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Article

Not the Last Piece of the Puzzle: Niphargus Phylogeny in Hungary

by
Gergely Balázs
1,2,*,
Špela Borko
3,
Dorottya Angyal
4,
Valerija Zakšek
3,
Anna Biró
1,2,5,
Cene Fišer
3 and
Gábor Herczeg
1,2
1
ELKH-ELTE-MTM Integrative Ecology Research Group, 1117 Budapest, Hungary
2
Department of Systematic Zoology and Ecology, Institute of Biology, ELTE Eötvös Loránd University, 1117 Budapest, Hungary
3
SubBio Lab, Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia
4
Calle 31 #436A x 36 y 36A Colonia Jesús Carranza, Mérida 97109, Mexico
5
Doctoral School of Biology, Institute of Biology, ELTE Eötvös Loránd University, 1117 Budapest, Hungary
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(2), 223; https://doi.org/10.3390/d15020223
Submission received: 22 December 2022 / Revised: 17 January 2023 / Accepted: 30 January 2023 / Published: 3 February 2023
(This article belongs to the Special Issue Recent Advances in the Diversity and Taxonomy of Subterranean Arthropods)
Browse Figures
Versions Notes

Abstract

:
The Palaearctic genus Niphargus is a promising model system to understand subterranean fauna genesis in Europe. The Pannonian Plain (mainly covered by Hungary) in Central Europe, once being the area of the Paratethys, is a key area for Niphargus diversification. However, our knowledge on Hungarian species of Niphargus is primarily based on sporadic taxonomical works from the pre-molecular era. Here, we studied 14 localities, covering the eight valid Hungarian species of Niphargus and including nine previously unstudied populations. Based on sequences of three gene fragments, we reconstructed their phylogeny using maximum likelihood and Bayesian approaches. We found that not all Hungarian species of Niphargus are closely related, and even species sampled at the same localities can belong to different clades. Some Hungarian species form monophyletic clades, while others are nested in various non-Hungarian lineages. The new populations are all genetically distinct from the known species. Our results suggest that the Hungarian Niphargus fauna has originated from seven unrelated clades and its diversity is underestimated due to unknown populations and cryptic species. The detection of genetically distinct species of Niphargus from non-carbonate regions calls for further research efforts. The high diversity and the number of putative new species in the N. tatrensis clade warrants further, high-resolution phylogenetic studies.

