Checklist of amphipod crustaceans (Crustacea: Amphipoda) in Slovenia

. Amphipods are among the most species-rich orders of peracarid crustaceans, inhabiting marine and fresh waters, including subterranean waters, as well as semi-terrestrial environments. Data on amphipods from Slovenia are scattered among published and unpublished references. We gathered all available data on amphipods in the country, using published and unpublished records from two databases, supplemented by additional published information and unpublished records from two collections of two institutions. All data were critically evaluated and species names updated to the latest taxonomically valid name. In total, we listed 198 species, belonging to 85 genera and 41 families. About two thirds of the species are marine, while the rest are semiterrestrial, brackish and freshwater species. Among the latter, subterranean species dominate. In total, 26 species endemic to Slovenia were identified, one of which lives in surface freshwater, and the rest in groundwaters. Surprisingly, no presence of invasive freshwater amphipod species has currently been confirmed. We provided critical comments on nine amphipod species that were listed erroneously for the country. Our work revealed two major knowledge gaps. Marine species records come from occasional sampling campaigns, and we suggest that this group calls for a more systematic research. More work has been done on freshwater species, which revealed many taxonomic uncertainties that cannot be resolved at present and call for additional taxonomic research.


Introduction
With over 10,000 species, the order Amphipoda is one of the largest groups in the superorder Peracarida in the classis Crustacea (Horton et al. 2021). Amphipods are distributed in all aquatic and semiterrestrial habitats around the globe. About 80% of species live in marine environments (Väinölä et al. 2008), and span between supralittoral to deepest trenches, where they represent an important fraction of deep-sea communities (Jamieson et al. 2010). The remaining 20% are freshwater (Väinölä et al. 2008), living in all types of freshwater including groundwater (Sket 1999a, Zagmajster et al. 2014.
Amphipods represent an ecologically important group that contributes to nutrient cycling. They act as detritivores, shredders, suspension feeders, scavengers, parasites or predators (Kaestner 1967, MacNeil et al. 1997, Ruffo et al. 1998) -and constitute an important food source for fish. Some species are intermediate hosts to acanthocephalan parasites (Westram et al. 2011, Shaw et al. 2020, some are hosts to ciliates (Gudmundsdóttir et al. 2018) or temnocephalid flatworms (Matjašič 1990), while several species live in associations with other animals, such as other crustaceans, bryozoans, mollusks, or cnidarians (Lörz et al. 2014, Vader & Myers 1993, Vader & Tandberg 2013, 2020. Because of the absence of dispersal larvae (Myers & Lowry 2009), many species are narrow endemics (Trontelj et al. 2009, Bregović et al. 2019, Esmaeili-Rineh et al. 2020 and an important part of natural heritage. Yet, few species have been rapidly expanding their original distributional ranges, and as invasive species remarkably modified native freshwater communities across Europe (Jazdzewski et al. 2004, Grabowski et al. 2012, Dodd et al. 2014. Importantly, many amphipod species have been included into ecotoxicological bioassays and used in monitoring schemes of water quality (Feckler et al. 2012, Major et al. 2013.
Amphipods thus constitute an important group of crustaceans for basic and applied research. Successful implementation of amphipod crustaceans into diverse research programs requires a resolved taxonomy, an easy identification system, good understanding of species' distributions, an estimation of potential threats and species' conservation status. On many occasions, this information is incomplete or completely lacking.
Slovenia has a long tradition in amphipod research. Yet, biological data on amphipod species are scattered in many sources or sometimes unpublished. As a consequence, we are facing larger or smaller knowledge gaps on the taxonomy, distribution, ecology and biology of many species. An updated checklist of species present in the country is the first step towards systematic studies of amphipods. Such a list is the most elementary document needed for inventory and planning of future research, be it basic or applied. The hitherto published lists of Slovenian amphipods (Karaman 1974, Sket 1996, Fišer 2002 have long been outdated and need to be revised and corrected. To progress towards proper and complete inventory of amphipods in Slovenia, we assembled all available published and unpublished data on amphipods and compiled the first comprehensive national checklist of all amphipods in coastal and inland waters.

