Fig. 5C—E) are more or less convex and bear many short setae, anterior margins with 3—7 small spines, and no setae present on the inner surfaces of the basal segments; no spine exists in the postero-ventral corner of the basal segment of pereopod 7. Pereopod 7 bears no setae along the anterior margins of merus and carpus, while pereopods 5 and 6 have a few setae 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. Epimeral plates: They are slightly pointed. Epimeral plate 1 (Fig. 6C) bears 10 long setae along the antero- ventral margin, and the postero-ventral corner is angu- lar. Epimeral plate 2 (Fig. 6B) bears five setae in the an- teroventral corner; the ventral margin is armed with two spines; the posterior margin with 4—5 setules; the poste- ro-ventral corner is pointed. Epimeral plate 3 (Fig. 6A) is pointed; the anteroventral corner bears two setae; the ventral margin is armed with three spines in addition to a seta; the posterior margin bears 5—6 setules. Urosomites: Not elevated (Fig. 2). Each segment bears a median and two dorsolateral groups of armaments; each of them consists of 1—2 spines and 3-4 accompanying setae. Uropods: Uropod | (Fig. 6E) has a spine in the outer margin of the base; inner margins bear 4+5 spines; the pe- duncle is longer than rami; the length ratio is about 1:0.7. Peduncle with a spine in the outer margin of the proximal part in addition to three spines along the inner margin and three spines in the distal part. The inner ramus 1s slightly longer than the outer ramus and bears three spines along their inferior margin in addition to 4—5 distal spines. The outer ramus has two and three spines along the inferior and outer margins in addition to 4—5 distal spines, respectively. Uropod 2 (Fig. 6D) is smaller than the first one; the length ratio is about 1:0.6; the peduncle segment is slight- ly longer than the rami and bears 2+1 spines along the inner margin and the distal part, respectively. The outer margin is bare. The length and armaments of both rami are similar to each other; they bear two spines along their Zoosyst. Evol. 100 (3) 2024, 989-1004 inner margins in addition to 4—5 longer spines on their distal tips. Uropod 3 (Fig. 6F) is setose and bears both simple and plumose setae. The peduncle segment is much shorter than the outer ramus, and the length ratio is about 1:0.48. The outer ramus is two articulated and densely setose along both margins; the outer margin bears three groups of spines accompanied by groups of long simple setae; the inner margin with plumose setae; the second article is well developed and longer than the surrounding distal spines. The inner ramus is about 0.78 times the length of the outer ramus. It bears one spine in the proximal part of the outer margin in addition to groups of simple and plumose setae; the inner margin bears both simple and plumose setae. Telson: Telson (Fig. 6G) lobes cleft; each lobe bears two spines and 5—6 simple setae in their distal parts. The setae are twice as long as the spines. There are 3-4 groups of short setae 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. Description of females. Smaller than males. Except for the sexual dimorphism indicated for the genus Gam- marus, females do not show obvious differences from males. At first glance, the morphological differences be- tween the female allotype and the male holotype can be listed as follows: More setose antenna 2, less setose and small gnathopod 2, more setose pereopod 4; more setose anterior margins of pereopods 5 to 7 (Figs 7, 8). Variability. Some of the paratypes are immature. The eyes are kidney-shaped, or elongated, and oval. The