Description of a neotype for Asellus aquaticus Linné, 1758 (Crustacea: Isopoda: Asellidae), with description of a new subterranean Asellus species from Europe

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Description of a neotype for Asellus aquaticus Linne´, 1758 (Crustacea: Isopoda: Asellidae), with description of a new subterranean Asellus species from Europe Rudi Verovnik, Simona Prevorcˇnik, Jure Jugovic University of Ljubljana, Biotechnical Faculty, Department of Biology, Vecˇna pot 111, 1000 Ljubljana, Slovenia Received 3 March 2009; accepted 3 March 2009 Corresponding Editor: S. De Grave

Abstract Asellus aquaticus is one of the most common and well-studied freshwater macroinvertebrates in Europe, but its current taxonomic description is inadequate. Therefore, a neotype is designated and described to allow a comparison with a newly described and illustrated species, Asellus kosswigi sp. n. While several troglomorphic Asellus species are known from Japan, this is the first subterranean species of the genus in Europe. It is morphologically, as well as genetically, distinct from all other, local, surface and subterranean populations. Its species status is confirmed by its syntopic occurrence with Asellus aquaticus without any sign of gene flow. r 2009 Elsevier GmbH. All rights reserved. Keywords: Asellidae; Asellus aquaticus; Neotype; Asellus kosswigi sp. n.; Speciation; Phylogeny; Morphology; Troglobiont

1. Introduction Although Asellus aquaticus Linnaeus, 1758 is one of the most common and widespread freshwater crustaceans in Europe there is no accurate description of the species available. The original description of ‘Oniscus aquaticus’ was ‘‘Oniscus cauda rotundata, stilis bifurcis’’ from ‘‘aquis puris’’. Racovitza (1919) provided a new but still incomplete description, based on material from France, Great Britain and Slovenia. Since then many authors have tried to clarify the systematics of A. aquaticus; with all references until 1951 summarized by Gruner (1965, p. 96). Because morphometric data, as well as data about the morphological variation within the species were inadequate in previous descriptions, a Corresponding author.

E-mail address: [email protected] (R. Verovnik). 0044-5231/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2009.03.001

re-analysis of the name-bearing type appears necessary in order to provide a more objective description. However, the type material from Linnaeus, appears to be lost, as is any specific information on the type locality. Our search for the name-bearing specimen was in vain, it not being located in the Linnaeus collection in the Museum of Evolution (Uppsala), nor the Swedish Museum of Natural History (Stockholm). Neither was it present in Racovitza’s collection, kindly provided to us by G. Magniez. Therefore it is imperative to designate a neotype for this important taxon, the surrounding area of Uppsala being the historically most justified locality. Several troglomorphic species in the genus Asellus Geoffroy, 1762 are known all described from Japan, a centre of speciation of this genus (Matsumoto 1963; Henry and Magniez 1970). As all these 12 endemic species are subterranean, this high level of endemism can be attributed to the reduced dispersal abilities associated

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with this mode of life (Trontelj et al. 2008). In contrast, all European populations are believed to belong to a single species (Birsˇtejn 1951). A. aquaticus inhabits a wide array of different freshwater habitats, including the subterranean (Sket 1994). Nevertheless, as the morphology of A. aquaticus is highly variable, many subspecies and forms have been described throughout its range (summarized in Sket 1994). While most of the range is inhabited by the nominotypical A. a. aquaticus, five additional subspecies have been described from the Dinaric Region of the western Balkan Peninsula (Racovitza 1925; Karaman 1952; Sket 1965). These subspecies predominantly inhabit surface waters of isolated karst basins. Only A. aquaticus cavernicolus Racovitza, 1925 and A. a. cyclobranchialis Sket, 1965 from the northwestern Dinaric Region are subterranean and highly (the former) or slightly (the latter) troglomorphic (Sket 1994). Previous molecular studies have dealt with their relationship to surface populations (Verovnik et al. 2003, 2004) and their origin (Verovnik et al. 2005). The troglomorphic population living in the subterranean part of the river Reka/Timavo (cave Grotta di Trebiciano/Labodnica near Trieste/Trst in NW Italy) has been attributed to A. a. cavernicolus (Stammer 1932; Stoch 1984; Sket 1994), but was shown to be morphologically unique (Prevorcˇnik et al. 2004) and genetically distinct from other Dinaric Karst populations (Verovnik et al. 2003, 2004, 2005), on the basis of six specific RAPD fragments (Verovnik et al. 2003), and monophyly by nuclear and mitochondrial DNA sequences (Verovnik et al. 2004, 2005). Troglomorphic individuals can be occasionally found in a resurgence of the Reka/Timavo River together with animals belonging to the nominotypical subspecies (Stoch 1984). After several attempts, specimens of both taxa were collected from the resurgence enabling testing for potential gene flow and/or reproductive isolation and description of a new subterranean species of Asellus.

2. Material and methods 2.1. Morphology Body length, length and width of the head, pereomere V and the pleotelson were measured under a dissecting microscope, using an ocular micrometer. After dissection, pereopods I, IV and VII from one side of the body were heat-treated in a KOH solution, dyed with chlorazole black E and mounted in glycerine-gelatine on slides together with antennae I, II, pleopods, uropods and pleotelson, for examination and drawing under a compound microscope with camera lucida. Drawings were measured using a Genius graphic tablet (GT-1212B Series) and the Windows-supported program MERE

(by G. Sket). Alternatively, specimens from Sweden were photographed and measured using a Sony DXC390P digital camera mounted on a stereomicroscope or microscope (depending on the size of the structure), and measured with analySISs 3.1. The remains of the dissected specimens were transferred to 70% ethanol for storage. In one of the specimens from both type localities, all appendages were dissected and prepared for drawing. All pereopods, as well as the trunk were heat-treated and dyed as described above, and then mounted in glycerine-gelatine on one slide, alongside the rest of dissected specimen (mouth appendages, antennae, pleopods, uropods). In males of both species, 91 morphometric characters (listed in Prevorcˇnik et al. 2004, Appendix II) were recorded describing body proportions (trunk, appendages), characterization of cuticular and sense structures (number and length of spines, setae, aesthetascs). In antenna II only the last two articles of the antennal basis (fourth and fifth) were included in the total antennal length together with the length of the flagellum. In pereopods IV and VII, length of the coxa was not included in the total pereopod length. Additionally, pigmentation of the body and eyes was recorded, as well as the number of spines on the capitulum of pleopod II endopodite. In females, 9 morphometric characters describing body proportions (trunk, appendages), and the state of pigmentation were recorded. A single reported value in the description is based on the holotype or neotype, a range of values (in parenthesis) refers to the males from the type material.

