Zoosyst. Evol. 99 (1) 2023, 15-27 | DOI! 10.3897/zse.99.89957 ee BERLIN A new freshwater amphipod (Amphipoda, Gammaridae), Gammarus tumaf sp. nov. from the Gékgél Cave, Tiirkiye Murat Ozbek!, Ismail Aksu2, Hazel Baytasoélu? 1 Department of Hydrobiology, Faculty of Fisheries, Ege University, TR-35100 Bornova, Izmir, Turkey 2 Recep Tayyip Erdogan University, Faculty of Fisheries and Aquatic Sciences, 53100 Rize, Turkey https://zoobank. org/96DES 1C2-55DD-4E90-8186-ADD75AD2F8D9 Corresponding author: Murat Ozbek (ozbekm71@gmail.com) Academic editor: Luiz F. Andrade # Received 5 July 2022 Accepted 8 October 2022 Published 6 January 2023 Abstract A new amphipod species belonging to the genus Gammarus 1s described from the Gokg6l Cave, Zonguldak Province, Tiirkiye. The newly-identified species is relatively small (13 mm) and is a member of the Gammarus pulex-group by the presence of numerous long setae along the posterior margins of pereopods 3 and 4. The specimens were sampled from a shallow pond located in the dark zone (about | km inside the entrance) of the cave. Minute eyes, setose (both peduncle and flagellar segments) second antenna, slight- ly swollen flagellar segments of the second antenna, setose pereopods 3 and 4 and relatively short endopod/exopod ratio of the third uropod are the character combination of the newly-identified species in addition to lacking body pigmentation. The molecular phy- logeny, based on the concatenated dataset (28S+COI, 1495 bp) indicated that the new species was resolved from the other Gammarus species by high bootstrap (NJ: 100, ML: 100). In addition to Gammarus tumaf sp. nov., mtDNA COI and nuclear DNA 28S gene data of Gammarus baysali Ozbek et al., 2013 were recorded for the first time. The newly-identified species was well-differentiated from the genetically closest species, G. baysali, with genetic distance of 12.22% and 0.55% for the COI and 28S genes, respectively. Detailed descriptions and drawings of the extremities of the holotype male were given and the morphology of the newly-identified species 1s compared with its relatives. Key Words benthos, cave, identification key, invertebrate, molecular identification, new species Introduction Gammarus Fabricius, 1775 is one of the richest genera of the Gammaridae Leach, 1814 family with more than 225 species worldwide (Vainola et al. 2008). The members of the genus are widely distributed in the Palearctic and Hol- arctic Regions. They inhabit both epigean (seas, lakes and streams) and hypogean (caves, wells and groundwater) habitats (Karaman and Pinkster 1977). The first study on the Gammarus genus in Turkish inland waters started with the identification of Gammarus argaeus Vavra, 1905 from Ercityes Mountain by Vavra (1905). In the last two decades, many studies have been reported by both foreign and native researchers, increasing compared to previous years. As a general result of these studies, knowledge about the distribution of the Gammarus species in Turkish inland waters has increased. In addition, new Gammarus species, most of which are endemic, have been identified from both ground and surface waters. To date, 51 Gammarus taxa have been recorded from the inland waters of Turkiye and 30 of them are endemic to the country (Ipek and Ozbek 2022). DNA barcoding, one of the molecular techniques devel- oped in recent years, has brought an integrative approach by contributing to species identification, based on tradi- tional taxonomy (Dayrat 2005). DNA barcoding studies, first introduced by Arnot et al. (1993) and later gained pop- ularity with the work of Hebert et al. (2003), have allowed speedy, reliable and cost-effective species identification, based on specific nucleotide sequences on the genome. 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. 16 Some mitochondrial (such as COI, Cytb, 16S, 12S) and nuclear DNA (suchas 18S, 28S, ITS, EF1-alpha) genes are powerful markers for deducing evolutionary relationships at the species, genera, family and higher levels (Johns and Avise 1998; Englisch and Koenemann 2001; Cristescu and Hebert 2005; Kartavtsev and Lee 2006; Witt et al. 2006; Hou et al. 2007, 2011; Costa et al. 2009; Hupato et al. 2020; Morhun et al. 2022). Studies to date have confirmed that molecular techniques are a powerful tool for the discov- ery of cryptic species, as well as revealing speciation and population diversity within the genus Gammarus (Meyran et al. 1997; Meyran and Taberlet 1998; Muller 2000; Hou et al. 2007; Lagrue et al. 2014; Mamos et al. 2014; Weiss et al. 2014; Wysocka et al. 2014; Copilas-Ciocianu et al. 2018; Copilas-Ciocianu et al. 2019). This study aims to examine the individuals collected from Gokgol Cave, Zonguldak Province, Turkiye in terms of morphological and molecular features. Additionally, the molecular analysis of Gammarus baysali Ozbek et al., 2013, which was reported from another cave (Cumayan1 Cave) located in a geographically close location, was also carried out. Detailed descriptions and drawings of the extremities of the holotype male are given and the morphology of the newly-identified species is compared with its relatives. Materials