Materials and methods
To assemble the list, we first used the records of all previously published checklists (Karaman 1974, Sket 1996, Fišer 2002, and supplemented it with data from literature published afterwards. For this, we used already extracted data stored in two databases, BioPortal (BioPortal, Centre for Cartography of Fauna and Flora -CKFF) and SubBioDB (SubBio Database, Subterranean Biology Laboratory at Department of Biology, Biotechnical Faculty, University of Ljubljana -SubBioLab). Additionally, we added new information from the specimen collections of the Marine Biological Station of Piran of the National Institute of Biology (hereinafter referred to as MBP) and of the SubBioLab. While preparing the checklist, we specifically considered the following points.
Names and taxonomy. We used the names evaluated as »accepted« in the World Register of Marine Species (Horton et al. 2021), with one exception, when we considered the relevant publication (Sket & Hou 2018 synonymized the genera Chaetogammarus and Echinogammarus into Homoeogammarus); all other names were discarded as synonyms and were not added to the checklist. We listed only formally described species, even though molecular analyses in many cases imply that morphological species comprise morphologically indistinguishable species complexes. These cryptic species (e.g., Copilaş-Ciocianu & Petrusek 2015, Mamos et al. 2016, Csapó et al. 2020, Hupało et al. 2020 are not considered in this checklist, but we do discuss them when relevant. The possible new species candidates and problematic taxonomic cases are beyond the scope of this overview. The higher taxonomy of amphipods above the family rank has been challenged (Copilaș-Ciocianu et al. 2020). To keep the checklist simple and insensitive to taxonomic changes, we excluded ranks between order and family.
Ecology. Each species in the checklist is labelled according to its habitat where it can be primarily found. We introduced categories »marine«, »brackish«, »freshwater« (i.e. surface freshwater), »groundwater« (i.e., subterranean freshwater) and »semiterrestrial«, but do not make distinction among habitats within these categories (Trontelj et al. 2012, Culver & Pipan 2014. Species from springs, which some consider an ecotone between groundwater and surface waters, were assigned to the habitat typical for that genus. Endemism. Present knowledge of many species is not sufficient to construct detailed maps of their distribution. We, however, indicated whether species are living within the political borders of Slovenia (endemics), or whether they are distributed also beyond the country borders.
Citation. Every species record is linked to the reference, supporting its presence in the country -either being a literature reference or a database. Therefore, the list of references should not be considered as a complete list of all references on amphipods of Slovenia. We selected the most comprehensive resources, to keep the list of supporting references at minimum.

Results
The checklist of amphipods of Slovenia encompasses a total of 198 species, belonging to 41 families and 85 genera (Tabs. 1, 2). Of all the species in the list, 77 are listed for the country for the first time, based on data coming from the collections of UL and MBP (Tab. 2). Table 1. Numerical summary of all amphipod species confirmed to occur in Slovenia, according to the habitat they occur in. All three brackish species can also be found in surface freshwaters (to simplify, here referred to only as freshwater). 1 Members of some families and genera live in more than one environment, therefore the numbers do not simply sum up. The term endemic relates to species, occurring within Slovenian political borders only. Tabela 1. Številčni povzetek vseh postranic, ki se potrjeno pojavljajo v Sloveniji, glede na habitate, kjer se pojavljajo.
Vse tri brakične vrste so našli tudi v površinskih sladkih vodah. 1 Predstavniki nekaterih družin/rodov žive v več kot enem habitatu, zato se številke teh ne seštejejo. Oznaka »endemic« se nanaša na vrste, ki žive izključno znotraj političnih meja Slovenije. Uristidae Tmetonyx nardonis (Heller, 1867) marine no Heller 1866 About two thirds of species (127 species, 73 genera and 36 families) are marine, one family with four genera totalling four species are semiterrestrial, three species can be found in both brackish and freshwaters, whereas the rest of the species (65) live in freshwater only. Among the latter, subterranean species (57 species) predominate. There are 26 species endemic to the country, of which only one is from surface freshwater, while all the rest are from groundwater (Tabs. 1, 2). We detected two alien marine species, of which one is invasive, but found no alien freshwater species.

Number of species
Nine species are considered as erroneously listed for Slovenia, with detailed explanations on the reasoning behind exclusion from the national list of amphipods explained in Tab. 3. Table 3. A list of amphipod species erroneously listed as present in Slovenia in previous papers. In a separate column, we justify their removal from the Slovenian checklist. Tabela 3. Seznam vrst postranic, ki jih starejša dela zmotno navajajo kot del slovenske favne -kar je napaka. V ločenem stolpcu utemeljujemo njihovo odstranitev s seznama slovenske favne.