2.2. Molecular phylogenetic analysis Sixteen population samples of Asellus were included in the molecular analysis (Table 1), including the neotype population of A. a. aquaticus, the newly described species Asellus kosswigi, A. aquaticus populations sampled from the Adriatic drainage, and populations from other parts of Europe. All material was collected with a dip net and preserved in 96% ethanol. A. kosswigi was collected from the type locality in the cave Grotta di Trebiciano/Labodnica in NW Italy and from the resurgence of the river Reka/Timavo (details in descriptive part) where two females were found among A. a. aquaticus specimens. Asellus hilgendorfii Bovallius, 1886 from Japan, Baikaloasellus sp. from Siberia, and Proasellus coxalis Dollfus, 1892 from Slovenia were used as out-groups for the phylogenetic analysis. Total DNA was extracted from specimens preserved in 96% ethanol using the GenElute Mammalian Genomic DNA minprep kit from Sigma-Aldrich. For each of the 122 specimens (including the out-group taxa) an approximately 700 bp fragment of the first subunit of cytochrome oxidase mitochondrial gene (COI) was

Table 1. List of sampling sites with corresponding information about sequences (abbreviated – first two letters of the sampling site or country), and sequence accession numbers. 28s rRNA alleles

Sample size

Accession nos. COI

Accession nos. 28s

( Alborg, Nordjylland, DK Osp, Koper, SI S. Giovanni al Timavo, Monfalcone, IT

AA1-AA3 OSP1, OSP2, OSP4 TM1, TM4, TM10

a 5 6

Rak channel, Planinska jama, Planina, SI San Pancrazio, Verona, IT Rakov Sˇkocjan, Cerknica, SI

RR1, RR8, CP-RR IT1, IT3, IT5, IT6 RS1, RS4, PP-RS, CPRS-PR, RS-CR

EUR-CP EUR-CP EUR-CP, LB-TM, TM11 PR-RR EUR-CP VR-RS-PP

DQ144746 DQ144746 DQ144746, FJ749281, FJ749280 DQ144742 DQ144746 DQ144743

Otok, Cerknica, SI

CR1, CR2, CR5, CR8, RS-CR CP1, CP2, CP8, CPRR, CP-RS-PR

DQ144816–DQ144818 DQ144769–DQ144771 DQ144792, DQ144793, FJ749275 AY531802–AY531804 DQ144779–DQ144782 AY531768, AY531805–AY531807, AY531820 AY531811–AY531813, AY531819, AY531820 AY531759, AY531760, AY531796, AY531802, AY531807 AY531780–AY531782 AY531798, AY531761, AY531806 AY531775, AY531799–AY531801, AY531807 AY531762–AY531765, AY531791–AY531793 AY531769–AY531771 AY531814–AY531818 DQ144821–DQ144822 FJ749276–FJ749279 DQ144833, DQ144834, AY531784 AY531829 DQ144778 DQ144777, DQ144752

DQ144750 DQ144737 DQ144751

Veliki Otok, Postojna, SI

10 5 10

10 EUR-CP

9

Virsˇnica jama, Velika Racˇna, Grosuplje, SI Planina, Rakek, SI

VR1, VR2, VR5 PP1, PP2, PP-RS

VR-RS-PP VR-RS-PP

5 10

Pivka channel, Planinska jama, Planina, SI

PR1, PR3, PR5, PR6, CP-RS-PR

PR-RR

10

Trebiciano cave, Trieste, IT

LB1-LB5, LB8, LB9

LB-TM

9

Dolga vas, Kocˇevje, SI Zbilje Reservoir, Ljubljana, SI Olsztyn, PL Ulva near Uppsala, SW Neuville sur Saone, Lyon, FR

KO1, KO2, KO5 ZB1-ZB5 PL1, PL4 UP1, UP2, UP3, UP4 LY1, LY2, LY4

KO2 ZB3 PL1 EUR-CP

5 5 5 4 5

Outgroups Asellus hilgendorfii Baikaloasellus sp. Proasellus coxalis

JP2 BA4 PA4, PA5

JP1 BA4 PA4

1 1 2

DQ144746

DQ144743 DQ144743 DQ144742

DQ144740 DQ144741 DQ144744 DQ144748 DQ144746

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COI haplotypes

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amplified using primers LCO1490 and HCO2198 (Folmer et al. 1994). A 780–820 bp fragment of the 28S rRNA gene (28S) was amplified using primers CAAGTACCGTGAGGGAAAGTT-30 and 50 -AGGGAAACTTCGGAGGGAACC-30 designed by comparing available invertebrate 28S rDNA sequences. With this gene, amplification was unsuccessful in specimens from Cerknica (CR) and Uppsala (UP). PCR products were eluted directly from agarose gels and sequenced under BigDye terminator cycling conditions, purified by ethanol precipitation, and run on an Applied Biosystems 3730xl sequencer by Macrogen. All COI sequences were of equal length (653 bp) and could thus be unambiguously aligned by hand. The 28S sequences were of unequal length and were aligned under a range of ClustalX (Thompson et al. 1997) gap opening and gap extension penalties (7–19 for opening, and 3–9 for extension, both by steps of two). The robustness of the alignment was explored as described by Trontelj and Utevsky (2005). All sequences have been deposited in the GenBank Nucleotide Sequence database (see Table 1 for accession numbers). Both COI and nuclear 28S rDNA sequence data were analyzed using Bayesian inference. The program MrBayes, version 3.0b4 (Huelsenbeck and Ronquist 2001) was used. Hierarchical likelihood test (Posada and Crandall 1998) was employed in order to test alternative models of evolution, using Modeltest 3.06b (Posada and Crandall 1998). A HKY85 model of nucleotide sub-

