Org Divers Evol (2010) 10:5–22 DOI 10.1007/s13127-010-0006-2

ORIGINAL ARTICLE

Old museum samples and recent : A taxonomic, biogeographic and conservation perspective of the tatrensis species complex (Crustacea: )

Cene Fišer & Charles Oliver Coleman & Maja Zagmajster & Benjamin Zwittnig & Reinhard Gerecke & Boris Sket

Received: 13 December 2008 /Accepted: 21 July 2009 /Published online: 9 March 2010 # Gesellschaft für Biologische Systematik 2010

Abstract Natural history museum collections harbour tional criteria reveal northern and southern diagnosable and valuable information on species. The usefulness of such exclusive lineages identified here as N. tatrensis data critically depends on the accurate identification of Wrześniowski, 1888 and N. scopicauda sp. n., respectively. species, which has been challenged by introduction of The remaining populations represent the non-exclusive N. molecular techniques into taxonomy. However, most aggtelekiensis Dudich, 1932, which occurs from the eastern collections may suffer from DNA degradation, due to age Alps to Hungary. In the entire complex, altitudinal and/or improper preservation; hence the identification of distribution is largely limited to areas above 400 m, where specimens depends solely on morphological features. This the mean annual temperature never exceeds 9°C. Seemingly study explores how and to what extent morphological data well-defined distributional ranges of N. tatrensis and N. can help to solve ambiguous taxonomic cases based on aggtelekiensis are fragmented in an ecological sense, which selected species concepts and with the use of operational raises the question whether two of the three species criteria in a species-hypothesis testing procedure. The recognised here actually consist of several unidentified studied taxon, the Niphargus tatrensis species complex, taxa. Morphological similarity between the species, numer- comprises freshwater subterranean amphipods, distributed ous polymorphic features, and the association with cool across Central Europe, the taxonomic status of which was temperatures lead to a hypothesis in which fragmentation of debated extensively between 1930 and 1960. Using the the ancestral range occurred during post-Pleistocene climate general species concept, character- and tree-based opera- warming, reducing gene flow across lowland populations due to niche conservatism of the ancestral species and/or to invasion of competitive species along the Danube and Drava rivers. The results are discussed regarding how old C. Fišer (*) : M. Zagmajster : B. Sket museum samples are conducive to more detailed molecular- Oddelek za Biologijo, Biotehniška Fakulteta, taxonomic and conservation studies. Univerza v Ljubljani, PO box 2995, 1001 Ljubljana, Slovenia Keywords General species concept . Population aggregation e-mail: [email protected] analysis . Tree based species delimitation . Mantel test . C. O. Coleman Museum collections . Polymorphic characters Museum für Naturkunde der Humboldt-Universität zu Berlin, 10115 Berlin, Germany

B. Zwittnig Introduction Akademska in raziskovalna mreža Slovenije, PO box 7, 1001 Ljubljana, Slovenia Taxonomy has progressed remarkably in the past 50 years, both conceptually and technically. Molecular R. Gerecke Biesingerstr. 11, techniques have been found to be a powerful tool for the 72070 Tübingen, Germany detection of morphologically similar (‘cryptic’ or ‘sib- 6 C. Fišer et al. ling’) species, and we are about to realize how much the unsatisfactory (Lefébure et al. 2006, 2007; Trontelj et al. diversity of some groups has been underestimated (e.g. 2009). Morphological characters either are highly homo- Bickford et al. 2007). As a result, many previous plastic (Fišer et al. 2006a, 2007a), were overlooked or their identifications of old samples appear questionable—at taxonomic importance was underestimated (Fišer and least in some groups—and their value for science becomes Zagmajster 2009); many cryptic species may be expected limited (Graham et al. 2004). Advances in molecular (Trontelj et al. 2009). Furthermore, taxonomist-subjective techniques allow DNA extraction from rather old samples assignment of species rank (Fišer et al. 2009b) can be a (Stuartetal.2006; Wandeler et al. 2007), but only if the source of bias in subsequent ecological, biodiversity and latter were preserved properly.Thisisusuallynotthecase conservation studies (e.g. Agapow et al. 2004). with old samples; to our knowledge DNA The primary targets in niphargid taxonomy, therefore, are extraction has been unsuccessful in many such cases ‘species’ with large ranges that possibly represent complexes (authors’ experience; pers. comm. from other researchers). of morphologically similar species (Trontelj et al. 2009). The The same is true for all samples preserved at high focal ‘species’ of this study is N. tatrensis Wrześniowski, temperatures or with inappropriate preservatives such as 1888, originally described from southern , which is formaldehyde. On the other hand, museum collections important for several reasons. Its name was established harbour specimens that are rare and hard to be matched by early, thus has a long and stable standing in nomenclature. fresh samples; the many reasons for this include taxon Second, the wide range of the taxon encompasses the extinction as the result of “a changing world” (Wheeler territories of Poland, , the Czech Republic, Austria 2004;Winston2007). Consequently, the key to obtaining and Hungary. Finally, the variability of the ‘species’ has biologically relevant information from such specimens is attracted the attention of taxonomists, who have disagreed the comparative analysis of their morphology (Wiens about its taxonomic status. After the original description, 2004a). Progress in debates of species concepts (e.g. de seven ‘forms’ from Austria, Poland and Hungary were Queiroz 2007) and the development of operational criteria distinguished (Schellenberg 1935, 1937, 1938). [The names for species delimitation (DeSalle et al. 2005; Sites and newly proposed for these ‘forms’ by Schellenberg (1935, Marshall 2003, 2004;Wiens1999, 2007) set a framework 1937) unequivocally denoted infrasubspecific entities. Since that should help interpret ambiguous patterns of morpho- they were never used as valid for a species or subspecies logical data. It is expected that a systematic study of the prior to 1985, they are nomenclaturally unavailable under morphology of specimens from old collections should lead ICZN (1999) Code Article 45.6.4.] Schellenberg’s divisions to more convincing taxonomic conclusions, or at least to a were criticized by Jersche (1963), who explicitly rejected better understanding of the geographic distribution of three forms from the eastern Alps, and by Micherdziński morphological variation. In this paper we explore how (1956), who recognised only the “North-Alpine”, “Silesian much and what kind of information can be grasped from (Sudety)” and “Tatry” forms. Both authors based their old museum samples that were taxonomically re-evaluated conclusions on “considerable plasticity and wide range of using the general species concept (GSC) and operational variability” (Micherdziński 1956) that is larger within than criteria developed since the early 1990s. between populations. More recently, Sket (1994) reported The material studied belongs to the amphipod genus on an undescribed niphargid from northern Slovenia, Niphargus Schiödte, 1849, the members of which mainly informally named N. “scopicauda n.n.”, that proved to be inhabit groundwater in Europe and the Middle East (Fišer et morphologically and genetically similar to N. tatrensis al. 2009a; Karaman and Ruffo 1986). Apart from evolu- (Fišer et al. 2008b; Trontelj et al. 2009). tionary and ecological issues, the high number of species in The first aim of the present study is a taxonomic (re-) this genus (Väinölä et al. 2008) and the high degree of evaluation of variation in the ‘species’ considered as N. endemism in most of them (Fišer et al. 2006b; Trontelj et tatrensis and populations informally named N. scopicauda. al. 2009) suggest that Niphargus could play an important We relied on the general species concept (GSC; de Queiroz role in conservation planning for European groundwaters 1998, 2007) that was tested with character- and tree-based (see, e.g., Bininda-Emonds et al. 2000; Funk et al. 2002; operational criteria. In a second step, the taxonomic Myers et al. 2000). Moreover, the capacity of niphargids to conclusions are critically reviewed with the aid of correla- accumulate heavy metals implies that they could be used as tion distance matrix analysis, and with an analysis of the biological indicators (Coppellotti Krupa and Guidolin distribution of some recent ecological factors. The second 2003). aim is to address the role of collections. Based on the These topics have remained virtually unexplored mainly taxonomic conclusions drawn here, we illustrate how due to the problematic taxonomy of the genus (Ruffo morphology-based taxonomic conclusions affect further 1972). The genetic diversity in Niphargus implies that the conservation studies, and what the current impediments to taxonomy, as inferred from morphology so far, is largely morphology-based taxonomy are. Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 7