1. Introduction

The genus Niphargus Schiødte, 1849 (Crustacea: Amphipoda) is the most diverse subterranean amphipod genus [1]. This taxon with more than 400 known and numerous undescribed species is the most salient element of the subterranean fauna in the Western Palaearctic [2,3]. The genus is an important model system that may help answer several questions addressing the origin of subterranean fauna in Europe. Niphargus originated in an area that nowadays belongs to Western Europe, and subsequently dispersed eastward, with at least five radiations [4,5]. High species richness of the genus can be attributed to speciation that followed the colonisation of new ecological niches [6], but also to fragmented karstic systems that enabled speciation within the same environment in micro-allopatry [7], eventually resulting in numerous morphologically indistinguishable or very similar species, called cryptic species [8,9]. Bursts of speciation can be tentatively attributed to the colonisation of the novel regions, such as karstic masses that emerged from ancient seas, or to major geological events that reshuffled dispersal routes [10].
Accurate reconstruction of the speciation and biogeography of the genus, however, depends on completeness of the taxonomic inventory. The taxonomic structure of the genus is far from resolved. The results of the attempted large-scale taxonomic revisions of the genus evidently showed [4,11,12] that smaller-scale local revisions are needed [13], as they can point out taxonomic uncertainties, so revealing important taxa missed in large-scale studies. Molecular studies imply that on average every described species in fact comprises two-three cryptic, yet undescribed species, which often show extremely limited distribution [11]. Sampling effort is highly uneven across the range of the genus. While some regions have been thoroughly sampled, some countries remained generally underexplored, or were not explored with molecular methods. Insufficient sampling is particularly critical in regions with turbulent geological history that in the past acted as major dispersal routes or speciation centres. Such are the Pannonian lowlands and some karstic regions within it, which belonged to the ancient Paratethys. The current area of Hungary occupies the central area of the former Pannonian Sea, which was part of the ancient Paratethys. The regression of the Paratethys prompted speciation in surface waters [14,15,16] and after the Pannonian Sea got isolated from it and became the shallow Pannonian Lake [17], it most likely remained a dispersal route for aquatic animals [18].
Research on Niphargus in Hungary had its peak in the first half of the 20th century when most of the taxonomic works along with species descriptions were published e.g., [19,20,21,22]. In the following decades, the number of publications declined and only one species description was published [23]. During this period, studies on Niphargus were flourishing at the international level and our knowledge about the genus increased considerably along with the number of species and the number of morphological traits used for species identification [24]. A previous study based on literature data and morphotaxonomy [25] showed that although there are more than 20 species of Niphargus reported from Hungary [22,26], only eight are currently valid: N. tatrensis (Wrzesniowsky, 1890); N. molnari Méhelÿ, 1927; N. aggtelekiensis Dudich, 1932; N. hrabei S. Karaman, 1932; N. valachicus Dobreanu and Manolache, 1933; N. gebhardti Schellenberg, 1934; N. hungaricus Méhelÿ, 1937; N. forroi G. Karaman, 1986. Most of the taxonomic work on Hungarian species of Niphargus had been done prior to genetic methods becoming available. Therefore, the insights into genetic diversity and phylogenetic structure of the Hungarian species are limited to a small number of species [15,27,28].
In the present paper, we studied the phylogenetic relationships of the known Hungarian taxa of Niphargus (including six subterranean and two surface-dwelling species). We also included numerous new samples representing previously unknown Hungarian populations. We were particularly interested in whether (i) the Hungarian species of Niphargus are closely related, or belong to phylogenetically distinct clades, (ii) the clades incorporating the Hungarian species/populations are exclusive to Hungary, or the Hungarian species are members of clades with larger distribution and (iii) the new populations belong to known species or are genetically distinct from them.

2. Materials and Methods

2.1. Sample Collection

Most of the valid Hungarian species (N. molnari, N. gebhardti, N. aggtelekiensis, N. hungaricus, N. forroi) were described from Hungarian type localities. To secure species identity, we sampled at type localities of the valid species where it was possible (Figure 1 and Table 1). In the case of two out of the above five species, sampling at the type locality was not possible. The type locality of N. hungaricus is the Jávor Spring (KőszegMts) [29], but due to changes in the morphology of the spring orifice, the local population became physically inaccessible. Therefore, we collected samples from the closest known location, the Borha Valley mine tunnel situated 300 m from the spring. The type locality of N. molnari is the Mánfai-kőlyuk Cave where the species is not present anymore, most probably due to artificial changes in connection with the utilisation of the cave as a water reservoir [30]. Therefore, a sample was collected from the closest known location, the Abaligeti Cave [27], which is 7.5 km away. In the case of N. aggtelekiensis, besides from the type locality (Baradla-Domica Cave System), we also collected samples from the hydrologically separate, yet geographically close Rákóczi No. 1 Cave. In the case of the two surface-dwelling species that have wide distribution ranges (N. hrabei, described from Slovakia [31], and N. valachicus, described from Romania [32]) we collected samples from known Hungarian locations (one sample per species). According to previous studies, these species show very limited genetic variation throughout their range [15,28], therefore we assumed that the sampling locality does not affect our results. The occurrence of N. tatrensis in Hungary is doubtful [25]. In this case we included a sample from the Kecske-lyuk Cave in the Bükk Mts, which was identified as N. tatrensis based on morphological characters. We also included samples from two undescribed, but morphologically distinct species known only from the Molnár János Cave. The sampling of valid Hungarian species and the undescribed species inhabiting the Molnár János Cave was carried out between 2013 and 2016 (for details see [15,25,27,30,33]). Ever since the beginning of studies on Niphargus in Hungary [34], the research focus was always on caves, thus almost all the species of Niphargus described from Hungary (with the sole exception of N. hungaricus) are known from karstic areas. To get a more detailed view on the Niphargus fauna of the country, we carried out extensive sampling in non-karstic mountainous areas too. Between 2016 and 2022, we visited over 80 mines and springs in the Visegrád Mts, Börzsöny Mts, Mátra Mts, Balaton Uplands, Bakony Mts and Zemplén Mts. This effort resulted in sites with new findings of Niphargus out of which—based on morphological traits and/or preliminary genetic data—we detected putative new species to science. Samples representing these putative new species to science (from Dömös, Vasbánya Spring, Kánya Spring, Werbőczy Spring, Gejzír Spring 1, Gejzír Spring 2) were also included in the present analysis. Collected adults were stored in 96% ethanol at 10 °C until DNA extraction.