number of flagellar segments in antenna | varies between 26 and 29. Similarly, there are 10—11 flagellar segments in antenna 2. Etymology. The species epithet is derived from the name of our dear friend Prof. Dr. Murat Sezgin (R.LP.), who made valuable contributions to the marine amphipod species in Turkiye. Results of molecular data analyses We tested the new species with molecular methods as well as morphological characters. For this, firstly, the COI (573 bp.) and 28S (911 bp.) genes of the new species from type and paratypes were amplified and sequenced. The obtained sequences were deposited in Genbank with the corresponding accession numbers: PP457381— PP457385 for COI and PP456724—PP456728 for 28S. For molecular comparison, sequences of topotype sam- ples of valid congeners of the new species or otherwise correct sequences of valid species were downloaded from GenBank (see Table 1) and used in all analyses. The pairwise genetic distance amongst Gammarus spe- cies based on the COI was calculated to range from 5.24% (G. stankokaramani G. Karaman, 1976 - G. salemaai G. Karaman, 1985) to 28.97% (G. sezgini sp. nov. - G. roe- selii Gervais, 1835). The species most closely related to G. sezgini sp. nov. is G. tumaf Ozbek, Aksu & Baytaso$lu, 2023, with 17.10%, approximately three times larger than 999 the minimum genetic distance. The pairwise genetic dis- tance amongst Gammarus species based on the 28S was calculated to range from 0.11% (G. halilicae G. Karaman, 1969 - G. pljakici G. Karaman, 1964) to 7.73% (G. ram- bouseki G. Karaman, 1931 - G. stojicevici (S. Karaman, 1929)). The species most closely related to G. sezgini sp. nov. 1s G. kesslerianus Martynov, 1931, with 0.88%, eight times larger than the minimum genetic distance. All pairwise genetic distance values calculated with the p-dis- tance model based on COI and 28S genes amongst Gam- marus species are given in Suppl. material 1. Phylogenetic trees constructed with ML, NJ, and BI methods based on the concatenated dataset (28S+COI) showed similar topologies with a few exceptions and had high bootstrap (ML and NJ BP>70%; Fig. 9) and poste- rior probability (BI PP>0.7; Fig. 9) support for a large number of nodes. In the phylogenies constructed accord- ing to all three methods, the newly identified species, G. sezgini sp. nov., formed the sister clade of the G. kunti Ozbek, Baytasoglu & Aksu, 2023, G. tumaf, and G. bay- sali Ozbek, Yurga & Kulkoyltoglu, 2013 (G. sezgini sp. nov., (G. kunti, (G. tumaf+ G. baysali))) and was resolved from it with strong support (ML BP: 78%, NJ BP: 70%, and BI PP: 1.0; Fig. 9). The species delimitation analysis we performed ac- cording to the ASAP method identified 27 MOTUs for 27 morphologically valid species (Fig. 9). The best ASAP score was 3.0 (p = 0.01) at a threshold distance of 0.079053. The analysis identified the species G. stanko- karamani and G. salemaai as a single MOTU, while the Bulgarian and Iranian samples of G. komareki were iden- tified as separate MOTUs. The PTP method identified 28 MOTUs for 27 species (Fig. 9). p=0.001, null-model score: 84.937039, best score for single coalescent rate: 95.647569. Similar to ASAP, Bulgarian and Iranian sam- ples of G. komareki formed separate MOTUs, while un- like ASAP, G. stankokaramani and G. salemaai species also formed separate MOTUs. Gammarus sezgini sp. nov. formed a single MOTU independently of other species according to both methods (Fig. 9). Discussion The consensus of morphological and molecular findings has shown that the Balat, Tasli, and Yesildere streams at Rize province populations of Gammarus are distinct from their congeners and