Cryptic species from cryptic space: the case of Niphargus fongi sp. n. (Amphipoda, Niphargidae)

CRYPTIC SPECIES FROM CRYPTIC SPACE: THE CASE OF NIPHARGUSFONGI SP. N. (AMPHIPODA, NIPHARGIDAE)

BY

CENE FIŠER1,2) and MAJA ZAGMAJSTER1,3)1) Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111,

SI-1000 Ljubljana, Slovenia

ABSTRACT

Niphargus fongi sp. n. (Amphipoda, Niphargidae) is described from a cave in the Dinaric karst ofsoutheastern Slovenia and its morphology is reported in detail. Its occurrence in pools of percolatingwater, some of which temporarily dry up, and its narrow distribution range suggest that thespecies lives in limestone fissures, possibly in the epikarst ecotone. Small though distinct diagnosticmorphological characters support the species hypothesis based on molecular evidence. Re-evaluationof the degree of morphological differentiation in the light of molecular findings implies that the term“cryptic species”, at least in some less well-studied taxa, may also depend on taxonomic evaluationof morphologically distinct populations. Additionally, a species described from a poorly sampledhabitat constitutes a sample case of where taxonomic as well as ecological influences on biodiversitybecome apparent. Both incomplete taxonomy as well as insufficient sampling affect estimates ofbiodiversity in subterranean habitats.

RÉSUMÉ

Niphargus fongi sp. n. (Amphipoda, Niphargidae) est décrit d’une grotte du karst dinariquede Slovénie sud-orientale et sa morphologie est décrite en détail. Sa présence dans les bassinsd’eaux de percolation, dont certains s’assèchent temporairement, et son aire de répartition étroitesuggèrent que l’espèce vit dans les fissures du calcaire, et peut-être dans l’écotone épikarst. Depetits, bien que distincts, caractères morphologiques diagnostiques supportent l’hypothèse d’espèceà partir de l’évidence moléculaire. La réévaluation du degré de différenciation morphologique àla lumière des résultats moléculaires impliquent que le terme “espèce cryptique”, au moins chezquelques taxons moins bien étudiés, peut aussi dépendre de l’évaluation taxonomique de populationsmorphologiquement distinctes. De plus, une espèce décrite d’un habitat peu échantillonné constitueun cas dans lequel les influences taxonomiques comme écologiques sur la biodiversité deviennentapparentes. Une taxonomie incomplète et un échantillonnage insuffisant affectent tous deux lesestimations de la biodiversité dans les habitats souterrains.

2) e-mail: [email protected]) e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2009 Crustaceana 82 (5): 593-614Also available online: www.brill.nl/cr DOI:10.1163/156854009X407704

594 CENE FIŠER & MAJA ZAGMAJSTER

INTRODUCTION

Research on cryptic species, that is, species with a few or no conspicuousdifferences in external appearance, has increased exponentially over the past twodecades (overview in Bickford et al., 2007). Discoveries of cryptic species affectall fields of biology, including taxonomy, evolution, pharmacology, biodiversity,ecology including applied ecology, and conservation. It seems reasonable to expectthat similar selection pressures in extreme environmental conditions constrain theevolution of morphology (Bickford et al., 2007; but see Pfenninger & Schwenk,2007). In the field of speleobiology (biology dealing with subterranean life),it has long been assumed that convergent morphological characters, so calledtroglomorphies (e.g., Christiansen, 1962; Langecker, 2000; Aden, 2005), may beexpected in different subterranean species (so called troglobionts) as adaptationsto severe ecological constraints (Culver, 1982). Thus, morphological similaritybetween troglobionts either represents shared attributes inherited from a commonancestor, or has evolved among non-related species convergently. The former arereferred to as sibling species or cryptic species type I, and the latter are calledsimply cryptic species or cryptic species type II (terminology refers to Bickford etal., 2007; and to Trontelj et al., in press).