2.2. DNA Extraction, PCR, and Analysis

We extracted genomic DNA with QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) or GenElute Mammalian Genomic DNA Miniprep Kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s specification using the pereiopods and antennae of the animals. We amplified the fragments of three genes: two fragments of 28S rRNA gene, the histone 3 subunit A (H3), and a fragment of mitochondrial cytochrome oxidase I (COI). The fragments of 28S rRNA gene were amplified using primers 28Slev2, 28Sdes2 [35], 28Slev3, and 28Sdes5, and the corresponding internal primers [36]. The histone H3 gene was amplified using primers H3AFR and H3AR2 [37], and COI was amplified using primers LCO1490 and HCO 2198 [38] applying PCR cycler settings as described in Angyal et al. [27]. The PCR products were purified using Roche High Pure Purification Kit (Merck, Darmstadt, Germany) or Exonuclease I and Fast AP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced at Macrogen Europe (Amsterdam, Netherlands) or at the Molecular Taxonomy Laboratory of the Hungarian Natural History Museum (Budapest, Hungary) using the same primers as for amplification. The chromatograms were assembled and edited in Geneious 11.0.3 (Biomatters Ltd., Auckland, New Zealand). To identify the phylogenetic relationships of the species of Niphargus from Hungary we compiled a dataset comprising 121 specimens (including Carinurella paradoxa Sket, 1964, Haploginglymus geos Jurado-Rivera, 2017, and Haploginglymus morenoi Ianilli, Minelli and Ruffo, 2009) that well cover the phylogenetic diversity of Niphargus in the entire distribution range [3,39]. Pseudoniphargus gorbeanus Notenboom, 1986 was used as an outgroup [5]. The collected material is deposited at the Department of Systematic Zoology and Ecology, Eötvös Loránd University, Budapest, Hungary. The list of the species studied, origin of samples, and the GenBank accession numbers are available in Supplementary Material, Table S1.
We aligned the sequences in MAFFT 7.388 [40] using the E-INS-I algorithm with the scoring matrix 1PAM/k = 2 and with the highest gap penalty. Alignments were concatenated, partitioned by gene and codon position, and the optimal substitution model for each partition was chosen using Partition Finder 2 [41,42] under the corrected Akaike information criterion (AICc). The optimal substitution models were GTR+I+Γ for the second and third codon position of COI and the first and third codon position of 28S 22 and H3; SYM+I+Γ for the first codon position of 28S 35 and COI and JC+I for the second codon position of H3. We reconstructed the phylogenetic relationships with Bayesian inference (BI) in MrBayes v3.2.6 [43], where we used the optimal substitution models inferred with Partition Finder; and the maximum likelihood (ML) method in IQ-TREE 2.2.0 [44], where we used built-in automatic best fit substitution model search [45].
A Bayesian MCMC tree search was run for 20 million generations with two independent runs with four chains for each run. Trees were sampled each 2000th generation. After reaching the stationary phase, the first 25% of trees were discarded as burn-in, and from the remaining trees we calculated the 50% majority rule consensus tree. The ML phylogenetic analysis was run using simultaneous optimisation of substitution models, with ultrafast bootstrap approximation (UFBoot; [46]). Support values were calculated using the Shimodaira–Hasegawa approximate likelihood ratio test (SH-aLRT) approach [47]. Phylogenetic analyses were run on the CIPRES Science Gateway (https://www.phylo.org; [48]).