Material and methods extended through time or ancestral-descendant series of time-limited (instantaneous) populations. In this view, de Samples Queiroz identified a separate evolution of metapopulation lineages as a property necessary for the postulation of a The specimens examined derived from the Schellenberg species hypothesis, whereas any of the properties required collection held in the Museum für Naturkunde Berlin by different concepts become a sufficient pointer for (totalling 45 samples from Poland, the Czech Republic, postulation of a species hypothesis. The focus of the Austria and Hungary), and from the collection of Odd- analyses in the present work is the identification of two or elek za biologijo, Biotehniška fakulteta Univerza v more metapopulations, among which gene flow has been Ljubljani (10 samples from Slovenia, Austria and the restricted. The operational criteria for species delimitation type locality of N. tatrensis). Distribution data were used together with underlying assumptions are outlined in obtained from the collections and supplemented with the next section. information from the literature (Micherdziński 1956; Skalski 1972). Several samples consisted of juvenile Assumptions and operational criteria specimens, which together with literature data remained identified only to the level of a species group. Altogether All approaches outlined below require an assumption that 17 populations distributed across the entire range of the each sample (population) from each locality consists of a complex were re-examined morphologically. single species. The assumption is based on the premise that In the first phase of research, the type population of N. two morphologically similar, closely related species com- tatrensis, voucher material for the seven ‘forms’ described pete for the same food and space, which will result in a by Schellenberg (1935, 1937, 1938), and one sample of N. competitive exclusion of the weaker competitor in a scopicauda (Table 1) were reviewed for a large set of relatively short period of time (Fišer et al. 2007b; Ginet morphological characters traditionally considered as infor- 1965; but see also Fišer 2006b). Next, as pointed out, the mative in niphargid taxonomy (Fišer et al. 2009b, c; see term monophyly may not be applicable at the species level; also http://niphargus.info). In order to minimize variation for this reason we operate with the related term ‘exclusive’ due to differences in age or sex (Fišer et al. 2008a), only (e.g. Graybeal 1995). adult specimens were examined (males with differentiated The species hypotheses were built using the following uropods III; females with oostegites). Only characters that protocol. In the first step we separately postulated species showed between-population variation (Table 1)were hypotheses using character-based and tree-based opera- reviewed for a larger number of adult individuals. We tional criteria described in more detail below. The final present and discuss these characters in the “Results” section species hypotheses are the conservative consensus of below, together with remarks on how observations were results in order to minimize the flaws of both methods. treated in the character-coding process. In the second step, we searched for additional support for species hypotheses in correlation between morphological Species concepts variation and geographic distribution. Finally, the avail- able ecological information was used to explain current Sites and Crandall (1997) emphasized the importance of morphological variation and geographic distribution of viewing the diagnosis of species boundaries as a the complex within a historical perspective and the hypothesis-testing procedure, with clear statements of the niche-conservatism concept (Wiens 2004b;Wiensand criteria used. Despite the long history of dispute over Graham 2005). species concepts, Mayden (1997) and de Queiroz (1998, 2005a, b, 2007) found that all contemporary species Character-based species delimitation concepts and definitions share common a element related to the speciation process that (according to de Queiroz Character-based species delimitation has been formalised in a 1998, 2005a, b, 2007) is a key to reconciling the alternative procedure called population aggregation analysis (PAA; Davis species concepts. As pointed out, differences among species and Nixon 1992). PAA involves systematically comparing concepts arise on account of different defining (necessary) character state distributions among populations, aggregating properties that function as cut-off criteria in species sets of populations which differ only in polymorphic traits, delimitation. By contrast, all of the concepts explicitly or and considering sets of populations which differ by at least implicitly equate species with separately evolving (seg- one seemingly fixed difference (or which shares no states for ments of) metapopulation lineages, where a metapopulation that character) to be different species. The usage of the is an inclusive population made up of a set of subpopula- method has been extended to the nonoverlapping ranges of tions, and a lineage (at the population level) is a population quantitative characters (Wiens and Penkrot 2002). The 8 Table 1 Distribution of character states in the Niphargus tatrensis aggregate

Species Population Locality (number of specimens Maxilliped Pp IIIb Pp IVb Pp Vc Pp VI Pp VII Telson Telson Telson Telson Telson examined)a inner lobe apical lateral mesial dorsal lobes setae extrasp. extrasp. extrasp. extrasp. extrasp. spines (A, spines (A, spines (A, spines apical (A, R) (F) (F) (F) (F) (F) R) R) R) (A, R) shape

N. tatrensis type Zakopane (3) 3.33 (3–4) 0.00 0.00 0.00 0.67 0.67 3.50 (3–4) 1.75 (0–3) 0.33 (0–1) 1.00 (1) narrow Byči Skala (15) 3.50 (3–4) 0.72 0.76 0.00 0 0.16 3.00 (2–4) 1.47 (1–2) 0.83 (0–2) 1.07 (1–2) narrow Demanowska Höhle (15) 3.00 (3–4) 0.08 0.14 0.00 0.04 0.45 3.04 (2–4) 1.64 (1–2) 0.07 (0–1) 1.57 (1–3) narrow “bei Rajec” (15) 3.50 (3–5) 0.07 0.03 0.00 0 0.29 3.03 (3–4) 1.73 (1–3) 0.07 (0–1) 1.17 (1–3) narrow “f. Reyersdorfer Höhle (16) 3.00 (1–3) 0.30 0.30 0.00 0 0 3.10 (3–4) 1.30 (1–2) 0.40 (0–1) 1.03 (1–2) narrow reyersdorfensis” “f. Patzelt Höhle (10) 3.00 (3–4) 0.00 0.00 0.00 0.18 0.8 3.00 (2–4) 1.85 (1–3) 0.00 (0) 1.00 (1) narrow schneebergensis” N. aggtelekiensis type* Aggteleker Höhle (18) 4.50 (4–5) 0.84 0.95 0.06 0.04 0.69 3.00 (3) 0.51 (0–2) 0.00 (0) 1.03 (1–2) wide Ostmark (6) 2.80 (2–3) 0.08 0.00 0.00 0 0.25 4.00 (4) 0.92 (0–2) 0.08 (0–1) 1.08 (1–2) wide Koppenbrueller Sinonykapelle 3.10 (3–4) 0.00 0.00 0.00 0 0 4.40 (4–5) 1.05 (0–2) 0.20 (0–1) 1.00 (1) wide (10) Gesäuse National Park (8) 3.00 (3) 0.73 0.71 0.10 0 0.5 3.88 (3–5) 0.81 (0–2) 0.31 (0–1) 1.00 (1) wide “f. lunzensis” Lunz, 4 samples (17) 2.75 (2–3) 0.97 0.97 0.31 0.65 0.97 3.79 (3–5) 0.83 (0–3) 0.37 (0–1) 1.94 (1–3) wide “f. lurensis” Lurhöhle, Semriach (16) 2.00 (1–3) 0.88 0.87 0.00 0.97 0.97 4.25 (3–5) 1.25 (1–3) 0.07 (0–1) 1.00 (1) wide “f. oetscherensis” Oetscher Höhle (3), Nixhöhle 2.50 (2–3) 0.67 0.77 0.23 0.25 0.82 3.86 (3–4) 0.57 (0–2) 0.00 (0) 1.14 (1–2) wide (6) “f. salzburgensis” Schenkofenhöhle, Sulzak (4) 3.00 (3–4) 0.72 0.72 0.07 0 0.40 4.00 (4) 1.25 (1–2) 0.80 (0–1) 1.08 (1–2) wide N. scopicauda Belojača (4) 3.00 (3) 0.13 0.00 0.00 0.40 0.43 3.88 (3–4) 0.38 (0–1) 0.00 (0) 0.00 (0) wide Habidova grapa (3) 2.50 (2–3) 0.00 0.00 0.00 0.00 0.00 4.25 (4–5) 0.38 (0–1) 0.00 (0) 0.00 (0) wide Huda Luknja (10) 2.50 (2–3) 0.04 0.04 0.00 0.18 0.48 4.30 (4–5) 0.26 (0–1) 0.04 (0–1) 0.00 (0) wide

(A, R) average or range, extrasp. extraspine, (F) frequency, Pp pereopod a We reviewed the left and right side of each specimen; individuals in some populations are extremely asymmetrical b Here we present raw frequencies. In the analysis we treated extraspines on pereopods III–IV as a single character, calculated as average frequency (see text) c These frequencies of extraspine presence on pereopod V seem to be too low to be informative, thus were excluded from further analyses .Fi C. š re al. et er Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 9 approach is advantageous in that there is a clear relationship strong and spiniform setae on the pereopod dactyls and the between fixed differences and gene flow—if the diagnostic shape of the telson were treated as qualitative characters, traits are genetically based and are truly fixed in each which were expressed in frequencies, whereas the numbers species, there is unlikely to be any gene flow between the of strong and spiniform setae on maxilliped and telson were species. However, given finite sample sizes, determining treated as quantitative traits and expressed as value ranges. with certainty whether traits are truly fixed is almost Overlap in trait values between populations was treated as impossible. We followed Wiens and Servedio (2000)in equivalent to polymorphism in qualitative traits. order to evaluate statistical confidence in distinctness of sets of populations that were delimited using the character-based Tree-based analyses approach. Hence, we tested whether sample sizes (for a given set of populations) were large enough to argue that at Davis and Nixon (1992) stated that phylogenetic analysis least one of the diagnostic characters was not polymorphic should not be attempted below the species level, because above a selected frequency cut-off. The maximum frequency gene flow among populations and recombination among of the alternative character state allowed in the diagnostic characters should break up hierarchical patterns within character used as a cut-off value was 0.1. Failing this test species inferred from morphological data. By contrast, meant that there was greater than 5 % probability that all of Wiens and Penkrot (2002) advocated a tree-based approach, the diagnostic characters for a given taxon were actually using the assumption that the absence of hierarchic polymorphic, with the unobserved character state occurring relationships below the species level leads to weakly at a frequency of 0.1 or greater. supported population-level trees that are discordant with The characters used in our study are presented in the geography, and that this lack of signal can be detected and “Results” section and in Fig. 1. The presence of additional used to infer species limits in cases where these limits are

