Two New Species of Stygobiotic Amphipod Niphargus (Amphipoda: Niphargidae) and their Phylogenetic Relationship with Other Congeners from Iran

© 2024 Academia Sinica, Taiwan Open Access Two New Species of Stygobiotic Amphipod Niphargus (Amphipoda: Niphargidae) and their Phylogenetic Relationship with Other Congeners from Iran Mahmoud Mamaghani-Shishvan1, Vahid Akmali2,* , Cene Fišer3, and Somayeh EsmaeiliRineh4,* 1Department of Biology, Faculty of Science, Razi University, Bagabrisham 6714967346, Kermanshah, Iran. E-mail: [email protected] (Mamaghani-Shishvan) 2Department of Biology, Faculty of Science, Razi University, Bagabrisham 6714967346, Kermanshah, Iran. *Correspondences: E-mail: [email protected] (Akmali) 3Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000, Ljubljana, Slovenia. E-mail: [email protected] (Fišer) 4Department of Biology, Faculty of Science, Razi University, Bagabrisham 6714967346, Kermanshah, Iran. *Correspondences: E-mail: [email protected] (Esmaeili-Rineh) Received 1 July 2023 / Accepted 10 April 2024 / Published 4 September 2024 Communicated by Benny K.K. Chan Two new amphipod species from Iran, Niphargus sahandensis sp. nov. and Niphargus chaldoranensis sp. nov., are described based on their morphological characteristics and molecular analyses. Bayesian inference analyses of COI and 28s rDNA sequence data provided evidence for the validity of the two species and their placement in the Niphargus genus. N. sahandensis sp. nov. primarily differs from similar species by having more than two hook-like retinacles on the inner surface of pleopods I–III, the presence of two spines at the base of uropod I and rectangular-shaped propodi in both gnathopods. N. chaldoranensis sp. nov. is distinguished by the trapezoidal-shaped propodi in gnathopods I to II, the equal sizes of pereopods V and VI, and the proportional size of periopod VII in relation to the total body (60%). Morphological descriptions with illustrations of the new species, as well as a DNA-based phylogeny generated from analyses of a multigene dataset, are provided to better understand species relationships. Key words: Niphargus sahandensis sp. nov., Niphargus chaldoranensis sp. nov., Morphological characters, COI and 28s rDNA, Iran BACKGROUND Groundwater ecosystems are physically heterogenous and complex systems recognized by low oxygen levels, constant temperature, limited space, and scarcity of food and energy sources (Macario-González et al. 2021; Danielopol et al. 2000). Resident species have been able to occupy groundwater environments through specific physiological, morphological and behavioral adaptations, such as low metabolic rates, vermiform body shape, reduced or complete loss of vision and pigments, and enhancement of certain sensory structures (Sket 1985; Gibert et al. 1994; Langecker 2000; Parzefall 2000; Culver and Sket 2000). Amphipod crustaceans from the genus Niphargus are the largest genus of freshwater amphipods, most of which are found exclusively in groundwater (Petković et al. 2020). Their functional morphology and feeding habits likely played an important role in their evolution, leading to large morphological and ecological Citation: Mamaghani-Shishvan M, Akmali V, Fišer C, Esmaeili-Rineh S. 2024. Two new species of stygobiotic amphipod Niphargus (Amphipoda: Niphargidae) and their phylogenetic relationship with other congeners from Iran. Zool Stud 63:23. doi:10.6620/ZS.2024.63-23. Zoological Studies 63:23 (2024) doi:10.6620/ZS.2024.63-23 1 © 2024 Academia Sinica, Taiwan diversity within the genus (Borko et al. 2021). These characteristics make them an interesting ecological model system that can, at times, be used as a proxy for groundwater communities (Balázs et al. 2023). The taxonomy of Niphargus is unresolved. New species are continuously being found, both morphologically indistinguishable (so-called ‘cryptic species’) as well as morphologically distinct. However, accurate reconstruction and resolution