Org Divers Evol (2013) 13:569–581 DOI 10.1007/s13127-013-0141-7 ORIGINAL ARTICLE Spatial pattern of intraspecific mitochondrial diversity in the Northern Carpathian endemic spring snail, Bythinella pannonica (Frauenfeld, 1865) (Gastropoda: Hydrobiidae) Zoltán Fehér & Ágnes Major & Virág Krízsik Received: 25 September 2012 /Accepted: 21 May 2013 /Published online: 17 June 2013 # Gesellschaft für Biologische Systematik 2013 Abstract Bythinella is a species-rich genus of spring-snails, distribution of which has been shaped by fragmentation, as having a wide range in Europe and Asia Minor. The genus well as by short and long distance dispersal events. These two contains several endemic species with narrow ranges, creat- clades have not been found syntopically but, as a peculiar ing interest from a conservation perspective as well as their feature of this taxon, they are able to persist in nearby habitats. use as a model for research into biogeographical patterns and We have demonstrated that the patchy distribution of suitable evolutionary development. Most of the species of the genus habitats and the restricted, but non-zero gene flow amongst Bythinella are difficult to distinguish by traditional methods the populations might play a key role in maintenance of the due to their similar shell morphology. In previous studies, observed genetic structure of this species. molecular approaches often came to conclusions that contradicted those from a morphology-based approach, Keywords Intraspecific diversity . COI . Barcoding . hence the classification of species of Bythinella has been in Phylogeography . Species delimitation . Cryptic lineage dispute. Bythinella pannonica has clearly distinct shell mor- phological features, and consequently is one of the few species of undisputed taxonomic status within this genus. Introduction As an important step towards clarifying the systematics of this genus, we have attempted to describe the structure and spatial As species is the fundamental unit of taxonomy, biogeography, pattern of diversity of the mitochondrial COI marker within ecology and conservation biology, species delimitation is a this species, in the hope of applying these findings generally to crucial question in supra-individual biology (de Queiroz the whole genus. Molecular results support the monophyly of 2007;Flotetal.2010; Petit and Excoffier 2009;Ponsetal. B. pannonica within the genus. The largest pairwise intraspe- 2006; Rissler and Apodaca 2007; Sites and Marshall 2003). cific COI sequence difference was almost 5 %, much larger Over ten thousand new taxa are described every year, most of than the value of 1.5 % previously proposed as a barcoding them by traditional, morphology-based methods (Zhang 2008). threshold for delimitation of Bythinella species. This finding Morphology, however, does not always reflect phylogeny, and suggests that incautious application of barcoding might lead to those taxa for which morphology-based methods are of limited incorrect taxonomic conclusions. Within B. pannonica there use are a challenge for taxonomists (Bickford et al. 2007; are two deeply divergent intraspecific clades, the spatial Havermans et al. 2010;Poisotetal.2011; Weigand et al. 2011). Among hidrobiid gastropods there are several problemat- ic groups, including the most widespread and species-rich Ágnes Major is deceased. European spring-snail genus, Bythinella Moquin-Tandon, Z. Fehér (*) 1856. The majority of Bythinella species are quite similar Department of Zoology, Hungarian Natural History Museum, in terms of their shell morphology, habitat preference, repro- 1088 Baross u 13, Budapest, Hungary ductive biological and behavioural characteristics (Wilke e-mail: [email protected] et al. 2010) and, due to the slightly differing and largely : overlapping features of the genital organs, not even the Á. Major V. Krízsik genital morphology provides clear and unambiguous solu- Laboratory of Molecular Taxonomy, Hungarian Natural History Museum, 1083 Ludovika tér 2, tion for taxonomic problems (Falniowski and Szarowska Budapest, Hungary 2009; Giusti and Pezzoli 1977). In recent years, this genus 570 Z. Fehér et al. has attracted increasing attention, as indicated by the number We aimed (1) to test whether or not COI-based phylogeny of new species descriptions (Boeters and Falkner 2008; is concordant with the morphology-based species delimita- Falniowski et al. 2009a; Georgiev 2009; Glöer and Georgiev tion (i.e. B. pannonica is monophyletic); (2) to measure the 2009, 2011; Glöer and Pešić 2006, 2010; Prié and Bichain intraspecific variability of this marker and to test the appli- 2009). Due to its Europe-wide range (Bank 2012) and spe- cability of the 1.5 % barcoding threshold value proposed by cial habitat preference, this