Fig. 1 Morphological charac- ters included in the analysis. (a–c) Inner lobe of maxilliped, showing flattened spines; a Niphargus aggtelekiensis “f. lurensis”, b N. tatrensis “f. schneebergensis”, c N.. aggtele- kiensis, topotype. (d, e) Dactylus with (d) and without (e) addi- tional spiniform seta. (f–h) Niphargus tatrensis, telson nar- rowed and concave in apical part (f = “forma schneebergen- sis”, g = topotype, h = “forma reyersdorfensis”). (i–l) Niphar- gus aggtelekiensis, telson broad convex (i = topotype, j = “forma oethscerensis”,k=“forma lun- zensis”,l=“forma salzburgensis”) 10 C. Fišer et al. unknown. Sets of populations that were strongly supported analysis with morphological distances includes only those as ‘exclusive’, and were geographically coherent, were 17 populations, from which sufficiently large samples recognized as potentially distinct species. This approach allowed an estimation of morphological variation. uses properties of populations rather than properties of Dissimilarity coefficients for pairs of populations were individuals as terminal units, because morphological char- summed from standardized absolute differences in frequen- acters are generally thought to be genetically unlinked, and cies (quantitative characters) and average values (qualita- using individuals will inappropriately treat all polymor- tive characters) for all characters using the program phisms shared between populations as homoplasies rather package DELTA (Dallwitz et al. 2007). Geographic than potential synapomorphies. distances among localities were calculated with Hawth’s We followed the above assumptions. Qualitative poly- Analysis Tool, ver. 3.27 (Beyer 2008) in ArcMap (ArcGIS, morphic characters were coded using the frequency-based ver. 9.1; ESRI). step-matrix approach (for performance see Wiens 1995; To test whether specimens closer to each other in space Wiens and Servedio 1998); quantitative characters were are more alike we calculated correlation of the two matrices coded using step-matrix gap-weighting (Wiens 2001). The using a Mantel test. We used the ADE4 package (Chessel et method uses absolute differences between the standardized al. 2008) in the R environment (www.r-project.org). raw data (summarized as an average value) as weights. The Statistical significance was tested with 1000 randomizations maximum number of steps for any step-matrix character was (Chessel et al. 2008). 100. In order to maintain balanced character weights, i.e. the same cost for a transition between fixed character states for Use of ecological data all morphological and molecular characters, all other charac- ters were weighted by 100. Step matrices were constructed In this part of the analyses we included and mapped using a Pearl script (available at http://www.niphargus.info/). additional localities from the literature. Relying on pub- As bootstrapping of nine characters makes virtually no lished data and preliminary analyses we eliminated param- sense, we tested clade support by modifying weights eters that do not affect the distribution of the species between character states in a step matrix by modification complex—presence of karst (own data); pH and carbonate of difference between raw data by square and root hardness (Micherdziński 1956)—and constrained this part functions. Furthermore, in accordance with existing molec- of the analyses to the temperatures. The temperature of the ular studies (Fišer et al. 2008b; Trontelj et al. 2009)we subterranean environment roughly corresponds to the mean included Niphargus schellenbergi S. Karaman, 1932 as the annual temperature of the area (Gams 1974), a parameter sole outgroup taxon. related to altitude and latitude. We overlaid the distribu- tional data over a map of mean annual temperature layers Correlated distance matrices for Europe (Hijmans et al. 2005; layers available via http:// www.worldclim.org/) in order to see whether distribution We tested whether morphological similarity and geographic corresponds with either of the two parameters. The spatial distance matrices correlate with each other. Our two-step resolution of the original layer is a square kilometre. The procedure was based on two assumptions. (1) Phenetic temperatures at localities that could not be georeferenced diversity, when significantly correlated with geography, precisely were obtained by averaging the mean annual may present evidence either for a morpho-cline or for temperature values within a diameter of 10 kilometres in separate metapopulations consistent with geography. (2) order to minimize possible error. The absence of significant correlation may indicate panmictic populations. In the first step, calculations were performed on the whole set of data that were morpholog- Results ically examined, whereas in the second step correlations were repeated for the populations of the three putative Characters species, namely N. tatrensis, N. aggtelekiensis and N. scopicauda. The morphological characters reviewed in this section Localities were georeferenced as precisely as possible showed outstanding between-population variation and form with the use of various cartographic sources. The studied the basis of all analyses. The maxilliped consists of an inner region was projected in Lambert Conformal Conical lobe, outer lobe and palpus. In the apical part of the inner Projection for Europe (central meridian 10°, standard lobe there are two types of setae: cylindrical ones with fine parallels 43° and 62°, latitude of origin 30°), which was hair, and stronger flattened setae (character 1; Fig. 1a). The used for measures of geographic distances. In total, 27 number of the latter varies between 2 and 5 per lobe; it is localities were georeferenced (Appendix A). Correlation lower in juvenile than in adult specimens and can differ Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 11 between the left and right body sides of a specimen. Species hypotheses However, Schellenberg (1938) reported an increased num- beroftheseflattenedsetaeinN. tatrensis “forma” The studied characters are highly variable (Table 1). The aggtelekiensis. PAA merged populations into three groups on the basis of The dactyls of pereopods III–VII are regularly armed two characters. Populations aggregated into N. tatrensis with a single dorsal (convex side) plumose seta and with sensu stricto share narrowed and apically concave telson one weak and one spiniform seta close to the insertion of lobes (Fig. 1c, d), whereas in the remaining populations the the nail. In addition, one, rarely two additional spiniform apical lobes are wide and convex. The three southernmost setae can be developed in the proximal part of the dactylus populations treated as N. scopicauda, in contrast to the rest in the N. tatrensis aggregate (Fig. 1b). Schellenberg’s of the populations, lack strongly spiniform setae on the (1935, 1937) definitions of his ‘forms’ were largely based dorsal surface of the telson. A weak point of these results is on the presence of these additional spiniform setae. the number of specimens analysed: According to the However, close examination has revealed that their pres- probabilistic approach by Wiens and Servedio (2000), a ence varies even in fully developed (i.e. the least variable) minimum of 30 specimens should be analysed to achieve specimens. Additional spiniform setae develop on different 95 % confidence that seemingly rare frequencies of the two pereopods at different frequencies; the armature of the left out of nine studied characters are fixed below the value of and right appendages can differ. A comparison with other, 0.1. Even when the number of specimens was increased unrelated niphargid species shows that such extra setae artificially by treating left and right sides of the develop either on all pereopods (pp), exclusively on pp III separately, the number of analysed individuals still was and IV, on pp V–VII, on pp VI and VII, or exclusively on insufficient in nine out of seventeen populations. However, pereopod VII (e.g. Fišer et al. 2006a). Assuming indepen- assuming that aggregated populations constitute the same dent evolution of extra setae on different pereopods, we metapopulation, the total number of the analysed specimens treated pp III–IV, pereopod VI and pereopod VII as three is high enough to assume fixations of the two diagnostic separate, independent characters (characters 2–4) rather characters. than as a single putative serial homologue. Pereopod V was All five parsimony analyses resulted in a similar hierarchic omitted from the analyses, since it rarely carries extra setae structure (Fig. 2; linear weighting: length = 1925.3, CI = (Table 1). We examined the left and right sides of each 0.467, RI = 0.661; quadrate weighting: length = 1723.3, CI = ; each extra seta was counted as ‘present’. Using 0.522, RI = 0.665; square-root weighting: length = 2016.8, these data we estimated the frequency of presence of extra CI = 0.446, RI = 0.655). Regardless of the weighting spiniform setae (or extraspines) for each character in all scheme, the aggregate consists of a southern (N. scopicauda) populations. Frequencies calculated for pereopods III and and a northern lineage. The latter is further split in two IV differed minimally; hence, character 2 is the average of populations and two large groups; one of them being N. the frequencies calculated for pereopods III and IV. tatrensis s. str. Considering the size of the dataset, the The telson is bilobed. The shape of the lobes differs question arises whether exclusivity assumed for these five among populations (Schellenberg 1937). The apical part of lineages is true or not. a lobe is either broad with clearly convex outer margins or PAA and parsimony analysis agree that the northernmost more or less narrowed with concave or ‘broken’ outer and the southernmost populations are diagnosable and margins (character 5; Fig. 1c, d). Furthermore, four groups exclusive. We find it plausible to consider the two groups of spiniform setae that seem to have evolved independently of populations as separate species, to which we therefore (Fišer et al. 2006a) can be identified on the telson (Fig. 1d): apply the names N. tatrensis and N. scopicauda, respec- an apical (character 6), lateral (character 7), mesial (located tively. The remaining populations constitute three para- in the telson’s cleft; character 8) and a dorsal group phyletic lineages. In contrast to the results from the (character 9). The number of spiniform setae depends on parsimony analysis, the PAA implies gene flow between the specimen’s age and can be left-right asymmetric. these presumable lineages. Therefore, they could be Schellenberg (1935, 1937) noted varying numbers of dorsal considered as a non-exclusive species (“ferespecies” sensu spiniform setae, whereas the remaining spiniform setae Graybeal 1995), for which the name N. aggtelekiensis remained unstudied. In contrast with pereopod dactyls, for Dudich, 1932 would be available. which we express the presence of extra spiniform setae in Some additional support for the consensus between the frequencies, the numbers of spiniform setae on (the left and generated character- and tree-based species hypotheses is right lobe of) the telson in a population were pooled and provided by the geographic distribution of the putative subsequently treated as average values (tree-based opera- species (Fig. 3). Niphargus tatrensis is distributed north- tional criteria) or ranges (character-based operational ward from the Danube (Donau) river, across mountain criteria). ridges along the southern Polish and northern Czech and 12 C. Fišer et al.