and methods Gokgol Cave is located on the road around Uztilmez Re- gion at the 4" km of Ankara highway, to the southeast of the city, in Ercek Village, NW Anatolia, Turkiye and is an active cave with a length of 3,350 m (Fig. 1) (Yamac et al. 2021). The deepest point from the cave entrance is -5 m and the highest point is +35 m. From the entrance to the 830" metre of the cave, it is open to touristic activities. The main branch of the Cave lies down in the east-west direction and the Cave has four lateral branches extending in the north-south direction. Two of them extend in the north direction and the other ones in the south direction. The waters coming from the siphon, which is located at the end of the main branch, continue to flow from the first branch that develops in the south direction (Fig. la, b). Water flow is present throughout the year in the Cave. Additionally, a water inflow in the form of leakage from the cracks in the ceiling and walls of the Cave is observed. Alive amphipod specimens were photographed and sampled with the help of a hand aspirator for the taxo- nomical investigation (Fig. Ic). To measure the body length, individuals were straight- ened with forceps under a stereomicroscope and the distance between the rostrum and the base of the telson was measured. Permanent slides of the holotype individual were pre- pared using the high-viscosity mount, CMCP-10. Photo- graphs of the extremities were taken with a digital camera connected to a microscope (Olympus CX41). Photos were processed with image processing programmes and a stan- dart pen. A digitiser board (Wacom PTH-451) and stan- zse.pensoft.net Ozbek, M. et al.: Gammarus tumaf sp. nov. from the Gdkgél Cave, Turkiye dard pen connected to a PC were used for detailed draw- ings of the extremities. Scaled drawings of the extremities were made on the photographs (Coleman 2003). Some of the collected samples are kept in Eskisehir Technical Uni- versity Zoology Museum and some others in the Museum of the Faculty of Fisheries, Ege University (ESFM). DNA extraction, PCR amplification and sequencing Genomic DNA of Gammarus specimens was extract- ed on the Qiacube Automated DNA Isolation Device (Qiagen, Valencia, CA) according to the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) protocol. Mi- tochondrial cytochrome c oxidase subunit I gene (COI) and the nuclear large subunit ribosomal RNA gene (28S) were amplified from the extracted DNA. Amplification of the COI marker was performed with the primers UCOIF (5’- TAWACTTCDGGRTGRCCRAAAAAYCA-3’) and UCOIR (5’- ACWAAYCAYAAAGAYATYGG-3’) ac- cording to the PCR protocol of Costa et al. (2009). Ampli- fication of the 28S marker was performed with the primers 28F (5’- TTAGTAGGGGCGACCGAACAGGGAT-3’) and 28R (5’- GTCTTCGCCCCTATGCCCAACTGA-3’) according to the PCR protocol of Hou et al. (2007). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and one-directional sequencing of PCR products was performed with an ABI PRISM 37301 Genetic Analyser using a BigDye Terminator 3.1 cycle sequencing ready reaction kit (Applied Biosystem) at Macrogen Europe according to the Sanger method. Molecular data analyses In the present study, a total of one individual of Gammarus tumaf sp. nov. and one of Gammarus baysali Ozbek et al., 2013 were sequenced. In addition, previously-published sequences of 12 species as in-groups and one species as an outgroup from the GenBank (http://www.ncbi.nih. gov) containing both the COI and 28S sequences were downloaded for use in molecular analyses. GenBank accession numbers, locations and reference information of the sequences used in molecular analyses are given in Table 1. The raw COI and 28S sequences generated in the pres- ent study were initially edited by checking their chro- matograms in the Bioedit 7.2.5 programme (Hall 1999). To perform the molecular analyses, the COI and 28S sequences, both newly generated and downloaded from GenBank, were added end-to-end to obtain a concatenated dataset (28S+COI). 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 inter-specific pairwise genetic distances for both markers were calculated separately, according to the uncorrected p-distance in MEGA X software (Kumar et al. 2018). Phylogenetic relationships amongst the Zoosyst. Evol. 99 (1) 2023, 15-27 17 BLACK istanbul Gékg6él Cave Ankara - aN = Antalya ff MEDITERRANEAN SEA 28° 30° 328 Trabzon we ey ‘ Erzurum ee ae Diyarbakir Figure 1. Inside the Gékgél Cave (a); type locality of Gammarus tumaf sp. nov. (b); photo of an alive specimen (c); habitus of the holotype male (d) and the geographical location of the Gdkg6l Cave (Cave photos: M. Elverici). Gammarus species were estimated by using Neighbour- Joining (NJ) and Maximum Likelihood (ML) methods in MEGA X software. The NJ tree was generated according to the p-distance model. The ML tree was generated according to the GTR+G+I model (Tavaré 1986) and the best-fit substitution model was selected with the lowest Akaike Information Criterion (AIC) score in jModelTest 0.1.1 (Posada 2008). Confidences of the NJ and ML analyses were estimated by the bootstrap test (Felsenstein 1985) using 1000 replicates. zse.pensoft.net 18 Results Gammarus tumaf sp. nov. https://zoobank. org/654377E5-2984-4 189-B 1 EF-7D7346A4 14EF Figs 1-5 Holotype. Male, 12.6 mm (ESFM-MALI/20-10), Zongul- dak Province, Turkiye (41°26'26.42"N, 31°49'57.48"E), 03.1x.2020; collected by M. Elverici. Paratypes. 