The first records of Niphargus hrabei and N. potamophilus in Ukraine and Bulgaria significantly enlarge the ranges of these species

Abstract Niphargus comprises hundreds of narrowly-endemic West Palaearctic subterranean taxa. However, a few exceptional species inhabit surface waters and have remarkably large ranges. Herein, based on morphological and molecular analyses, we provide important new records for two of these species. N. potamophilus, previously known from the eastern Azov Sea lowlands, is reported for the first time from Ukraine and Bulgaria from localities adjacent to the Black Sea. These findings expand its range westward by more than 1000 km along the coastline. From Bulgaria, we also report for the first time N. hrabei, a species previously known to occur along the middle and lower Danube lowlands and in isolated populations at the foothills of the Northern Caucasus. Our new record thus extends its range southwards by more than 150 km. Both species contained unique haplotypes at all of the sampled localities. These were, nevertheless, not very divergent from more distant populations, emphasizing their good dispersal ability. Ecologically, the sampling localities were generally characterized by stagnant to low running water, dense vegetation, and muddy substrate. Overall, our results bring important insights, shedding more light on the biogeography and ecology of Niphargus.

Remarkably, a few species of Niphargus are known almost exclusively from epigean habitats, and they also have the widest known ranges of any other niphargid, despite being blind and depigmented (Copilaș-Ciocianu et al. 2017). The best known are Niphargus valachicus Dobreanu &Manolache, 1933 andN. hrabei S. Karaman, 1932, both inhabiting the middle and lower Danube lowlands as well as being occasionally reported around the Black Sea lowlands at the foothills of the Caucasus or the Anatolian Peninsula (Cărăușu et al. 1955;Nesemann et al. 1995;Copilaş-Ciocianu et al. 2014;Copilaș-Ciocianu et al. 2017;Palatov & Marin 2021). Although both are similar from a morphological, ecological and biogeographic perspective, they are not closely related (Copilaş-Ciocianu et al. 2018;Palatov & Marin 2021).
Niphargus potamophilus (Birstein 1954) is another lowland species that inhabits similar habitats and has a similar morphology to N. hrabei and N. valachicus, being closely related to the latter (Birstein 1954;Palatov & Marin 2021). These species have trapezoidal gnathopods, acutely produced infero-posterior corners of epimeres 2 and 3, additional spines on pereopod 3-4 dactily, nonsexually dimorphic 3rd uropod and a spoonshaped appendix on the distal end of the uropod 1 peduncle. Niphargus potamophilus was originally described based on the specimens collected from the Don River delta, from a floodplain reservoir nearby Rostov-on-Don City, and from fish farms in Atkhyr, Kuban River delta, ca. 300 km southwest from the Don delta (Birstein 1954). The recognized range of this species is much smaller than that of N. hrabei and N. valachicus, for now being reported along the eastern Azov Sea shore (Palatov & Marin 2021), but it was suggested that its distribution might be much wider, covering southern Ukrainian rivers (Birstein 1954).
In the present study, we report N. potamophilus and N. hrabei from new territories and river basins in Bulgaria and Ukraine. Our combined molecular and morphological approach confirmed that these species are more widespread than previously known.

Sampling
Specimens were collected with a hand net during sampling campaigns in Ukraine in 2018 and in Bulgaria in 2014-2018 (for details see Table I). Animals were fixed in 96% ethanol. The material is stored in the collection of invertebrates at the Department of Invertebrate Zoology and Hydrobiology (University of Lodz, Poland) and Nature Research Center (Lithuania).

DNA extraction, amplification, and sequencing
Samples were processed either at the University of Lodz (Poland) or at the Nature Research Centre (Lithuania) (see Supplemental Table SI for details). For samples analyzed at the University of Lodz, total DNA was extracted from the leg of each specimen using the Chelex procedure (see Casquet et al. 2012, Morhun et al. 2022 for details). For samples analyzed at the Nature Research Center DNA was extracted from the dorsal side using the Quick-DNA Miniprep Plus Kit (Zymo Research) (see Copilaş-Ciocianu et al. 2022 for details). The standard animal DNA barcode gene region (COI) (Hebert et al. 2003) was amplified using the primers LCO1490, 5′-GGTCAACAAATCATAAAGATAT TGG-3′ and HCO2198, 5′-TAAACTTCAGGGT GACCAAAAAATCA-3′ (Folmer et al. 1994) with the PCR protocol described by Hou et al. (2007) or with LCO1490-JJ, 5′-CHACWAAYCATAAAGAT ATYGG-3′ and HCO2198-JJ, 5′-AWACTTCVGG RTGVCCAAARAATCA-3′ (Astrin & Stüben 2008) with the PCR protocol described by Copilaş-Ciocianu  (Tamura et al. 2013). Sequence divergences for the COI-5P barcode region (mean and maximum intraspecific variation and minimum genetic distance to the nearestneighbor species) were calculated using the analytical tools of the BOLD workbench ("Barcode Gap Analysis"), employing the Kimura 2-parameter model (K2P; Kimura 1980). We estimated the haplotype number, using the DnaSP v5 software (Librado & Rozas 2009). The genetic structure within both species was visualized through a Median-joining network using PopART (Leigh & Bryant 2015). Phylogenetic analyses were performed in MEGA 6 (Tamura et al. 2013) using the Neighbor-Joining algorithm with Kimura 2-parameter distances, and 1000 bootstrap replicates to visualize relationships among haplotypes.