stitution with gamma distributed rate heterogeneity and a significant proportion of invariable sites was selected for the COI sequence data and HKY+G model of nucleotide substitution with gamma distributed rate heterogeneity and a significant proportion of invariable sites for the nuclear 28S rDNA sequence. Uniform or fixed default prior settings were used. In both analyses a Markov chain Monte Carlo search was run with four chains for 4  106 generations, taking samples every 100 generations. The approximate number of generations needed to obtain stationarity of the likelihood values (‘‘burn-in’’) of the sampled trees was estimated graphically to 5000 and 2000 trees. From the remaining trees posterior probabilities were assessed for individual clades based on their observed frequencies.

3. Taxonomy Asellidae Sars, 1897 Asellus E. L. Geoffroy, 1762 Asellus aquaticus Linnaeus, 1758, n. def. (Figs. 4–8) Material examined. Neotype (UUZM 60171): mature male, 8.3 mm, river Fyrisa( n downstream from the old mill *Ulva kvarn), village Ulva N of Uppsala, Uppland, Sweden, lat: 591540 45.6600 N, long: 171340 32.700 E (WGS 84), 15.4.2007, collected by K. Larsson, P. Kloss and B. Frajman; animal partly dissected and mounted in

Fig. 1. Map showing the positions of sampling sites of the sampled cave and surface populations of Asellus. The position of the enlarged part (left) is given by the black rectangle. Each locality/sample is abbreviated as in Table 1.

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Fig. 2. Phylogenetic relationship of Asellus aquaticus and A. kosswigi COI haplotypes derived from 15,000 Bayesian trees. Values on major branches are Bayesian posterior probabilities. Haplotypes are coded as in Table 1. The haplotypes of the syntopic samples from S. Giovanni al Timavo are highlighted.

Fig. 3. Phylogenetic relationship of Asellus aquaticus and A. kosswigi nuclear 28S rDNA sequences derived from Maximum likelihood analysis. Values on branches are bootstrap values (500 replicates). Haplotypes are coded as in Table 1. The sequences of the syntopic samples from S. Giovanni al Timavo are highlighted.

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glycerine-gelatine, rest preserved (as other specimens from the same locality) in 70% ethanol; deposited in the collection of the Museum of Evolution, Zoology (Uppsala), Sweden (UUZM). Other specimens examined from the neotype locality (SMNH 103932–103935; UUZM 60172–60175; SE-UL A0001–A0004): 9 mature males, 8.1–9.8 mm; 6 mature females (OBBFUL A0004–A0009) 7.2–9.8 mm, 4 of them ovigerous, same data as neotype. Glycerinegelatine slides and rests preserved in 70% ethanol and deposited in the collection of the Swedish Museum of Natural History (Stockholm) (SMNH), Museum of Evolution, Zoology (Uppsala) (UUZM) and in the zoological collection of the Slovenian Univerza v Ljubljani, Biotehnisˇka fakulteta, Oddelek za biologijo (OBBFUL). Additional 4 mature non-ovigerous females, same data as neotype, were used in the genetic analyses. Remains in 98% ethanol are deposited in the Slovenian zoological collection (OBBFUL). Other specimens examined: 8 mature males (10.1–12 mm), Uppsala in Uppland region, Sweden, leg. unknown; det. Wahlberg, collection of the Swedish Museum of Natural History (SMNH 89525); partly dissected and mounted in glycerine-gelatine, rest preserved in 70% ethanol; 2 mature males (10.2–10.8 mm), Uppsala in Uppland region, Sweden, leg. C. Bovallius 1884; det. Wahlberg (SMNH 89693); partly dissected and mounted in glycerine-gelatine, rest preserved in 70% ethanol. Diagnosis of male: Species of Asellus with pigmented oval body, about 2.8 times as long as wide. Head more than 2.2 times as wide as long, with two large bright patches at the posterior margin separated by a darker median line; frontal margin bisinuate, medially concave, lateral margins rounded, each with posterolateral setous prominence. Eyes black pigmented, of 3 ommatidia. Pereonites I–III with distinctively convex lateral margins, pereonites IV–VII with straight lateral margins and rounded antero- and posterolateral angles, all fringed with dense long and short spiniform setae. Pleomere I–II width about 45% of pereonite VII width. Pleotelson rounded subrectangular, lateral margins densely fringed with short and long spiniform setae, terminal edge bisinuate with obtusely triangular median prominence and with numerous short simple setae. Antennae I and II lengths about 30% and 95% of body length and with 11–14 and 50–82 flagellar articles, respectively. Propodus I wide triangular, with strong apophysa on palmar margin. Propodus IV superior margin and submarginal surface with 3–6 short simple setae. Length of longest spiniform robust seta on propodus VII inferior margin 18–21% of pereopod VII length. Pleopod I exopodite with well expressed, asymmetrical concavity on lateral margin, its depth 10–17% of exopodite width. Pleopod II architecture following typical ‘‘Asellus pattern’’ sensu Henry and Magniez (1968), endopodite usually with 2

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Fig. 4. Asellus aquaticus, river Fyris(an, village Ulva N of Uppsala, Uppland, Sweden, male, 8.2 mm: A I, II, antennae I and II; B, body dorsal view; H, head pattern, dorsal view; Plt, pleotelson half, ventral view.