of their phylogenetic relationships as well as reconstructions of their ecology and biogeography require a complete taxonomic structure (Mammola et al. 2019; EsmaeiliRineh et al. 2020; Petković et al. 2020). Due to numerous morphologically cryptic species, the taxonomy of Niphargus should rely on both molecular and morphological analyses. By combining these approaches, scientists can achieve a more accurate and comprehensive understanding of the taxonomy and evolutionary history of the Niphargus genus, as well as other groups of organisms found in groundwater ecosystems (Esmaeili et al. 2020; Balázs et al. 2023). Niphargus is distributed between Ireland and Iran. Taxonomic research of this genus in Iran has begun relatively recently and so far relatively few species are known from Iran (Esmaeili et al. 2015). This is in stark contrast with the fact that the Alborz and Zagros mountain ranges in Iran have high biodiversity and are considered hotspots. New cave explorations, however, continuously unveil new findings of Niphargus. In this study, we present three populations of the Niphargus genus collected in northwest Iran and morphological and molecular evidence that suggests they should be treated as new species. MATERIALS AND METHODS Morphological and morphometric studies The samples were provided from Hargalan Spring in East Azerbaijan Province, and the Salmas and Shoan Springs in West Azerbaijan Province, Iran (Fig. 1). The animals were collected using a hand net. Shoan and Salmas Springs are located in the proximity of Chaldoran and Salmas cities, respectively, in West Azerbaijan. The distance between the localities is about Fig. 1. Distribution map of the genus Niphargus Schiödte, 1849 in North and Northwest Iran. page 2 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan 120 km in a straight line. The materials were examined morphologically and mounted on slides in Euparal® medium. An Olympus LABOMED iVu7000 camera fitted on a LABOMED Lx500 stereo microscope was used to take the digital photos. The computer program ProgRes Capture Pro ver. 2.7 was used to perform the measurements and the counts. We measured and counted characters from six individuals. All materials were deposited in the Zoological Collection, Razi University (ZCRU). Phylogenetic analyses and molecular divergence For the molecular analyses, we extracted the total genomic DNA from part of an animal using Tissue Kits (GenNet Bio™) following the manufacturer’s instructions (Seoul, South Korea). Mitochondrial COI gene was amplified using the modified primer pair LCO1490-JJ and HCO2198-JJ (Astrin and Stüben 2008). The amplification and sequencing of the first fragment of 28S ribosomal DNA (rDNA) were performed using the forward primer proposed by Verovnik et al. (2005) and the reverse primer used by Zakšek et al. (2007). Each 25 µl reaction consisted of optimized amounts of PCR water, 12.5 μl of Master Mix kit (Ampliqon), 0.2 μl of each primer (10 µM), and 50–100 ng of genomic DNA template. For COI gene amplification, an initial denaturation step at 94°C for 3 minutes was followed by 36 cycles of 40 seconds at 94°C, 40 seconds at 52.5°C and 2 min at 65°C with a final extension step for 8 minutes at 65°C. Cycling parameters for the 28S rDNA gene were as follows: Initial denaturation of 94°C for 7 minutes, 35 subsequent cycles of 94°C for 45 seconds, 55°C for 30 seconds, 72°C for 1 minute, and a final extension of 72°C for 7 minutes. Purification of PCR products and sequencing were commercially performed by Microsynth AG (Swiss). Sequencing was performed with both primers mentioned above. In order to identify the phylogenetic position of the newly discovered materials, the acquired sequences (with GenBank accession numbers PP492996– PP493003, and PP492709–PP492716 for COI and 28S rDNA gent respectively) were analyzed within the data set of Esmaeili-Rineh et al. (2015 2017a) and Bargrizaneh et al. (2021). The NCBI sequences of Synurella ambulans, Pontogammarus