3 males and 3 females, (ESFM-MA- LI/20-11), same data as holotype. Diagnosis. A medium-large species with a smooth body, lacking body pigmentation, minute eyes, setose (both peduncle and flagellar segments) second antenna, slightly swollen flagellar segments (second antenna), se- tose pereopods 3 and 4 and relatively short endopod/exo- pod ratio of the third uropod. Description of holotype male. Head: Rostrum ab- sent, inferior antennal sinus deep, rounded. Eyes small, ovoid; shorter than the diameter of the first peduncular segment of antenna | (Fig. 1d). Antennae: Antenna | is longer than half of the body length; the length ratio of the peduncular segments is 1:0.7:0.5; 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 30 segments; each segment bears a few short setae in distal side; aesthetasc absent; accessory flagellum 5 seg- mented (Fig. 3D). Antenna 2 is shorter than antenna 1 (ra- tio 1:0.56); the antennal gland cone is straight and short; 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 longer than the dorsal ones and can be up to 1.5 times lon- ger than the diameter of the segment; flagellum consists of 13 segments; flagellar segments are setose and swol- len; each segment bears many long setae on both dorsal and ventral sides; calceoli absent (Fig. 3A). Mouthparts: Left mandible (Fig. 2H) with 5-toothed incisor, lacinia mobilis with 4 dentitions, molar tritura- tive. The first article of palp without setae, the second one bears 13 setae; the setae become shorter from distal to proximal. The third segment has 28 D-setae, 4—5 E-setae, one group of A- and one group of B-setae. C-setae absent. Right mandible (Fig. 21) has a 3-toothed incisor and bifurcate lacinia mobilis. Right maxilla 1 (Fig. 2D) is asymmetric to the left, it has 20 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 five 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 5 simple setae on its distal part and no setae along the outer margin. Lower lip (Fig. 2B) has no inner lobe and bears numerous small simple setae along the distal margins of both lobes. zse.pensoft.net Ozbek, M. et al.: Gammarus tumaf sp. nov. from the Gdkgél Cave, Turkiye Upper lip (Fig. 2A) with numerous minute setules in the distal part. Maxilla 2 (Fig. 2F, G) has 20—25 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 8—10 simple setae in the distal part in addition to 20 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 6 plumose setae along the inner margin of the lobe. Outer plate armed with 4-5 serrate stout setae in the distal part and 13 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 addi- tion to some tiny setules along the ventral margin (Fig. 3B). Coxal plate 2 is in the shape of an elongated rectangle, dis- tal part narrower than the proximal, the ventral margin is highly convex and setation is similar to that of coxal plate 1 (Fig. 3C). Coxal plate 3 is similar tn shape and setation to coxal plate 2, with less narrowing in the distal part (Fig. 4A). The ventral edge of the fourth coxal plate is almost straight and bears 4 and 7 setae along the anteroventral and posterior margins, respectively (Fig. 4B). Coxal plates 5 and 6 bilo- bate, each one having one seta in the anterior and 4 setae in the posterior lobes (Fig. 4D, E). Coxal plate 7 with 34 setae on the posteroventral margin (Fig. 4C). Gnathopods: Basal segment of gnathopod 1 bears many long setae along both margins, the length of the se- tae can be more than twice the diameter of the segment. Ischium bears a group of setae in posteroventral corner. Posterior margin of the merus with 4 groups of setae. Carpus 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: 0.34, anterior margin with three groups of setae, medial palmar spine is present, posterodistal cor- ner armed with a strong spine in addition to some small spines, posterior margin bears 4—5 groups of setae. Dac- tylus reaches the posterodistal 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. 3B, B’). Basis and ischium of gnathopod 2 have a similar seta- tion to that of gnathopod 1. Merus and carpus are more setose than those of gnathopod 1. Carpus triangular, densely setose along the posterior margin in addition to three groups of setae along the anterior margin. Propo- dus is densely setose and has a sub-rectangular shape, the length/width ratio is 1: 0.55, anterior margin bears 4 groups of setae, some setae have curled distal tips, pos- terior margin with many groups of setae, medial palmar spine is present, posterodistal corner armed with three strong spines in addition to some small spines. Dacty- lus reaches the posterodistal 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. 