3. Results

Phylogenetic relationships of the species of Niphargus from Hungary showed that most are not closely related and belong to different clades, sometimes even if they are from the same locality (Figure 2). Some of the Hungarian species form monophyletic clades, while others are nested in various non-Hungarian lineages. The structure of the ML (Figure 2A) and Bayesian tree (Figure 2B) differs considerably reflecting weak support among the well-supported clades. Sister relationships among the species are generally well-supported and congruent in both trees. The structural differences and uncertainties are most probably the results of a lack of genetic information at certain splits, which were reconstructed differently (albeit with no support) in both approaches [49]. Although the phylogenetic position of larger clades that include Hungarian species remain unclear, some cautious conclusions can be drawn based on our results.
Below we overview the results of phylogenetic analysis clade by clade. The first clade comprises N. molnari from the Mecsek Mts, two undescribed species from the Molnár János Cave, Budapest and two species hitherto not recorded from Hungary, Niphargus kieferi Schellenberg, 1936 and N. inopinatus Schellenberg, 1932.
Niphargus gebhardti, another species from the Mecsek Mts, is nested within a clade which includes species scattered from Italy to the Crimean Peninsula.
Five samples, (N. tatrensis from Kecske-lyuk Cave (Bükk Mts), Gejzír Spring 2 (Zemplén Mts), Vasbánya Spring (Börzsöny Mts), and two samples of N. aggtelekiensis) are part of the N. tatrensis clade together with many other specimens from Slovenia, Austria and Slovakia, including the relatively recently described species from the N. tatrensis species group (Niphargus scopicauda Fišer, Coleman, Zagmajster, Zwittnig, Gerecke and Sket, 2010, N. salzburgensis Schellenberg, 1935, N. moogi Stoch, Christian and Flot, 2020). The specimen identified as N. tatrensis living in the Kecske-lyuk Cave is clearly separated from N. tatrensis from its type locality (labelled as N. tatrensis A), indicating that the Hungarian population belongs to a new putative species. The position of N. aggtelekiensis from the type locality (N. aggtelekiensis A Baradla cave) further confirms the valid status of the species. Interestingly, our other N. aggtelekiensis sample from the Rákóczi No. 1 Cave shows clear genetic differentiation from the topotype. The Gejzír Spring 2 and the Vasbánya Spring samples are both from taxa that require further investigations.
Four samples, (N. forroi, Gejzír Spring 1 (Zemplén Mts), Kánya Spring (Mátra Mts), and Werbőczy Spring (Mátra Mts)) form a well-defined and separate clade together with a taxon from Southeast Slovakia (N. loc. Brdo). Genetic distinctness of the three Hungarian populations indicate that each of them represents most likely a new putative species.
Niphargus hungaricus and the sample from Dömös mine tunnel (Visegrád Mts) form a well-supported monophyletic clade, although its position in the entire phylogeny remains uncertain, hence the sister clades and the origin of the clade cannot be defined.
The position of the two surface-dwelling species, N. hrabei and N. valachicus, proves their different origins, which is in accordance with the results of previous studies [15,28].