The recent introduction of molecular techniques into taxonomic research offersa new perspective on hidden subterranean biodiversity. Particularly important inthis respect are the studies on the largest genus of freshwater amphipods, Niphar-gus Schiœdte, 1847 (cf. Väinölä et al., 2008). This ubiquitous, monophyletic genus(Trontelj et al., in press) consists of species living mainly in various types of sub-terranean waters (Karaman & Ruffo, 1986; Sket, 1999a). The existence of bothsibling species and cryptic species has been well documented across several lin-eages within the genus (Mathieu, 1997; Lefébure et al., 2006, 2007; Trontelj et al.,in press), suggesting that the taxonomy of the genus is far from being complete.

In this study we searched for morphological support for a species informallynamed as “N. cf. tauri 4” that was postulated from molecular data (Trontelj et al., inpress). In addition, we present data from which we infer that the species is narrowlyendemic, and ecologically specialized for water-filled fissures in the rock. Usingthe data on the newly described species’ morphology, distribution, and certainaspects of its ecology, we tend to demonstrate the following two points. First,a diagnosis based on a few, small morphological characters shows that the term“cryptic” possibly reflects the current state of taxonomy in the group. Secondly,using this case we attempt to demonstrate that the study of biodiversity shouldaccount for both the cryptic species’ presence (Bickford et al., 2007) as well as forthe problem of sampling adequacy (Gotelli & Colwell, 2001).

NIPHARGUS FONGI NOV. 595

MATERIAL AND METHODS

Description of the sampling sites and sampling

The material examined was collected in two caves, situated in the northwesternpart of the Dinaric karst in the western Balkan Peninsula (fig. 1). Both arelocated in the limestone hill Mala Gora, north from the town of Kocevje insoutheastern Slovenia. Dolga Jama pri Koblarjih (45◦42′43.9′′N 14◦49′48.2′′E,altitude of entrance 640 m, later on referred to as “Koblarska Jama”) is a cave withapproximately 210 m of cumulative passage length and a lowest point approx. 17 mbelow the entrance (Stepišnik, 2006). Vanceva Jama (45◦42′11.2′′N 14◦49′21.5′′E,altitude of entrance 555 m) is a cave located at approximately 1.2 km aerial

Fig. 1. Localities of: 1, 2, Niphargus fongi sp. n.; 5, the phylogenetically related N. carniolicus (Sket,1960); 3, “N. cf. tauri 3”; and, 1, 2, 4, the unrelated N. stygius brachytelson S. Karaman, 1952 insoutheastern Slovenia. Phylogenetic relationships of the three taxa are adopted from Trontelj et al.(in press) (lengths of the branches not to scale). 1, Dolga Jama pri Koblarjih; 2, Vanceva Jama;

3, Prepadna Jama; 4, Lukova Jama pri Zdihovem; 5, Jama pod gradom Luknja.


distance from Koblarska Jama, with nearly 160 m of cave passages and the lowestaccessible point 35 m below the entrance (Belšak, 1973).

There is no surface water in the vicinity of the two caves. The nearest intermit-tent surface streams are located about 1700 m aerial distance south of KoblarskaJama and 650 m south of Vanceva Jama, being about 200 and 100 altitudinal me-ters, respectively, lower than the two caves. Pools with percolating water of dif-ferent sizes, most of which temporarily dry out, can be found in both caves (fivein Koblarska Jama and six in Vanceva Jama). Our rough estimate of the cave ceil-ing height above the pools in Koblarska Jama varies between 6 and 13 m and inVanceva Jama between 8 and 16 m.

Both caves were regularly visited between October 2004 and April 2005 in orderto characterize their fauna. Sampling was not focused on any particular animalgroup. The aquatic animals were collected by hand or with a hand net. Altogether,eleven and ten samples of niphargids were collected during six visits to KoblarskaJama and four visits to Vanceva Jama.

Taxonomic part

Selected specimens of the presumably new species were partly dissected inglycerol and mounted on slides in glycerol gelatine medium for detailed analysis.Digital photos were taken with an Olympus DP10 camera mounted on an OlympusSZX9 stereomicroscope. Measurements and counts were made using the computerprogram Olympus DP–soft. Finer details were examined using a Zeiss microscopewith magnifications of 100-400×. The description was generated with the help ofthe program package DELTA (CSIRO, Dallwitz et al., 2007). Raw data needed forcomparative purposes are embedded into a data-matrix (DELTA program package)together with data on over 70 other Niphargus species, and are accessible viahttp://niphargus.info. Anybody using data related to Niphargus fongi sp. n. iskindly asked to refer to this paper.