(1) basal spines next to anterior lobe and mostly with 2 (3) spines on capitulum. Pleopod IV and V with small respiratory areae, linea areae beginning and ending at the distal exopodite margin. Description of neotype and male type material (values in parenthesis): Body (Fig. 4B) about 2.8 (2.6–3.0) times as long as wide, oval. Head (Fig. 4(H) light brown with two large bright patches at the posterior margin, separated by a darker median line, its width 2.3

(2.2–2.8) times of its length. Frontal margin bisinuate, medially concave, without rostral process, lateral margins rounded, each with small posterolateral protuberance, with one long and 2–3 short stiff setae. Eyes black pigmented, of 3 ommatidia each. Pereonites (Fig. 4B) light brown medially and with some irregularly shaped brighter patches laterally, anterolateral, lateral and posterolateral margins densely fringed with long and short spiniform setae. Pereonites I–III with

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Fig. 5. Asellus aquaticus, river Fyris(an, village Ulva N of Uppsala, Uppland, Sweden, female, 9.8 mm: Plt (v), pleotelson with pleopods, ventral view. Male, 8.2 mm: L, lower lip; MdbL, left mandible with lacinia mobilis and palp; MdbR, right mandible with palp.

distinctively convex lateral margins, pereonites IV–VII with straight lateral margins and rounded angles. Last two pereonites with antero- and posterolateral angles slightly protruding backwards. Pereonite VI widest. Coxopods well developed, margins of all epimerae dorsally visible, last three the most prominent. Pleomere I–II small (Figs. 4B and 5Plt(v)), their width only 45% of pereonite VII width, forming a stalk largely covered by posterior margin of pereonite VII. Pleotelson (Fig. 4B and Plt) rounded subrectangular, its width 1.2 (1.1–1.3) times of its length, terminal edge bisinuate

with obtusely triangular median prominence between uropods. Lateral margins in their anterior quarters with few short setae, other three quarters with dense spiniform setae, approximately two or three shorter setae placed between two longer ones. Terminal edge with numerous short setae and with two longer setae medially, subterminal margin with scarce short setae. Dorsal surface covered with numerous short delicate setae. Antenna I length (Fig. 4AI) 29% (27–31%) of body length, with 3 peduncular articles. First article robust,

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Fig. 6. Asellus aquaticus, river Fyris(an, village Ulva N of Uppsala, Uppland, Sweden, male, 8.2 mm: Mx I, II, maxillae I and II; Mxlp (v), (d), maxilliped ventral and dorsal view.

with curved superior (longer) and inferior (shorter) margin, other two articles cylindrical. Second article 1.5 (1.2–1.9) times as long as first and 1.5 (1.1–1.7) times as long as third. Longest setae on articles 1 and 2 about as long as article 1. Flagellum of 12 articles (11–14 articles), usually 4 (1–4) distal articles with one aesthetasc each. Proximal aesthetascs mainly as long as their parallel articles. Antenna II length (Fig. 4AII) 106% (85–106%) of body length, with 6 peduncular and 82 (50–82) flagellar articles. Sixth peduncular article 1.6 (1.5–1.7) times as long as fifth, both with long and short setae on

superior margins, inferodistal and superior distal angles, but with only short setae on inferior margins. Flagellum length 78% (72–79%) of antenna II length. Mandibullae robust (Fig. 5MdR and MdL): Pars molaris (molar process) U-shaped, with toothed margin and wrinkled crushing surface. Pars incisiva (incisor) formed by 4 blunt cusps arranged in semi-circle. Left lacinia mobilis with 5 indistinct cusps, spine row of 16 biserrate setae, right mandible without lacinia mobilis. Palp of three articles. First article widest, with 3 simple setae along internal margin and 4 extero-subapical ones.

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Fig. 7. Asellus aquaticus, river Fyris(an, village Ulva N of Uppsala, Uppland, Sweden, male, 8.2 mm: Plp I–V, pleopods I–V; U, uropod. Female, 9.8 mm: Plp II (F), pleopod II.

Second article 1.4 times as long as first, with 2 simple setae and about 20 robust long and short biserrate setae along external margin. Third article length 0.7 times of second, with row of about 20 (22) robust biserrate setae along external margin, terminal two being longest. Maxilla I (maxillule; Fig. 6MxI) lateral lobe with 14 distal spines; the outer 5 cone-shaped, smooth, forming a semi-circle, inner spines slender and weakly serrated (2–8 teeth). Inner margin of the lobe with scarce short setae and one long submarginal seta, distal part of outer margin with 1 long slender seta, proximal part scarcely serrated. Mesial lobe with 3 robust long cirkumplumose

setae and 1 long slender plumose seta. Inner margin of the lobe with scarce short setae. Maxilla II (Fig. 6MxII) lateral and middle lobe with 17 and 13 curved pectinate robust setae, respectively, mesial lobe with about 16 biserrate setae and parallel row of about 26 long simple setae along inner margin. Maxilliped (Fig. 6 Mxlp (v)) endite distal margin with about 14 biserrate robust setae and long simple setae. Mesial margin (Fig. 6Mxlp (d)) curved dorsally, with row of about 10 biserrate setae, distomesial margin with setulose fringe and 6 (5–6) coupling hooks, lateral margin with dense setulose fringe. Palp of five articles. First article short, with 3

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Fig. 8. Asellus aquaticus, river Fyris(an, village Ulva N of Uppsala, Uppland, Sweden, male, 8.2 mm: Pp I, IV, VII, pereopods I (distal articles), IV and VII. Female, 9.8 mm: Pp I, IV (F), distal articles of pereopods I, IV.

short setae apically on outer margin, second about 1.6 times as long as first, subtrapezoidal, with 5 long stiff setae on outer margin and row of about 25 longer medially directed simple setae on inner margin, medio-distally with 3 distally directed setae. Third article length 75% of second article length, less broad, with 3 long stiff setae on outer margin and row of about 11 setae (as on second article) on inner margin. Fourth article 1.6 times as long as third, slender, with row of 8 and about 16 long slender setae along outer and inner margin, respectively. Fifth article length 60% of fourth article length, ovoid, fringed with 12 long slender setae and 2 stiff simple apical setae. Epipodite subrectangular, lateral margin fringed with about 19 short setae.