4. Discussion

The result of the first genetic insights into Hungarian species of Niphargus brought up several important conclusions. First, the Hungarian Niphargus fauna is phylogenetically diverse. Species originated in seven unrelated clades. Most of these clades show a west-east distribution. This pattern was probably shaped by the geological history of the Paratethys (N. tatrensis, N. gebhardti and N. molnari clades, see [50] and [5] for comparisons). Second, the diversity of Niphargus in Hungary is underestimated. We detected almost twice as many (putative) species of Niphargus than known from the most recent checklist [25]. Some species show distinct morphology (e.g., species from the Molnár János Cave), but some are morphologically similar (e.g., N. aggtelekiensis from the Rákóczi No. 1 Cave) and should be taxonomically evaluated in focused molecular studies. Third, the detection of Niphargus in non-carbonate regions calls for additional sampling. We have no samples from riverine interstitials, a habitat type that should be systematically studied in the future. Moreover, sampling in carbonate and non-carbonate regions—despite our effort—might be far from exhaustive and should be intensified to properly define species distributions and their population structure.
Phylogenetic analysis with the present data has certain limitations. The position of the clades with Hungarian taxa of Niphargus is in many cases weakly supported and in this respect, considerable differences can be observed between the ML and Bayesian trees. Consequently, we cannot define the absolute position of the Hungarian taxa within the genus. Our phylogeny trees contain weak support values in many cases, especially at basal splits. We can think of various reasons behind the low values. In general, we can assume that by increasing the number of samples and the number of genes, better results can be expected. This is only true if the markers are representing similar phylogeny, and there is no considerable difference in the speed of genetic changes between lineages [51,52,53]. Moreover, by including more genes, the amount of phylogenetically noninformative information could increase by increasing the chance for inclusion of genes under environmental selection. In such case, the analysis would result in well-supported, but incorrect trees [54]. Although it is possible that we could obtain better results by using other or additional markers, our options were rather limited as the markers we could use were strongly determined by the already available sequences for comparison. More even sampling distribution is also a good way to improve tree reliability [55,56]. Unfortunately, in the case of subterranean species, this possibility is severely constrained due to the limited number of known and accessible locations. Despite these limitations, the composition of the clades with Hungarian samples are well supported in most cases and consistent on both the ML and Bayesian trees. Taken together, we can state that even though the results of our analysis cannot be used to draw unquestionable conclusions about the phylogeny of the studied species, they can provide solid background for further, more specific studies.
The clade including N. molnari and the two species from the Molnár János Cave contains two other species. N. kieferi is a widely distributed species that lives in groundwater habitats in Germany and France, while N. inopinatus also lives in non-karstic habitats from Germany to Slovakia [57] (Figure 3). Both species have wide distribution ranges and were found in interstitial habitats in the Danube basin and could be expected in Hungary as well.
Niphargus gebhardti is clustered together with species from Crimea (N. dimorphus Birstein, 1961, N. vadimi Birstein, 1961), Romania (N. bihorensis Schellenberg, 1940), and Italy (N. ambulator G. Karaman, 1975) that form a well-defined clade. Interestingly, although the clade covers a wide geographic range, the occurrence of its members is limited to a narrow latitudinal range between 44° and 46° (Figure 3). The sister clade to the N. gebhardti clade, containing species from Slovenia, Bulgaria, Romania, and Iran is also scattered along a west-east line. The distribution of the species in the N. gebhardti clade and the species in the sister clade suggest the North Dinaric origin of the lineage.
The clade which contains samples identified as N. aggtelekiensis and N. tatrensis from Hungary exhibits many uncertainties, yet its members are all from the Western and Eastern Carpathians (Figure 3). Niphargus aggtelekiensis from the type locality is assuredly separated from other taxa, but the other supposed N. aggtelekiensis sample from the nearby Rákóczi No 1 Cave also appears to be genetically distinct. While the geographical distance between the two locations is only 15 km, the genetic distance is quite substantial (16% uncorrected distance for COI). Although genetic distance on a mitochondrial marker alone is not sufficient to draw any firm conclusions, this result certainly calls for further investigation. The positions of the relatively recently identified species from the N. tatrensis group (N. scopicauda, N. moogi, N. salzburgensis) are not surprising and are in accordance with previous studies [46,54]. Although the phylogenetic information provided by this study on the other Hungarian samples in the N. tatrensis clade is not sufficient to make secure taxonomic conclusions, we believe that some remarks are worth mentioning. While the taxonomic status of N. tatrensis from the Bükk Mts is unclear, we can state that the population living in the Kecske-lyuk Cave does not belong to the N. tatrensis. The samples from Gejzír Spring, Zemplén Mts (Gejzír 2) and Vasbánya Spring, Börzsöny Mts most probably also represent putative new species. The sample from Vasbánya Spring is genetically almost identical to Niphargus found near Bratislava, West Slovakia [58]. In general, many unidentified taxa can be found in the N. tatrensis clade. Their relative position to the topotype suggests that some Northern and Western Carpathian samples included in our analysis might represent additional putative new species. Taken together, we can say that while the Hungarian samples turned out to be important pieces in the N. tatrensis clade, unfortunately, our results have only further complicated the long-lasting problematic taxonomy of the group [50,59]. Based on our results, we think that the solution to overcome the difficulties is to increase the resolution with denser sampling in smaller geographical areas and to use higher number of genetic markers in future studies.