3C, C’). Zoosyst. Evol. 99 (1) 2023, 15-27 19 / @ Figure 2. Mouth parts of Gammarus tumaf sp. nov., (holotype male). A. Upper lip; B. Lower lip; C. Left maxilla 1; D. Right maxilla 1; E. Maxilliped; F. Left maxilla 2; G. Right maxilla 2; H. Left mandible; I. Right mandible. zse.pensoft.net nov. from the Gokgol Cave, Turkiye Ozbek, M. et al.: Gammarus tumaf sp. Sr or }—§{}— ae Wo Se oS —> a D5 ae A __| ae eee aI | tS VS a: A Ss = (f => = Jo as D a een | 1» eo a oe Oe ee ees Ge ee, 1A \_-# ne - \\ \ \W i L a Pa z = ff — Ps Cr y ty — S irene a ———— false | \\ . y —— ne SS Wy | ay a Ne ——= on () Lhe WS . SUVS \ NN aN Ete Za. oa , . —— > UU ss, > g ‘ i 1 i / ™ = = aa. n ’ z H t Ja. H ¢ i / \ =a \ 'G / / IN SS Se = SS XQ é / / Sa phe Wi ; —== Seen i; Ped Pd ~ ” S i XN \ \ ret Pra = z= v., (holotype male). A. Antenna 2; B. Gnathopod 1; B’. Palm of gnathopod 1; tumaf sp. no C. Gnathopod 2; C’. Palm of gnathopod 2; D. Antenna 1. et Figure 3. Extremities of Gammarus oft.n zse.pens Zoosyst. Evol. 99 (1) 2023, 15-27 Pereopods: Anterior and posterior margins of the pereopod 3 bear 4—6 groups of setae, the setae along the posterior margin are much longer than those in the anteri- or margin and posterior margins of the merus, carpus and propodus bear long and slightly curved setae, the setae can be three times longer than the diameter of the seg- ment 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 IN 2 A Lee = = SSE CLES SS | = 21 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 set- ules (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; there is a spine in the posteroventral 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 — >, Ye: - —— Sy yee WS eee, = SS / Figure 4. Extremities of Gammarus tumaf sp. nov., (holotype male). A. Pereopod 3; B. Pereopod 4; C. Pereopod 7; D. Pereopod 6; E. Pereopod 5. zse.pensoft.net 22 spines along with the mentioned segments. Propodus of pereopod 5 to 7 with 4-5 groups of long setae groups along their outer margins in addition to 5—6 groups of small spines along their inner margins. Setae on the out- er margins of the propodus of pereopod 7 are shorter than those in pereopod 5 and 6. Dactylus slim, a minute plumose seta occurs on the outer margin; the inner margin with two small setules (Fig. 4C—E). Epimeral plates: They are neither curved nor sharply pointed. Epimeral plate 1 bears 5—6 long setae in addition to a few setules along the anterior margin and posteri- or margin with 6—7 tiny setae. The posteroventral corner is angular (Fig. 5E). Epimeral plate 2 bears 3-4 setae in the anteroventral corner, the ventral margin is armed with 3 spines and the posterior margin with 4—5 setules. The posteroventral corner is angular (Fig. 5F). Epimeral plate 3 is slightly pointed; the anteroventral corner bears 6—7 setae; the ventral margin is armed with 4 spines; the pos- terior margin bears 6—7 setules (Fig. 5G). 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 distoventral comer of the base; the peduncle is longer than rami; the length ratio is about 1:0.75. Peduncle with a spine in the outer margin of the proximal part in addition to 6 spines along the inner margin and 2 spines in the distal part. Both rami are of equal size and bear 4—5 spines along their in- ferior margins in addition to 4—5 distal spines (Fig. 5D). Uropod 2 is smaller than the first one; the length ratio is about 1:07; the peduncle segment is longer than the rami and bears 4 spines along the inner margin and the outer margin is bare. The length and armaments of both rami are similar to each other, they bear 2—3 spines along their inner and outer margins in addition to 4-5 longer spines on their distal tips (Fig. 5C). Uropod 3 is setose and bears simple and plumose se- tae. The peduncle segment is much shorter than the outer ramus and the length ratio is about 1:0.38. The outer ra- mus has two articulated and densely setose along both margins; the outer bears 4 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 ra- mus 1s about 0.75 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. 5B). 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 some setae on the dorsal surface of the lobes in addition to two plumose setules. The length/width ratio of each is about 1:0.5 (Fig. 5A). Etymology. The species epithet (tumaf) 1s the abbrevi- ation of the Turkish Caving Federation. Description of females. Smaller than males. Except for the sexual dimorphism indicated for the genus zse.pensoft.net Ozbek, M. et al.: Gammarus tumaf sp. nov. from the Gdkgél Cave, Turkiye Gammarus, females do not show obvious differences from males. Setation and armaments of the extremities are more or less similar to those of males. Variability. In some individuals, the size of the eyes is slightly smaller than in the holotype. The number of flagellar segments in Antenna | and Antenna 2 can be variable. The number of flagellar segments of Antenna 1 in paratype individuals ranged from 32 to 37. Similarly, the number of flagellar segments of Antenna 2 varied be- tween 11 and 14. Results of molecular data analyses We produced the partial sequences of the COI and 28S genes of Gammarus tumaf sp. nov. and Gammarus baysali Ozbek et al., 2013 (from Cumayani Cave) and performed molecular analyses, based on concatenated data. A concatenated dataset with a total length of 1495 bp (for the COI