Corresponding author: Somayeh Esmaeili-Rineh ( email@example.com )
Academic editor: Oana Moldovan
© 2017 Somayeh Esmaeili-Rineh, Seyyed Ahmad Mirghaffari, Mozafar Sharifi.
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.
Citation: Esmaeili-Rineh S, Mirghaffari SA, Sharifi M (2017) The description of a new species of Niphargus from Iran based on morphological and molecular data. Subterranean Biology 22: 43-58. https://doi.org/10.3897/subtbiol.22.11286
The freshwater amphipods of the genus Niphargus Schiödte, 1849 are widespread in subterranean waters of the western Palearctic. The eastern half of the genus range has been incompletely studied despite the presence of karstic areas and large aquifers. In this paper, we describe a new species from Hamedan Province in Iran and name it as N. hakani sp. n. This species hypothesis is based on the analysis of morphological characters and 28S ribosomal DNA sequences. The taxonomic status of the new species within the genus is discussed in comparison to the known Iranian species. Results revealed that this species is phylogenetically close to N. khwarizmi and is morphologically similar to N. borisi.
Niphargus, Hamedan Province, Taxonomy, 28SrDNA, Iran
The members of genus Niphargus Schiödte, 1849 are widespread in subterranean freshwaters of West Palearctic (Meleg et al. 2013). Most of the species of this genus occur in subterranean waters and constitute an important part of biodiversity in this environment (Fišer 2012). Few studies of this genus in the Middle East indicate that about 24 species live in this area, representing only a small fraction of over 350 known species.
Iran presents the eastern-most boundary of the Niphargus range. The first species is record of N. valachicus Dobreanu & Manolache, 1933 in Iran, a widely distributed species (
The specimens were collected using a small hand net in Kheder-Goli Spring in Hakan Village close to Razan City (see Figure
Genomic DNA was isolated from a part of animal using the Tissue Kits (GenNetBio™), according to the manufacturer’s instructions (Seoul, South Korea). Amplification and sequencing of the 28S ribosomal DNA (rDNA) fragment were performed using the forward primer from
PCR amplifications were done in 25μl volumes, containing, 2.5 μl of 10× PCR buffer (100 mM Tris-HCl (pH 8.8), (500 mM KCl), 0.2 μl of each primer (10 μM), 0.5 μl of dNTP (10 mM), 0.75 μl MgCl2 (50 mM), 50–100 ng of genomic DNA template, and 1 unit of Taq DNA polymerase. Cycling parameters were as follows: initial denaturation of 94°C for 7 min, 35 subsequent cycles of 94°C for 45 s, 55°C for 30 s and 72°C for 1 min, and a final extension of 72°C for 7 min. Sequencing reactions were done in Macrogen Korea Laboratories.
In order to figure out the phylogenetic position of the new discovered species, we analyzed the acquired sequences (GenBank accession numbers are KY629001 and KY629002) within the data set of
Phylogenetic reconstruction was performed using the Bayesian inferences in MRBAYES, version 3.1.2 (
To assess interspecific divergence between the Iranian species of Niphargus, we calculated the genetic distances corrected with Kimura two-parameter (K2P) model (
Bayesian Inference was performed with two simultaneous runs and four search chains within each run (three heated chains and one cold chain) for 10,000,000 generations, sampling trees every 1000 generations. The first 2500 sampled trees were discarded as burn-in, and subsequent tree likelihoods were checked for convergence in Tracer 1.5.0 (
Holotype, male specimen (9 mm) from Kheder-Goli spring, Razan city, Hamedan Province, Iran; coordinates 35°27'N, 49°07'E. Specimens were collected by S. A. Mirghaffari in September 2014. Holotype with two paratypes is stored under catalogue number ZCRU Amph.1010 in the Zoological Collection of Razi University, Iran (ZCRU).
At the base of uropod I observed only one robust seta. The palpus of maxilla I is slightly longer than outer lobe. Urosomites I–III bear two, five and two robust setae on dorso-lateral margin. Epimeral plates distinctly pointed and have three and four robust setae in ventral margin of plates II-III. The propodi of gnathopods I-II are trapezoid. Dactylus of gnathopod I dose not reach to posterior margin of propodus. Third article of mandibular palp bears a single group of two A-setae.
Total length of specimen 9 mm. Body strong and stout. Head length 11% of body length (Figure
Niphargus hakani sp. n., male 9 mm (holotype, ZCRU Amph.1010). A Antenna I B Antenna II C Head D–E Maxilla I F Left mandible. G Right mandible. H Mandibular palp. Scale bars: 1=0.25 mm (F–G). 2=0.5 mm (C–E, H). 3=1mm (A–B).