RESULTS

Niphargus fongi sp. n. (figs. 2-8)

Type locality and material examined. — Dolga Jama pri Koblarjih (shorter Koblarska Jama),Kocevje, Slovenia (Cadastre number 94, Slovenian Cave Cadastre, Institute for Karst Research,Postojna); examined were six samples collected between October 2004 and April 2005 (leg.M. Zagmajster and L. Ramšak). Holotype male (5.9 mm) and paratypes (subadult male 5.3 mm andfemale 8.8 mm) dissected and mounted on slides; additional 12 specimens not dissected. Holotypeand paratypes deposited in the collection of Oddelek za Biologijo, Biotehniška Fakulteta, Univerza


v Ljubljani (Dept. of. Biology, Biotechnical Faculty, University of Ljubljana), with collection nos.:OBBFUL_NA1001-OBBFUL_NA1004.

Etymology. — The species epithet is dedicated to our colleague Daniel Fong,speleobiologist from the U.S.A., involved in various aspects of cave biology andamphipod research. The name thus is a noun in the genitive singular.

Terminology. — The usage of the terms “seta” and “spine” do not agree withterminology introduced by Watling (1989). However, since no cuticular teeth(usually referred to as spines or thorns) exist in the described species, we use theterm “spine” for thick and stiff setae and the term “seta” for a thin and flexiblestructure, in order to avoid overburdening of the text.

Diagnosis. — Small to middle-sized niphargid; postero-dorsal margins ofpleonites I-III with 2-4 slender setae; epimeral plates subrounded; postero-dorso-lateral margin of urosomite I with 2 setae, postero-dorso-lateral margin of uro-somite II with 3-4 spines; telson with 3-5 apical spines (per lobe) of 0.45-0.55 tel-son length; 1-2 groups of spines with 1-2 spines each along lateral margin; telsoncleft and dorsal surface without spines. Maxilla I distal palp article with 4-7 apicaland subapical setae; outer lobe with 7 uni-, or multi-toothed spines; inner lobe with1-2 setae. Propodi of gnathopods I-II middle-sized and quadrate in shape; dactyli ofgnathopods with single setae; gills II-IV narrow; pleopods I-III with 4-7, 3-5, and4-5 hooks in retinacles; length ratio endopod : exopod of uropod I is 1 : 1.00-1.05;exopod of uropod III rod-shaped, distal article up to 0.55 of the proximal articlelength.

Description of holotype and dissected paratypes. — Head and trunk (fig. 2, 8).Body length up to 9 mm. Head length up to 10% of body length; rostrum absent.Pereonites I-IV without setae; pereonites V-VII with 1 slender postero-ventral seta.

Pleonites I-III with 2-4 setae along dorso-posterior margin. Epimeral plateII subrounded, posterior and ventral margin convex, ventro-postero-distal cornerdistinguishable only by presence of seta; along ventral margin 2 spines; alongposterior margin 5-8 setae. Epimeral plate III subrounded, posterior and ventralmargin convex, ventro-postero-distal corner distinguishable only by presence ofseta; along ventral margin 1-3 spines; along posterior margin 5-7 setae.

Urosomite I postero-dorso-laterally with 2 setae, rarely spines (on each side);urosomite II postero-dorso-laterally with 3-4 spines and/or setae (on each side);urosomite III without setae. At base of uropod I there is 1 spine.

Telson length : width ratio is 1 : 0.80-0.85; cleft 0.70-0.80 of length. Telsonspines (per lobe): 3-5 apical spines of 0.45-0.55 telson length; 1-2 groups of spineswith 1-2 spines each along lateral margin; telson cleft and dorsal surface withoutspines. Pairs of plumose setae inserted mid-laterally.