should be recognized as a sep- arate species. Gammarus sezgini sp. nov. belongs to the G. komareki-group due to the characteristic setation of the posterior part of pereopod 3 and 4, the setation of an- tenna 2 and uropod 3 (Karaman and Pinkster 1977). At first glance, the newly identified species looks simi- lar to G. komareki by the setation of the antenna 2, pereo- pod 3, and uropod 3, by the presence and the shape of the eyes; but G. sezgini sp. nov. differs from G. komareki by having a longer antennal gland cone, having fewer D-setae (33) in the third segment of the mandible palp, zse.pensoft.net 1000 Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye 1mm 0.5 mm (E) Figure 8. Appendages of the allotype female of Gammarus sezgini sp. nov. A. Pereopod 5; B. Pereopod 6; C. Pereopod 7; D. Uro- pod 3; E. Telson; F. Uropod 1; G. Uropod 2; H. Pleopod 1 and Epimeral Plate 1; I. Pleopod 2 and Epimeral Plate 2; J. Pleopod 3 and Epimeral Plate 3. having shorter setae on the ventral part of the peduncular Gammarus komareki has been recorded from the Black segment of the antenna 2, and having a longer antennal, Sea coasts, Eastern Europe, the Balkans, and Iran in pre- having fewer setae along the posterior margins of pereo- vious studies (Copilas-Ciocianu et al. 2014; Grabowski pod 3 and 4, by the absence of setae along the anterior and PeSic 2007; Zamanpoore et al. 2011). The species margins of merus and carpus of pereopod 7. was reported from the Zonguldak, Trabzon, Sinop, and zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 989-1004 85/93/- PP456724+PP457381 Gammarus sezgini 96/96/- PP456725+PP457382 Gammarus sezgini 100/100/1 100/100/1 PP456727+PP457384 Gammarus sezgini 78/70/1 PP456726+PP45/383 Gammarus sezgini 1001 100/99/1 89/8$3/0.99 100/100/1 $3/83/1 -/-10.95 100/100/1 80/85/0.71 9$/98/0.99 100/100/0.97 100/100/1 -/-/0.88 73/77/0.82 98/98/0.52 7-40.96 | “EE ofa -/-/0.69 PP456728+PP457385 Gammarus sezgini OP650556+OP642558 Gammarus kunti ON751931+ON749780 Gammarus tumaf ON751932+ON749781 Gammarus baysali JF965721+JF965909 Gammarus kesslerianus JF965725+JF965913 Gammarus komareki JF965/23+JF965911 Gammarus komareki JF965771+JF965947 Gammarus roeselii JF965770+JF9659465 Gammarus rambouseki JF965696+JF965886 Gammarus fossarum JF965817+JF965986 Gammarus uludagi MT999102+MT999049 Gammarus plaitisi JF965738+JF965923 Gammarus monspeliensis JF965767+JF965943 Gammarus pulex JF965713+JF965901 Gammarus ibericus JF965728+JF965915 Gammarus lacustris JF965716+JF965904 Gammarus italicus JF965818+JF965987 Gammarus varsoviensis MG9875294+MG987571 Gammarus kischineffensis JF965801+JF965971 Gammarus spelaeus JF965640+JF965834 Gammarus balcanicus JF965680+JF965872 Gammarus bosniacus JF965734+JF965919 Gammarus leopoliensis JF965808+JF965978 Gammarus stojicevici JF965711+JF965900 Gammarus halilicae 100/100/1 JF965758+JF965936 Gammarus pljakici 96/85/0.69 EB 100/100/1 JF965780+JF965955 Gammarus salemaai JF965822+JF965990 Pontogammarus robustoides f=! MLI/NJ/BI 0,050 JF965806+JF965976 Gammarus stankokaramani wor RRR RRR eee sv? HERR RRR eee ? BERR RRR eee Figure 9. Phylogenetic relationships of Gammarus species reconstructed with the ML method based on the concatenated data set (28S+COI). Since the ML, NJ, and BI methods generally yield similar topologies, only the ML phylogeny is shown. The nodes (ML, NJ, and BI) show the Bayesian posterior probabilities and the bootstrap percentage. For the support values of the nodes, ML > 70%, NJ = 70%, and BI > 0.70 are shown. Black bars indicate OTUs. The first column shows morphology-based results, the second column shows ASAP results, and the third column shows