Redescription of two subterranean amphipods Niphargus molnari Méhely, 1927 and Niphargus gebhardti Schellenberg, 1934 (Amphipoda, Niphargidae) and their phylogenetic position

Abstract A detailed redescription of two endemic, cave-dwelling niphargid species of the Hungarian Mecsek Mts., Niphargus molnari Méhely, 1927 and Niphargus gebhardti Schellenberg, 1934 is given based on newly collected material. Morphology was studied under light microscopy and with scanning electon microscopy. Morphological descriptions are complemented with mitochondrial cytochrome c oxidase subunit I (COI) sequences as barcodes for both species and with notes on their ecology. Using three independent molecular markers we showed that Niphargus gebhardti belongs to the clade distributed between Central and Eastern Europe, whereas phylogenetic relationship of Niphargus molnari to the rest of Niphargus species is not clear. The two species from the Mecsek Mts. are phylogenetically not closely related. Both species need to be treated as vulnerable according to IUCN Red List of Threatened Species.


Introduction
Fragmented mountain areas in East-Central Europe had been suggested to be centres of endemisms that evolved through a complex geological history including Eocene marine regression-transgression cycles and Pleistocene glacial cycles (Hou et al. 2013, Meleg et al. 2013, Mamos et al. 2014. The Mecsek is one of these isolated mountain ranges, that is situated in Southern Hungary and surrounded by Pannonian plains. The closest mountain ranges are the Croatian Papuk Mts. (80 km) and the Hungarian Transdanubian Mts. (150 km) (Fig. 1). The area is small of approximately 545 km². In biological sense, it is populated by numerous endemic species the origin of which may date back to Tertiary and which therefore apparently have survived mass extinctions in glacial periods. The upper geological layers comprise of Triassic and Jurassic limestones and dolomites, where extensive karstification has created over 200 caves. The subterranean environment of the area harbours numerous terrestrial and aquatic highly endemic invertebrates, known only from one or a few caves. Although the region apparently harbours an important piece of European and Hungarian natural heritage, until now only one species, the Hungarian blind snail (Bythiospeum hungaricum (Soós, 1927)) has been protected by law. A serious impediment for conservation biology is that our knowledge of species is only limited, beginning with poor taxonomic descriptions. The aim of this study is to bridge this gap at the most basic level. We morphologically redescribe and present phylogenetic relationships of two amphipod species from the genus Niphargus, both endemic to this area.
Niphargus molnari Méhely, 1927 was described from the stream of the Mánfai-kőlyuk Cave (Méhely 1927). The description is not detailed, as it contains only a few information about the body lenght, the pereonits, the pleon segments, the first antenna, the uropods and the telson, and two drawings about the epimeral plates and the pereion segments. Further drawing of the right lacinia mobilis can be found in Méhely's summarizing work (Méhely 1941). At approximately the same period the species was also studied by Schellenberg, who analysed samples fom Abaligeti Cave. In his early study he first treated it as N. leopoliensis molnari (Schellenberg, 1933), but later he acknowledged its species status and supplemented description with data about the seta number of the palpus of the first maxilla (Schellenberg 1935). The species was found in the Mánfai-kőlyuk Cave (Gebhardt 1933(Gebhardt , 1934(Gebhardt , 1963(Gebhardt , 1967 and in the stream of the Abaligeti Cave too (Gebhardt 1934(Gebhardt , 1963(Gebhardt , 1967. Recently, the species was found in other two localities, the Spirál Sinkhole and the Vadetetős Sinkhole (Angyal and Balázs 2013). During our research in the caves of the Western Mecsek between 2010 and 2013, the species could not have been re-collected on the type locality, which is supposedly related to the artificial utilization of the Mánfai-kőlyuk Cave. The intrusive introduction of waterworks in the 1960-s and 1970-s has caused irreversible changes in the cave's character, hidrology and ecosystem (Angyal 2012).
Niphargus gebhardti Schellenberg, 1934 was described from the pools formed by dripping water of the Abaligeti Cave, originally as Niphargus foreli gebhardti (Schellenberg 1934). Brief description reports on only few characters, like the pereopods, the antennae and the mouth parts, and two drawings about the second gnathopod's propodus and the telson. Later the author gave additional data on the body length and the telson (Schellenberg 1935). Gebhardt mentioned the species'distribution from pools of the Abaligeti Cave's main passage in various papers (Gebhardt 1934(Gebhardt , 1963(Gebhardt , 1967. The species rank was proposed for the first time in Méhely's synthetic work (Méhely 1941), wherein a drawing of the pleopod's retinacles and some data about the lacinia mobilis are also presented. Dudich (1941) discussed 'Niphargus foreli gebhardti' from the Abaligeti Cave as a fauna element of the historical Hungary. More recent sampling revealed new records of the species from Vadetetős Sinkhole, Szajha-felső Sinkhole, Spirál Sinkhole, Gilisztás Cave and Trió Cave (all Mecsek Mts.; see Angyal and Balázs 2013).
The holotypes of both species are either in an unknown place or had been destroyed. Although we identified the distinguishing characters of N. gebhardti and N. molnari, and presented comparative drawings of them (Angyal and Balázs 2013), the morphology of both species is unsuficiently known and cannot be used in a broader comparative research of Niphargus. In order to follow modern trends in taxonomy, we revised all possible sources of data that might increase the robustness of taxonomic conclusions (Padial et al. 2010). We provide a detailed and richly illustrated redesription of N. molnari and N. gebhardti with cytochrome c oxidase subunit I (COI) sequences as barcodes. We also present comparative scanning electron micrographies which are -to our knowledge -the first comparative micrographies of Niphargus. Moreover, we present phylogenetic relationships of both species within the genus Niphargus using three independent molecular markers and summarize field observations that may indicate species' ecology.