Fig. 2 Most parsimonious tree based on morphological charac- ters; except for branch-length differences, identical topology was obtained regardless of the weighting scheme

Slovakian borders; N. aggtelekiensis is widespread in the The subset of N. scopicauda was too small, thus was not eastern Alps (south of the Danube), with an isolated analysed further. population in northwestern Hungary (north of the Danube). The present-day distribution corresponds to altitudes Niphargus scopicauda is limited to a narrow prealpine area above 400 m (with minor exceptions), at which the mean in northeastern Slovenia, south of the Drava (Drau) river. annual temperature never exceeds 9°C (Fig. 3, Appendix A; Interestingly, the distance matrix based on morphological note that two localities at which temperatures exceed 8°C variation showed a highly significant correlation (coeffi- refer to Salzburg, not to any in the region). cient = 0.418, p=0.00099) with the distance matrix based Considering low temperatures as a factor affecting distri- on geographic distribution, when all 17 populations were bution (see “Discussion” for different views of this pattern), included. By contrast, the correlations largely decreased the ranges of N. tatrensis and N. aggtelekiensis appear and became non-significant when populations of N. fragmented. Distributional data thus unambiguously sup- tatrensis (coefficient = 0.009, p=0.411) and N. aggtele- port only the species hypothesis for N. scopicauda, and to a kiensis (coefficient = 0.227, p=0.240) were analysed alone. lesser extent the species hypothesis for N. tatrensis, Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 13

Fig. 3 Distribution of the Niphargus tatrensis aggregate in relation to mean annual temperature (below 8°C in the shaded areas) whereas the status of N. aggtelekiensis is questionable. distribution of the aggregate that corresponds to low temper- Compared to the Alpine populations the Hungarian one atures. Morphological similarity and lack of exclusivity in N. exhibits a higher number of flattened spines on the inner aggtelekiensis favour the hypothesis of relatively recent lobe of the maxilliped (4–5and2–3, only rarely 4, emergence two exclusive lineages (N. tatrensis, N. scopi- respectively), but independent evolution of the two (groups) cauda) over the alternative view, i.e. that speciation took of populations is not supported by a clear and unambiguous place in ancient karstic massifs. Low-temperature dependent gap in morphology. Unfortunately, from that part of the geographic distribution implies that Quaternary glaciations aggregate range we had a single sample of adult specimens, played an important role in diversification of the N. tatrensis which does not allow us to test the exclusivity. complex. The present data are insufficient to resolve whether To summarize, we hypothesize the existence of three individual populations survived ice ages under glaciers species, keeping in mind that the diversity of the aggregate (Kristjánsson and Svavarsson 2004) or recolonized the might be underestimated. No data reject the hypothesis of empty niches after retreat of the glaciers (Folquier et al. Niphargus scopicauda as a separate species which is 2008). Nevertheless, some hypotheses on causes for the formally established in Appendix B. separation of lineages can be discussed. The most straight- forward explanation assumes that temperatures act as a distribution-limiting ecological factor, so that post- Discussion Pleistocene climate warming negatively affected fitness of lowland populations and reduced gene flow across these Evidence for recent speciation areas. “Niche conservatism” (Wiens 2004b; Wiens and Graham 2005)ofN. tatrensis—inability to adapt to high Our view on the history of the aggregate is built on (1) temperatures that limits dispersal—explains its distribution. morphological similarity among lineages coupled with a high This hypothesis, however, is in strong contrast to the number of polymorphic characters, and (2) geographic general view that niphargids, including N. tatrensis (Jersche 14 C. Fišer et al.

1963;Micherdziński 1956), are generalists. Although species hypotheses presented here would also fulfil the temperature preferences have never been studied in detail, criteria of related evolutionary species concepts (e.g. researchers have argued that various niphargid species are Mayden 1997) or of (some) phylogenetic species concepts euryhaline (Jersche 1963; Sket 1977) or able to cope with (Davis and Nixon 1992), whereas the evaluation whether scarce food (Hervant et al. 1997, 1999a), low oxygen the members of the three groups of populations could concentrations (Hervant and Mathieu 1995; Hervant et al. interbreed successfully (biological species concept) remains 1999b; Sket 1977) and low pH (Simčič and Brancelj 2006). a challenge to future research. By contrast, niphargids seem to be poor competitors (Fišer The most important taxonomic issue that needs to be et al. 2007b;Sket1981), which supports an alternative resolved is the status of N. aggtelekiensis. Ecological data, hypothesis to explain fragmentation of the ancestral especially when interpreted within the historical context, population. This view assumes a widely distributed ances- disagree with the conclusions based on morphology alone. tral population living in both surface and subterranean This discrepancy can be the result either of incomplete waters (Fišer et al. 2006b; b;Sket1958; 1981). Post- sampling in the lowland area and an inappropriate Pleistocene warming could have resulted in extensive changes temperature-dependence hypothesis, or of incomplete tax- to the crustacean fauna along the Danube and Drava rivers, onomic conclusions which underestimated both, the ob- including the arrival of ‘paleo-invasive’ species. The repro- served morphological differences between Hungarian and ductive potential of gammarid species seems to be higher than Austrian populations (number of flattened spiniform setae that of niphargids (Fišer et al. 2007b), which could have on maxilliped inner lobes) and the slightly higher temper- formed the basis for competitive exclusion of the studied ature regime at the Hungarian locality (see Raxworthy et al. niphargids (Fišer et al. 2007b; Grabowski et al. 2007; 2007 for species identification using the accuracy of Savage 1981) and consequently led to fragmentation of the ecological models). While the first alternative seems less range of the ancestral population. Other Niphargus species probable, the second could be tested with molecular and/or within the area (Nesemann et al. 1995) also could have ecological data. Similarly, the postulated range of N. played an important role in competition by occupying tatrensis is fragmented in an ecological sense. The question possible refugia or distributional pathways such as interstitial whether this fragmentation affects the taxonomic conclu- spaces in large rivers. The cessation of gene flow between sions presented here remains unsolved. subpopulations of the ancestral population could have been a In short, even in taxonomic puzzles such as Niphargus, result of interactions resembling those observed today morphology alone renders some taxonomic conclusions. A between invasive and native species (Jażdżewski et al. formalized species concept / operational criteria procedure 2004; Sax et al. 2007; Vellend et al. 2007). seems to be a key to unlocking the doors of collections with material of even the most problematic taxa the DNA of The collections and modern taxonomy: implications which has been damaged. From that point of view we welcome both, additional development of methodology and Whenever dealing with poorly differentiated species, projects promoting the exploration of collections, such as taxonomic conclusions largely depend on the selection Synthesys (http://www.synthesys.info/). and number of observed characters, abundance and number of samples, as well as on species concepts and methods The role of collections: guidelines for conservation used. The latter two factors play rather important roles— after all, we reanalysed Schellenberg’s samples using In conservation biology, the present study yields the most virtually the same characters as Schellenberg (1935, 1937, interesting effects if the subject of conservation is variation 1938) had. Cases in which the evidence for separation of on a subspecific level (evolutionary significant units; see lineages is weak will always be debatable and will largely Bininda-Emonds et al. 2000; Crandall et al. 2000; Moritz depend on the particular species concept applied. The 1994). The major concern with this approach is the general species concept advocated by de Queiroz (1998, maintenance of species fitness and its capacity for evolu- 2005a, b, 2007) minimizes the number of properties needed tionary response to environmental change. As pointed out, for the definition of species, and therefore inherently forces subspecific variation depends on long-term historical a splitter’s taxonomy. It can be expected that some isolation (vicariance) and adaptive evolution (phenotypic taxonomists will be critical of taxonomic conclusions variation) (Moritz 1994; 2002). (However, it remains relying on the GSC. On the other hand, rather than unclear how long-isolated populations defined as recipro- discussing the appropriateness of various species concepts, cally monophyletic groups differ from some, e.g. phyloge- paying more attention to indications of lineage separation netic, species concepts. The GSC—if accepted—may might result in higher ranking of taxonomy among simplify a starting position in this branch of conservation hypothesis-driven fields in biology. For instance, the biology.) We share the opinion of Crandall et al. (2000) Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 15 who state that “the potential for species evolutionary success and two anonymous reviewers critically commented on an earlier is maximized through the maintenance of adaptive diversity, version of the manuscript. The first author received support from the SYNTHESYS Project (http://www.synthesys.info/) financed by Euro- by preserving the maximum diversity of functionally diver- pean Community Research Infrastructure Action under the FP6 gent gene copies across the geographic range of a species.” “Structuring the European Research Area” Programme (DE-TAF- However, if the entire aggregate is treated as a single species, 3960; visit to the Museum für Naturkunde der Humboldt-Universität the orientation of phenotypic diversity follows a north-south zu Berlin). direction. By contrast, considering three groups of populations as three species, the longest axes of their ranges are reoriented roughly perpendicularly to follow a west-east direction. Appendix A Although the studied characters show no geography- dependent subspecific variability, it can be assumed that Geographic origin, temperature and voucher information genetic diversity increases with geographic distance, thus for the known material along the longest axis in the range. This view strengthens habitat patches arranged in a west-east direction with Asterisks denote samples used for analyses in the present favourable temperatures occupied by N. tatrensis.Fromthis study. Locality names are given exactly as by the respective point of view, reconsideration of old museum samples original source (reference or label); any additions are changes both, sampling strategy in studies aiming to evaluate enclosed in square brackets. Georeferences often refer to genetic diversity of species and eventual conservational acts. larger geographic entities rather than to the detailed locality. Abbreviations for country names: A = Austria, CZ = Czech Acknowledgements Our thanks go to Elzbieta Dumnicka (Krakow, Republic, H = Hungary, POL = Poland, SK = Slovakia, Poland) for providing additional samples from Poland. Peter Trontelj SLO = Slovenia.