PTP results. Rize provinces previously (Karaman 2003; Ozbek 2011). Additionally, it was reported from the inland waters of Gokceada Island (Ozbek and Ozkan 2017), from the lakes of the Western Black Sea Region and the Sakarya River Basin (Ozbek 2008), from the inland waters of Sin- op and Samsun provinces (Akbulut et al. 2009), and from the inland waters of Ordu (Ekinci and Miroglu 2016). Due to the lack of detailed sampling in the rivers in the Eastern Black Sea Basin, it is likely that the species G. sezgini sp. nov. has been diagnosed as G. komareki or has not been reached. The present study reveals the mor- phological and molecular differences between the two Species in detail. Gammarus obruki Ozbek, 2012, G. baysali, G. tumaf, and G. kunti have been recently identified from four dif- ferent caves (Inderesi Cave/Bartin province, Cumayan1 Cave/Zonguldak province, Gokg6l Cave/Zonguldak province, and Fakill1 Cave/Duizce province, respective- ly) within the Western Black Sea Basin (Ozbek 2012; Ozbek et al. 2013; Ozbek et al. 2023a, 2023b). They are the closest relatives of the newly described species due to their presence in geographically close localities and their morphological similarities. The four species men- tioned above, including Gammarus sezgini sp. nov., are morphologically very similar to G. komareki, especially due to the dense setation on the flagellum and peduncle zse.pensoft.net 1002 segments of antenna 2. As a result of the study, morpho- logical and genetic similarities and differences were de- fined in detail (Table 2, Fig. 9). Gammarus sezgini sp. nov. is similar to G. obruki in that it has a yellowish body color, kidney-shaped eyes, and densely setose flagellum and peduncle segments of antenna 2. But the newly described species is almost half the size of G. obruki, has a much shorter antenna 1 (52 vs. 32 segments), and no elongated extremities. Additionally, the inner ramus of the uropod 3 is shorter in the newly identified species (Table 2). Although the newly described species is similar to G. baysali in having setose antenna 2, it is quite different from it in terms of both morphological characters and hab- itat. G. baysali, like G. obruki, 1s a large species and 1s ap- proximately 2 times larger than the newly described spe- cies. Additionally, G. baysali is a hypogean eyeless species and has elongated extremities. Gammarus sezgini sp. nov. is an epigean species with well-developed kidney-shaped eyes and does not have elongated extremities. Although there are spines and setae on the anterior margins of pereo- pods 6 and 7 in G. baysali, the newly described species has no setae along the mentioned margins. While the inner lobe/outer lobe length ratio is 0.9 in the third uropod of G. baysali, this ratio is 0.78 in G. sezgini sp. nov. (Table 2). The newly described species is similar to G. Aunti in hav- ing kidney-shaped eyes and setose antennae | and 2, but differs from it in the following features: Its body has a yel- Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye lowish rather than whitish color; it bears one seta instead of two along the anterior margin of the inner lobe of the right maxilla 1; the inner lobe of the right maxilla bears more than 14 plumose setae. In addition, the 2"! and 3 epimeral plates are more pointed, and the telson lobes bear more and longer setae on the dorsal surface and the distal part (Table 2). Gammarus sezgini sp. nov. differs from G. tumaf by having kidney-shaped eyes, a less setose inner lobe of right maxilla 1 (16-17 vs. 20), the armaments of the palp of maxilla 1, having fewer plumose setae on maxilla 2 (14-15 vs. 20), and