Field research
In the Dniester basin (Ukraine), N. potamophilus was collected in 2018 from the flooded meadow area beside the river (near Mayaki), in shallow, wellwarmed floodplain puddles (both temporary and permanent ones) with almost no water flow (Figure 1(a,b). Amphipods co-occurred alongside A. aquaticus and diverse fauna of freshwater mollusks and leeches. It should be noted that this floodplain meadow has recently been greatly reduced in size due to ongoing urbanization, and the natural floodplain puddles (Figure 1(b)) are rare now.
Niphargus potamophilus in the delta of the Dnipro River (Ukraine) was found in 2018 in a shallow stream (near Stara Zburivka) with a lot of decaying vegetation, that flows over an alder forest, in a place where the spring starts its outflow (Figure 1(c)). The water in this place was colder than in the surroundings and was flowing slowly. The amphipods cooccurred alongside A. aquaticus and Haemopis sanguisuga (Linnaeus, 1758). This stream is part of the channel that connects the estuarine bay to a system of fish breeding ponds. The amphipods were found in the least anthropogenically transformed part of a former floodplain channel.
In Bulgaria in the Tankovo area, both Niphargus hrabei and N. potamophilus were sampled four years apart. Niphargus hrabei was collected in 2018 from a ditch with apparently temporary water (Figure 1(d)). The substrate was muddy with a lot of decaying vegetation. The riparian vegetation was very abundant. The density of the animals was rather high as several tens of individuals were sampled, including 21 females (a few ovigerous), 52 males, and 16 juveniles. The water louse Asellus aquaticus (Linnaeus, 1758) was also highly abundant. This sampling point represents the southernmost record of this species, being situated ca. 150 km south of the previously known locality (Hagieni, Romania; Flot et al. 2014). Niphargus potamophilus was sampled in 2014 from Hadjiyska stream, a few tens of meters eastward from where N. hrabei was collected (Figure 1(e)). It occurred among reeds on the shallow and muddy shores. Few adult specimens were sampled including both males and females. Accompanying crustacean fauna consisted of Gammarus cf. arduus G. Karaman, 1975, Pontogammarus robustoides (Sars, 1894, and A. aquaticus. This sampling point is the furthest locality from the previously known records (ca. 1000 km) (Palatov & Marin 2021).

Morphology of specimens
All of the examined morphological characters of our material fell within the range indicated for N. hrabei and N. potamophilus species by Birstein (1954) and the recent redescription of the latter species by Palatov and Marin (2021). Both species are characterized by trapezoidal gnathopod propodi, acutely produced infero-posterior corners of epimeres 2 and 3, additional spines on dactyly of pereopods 3 to 4, uropod 3 non-sexually dimorphic, and the presence of a spoon-shaped appendix on the basis of uropod 1 in males (Figures 2 and 3). The two species can be distinguished from one another by the lack of dorsal spines on telson lobes in N. hrabei (present in N. potamophilus), presence of lateral setae on peduncle of pleopod 3 in N. hrabei (absent in N. potamophilus), inner lobe of maxilla 1 with 3 seate in N. hrabei (one seta in N. potamophilus) (Palatov & Marin 2021) (Figures 2 and 3). The morphological identification is in full agreement with the molecular identification through the COI mtDNA barcode.

Molecular studies
We generated four new sequences for Niphargus hrabei (representing two new haplotypes), and nine new sequences for Niphargus potamophilus (representing six new haplotypes). Analyzing the whole assembled dataset, we identified 15 haplotypes within 48 sequences for N. hrabei, with a 0.41% mean, and 1.31% max intraspecific K2P distance; all sequences were assigned to a single BIN -BOLD:ACQ4737. For N. potamophilus we identified 10 haplotypes within 13 sequences, with a 1.15% mean, and 2.85% max intraspecific K2P distance; all sequences were assigned to a single BIN -BOLD:AEB5889. K2P distance between these two species was 17.25%, clearly indicated in the Neighbor-Joining tree (Figure 4). The Median-Joining network of N. hrabei shows that individuals from Russia and Bulgaria have only private haplotypes ( Figure 5(a)). The network for N. potamophilus shows quite different private haplotypes from Russia, with a minimum distance of six substitutions, to the closest haplotypes from Bulgaria and Ukraine ( Figure 5(b)).