The N. forroi clade appears to be well-defined on both trees. It contains N. forroi from the type locality and four putative new species. It is worth mentioning that the geographical distance between Kánya Spring and Werbőczy Spring is only 700 m, yet the two samples show genetic distinctness. In one of his works, Méhelÿ [22] mentioned a Niphargus species he found in the Mátra Mts and named it N. matrensis. It is possible that one of the taxa revealed in our study refers to this species, yet, as the author did not provide a morphological description and precise type locality, the species has to be treated as species inquirenda. While relationships of taxa within the N. forroi clade are not well-supported, the clade itself is clearly monophyletic. Bearing in mind that N. forroi is a relatively small species typically found in infiltrating waters in the caves of the Bükk Mts and that the other Hungarian specimens from the clade are also small and elongated and from non-karstic springs, it is plausible that this lineage contains “small-pore” species (for additional information on morphotypes of Niphargus see [60]). Members of the N. tatrensis clade are relatively big and typically found in cave streams. Therefore, we can assume niche partitioning, where species from the N. tatrensis and N. forroi clades can be found together, as in the caves of the Bükk Mts and in the subterranean aquatic habitat sampled via the Gejzír Spring, Zemplén Mts.
Niphargus hungaricus and the sample from Dömös mine tunnel (Visegrád Mts) form a well-supported monophyletic clade. Both sampling locations are situated in non-karstic areas. The low number of species in the clade can be explained by the insufficient sampling of Niphargus in the non-karstic areas. Our results suggest that the Danube River can act as a strong dispersal barrier, as previously it was also suggested by Fišer et al. [59]. While numerous members of the N. tatrensis clade can be found north and east of the Danube River, the lineage is completely missing from Transdanubian locations (Figure 3). Likewise, members of the N. hungaricus clade can only be found in the Transdanubian region. This pattern is the most obvious in the case of the Dömös and Vasbánya Spring sample pair, as the Börzsöny and the Visegrád Mts are actually two parts of a single geological unit divided by the Danube River [61].
The closest relative of N. hrabei is the widely distributed N. plateaui Chevreux, 1901 from France. This result is consistent in both trees and in accordance with a previous study [28]. In the case of these two species, the clade is rather weakly defined and can only be outlined on the ML tree. Based on the sister species found on the ML tree, we can cautiously assume the western and eastern expansion of the clade from Mediterranean refugia.
The position of N. valachicus on the Bayesian tree does not provide any information on its relatives. Based on the sister species revealed by the ML tree, we can suggest its northern Dinaric origin, which is in line with the theory proposed by Copilaș-Ciocianu et al. [28], even though the sister species are only partially overlapping.
Based on the results, we can draw some conclusions regarding the Niphargus fauna of different regions in Hungary. The two species inhabiting the caves of the Mecsek Mts, N. gebhardti and N. molnari, are from different lineages and their co-occurrence is most likely a result of independent colonisation. The taxa found in the North Hungarian Mountains (except the Visegrád Mts) are from two major lineages—the N. tatrensis and the N. forroi clades. The high number of taxa in the N. tatrensis clade is probably a result of an expansion followed by allopatric speciation. In a previous study focusing on the phylogeny of the N. tatrensis group, multiple secondary contacts were also proposed based on the difference of the variability of COI and ITS sequences [50]. The two undescribed species of Niphargus from the Molnár János Cave are obviously closely related, but due to the relatively weak support values it is not clear whether sympatric or allopatric speciation is more likely.
While Hungary is not renowned for its extensive karstic areas, picturesque limestone caves or rich subterranean fauna [62], this study has shown that the subterranean fauna of this country is understudied. This is rather unfortunate, given that the geographic position of Hungary falls within the area of the ancient Paratethys. Moreover, during the regression of the Paratethys, the Pannonian Sea and later the Pannonian Lake with their occasionally decreased salinity [17] could serve as a dispersal route for freshwater animal species. Indeed, the results show that species found across the karstic and non-karstic regions of Hungary may improve the biogeography of some clades and may be of utmost importance in future reconstruction analyses. We hope this preliminary study might prompt future research on the subterranean fauna of the country.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d15020223/s1. Table S1: Data on the taxa, sample locations and sequences used in this study.