Niphargus hakani sp. n., male 9 mm (holotype, ZCRU Amph.1010). A Gnathopod I B Gnathopod II C Maxilliped D Labium E Maxilla II. Scale bars: 1=0.5 mm (C–E). 2=1 mm (A–B).
Incisor in left mandible with five teeth, lacinia mobilis with four teeth; seven setae with lateral projections between lacinia and triturative molar (Figure
Maxilliped with short inner plate on which are four distal robust setae intermixed with five distal simple setae; outer plate exceeding half of the posterior margin of palp article 2, with 11 robust setae along inner margin and three simple setae distally. Palp article 3 of maxilliped with one proximal, inner and outer group of long simple setae at outer margin; terminal article of palp with one simple seta at outer margin, nail shorter than pedestal (Figure
Coxa of gnathopod I shorter than gnathopod II. Coxa I rectangular, longer than broad, ventral to anterior margin with four and three simple setae, respectively. Basis with setae on anterior and posterior margins; ischium and merus with posterior group of setae. Carpus with one group of three setae anterodistally, a bulge with long simple setae; carpus 0.42 of basis length and 0.79 of propodus length. Propodus slightly longer than broad; anterior margin with seven setae in two groups in addition to anterodistal group of six simple setae. Palm slightly convex, with one strong long palmar robust seta, one short supporting robust seta on inner surface and two robust setae with lateral projections on outer surface; two simple setae under supporting robust seta in palmar corner. Dactylus not reaching posterior margin of propodus, outer and inner margins with a row of three and five simple setae, respectively; nail short, 0.23 of total dactylus length (Figure
Coxa of gnathopod II slightly rounded, with six setae along antero-ventro-posterior margins. Basis with setae in groups and single setae along anterior and posterior margins; posterior margins of ischium and merus with one posterior group of setae each. Carpus 0.43 of basis length and 0.67 propodus length. Carpus with one group of four setae anterodistally. Propodus in gnathopod II larger than gnathopod I, trapezoid shape and broader than long; anterior margin with seven setae in one group in addition to anterodistal group of six simple setae. Palm nearly convex, with one strong palmar robust seta, one supporting robust seta without lateral projections on inner surface, and one robust seta with lateral projections on outer surface; two setae under supporting robust setae in palmar corner. Dactylus reaching posterior margin of propodus, outer and inner margins of dactylus with three and four simple setae, respectively. Nail length 0.25 of total dactylus length (Figure
Coxa III rectangular, length to width ratio as 1.29 : 1; antero-ventral margin with five simple setae. Coxa IV rectangular, length to width ratio as 1 : 1.04, antero-ventral margin with seven simple setae, posterior concavity shallow and approximately 0.1 of coxa width (Figure
Niphargus hakani sp. n., male 9 mm (holotype, ZCRU Amph.1010). A Pereopod III B Pereopod IV C Pereopod V D Pereopod VI E Pereopod VII. Scale bars: 1mm (A–E).
Pereopod III : IV lengths ratio as 1 : 1.35 (Figure
Epimeral plates I–III (Figure
Niphargus hakani sp. n., male 9 mm (holotype, ZCRU Amph.1010). A Pleopod I B Pleopod II C Pleopod III D Uropod I E Uropod II F Uropod III. G Epimeral plates H Telson. Scale bars: 1=0.5 mm (G–H). 2=1 mm (A–E). 3=2mm F.
Pereonites I–VI without setae. Pereonite VII with two simple setae. Pleonites I–III each with one long simple seta along dorsal surface. Urosomites I-III with two, five and two robust setae laterally, respectively.
Peduncle of uropod I with six and three large robust setae along dorsolateral and dorsomedial margins, respectively. Rami of uropod I with equal length (ratio 1 : 1); inner ramus with three groups of robust setae laterally and five robust setae distally; outer ramus with three groups of six robust setae laterally and five robust setae distally (Figure
The name “hakani” refers to Hakan village where specimens were found.
This species is nested within the main Iranian clade and apparently shares the nearest common ancestor with N. khwarizmi, N. khayyami, N. kermanshahi and N. bisitunicus (Figure
Bayesian consensus tree of 49 Niphargus species (48 taxa from
Diagnostic characters in identification of Iranian species of the genus Niphargus.