With the exception of the first and the fourth pair, seven pairs of pereopods ambulatory and similar in construction, increasing in length towards posterior pairs. Pereopod I (Fig. 8PpI) grasping, subchelate. Propodus I (article 6) broadly triangular, up to 1.7 times (1.3–1.7 times) as long as wide, inferior margin with well-developed proximal apophysa, armed with 3 (3–4) robust spiniform setae. Mesial surface with 2 rows of 21 (19–24) long and 18 (16–19) shorter simple setae. Dactylus I length (article 7) about two-thirds of propodus length, with 11 (11–12) densely packed short (their length about half of dactylus greatest width) stiff robust setae along inferior margin. Pereopod IV (Fig. 8PpIV) grasping, with parallel, visibly curved superior and inferior margins of propodus. Pereopod IV

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length 43% (39–48%) of body length, length relations of articles from ischium (article 2) to dactylus (article 7): 1: 0.7 (0.6–0.8): 0.5 (0.4–0.6): 0.9 (0.7–1.0): 0.8 (0.7–1.0): 0.6 (0.4–0.6), unguis length 50% (39–57%) of dactylus length. Propodus IV inferior margin with row of 9 (9–17) acute stiff robust setae, longest robust seta 9% (8–13%) of propodus length, mesial surface with row of 15 (10–15) submarginal simple setae, inferodistal angle with 1 stiff acute robust and 3 simple setae, superior margin and submarginal surface with 4 (3–6) short simple setae and with 3 (1–3) penicilate setae, superior distal angle with 2 (2–7) long simple setae and 1 penicilate seta. Dactylus IV inferior margin with 5 (3–6) robust stiff setae, superior margin distally with 2–5 simple setae. Pereopod VII (Fig. 8PpVII) with long slender articles, its length 87% (87–103%) of body length, length relations of articles along pereopod VII (given as in pereopod IV): 1: 1 (0.8–1.0): 0.7 (0.6–0.7): 1 (0.7–1): 1.2 (1.0–1.2): 0.4 (0.3–0.4), unguis length 54% (38–54%) of dactylus length. Propodus VII inferior margin with row of 10 (7–10) acute stiff robust setae, longest robust seta 21% (18–21%) of propodus length, mesial surface with row of 10 (7–11) submarginal simple setae, inferodistal angle with 1 stiff acute robust and 2 (1–4) simple setae, superior margin and submarginal surface with 8 (8–12) short simple setae and with 3 (3–5) penicilate setae, superior distal angle with 1 (1–2) simple setae and 1 penicilate seta. Dactylus VII inferior margin with 5 (5–6) robust stiff setae, superior margin distally with 2–5 simple setae. Pleopod I (Fig. 7PlpI) protopodite rounded rectangular, 1.03 (0.9–1.1) times as wide as long, retinacle on medial margin of 3 (3–6) hooks. Exopodite elongated ovoid, its width 57% (51–60%) of its length, with 15 (12–19) simple setae among 14 (8–14) plumose terminal marginal setae and 3 (3–6) acute simple setae on proxomedial angle. Concavity on lateral margin well expressed, its depth 14% (10–17%) of exopodite width, with 4 (3–12) asymmetrically positioned simple setae. Concavity proximally shallow and narrow, distally deeper and flattened; flattened part perpendicular to equally flattened lateral exopodite margin. Pleopod II (gonopod; Fig. 7PlpII) protopodite subtrapezoidal, its width 90% (81–92%) of its length. Lateral and medial margins with 1 (1–3) and 3 (2–5) simple spiniform setae, respectively. Exopodite suboval, 1.4 (1.2–1.7) times as wide as long, lateral and medial margins fringed with 13 (9–14) simple and 6 (5–8) long plumose setae, respectively. Endopodite elongated ovoid, its length 65% (60–71%) of protopodite length, with horn-shaped basal spur (processus calcariformis). Processus cylindriformis club-like and wrinkled distally. Anterior lobe transversely elongated with few serrated scales on its lateral surface, next to anterior lobe usually 2 (1) short basal spines. Capitulum helical, mostly with 2 (3) conical spines, squamiferous membrane (membrana squamifera)

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minute, cannula short and deeply hidden. Pleopod III (Fig. 7PlpIII) exopodite subtriangular, about 1.5 (1.4–1.5) times as long as wide, with almost straight medial margin. Terminal margin with medio- and latero-distal angles fringed with 19 (15–24) long plumose setae. Lateral margin with 14 (13–23) short simple setae, lateral and terminal surface with 13 (13–25) short setae. Endopodite about 1.7 (1.3–1.8) times shorter than exopodite. Pleopod IV (Fig. 7PlpIV) exopodite broadly ovoid, about 1.3 (1.3–1.6) times as long as wide, its area equally shaped as in pleopod V, lateral margin proximally with 7 (7–11) simple setae. Endopodite subrectangular, about 1.4 (1.2–1.7) times as long as exopodite. Pleopod V (Fig. 7PlpV) exopodite ovoid, 1.4 (1.4–1.6) times as long as wide, lateral margin proximally with 6 (5–10) simple setae. Respiratory area small, its surface 31% (30–39%) of exopodite surface, linea areae beginning and ending on the distal exopodite margin. Endopodite suboval, its length 97% (93–113%) of exopodite length. Uropod (Fig. 7U) length 34% (25–40%) of body length. Proto-, endo- and exopodite length relations: 1:1.96 (1.8–2.5):2.02 (1.7–2.5).