Author Contributions

Conceptualization, G.B. and D.A.; methodology, G.B., D.A., A.B., Š.B. and V.Z.; validation, G.B. and Š.B.; formal Analysis, Š.B.; resources, G.B., G.H. and C.F.; data curation, G.B., D.A., A.B. and Š.B.; writing—original draft preparation, G.B., C.F. and A.B.; writing—review and editing, G.B., A.B., Š.B., V.Z., G.H. and C.F.; visualization, G.B.; supervision, G.H.; project administration, G.H.; funding acquisition, G.H. and C.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the joint Hungarian–Slovenian grant for international cooperation (Hungarian National Research, Development and Innovation Fund: #SNN-125627; Slovenian Research Agency: #N1-0069 and #N1-0096) and the Research Core Funding (Slovenian Research Agency: #P1-0184, J1-2464). GB was supported by the ÚNKP-20-4 New National Excellence Program of the Ministry of Innovation and Technology, Hungary from the source of the National Research, Development and Innovation Found.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We are truly grateful for all the cavers who helped our work in the field. We would also like to thank the people in SubBio Lab (Ljubljana, Slovenia) and Bracken–Grissom Lab (Florida, USA) for their help with this study. Sampling would not be possible without the help of colleagues and officials, namely Péter Borza (Institute of Aquatic Ecology, Budapest, Hungary), Péter Gruber (Aggtelek National Park, Jósvafő, Hungary), Gergely Ferenczy (Bükk National Park, Miskolc, Hungary) and Csaba Egri (Ministry of Agriculture, Budapest, Hungary).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Sampling locations in Hungary (coloured circles) and the samples from surrounding regions (white circles). Colour codes for the Hungarian samples are the same as in Figure 2 and Figure 3, representing clades revealed by this study.
Figure 1. Sampling locations in Hungary (coloured circles) and the samples from surrounding regions (white circles). Colour codes for the Hungarian samples are the same as in Figure 2 and Figure 3, representing clades revealed by this study.
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Figure 2. Phylogenetic relationships of the species in the genus Niphargus inferred from maximum likelihood (A) and Bayesian (B) approaches, including ultrafast bootstrap (UFBoot), Shimodaria–Hasewaga approximate likelihood ratio test (SH–aLRT) and Bayesian posterior probability support values. Outgroup species have been removed. The specimens from Hungary are in bold and the clades including specimens from Hungary are colour coded using the same colours as on Figure 1 and Figure 3.
Figure 2. Phylogenetic relationships of the species in the genus Niphargus inferred from maximum likelihood (A) and Bayesian (B) approaches, including ultrafast bootstrap (UFBoot), Shimodaria–Hasewaga approximate likelihood ratio test (SH–aLRT) and Bayesian posterior probability support values. Outgroup species have been removed. The specimens from Hungary are in bold and the clades including specimens from Hungary are colour coded using the same colours as on Figure 1 and Figure 3.