having more setose telson. Addition- ally, the newly identified species has 33 D-setae, while the number 1s 28 in G. tumaf (Table 2). Although the newly described species is similar to G. kesslerianus in having a setose second antenna 2, Gammarus sezgini sp. nov. differs from it in having al- most half a smaller body length (20 mm vs. 9.8 mm), a shorter flagellum of antenna 2 (13 vs. 17 segments), and a shorter inner lobe of uropod 3. Anatolia is a peninsula very rich in biodiversity, as it is located at the intersection of three different biodiver- sity hot spots. The presence of several unique habitats is an important factor in increasing the number of endemic species on the peninsula. Turkiye’s Black Sea region hosts fast-flowing streams that are generally fed by snow water. The authors believe that G. komareki, which is a typical species of these types of habitats, still contains many cryptic and undefined species and should be ex- Table 2. Some morphological features of Gammarus sezgini sp. nov. and its sister species (G. baysali, G. tumaf, G. kunti) and G. obruki (reproduced from Ozbek et al. 2023a). Characters Gammarus sezgini G. obruki G. baysali G. tumaf G. kunti G. komareki sp. nov. Body length 9.8 mm 21.0 mm 12.6 mm 11.5 mm 15mm Eyes kidney-shaped kidney-shaped eyeless minute kidney-shaped reniform Body color yellowish yellowish colorless, whitish whitish whitish Antenna 1 32+5 flagellar 52+6 flagellar 41+6 flagellar 30+5 flagellar 32+6 flagellar 39+5 flagellar segments segments segments segments segments segments Antenna 2 peduncular and fifth peduncular and peduncular and peduncular and peduncular and peduncular and flagellar segments flagellar segments flagellar segments flagellar segments flagellar segments flagellar segments densely setose; densely setose; setose; flagellum 20 densely setose; densely setose; densely setose; flagellum 12 flagellum 17 segmented flagellum 13 flagellum 15 flagellum 13 segmented segmented segmented segmented segmented Antennal gland cone Inner lobe of right straight, reaches to the distal end of the third peduncular segment with 16 (17) plumose straight, not reaches | straight, reaches to the | straight, reaches to the to the distal end of the third peduncular segment with 18 plumose setae | with 19 plumose setae | with 20 plumose setae distal end of the third peduncular segment distal end of the third peduncular segment straight, reaches to the distal end of the third peduncular segment with 14 plumose setae Short, about half as long as the third peduncle segment No data in original maxilla 1 setae description Palp of right 6 stout spines, 1 seta | 6 stout spines, 3 setae] 6 stout spines, 4 setae|5 stout spines, 2 setae | 6 stout spines, 2 setae} No data in original maxilla 1 along the anterior along the anterior along the anterior along the anterior along the anterior description margin margin margin margin margin Maxilla 2 inner lobe with 14-15 | inner lobe with 21 inner lobe with 21 inner lobe with 20 inner lobe with 15 No data in original plumose setae plumose setae plumose setae plumose setae plumose setae description Number of D-setae 33 37 34 28 28 40 Pereopods not elongated slightly elongated elongated not elongated not elongated No data in original description Pereopods 6-7 Uropod 3 Telson (each lobe) zse.pensoft.net anterior margins without setae setose, inner/outer lobe ratio: 0.78 with 2 distal spines and 5-6 longer setae; IW ratio 1:0.5 anterior margins without setae setose, inner/outer lobe ratio: 0.9 with 1-2 distal spines and 3-4 longer setae; lw ratio 1:0.5 anterior margins with setae setose, inner/outer lobe ratio: 0.9 with 