Species Lat, Long Temperature Source / reference Country: locality (WGS84) mean (10 km range) Niphargus aggtelekiensis Dudich A: Gesaeuse National Park [several samples]* 14.726342, 47.599389 5.9 (5.6–6.0) BF A: Koppenbrueller Höhle, Dachstein Gebirge* 13.719097, 47.568692 7 (6.6–7.0) ZMB 25100 A: Koppenbrueller Sinonykapelle 13.719097, 47.568692 7 (6.6–7.0) ZMB 25546, ZMB 25481, ZMB 25482 A: Herdengelhöhle, Lunz am See 15.050000, 47.850000 5.3 (5.1–5.5) ZMB 25479 [“Ostmark” in publications]* A: Lunz, aus einem Seitenarm des Seebaches* 15.050000, 47.850000 5.3 (5.1–5.5) ZMB 24487, ZMB 24488 A: Lunz 15.050000, 47.850000 5.3 (5.1–5.5) ZMB25240 A: Wilhelminenhöhle bei Lunz 15.050000, 47.850000 5.3 (5.1–5.5) ZMB 24489, ZMB 25244, ZMB 25483, ZMB 24865 A: Semiriacher Eingang, Lurhöhle* 15.402756, 47.217814 6.3 (6.1–6.5) ZMB 24484 A: Lurhöhle bei Peggau 15.350000, 47.200000 8 (7.6–8.0) ZMB 26239 A: Lurhöhle b. Semiriach 15.402756, 47.217814 6.3 (6.1–6.5) ZMB 25245, ZMB 25272, ZMB 25181 A: “Nischöhle” :(=Nixhöhle?) 15.316667, 47.966667 6.9 (6.6–7.0) ZMB 25480 A: Nixhöhle b. :Frankenfels 15.316667, 47.966667 6.9 (6.6–7.0) ZMB 24839 A: Ötscher Höhle, :Nieder Oesterreich* 15.100000, 47.933333 7.4 (7.1–7.5) ZMB 24490 A: Schenkofenhöhle :bei Sulzau 13.166667, 47.500000 7.6 (7.6–8.0) ZMB 24491 A: Schenkofenhöhle, :Hage-Gebirge bei Salzburg* 13.033333, 47.800000 8.9 (8.6–9.0) ZMB 25730 A: Steinkeller bei :Lunz; Gang beim Wasser 14.745000, 46.984722 4.7 (4.6–5.0) ZMB 24492 A: Ybbs Gebiet (Weis :Ois, Aquelle Langau, Muhlbach) 14.916667, 47.800000 6.6 (6.6–7.0) ZMB 25817 A: Baernhoehle, :Hagen-Gebirge bei Salzburg 13.033333, 47.800000 8.9 (8.6–9.0) ZMB 25730 H: Aggteleker Höhle* 20.483333, 48.483389 8.3 (8.1–8.5) ZMB 24864 H: Höhle von Aggtelek 20.483333, 48.483389 8.3 (8.1–8.5) ZMB 25793 SK: Domica Höhle [Jaskyna Domica] 20.472500, 48.476667 8.4 (8.1–8.5) ZMB 24865 16 C. Fišer et al.

N. scopicauda sp. n. SLO: Belojača* 15.654633, 46.299612 8.8 (8.6–9.0) BF SLO: Habidova Grapa* 15.529364, 46.567097 8.1 (8.1–8.5) BF SLO: Huda luknja* 15.174250, 46.414464 7.2 (7.1–7.5) BF N. tatrensis Wrześniowski CZ: Höhle Byči skala [Morawsko]* 16.694722, 49.307500 7.9 (7.6–8.0) ZMB 24790, ZMB 24857, ZMB 24889 POL: Hausbrunnen [house well] in 16.879717, 50.350000 7 (6.6–7.0) ZMB 25181 Reyersdorf [Radechów]* POL: Patzelt Höhle, Glatzer Schneeberg [Patzeltova 16.799619, 50.122667 6.1 (6.1–6.5) ZMB 23906 jeskina, Śnieżnik Kłodzki (POL) or Králický Sněžník (CZ)]* POL: Quarzloecher Glatzer Schneeberg 16.799619, 50.122667 6.1 (6.1–6.5) ZMB 23989 POL: Reyersdorfer Höhle [also reported as Reyersdorfer 16.879717, 50.350000 7 (6.6–7.0) ZMB 24486 Tropfsteinhöhle, Bad Landeck, Schlesien; now Jaskinia Radochowska, Lądek-Zdrój] POL: Salzlöcher b. Habelschwerdt [= Bystrzyca Kłodzka] 16.650000, 50.300000 7.3 (7.1–7.5) ZMB 19368, ZMB 24908 POL: Zakopane* 19.948694, 49.300239 4.8 (4.6–5.0) BF SK: Demänovska Höhle [Demänovská jaskyňa, Tatry]* 19.584233, 48.997700 4.1 (4.1–4.5) ZMB 24866 SK: südl. Tatra u. bei Rajec* 18.633333, 49.083333 7.5 (7.1–7.5) ZMB 25168/71 N. tatrensis group POL: Jaskinia Mrozna 19.871111, 49.269444 4.5 (4.1–4.5) Micherdziński (1956) POL: Lodowe źrodło[“Icy Spring”], Kościeliska dolina 19.869514, 49.261667 4.5 (4.1–4.5) Micherdziński (1956) POL: Jaskinia Malinowska 18.986111, 49.655556 4.3 (4.1–4.5) Micherdziński (1956) POL: Jaskinia Miŕtusia 19.906944, 49.263333 3.5 (3.0–3.5) Micherdziński (1956) POL: Babia gora, Beskidy Mountains 19.566292, 49.584375 3.3 (3.0–3.5) Skalski (1972) POL: Poronin 20.006667, 49.335833 5.9 (5.6–6:.0) Skalski (1972) POL: Jurkow 20.234047, 49.683506 7 (6.6–7.0) Skalski (1972) POL: Wielka Puszcza 19.259167, 49.821389 6.1 (6.1–6.5) Skalski (1972) POL: Pewel Mala 19.277778, 49.662500 7.8 (7.6–8.0) Skalski (1972) POL: Bialka Tatrzanska 20.105556, 49.387778 6.2 (6.1–6.5) Skalski (1972) POL: Romanka 19.277778, 49.662500 7.8 (7.6–8.0) Skalski (1972) POL: Menczot 19.277778, 49.662500 7.8 (7.6–8.0) Skalski (1972) POL: Barania Gora 18.981497, 49.601364 4.4 (4.1–4.5) Skalski (1972) POL: Leskowiec estimated from figure 6.8 (6.6–7.0) Skalski (1972) POL: Gorce estimated from figure 3.1 (3.0–3.5) Skalski (1972)

Appendix B Taxonomic section dorso-posterior margin. Epimeral plates II–III with distinct postero-distal corner, but never produced. Uro- Here we present (1) characteristics needed for diagnosing somite I postero-dorso-laterally with 1 weak seta; the entire aggregate and (2) a full description of N. urosomite II postero-dorso-laterally with 2–4 spiniform scopicauda sp. n. Terminology and detailed descriptions setae; urosomite III without setae. Telson roughly of landmarks used in measurements follow Fišer et al. quadrangular (length/width = 0.80–0.95), with deep cleft (2009c). All morphological data presented here are freely (65–75% of length); lobes apically of variable shape. available at http://www.niphargus.info/, as are photographs Telson armed with long spines (40–60% of telson of specimens from the populations of Schellenberg’s length); apical and lateral strong, spiniform setae always ‘forms’ of N. tatrensis. Anyone using data referring to this present, mesial and dorsal spiniform setae only in certain study is kindly asked to cite the present paper. species/populations (Table 1). Antenna I 40–50% of body length, maxilla I with 7 uni-, bi- or pluri-toothed stout Niphargus tatrensis species complex setae; inner lobe with 2–3 (rarely 4) setae. Propods of gnathopods I–II small to middle-sized (gnathopod II Diagnosis propodus length + palm length + diagonal length = 15– 20% of body length), subquadrate, palm convex and Body length up to 20 mm. Pereonite VII with 2–4 slender slightly inclined; propodusIIlargerthanpropodusI. postero-ventral setae, pleonites I–III with 6–8 setae along Gnathopod dactyls with a row of single setae along Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 17 anterior margins. Pereopod dactyli III–VII ventrally with Niphargus tatrensis Wrześniowski, 1888 1, occasionally 2 spiniform setae. Coxa VII with 2–4 posterior setae. Bases V–VII narrow with straight poste- Diagnosis rior margins, length/width = 0.50–0.65; pereopod VII 40– 50% of body length. Pleopods I–III with 2-hooked Member of the N. tatrensis aggregate with apically concave retinacles. Uropod I endopodite/exopodite length ratio = and narrowed telson lobes; telson always with dorsal 0.90–1.05; endopodite distally with bunches of long spiniform setae. Distributed in Poland, the Czech Republic setae. Male uropod III up to 45% of body length. and Slovakia. Comprises the population at the type loaclity, Endopodite 40–50% of protopodite length; exopodite of and Schellenberg’s(1935, 1937) ‘forms’“f. reyersdorfen- uropod III rod-shaped. Endopodite distal article 40–55% sis” and “f. schneebergensis”. The taxonomy of the species of proximal article length in females, 40–100% in males. may be incomplete (see Discussion).