Antennae I-II (fig. 3). Antenna I is 0.45-0.35 of body length. Flagellumwith up to 17 articles; each article with 1 long aesthetasc. Peduncle articles in


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Fig. 3. Niphargus fongi sp. n., holotype: aI, II, antenna I and II, respectively; ul, upper lip; lb, labium;md-L, R, left and right mandibula, respectively; plp, mandibular palp; mxI, II, maxilla I and II,

respectively; mxpe, maxilliped.

ratio 1 : 0.75-0.80 : 0.40-0.45. Proximal article of peduncle dorso-distally slightlyproduced. Accessory flagellum biarticulate; distal article shorter than half thelength of proximal article.

Length ratio antenna I : II as 1 : 0.40-0.45. Flagellum of antenna II with 6-7 articles; each article with setae and elongate sensilla of unknown function.Length of peduncle articles 4 : 5 is 1 : 0.85-0.90; flagellum 0.60-0.75 of pedunclelength (articles 4 + 5).

Mouthparts (fig. 3). Inner lobes of labium longer than half of outer lobes.Left mandible: incisor processus with 5 teeth, lacinia mobilis with 4 teeth; long

seta at base of molar; between lacinia mobilis and molar a row of serrated spines.


Right mandible: incisor process with 4 teeth, lacinia mobilis with several smalldenticles, between lacinia and molar a row of serrated spines. Ratio of mandibularpalp article 2 : article 3 (distal) is 1 : 1.05-1.15. Proximal palp article without setae;second article with up to 5 setae (holotype has additional dorso-distal group ofsetae); distal article with 1 A group of 2-4 setae; 1-2 B setae; 9-15 D setae; 3-4 Esetae.

Maxilla I distal palp article with 4-7 apical and subapical setae. Outer lobe ofmaxilla I with 7 uni-, or multi-toothed spines; inner lobe with 1-2 setae.

Maxilla II inner lobe slightly smaller than outer; both of these setose apicallyand subapically.

Maxilliped palp article 2 with 5 rows of setae along inner margin; distal articlewith a dorsal seta, a group of small setae at base of nail. Maxilliped outer lobewith 7-11 flattened spines and 4-8 serrated setae; inner lobe with 3-4 flattenedapical spines and 5 serrated setae.

Coxal plates, gills (figs. 4-7). Coxal plate I of flattened rhomboid shape, antero-ventral corner subrounded; anterior and ventral margin of coxa I with 5-7 setae.Coxal plate II width : depth is 1 : 0.90-1.00; anterior and ventral margin with 4-6 setae. Coxal plate III width : depth ratio is 1 : 0.80-1.00; along antero-ventralmargin 4-6 setae. Coxal plate IV width : depth ratio is 1 : 1.05-1.10; posteriorlyslightly concave (0.10-0.15 of coxa width); along antero-ventral margin 4-7 setae.Coxal plates V-VI: anteriorly developed lobe; along posterior margin 2-3 setae.Coxal plate VII semicircular, along posterior margin 2 setae and/or spines. GillsII-IV narrow and distal part of appendage basis; gills V-IV ovoid and reaching tomid-basis.

Gnathopod I (figs. 4-5). Ischium with 2-4 postero-distal setae. Carpus 0.55-0.60 of basis length and 0.90-1.15 of propodus length. Anterior margin of carpuswith distal group of setae; posterior margin of carpus with transverse rows ofsetae proximally and with a row of lateral setae; postero-proximal bulge large(1/3 of carpus length), positioned proximally. Propodus subquadrate, palm short,convex, and slightly inclined. Along posterior margin 3-7 rows of denticulatedsetae. Anterior margin with 4-10 setae in 2 groups, antero-distal group with 6-12 setae. Group of 2-3 facial setae proximally of palmar spine; individual surfacesetae present. Palmar corner with strong palmar spine, single supporting spine oninner surface and 1 denticulated spine on outer side. Nail length 0.30-0.35 of totaldactylus length; along anterior margin a single seta; along inner margin a few longsetae present.