2 distal spines and 4-5 longer setae; W ratio 1:0.5 anterior margins without setae setose, inner/outer lobe ratio: 0.75 with 2 distal spines and 3-4 longer setae; IW ratio 1:0.5 anterior margins without setae setose, inner/outer lobe ratio: 0.77 with 2 distal spines and 3-4 longer setae; Ww ratio 1:0.5 anterior margins without setae setose, inner/outer lobe ratio: 0.75 with 2 distal spines and 3-4 longer setae; w ratio 1:0.5 Zoosyst. Evol. 100 (3) 2024, 989-1004 amined in detail from both molecular and morphological perspectives. Such an integrative approach would help highlight the true biodiversity of gammarids in Anatolia. Acknowledgments This work was financially supported by TUBITAK (Pro}- ect No. 119Y006). The authors would like to thank TU- BITAK for their financial support. References Akbulut M, Ustaoglu MR, Celik ES (2009) Freshwater and Brackish Water Malacostraca (Crustacea-Arthropoda) Fauna of Sinop and Samsun and Their Ecology. Journal of the Black Sea/Mediterranean Environment 15: 47-60. Albayrak E, Ozulu% O (2016) Danamandira Golu (Silivri-Istanbul) Bentik Makro Omurgasizlari. Turkish Journal of Aquatic Science 31: 51-58. https://doi.org/10.18864/TJAS201606 Bacescu M (1954) Fauna Repulicii Populare Romine, Crustacea 4(3). Academia Republicii Populare Romine, 126 pp. Balik S, UstaoZlu MR, Ozbek M, Tasdemir A, Yildiz S (2004) Buldan Baraj Golw’ niin (Denizli-Turkiye) Bentik Faunasi. Ege Universitesi Su Urunleri Dergisi 21: 139-141. Baytasoglu H, Gozler AM (2018) Seasonal Changes of Malacostraca (Crustacea) Fauna of the Upper Coruh River Basin (Bayburt Prov- ince, Turkey) and Its Ecological Characteristics. Turkish Journal of Fisheries and Aquatic Sciences 18(3): 367-375. https://doi. org/10.4194/1303-2712-v18_ 3 02 CoifmanI(1938)Nota Sul Potamon Edule, Dell’ anatolia. BollettinodiZoo- logia 17(1): 223-225. https://doi.org/10.1080/11250003809437002 Coleman CO (2003) “Digital inking”: How to make perfect line draw- ings on computers. Organisms Diversity & Evolution. Organisms, Diversity & Evolution 3(4, Supplement 14): 1-14. https://doi. org/10.1078/1439-6092-0008 1 Copilas-Ciocianu D, Grabowski M, Parvulescu L, Petrusek A (2014) Zoogeography of Epigean freshwater Amphipoda (Crustacea) in Romania: Fragmented distributions and wide altitudinal variability. Zootaxa 2: 243-260. https://doi.org/10.11646/zootaxa.3893.2.5 Copilas-Ciocianu D, Zimta AA, Grabowski M, Petrusek A (2018) Sur- vival in northern microrefugia in an endemic Carpathian gammarid (Crustacea: Amphipoda). Zoologica Scripta 47(3): 357-372. https:// doi.org/10.1111/zsc.12285 Costa FO, Henzler CM, Lunt DH, Whiteley NM, Ock J (2009) Prob- ing Marine Gammarus (Amphipoda) Taxonomy with DNA Bar- codes. Systematics and Biodiversity 7(4): 365-379. https://doi. org/10.1017/S 1477200009990 120 Ekinci M, Miroglu A (2016) Ordu Ili (Turkiye) Tatlisu Gammaridea (Crustacea, Amphipoda) Uzerine Arastirma. Ordu Universitesi Bilim ve Teknoloji Dergisi 6: 158-169. Felsenstein J (1981) Evolutionary trees from DNA sequences: A max- imum likelihood approach. Journal of Molecular Evolution 17(6): 368-376. https://doi.org/10.1007/BF01734359 Felsenstein J (1985) Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution; International Journal of Organic Evolution 39(4): 783-791. https://doi.org/10.2307/2408678 1003 Grabowski M, PeSic V (2007) New Data on the Distribution and Check- list of Fresh- and Brackishwater Gammaridae, Pontogammaridae and Behningiellidae (Amphipoda) in Bulgaria. Lauterbornia 59: 53-62. Hall TA (1999) BioEdit: A user—friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. Horton T, Lowry J, De Broyer C, Bellan-Santini