fragment 573 bp and for the 28S fragment 922 bp including gaps) were sequenced. While no stop codon, insertion, deletion and a gap were detected in the protein-coding mtDNA COI gene, there are insertions and deletions in the nuclear 28S gene. Additionally, newly-generated sequences are deposited in GenBank accession numbers, for COI ON749780—ON749781 and 28S ON751931—ON751932. We performed phylogenetic and genetic distance anal- yses with the topotype sample sequences of the nominal taxa that we could find especially in GenBank. Other- wise, sequences considered representative of the species were preferred (Table 1). For the COI gene, the genetic distance amongst the species ranged from a minimum of 12.22% (Gammarus tumaf sp. nov. — G. baysali) to a maximum of 16.00% (G. kesslerianus — G. plaitisi). The next minimum genetic distance value is 16.06% (G. uludagi — G. plaitisi). For the 28S gene, it ranged from a minimum of 0.55% (Gammarus tumaf sp. nov. — G. baysali) to a maximum of 7.38% (G. roeselii — G. balcanicus Kolasin Montene- gro). The next minimum genetic distance value is 0.66% (G. pulex — G. plaitisi) (Table 2). According to phylogenetic results, NJ and ML methods provided similar topologies for the Gammarus species. Many species lineages were supported by high bootstrap values. Gammarus tumaf sp. nov. is closely related to G. baysali in phylogenetic trees, but differs from it (Fig. 6). Discussion Gammarus tumaf sp. nov. belongs to the Gammarus pulex-group because of the presence of long setae along the posterior margins of the merus and carpus of pereopods 3 and 4 (Fig. 4A, B). The newly-identified species shows some common characteristics seen in many other members of the group. Gammarus tumaf sp. nov. is very similar to Gammarus komareki Schaferna Zoosyst. Evol. 99 (1) 2023, 15-27 23 | i xe aa JE SS = SES ~~ “SAREE N SSS x = <—S . ————— = -— Figure 5. Extremities of Gammarus tumaf sp. nov., (holotype male). A. Telson; B. Uropod 3; C. Uropod 2; D. Uropod 1; E. Epu- meral plate 1; F. Epimeral plate 2; G. Epimeral plate 3; H. Urosomites. zse.pensoft.net 24 Table 1. List of samples used in molecular analysis. Species Gammarus tumaf sp. nov. (T) baysali (T) balcanicus (T) balcanicus fossarum (T) kesslerianus (T) kischineffensis (T) Locality Gokgol Cave, Turkiye Cumayan! Cave, Turkiye Kolasin, Montenegro Virpazar, Montenegro Regensburg, Germany Simferopol, Crimea, Ukraine Targu Bujor, Romania Mazandaran, Iran G. G. G. G. G. G. G. komareki G. G. G: G. G. G. komareki (T) ca 200km SE Sofia, Bulgaria lacustris Bled, Slovenia plaitisi Tinos, Komi, Greece pulex (T) Slovenia roeselii Netherlands uludagi Evia, Greece Ozbek, M. et al.: Gammarus tumaf sp. nov. from the Gdkgél Cave, Turkiye 28S Col References ON751931 ON749780 This study ON751932 ON749781 This study JF965640 JF965834 Hou et al. (2011) JF965654 JF965848 Hou et al. (2011) JF965696 JF965886 Hou et al. (2011) JF965721 JF965909 Hou et al. (2011) MG987529 MG987571 Copilas-Ciocianu et al. (2018) JF965723 JF965911 Hou et al. (2011) JF965725 JF965913 Hou et al. (2011) JF965728 JF965915 Hou et al. (2011) MT999102 MT999049 Hupato et al. (2020) JF965767 JF965943 Hou et al. (2011) JF965771 JF965947 Hou et al. (2011) JF965817 JF965986 Hou et al. (2011) JF965822 JF965990 Hou et al. (2011) Pontogammarus robustoides Delta Volgi, Russia Note: (T) Topotype samples of nominal taxa. 1922, which is distributed over a large geographical area including the Balkans, Black Sea coasts, northern parts of Turkiye and Iran (Karaman and Pinkster 1977; Zamanpoore et al. 2011; Ipek and Ozbek 2022). The characteristic densely setose peduncular and flagellar segments of antenna 2 and many other morphological features of the newly-identified species suggest that it can be closely related to G. komareki. Molecular analysis results also support this assumption. On the other hand, it differs from G. komareki by the following features: a. having fewer setae on the first segment of the man- dible palp, b. having fewer D-setae on the mandible palp. c. absence of long setae along the anterior margins of pereopods 5-7, d. having a shorter inner lobe of uropod 3. Karaman and Pinkster (1977) state that the setation of the anterior margins of pereopods 5 to 7 is a variable fea- ture, even in some populations there are no setae along the anterior margins of pereopods 5—7 in some populations. We included two G. komareki reference sequences in molecular analyses. The two most distant tndivid- uals constituting the geographical distribution line of G. komareki species are Sofia, Bulgaria (topotype sam- ple) and Mazandaran, Iran samples. The newly-identified species was resolved in the phylogenetic tree with high support values (NJ: 95-100, ML: 95-100) from these two G. komareki samples. Gammarus tumaf sp. nov. differs from both samples with pairwise genetic distance values of 18.15% and 22.34% for the COI gene and 1.75% and 1.75% for the 28S gene, respectively. Gammarus tumaf sp. nov. also shows morphological similarities with Gammarus baysali which was reported from another geographically close cave. However, G. baysali has 4 setae along the outer margin of