Gnathopod II (figs. 4-5). Basis width : length is 1 : 0.25-0.35. Ischium with 2-3postero-distal setae. Carpus 0.50-0.60 of basis length and 0.85-1.00 of propoduslength. Anterior margin of carpus with distal row of setae; posterior marginof carpus with transverse rows of setae proximally and with a row of lateral


Fig. 4. Niphargus fongi sp. n., holotype: gI, gnathopod I; gII, gnathopod II.

setae; postero-proximal bulge large (1/3 of carpus length), positioned proximally.Propodus middle-sized (up to 0.20 of body length) and larger than propodus ofgnathopod I (1 : 0.65-0.75). Propodus trapezoid, palm short, convex and moreinclined than palm of gnathopod I. Posterior margin with 6-10 rows of denticulatedsetae. Anterior margin with 3-9 setae in 2 groups; antero-distal group with 6-12setae. Group of 3-5 facial setae proximally of palmar spine; individual surfacesetae present. Palmar corner with strong palmar spine, single supporting spine on


Fig. 5. Niphargus fongi sp. n., pm and pf, male and female paratypes, respectively: gI, gnathopod I;gII, gnathopod II.

inner surface and 1 denticulated spine on outer side. Nail length 0.30-0.35 of totaldactylus length. Along anterior margin a single seta; along inner margin a few longsetae present.

Pereopods III-IV (fig. 6). Length of pereopods III : IV equal to 1 : 0.95-1.05.Dactylus IV is 0.45-0.53 of propodus IV; nail length 0.45-0.55 of total dactyluslength. Dactyli III-IV with dorsal plumose seta; at base of nail two tiny setae.


Fig. 6. Niphargus fongi sp. n., holotype: ppIII, IV, pereopods III and IV, respectively; p-f ppIV,female paratype pereopod IV.

Pereopods V-VII (fig. 7). Length of pereopods V : VI : VII is 1 : 1.35-1.40 : 1.40-1.45. Pereopod VII length 0.40-0.50 of body length.

Bases V-VII of medium width, length : width is 1 : 0.65-0.75; posterior marginstraight, without distal lobes; posteriorly 6-10, 7-12, and 7-12 setae, respectively;anteriorly 5-7, 6-7, and 4-7 slender spines, respectively. Dactylus VII length is0.35-0.40 of propodus VII length; nail length 0.35-0.40 of total dactylus length.Dactyli V-VII with dorsal plumose seta; at base of nail two tiny setae.

Pleopods and uropods (fig. 8). Pleopods I-III with 4-7, 3-5 and 4-5-hookedretinacles, respectively. Pleopod II rami of 6-9 articles each.

Uropod I protopod with 4-5 dorso-lateral spines; 2-3 dorso-medial spines.Endopod : exopod length 1 : 1.00-1.05; rami straight. Endopod with 1-2 slenderspines; apically 5 spines. Exopod with 0-7 setae in 0-2 groups; apically 5 spines.

Uropod II endopod : exopod length 1 : 1.00-1.10.Uropod III up to 0.3 of body length. Protopod with no lateral setae and 5-7

apical spines and setae. Endopod 0.45-0.55 of protopod length, apically with 1-2spines; laterally 1-3 spines. Exopod of uropod III rod-shaped, distal article up to


Fig. 7. Niphargus fongi sp. n., holotype: ppV-VII, pereopods V-VII, respectively.

0.55 of proximal article length. Proximal article with 4-6 groups of setae, plumosesetae, and spines along inner margin; 4-5 groups of spines and setae along outermargin. Distal article with 1-2 lateral groups of setae along each side; apically 4-6setae.

Variability. — Body length of the largest female significantly exceeds the bodylength of any male, similarly to many unrelated troglobitic niphargids (unpubl.obs.). Females have oostegites on pereopods II-V and an undifferentiated distalarticle of exopod of uropod III. Younger specimens have fewer spines, fewerarticles in the antennal flagella and the pleopod rami, gnathopods slightly producedin palmar corner, and distal article of exopod of uropod III not elongated. Thenumber of hooks of the retinacula is variable, but consistently higher than inN. carniolicus (Sket, 1960) (see below).