Sampling sites and sampling
Samples for the redescription were collected in the Abaligeti Cave (N46°8'11.89", E18°6'59.40"), which is located in Southern Hungary, Western Mecsek in Abaliget village, near Pécs city. The altitude of the cave entrance is 219 m above sea level. With its three collaterals and the main passage, the total length of the cave is 2000 m. Its lowest point below the entrance is 10 m, while its highest point is 38 m. Shallow pools of water in the cave are of two types: some are formated by dripping water of the dripstones whereas others are filled during floods and contain residual water. The cave was regulary visited between 2010 and 2013 to characterize its fauna. For the morphological and molecular taxonomic analysis in total 18 and 20 specimens of N. molnari and N. gebhardti respectively were collected on 23 March 2013. Niphargus molnari was found in the stream of the Western 2. collateral and N. gebhardti was collected from a permanent pool in a lateral chamber of 'Karthago romjai' hall in the main passage and from a pool at the end of Western 2. collateral, near Akácos Sinkhole's entrance (Fig. 2). An additional specimen of N. gebhardti for molecular studies was collected from a pool of the Szajha-felső Sinkhole (46°8'5.4"N, 18°7'8.22 E) 30 m vertical distance and 100 m horizontal distance from the entrance. The cave is situated in the area of a platform right above the Abaligeti Cave, 283 m above sea level. The two caves are supposedly connected, their entrances are approximately 1 km from each other (Dezső 2011). Specimens were collected using entomological (soft) forceps and were fixed and stored in 96% ethanol.

Morphological studies
Cleared and stained exoskeletons of 10 (N. molnari) and 11 (N. gebhardti) specimens were dissected under a Leica MZ75 and a Leica M125 stereomicroscope. Slides were examined using a Leica DM 1000 light microscope. Drawings were made using a drawing tube mounted on the light microscope. Measurements were made using the AnalySIS Program Package, the computer was connected with a Zeiss Axioscope II light microscope. In total 230 morphological characters on each speciemens were examined according to the characters of the DELTA program package ) which were recorded in an Excel data matrix. Scanning micrographs of two individuals of each species about the main characters were made with a HITACHI S-2600 N scan-ning electron microscope. Specimens were placed in absolute alcohol for one day, then cleaned in an EMAG Emmi-16 Ultrasonic Cleaner and dried out on air. Dry samples were sticked onto holders and were sputter-coated by gold-palladium. Micrographs were digitally edited.

Molecular studies
DNA extraction was performed using QIAamp DNA Microcit® (Qiagen) or Sigma Aldrich GenElute Mammalian Genomic DNA Miniprep Kit® following the manufacturer's instructions. Only a few pereopods were used for DNA isolation of each animal. The following primer pairs were used for PCR amplifications of COI, 28S rDNA fragment and histone (H3). For COI: LCO 1490 -HCO 2198 (Folmer et al. 1994), for 28S rDNA: 28S lev2 -28S des2 or 28S rtest2 (Verovnik et al. 2005, Zakšek et al. 2007) and H3aF2-H3aR2 (Colgan et al. 2000) for histone (H3). Details on PCR conditions are listed in Suppl. material 1. PCR products were cleaned using Roche High Pure Purification Kit® or Exonuclease I and Alkaline Phosphatase (Fermentas, Germany) according to manufacturer's instruction. The fragments were sequenced in both directions using PCR amplification primers using ABI 3130 sequencer in the Laboratory of Molecular Taxonomy in Budapest or Macrogen Europe (Amsterdam, The Netherlands). Contigs were assembled and sequences were edited using Geneious Pro 5.5.6. (Biomatters, New Zeland).

