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Demographic history, range size and habitat preferences of the groundwater amphipod Niphargus puteanus (C.L. Koch in Panzer, 1836) Dieter Weber1,2, Jean-François Flot1,3, Hannah Weigand2, Alexander M. Weigand2* 1 Université Libre de Bruxelles, Evolutionary Biology & Ecology group, Avenue F.D. Roosevelt 50, B- 1050 Brussels - Belgium 2 Musée National d'Histoire Naturelle Luxembourg, Section de zoologie, 25 Rue Münster, L-2160 Luxembourg, Luxembourg 3 Interuniversity Institute of Bioinformatics in Brussels – (IB)2, ULB-VUB, La Plaine Campus, Triomflaan, C building, 6th floor, CP 263, 1050 Brussels, Belgium *corresponding author: [email protected] Abstract Niphargus puteanus is the oldest described species of its genus and, in the past, was used as a taxonomic annotation for any subterranean amphipod record. For that reason, no clear knowledge exists about its actual range size and habitat preferences. We here applied a molecular taxonomic and phylogeographical approach based on mitochondrial and nuclear DNA to shed light on its distribution and to infer its demographic history. Furthermore, we analysed aquifer types and water flow regimes to provide a clearer picture of the species’ ecological requirements. Our results indicate that N. puteanus is widely distributed north of the Alps, having its core range in the geomorphological natural region of the ‘South German Scarplands’ (SGS). Additionally, isolated satellite populations exist in the Taunus and the Sauerland, and two single individuals were collected in Luxembourg and in Austria, respectively. The species’ maximal distribution range reaches 756 km between the two single-specimen records and 371 km within the SGS. A very high haplotype diversity was observed, revealing the presence of seven haplotype groups. All the haplogroups were present in the SGS and exhibited distinct spatial-genetic patterns. We thus inferred a high degree of population isolation, with the SGS being a potential long-term refugium for N. puteanus. Historical drift of specimens along major riverine networks (Rhine, Danube) may provide an evolutionary dispersal mechanism explaining the formation of satellite populations. From an ecological perspective, populations of N. puteanus were found to be closely associated with fast-flowing spring environments within fissured aquifers. This species should therefore be classified as both crenophile and stygophile, effectively exploiting resources of surface as well as subsurface spring waters. keywords: distribution; genetic diversity; phylogeography; ecological classification; DNA taxonomy; Niphargus stadleri; Niphargus puteanus Introduction Groundwater comprises the largest fraction of unbound freshwater on Earth and represents the primary drinking water source for half of the global human population (Tushaar, 2007). Despite its paramount importance for human well-being, the recognition of groundwater as an aquatic ecosystem and home for often enigmatic species is still at an early stage. Nevertheless, some 2,000 obligate groundwater metazoans are known from Europe (Gibert and Culver, 2004). These so-called stygobionts are typically characterised by a lack of eye and body pigmentation and a considerably prolonged lifespan compared to their surface-water counterparts (Bellan-Santini, 2015). Stygobionts often have small distribution ranges or are even sometimes known from a single locality only (Trontelj et al., 2009; Fišer et al., 2016). Because of limited resource availability, the subterranean environment presumably supports small local communities (Gibert and Deharveng, 2002). Furthermore, geographically separated communities are often quite distinct in their species pools, suggesting that distribution patterns result from past vicariance events rather than from active migration (Stock, 1993; Trontelj et al., 2009; Culver et al., 2009). The detection of cryptic diversity – in particular for morphospecies assumed to be geographically widespread – is a frequent phenomenon in molecular studies dealing with stygobionts (Zakšek et al., 2009; Camacho et al., 2011; Niemiller et al., 2012; Délic et al., 2017; Eme et al., 2018), where morphospecies are often revealed to harbour more than one distinct molecularly defined species. As a result, estimations about the global number of stygobiont species are in the range of 50,000 – 100,000 (Culver and Holsinger, 1992). Members of the amphipod genus Niphargus are among the most common representatives of groundwater communities of Europe. More than 425 species have been described for the West Palearctic (Horton et al., 2018), with the large majority being strictly stygobiont and inhabiting diverse groundwater habitats. Prominent exceptions comprise Niphargus hrabei and Niphargus