(number of dorso-postero-lateral setae)
|Telson setal pattern (A – apical robust setae, L – lateral robust setae)||Size of coxa of GNI to GNII||Maxilla I -number of denticles on R. S of outer plate (from outer towards inner)||Uropod I (ratio of uropod rami)|
|N. hakani sp. n.||I:2 R.S
II: 5 R.S
III: 2 R.S
|3 A, 1 L||smaller||0,0,0,2,3,4,more than 5||Inner>outer|
II: 3 S.S, 2R.S
III: No setae
|3 A, 1 L||of similar size||0,1,1,1,2,2,5||Inner<outer|
|N. alisadri Esmaeili-Rineh & Sari, 2013||I-III: 6 R. S||3 A, 2 L
|N. daniali Esmaeili-Rineh & Sari, 2013||I-II: 1 S.S
III: No setae
|4 A, 2 L||smaller||0,0,0,1,1,1,3||Inner>outer|
|N. valachicus Dobreanu and Manolache, 1933||I-II: 1 R.S
III: No setae
|3 A, 1 L||smaller||1,1,1,1,1,1,1||Inner<outer|
||I: 1- 2 S.S
II: 2- 3 S.S
|3 A||smaller||1,others more than 5||Inner>outer|
II: 2 R.S
III: No setae
|3 A, 2 L||smaller||0,1,1,1,1,1,1||Inner<outer|
|N. sharifii Esmaeili-Rineh et al. 2015||I:1 S.S
II: 3 R.S
III: No seta
|3 A||smaller||5,5,5, others more than5||Inner>outer|
|N. bisitunicus Esmaeili-Rineh et al. 2015||I:1R.S,2S.S
III: No setae
|3 A, 1 L||smaller||0,0,1,1,1,1,2||Inner<outer|
|N. borisi Esmaeili-Rineh et al. 2015||I-II: 4 R.S
III: 2 R.S
|3 A, 1 L||smaller||0,0,0,1,1,2,2||Inner>outer
|N. darvishi Esmaeili-Rineh et al. 2015||I-II:2R.S, 2S.S||3 A||smaller||0,2,2,3,3,4,5||Inner>outer|
|Characters||The number of supporting robust setae in palmar corner of GNII||Length of maxilla I palpus||Shape of propods of GNI||Shape of propods of GNII||Uropod III (length of distal article of outer ramus.)|
|N. hakani sp. n.||1||longer than outer lobe of maxilla I (without R. S)||trapezoid||trapezoid||approximately 0.52 of the first article|
|N. kermanshahi Esmaeili-Rineh et al. 2016||2||shorter than outer lobe of maxilla I (without R. S)||trapezoid||trapezoid||approximately 0.5 of the first article|
|N. alisadri Esmaeili-Rineh & Sari, 2013||3||as long as outer lobe of maxilla I (without R. S)||rectangular||triangular||as long as the first article.|
|N. daniali Esmaeili-Rineh & Sari, 2013||1||longer than outer lobe of maxilla I (without R. S)||rectangular||sub quadrangular||approximately 0.33 of the first article|
|N. valachicus Dobreanu and Manolache, 1933||1||longer than outer lobe of maxilla I (without R. S)||trapezoid||trapezoid||approximately 0.33 of the first article|
|N. khayyami Hekmatara et al. 2013||1||as long as outer lobe of maxilla I (without R. S)||trapezoid||rectangular||0.1 of the first article|
|N. khwarizmi Hekmatara et al. 2013||1||longer than outer lobe||trapezoid||trapezoid||0.65 of the first article|
|N. sharifii Esmaeili-Rineh et al. 2015||2||as long as outer lobe||rectangular||rectangular||0.16 of the first article|
|N. bisitunicus Esmaeili-Rineh et al. 2015||2||longer than outer lobe||rectangular||rectangular||approximately 0.33 of the first article|
|N. borisi Esmaeili-Rineh et al. 2015||3||shorter than outer lobe of maxilla I (without R. S)||rectangular||rectangular||0.14 of the first article|
|N. darvishi Esmaeili-Rineh et al. 2015||1||longer than outer lobe of maxilla I (without R. S||trapezoid||trapezoid||0.08 of the first article|
Niphargus hakani sp. n. is a member of the main Iranian clade (
An important diagnostic traits of N. hakani sp. n. is the presence of robust setae on urosomite III. This character was observed in N. borisi and N. alisadri from Iran and some species from Europe including N. croaticus Sket, 1958, N. trullipes (Jurinac, 1887), and N. hercegovinensis S. Karaman, 1950 (
We described new taxon of Iranian Niphargus. Iranian niphargid fauna now counts 11 species, all but N. valachicus being endemic to Iran. We expect that further explorations will unveil additional new species from rich biodiversity in this area.
This project was funded by the Iran National Science Foundation (INSF-92033591). Authors are grateful to Razi University for providing the fieldwork facilities. We are especially grateful to two anonymous referees for their valuable comments.