Phylogenetic analysis
In order to recover phylogenetic relationships of N. molnari and N. gebhardti within the genus Niphargus, a dataset of three molecular markers were complied, using available Niphargus sequences from previous studies (see Suppl. material 2 for references) and Synurella ambulans as outgroup taxon (Švara et al. submitted, Meleg et al. 2013). Altogether 104 taxa were included in the final dataset. List of taxa and sequences with GenBank accession numbers used in the analyses are listed in Suppl. material 2. The sequences were aligned using MAFFT 7 (Katoh and Standley 2013). Each sequence alignment was concatenated to the joint dataset and analysed as a single dataset in phylogenetic analysis. The length of combined dataset, including sequences of COI, 28S rDNA and H3 was 2068bp. A general time-reversible model with a proportion of invariant sites and a gamma distribution of rate heterogeneity (GTR+I+Γ) assuming six discrete gamma categories was chosen as the most appropriate model according to AIC and BIC criteria, using ModelGenerator (Keane et al. 2006). Phylogenetic relationships were reconstructed with Bayesian inference (BA) using MrBayes v3.2 (Ronquist and Huelsenbeck 2003). Two parallel searches with four chains each were run for 20 million generations, sampled every 1000 th generation. After discarding the first 25% of the sampled trees, the final tree was constructed according to the 50% majority rule. MrBayes phylogenetic analysis was run on the CIPRES Science Gateway, www.phylo.org (Miller et al. 2012). Méhely, 1927 Order Amphipoda Latreille, 1816 Suborder Gammaridea Latreille, 1802 Family Niphargidae G. Karaman, 1962 Genus Niphargus Schiödte, 1849
Maxilla II inner lobe slightly smaller than outer lobe; both of them setose apically and subapically, number of setae is approximately 13-23 per lobe (Fig. 5).
Inner lobes of labium longer than half of outer lobes (Fig. 12).
Maxilla II inner lobe slightly smaller than outer lobe; both of them setose apically and subapically, number of setae is approximately 6-11 on inner lobe and 8-12 on outer lobe (Fig. 12).