crassus and Gammarus fossarum were used as out-groups (accession numbers: KF719240, KF719242 and KF71924). All sequences were edited and aligned using ClustalW (Thompson et al. 1994), as implemented in the Bioedit program sequence alignment editor (Hall 1999) using the default settings. Phylogenetic reconstruction was performed using the Bayesian inferences in MrBayes, version 3.1.2 (Ronquist and Huelsenbeck 2003). Bayesian analyses were run for 20 million generations, under GTR+G and TIM3+I+G models (jModelTest, version 0.1.1, Posada 2008) for 28S and COI genes, respectively. We run four chains, and the trees were sampled every 1000 generations. The first 5000 sampled trees were discarded as burn-in, and the subsequent tree likelihoods were checked for convergence in Tracer 1.5.0 (Rambaut and Drummond 2009). A fifty percent majority rule consensus tree was computed using the remaining trees and visualized by FigTree v1.4.0 software. The data on the analyzed species can be found in the Electronic Supplement of Esmaeili-Rineh et al. (2015 2020) and Bargrizaneh et al. (2021). To evaluate the divergence from other previously described Iranian species of Niphargus, we calculated the genetic distances using the Kimura two-parameter (K2P) model (Kimura 1980), which was implemented in MEGA ver. 5 (Tamura et al. 2011). RESULTS Phylogenetic position of the new species and their genetic distinctness We sequenced and analyzed eight new individuals; namely, three from Salmas, three from Shoan, and two from Hargalan springs. The two specimens from the Hargalan population showed a unique haplotype for a 902 base pairs long fragment of 28S ribosomal DNA gene, and two haplotypes for 513 base pairs of COI gene. However, the six specimens from the Salmas and Shoan populations showed a unique haplotype for the 28s gene, while two haplotypes for the COI gene, one was found in Salmas and one in Shoan. The phylogenetic analyses of 62 specimens consistently placed the two new species into two different clades. One of the species was nested in a clade sister to the Middle East clade, while the other species was placed in a European clade. The phylogenetic relationships of this clade and other Middle Eastern clades remained mainly unresolved based on the combined data of 28S and COI gene fragments (Fig. 2), and therefore the accurate phylogenetic position of the Salmas and Shoan clade cannot be determined. The two new species are genetically distinct from all other Iranian species. N. sahandensis sp. nov. is most genetically similar to N. daniali (3.59% based on 28s rDNA genes fragment) and the most divergent from N. sarii (11.82%) and N. alisadri (23.07%) based on both 28 rDNA and COI genes. N. chaldoranensis sp. nov. is most genetically similar to N. alisadri (2.31%) and page 3 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan Fig. 2. Bayesian consensus tree of 59 Niphargus specimens, based on the 28S ribosomal DNA and COI gene sequences. Species are identified and named according to available taxonomic descriptions. Posterior probabilities are indicated on branches. page 4 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan N. fiseri (11.37%) for 28srDNA and COI, respectively. Also, N. chaldoranensis sp. nov. is the most genetically divergent species from N. daniali (22.96% and 11.93%) for the studied COI and 28s rDNA genes fragments, respectively. All pairwise Kimura two parameter genetic distances of the Iranian taxa are shown in table 1. Order Amphipoda Latreille, 1816 Suborder Senticaudata Lowry and Myers, 2013 Family Niphargidae Bousfield, 1977 Genus Niphargus Schiödte, 1849 Niphargus sahandensis sp. nov. (Figs. 3–6) urn:lsid:zoobank.org:act:B233ED2D-3CC8-47C6-AFD0B88950D19464 Type locality and Material examined: Holotype, Male specimen (7 mm) from Hargalan Spring, Ajabshir City, East Azerbaijan Province, Iran, coordinates (N 37°37'35", E46°09'50"). Specimens collected by M. Mamaghani-Shishvan; 11 Aug 2022. Holotype