Fig. 9. Asellus kosswigi sp. nov., river Reka, cave Grotta di Trebiciano/Labodnica, Trieste/Trst, Italy, male, 9 mm: A I, II, antennae I and II; B, body dorsal view; Plt (v), pleotelson with pleopods, ventral view.

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Endopodite (terminal margin excluded) with 11 (10–15) spiniform simple setae and 1 (1–3) penicilate setae, longest simple seta length 20% (16–24%) of endopodite length. Female type material. Body length 7.2–9.8 mm (2.5–2.8 times of body width), almost identical to male except, antenna I length 24–29% of body length, with 10–13 flagellar articles; antenna II with about 61–71 flagellar articles. Pereopod I (Fig. 8PpI(F)) with less numerous, slender simple setae, propodus with less expressed proximal apophysa. Pereopod IV (Fig. 8PpIV(F))not for grasping, ambulatory, little longer than the preceding pairs, with less numerous setae along mesial surface and inferior and superior margins. Pleopod II (Fig. 5Plt(v), Fig. 7PlpII(F)) suboval, about 1.3 (1.2–1.4) times as long as wide, with 17–25 long marginal plumose setae. Remarks: Due to the extreme diversity of the species’s external morphology and colouration, 10 subspecies have been described throughout the species range

Fig. 10. Asellus kosswigi sp. nov., river Reka, cave Grotta di Trebiciano/Labodnica, Trieste/Trst, Italy, male, 9 mm: C (l), (v), clypaeus lateral and ventral view; L, lower lip; MdbL, left mandible with lacinia mobilis and palp; MdbR, right mandible with palp.

(see Introduction and Sket 1994). While four subterranean, slightly to strongly troglomorphic subspecies (A. a. cavernicolus Racovitza, 1925, A. a. infernus Turk–Prevorcˇnik et Blejec, 1998, A. a. cyclobranchialis Sket, 1965 and A. a. strinatii Chappuis, 1955) differ in the degree of expressed troglomorphic and paedomorphic traits, six surface subspecies (A. a. aquaticus, A. a. carniolicus Sket, 1965, A. a. carsicus Karaman, 1952, A. a. irregularis Sket, 1965, A. a. longicornis Sket, 1965 and A. a. meserianus Birsˇtejn, 1945) differ in pigmentation, body and appendage proportions, and the number of spines and setae on the structures. According to extensive multivariate statistical analyses of geographic variation of 59 morphometric characters in 183 A. aquaticus samples (Prevorcˇnik et al. 2004), the most discriminative trait in surface A. aquaticus is the size of the respiratory area (non-sclerotized part, limited by linea areae) on the exopodite of pleopod V. The A. aquaticus neotype, and thus the nominotypical subspecies, morphologically belongs with certainty to the ‘‘small’’ respiratory area morph (sensu Prevorcˇnik et al. 2004), together with the troglomorphic A. a. infernus and, presumably, A. a. strinatii and

Fig. 11. Asellus kosswigi sp. nov., river Reka, cave Grotta di Trebiciano/Labodnica, Trieste/Trst, Italy, male, 9 mm: Mx I, II, maxilla I and II; Mxlp, maxilliped ventral view.

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A. a. meserianus. The respiratory area of this morph covers less than 40% of pleopod exopodite surface, with the linea areae beginning and ending on opposite parts of the distal exopodite margin. Due to inconsistency in the presently applied subspecific classification of A. aquaticus with morphometric (Prevorcˇnik et al. 2004), as well as genetic analyses (Verovnik et al. 2003, 2004, 2005), a full taxonomic revision of the species would be beneficial. Asellus kosswigi sp. nov. (Figs. 9–13) Asellus aquaticus f. cavernicola Stammer 1932 p.p. Asellus aquaticus cavernicolus Stoch 1984, Sket 1994 p.p. Material examined. Holotype (OBBFUL A0006): mature male, 9.0 mm, river Reka, cave Grotta di Trebiciano/Labodnica, Trebiciano/Trebcˇe, Trieste/Trst, Italy, 15 June 1996, collected by S. Dolce; animal partly dissected and mounted in glycerine-gelatine, rest preserved in 70% ethanol; deposited in the collection of the Slovenian Museum of Natural History (SMNH). Paratypes (OBBFUL A0007–A0027): 21 mature males (6.5–12.0 mm), 4 mature non-ovigerous females

Fig. 13. Asellus kosswigi sp. nov., river Reka, cave Grotta di Trebiciano/Labodnica, Trieste/Trst, Italy, male, 9 mm: Pp I, IV, VII, pereopods I (distal articles), IV and VII.

Fig. 12. Asellus kosswigi sp. nov., river Reka, cave Grotta di Trebiciano/Labodnica, Trieste/Trst, Italy, male, 9 mm: Plp I–V, pleopods I–V; U, uropod. Female, 6 mm: Plp II (F). pleopod II.

(5.1–6.3 mm) and 2 juveniles (4.0 and 4.5 mm), data as for holotype. Other specimens examined (OBBFUL A0032): 7 mature males, 4 mature non-ovigerous females and 2 juveniles, same locality, 2 July 1996, collected by B. Sket and S. Prevorcˇnik; (OBBFUL A0028–A0031): 4 mature males (6.7–7.7 mm), data as above, partly dissected and mounted in glycerine-gelatine, rest preserved in 70% ethanol; (OBBFUL A0033):10 mature males, same locality, 23. January 2000, collected by R. Verovnik and C. Fisˇer, animals used in molecular analyses, rests preserved in 98% ethanol; (OBBFUL A0034–A0035): 2 females, resurgences of the river Timavo/Reka, Monfalcone, Italy, 17. December 2006, collected by B. Sket; pleotelsons with pleopods preserved in 70% ethanol, rest used in molecular analyses. OBBFUL A0007–A0011 deposited in the collection of the Slovenian Museum of Natural History, all other specimens in the zoological collection of the Slovenian Univerza v Ljubljani, Biotehnisˇka fakulteta, Oddelek za biologijo (OBBFUL).