Comparison with phylogenetically related and geographically close species
N. molnari and N. gebhardti share few main traits (the same body size class, slender body, sexually dimorphic uropod III but not uropod I), but differ from each other in the shape of epimeral plates, the size of gnathopod propodi, in denticulation of spines on outer lobe of maxilla I and in the number of retinacles (Angyal and Balázs 2013). Keeping these differences in mind we compare both species to the species that are either closely related according to molecular phylogeny, or to the species that live in the same geographic area.
Niphargus vadimi Birstein, 1961 is known from Crimea. Despite its close position suggested by the presented molecular tree, this species differs from phylogenetically related N. gebhardti and non-related N. molnari in considerably larger body size and much larger gnathopods.
High morphological similarity to the focal pair of species reveal another four species phylogenetically related to N. gebhardti, namely Niphargus bihorensis Schellenberg, 1940, Niphargus fongi Fišer & Zagmajster, 2009, Niphargus carniolicus Sket, 1960, and Niphargus dobati Sket, 1999. Epikarstic N. bihorensis is known from Romania and Italy, whereas the latter three are known from epikarst and karst river beds from Slovenian caves. All four species share with focal species main traits (body size, slender body, sexually dimorphic uropod III but not uropod I).
N. bihorensis and N. fongi differ from the focal species in the shape of gills (being narrow instead of ovoid as in focal species) and in higher number of retinacles on pleopods. In addition, N. fongi differs from N. molnari and N. gebhardti by (i) the elevated number of setae along posterior margin of epimeral plate III, (ii) the longer apical telson spines, (iii) and the reduced number of denticulated spines in palmar corners of both gnathopods. N. bihorensis, which is a complex of at least two morphologically indistinguishable species (Meleg et al. 2013), differs from the focal species by (i) reduced number of spines on maxilla I outer lobe (only 6), (ii) more numerous setae on maxilla I palpus (7-8), (iii) and by more numerous retinacles.
N. carniolicus and N. dobati differ from the focal pair of species in the length of rami of uropod I (expopodite equal to or slightly longer than endopodite versus exopodite consistently shorter to endopodite in focal species). In addition, N. carniolicus differs from N. molnari and N. gebhardti by (i) shorter apical spines on telson, and (ii) fewer denticulated spines on palmar corner of gnathopods. N. dobati differs from the two focal species by (i) the elevated number of spines on uropod I basipodite, (ii) the length of pereopod V and VI (which are longer comparing with pereopod VII), and the (iii) elevated number of mandibular palp 'D seta'.
Phylogenetic relationship of N. molnari to the rest of Niphargus species is not clear, however a few morphologically similar species, like Niphargus schellenbergi S. Karaman, 1932 are known. It differs from N. molnari and N. gebhardti by (i) the differently ornamented telson (5-7 long apical spines and 2-5 lateral spines in N. schellenbergi, respectively), (ii) more numerous apical setae on uropod III endopodit, (iii) elevated number of pleopod retinaculi, (iv) by the length of uropod I exopodite, which is slightly longer than endopodit, (v) by several setae along outer margin of gnathopod dactyli, and (vi) by bigger body size (>10 mm).
The following species are compared with N. molnari and N. gebhardti due to their geographical vicinity. Niphargus forroi G. Karaman, 1986 was described from Northeast Hungary, and is known from only a couple of caves from the Bükk Mts. Beside the close body size, N. forroi agree with N. molnari by the similar seta numbers and arrangement on the gnathopods, by the telson spine-pattern, as well as by the number of different spine and seta types on pereopod dactyli. N. forroi differs from N. molnari by (i) the subrounded posteroventral corner of the epimeral plates, (ii) the lower number of mandibular palp 'D setae' and by (iii) the reduced number of maxilla distal article apical seta. N. forroi differs from both N. molnari and N. gebhardti by the number of posterior margin setae on pereopods V-VII. The description of Niphargus hungaricus Méhely, 1937 (endemic species of the Kőszegi Mts.) contains no drawings and not enough characters that would be needed for proper comparison. A later work of Méhely (1941) is only partially filling this gap by containing a drawing on the first gnathopod and some additional data on its seta arrangement. According to the available information, N. hungaricus differs from N. molnari and N. gebhardti by (i) the setae number of gnathopods dactyli outer margin (always more than 1 seta of N. hungaricus) and by (ii) the length of male's uropod I endopodite (inner ramus is elongated and two times long as outer ramus in N. hungaricus). There are different Niphargus populations in the Bükk Mts. and in the Aggtelek Karst belonging to the Niphargus tatrensis Wrzesniowsky, 1888 species group including Niphargus aggtelekiensis Dudich, 1932. Although the taxonomic status of these populations is not clear, the complex shares several distinct morphological characters that can be compared with the focal species. Populations of N. tatrensis -N. aggtelekiensis complex differ from N. molnari and N. gebhardti by (i) larger body size (>15 mm), (ii) the elevated number of setae along outer margin of gnathopods dactyli (there are more than one), (iii) the lower mandibular 'A' and 'D seta' number and (iv) the elongated distal article of uropod III of both gender. Main diagnostic characters are presented in Table 1.

Molecular taxonomy
Phylogenetic relationships within the genus Niphargus (Fig. 17) showed that the two redescribed species of Niphargus from Hungary are not phylogenetically closely related. Phylogenetic relationship of N. molnari to the rest of Niphargus species is unclear; species is nested within basal polytomy. N. gebhardti belongs to the clade of Central to Eastern European species. The focal species is in sister relationship with a pair of morphologically cryptic species endemic to Western Carpathian (N. bihorensis, see Meleg et al. 2013). Other closely related species include N. vadimi from Crimea, Pontoniphargus racovitzai from Eastern Romania and a clade of epikarstic and interstitial species from Southern Slovenia (N. fongi, N. carniolicus, N. wolfi and N. dobati