with two paratypes are stored under catalogue number ZCRU Amph.1604. Etymology: The name “sahandensis” refers to Sahand Mountain in East Azerbaijan (Iran). Hargalan Spring is located on its slope. Diagnosis: Peduncle of pleopods I to III with 3–4 hooked retinacles at distal part of inner margin. At the base of uropod I peduncle with two spines. Maxilla I palp long, reaching beyond the tip of the outer lobe. A relatively equal size of coxae of gnathopods I–II. The propodi of gnathopod I with two supporting spines in palmar corner. Ventro-posterial corner in epimeral plates I to III not produced. Rectangular shape of propodi in both gnathopods. Outer ramus of uropod I slightly shorter than inner ramus. Description of holotype: Measurements: The total length of the holotype is 7 mm. Head represents 12% of the total body length (Fig. 3A). Antennae: Antenna I is 0.40 times body length. Peduncular articles 1–3 progressively shorter; length of peduncular article 3 exceeds half of peduncular article 2 (ratio 1.00 : 1.32). Main flagellum with 14 articles (most with short setae). Accessory flagellum bi-articulated and reaching 0.5 of article 4 of main flagellum; both articles with two and three setae, respectively (Fig. 3B). Antenna II with flagellum formed of 9 articles, approximately 0.80 times as long as antenna I. Flagellum length is 0.84 times the length of peduncle articles 4 + 5. Peduncular article 4 of antenna II is longer than article 5 (1.12 : 1.00), peduncle articles 4 and 5 with seven and eight groups of setae, respectively (Fig. 3C). Mouth parts: Labium (Fig. 4D) bi-lobate; with fine setae on tip of outer lobes. Inner plate of maxilla I Table 1. Kimura 2-parameter-distances (K2P) between Iranian species and new collected populations of the genus Niphargus Schiödte, 1849 (based on 28S ribosomal DNA gene (below diagonal) and mtDNA (COI) gene (above diagonal) 1234567891011 12 13 14 15 16 17 18 19 20 21 22 23 24 1: N. sahandensis (Hargalan) 20.80 20.80 23.07 18.29 20.32 20.64 19.02 19.78 20.86 17.77 20.12 17.76 18.28 20.62 19.27 19.52 21.16 20.34 20.88 * * * * 2: N. chaldoranensis (Salmas) 11.79 0.39 13.36 11.90 12.53 22.96 12.28 11.37 14.67 12.79 13.24 12.08 13.06 13.50 13.71 13.72 16.67 12.08 12.82 * * * * 3: N. chaldoranensis (Shoan) 11.79 0.00 - 13.36 11.90 13.01 23.51 12.75 11.37 14.67 13.27 13.24 12.08 13.54 13.50 14.19 14.21 17.18 12.08 13.30 * * * * 4: N. alisadri 11.06 2.31 2.31 11.03 16.88 20.91 12.35 12.16 11.90 13.08 12.38 12.10 12.36 15.41 12.36 15.76 14.15 12.15 15.93 * * * * 5: N. bisitunicus 11.21 3.37 3.37 1.15 14.70 21.67 10.49 10.72 10.51 11.16 11.16 12.07 12.80 14.90 8.70 14.71 13.60 9.80 13.50 * * * * 6: N. borisi 11.78 4.29 4.29 2.17 2.70 23.23 13.19 14.12 17.07 15.34 15.82 14.88 14.37 10.04 13.90 12.76 17.86 13.91 7.38 * * * * 7: N. daniali 3.59 11.93 11.93 10.89 11.35 11.33 17.12 19.62 22.18 21.96 19.89 19.87 17.06 23.05 17.82 22.18 21.70 18.59 21.93 * * * * 8: N. darvishi 11.37 3.10 3.10 0.76 1.40 2.69 11.20 9.58 12.08 10.27 9.82 12.27 10.01 13.65 3.82 14.86 13.33 8.69 11.80 * * * * 9: N. fiseri 11.02 2.56 2.56 2.57 2.96 4.29 11.16 3.09 12.52 12.98 11.37 11.59 11.37 12.98 10.02 13.95 13.78 7.62 15.14 * * * * 10: N. hosseiniei 11.82 3.36 3.36 1.40 1.27 2.82 11.96 1.40 2.83 13.99 12.10 13.22 14.18 13.44 12.09 15.65 9.65 13.70 14.16 * * * * 11: N. ilamensis 11.06 3.23 3.23 1.27 1.15 2.83 11.51 1.27 2.83 0.89 8.98 12.52 11.87 15.61 10.07 14.67 13.31 10.02 14.42 * * * * 12: N. khwarizmi 11.52 2.97 2.97 1.02 1.15 2.56 11.66 1.27 2.83 1.14 1.02 11.61 10.52 13.43 8.95 15.85 15.53 9.13 16.09 * * * * 13: N. kurdistanensis 10.90 2.97 2.97 0.63 1.27 2.04 10.73 1.14 2.96 1.66 1.66 1.40 9.78 13.70 11.58 14.70 15.44 12.05 14.42 * * * * 14: Niphargus sp. (Lebanon) 11.23 2.97 2.97 0.63 1.53 2.04 11.06 1.14 2.97 1.66 1.66 1.40 0.25 15.82 9.57 15.61 16.16 9.82 14.62 * * * * 15: N. nasrullahi 11.21 3.23 3.23 0.89 1.53 1.78 11.04 1.40 3.23 1.79 1.66 1.40 1.27 1.28 13.90 14.99 16.94 13.22 8.98 * * * * 16: N. persicus 