Species Reason to remove Source Hadziidae
Hadzia fragilis stochi Karaman, 1989 Present on Carso Goriziano, Italy, but its presence in Slovenia was never confirmed. Also Sket (1996) listed its presence as doubtful, with question mark added. Sket 1996 Gammaridae Gammarus wautieri Roux, 1967 A species distributed in France, identified using the identification key of Karaman and Pinkster (1977). According to molecular evidence, this is an error; it likely represents an unknown species from the complex Gammarus fossarum.  Sket 2000, Rejic 1958 Niphargus croaticus (Jurinac, 1887) This species lives in Croatia and Bosnia and Herzegovina (Delić et al. 2016, Zakšek et al. 2019. Before the revision, it was often erroneously confused with another species, N. arbiter (Karaman 1984). In Sket (2000) it is listed as N. cf. croaticus, which may lead to wrong conclusion of N. croaticus presence in Slovenia.

Family Species Reason to remove Source
Niphargus kochianus This species is one of the first described Niphargus species. It lives in Northern and  Sket 1972, Sket 1979, Sket & Velkovrh 1981, Sket 2000 Niphargus jovanovici This species lives in Macedonia and Greece (Karaman 2017), but several species from Slovenia were assigned as closely-related species or subspecies (N. grandii, N. multipennatus).

Sket 1972
Niphargus hrabei S. Karaman, 1932 This species is distributed throughout the Pannonian basin and could be expected in Slovenia (Copilaș-Ciocianu et al. 2017). However, its presence has never been confirmed, even though a related undescribed species lives in Krakovski gozd (Borko et al. 2021). Sket (1996) mentiones this species with question mark.

Sket 1996
Niphargus puteanus (Koch & Panzer, 1836) Niphargus puteanus is distributed in the Rhine and upper Danube basins and Luxembourg (Weber et al. 2020). However, this is the first Niphargus species to be described, and many other species were originally described as its subspecies. In Slovenia, N. spoeckeri and N. krameri were probably confused with this species. Gherlizza 1999, Megušar 1914, Perko 1910 Niphargus tauri This species lives in Turkey, but similar species were reported from Western Balkans, including Slovenia. N. carniolucus was initially affiliated to this species. It was reported as N. tauri ssp. or as N. tauri gr.