Remarks and affinities. — Niphargus fongi sp. n. is sister species to “N. cf.tauri 3” from Prepadna Jama, a cave about 20 km south of the caves here studied(fig.1; Trontelj et al., in press) and both of them share a common ancestry withthe morphologically similar N. carniolicus. The lack of sufficient specimens doesnot allow the description of “N. cf. tauri 3”. However, preliminary observations


Fig. 8. Niphargus fongi sp. n., h, holotype: plp, pleopod II; upI-III uropods I-III; t, telson; pm and pf,male and female paratypes, respectively: t, telson; upIII, uropod III. All scale bars 0.2 mm.

suggest that the latter species differs from N. fongi in the number of retinacularhooks on the pleopods and in the number of spines on urosomites I-II. Nipharguscarniolicus differs from N. fongi in (i) fewer hooks in the pleopod retinacula (inN. carniolicus invariably 2-3-3); (ii) fewer spines on urosomites I-II (a single setaper segment in N. carniolicus); and (iii) in a more elongated distal article of theexopod of uropod III (up to 0.75 of the proximal article in N. carniolicus). Allthree species are nested within a morphologically diverse clade. The remainingspecies within the clade that are sharing some diagnostic characters with N. fongi,are N. aquilex dobati Sket, 1999 (Rak River, Slovenia), N. strouhali alpinusG. Karaman & Ruffo, 1989 (Germany), and five undescribed species labelled as“N. cf. tauri 1” (Eskisheir, Turkey), “N. cf. tauri 5” (Litija, Slovenia), “N. cf.aquilex 3” (Huda luknja Cave, Slovenia), “N. cf. aquilex 4” (Podutik, Ljubljana),and “N. cf. aquilex 5” (Lavreilles, Belgium) (Trontelj et al., in press). Thelast five have not been examined morphologically yet, mainly due to the smallnumber of specimens available. Niphargus a. dobati differs from N. fongi by (i)differently ornamented urosomites I-II (0 and 1 seta, respectively in Niphargus a.


dobati); (ii) shorter and less numerous telson spines; (iii) the absence of lateraltelson spines; (iv) smaller propodi of gnathopods I-II; (v) fewer hooks in theretinacula; and (vi) in a more elongated distal article of the exopod of uropod III(up to 0.75 of the proximal article in Niphargus a. dobati). Niphargus strouhalistrouhali Schellenberg, 1933 (not included in the study of Trontelj et al., in press)and N. s. alpinus differ from N. fongi by (i) differently ornamented urosomitesI-II (1 and 1 seta, respectively in N. strouhali subspecies); (ii) shorter and lessnumerous telson spines; (iii) the absence of lateral telson spines; (iv) severalmultitoothed spines on the outer lobe of maxilla I; (v) smaller and differentlyshaped gnathopods; (vi) fewer hooks in the retinacula (nominal subspecies 3,N. s. alpinus has 2); (vii) less elongated article of the exopod of uropod III (atmost 0.25 of the proximal article in N. strouhali subspecies). (Please note also thatN. s. alpinus was described from caves of Southern Limestone Alps, thus Germansamples used in study of Trontelj et al., in press, despite identical with description,may present the third taxon within the “N. strouhali” complex.)

The distinctness of N. fongi from the rest of the Niphargus species that sharewith the newly described species: (i) subrounded epimeral plates; (ii) a single setaon dactylus of gnathopods I-II; and (iii) subequal rami of uropods I, is summarizedin table I. Phylogenetic relationships between these species and N. fongi are as yetunknown as they have not been analysed.

Remarks on ecology and distribution range

In both caves we found two species of amphipods: N. fongi sp. n. and N. stygiusbrachytelson S. Karaman, 1952. In Koblarska Jama, N. fongi was collected insix out of eleven samples at three sites. It occurs most commonly in a poolthat occasionally dries up. Niphargus s. brachytelson was found in five pools,altogether in seven samples. In Vanceva Jama N. s. brachytelson predominated.It was collected in nine samples at four sites, while N. fongi was found only twicein two remote pools.

Both species inhabit water pools. However, they were sampled together onlytwice at two sites, and it seems that the micro-distributions of the two speciesdiffer. In both caves, N. fongi was found exclusively in pools on limestone withsome soil and some organic debris, while N. s. brachytelson was also frequentlyfound in muddy pools.

It is highly unlikely that pools are the main habitat of N. fongi. The poolsare small, not connected to each other, and occasionally dry up. Although thespecies could survive the dry period buried in clay (Mathieu & Turquin, 1992),the spatio-temporal sporadic occurrence of specimens in individual pools impliesthey are vagrants from a hidden and possibly more evenly distributed population.