11.52 3.23 3.23 0.89 1.53 2.82 11.35 0.25 3.23 1.53 1.40 1.40 1.27 1.27 1.53 14.87 14.33 8.90 12.98 * * * * 17: N. sharifii 10.90 2.97 2.97 0.63 1.02 1.78 10.73 1.14 2.96 1.53 1.40 1.15 0.76 0.76 0.76 1.27 15.71 13.70 14.48 * * * * 18: N. sohrevardensis 11.64 3.09 3.09 1.02 0.89 2.56 11.78 1.01 2.56 0.38 0.51 0.76 1.40 1.40 1.40 1.14 1.15 14.72 16.42 * * * * 19: N. urmiensis 11.02 3.52 3.52 2.33 2.20 3.79 11.47 2.59 2.06 2.59 2.60 2.46 2.46 2.47 2.73 2.73 2.20 2.46 13.47 * * * * 20: N. yasujensis 11.05 3.77 3.77 1.66 2.31 1.14 11.20 2.18 4.03 2.57 2.44 2.18 1.79 1.79 1.27 2.31 1.27 2.18 3.26 * * * * 21: N. hakani 11.64 3.09 3.09 2.43 2.56 3.88 12.08 2.95 2.83 2.56 2.43 2.30 2.83 2.83 2.83 3.09 2.56 2.17 2.20 3.35 * * * 22: N. hegmatanensis 11.21 2.57 2.57 0.25 1.15 1.91 11.20 0.76 2.83 1.40 1.27 1.02 0.63 0.63 0.89 0.89 0.63 1.02 2.33 1.40 2.43 * * 23: N. kermanshahi 11.21 3.10 3.10 0.89 0.51 2.43 11.35 1.14 2.96 1.01 0.89 0.63 1.02 1.28 1.27 1.27 1.02 0.63 2.46 2.05 2.30 0.89 * 24: N. lorestanensis 11.21 2.70 2.70 0.38 1.27 2.56 11.04 0.38 2.70 1.27 1.15 1.15 1.02 1.02 1.27 0.50 1.02 0.89 2.46 2.05 2.83 0.63 1.02 25: N. sarii 11.82 3.63 3.63 1.40 1.27 2.96 11.96 1.40 3.23 0.76 1.14 1.14 1.79 1.79 1.79 1.53 1.53 0.63 2.73 2.57 2.82 1.40 1.01 1.27 page 5 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan Fig. 3. Niphargus sahandensis sp. nov., Hargalan Spring, male 7 mm (holotype). A, Head; B, Antenna I; C, Antenna II; D, Mandibular palp; E, Maxilla I; F, Left mandible; G, Right mandible. Scale bars: 1 = 0.25 mm (F–G); 2 = 0.5 mm (A, D–E); 3 = 1 mm (B–C). page 6 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan with one long apical seta, outer plate with seven long spines with 3-1-1-2-1-1-1 lateral projections; palp biarticulated, long and reach the tip of outer lobe, with three apical setae (Fig. 3E–F). Both plates of maxilla II with numerous long distal setae (Fig. 4E). Mandibles: left mandible with five teeth on incisor process, lacinia mobilis with four teeth, between lacinia and molar a row of seven setae with lateral projections (Fig. 3F). Right mandible with four teeth on incisor process, lacinia mobilis pluritooth, between lacinia and molar a row of five setae with lateral projections (Fig. 3G). Mandibular palp articles 1:2:3 represent 21%, 38% and 41% of total palp length, respectively. Proximal article without setae; second article with five setae along inner margin and third article with one group of one A-seta, two groups of B-setae, no C-setae, 12 D-setae and five E-setae (Fig. 3D). Maxilliped with short inner plate bearing four distal spines intermixed with six distal setae; outer plate less than half of palp article 2, with 9 spines along inner margin and 4 setae distally; maxilliped palp article 3 at outer margin with one proximal and one distal group of long setae; palp terminal article with one seta at outer Fig. 4. Niphargus sahandensis sp. nov., Hargalan Spring, male 7 mm (holotype). A, Gnathopod I; B, Gnathopod II; C, Maxilliped; D, Labium; E, Maxilla II. Scale bars: 1 = 0.5 mm (D–E); 2 = 1 mm (A–C). page 7 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan margin and two setae at base of nail, nail shorter than pedestal (Fig. 4C). Gnathopods: Coxal plates of gnathopods I–II almost equal in size. Coxa of gnathopod I trapezoid, antero-ventral margins with four marginal setae. Basis with setae on anterior and posterior margins; ischium and merus with posterior group of setae. Carpus with one group of four setae antero-distally, bulge with long setae; carpus 0.61 times basis length and 0.65 times propodus length. Propodus of gnathopod I rectangular in shape and longer than broad; anterior margin with four setae in one group in addition to antero-distal group of four setae. Palm convex, defined on outer surface by one strong long corner S-seta accompanied laterally by two L-setae with lateral projections, on inner surface by two short sub-corner R-setae. Dactylus reaches posterior