genus is a promising model taxon Bichain et al. (2007) for this genus; (3) to explore the spatial to study Pleistocene refugial patterns in Europe (Benke et al. pattern of the intraspecific variability; and (4) to gain some 2009) and non-adaptive radiation in freshwater systems insight into the biogeographic/demographic history of this (Wilke et al. 2010). Due to their narrow ranges, several species. Bythinella species are of special conservation concern (Cuttelod et al. 2011). For the above reasons, clarification of the species boundaries and systematic relationships within Material and methods Bythinella is more than a fundamental research objective in itself. The study species So far, the majority of phylogenetic studies related to European hidrobiids have focused on the most popular B. pannonica is endemic to the southeastern part of the barcoding sequence, the mitochondrial cytochrome c oxi- Northern Carpathian Mountains. As characteristic to the dase subunit I gene (COI). In related genera, the application whole genus, it prefers clear, oligotrophic waters and in- of this marker gave somewhat inconsistent results. For in- habits springs and upper stream sections. Unlike other spe- stance, COI performed well for Adriohidrobia (Wilke and cies of the genus, occurring in the Northern and Eastern Falniowski 2001) but it was hardly convenient for Hauffenia Carpathians, B. pannonica exclusively inhabits limestone (Šteffek et al. 2011). Studies on Bythinella (Bichain et al. habitats. It is distributed in two well distinguishable sub- 2007; Falniowski et al. 2009b, c, 2012; Falniowski and areas; a southern one in the Bükk Mts. (Hungary) and a Szarowska 2011; Haase et al. 2007) have shown a certain northern one, which involves the Aggtelek Karst (Hungary) degree of incongruence between COI-based phylogeny and and the neighbouring Slovak Karst (Slovakia) (Fig. 1). morphology-based species delimitations. This phenomenon Aggtelek and Slovak Karst form one continuous biogeo- might be explained by the inadequacy of the marker used; graphical subregion, also known as Gömör-Torna Karst however, a more concrete reason is difficult to define in a (hereafter referred to as Karst). The two subareas are group like Bythinella. As the delimitation of Bythinella spe- cies is based largely on morphological characters of limited applicability, one might rather suppose that the incongruence is the result of incorrect taxonomy instead of an inappropri- ate marker. In order to test the applicability of the molecular marker used, reliably delimited taxa are needed. As an important step towards the better understanding of the uses and limita- tions of the COI marker in the phylogenetic reconstruction of Bythinella and the clarification of the systematics of this genus, we aimed to investigate the intraspecific variability of such a species. Bythinella pannonica (Frauenfeld, 1865) seemed to be the most appropriate study system for this purpose. This is one of the few species within this genus that is distinguishable unambiguously by morphological fea- tures. By shell characters, B. pannonica is so strikingly different from other Bythinella species that, prior to the revision of Szarowska and Wilke (2004), it was classified in another genus, Sadleriana Clessin, 1890. Its distinct spe- cies status is, therefore, generally and undisputedly accepted Fig. 1 Relief map of the northeastern part of the Carpathian Basin (Falniowski and Szarowska 2009). At the same time, the showing collection sites and the geographic distribution of intraspecific number of known populations (>50) and the subdivided clades [at the level at which significant phylogenetic structure was geographical range makes it feasible to get an insight into found by nested clade phylogeographic analysis (NCPA)]. Clade names correspond to those in Figs. 2 and 3. More precise locality data are given the structure and the spatial pattern of the intraspecific in Table 1. Thick solid line Political border between Hungary and diversity. Slovakia Mitochondrial diversity in Bythinella pannonica 571 separated by a 25–30 km wide non-limestone zone. The (BIC). Molecular clock likelihood ratio test (LRT) was number of known populations is>50 (Domokos 1992; performed by Tree-Puzzle version 5.2 (Schmidt et al. 2002) Farkas 2005; Lisicky 1991; Lukács 1954; Pintér and Suara to test the clock hypothesis. 2004; Szabó 1984; Wagner 1937). Its geographic range over- Using all Bythinella COI sequences available in laps with that of B. austriaca (Frauenfeld 1857) but, except GenBank, a preliminary NJ tree with 1,000 bootstrap repli- for some rare occasions, the two species are not syntopic (see cates was constructed by MEGA version 5 (Tamura et al. also Wilke et al. 2010). 2011) to test the monophyly of