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No springs appear above the two caves and the boundary with the saturated(phreatic) zone is at least 100 m below the caves as indicated by downhill springs.A connection with permanent groundwater thus seems to be highly questionableand it is reasonable to infer that N. fongi primarily inhabits the fissure systemin the rock. The shallow ceiling of the cave points to proximity of the boundaryzone between the rock and soil, rich in organic material, generally referred to asthe epikarst (e.g., Bakalowicz, 2005; Brancelj & Culver, 2005), where the speciescould reach higher abundances.

Niphargus fongi probably has a small distribution range. The co-occurringspecies N. s. brachytelson was also found in the cave Lukova Jama pri Zdihovem(Karaman, 1952), located about 20 km south of the two caves studied (fig. 1),while N. fongi is absent from this cave. Additionally, in Prepadna Jama, a caveclose to Lukova Jama, “N. cf. tauri 3” inhabits water pools that are separatedfrom the phreatic zone. About 20 km towards the northeast, in Jama pod GradomLuknja, N. carniolicus inhabits pools of percolated water. The similar morphologyof the species and their possible occupancy of the same habitat may suggest mutualcompetitive exclusion of the three species, and consequently imply the hypothesisthat the species’ ranges only minimally overlap. The distribution range of N. fongitoward the northwest is not known, but it probably does not extend over the non-karstic dolinas at the end of the karstic ridge Mala Gora, which lies approx. 25 kmfrom the two caves studied (fig. 1).

DISCUSSION

How cryptic are cryptic species?

As is apparent from the description, Niphargus fongi can well be diagnosedwith morphological characters. Despite this, the species was identified onlyas a morphological variation of “N. tauri” prior the molecular analysis. Suchconservative conclusion, i.e., erroneous rejection of the species hypothesis, may bea common problem in taxonomy. This case satisfactorily illustrates the impact oftaxonomic history and personal affiliations of each taxonomist toward recognizingdistinct species (splitters versus lumpers), which affects the rate at which (cryptic)species are discovered (Bickford et al., 2007).

It seems reasonable that cryptic species found in better-studied groups may haveless morphological differences (e.g., bats: see Mayer et al., 2007) while taxonom-ically significant morphological characters may remain overlooked in less studiedgroups. What can be even more problematic, is the absence of centralized and com-parable datasets on morphology (e.g., Godfray, 2002), which represents a seriousimpediment for taxonomic evaluation of detected morphological variation. To be


more explicit, several authors noted and clearly reported on morphological varia-tion of certain niphargid populations. However, these differences were attributed togeographic variation and left unevaluated taxonomically (Karaman, 1982, 1989b;Ginet, 1985; Fišer et al., 2006), which seems to be (at least in three cases) un-justified in the light of additional molecular findings (Trontelj et al., in press) anddifferences found in developmental processes (Fišer et al., 2008).

Elements of hidden diversity reconsidered: cryptic species, endemism, the natureof habitats, and sampling effort

Niphargus fongi seems to be an inhabitant of the fissure system in the rockwithin a narrow geographic area. This habitat can certainly be considered as having“extreme” environmental conditions (absence of light, rare food supply, narrowfissures, etc.), where cryptic species may be expected (Bickford et al., 2007). Suchhabitats can be rather inaccessible to man and consequently subjected to difficultiesin developing effective sampling strategies. Sporadically present in only two caveswhere it was collected in its secondary “habitat” (pools of percolating water),N. fongi may be an example of this kind of problem. The issue “to what extentbiodiversity remains unexplored on account of cryptic species” (Bickford et al.,2007) can be supplemented by another question, “whether ‘hidden’ biodiversityis influenced more by the presence of cryptic species, or inadequate sampling,or both”. This is especially apparent in studies of subterranean organisms. Forexample, explicit reports on niphargids found in percolating water are exceptional(e.g., Sket, 1981). Niphargus fongi is the fifth species reported from pools ofpercolating water (a rough estimate of the number is based on own unpublisheddata, but see Sket, 1999a) out of 42 species of Niphargus found in Slovenia, eventhough the genus is considered well studied in the country. No data for fissureinhabiting amphipods from the rest of Europe exist according to our knowledge.By contrast, 45 out of 100 species of the North American subterranean amphipodgenus Stygobromus were recognized as inhabitants of an epikarst that presentspart of a rock fissure system (Brancelj & Culver, 2005). Rather than pointing tointercontinental differences, the case implies that the European amphipod faunaof the fissure systems might be incompletely studied and/or ecologically as yetuncharacterized.