margin of propodus, outer and inner margins of dactylus with one and three simple setae, respectively. Nail length 0.25 times total dactylus length (Fig. 4A). Coxal plate of gnathopod II with square, ventral margins with four setae. Basis with setae on anterior and posterior margins; ischium and merus with posterior group of setae. Carpus with one group of two setae antero-distally, bulge with long setae; carpus 0.61 times basis length and 0.85 times propodus length. Propodus longer than broad; anterior margin with two setae in one group in addition to antero-distal group of three setae. Palm slightly convex, defined on outer surface by one strong, long corner S-seta accompanied laterally by two L-setae with lateral projections, on inner surface by one short sub-corner R-seta. Dactylus reaching posterior margin of propodus, outer and inner margins of dactylus with one and four simple setae, respectively; nail short, 0.28 times total dactylus length (Fig. 5B). Pereopods: Coxal plate III rectangular, length to width ratio is 1.13: 1; anterio-ventral margin with four setae. Coxal plate IV quadrate, anterio-ventral margin with four setae (Fig. 5A–B). Coxal plate V with posterior lobe, with two setae each on anterior and posterior lobes. Coxal plate VI with anterior lobe, with two simple setae on anterior lobe and one simple seta on posterior lobe. Coxal plate VII with one simple seta on posterior lobe (Fig. 5E). Pereopod III: IV length ratio is 1.06: 1. Dactylus IV short, dactylus length 0.32 times propodus length, nail shorter than pedestal (Fig. 5A–B). Pereopods V: VI: VII length ratios 1: 1.37: 1.49, respectively. Pereopod VII is 0.61 times the total body length. Pereopod bases V and VII each with four groups of spines along the anterior margins and five and six groups of setae along the posterior margins, respectively. Pereopod basis VI with five groups of spines and seven groups of setae along the posterior and anterior margins, respectively (Fig. 5C–E). Postero-ventral lobe of ischium in pereopods V–VII developed. Ischium, merus and carpus in pereopods V–VII with several groups of spines and setae along the anterior and posterior margins, dactyli of pereopods V–VII with one spine at base of nail on inner margin and one short seta on outer margin in pereopods V–VI. Nail length of pereopod VII 0.40 times the total dactylus length (Fig. 5C–E). Epimeral Plates: With angular postero-ventral corner, postero-ventral corners of plates I–III posteriorly with three, four and three setae and spines, respectively. Epimeral plates II–III each have two spines along the ventral margins (Fig. 6H). Pleopoda: Peduncle of pleopods I have three hooked retinacles at distal part of inner margins; peduncles of pleopods II-III each have four hooked retinacles at distal part of inner margins. Peduncle of pleopod III with one seta along outer margin. Rami of pleopods I-III with five to nine articles (Fig. 6A–C). Urosomites: Urosomites I–II with two and three setae on dorso-lateral margins, respectively. Urosomite III lacks setae. Urosomite I with two spines at base of uropod I. Uropods: Peduncle of uropod I with seven and one large spines along dorso-lateral and dorso-medial margins, respectively. Outer ramus of uropod I slightly shorter than inner ramus (ratio 1 : 1.02); inner ramus with two groups of two spines laterally and five spines distally; outer ramus with two groups of spines laterally and five spines distally (Fig. 6D). Inner ramus in uropod II longer than outer, both rami with lateral and distal long spines (Fig. 6E). Uropod III long, almost 0.31 times body length. Peduncle of uropod III with five spines, outer ramus bi-articulated, distal article 0.17 times proximal article. Proximal article of outer ramus bearing four and five groups of spines along outer and inner margins, respectively (Fig. 6F); distal article has lateral and distal setae. Inner ramus short, with one distal spine. Telson: Longer than broad, lobes slightly