Remarks Niphargus aggtelekiensis Dudich, 1932

The combination of characters listed above is unique, but Diagnosis each of the individual character states can be found in other niphargid species as well. Some features are less Member of the N. tatrensis aggregate with apically wide, frequent among the remaining niphargid species than convex telson lobes; telson always with dorsal spiniform others. Noteworthy is the elongated distal article of the setae. Distributed in Austria, Slovakia and Hungary. Com- uropod III exopodite in females. The elongation of the prises the population at the type locality, and Schellenberg’s distal article in uropod III is common among niphargids, (1935, 1937) ‘forms’ N. tatrensis “f. lurensis”, N. t. “f. but occurs almost exclusively in males. Although the N. lunzensis”, N. t. “f. ötscherensis” and N. t. “f. salzburgensis”. tatrensis species complex has retained sexual dimorphism The taxonomy of the species may be incomplete (see in this character, the degree of elongation of this article Discussion). appears to be exceptional in females. According to our observations (75 species) and published information Niphargus scopicauda sp. n (unfortunately often incomplete), the length of the distal article in females does not exceed 30% of the proximal Etymology article, thus is much shorter than in the species studied here. The species epithet refers to the bunches of long setae on ‘ ’ ‘ ’ The monophyly of the aggregate is well supported the rami of uropod I (Latin scopa meaning broom or ‘ ’ morphologically as well as by sequences of 28 S DNA brush ). It is to be treated as a noun in apposition for the fragments (N. tatrensis: from type locality; N. scopicauda: purposes of nomenclature. from type locality and Habidova grapa). The sister-taxon of Material examined this species complex remains unknown. Schellenberg (1937) clarified similarities and differences between N. Holotype. Male, 14.5 mm; SLOVENIA, puddles in Huda tatrensis and N. stygius Schiödte, 1847; Jersche (1963) luknja near Gornji Dolič, 16.11.2002, leg. C. Fišer; noted similarities with N. foreli Humbert, 1876. The latter deposited in the collection of Oddelek za biologijo, Biotehniška species shows not only morphological but also some fakulteta, Univerza v Ljubljani (Department of Biology, Bio- ecological similarities with the N. tatrensis aggregate. It technical Faculty, University of Ljubljana). Paratypes.5males shares with N. tatrensis the long spiniform setae on the and 5 females from type locality (undissected); male and female telson, and narrowed pereopod V–VII bases. It is distrib- from Habidova grapa (dissected); all other data as for holotype. uted across Italy, Switzerland and Germany within the Alpine range, thus within the area with low mean annual Diagnosis temperatures (Karaman and Ruffo 1993). In addition, the morphologically related N. setiferus Schellenberg, 1937 Member of the N. tatrensis aggregate with apically wide, (originally described as N. foreli setiferus) from the western convex telson lobes; telson never with dorsal spiniform Alps has similar bunches of setae on the apical part of the setae. Distributed in Slovenia. rami of uropod I. However, neither N. foreli nor N. stygius appeared to be more closely related to the N. tatrensis Description of holotype and dissected paratypes aggregate than any other niphargid species in morphology- based and DNA-based phylogenetic analyses, respectively Head and trunk (Figs. 4, 9). Body length up to 14.5 mm. (Fišer et al. 2008b). Head length up to 10% of body length; rostrum absent. 18 C. Fišer et al.

Fig. 4 Niphargus scopicauda sp. n.; holotype, pleopods omitted

Pereonites I–VI without setae; pereonite VII with 2–4 setae. Proportions of mandibular palp articles 2 : 3 (distal) = slender postero-ventral setae. 1.0 : 1.15–1.20. Proximal palp article without setae; second Pleonites I–III with 6–8 setae along dorso-posterior article with up to 5–7 setae; distal article with 1 group of 5– margin. Epimeral plate II ventral and posterior margins 7 A setae; 3–4 groups of B setae; 20–28 D setae; 4–5E roughly perpendicular, posterior and ventral margins con- setae. vex, ventro-postero-distal corner distinct but blunt and not Maxilla I distal palp article with 5–7apicaland produced; along ventral margin 1–2 spiniform setae; along subapical setae. Outer lobe of maxilla I with 7 uni-, bi-or posterior margin 5–8 thin setae. Epimeral plate III ventral pluri-toothed spines; inner lobe with 2 setae. and posterior margins at sharp to perpendicular angle, Maxilla II inner lobe slightly smaller than outer lobe; posterior and ventral margin slightly convex, ventro- both of them setose apically and subapically. postero-distal corner distinct but not produced; along Maxilliped palp article 2 with 6–9 rows of setae along ventral margin 2 spiniform setae; along posterior margin inner margin; distal article with dorsal seta and group of 7–8 thin setae. Urosomite I postero-dorso-laterally with 1 weak seta; urosomite II postero-dorso-laterally with 2 spiniform setae; urosomite III without setae. Near insertion of uropod I 1 spiniform seta. Telson length : width as 1.0 : 0.80–0.95 ; cleft 65–75% of length; lobes apically widely rounded. Telson spines (per lobe): 4–5 apical spines of 40–55% telson length; lateral and mesial margins exceptionally with 1 spine; dorsal surface without spines (see also Table 1). Pairs of plumose setae inserted mid-laterally. Antennae (Fig. 5). Antenna I 35–40% of body length. Flagellum with up to 25 articles; each article with 1 long aesthetasc. Peduncle article proportions 1.0 : 0.90 : 0.35– 0.40. Proximal article of peduncle dorso-distally slightly produced. Accessory flagellum biarticulated; distal article shorter than one-half of proximal article. Lengths of antennae I : II as 1.0 : 0.50. Flagellum of antenna II with 10–11 articles; each article with setae and elongate sensilla of unknown function. Lengths of peduncle articles 4 : 5 as 1.0 : 0.90–0.95; flagellum 70–80% of peduncle length (articles 4 + 5). Mouthparts (Fig. 5). Inner lobes of labium longer than half of outer lobes. Left mandible: incisor with 5 teeth, lacinia mobilis with 4 teeth; between lacinia and molar a row of thick, serrated setae, long seta at base of molar. Right mandible: incisor Fig. 5 Niphargus scopicauda sp. n.; holotype, antennae (aI, aII) and processus with 4 teeth, lacinia mobilis with several small mouthparts (lb = labium; mdL, mdR = left and right mandibles; mxI, denticles, between lacinia and molar a row of thick, serrated mxII = maxillae I and II; mxpe = maxilliped) Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 19

Fig. 8 Niphargus scopicauda sp. n.; holotype, pereopods (pp) VI and Fig. 6 Niphargus scopicauda sp. n.; holotype, gnathopods (gp) I and II VII

Fig. 9 Niphargus scopicauda sp. n.; holotype, pleon details (f-upIII = uropod III of female paratype; plp = pleopod II; T = telson; up = Fig. 7 Niphargus scopicauda sp. n.; holotype, pereopods (pp) III and IV uropod) 20 C. Fišer et al. small setae at base of nail. Maxilliped outer lobe with 9–10 dorsal plumose seta; at base of nail a tiny seta and a flattened, thick setae and 5–7 serrated setae; inner lobe with spiniform seta; additional spiniform seta present in less than 3–4 flattened, thick setae apically and 6–8 serrated setae. 5% of individuals (see Table 1). Coxal plates, gills (Figs. 4, 6, 7 and 8). Coxal plate I of Pereopods V–VII (Fig. 8). Proportions of pereopods V : flattened rhomboid shape, antero-ventral corner sub- VI : VII as 1.00 : 1.40–1.45 : 1.55. Pereopod VII length rounded; anterior and ventral margin of coxa I with 6–9 40–45% of body length. setae. Coxal plate II width : depth as 1.00 : 1.00–1.20; Bases V–VII narrow, with straight or slightly concave anterior and ventral margin with 7–10 setae. Coxal plate III posterior margins, length : width as 1.00 : 0.55–0.65; width : depth as 1.00 : 1.10–1.25; along antero-ventral posterior margin straight, without distal lobes; posteriorly margin 6–7 setae. Coxal plate IV width : depth as 1.00 : 9–10, 10–14 and 13–15 setae, respectively; anteriorly 5, 5– 1.10–1.30; posteriorly slightly concave (5–10% of coxa 6 and 5–6 slender spiniform setae, respectively. Dactylus width); along antero-ventral margin 7–6 setae. Coxal plates VII length 25–30% of propodus VII length; nail length 30– V–VI: anteriorly developed lobe; along posterior margin 3–4 35% of total dactylus length. Dactyli V–VII with dorsal setae. Coxal plate VII half-egg shaped, along posterior plumose seta; at base of nail a tiny seta and a spinform seta; margin 2–3 setae. Gills II–VI ovoid, reaching to mid-basis. up to 50% of specimens have an additional spinifom seta Gnathopod I (Fig. 6). Ischium with up to 11 postero- on dactyli VI–VII (Table 1). distal setae. Carpus length 60–70% of basis and 75–80% of Pleopods and uropods (Fig. 9). Pleopods I–III with 2- propodus. Anterior margin of carpus with distal group of hooked retinacles. Pleopod II rami of 9–13 articles each. setae; carpus posteriorly with transverse rows of setae Uropod I protopodite with 7 dorso-lateral and 3–4 dorso- proximally and a row of lateral setae; postero-proximal medial spinifom setae. Length ratio endopodite : exopodite as bulge large (1/3 of carpus length), positioned proximally. 1.00 : 0.90–1.00; rami straight. Endopodite with 9–26 long Propodus subquadrate, palm convex and slightly inclined. setae in 5–8 groups; apically 5 spinifom setae. Exopodite with Along posterior margin 7–8 rows of denticulated setae. 9–38 setae in 5–15 groups; apically 5 spinifom setae. Anterior margin with 18–23 setae in 4 groups, antero-distal Uropod II endopodite : exopodite length as 1.00 : 1.05– group with 12–13 setae. Group of 4–5 facial setae below 1.15. (proximal of) palmar spine; several groups of surface setae Uropod III up to 45% of body length. Protopodite with present. Palmar corner with strong palmar spine, single 6–12 lateral setae and 8–12 apical spiniform and thin setae. supporting spine on inner surface, and 3 denticulated, thick Endopodite 40–50% of protopodite length, apically with 2– spiniform setae on outer side. Nail length 30–35% of total 4 thin-flexible and spiniform setae; laterally 1–3 thin and dactylus length; along anterior margin 5–6 single setae; spiniform setae. Exopodite of uropod III rod-shaped, distal along inner margin a row of short setae. article 55–100% of proximal article length. Proximal article Gnathopod II (Fig. 6). Basis width : length as 1.0 : 0.30– with 5–6 groups of plumose, thin-flexible and spiniform 0.33. Ischium with 4–7 postero-distal setae. Carpus length 55– setae along inner margin; 4–6 groups of thin-flexible and 60% of basis and 80–85% of propodus. Anterior margin of spiniform setae along outer margin. Distal article with 2–5 carpus with distal row of setae; carpus posteriorly with lateral setae groups along each side; apically 3–5 setae. transverse rows of setae proximally, a row of lateral setae; postero-proximal bulge large (1/3 of carpus length), posi- tioned proximally. Propodus medium-sized (sum of length, References diagonal and palm length measures up to 20% of body length) and larger than propodus of gnathopod I (1.0 : 0.85). Propodus Agapow, P. M., Bininda-Emonds, O. P. R., Crandall, K. A., Gittleman, rectangular, palm convex and more inclined than palm of J. L., Mace, G. M., Marshall, J. C., et al. (2004). The impact of gnathopod I. Posterior margin with 10–12 rows of denticulat- species concept on biodiversity studies. The Quarterly Review of – – Biology, 79, 161–179. ed setae. Anterior margin with 10 24 setae in 3 5groups; ’ – – Beyer, H. L. (2008). Hawth s Analysis Tools for ArcGIS. SpatialEcol- antero-distal group with 11 16 setae. Group of 4 6facial ogy.com. http://www.spatialecology.com/htools. Accessed 15 setae below (proximal of) palmar spine; individual surface June 2008. setae present. Palmar corner with strong palmar spine, single Bickford, D., Lohman, D. J., Sodhi, N. S., Ng, P. K. L., Meier, R., supporting spine on inner surface, and 2 denticulated, thick Winker, K., et al. (2007). Cryptic species as a window on – diversity and conservation. Trends in Ecology and Evolution, 22, spiniform setae on outer side. Nail length 30 35% of total 148–155. dactylus length. Along anterior margin 5–7 single setae; along Bininda-Emonds, O. R. P., Vázquez, D. P., & Manne, L. L. (2000). inner margin few short setae. The calculus of biodiversity: integrating phylogeny and conser- – Pereopods III–IV (Fig. 7). Proportions of pereopods III : vation. Trends in Ecology and Evolution, 15,92 94. – Chessel, D., Dufour, A.-B., & Dray, S. (2008). The ade4 Package. IV as 1.0 : 0.95. Dactylus IV 40 45% of propodus IV; nail Biometry and Evolutionary Biology Lab, University Lyon 1. length 50–60% of total dactylus length. Dactyli III–IV with http://pbil.univ-lyon1.fr/ADE-4. Accessed 20 June 2008. Old museum samples and recent taxonomy: A taxonomic, biogeographic and conservation perspective 21

