DOI: 10.15468/q7bard
title: A new cave-dwelling hadzioid amphipod (Senticaudata, Hadzioidea, Melitidae) from sulFIdic groundwaters in Iran

description: This dataset contains the digitized treatments in Plazi based on the original journal article Malek-Hosseini, Mohammad Javad, Brad, Traian, Fatemi, Yaser, Kuntner, Matjaž, Fišer, Cene (2024): A new cave-dwelling hadzioid amphipod (Senticaudata, Hadzioidea, Melitidae) from sulFIdic groundwaters in Iran. Contributions to Zoology 93 (2): 107-126, DOI: 10.1163/18759866-BJA10054, URL: http://dx.doi.org/10.1163/18759866-bja10054AbstractCave systems fed with hydrogen sulfide-rich groundwater are unique chemoautotrophy-dependent ecosystems. Although globally widespread and known to harbor unique subterranean metazoan communities, they have mostly been studied in Europe and North America, less so in Asia. Here, we report on a discovery of a new species of amphipod crustacean from sulfidic waters of Tashan-Chah Kabootari aquifer from Zagros Mountains in Iran. The new species corresponds morphologically to the melitid genus Tegano Barnard & Karaman, 1982, and Tegano tashanensis sp. nov. is the first amphipod from the superfamily Hadzioidea found exclusively in sulfidic water. Phylogenies derived from the mitochondrial COI and the nuclear 28S rRNA sequences recover T. tashanensis sp. nov. in a clade that contains representatives of the currently para- or polyphyletic genera Barnardomelita, Brachina, Josephosella, and Tegano indicating the need for a revised melitid genus level systematics. This clade comprises marine, freshwater and subterranean species globally distributed in regions that were connected by the ancient Tethys Sea. As such, these taxa may be suitable biogeographic models for studying past dispersal, vicariance and multiple colonization of inland groundwaters.KeywordsAmphipoda ‒ groundwater ‒ Iran ‒ Melitidae ‒ phylogenyIntroductionGroundwater represents the largest freshwater body in the world (Gibert & Deharveng, 2002). Yet, due to being remote and hard to access (Ficetola et al., 2019), groundwater remains one of the most unexplored habitats in the world (Larned, 2012). Groundwater is not a homogenous habitat but rather a palette of diverse habitats (Culver & Pipan, 2019). One of the most interesting groundwater habitats are waterbodies rich in hydrogen sulfide. While the sulfide is toxic to most metazoans, it is also a source of energy to chemolitho-autotrophic bacteria, resulting in primary production in the absence of light (Sarbu et al., 1996). On some occasions, chemolithoau-totrophic primary production is sufficient to establish and maintain isolated, local ecosystems (Sarbu, 2000). These caves are not as oligotrophic as freshwater caves; despite sulfide toxicity these caves often harbor species-rich communities (Sarbu, 2000; Hutchins et al., 2016). Apparently, while H2S may be toxic for most animals, other organisms cope well with it and even thrive in these habitats. To date, more than 19 caves with sulfidic hypogean waters have been reported worldwide (Culver & Pipan, 2019). Metazoan faunas of sulfidic caves have been studied mostly in Europe and North America, including Movile Cave in Romania (Sarbu, 2000; Brad et al., 2021), Frasassi Cave System in Italy (Sarbu et al., 2000), Melissotrypa Cave in Greece (Vaxevanopoulos, 2006; Borko et al., 2019; Popa et al., 2019) and Cueva del Azufre in Mexico (Tobler et al., 2008, 2011; Riesch et al., 2015). In Asia, to our knowledge, metazoan fauna from sulfidic groundwater has only been studied from Ayyalon Cave in Israel (Por et al., 2013) and Tashan Cave in Iran (Fatemi et al., 2019).The exploration of Tashan Cave in Iran has begun relatively Downloaded recently from. Brill So. far com, 07 three /10/2024 species01:59:59PMvia Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ have been described from the cave, including a cave fish Garra tashanensis Mousavi-Sabet, Vatandoust, Fatemi & Eagderi, 2016, an isopod Stenasellus tashanicus Khalaji-Pirbalouty, Fatemi, Malek-Hosseini & Kuntner, 2018, and a gastropod Trogloiranica tashanica Fatemi, Malek-Hosseini, Falniowski, Hofman, Kuntner & Grego 2019 (Mousavi-Sabet et al., 2016; Khalaji-Pirbalouty et al., 2018; Fatemi et al., 2019). Presence of the Tashan Cave fish in outflows of a seasonal karstic spring, five km to the east of its type locality, in syntopy with the stygobiotic form of Garra rufa (Heckel, 1843), shows the presence of a unique and big aquifer in that area (Malek-Hosseini et al., 2023). The Tashan-Chah Kabootari aquifer comprises also the Chah-Kabootari Cave. It is in this cave that eyeless and depigmented amphipod crustaceans were found, which we describe in this article.Crustaceans often dominate metazoan subterranean aquatic communities (Sket, 1999). Amphipods represent a substantial fraction of subterranean crustacean diversity (Väinölä et al., 2008; Zagmajster et al., 2014; Eme et al., 2017). In the Western Palearctic, most subterranean amphipod species belong to the genus Niphargus (SchiØdte, 1847). Niphargus, known from all European sulfidic caves (Flot et al., 2010, 2014; Brad et al., 2015; Fišer et al., 2015; Borko et al., 