narrowing; each lobe with three spines distally, with one plumose seta laterally (Fig. 6G). Order Amphipoda Latreille, 1816 Suborder Senticaudata Lowry and Myers, 2013 Family Niphargidae Bousfield, 1977 Genus Niphargus Schiödte, 1849 Niphargus chaldoranensis sp. nov. (Figs. 7–10) urn:lsid:zoobank.org:act:6FC80139-410C-418D-B5C3929A95A3F70C Type locality and Material examined: Holotype, Male specimen (8 mm) from Shoan Spring, Chaldoran page 8 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan City, West Azerbaijan Province, Iran, coordinates (N39°04'13", E44°09'28"). Specimens collected by M. Mamaghani-Shishvan; 6 Aug 2022. Holotype with three paratypes are stored under catalogue number ZCRU Amph.1602. Material examined: One male specimen (holotype) and two paratypes from Shoan Spring. Three male specimens were examined from Salmas Spring, close Fig. 5. Niphargus sahandensis sp. nov., Hargalan Spring, male 7 mm (holotype). A, Pereopod III; B, Pereopod IV; C, Pereopod V; D, Pereopod VI; E, Pereopod VII. Scale bars = 1 mm. page 9 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan III long, almost 0.36 times body length. Peduncle of uropod III with four spines, outer ramus bi-articulated, distal article 0.76 times proximal article. Proximal article of outer ramus bear four and five groups of setae and spines along inner and outer margins, respectively (Fig. 10F); distal article with four setae distally. Inner ramus short, with one distal spine and one distal seta. Telson: Longer than broad, lobes slightly narrowing; each lobe with three spines distally and two plumose setae laterally (Fig. 10G). Interpopulational variation Although differences between species, however small, can be important, differences between populations of the same species are also significant. A total of six individuals from the two populations of Shoan and Salmas were examined and compared. Many taxonomic traits seem to be stable; however, we observed some differences that may be taxonomically important. In particular, there are notable differences in the number of supporting spines in the palmar corner of gnathopod propodi II (between 1–2), the number of spines with lateral projections on the outer surface in the palmar corner of gnathopod propodi II (between 1–2), the shapes of gnathopod II propodi (rectangular to trapezoid), and the ratio of inner to outer ramus of uropod I (similar size to longer). DISCUSSION In this study, three populations of the genus Niphargus were collected from northwest Iran and examined using morphological and molecular characteristics. DNA sequences support the species status of two new species, N. sahandensis sp. nov. and N. chaldoranensis sp. nov. Furthermore, the Bayesian analysis showed that the two new species are phylogenetically distinct, independent and different from all other related species. N. sahandensis has the most dissimilar COI sequence with N. alisadri (distance after K2P correction = 23%) and the most similar COI sequence with N. ilamensis and N. kurdistanensis (distance after K2P correction = 17.7%) (Table 1) (Esmaeili-Rineh et al. 2017b; Mamaghani-Shishvan et al. 2017). Differences in nuclear 28S are smaller; N. sahandensis sp. nov. is the most dissimilar to N. hosseiniei and N. sarii (11.12%) and the most similar to N. daniali (3.5%) (EsmaeiliRineh and Sari 2013; Esmaeili-Rineh et al. 2017b; Esmaeili-Rineh et al. 2018). Up to this study, N. daniali was the only species from the Middle East that was nested within the clade with many species from Europe. Our study suggests that N. sahandensis sp. nov. is a member of the European clade. Quite expectedly, the newly described species from Hargalan is genetically the most similar to N. daniali (Esmaeili-Rineh and Sari 2013). Morphological examination shows that although both species have similarities in the propodi shape of gnathopods I to II, the ratio of palpus to outer plate in maxilla I, the size of the inner to outer ramus in uropod I, and the