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Figure 3. Geographic distribution of the clades that include Hungarian species. Colour codes are the same as in Figure 1 and Figure 2.
Figure 3. Geographic distribution of the clades that include Hungarian species. Colour codes are the same as in Figure 1 and Figure 2.
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Table
Table 1. Sampling locations of Hungarian taxa of Niphargus.
Table 1. Sampling locations of Hungarian taxa of Niphargus.
Sample NameSampling LocationRegion, SettlementLatitude
(N)		Longitude (E)
Niphargus aggtelekiensis A Baradla caveBaradla-Domica Cave System *Aggtelek Karst, Aggtelek48.483120.5440
Niphargus aggtelekiensis B Rákóczi caveRákóczi No. 1 CaveAggtelek Karst, Bódvarákó48.520820.7489
Niphargus forroiDiabáz Cave *Bükk Mts, Bánkút48.095520.4822
Niphargus gebhardtiAbaligeti Cave *Mecsek Mts, Abaliget46.137418.1158
Niphargus hrabeiGödPest Plain, Göd47.715919.1409
Niphargus hungaricusBorha Valley, mine tunnelKőszegi Mts, Kőszeg47.35116.4843
Niphargus molnariAbaligeti CaveMecsek Mts, Abaliget46.137418.1158
Niphargus tatrensis
Kecske-lyuk cave		Kecske-Iyuk CaveBükk Mts, Alsóhámor48.117520.6316
Niphargus valachicusFarmosPest Plain, Farmos47.360819.8269
Niphargus 1 loc.
Molnár János cave		Molnár János CaveBuda Thermal Karst, Budapest47.518119.0358
Niphargus 2 loc.
Molnár János cave		Molnár János CaveBuda Thermal Karst, Budapest47.518119.0358
Niphargus loc.
Vasbánya spring		Vasbánya SpringBörzsöny Mts, Szokolya47.885019.0368
Niphargus loc.
Dömös		Dömös, mine tunnelVisegrád Mts, Dömös47.754818.9087
Niphargus loc.
Kánya spring		Kánya SpringMátra Mts, Galyatető47.926819.9131
Niphargus loc.
Werbőczy spring		Werbőczy SpringMátra Mts, Galyatető47.920719.9167
Niphargus 1 loc.
Gejzír spring		Gejzír SpringZemplén Mts, Telkibánya48.482021.3584
Niphargus 2 loc.
Gejzír spring		Gejzír SpringZemplén Mts, Telkibánya48.482021.3584
Type localities are marked with ”*”.
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Balázs, G.; Borko, Š.; Angyal, D.; Zakšek, V.; Biró, A.; Fišer, C.; Herczeg, G. Not the Last Piece of the Puzzle: Niphargus Phylogeny in Hungary. Diversity 2023, 15, 223. https://doi.org/10.3390/d15020223

AMA Style

Balázs G, Borko Š, Angyal D, Zakšek V, Biró A, Fišer C, Herczeg G. Not the Last Piece of the Puzzle: Niphargus Phylogeny in Hungary. Diversity. 2023; 15(2):223. https://doi.org/10.3390/d15020223

Chicago/Turabian Style

Balázs, Gergely, Špela Borko, Dorottya Angyal, Valerija Zakšek, Anna Biró, Cene Fišer, and Gábor Herczeg. 2023. "Not the Last Piece of the Puzzle: Niphargus Phylogeny in Hungary" Diversity 15, no. 2: 223. https://doi.org/10.3390/d15020223

APA Style

Balázs, G., Borko, Š., Angyal, D., Zakšek, V., Biró, A., Fišer, C., & Herczeg, G. (2023). Not the Last Piece of the Puzzle: Niphargus Phylogeny in Hungary. Diversity, 15(2), 223. https://doi.org/10.3390/d15020223

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