Discussion
In this contribution, we are increasing the number of registered species for Slovenia, as we list 77 (nearly 39%) of all amphipod species for the first time for our country. The large majority of species (131 species, 66% of all) is marine, found in the Slovenian part of the Northern Adriatic Sea. Two species (Caprella scaura, Jassa marmorata) are alien and one (Monocorophium sextonae) is cryptogenic (EASIN 2021). The marine species represent approximately 29% (127 out of 451) of all Mediterranean species (Ruffo et al. 1998). This is a relatively large share of regional species richness even though the Northern Adriatic Sea represents only a small fraction of the entire Mediterranean Sea, with Slovenian sea representing even smaller fraction. The share of marine against all Slovenian amphipods (64%), however, is somewhat lower than their respective share on a global scale (80%, Horton et al. 2021). This deviation can be in part explained by the fact that marine amphipods have not been systematically studied, in contrast to intensively studied freshwater amphipods. We believe that more intense research should expand the list of inhabitants of Slovenian sea. For example, the monograph of Mediterranean amphipods reported six species from the sea near Rovinj (Croatia) and Trieste (Italy), i.e., south and north of Slovenian coast, respectively, the presence of which can be highly expected in the Slovenian sea as well (Krapp-Schickel & Zavodnik 1996, Ruffo et al. 1998). Finally, we found some individuals that may belong to alien species, but need a confirmation from a specialist for these families.
Among freshwater amphipod species, subterranean amphipod species (groundwater species) represent the main share of species (57 out of 64 freshwater species). Subterranean amphipods comprise species from currently six genera, Bogidiella, Carinurella, Ingolfiella, Niphargus, Niphargobates and Synurella. Two of them, Niphargobates and Carinurella, are in need of taxonomic revision, as they are nested in phylogenetic trees within the genus Niphargus (Esmaeili-Rineh et al. 2015, Borko et al. 2021. Species of the genus Niphargus dominate in groundwaters (53 species). The high number of Niphargus species seems to be a result of turbulent geological history that in a broader region of the Western Balkans prompted multiple evolutionary radiations, descendants of which dispersed into the territory that is nowadays called Slovenia (Borko et al. 2021). The number of current species of this genus is rather underestimated since the morphological identification of Niphargus species is challenging and the taxonomy of the genus incomplete. We estimate there might be between 30-50% additional and yet undescribed species (Borko et al., in revision).
The only Synurella species listed in the checklist needs further clarifications. Slovenian populations of S. ambulans colonized a number of Slovenian caves, with each subterranean population differing from neighbouring surface populations and hence suggesting multiple independent colonization events and probable ongoing speciation (Pekolj 2020). Some authors considered morphologically differentiated subterranean populations as a distinct subspecies named S. ambulans subterranea (Karaman 1931). Subterranean Synurella resembles the evolutionary history of the isopod species Asellus aquaticus (Linnaeus, 1758), which independently colonized subterranean waters several times, and molecular studies suggest that most of these cave populations deserve separate species status (Verovnik et al. 2009, Konec et al. 2015. By analogy, we assume that subterranean populations of Synurella represent distinct species, a hypothesis that should be tested using molecular tools. Most endemic species are groundwater species, although there is one endemic species in the surface freshwater. The term »endemic« requires a caution note. For the needs of this checklist, we considered »endemic« those species the distributional range of which falls completely within the political borders of Slovenia. However, there are species having small distribution ranges, which also occur in either of the neighbouring countries (Italy, Croatia). Even though they are not national endemics, they are endemic to very small geographic area (often called subendemic). A different case of missed national endemics in the current list are some large-ranged species that may have genetically distinct populations in Slovenia (e.g. N. liburnicus, N. krameri) and await to be taxonomically evaluated, formally described as species, and put on a list of national endemics. NATURA SLOVENIAE 23(2): 5-24 The most species from poor ecological categories are species from the surface water and species from brackish water. These include genera Homoeogammarus, Gammarus, Jugogammarus and Synurella. Taxonomically unambiguous is the monotypic species Jugogammarus kusceri, a narrow endemic species living in springs within the catchment of the Krka River (Sket 1996). The taxonomic status of species from other genera is highly questionable. Individuals characterized as nominal species G. fossarum, G. balcanicus, G. roeselii and S. ambulans in fact belong to species-rich complexes (Copilaş-Ciocianu & Petrusek 2015, Mamos et al. 2016, Csapó et al. 2020, Hupało et al. 2020. With rare exceptions (Alther et al. 2017), most of Slovenian freshwater gammarids have never been studied genetically and we thus cannot evaluate their taxonomic status nor the true number of potential species. Consequently, distribution of these species in Slovenia and their conservation status remains poorly known.
Contrary to our expectations, as of present time and up to our knowledge, there is no confirmed record of invasive freshwater amphipod species presence from Slovenia. Many Ponto-Caspian species of the genera Dikerogammarus, Chelicorophium and Obesogammarus spread across the continent and through rivers and artificial channels and have reached Poland, Germany, France and Switzerland (Altermatt et al. 2014, Copilaş-Ciocianu et al. 2021. These species were recorded in the Drava and Sava Rivers in the territories of Croatia and Serbia more than a decade and five decades ago, respectively (Karaman 1974(Karaman , Žganec et al. 2009). The probability that these species could be present also in Slovenia or could colonize Slovenian rivers, streams, and lakes in the near future is quite high. Thus, the here presented checklist offers a challenge to researchers, to look carefully into riverine benthos and check whether native fauna has encountered Pontocaspian newcomers.
Finally, while preparing and critically evaluating the checklist, several erroneous names resurfaced. Changes of species names are an integral part of taxonomic practice. Some species were in due revisions transferred to other genera during revisions, recognized as junior synonyms or erected from subspecies to species status. These names can be traced as synonyms on the World Register of Marine Species (Horton et al. 2021) and are not problematic. However, some sources of erroneous records can create confusion with the risk to be repeated when checklists are revised. First, some species were erroneously identified. As an example, in the past, individuals could have been identified as a widespread species (like Niphargus puteanus), but subsequent taxonomic work unveiled this individual belongs to another species (in this example, N. krameri) and the previously identified species (in this case N. puteanus) is not present in Slovenia (Weber et al. 2020). A similar confusion may derive from identification to the species level (e.g., N. aquilex) rather than to the subspecies level (in this case N. aquilex dobati), and subsequent taxonomic work raised subspecies to the species level (N. dobati); also in this example, nominal species does not live in the country (McInnerney et al. 2014). The last problem deals with dubious records, not backed with data. With the aim to remove such species from further lists, we decided to list them in this contribution, with the hope that they will no longer be listed as representatives of Slovenian amphipod fauna.