Discussion
The epigean Niphargus species from the Azov-Black Sea region, N. hrabei, N. potamophilus, N. valachicus, and N. magnus Birstein, 1940, have formerly been considered as members of the subgenus Phaenogammarus (Dudich 1941) based on their ecological and morphological similarity (Birstein 1954;Straškraba 1972;Sket 1981;Palatov & Marin 2021). However, recent molecular research has shown that this similarity is due to convergence, thus rendering Phaenogammarus obsolete (Borko et al. 2021). Nevertheless, regarding our focal species, a recent analysis showed that N. potamophilus, N. valachicus, and N. magnus are closely related, forming a well supported clade that is phylogenetically distant from N. hrabei (Palatov & Marin 2021 (Palatov & Marin 2021). Our findings of N. hrabei and N. potamophilus across the west part of the Black Sea basin significantly increased the range of these species, especially for the latter, confirming its westward distribution suggested in the original description (Birstein 1954). The previous and newly gathered ecological data indicates that N. hrabei, N. potamophilus and N. valachicus have similar environmental preferences, occupying a wide range of habitats ranging from springs to small and large streams, large deltaic lakes and channels, estuarine water bodies, and floodplains (Birstein 1954;Zorina-Sakharova 2017;Copilaş-Ciocianu et al. 2018;Palatov & Marin 2021;this study). Conversely, N. magnus is distributed both in subterranean and surface water. However, it seems to occupy a narrower spectrum of surface habitats, preferring well-shaded small forest ponds filled with fallen leaves (Birštein 1940(Birštein , 1952Palatov & Marin 2021). Such habitats are more isolated as evidenced by the pronounced genetic structuring within this species (Palatov & Marin 2021). This is probably the reason why it has a much smaller range than the other three species.
This increase in reports of epigean Niphargus species in the last decade around the Black Sea basin is noteworthy, and could either be a result of more active monitoring, or recent range expansion. The latter in unlikely given the changes in the last century in the condition of large river ecosystems, associated with the simultaneous effects of climate change and anthropogenic flow regulation, which affected the abundance of Ponto-Caspian species and communities (Gogaladze et al. 2021), and promoted the spread of non-native species. Furthermore, our molecular analysis showed that Ukrainian and Bulgarian populations of both species are characterized by unique haplotypes, not present anywhere else in the Pannonian or Black Sea basins.

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H. Morhun et al. As such, it is clear that N. hrabei and N. potamophilus are native species in these areas. It is, therefore, more likely that these species might have been overlooked previously in the Azov-Black Sea basin, and their dispersal across the marine/ brackish waters of the Black Sea might have occurred a long time ago, at the Last Glacial Maximum (30-25 thousand years ago), when this sea was a freshwater lake, isolated from the world ocean (Ryan et al. 1997;Bahr et al. 2006;Georgievski & Stanev 2006). At this time, the Pre-Danube, Pre-Don and Pre-Dnipro paleo-basins were merged into one common alluvial valley (Chepalyga 2007;Yanko-Homback et al. 2017) and lakes in the Syvash Gulf were desalinated (Olenkovsky 2012). This alluvial valley lasted until the latest Mediterranean transgression 16-9 thousand years ago (Yanko-Hombach & Yanina 2019: Figure 9). This palaeogeographic context most likely facilitated the dispersal of these freshwater amphipods around the Azov and Black Sea coastline. The phylogeography of both N. hrabei and N. valachicus is consistent with dispersal along the Black Sea coast during this time frame (Copilaş-Ciocianu et al. 2018). The subsequent flooding of the Black Sea with saline Mediterranean waters divided the common valley into separate estuaries of the Dnieper, Dniestr, and Danube River basins (Yanko-Homback et al. 2017: Figure 16.22), forming the modern Azov and Black Sea (7-9 thousand years ago) (Federov 1971;Ryan et al. 1997;Badertscher et al. 2011; Yanko-Hombach & Yanina 2019: Figure 9). Since then, all the major river basins have maintained their isolation. As such, N. hrabei, N. potamophilus and N. valachicus could be present across the entire north Black Sea basin for at least 9 thousand years. In conclusion, we provide the first morphological and molecular evidence of the presence of N. hrabei in Bulgaria, and N. potamophilus in Bulgaria and Ukraine. Our findings expand the known range of the former ca. 150 km southwards and for the latter species ca. 1000 km westwards. Molecular population structure shows that only Pannonian populations of N. hrabei shared haplotypes between sites and countries, and populations of both species along the Black Sea coast are genetically isolated. was supported by a grant from the Research Council of Lithuania (contract no. 09.3.3-LMT-K-712-19-0149).

Disclosure statement
No potential conflict of interest was reported by the author(s).

Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/24750263.2022.2126534