the palp of the right maxilla in addition to 6 blunt distal teeth, whereas the present species has 2 setae and 5 distal teeth (Ozbek et al. 2013). zse.pensoft.net In this study, in addition to Gammarus tumaf sp. nov., mtDNA COI and nuclear DNA 28S gene data of Gammarus baysali Ozbek et al., 2013 were recorded for the first time. It is important to create genetic records from type specimens to avoid confusion that may occur in species identification. The molecular phylogeny, based on the concatenated dataset (28S+COI, 1495 bp) indicated that the new species was resolved from Gammarus baysali species by high bootstrap (NJ: 100, ML: 100). Additionally, the newly-identified species are well differentiated from the genetically closest species, G. baysali, with a genetic distance of 12.22% and 0.55% for the COI and 28S genes, respectively. Gammarus tumaf sp. nov. 1s similar to G. kesslerianus by having setose antenna 2, but differs from it by having a shorter inner lobe of uropod 3. Gammarus tumaf sp. nov. and G. kesslerianus resolved in the phylogenetic tree with relatively strong bootstrap values (NJ:76, ML:65). The genetic distance between the two species is 17.45% and 1.42% for the COI and 28S genes, respectively. Phylogenetic analysis placed G. kes- Slerianus as a sister branch to G. tumaf and G. baysali species (Fig. 6). Gammarus tumaf sp. nov. 1s similar to Gammarus microps Pinkster & Goedmakers, 1975 by having min- ute eyes, but differs from it by having shorter extrem1- ties, fewer flagellar segments of antennae | and 2, less setose pereopod 4 and more setose carpus of pereopods 5 to 7 (Karaman and Pinkster 1977). Genetic compar- ison could not be made because molecular data were not available. The newly-identified species differs from Gammarus pulex pulex (L., 1758) by having more setae on the peduncle segments of antenna 2 and from Gammarus pulex polonensis Karaman & Pinkster, 1977 by having minute eyes. Gammarus tumaf sp. nov., morphologically described in the G. pulex-group, differs from the topotype sample of G. pulex with its high genetic distance (for COI: 26.18% and for 28S: 4.51%) and moderately-supported bootstrap values (NJ:74, ML:80). Zoosyst. Evol. 99 (1) 2023, 15-27 20 ON751931+ON749780 Gammarus tumaf n. sp. (Gokgol Cave Turkiye) ON751932+ON749781 Gammarus baysali (Cumayani Cave Turkiye) JF965721+JF965909 Gammarus kesslerianus (Simferopol Crimea Ukraine) JF965725+JF965913 Gammarus komareki (SE Sofia Bulgaria) JF965723+JF965911 Gammarus komareki (Mazandaran Iran) JF965771+JF965947 Gammarus roeselii (Netherlands) JF965696+JF965886 Gammarus fossarum (Regensburg Germany) JF965767+JF965943 Gammarus pulex (Slovenia) JF965817+JF965986 Gammarus uludagi (Evia Greece) MT999102+M1T999049 Gammarus plaitisi (Tinos Komi Greece) JF965640+JF965834 Gammarus balcanicus (Kolasin Montenegro) JF965654+JF965848 Gammarus balcanicus (Virpazar Montenegro) JF965722+JF965910 Gammarus kischineffensis (Harkiv Ukraine) JF965728+JF965915 Gammarus lacustris (Bled Slovenia) JF965822+JF965990 Pontogammarus robustoides (Delta Volgi Russia) 100 / 100 78 / 70 94/95 100 / 100 62/70 49 / 40 35/55 100 / 100 84/80 99 / 100 63 / 65 / 100 / 100 — 0.050 Figure 6. 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. The pairwise genetic distance values amongst the Gammarus species, based on the COI dataset (below the diagonal) and 28S dataset (above the diagonal). Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 Gammarus tumaf sp. nov. 0.0055 0.0142 0.0175 0.0175 0.0551 0.0464 0.0451 0.0441 0.0495 0.0670 0.0664 0.0638 0.0540 2. G. baysali 0.1222 0.0154 0.0165 0.0187 0.0530 0.0454 0.0442 0.0431 0.0463 0.0650 0.0622 0.0618 0.0531 3 G. kesslerianus 0.1745 0.1902 0.0142 0.0098 0.0518 0.0408 0.0418 0.0407 0.0440 0.0615 0.0586 0.0583 0.0474 4G. komareki (SE Sofia 0.1815 0.1728 0.1850 0.0197 0.0529 0.0442 0.0396 0.0386 0.0440 0.0593 0.0620 0.0540 0.0453 Bulgaria) 5 G. komareki (Mazandaran Iran) 0.2234 0.2094 0.2216 0.2112 0.0485 0.0353 0.0341 0.0330 0.0385 0.0582 0.0586 0.0550 0.0496 6 G. roeselii 0.2705 0.2810 0.2705 0.2688 0.2740 0.0475 0.0540 0.0518 0.0551 0.0738 0.0721 0.0673 0.0586 7 G. fossarum 0.2496 0.2653 0.2426 0.2321 0.2618 0.2391 0.0364 0.0353 0.0397 0.0695 0.0633 0.0631 0.0643 8 G. pulex 0.2618 0.2723 0.2792 0.2513 0.2583 0.2356 0.2304 0.0066 0.0099 0.0649 0.0653 0.0595 0.0552 9 G. plaitisi 0.2653 0.2653 0.2862 0.2653 0.2688 0.2618 0.2461 0.1745 0.0088 0.0639 0.0654 0.0585 0.0542 10 G. uludagi 0.2670 0.2757 0.2775 0.2513 0.2600 0.2391 0.2461 0.1640 0.1606 0.0681 0.0686 0.0628 0.0585 11 G. balcanicus (Kolasin 0.2635 0.2548 0.2513 0.2548 0.2461 0.2443 0.2443 0.2164 0.2513 0.2304 0.0365 0.0220 0.0441 Montenegro) 12 G. balcanicus (Virpazar 0.2548 0.2565 0.2653 0.2548 0.2757 0.2408 0.2461 0.2112 0.2304 0.2356 0.2042 0.0365 0.0576 Montenegro) 13 G. kischineffensis 0.2531 0.2478 0.2408 0.2391 0.2426 0.2461 0.2147 0.2059 0.2321 0.2234 0.1815 0.1955 0.0364. 