ratio of proximal to distal of the outer ramus in uropod III, the new species can be distinguished from N. daniali by the absence of a lateral spine and the presence of only three distal spines on the telson (two lateral and four distal spines in N. daniali), the absence of setae in the first article of the mandibular palp (two setae in N daniali), the presence of more than two hook-like retinacles in the pleopods I–III, and a greater number of supporting spines on the outer surface of the palpus (one spine in N daniali) (Esmaeili-Rineh and Sari 2013). In addition, N. sahandensis sp. nov. can be distinguished from N. ilamensis by a dactylus that extends to the outer corner of the gnathopod palps, a longer palp compared to the outer plate in maxilla I, and a lower number of supporting spines in gnathopods I and II. Also, N. sahandensis sp. nov. is distinguished from N. kurdistanensis by having a longer size of the inner to outer ramus ratio in uropod I, the presence of three distal spines, the shape of the propodus of gnathopod I, and the ratio of palp to outer plate in maxilla I (Esmaeili-Rineh et al. 2017b; MamaghaniShishvan et al. 2017). Further results of the analysis of genetic distances showed that N. chaldoranensis sp. nov. is the most dissimilar from N. daniali, with 22% and 11.93% K2P distances in COI and 28S genes, respectively. The lowest genetic distances between N. chaldoranensis and any other Iranian species is 11.37% (COI) with N. fiseri and 2.31% (28S) with N. alisadri. N. fiseri can be distinguished from N. chaldoranensis sp. nov. by the maxilla I palp, not reaching the tip of the outer plate in maxilla I, the smaller size of the inner to outer ramus in uropod I (the longer size of inner to outer ramus of uropod I in N. chaldoranensis), the presence of one lateral spine on the telson (absence of lateral spines in N. chaldoranensis), the presence of two supporting spines in gnathopod I (one supporting spine in N. chaldoranensis), and inclined angles in epimeral plates (slightly produced in N. chaldoranensis). Additionally, N. alisadri is distinguished from the new species by having equal size of the proximal to distal part of the outer ramus in uropod III (distal article 70% proximal article in N. chaldoranensis), equal lengths of the palp and outer page 16 of 18Zoological Studies 63:23 (2024) © 2024 Academia Sinica, Taiwan plate in maxilla I (The palpus is long and reaches beyond the tip of the outer lobe in N. chaldoranensis), and the presence of two lateral spines on the telson (Esmaeili-Rineh and Sari 2013; Mamaghani-Shishvan and Esmaeili-Rineh 2019). In conclusion, it should be noted that molecular and morphological data indicate that populations of Hargalan and Salmas-Shoan belong to two separate species. The grouping of N. sahandensis sp. nov. with N. daniali in the European clade suggests a closer evolutionary relationship between these two species and their European counterparts. This finding expands our understanding of the European clade and its diversity by including a new species from the Middle East. It also implies that there may have been historical migrations between the Middle East and Europe, leading to the grouping of these two species in the same clade. Overall, this discovery highlights the importance and need for continuation of research and exploration in understanding the evolution and diversification of species across different regions. Acknowledgments: This project was financially supported by Razi University as a part of the PhD dissertation of the first author. CF was supported by the Slovenian Agency for Research and Innovation through the programme P1-0184 and project J12464. We cordially thank Benny Chan and a reviewer who remarkably improved the early version of the manuscript. Authors’ contributions: VA, SE and CF designed the study. MM performed the field work and collected samples. MM and SE prepared the figures. 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