D, Copilas-Ciocianu D, Corbari L, Costello MJ, Daneliya M, Dauvin J-C, Fiser C, Gasca R, Grabowski M, Guerra-Garcia JM, Hendrycks E, Hughes L, Jaume D, Jazdzewski K, Kim Y-H, King R, Krapp-Schickel T, LeCroy S, Lorz A-N, Mamos T, Senna AR, Serejo C, Souza-Filho JF, Tandberg AH, Thomas JD, Thurston M, Vader W, Vainola R, Valls Domedel G, Vonk R, White K, Zeidler W (2024) World Amphipoda Database. Senticaudata. World Register of Marine Species. https://www.ma- rinespecies.org/aphia.php?p=taxdetails&id=7 19424 [on 2024-03-12] Hou Z, Fu J, Li S (2007) A molecular phylogeny of the genus Gam- marus (Crustacea: Amphipoda) based on mitochondrial and nucle- ar gene sequences. Molecular Phylogenetics and Evolution 45(2): 596-611. https://do1.org/10.1016/j.ympev.2007.06.006 Hou Z, Sket B, Fiser C, Li S (2011) Eocene habitat shift from saline to freshwater promoted tethyan amphipod diversification. Proceedings of the National Academy of Sciences of the United States of America 108(35): 14533-14538. https://doi.org/10.1073/pnas. 1104636108 Hupato K, Karaouzas I, Mamos T, Grabowski M (2020) Molecular data suggest multiple origins and diversification times of freshwater gammarids on the Aegean archipelago. Scientific Reports 10(1): 19813. https://doi.org/10.1038/s41598-020-75802-2 Ipek M, Ozbek M (2022) An updated and annotated checklist of the Malacostraca (Crustacea) species inhabited Turkish inland waters. Turkish Journal of Zoology 46(1): 14-66. https://doi.org/10.3906/ Z00-2109-12 Karaman GS (2003) New Data on Some Gammaridean Amphipods (Amphipoda, Gammaridea) from Palearctic. (Contribution to the Knowledge of the Amphipoda 245). Glasnik Odjeljenja Prirodnih Nauka. Crnogorska Akademija Nauka Umjetnosti Podgorica 15: 21-37. Karaman GS, Pinkster S (1977) Freshwater Gammarus species from Eu- rope, North Africa and Adjacent Regions of Asia (Crustacea-A mphi- poda), Part | Gammarus pulex-Group and related species. Bijdragen tot de Dierkunde 47(1): 1-97. https://doi.org/10.1163/26660644- 04701001 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis Across Computing Plat- forms. Molecular Biology and Evolution 35(6): 1547-1549. https:// doi.org/10.1093/molbev/msy096 Ozbek M (2008) Malacostraca (Crustacea) Fauna of Some Lakes West- ern Black Sea Region. Ege Universitesi Su Urinleri Dergisi 25: 311-314. Ozbek M (2011) An Oveview of the Gammarus Fabricius (Gammari- dae: Amphipoda) Species of Turkey, With An Updated Checklist. Zoology in the Middle East 53(1): 71-78. https://doi.org/10.1080/0 9397140.2011.10648863 Ozbek M (2012) A new freshwater amphipod species, Gammarus obruki sp. nov., from Turkey (Amphipoda: Gammaridae). Turkish Journal of Zoology 36(5): 567-575. https://doi.org/10.3906/zoo-1112-2 Ozbek M, Ozkan N (2017) Gokceada i¢sularinin Amphipoda (Crusta- cea: Malacostraca) faunasi. Ege Journal of Fisheries and Aquatic Sciences 34: 63-67. https://doi.org/10.12714/egejfas.2017.34.1.09 zse.pensoft.net 1004 Ozbek M, UstaoZlu MR (1998) Amphipoda (Crustacea-Arthropoda) Fauna of Izmir and Adjacent Areas Inland-Waters. Ege Universitesi Su Urinleri Dergisi 15: 211-231. Ozbek M, UstaoZlu MR (2001) Izmir [li ve Civari Tatlisu Malacostra- ca (Crustacea) Faunasi (Amphipoda Haric¢). Anadolu Universitesi Bilim ve Teknik Dergisi 2: 19-25. Ozbek M, UstaoZlu MR (2005a) Taxonomical Investigation of Lake District Waters Malacostraca (Crustacea-Arthropoda) Fauna. Ege University Journal of Fisheries and Aquatic Sciences 22: 357-362. Ozbek M, Ustao8lu MR (2005b) Géller Bolgesi I¢sularinin Malacostra- ca (Crustacea-Arthropoda) Faunasinin Taksonomik Acidan Incelen- mesi. Ege Universitesi Su Urinleri Dergisi 22: 