14 G. lacustris 0.2356 0.2269 0.2513 0.2304 0.2443 0.2234 0.2182 0.2094 0.2129 0.2164 0.2234 0.2007 0.1745 Gammarus tumaf sp. nov. differs from Gammarus uludagi Karaman, 1975 by the shape and armaments of telson in addition to the absence of long and curled setae on the palm of gnathopod 2. Similarly, the newly- identified species differs from Gammarus obruki Ozbek, 2012 by having smaller eyes, shorter antenna 1 and more setose peduncular segments of antenna 2 (Ozbek 2012). No molecular data were available for the G. obruki species. Gammarus tumaf sp. nov. is located far from the G. uludagi in the phylogenetic tree. They were resolved with moderately-supported bootstrap values (NJ:74, ML:80) from the common ancestor from which they originated. The genetic distance between the two taxa is also quite high (for COI: 26.70% and for 28S: 4.95%). Although the phylogeny of Gammarus 1s still not fully resolved, the species on the common branch from which the new species originated were well resolved with high bootstrap values and indicating that the new species is an independent branch. The newly-identified species is well supported by molecular data. In this study, morphological and molecular data which we handled with an integra- tive approach, strongly supported the taxonomic status of Gammarus tumaf as a new species. All of the studies on the taxonomy of amphipods that inhabited the inland waters of Turkiye so far have been based on the examination of morphological features only. Although these studies have contributed to species identi- fication or new species identification, the phylogenetic re- lationships between species are still not fully understood. This is the first study in which both morphological and molecular analyses have been used to define a new am- phipod species from the freshwaters of Turkiye. zse.pensoft.net 26 Taxonomic studies supported by molecular and DNA analyses help to understand the relationships of species. On the other hand, making detailed morphological defi- nitions also help other studies (ecological, taxonomic, population dynamics etc.), especially in accurate species determination. The authors agree that taxonomic studies taking into account both molecular analyses and detailed morphological features would be more beneficial. Acknowledgements The authors would like to thank Mert Elverici and Kadir Buga¢ 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 KulaksizoSlu (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). The samples studied in the present study were col- lected during the “Research Project for Some Caves in the Western and Eastern Black Sea Regions and Central Anato- lia Region” carried out within the scope of the Turkish Min- istry of Environment, Urbanisation and Climate Change. References Arnot DE, Roper C, Bayoumi RAL (1993) Digital codes from hyper- variable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates. Molecular and Biochemical Parasitology 61(1): 15—24. https://doi. org/10.1016/0166-685 1(93)90154-P Coleman CO (2003) “Digital inking”: How to make perfect line draw- ings on computers. Organisms, Diversity & Evolution 3(4): 303- 304. https://doi.org/10.1078/1439-6092-0008 1 Copilas-Ciocianu D, Zimta AA, Grabowski M, Petrusek A (2018) Survival in northern microrefugia in an endemic Carpathian gammarid (Crustacea: Amphipoda). Zoologica Scripta 47(3): 357-372. https:// doi.org/10.1111/zse.12285 Copilas-Ciocianu D, Zimta AA, Petrusek A (2019) Integrative taxono- my reveals a new Gammarus species (Crustacea, Amphipoda) sur- viving in a previously unknown southeast European glacial refugi- um. Journal of Zoological Systematics and Evolutionary Research 57(2): 272-297. https://doi.org/10.1111/jzs.12248 Costa F, Henzler CM, Lunt DH, Whiteley NM, Rock J (2009) Probing Marine Gammarus (Amphipoda) Taxonomy with DNA Barcodes. Systematics and Biodiversity 7(4): 365-379. https://doi.org/10.1017/ $1477200009990120 Cristescu ME, Hebert PD (2005) The” Crustacean Seas” an evolutionary perspective on the Ponto Caspian peracarids. Canadian Journal of Fisheries and Aquatic Sciences 62(3): 505-517. https://dot. org/10.1139/f04-210 zse.pensoft.net Ozbek, M. et al.: Gammarus tumaf sp. nov. from the Gdkgél Cave, Turkiye Dayrat B (2005) Towards integrative taxonomy. Biological Journal of the Linnean Society. Linnean Society of London 85(3): 407-415. https://doi.org/10.1111/).1095-83 12.2005.00503.x Englisch U, Koenemann §S (2001) Preliminary phylogenetic analysis of selected subterranean amphipod crustaceans, using small sub- unit rDNA gene sequences. Organisms, Diversity & Evolution 1(2): 139-145. https://doi.org/10.1078/1439-6092-00011 Felsenstein J (1985) Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution; International Journal of Organic Evo- lution 39(4): 783-791. https://doi.org/10.1111/).1558-5646.1985. tb00420.x Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/96/NT. Nucleic Acids Symposium Series 41: 95-98. Hebert PDN, Ratnasingham S$, DeWaard JR (2003) Barcoding animal life: cytochrome c oxidase subunit | divergences among closely related species. Proceedings of the Royal Society of London. Se- ries B: Biological Sciences 270: S96—S99. https://doi.org/10.1098/ rsbl.2003.0025 Hou Z, Fu J, Li S (2007) A molecular phylogeny of the genus Gammarus (Crustacea: Amphipoda) based on mitochondrial and nuclear gene sequences. Molecular Phylogenetics and Evolution 45(2): 596-611. https://dot.