Remarks on ecology and distribution
Among the studied two species, N. gebhardti was collected more frequently, as it was found in five other caves of the Western Mecsek in addition to the type locality, namely Trió Cave, Gilisztás Sinkhole, Szajha-felső Sinkhole, Vadetetős Sinkhole and Spirál Sinkhole (Fig. 18). In most of these, two types of water bodies exist: i) small pools of residual-or percolated/dripping water and ii) streams or minor streaming water. Amount of water in the caves is dependent on the rainfall in the surface. In all six caves, N. gebhardti specimens were found in isolated, shallow pools in limestone, sinter or clay, most likely formed by dripping water (Fig. 19). Specimens were never observed in streams or any other streaming waters. During our repeated visits between 2010 and 2013 (altogether 24 visits in the 6 caves), the same pools were checked every time and some specimens were always found in them (except when the pools dried out). Once it was observed that a group of N. gebhardti (approximately 20 specimens) were fed upon a dead Oxychilus snail in a pool.
N. molnari was observed in the Abaligeti Cave and in two sinkholes that the other species (N. gebhardti) was also inhabited, Spirál Sinkhole and Vadetetős Sinkhole (Fig. 18). Density of N. molnari was high in the stream of the Western 2 collateral of the Abaligeti Cave, however in the other two caves only a few specimens were found in streaming water, always in deeper parts of the caves. The two species were always spatially well segregated. In the Abaligeti Cave N. molnari coexisted with Protelsonia hungarica Méhely, 1924 (endemic aquatic troglobiont isopod of the cave) and with the troglomorph specimens of Gammarus fossarum Koch, 1836.

Discussion
Due to its protected geographical situation, since the Tertiary, the area of Mecsek may have played a refugial role during the alternating warmer and colder eras, preserving old lineages of Crustaceans. They presumably ensconced into subterranean aquatic habitats from searing creaks of the Paratethys Sea, that encompassed the islands of the Mecsek. Then, by degress, they had been adapted to the subterranean conditions in both physiological and morphological features (Méhely 1925). According to results of our phylogenetic analysis, N. molnari and N. gebhardti represent completely distinct lineages, which colonized the Mecsek area independently. The two species are spatially segregated within the same caves. N. gebhardti inhabits isolated pools of stagnant water, which fed by percolating water from the limestone fissures, so called epikarst. Interestingly N. gebhardti is apparently phylogenetically related to epikarstic species from Slovenia. On the contrary, N. molnari was always found in streaming waters.
The distribution range of the two endemic species is small, the most distant caves are seven kilometers far. These caves belong to three different catchment areas (Fig.  18). Despite of our repeated visits and careful searching, Niphargus specimens were not found in the Mánfai-kőlyuk Cave. N. molnari supposedly has gone extinct in its type locality as it is ruined due to the industrial utilization of the cave (Angyal 2012). Moreover, the type locality of N. gebhardti -which is a touristic cave with 80.000 annual visitors -may be also endangered. Considering the extremely narrow distributional range of the two species and the vulnerability of their populations, N. molnari and N. gebhardti are suggested to be placed into the 'Vulnerable (VU)' category according to the following criteria of IUCN Red List of Threatened Species (IUCN 2012): i) number of locations is ≤ 10 ('B2') and ii) area of occupancy is less than 20 km 2 ('D2'). ' Hidrologically connected caves are in quadrats.

Conclusions
Some highly endemic, troglobiont invertebrate taxa are known from the Southern Hungarian Mecsek Mts. Two of them, the blind amphipod Niphargus molnari Méhely, 1927 and Niphargus gebhardti Schellenberg, 1934 have been rediscribed, applying the modern approach of integrative taxonomy. Comparative scanning electron microscopy used for first time on niphargids, and it proved to be a rather useful method in analysing and illustrating of barely visible diagnostic characters. As contributions to the future molecular genetic studies on niphargids, cytochrome c oxidase subunit I (COI) sequences as barcodes of N. molnari and N. gebhardti are now available for the public. The phylogenetic analyses have shown that the two species -which are spatially segregated in caves where they coexist -represent completely distinct lineages and may have colonized the Mecsek area independently. Phylogenetic relationship of N. molnari to the rest of Niphargus species is for the present not clear. N. gebhardti is closely related to a clade of epikarstic species from Southern Slovenia and to cryptic species endemic to Western Carpathians. New localities of both species have been found. The two species are suggested for legal protection, they should be listed into 'Vulnerable' category of the IUCN Red List of Threatened Species.
linguistic checking of the manuscript. We also thank Dr. John Holsinger, Dr. Ronald Vonk and Dr. Oliver Coleman for their helpful and constructive commentaries that remarkably improved the early version of the manuscript.