2019), is represented in Iran with 23 species (Esmaeili-Rineh et al., 2015, 2017; Zamanpoore et al., 2019; Bargrizaneh et al., 2021). The shape of gnathopods, uropods and telson of the newly collected specimens from Chah Kabootari Cave suggests that this species does not belong to Niphargus, but rather to an unrelated superfamily Hadzioidea Bousfield, 1973 (Lowry & Myers, 2013; Copilaș-Ciocianu et al., 2020). Amphipods of the superfamily Hadzioidea live in marine and freshwater habitats, including groundwater. Subterranean hadzioid amphipods presumably colonized the subterranean realm from marine ancestors PM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ (Stock, 1980; Holsinger, 2000). Their global distribution in West Indies, Mediterranean, and the Pacific region implies that they may date back to the Tethys (Hou & Li, 2018; Zhao et al., 2021). The Chah-Kabootari Cave, situated proximally to the Persian Gulf, roughly falls within the Tethyan region, and is thus in geographic agreement with previous records of subterranean Hadzioids and our preliminary morphological identification.The taxonomic position of the herein described species is unclear. The superfamily Hadzioidea comprises eight families of controversial taxonomic status, including families Hadziidae and Melitidae (Lowry & Myers, 2013). Specimens from the Chah-Kabootari Cave could be considered either as a member of the family Hadziidae or, more likely, can be assigned to the melitid genus Tegano whose original diagnosis was proposed on a basis of series of reductions, including a minute accessory flagellum, absent inner lobes of the labium, absent mandibular palp, the loss of sexual dimorphism in gnathopods, and the loss of spine at the base of uropod I (Lowry & Myers, 2013); all these character states match the specimens from the Chah-Kabootari Cave. To clarify the taxonomic status of this unexpected finding, we studied the species morphologically and phylogenetically. We show that the new species falls into a clade that contains several paraphyletic melitid “genera”. In the absence of a thorough systematics revision of these genera, and to avoid creating a new monotypic genus that may subsequently need to be synonymized, we here describe the new species as Tegano tashanensis sp. nov.Materials and methodsThe studied cave Chah Kabootari Cave or Chal Kabootari in local language (Downloaded Persian) from means Brill.com“nest 07/10 of /2024 pigeons 01:59:59 ”. A part of the Tashan-Chah Kabootari cave system located close to Sarjowshar village, Tashan district, Behbahan county, Khuzestan Province, Zagros Mountains, southwestern Iran (30°52’10’‘N, 50°10’01’‘E, altitude 539 m above sea level) (figs. 1 and 2), it is a natural limestone cave containing sulfidic water. Several neighboring wells and springs are fed with sulfidic water from this vast underlying aquifer. At the time of sampling, the water in Chah Kabootari Cave had 25.6 ℃ and pH 7.1, while the air temperature was 25.3 ℃, the relative humidity was 99.8 %, and the air contained 768 ppm CO2 (0.08 %).We visited Tashan and Chah Kabootari caves during several expeditions, between 2016‒2023. The new amphipod species was collected from pools and streams of Chah Kabootari Cave (fig. 2) using a small hand net and a pipette. Specimens were also found in between the white biofilm floating on the sulfidic water in this cave. We preserved two samples in96% and the others in75% ethanol.Morphological analysisSpecimens were immersed in glycerol and the dissected body appendages were mounted on slides using Kaiser’s glycerol gelatin (Merck KGaA, Germany). The slides were examined under an Olympus SZX16 stereomicroscope equipped with an Olympus SC 180 camera, and under an Olympus BX 51 microscope with an Olympus SC50 camera. The drawings were performed upon printed photographs by manual inking and continuous inspection of the slides under the microscope. We followed the standard amphipod anatomic terminology (Fišer et al., 2009). The holotype and paratypes are deposited in the collection of Emil RacoviŢĂ Institute of Speleology, Cluj, Romania.FIGURE 1 Distribution map of the family Melitidae in Iran, (1) (green circle): Chah-Kabootari Cave, habitat of Tegano tashanensis sp. nov.; (2) (red circle): Qeshm Island, type locality of Melita persia Momtazi, Sari & Maghsoudlou, 2014; (3) (purple circle) Anzali Port, type locality of M. mirzajani Krapp-Schickel & Sket, 2015. Distribution of the genus Melita in Iran derived from Momtazi et al. (2014) and Krapp-Schickel and Sket (2015).Downloaded from Brill.com 07/10/2024 01:59:59PM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/FIGURE 2 Chah-Kabootari Cave, part of Tashan-Chah Kabootari aquifer, habitat of Tegano tashanensis sp. nov. (A) and (B): ponds at the first part and end of the cave respectively, where the specimens were collected.Molecular procedures Genomic DNA was extracted robotically from the pereopod muscle of three specimens using Mag MAX™ Express magnetic particle processor Type 700 with DNA Multisample kit (Thermo Fisher Scientific kit) following modified protocols (Vidergar et al., 2014). We employed PCR to amplify fragments of