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. Proceed- ings of the National Academy of Sciences 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 freshwa- ter gammarids on the Aegean archipelago. Scientific Reports 10(1): 19813. https://dot.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: 14-66. https://doi.org/10.3906/zoo- 2109-12 Johns GC, Avise GC (1998) A comparative summary of genetic dis- tances in the vertebrates from the mitochondrial cytochrome b gene. Molecular Biology and Evolution 15(11): 1481-1490. https://doi. org/10.1093/oxfordjournals.molbev.a025875 Karaman GS, Pinkster S (1977) Freshwater Gammarus speciesfrom Europe, North Africa and Adjacent Regions of Asia (Crustacea-Amphipoda). Part I. Gammarus pulex-group and related species. Blijdragen tot de Dierkunde 47: 1-97. https://doi.org/10.1163/26660644-04701001 Kartavtsev YP, Lee JS (2006) Analysis of nucleotide diversity at genes Cyt-b and Co-1 on population, species, and genera levels. Appli- cability of DNA and allozyme data in the genetics of speciation. Russian Journal of Genetics 42: 437-461. https://doi.org/10.1134/ $1022795406040016 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Mo- lecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549. https://doi. org/10.1093/molbev/msy096 Lagrue C, Wattier R, Galipaud M, Gauthey Z, Rullmann JP, Dubreuil C, Rigaud T, Bollache L (2014) Confrontation of cryptic diversity and mate discrimination within Gammarus pulex and Gammarus fossa- rum species complexes. Freshwater Biology 59(12): 2555-2570. https://doi.org/10.1111/fwb.12453 Zoosyst. Evol. 99 (1) 2023, 15-27 Mamos T, Wattier R, Majda A, Sket B, Grabowski M (2014) Morpho- logical vs. Molecular delineation of taxa across montane regions in Europe: The case study of Gammarus balcanicus Schaferna, (Crus- tacea: Amphipoda). Journal of Zoological Systematics and Evolu- tionary Research 52(3): 237-248. https://doi.org/10.1111/jzs.12062 Meyran JC, Taberlet P (1998) Mitochondrial DNA polymorphism among alpine populations of Gammarus lacustris (Crustacea, Amphipoda). Freshwater Biology 39(2): 259-265. https://doi.org/10.1046/,.1365- 2427.1998.00277.x Meyran JC, Monnerot M, Taberlet P (1997) Taxonomic Status and Phy- logenetic Relationships of Some Species of the Genus Gammarus (Crustacea, Amphipoda) Deduced from Mitochondrial DNA Se- quences. Molecular Phylogenetics and Evolution 8(1): 1-10. https:// doi.org/10.1006/mpev. 1996.0399 Morhun H, Copilas-Ciocianu D, Rewicz T, Son MO, Khomenko A, Huseynov M, Utevsky S, Grabowski M (2022) Molecular markers and SEM imaging reveal pseudocryptic diversity within the Ponto- Caspian low-profile amphipod invader Dikerogammarus bispinosus. The European Zoological Journal 89(1): 87-101. https://doi.org/10. 1080/24750263.2021.2018056 Miller J (2000) Mitochondrial DNA variation and the evolutionary histo- ry of cryptic Gammarus fossarum types. Molecular Phylogenetics and Evolution 15(2): 260-268. https://doi.org/10.1006/mpev. 1999.0740 Ozbek M (2012) A new freshwater amphipod species, Gammarus obruki sp. nov., from Turkey (Amphipoda: Gammaridae). Turkish Journal of Zoology 36: 567-575. https://doi.org/10.3906/zoo-1112-2 Ozbek M, Yurga L, Kiilk6yliioglu O (2013) Gammarus baysali sp. nov., a new freshwater amphipod species from Turkey (Amphipoda: Gammaridae). Turkish Journal of Zoology 37: 163-171. https://dot. org/10.3906/zoo0-1209-14 Posada D (2008) jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25(7): 1253-1256. https://doi.org/10.1093/ molbev/msn083 2/7 Tavaré S (1986) Some probabilistic and statistical problems in the anal- ysis of DNA Sequences. Lectures on Mathematics in the Life Sci- ences 17: 57-86. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight ma- trix choice. Nucleic Acids Research 22(22): 4673-4680. https://dot. org/10.1093/nar/22.22.4673 Vainola R, Witt JDS, Grabowski M, Bradbury JH, Jazdzewki K, Sket B (2008) Global diversity of amphipods (Amphipoda; Crusta- cea) in Freshwater. Hydrobiologia 595(1): 241-255. https://doi. org/10.1007/s10750-007-9020-6 Vavra V (1905) Rotatorien und Crustaceen. Annalen des K. K. Naturhis- torischen Hofmuseums in Wien 20: 106—113. Weiss M, Macher JN, Seefeldt MA, Leese F (2014) Molecular evidence for further overlooked species within the Gammarus fossarum com- plex (Crustacea: Amphipoda). Hydrobiologia 721(1): 165-184. https://doi.org/10.1007/s10750-013-1658-7 Witt JD, Threloff DL, Hebert PD (2006) DNA barcoding reveals ex- traordinary cryptic diversity in an amphipod genus: Implications for desert spring conservation. Molecular Ecology 15(10): 3073-3082. https://doi.org/10.1111/).1365-294X.2006.02999.x Wysocka A, Grabowski M, Sworobowicz L, Mamos T, Burzynski A, Sell J (2014) Origin of the Lake Ohrid gammarid species flock: Ancient local phylogenetic lineage diversification. Journal of Biogeography 41(9): 1758-1768. https://doi.org/10.1111/jbi. 12335 Yamag¢ A, Gilli E, Tok E, Tork K (2021) 1 Cave and Karst Systems of the World. Springer, 108 pp. https://doi.org/10.1007/978-3-030- 65501-3_ 1 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 zse.pensoft.net