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Diagnosis of male: Species of Asellus with completely reduced body pigmentation and eye pigmentation. Body elongated oval, about three times as long as wide. Head up to 2.3 times as wide as long, trapezoidal, with straight frontal and lateral margins, latter with two posterolateral setous prominences each. All pereonites with either straight or slightly concave lateral margins and, prominent only slightly rounded antero- and posterolateral angles, all fringed with simple short setae of about the same length. Pleomere I–II width about 57% of pereonite VII width. Pleotelson subrectangular, lateral margins densely fringed with simple short setae of about the same length and only sparse longer simple setae. Terminal edge bisinuate, without setae, with rounded median prominence. Antennae I and II lengths about 20% and 90% of body length and with 9–15 and 42–70 flagellar articles, respectively. Propodus I slender ovoid, with weak apophysa on palmar margin. Propodus IV superior margin and submarginal surface with 6–12 short simple setae. Length of longest spiniform robust seta on propodus VII inferior margin 8–13% pereopod VII length. Pleopod I exopodite either without, or with only minute symmetric concavity on lateral margin. Pleopod II arhitecture following typical ‘‘Asellus pattern’’ sensu Henry and Magniez (1968), endopodite with 0 or 1 basal spine next to anterior lobe and 0 or 1 spine on capitulum. Pleopod IV and V with large respiratory areae, linea areae beginning proximally on the lateral exopodite margin and ending at the transition from terminal to medial exopodite margin. Description of holotype and other male specimens (values in parenthesis). Body (Fig. 9B) about three (2.8–3.4) times as long as wide, elongated oval, without pigmentation. Head trapezoidal, two (1.9–2.3) times as wide as long. Frontal margin straight, without rostral process. Lateral margins straight, with two small setose protuberances posterolaterally, posterior ones being lateral expressions of maxillipedal segment. No eye structures visible. Pereonites I–III with straight lateral margins and weakly rounded antero- and posterolateral angles, slightly protruding forwards. Last two pereonites with antero- and posterolateral angles slightly protruding backwards. Pereonite VI widest. Anterolateral, lateral and posterolateral margins of pereonites with sparse short simple setae. Coxopods well developed, margins of all epimerae dorsally visible, last three largest. Pleomere I–II short but wide (Figs. 9B and 9Plt(v)), their width about 57% of pereonite VII width, forming a stalk largely covered by posterior margin of pereonite VII. Pleotelson (Fig. 9B) subrectangular, 1.2 (1.1–1.5) times as wide as long, terminal edge bisinuate with rounded median prominence between uropods. The anterior quarters of lateral margins with sparse short simple setae, rest three quarters with numerous short simple setae with only few little longer slender ones. Terminal median prominence without setae, subterminal

margin with scarce short setae. Dorsal surface covered with sparse simple setae of the same length as shortest setae on lateral margin. Antenna I (Fig. 9AI) length 19% (19–24%) of body length, with 3 peduncular articles. Article shapes as in type species. Second article 1.3 (0.9–1.5) times as long as first and 1.3 (1.2–1.9) times as long as third. Longest setae on peduncular article1 and 2 only half as long as first article. Fagellum of 11 articles (9–15 articles), 4 (3–6) distal articles with one aesthetasc each. Proximal aesthetascs mainly shorter as their parallel articles. Antenna II length (Fig. 9AII) 94% (75–99%) of body length, with 6 peduncular and 70 (42–70) flagellar articles. Sixth peduncular article 1.8 (1.3–1.8) times as long as fifth, both with only short setae, long setae present only on superior distal angles. Flagellum length 79% (71–80%) of antenna II length. Mandibullae (Fig. 10MdR and MdL) robust, as in type species, but with spine row of 14 biserrate setae. Second palpal article only 1.1 times longer than first, with 3 simple setae and only about 8 robust long and short short biserrate setae along external margin. Third palpal article, with row of about 16 robust biserrate setae along external margin. Maxilla I (maxillule; Fig. 11MxI) lateral lobe with 5 smooth and 9 weakly serrate (6–8 teeth) robust spines, rest as in type species. Maxilla II (Fig. 11MxII) lateral and middle lobe with 22 and 15 curved pectinate robust setae, respectively, mesial lobe with about 20 biserrate setae and parallel row of about 23 long simple setae along inner margin. Maxilliped (Fig. 11Mxlp) endite distal margin with about 11 biserrate robust setae, subapically with several rows of short simple setae. Mesial margin curved dorsally, with row of about 10 long biserrate setae, distomesial margin with setulose fringe and 6 (5–6) coupling hooks, lateral margin with dense setulose fringe. Palp of five articles. First article with 2 short setae apically on outer margin; second about 2.5 times as long as first, subtrapezoidal, with 4 long stiff setae on outer margin and row of about 22 longer medially directed simple setae on inner margin, medio-distally with 3 distally directed setae. Third article about as long as second, less broad, with 4 long stiff setae on outer margin and row of about 11 setae (as on second article) on inner margin. Fourth article almost twice as long as third, slender, with row of 6 and about 24 long slender setae along outer and inner margin, respectively. Fifth article as long as first, ovoid, fringed with 8 long slender setae and 3 stiff simple apical setae. Epipodite subrectangular, lateral margin fringed with about 16 short setae. With the exception of the first and the fourth pair, seven pairs of pereopods similar in construction and ambulatory, increasing in length towards posterior pairs. Pereopod I (Fig. 13PpI) grasping, subchelate. Propodus I (article 6) slender ovoid, twice (1.9–2.4 times) as long as wide, with weakly expressed proximal