two genes, the mitochondrial cytochrome c oxidase subunit 1 gene (COI) and the nuclear 28S rRNA gene (28S) following established protocols (Malek-Hosseini et al., 2021); for primer pairs, see supplementary table S1 and for PCR amplification protocols, see supplementary table S2. Each fragment was sequenced (Macrogen Europe, Amsterdam) in both directions using PCR amplification primers.To examine the phylogenetic position of the newly Downloaded described from Brill. species com, 07/10 we/2024 01 checked:59:59PM GenBank for both fragments of all melitid genera from previous publications available as of November 2022 (Tandberg et al., 2012; Chung et al., 2014; Park et al., 2020; King et al., 2022; Tomikawa et al., 2022). We obtained COI fragments for 11 melitid genera, totaling 31 species, and 28S rRNA fragments for six genera and 18 species including our original data (supplementary table S3). As outgroups, we selected two unrelated, freshwater amphipods, Synurella ambulans (F. Müller, 1846) (Crangonyctidae) and Carinurella Paradoxa (Sket, 1964) (Niphargidae), for which sequences were available (see also Copilas Ciocianu et al., 2020).via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/To assemble sequences, we used ChromasPro 2.1.3 (Technelysium, Tewantin, Australia). Alignment, the best substitution model and the optimal partitioning scheme for each marker were made using MEGA (Kumar et al., 2018). MUSCLE algorithm was used for alignments. The final length of aligned fragments was 657 bp for COI and 1052 bp for 28S rRNA. Fragments were concatenated in Mesquite version 3.70 (Maddison & Maddison, 2021). Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI). The optimal settings for our data involved two partitions, each using an independent nucleotide substitution model (COI: GTR+G+I; 28S rRNA:GTR+I) (supplementary alignments S1–S3). We calculated the ML tree using IQ-Tree v2.1.3 (Minh et al., 2020), and node support was assessed using 1000 ultrafast bootstrap replicates. BI tree and Bayesian posterior probabilities (PP) were estimated using MrBayes v3.2.7a (Ronquist et al., 2012). We ran two independent Bayesian runs, each with four MCMC chains, for 15 million generations with a sampling frequency of 3000. A relative burn-in was set to 25% and checked for the MCMC chain convergence in Tracer 1.7.1 (Rambaut et al., 2018). These anal-yses were performed for each gene fragment separately, as well as for a concatenated matrix.ResultsPhylogenetic analysesBoth, ML and BI yielded similar topologies.Detailed comparisons between gene trees are not possible due to different number of species used in different analyses. However, gene trees showed generally congruent topologies (supplementary figs. S1–S 5) and thus justify concatenated analysis. The concatenated analysis yielded an overall well supported phylogeny. Melitidae comprises three main clades, but our phylogeny indicates the paraphyly of the genera Melita, Josephosella, Megamoera and Quasimelita.SystematicsThe new species from Chah Kabootari Cave is nested in the well-supported clade comprising genera Josephosella, Barnardomelita, Tegano and Brachina (fig. 3 and supplementary figs. S1–S 5). The Iranian species represents its own, well supported lineage, that might justify the erection of a new genus. However, considering that the overall genus level systematics of the family needs to be revised within a much wider taxon sampling, we feel that an introduction of a new genus would further complicate, rather than resolve, its current taxonomic structure. For this reason, we tentatively place this new species in Tegano, although we freely admit that this placement is based on morphological similarities or even absences rather than presumed synapomorphies: the absence of mandibular palp, the shape of gnathopods, the absence of spine at the base of uropod I and of baso-facial spine at uropod I. For the above reason, we refrain from providing a taxonomic diagnosis of the genus Tegano. Taxonomy Downloaded from Brill.com 07/10/2024 01:59:59PM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ FIGURE 3 Phylogeny of the melitid amphipods including the new species of Tegano tashanensis sp. nov. from Chah Kabootari Cave. The topology derives from the Bayesian inference analysis on the concatenated matrix. Node support presents posterior probabilities obtained by Bayesian analysis in MrBayes (in black), and bootstrap support values (in blue) obtained by ML analysis in IQ-Tree. Order Amphipoda Latreille, 1816 Family Melitidae Bousfield, 1973 Genus Tegano Barnard & Karaman, 1982Downloaded from Brill.com 07/10/2024 01:59:59PMvia Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/FIGURE 3 Phylogeny of the melitid amphipods including the new species of Tegano tashanensis sp. nov. from Chah Kabootari Cave. The topology derives from the Bayesian inference analysis on the concatenated matrix. Node support presents posterior probabilities obtained by Bayesian analysis in MrBayes (in black), and bootstrap support values (in blue) obtained by ML analysis in IQ-Tree.Order Amphipoda Latreille, 1816Family Melitidae Bousfield, 1973Genus Tegano Barnard & Karaman, 1982