357-362. Ozbek M, Balik S, Ustao3lu MR, Berber S, Topkara ET (2004) Apoly- ont ve Iznik Golleri’nin Malacostraca (Crustacea) Faunas1. Anadolu Universitesi Bilim ve Teknoloji Dergisi 5: 291-295. Ozbek M, Balik S, Topkara E (2007) Turkiye Tatli Su Amphipod- larinin (Crustacea: Malacostraca) Dafilimlar1 ve Ekolojilerine Kat- kilar. Firat Universitesi Fen ve Muhendislik Bilimleri Dergisi 19: 455-461. Ozbek M, Yurga L, Kilkoylioglu O (2013) Gammarus baysali sp. nov., a new freshwater amphipod species from Turkey (Amphipoda: Gammaridae). Turkish Journal of Zoology 37(2): 163-171. https:// doi.org/10.3906/zoo0- 1209-14 Ozbek M, Baytasolu H, Aksu I (2023a) A new freshwater amphipod (Amphipoda, Gammaridae) from the Fakilli Cave, Duzce Turkiye: Gammarus kunti sp. nov. Zoosystematics and Evolution 99(2): 473- 487. https://doi.org/10.3897/zse.99. 108048 Ozbek M, Aksu I, Baytaso%lu H (2023b) A new freshwater amphipod (Amphipoda, Gammaridae), Gammarus tumaf sp. nov. from the Gokg6l Cave, Turkiye. Zoosystematics and Evolution 99(1): 15-27. https://do1.org/10.3897/zse.99.89957 Posada D (2008) jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25(7): 1253-1256. https://doi.org/10.1093/ molbev/msn083 Puillandre N, Brouille S, Achaz G (2020) ASAP: Assemble species by automatic partitioning. Molecular Ecology Resources 21(2): 609— 620. https://dot.org/10.1111/1755-0998. 13281 Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7. System- atic Biology 67(5): 901-904. https://do1.org/10.1093/sysbio/syy032 Rewicz T, Konopacka A, Bacela-Spychalska K, Ozbek M, Grabowski M (2016) First Records of Two Formerly Overlooked Ponto-Caspi- an Amphipods from Turkey, Echinogammarus trichiatus (Martynov, 1932) and Dikerogammarus villosus (Sovinsky, 1894). Turkish Jour- nal of Zoology 40: 328-335. https://doi.org/10.3906/zoo- 1505-31 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 zse.pensoft.net Baytasoglu, H. et al.: Gammarus sezgini sp. nov., a new freshwater amphipod from Turkiye Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolu- tion 4(4): 406-425. https://doi.org/10.1093/oxfordjournals.molbev. a040454 Sari HM, Balik S, Ozbek M, Aygen C (2001) Bafa Golw’niin Makro ve Meiobentik Omurgasiz Faunasi. Anadolu Universitesi Bilim ve Teknoloji Dergisi 2: 285-291. Selim S (2011) Akarsu Vadisindeki Insan Kaynakli Faaliyetlerin Eko- sistem Biuttnligtine Olasi Etkileri: CaSlayan Ornegi. Stileyman Demirel Universitesi Fen Bilimleri Enstittist Dergisi 15: 94-101. https://doi.org/10.19113/sdufbed.29642 Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10(3): 512-526. https://doi.org/10.1093/oxfordjournals.molbev. a040023 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improv- ing The Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice. Nucleic Acids Research 22(22): 4673-4680. https://doi.org/10.1093/nar/22.22.4673 Vavra (1905) Rotatorien Und Crustaceen. Annalen des K. K. Naturhis- torichen Hofmuseums in Wien 20: 106-113. Zamanpoore M, Grabowski M, Poeckl M, Schiemer F (2011) Taxo- nomic review of freshwater Gammarus (Crustacea: Amphipoda) from Iran. Zootaxa 3140(1): 1-14. https://Doi.Org/10.11646/zoot- axa.3140.1.1. https://doi.org/10.11646/zootaxa.3140.1.1 Zhang J, Kapli P, Pavlidis P, Stamatakis A (2013) A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29(22): 2869-2876. https://doi.org/10.1093/bioin- formatics/btt499 Supplementary material | The pairwise genetic distance values amongst the Gammarus species, based on the COI datasetand 28S dataset Authors: Hazel Baytasolu, Ismail Aksu, Murat Ozbek Data type: xlsx 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://doi.org/10.3897/zse.100.121692.suppl1