Coppellotti Krupa, O., & Guidolin, L. (2003). Responses of distribution of the obligate groundwater amphipod Niphargus Niphargus montellianus and Gammarus balcanicus (Crustacea, virei (). Journal of Biogeography, 35, 552–564. Amphipoda) from karst waters to heavy metal exposure. Journal Funk, V. A., Sakai, A. K., & Richardson, K. (2002). Biodiversity: the de Physique IV, 107, 323–326. interface between systematics and conservation. Systematic Crandall, K. A., Bininda-Emonds, O. R. P., Mace, G. M., & Wayne, Biology, 51, 235–237. R. K. (2000). Considering evolutionary processes in conservation Gams, I. (1974). Kras. Zgodovinski, naravoslovni in geografski oris. biology. Trends in Ecology and Evolution, 15, 290–295. Ljubljana: Slovenska Matica. Dallwitz, M. J., Paine, T. A., & Zurcher, E. J. (2007). DELTA— Ginet, R. (1965). Expérience de colonisation souterraine aquatique par DEscription Language for TAxonomy [computer software and Niphargus (Crustacé amphipode); premiers résultats biologiques. manuals]. DELTA Website. http://delta-intkey.com/. Accessed 15 Bulletin de la Société Zoologique de France, 90, 581–588. May 2007. Grabowski, M., Bacela, K., & Konopacka, A. (2007). How to be an Davis, J. I., & Nixon, K. C. (1992). Populations, genetic variation, and invasive gammarid (Amphipoda: Gammaroidea)—comparison of the delimitation of phylogenetic species. Systematic Biology, 42, life history traits. Hydrobiologia, 590,75–84. 421–445. Graham, C. H., Ferrier, S., Huettman, F., Moritz, C., & Townsend, DeSalle, R., Egan, M. A., & Siddall, M. (2005). The unholly trinity: P. A. (2004). New developments in museum-based informatics taxonomy, species delimitation and DNA barcoding. Philosoph- and applications in biodiversity analysis. Trends in Ecology and ical Transactions of the Royal Society, Series B, Biological Evolution, 19, 497–503. Sciences, 360, 1905–1916. Graybeal, A. (1995). Naming species. Systematic Biology, 44, 237– de Queiroz, K. (1998). General lineage concept of species, species 250. criteria, and the process of speciation. In D. J. Howard & S. H. Hervant, F., & Mathieu, J. (1995). Ventilatory and locomotory activities Berlocher (Eds.), Endless forms: Species and speciation (pp. 57– in anoxia and subsequent recovery of epigean and hypogean 75). Oxford: Oxford University Press. . Comptes Rendus de l’Académie des Sciences, Paris, Queiroz, K. de (2005a). Ernst Mayr and the modern concept of Série 3, Sciences de la vie / Life Sciences, 318,585–592. species. Proceedings of the National Academy of Sciences of the Hervant,F.,Mathieu,J.,Barré,H.,Simon,K.,&Pinon,C.(1997). United States of America, 102, 6600–6607. Comparative study on the behavioral, ventilatory, and respiratory de Queiroz, K. (2005b). Different species problems and their responses of hypogean and epigean crustaceans to long-term resolution. BioEssays, 27, 1263–1269. starvation and subsequent feeding. Comparative Biochemistry and de Queiroz, K. (2007). Toward an integrated system of clade names. Physiology A, Molecular & Integrative Physiology, 118,1277–1283. Systematic Biology, 56, 956–974. Hervant, F., Mathieu, J., & Barré, H. (1999a). Comparative study on Fišer, C., & Zagmajster, M. (2009). Cryptic species from cryptic the metabolic responses of subterranean and surface-dwelling space: the case of Niphargus fongi sp. n. (Crustacea, Amphipoda, amphipods to long-term starvation and subsequent refeeding. Niphargidae). Crustaceana, 82, 593–614. Journal of Experimental Biology, 202, 3587–3595. Fišer, C., Trontelj, P., & Sket, B. (2006a). Phylogenetic analysis of the Hervant, F., Garin, D., Mathieu, J., & Freminet, A. (1999b). Lactate Niphargus orcinus species-aggregate (Crustacea: Amphipoda: metabolism and glucose turnover in the subterranean crustacean Niphargidae) with description of new taxa. Journal of Natural Niphargus virei during post-hypoxic recovery. Journal of History, 40, 2265–2315. Experimental Biology, 202, 579–592. Fišer, C., Sket, B., & Stoch, F. (2006b). Distribution of four narrowly Hijmans, R. J., Cameron, S. E., Parra, J. L., Jones, P. G., & Jarvis, A. endemic Niphargus species (Crustacea: Amphipoda) in the (2005). Very high resolution interpolated climate surfaces for global western Dinaric region with description of a new species. land areas. International Journal of Climatology, 25,1965–1978. Zoologischer Anzeiger, 245,77–94. ICZN=International Commission on Zoological Nomenclature Fišer, C., Zakšek, V., Zagmajster, M., & Sket, B. (2007a). Taxonomy (1999). International Code of Zoological Nomenclature, fourth and biogeography of Niphargus steueri (Crustacea: Amphipoda). edition. International Trust for Zoological Nomenclature. http:// Limnology, 8, 297–309. www.iczn.org/iczn/index.jsp. Accessed 15 April 2009. Fišer, C., Keber, R., Kereži, V., Moškrič, A., Palandačić, A., Potočnik, Jażdżewski, K., Konopacka, A., & Grabowski, M. (2004). Recent H., et al. (2007b). Coexistence of two species of two amphipod drastic changes in the gammarid fauna (Crustacea, Amphipoda) genera: Niphargus timavi (Niphargidae) and Gammarus fossarum of the Vistula River deltaic system in Poland caused by alien (Gammaridae). Journal of Natural History, 41, 2641–2651. invaders. Diversity and Distributions, 10,81–87. Fišer, C., Bininda-Emonds, O. P. R., Blejec, A., & Sket, B. (2008a). Jersche, G. (1963). Zur Artfrage und Variabilität von Niphargus Can heterochrony help explain the high morphological diversity tatrensis Wrzesniowski (Ein Beitrag zur postembryonalen Verän- within the genus Niphargus (Crustacea: Amphipoda)? Organ- derung taxonomischer Merkmale). Zeitschrift für Zoologische isms, Diversity and Evolution, 8, 146–162. Systematik und Evolutionsforschung, 1, 240–276. Fišer, C., Sket, B., & Trontelj, P. (2008b). A phylogenetic perspective Karaman, G. S., & Ruffo, S. (1986). Amphipoda: Niphargus-group on 160 years of troubled taxonomy of Niphargus (Crustacea: (Niphargidae sensu Bousfield, 1982). In L. Botosaneau (Ed.), Amphipoda). Zoologica Scripta, 37, 665–680. Stygofauna mMundi (pp. 514–534). Leiden: E. J. Brill/Dr. W. Fišer, C., Çamur-Elipek, B., & Özbek, M. (2009a). The subterranean Backhuys. genus Niphargus (Crustacea, Amphipoda) in the Middle East: a Karaman, G. S., & Ruffo, S. (1993). Niphargus foreli Humbert, 1876 and faunistic overview with descriptions of two new species. its taxonomic position (Crustacea Amphipoda, Niphargidae). Bollet- Zoologischer Anzeiger, 248, 137–150. tino del Museo Civico di Storia naturale di Verona, 17,57–68. Fišer, C., Sket, B., Turjak, M., & Trontelj, P. (2009b). Public online Kristjánsson, B. K., & Svavarsson, J. (2004). Crymostygidae, a new databases as a tool of collaborative taxonomy: a case study on family of subterranean freshwater gammaridean amphipods subterranean amphipods. Zootaxa, 2095,47–56. (Crustacea) recorded from subarctic waters. Journal of Natural Fišer, C., Trontelj, P., Luštrik, R., & Sket, B. (2009c). Toward a History, 38, 1881–1894. unified taxonomy of Niphargus (Crustacea: Amphipoda): a Lefébure, T., Douady, C. J., Gouy, M., Trontelj, P., Briolay, J., & review of morphological variability. Zootaxa, 2061,1–22. Gibert, J. (2006). Phylogeography of a subterranean amphipod Folquier, A., Malard, F., Lefébure, T., Douady, C. J., & Gibert, J. reveals cryptic diversity and dynamic evolution in extreme (2008). The imprint of Quaternary glaciers on the present-day environments. Molecular Ecology, 15, 1797–1806. 22 C. Fišer et al.