valachicus, which have re-colonised surface freshwaters and occur over a wide geographical area (Copilaş- Ciocianu et al., 2018). Subterranean amphipods are frequently used as models in ecology and biogeography (Fišer, 2019). Over the last decade, it has become more and more obvious that our knowledge on the ecology and distribution of many niphargid species is heavily impacted by erroneous or ambiguous past taxonomic assignments (Fišer et al., 2016, 2018a, b). Species delimitation as well species identification based on potentially diagnostic morphological characters seems insufficient as intraspecific and interspecific variability ranges can largely overlap (Fišer et al., 2018b). Adding to this, several Niphargus morphospecies have been revealed to be complexes of cryptic or pseudocryptic species (e.g. McInerney et al., 2014). The latter refers to a population initially considered part of a morphospecies, but identified by molecular data as a separate entity and found out to be distinguishable by morphological characters upon re-investigation. It is thus not surprising that recent studies make use of integrative research designs applying a combination of morphological and molecular characteristics for Niphargus species identification. Molecular species identification using DNA taxonomy (Fontaneto et al., 2015) has the advantage to be applicable to developmental stages (e.g. juveniles) or sexes, which – in case they are not dealt with in taxonomic keys – are regularly omitted. An increase of high-quality occurrence data leads in turn to more robust datasets, which is one the major obstacles for current groundwater ecological and biodiversity research (Zagmajster et al., 2019; Mokany et al., 2019). Probably due to its relatively large body size of up to 2.2 cm in adult males, the first ever described niphargid species was Niphargus puteanus (C.L. Koch in Panzer, 1836) – at that time referred to as Gammarus puteanus. This species is commonly observed in aquatic subterranean biotopes (caves, mining galleries and interstitial) and groundwater discharges (springs and wells) in South-Western Germany (Baden-Württemberg), where it is considered to have its main distribution. Some isolated populations have been described from North and North-Western Germany (Sauerland, Pust, 1990; the Harz Mountains, Hartmann, 1978/79, 2004), Southern Poland (Arndt, 1923), Bulgaria (Bureš, 1929; Beron et al., 2004, referring to Fage, 1926), Switzerland (Moeschler, 1989), Belgium (Leloup and Jacquemart, 1963), France, Italy, Croatia and Slovenia (Wolf, 1934-38). However, morphology-based species records of the past can be questionable. For example, very early records of N. puteanus (or G. puteanus) may actually refer to any groundwater niphargid or even gammarid, as this taxonomic annotation was frequently used as a lumping bin for amphipod populations found in and adapted to the dark (see Brehm, 1955). Likewise, distribution data provided by Schellenberg (1932) remain unclear as he synonymised the species with Niphargus fontanus. Finally, repeated intensive sampling campaigns in the Hölloch cave system (Switzerland) did not re-detect the five previously reported Niphargus species, including N. puteanus (Fišer, et al. 2016): instead, the authors described three new niphargids from this location and emphasised that all past species records from there were probably misidentifications. The species was also historically reported from Belgium, but in their recent survey Fišer et al. (2018a) were not able to re-detect it. In the original description by Koch (1836), the type locality of N. puteanus comprises two wells near Regensburg (Germany, Bavaria) and a well in Zweibrücken (Germany, Rhineland-Palatinate). However, as carefully noted by previous authors (e.g. Wrzesniowski, 1890; Schellenberg, 1933), the two illustrated and investigated specimens were quite different from each other. Moreover, the original material of Koch (1836) seems to be lost (Goedmakers, 1972). For these reasons, Stock (1974) established a neotype and defined a neotype locality referring to the spring Weichselmühle (=Weichslmühle) near Regensburg (South-Western Germany). The present work aimed to provide robust and updated insights into the ecology, genetic diversity, distribution and demographic history of the groundwater amphipod N. puteanus. First, we performed molecular species delimitation to provide high-quality presence records for reconstructing the actual distribution range of the species. Second, the obtained dataset was used to generate insights into the genetic diversity of the species, and to investigate the connectivity (or lack thereof) of the populations sampled. The combined spatio-genetic data were then used to infer the plausibility and potential origin of isolated satellite