Insufficiently sampled amphipods from epikarst habitat may indicate that moreeffort is needed to collect them from fissure systems. The epikarst has been shownas especially rich with copepod species (Pipan & Culver, 2007a). Copepodologistsshowed that direct sampling of drips yields better results than sampling of pools(Pipan et al., 2006) and estimated that three to four months of continuoussampling of five drips finds 90% of epikarst copepods within the cave (Pipan &


Culver, 2007a). Zero to three specimens of amphipods versus several hundreds ofindividuals of copepods, collected at the same time and place (Brancelj & Culver,2005) clearly support the conclusion that amphipods are much less numericallyabundant than epikarst copepods. This can be understood in the light of biomassrelationships (Sket et al., 2004). What is important in terms of sampling strategiesis, that more sampling effort is needed for the study of epikarst amphipods, as wasshown for the drips.

Even when amphipods are not sampled directly from drips, more detailedobservations on the water situation in the cave may provide substantial supporttoward identifying a species as an element of the rock fissure community. Anyobservations on the possible permanent water flow in the cave, the distance fromthe saturated zone, positions of amphipod collection sites within the cave, and otherspecific local observations may constitute important support towards conclusionson the origin of the specimens collected. Unfortunately, such information israrely recorded with sufficient accuracy, or is totally neglected as the animals arecollected.

Accepting the hypothesis that selection pressures in the rock fissures habitatfavour speciation in the absence of conspicuous differences in morphology (Bick-ford et al., 2007), requires taxonomic (re-)evaluation of the fauna inhabiting thisspace. This approach would benefit from the introduction of molecular techniques,and is based on the premise that inhabitants of rock fissures are strict endemics (Pi-pan & Culver, 2007b; present study). In more general terms, it seems reasonable toinfer that specialized species regarded as widely distributed across discontinuouspatches of suitable habitat, instead represent aggregates of cryptic species and maybecome an interesting subject for taxonomic revisions. Such taxonomic practiceshould begin with an analysis of ecological and distributional information, andproceed with the inclusion of molecular techniques. This approach considerablydeviates from the traditional one, that builds a primary hypothesis on an analysisof the morphology and later on seeks support in distributional data or differencesin DNA sequences (DeSalle et al., 2005). Such “reversed” taxonomic practice maybecome important for taxonomy, and consequently for reliable estimates of thebiodiversity in ecologically extreme environments. Subterranean habitats may cer-tainly be regarded as such.

ACKNOWLEDGMENTS

We are grateful to the board of permanent members of DCBDC, who inspiredand supported us for this study. Lucija Ramšak was involved in general samplingresearch of the fauna of both caves. Many friends helped in the field, listed in al-phabetical order: Miloš Bartol, Milan Hornak, Klemen Koselj, Alenka Petrinjak,


Primož Presetnik, Aleksandra Privšek, Uroš Stepišnik, Marjeta Smrdel, and Mar-jetka Šemrl. We are very grateful to Miha Cekada from the Speleological Associ-ation of Slovenia (JZS), for his help in getting the latest map of Koblarska Jamafrom the Slovenian Cave Cadastre, stored in the cadastre of JZS. Access to othermaps from the Slovenian Cave Cadastre was enabled by DZRJL — Speleo ClubLjubljana. Boris Sket, David Culver, Gordan S. Karaman, J. C. von Vaupel Klein,and an anonymous referee gave valuable suggestions to improve an earlier versionof the manuscript. This work was funded by the Slovenian Research Agency.

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First received 9 April 2008.Final version accepted 1 August 2008.

Cryptic species from cryptic space: the case of Niphargus fongi sp. n. (Amphipoda, Niphargidae)

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