Lefébure, T., Douady, C. J., Malard, F., & Gibert, J. (2007). Testing nale sur le genre Niphargus, Verona 15–16 aprile 1969 (pp. 47– dispersal and cryptic diversity in a widely distributed groundwa- 53). Verona: Museo Civico di Storia Naturale di Verona. ter amphipod (Niphargus rhenorhodanesis). Molecular Phyloge- Sket, B. (1958). Prispevek k poznavanju naših amfipodov. Biološki netics and Evolution, 42, 676–686. Vestnik, 6,67–75. Mayden, R. L. (1997). A hierarchy of species concepts: the Sket, B. (1977). Niphargus im Brackwasser. Crustaceana Supplement, denouncement in the saga of the species problem. In M. F. 4, 188–191. Claridge, H. A. Dawah, & M. R. Wilson (Eds.), Species: the Sket, B. (1981). Distribution, ecological character, and phylogenetic units of biodiversity (pp. 381–424). London: Chapman and Hall. importance of Niphargus valachicus. Biološki Vestnik, 29,87–103. Micherdziński, W. (1956). Taksonomia i ekologia Niphargus tatrensis Sket, B. (1994). Distribution patterns of some subterranean Crustacea Wrześniowski 1888 (Amphipoda). Annales Zoologici, 16,81–134. in the territory of the former Yugoslavia. Hydrobiologia, 287, Moritz, C. (1994). Defining ‘Evolutionary significant units’ for 65–75. conservation. Trends in Ecology and Evolution, 9, 373–375. Stuart, B. L., Dugan, K. A., Allard, M. W., & Kearney, M. (2006). Moritz, C. (2002). Strategies to protect biological diversity and the Extraction of nuclear DNA from bone of skeletonized and fluid- evolutionary processes that sustain it. Systematic Biology, 51, preserved museum specimens. Systematics and Biodiversity, 4, 238–254. 133–136. Myers, N., Mittermeier, R. A., Mittermeier, C. G., da Fonseca, G. A. Trontelj, P., Douady, C., Fišer, C., Gibert, J., Gorički, Š., Lefébure, T., B., & Kent, J. (2000). Biodiversity hotspots for conservation et al. (2009). A molecular test for hidden biodiversity in priorities. Nature, 403, 853–858. groundwater: how large are the ranges of macro-stygobionts? Nesemann, H., Pöckl, M., & Wittmann, K. J. (1995). Distribution of Freshwater Biology, 54, 727–744. epigean in the middle and upper Danube (Hungary, Väinölä, R., Witt, J. D. S., Grabowski, M., Bradbury, J. H., Austria, Germany). Miscellanea Zoologica Hungarica, 10,49– Jażdżewski, K., & Sket, B. (2008). Global diversity of 68. amphipods (Amphipoda; Crustacea) in freshwater. Hydrobiolo- Raxworthy, Ch J, Ingram, C. M., Rabibisoa, N., & Pearson, R. G. gia, 595, 241–255. (2007). Applications of ecological niche modeling for species Vellend, M., Harmon, L. J., Lockwood, J. L., Mayfield, M. M., delimitation: a review and empirical evaluation using day Geckos Hughes, A. R., Wares, J. P., et al. (2007). Effects of exotic (Phelsuma) from Madagascar. Systematic Biology, 56, 907–923. species on evolutionary diversification. Trends in Ecology and Ruffo, S. (1972). Actes du Ier Colloque Internationale sur le genre Evolution, 22, 481–488. Niphargus, Verona 15–16 aprile 1969. Verona: Museo Civico di Wandeler, P., Hoeck, P. E., & Keller, L. F. (2007). Back to the future: Storia Naturale di Verona. museum specimens in population genetics. Trends in Ecology Savage, A. A. (1981). The Gammaridae and Corixidae of an inland and Evolution, 22, 634–642. saline lake from 1975–1978. Hydrobiologia, 76,33–44. Wheeler, Q. D. (2004). Taxonomic triage and the poverty of Sax, D. F., Stachowicz, J. J., Brown, J. H., Bruno, J. F., Dawson, phylogeny. Philosophical Transactions of the Royal Society of M. N., Gaines, S. D., et al. (2007). Ecological and evolutionary London, Series B, Biological Sciences, 359, 571–583. insights from species invasions. Trends in Ecology and Evolu- Wiens, J. J. (1995). Polymorphic characters in phylogenetic system- tion, 22, 465–471. atics. Systematic Biology, 44, 482–500. Schellenberg, A. (1935). Schlüssel der Amphipodengattung Niphar- Wiens, J. J. (1999). Polymorphism in systematic and comparative gus mit Fundortangaben und mehreren neuen Formen. Zoolog- biology. Annual Review of Ecology, Evolution, and Systematics, ischer Anzeiger, 111, 204–211. 30, 327–362. Schellenberg, A. (1937). Bemerkungen zu meinem Niphargus- Wiens, J. J. (2001). Character analysis in morphological phyloge- Schlüssel und zur Verbreitung und Variabilität der Arten, nebst netics: problems and solutions. Systematic Biology, 50, 689–699. Beschreibung neuer Niphargus-Formen. Mitteilungen aus dem Wiens, J. J. (2004a). The role of morphological data in phylogeny Zoologischen Museum in Berlin, 22,1–30. reconstruction. Systematic Biology, 53, 653–661. Schellenberg, A. (1938). Alters-, Geschlechts- und Individualunter- Wiens, J. J. (2004b). Speciation and ecology revisited: phylogenetic niche schiede des Amphipoden Niphargus tatrensis f. aggtelekiensis conservatism and the origin of species. Evolution, 58,193–197. Dudich. Zoologische Jahrbücher, Abteilung für Systematik, Wiens, J. J. (2007). Species delimitation: new approaches for Ökologie und Geographie der Tiere, 71, 191–202. discovering diversity. Systematic Biology, 56, 875–878. Simčič, T., & Brancelj, A. (2006). Effects of pH on electron transport Wiens, J. J., & Graham, C. H. (2005). Niche conservatism: integrating system (ETS) activity and oxygen consumption in Gammarus evolution, ecology, and conservation biology. Annual Review of fossarum, Asellus aquaticus and Niphargus sphagnicolus. Fresh- Ecology, Evolution, and Systematics, 36, 519–539. water Biology, 51, 686–694. Wiens, J. J., & Penkrot, T. A. (2002). Delimiting species using DNA Sites, J. W., & Crandall, K. A. (1997). Testing species boundaries in and morphological variation and discordant species limits in biodiversity studies. Conservation Biology, 11, 1289–1297. spiny lizards (Sceloporus). Systematic Biology, 51,69–91. Sites, J. W., & Marshall, J. C. (2003). Delimiting species: a Wiens, J. J., & Servedio, M. R. (1998). Phylogenetic analysis and renaissance issue in systematic biology. Trends in Ecology and intraspecific variation: performance of parsimony, likelihood, and Evolution, 18, 462–470. distance methods. Systematic Biology, 47, 228–253. Sites, J. W., & Marshall, J. C. (2004). Operational criteria for Wiens, J. J., & Servedio, M. R. (2000). Species delimitation in delimiting species. Annual Review of Ecology Evolution and systematics: inferring diagnostic differences between species. Systematics, 35, 199–227. Proceedings of the Royal Society of London / B, 267, 631–636. Skalski, A. W. (1972). Distribution des amphipodes souterrains en Winston, J. E. (2007). Archives of a small planet: The significance of Pologne, avec notes sur la variabilité du Niphargus tatrensis museum collections and museum based research in invertebrate Wrześniowski. In S. Ruffo (Ed.), Actes du Ier Colloque Internatio- taxonomy. Zootaxa, 1668,47–54.