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apophysa, armed with 3 slender stiff robust setae. Mesial surface with dense rows of shorter simple setae (about 40). Dactylus I (article 7) length about two-thirds of propodus length, with 8 (6–9) sparsely placed slender stiff robust setae along inferior margin (their length increasing towards unguis). Pereopod IV (Fig. 13PpIV) grasping, with parallel, weakly curved superior and inferior margins of propodus. Pereopod IV length 40% (40–50%) of body length, length relations of articles from ischium (article 2) to dactylus (article 7): 1:0.6 (0.5–0.6):0.3 (0.3–0.4):0.7 (0.6–0.7):0.7 (0.7–0.8):0.4 (0.3–0.4), unguis length 65% (50–80%) of dactylus length. Propodus IV inferior margin with row of 9 (6–10) acute stiff robust setae, longest robust seta 14% (10–15%) of propodus length, mesial surface with row of 18 (18–27) submarginal simple setae, inferodistal angle with 1 stiff acute robust and 3 (2–4) simple setae, superior margin and submarginal surface with 12 (6–12) short simple setae and with 5 (3–7) penicilate setae, superior distal angle with 6 (5–9) long simple setae and 1 penicilate seta. Dactylus IV inferior margin with 2 (2–3) robust stiff setae, superior margin distally with 2 (1–4) simple short setae. Pereopod VII (Fig. 13PpVII) with long slender articles, its length 80% (75–104%) of body length, length relations of articles along pereopod VII (given as in pereopod IV): 1:0.8 (0.8–0.9):0.6 (0.5–0.6):0.9 (0.8–1.0):1.2 (1.0–1.4):0.3 (0.2–0.3), unguis length 61% (45–75%) of dactylus length. Propodus VII inferior margin with row of 9 (7–10) acute stiff robust setae, longest robust seta 12% (8–13%) of propodus length, mesial surface with row of 13 (7–13) submarginal slender simple setae, inferodistal angle with 1 (2) stiff acute robust and 7 (5–9) simple setae, superior margin and submarginal surface with 16 (10–17) short simple setae and 7 (5–9) penicilate setae, superior distal angle with 7 (4–8) simple setae and 1 penicilate seta. Dactylus VII inferior margin with 2 (2–3) robust stiff setae, superior margin distally with 5 (4–8) simple short setae. Pleopod I (Fig. 12PlpI) protopodite 1.3 (1.2–1.6) times as wide as long, angles rounded, retinacle on medial margin of 5 (4–6) hooks. Exopodite elongated ovoid, its width 63% (56–69%) of its length, with 18 (16–22) simple setae among 12 (8–16) plumose terminal marginal setae and 4 (4–8) acute simple setae on proxomedial angle. Concavity on lateral margin hardly visible, when present, symmetric and extremely shallow, its depth only up to 2% exopodite width, with 3 (0–5) simple setae. Pleopod II (gonopod; Fig. 12PlpII) protopodite subtrapezoidal, with rounded angles, its width 92% (76–100%) of its length. Lateral and medial margins without and with up to two setae, respectively. Exopodite oval, 1.6 (1.2–1.8) times as wide as long, lateral and medial margins fringed with 8 (5–9) simple and 4 (3–5) long plumose setae, respectively. Endopodite elongated ovoid, its length 67% (51–70%) of protopodite length, with horn-shaped basal spur (processus

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calcariformis), processus cylindriformis as in type species. Anterior lobe transversely elongated with few serrated scales on its distolateral angle, next to anterior lobe usually 0 (1, randomly 2) short basal spines. Capitulum helical, mostly with 0 (2) apically serrated scale-like spines. Squamiferous membrane (membrana squamifera) minute, cannula short and deeply hidden. Pleopod III (Fig. 12PlpIII) exopodite rounded triangular, about twice as long as wide, with almost straight medial margin. Medio-distal, terminal and latero-distal margins with 16 (15–20) long plumose setae, lateral margin with 22 (18–26) short delicate simple setae, medial surface with 3 (2–5) and lateral/terminal surfaces with 12 (10–20) short setae, respectively. Endopodite length about 0.6 of exopodite length. Pleopod IV (Fig. 12PlpIV) exopodite broadly ovoid, about 1.4 times as long as wide, its area equally shaped as in pleopod V. Lateral margin proximally with 8 (6–9) simple setae. Endopodite subrectangular, its length about 0.6 of exopodite length. Pleopod V (Fig. 12PlpV) exopodite ovoid, 1.3 (1.3–1.5) times as long as wide, lateral margin proximally with 3 (3–5) simple setae. Area small, its surface 67% (55–68%) of exopodite surface, linea areae beginning proximally on the lateral exopodite margin and ending at the transition from terminal to medial exopodite margin. Endopodite suboval, hardly shorter than exopodite. Uropod (Fig. 12U) length 33% (23–37%) of body length; proto-, endo- and exopodite length relations: 1:1.96 (1.8–2.5):2.02 (1.7–2.5). Endopodite (terminal margin excluded) with 23 (15–25) simple setae and 5 (4–8) penicilate setae, longest simple seta length 12% (9–16%) of endopodite length. Female paratypes. Body length 5.1–6.3 mm (2.7–3.1 of body width), almost identical to male except, antenna I length 17–22% of body length, with 6–8 flagellar articles; antenna II with about 41 flagellar articles. Pereopod IV not for grasping, ambulatory, little longer as the preceding pairs. Pleopod II (Fig. 12PlpII(F)) suboval, about 1.3 times as long as wide, with 14–17 long marginal plumose setae. Remarks. In its general appearance and pleopod structure A. kosswigi is similar to A. aquaticus sensu lato, resembling in particular the Slovenian subterranean A. a. cavernicolus populations from the cave Planinska jama (Ljubljanica River basin). This is mainly due to a lack of pigmentation, however, less immediately obvious morphological differences exist between both subterranean taxa. Some troglomorphic traits are less expressed in the new species. Antenna II of A. a. cavernicolus is longer than the body (even with three shaft articles excluded) with more than 75 (mainly more than 90) articles in the flagellum. The difference in the pleopod I protopodite being narrower (only 0.9–1.2 times as wide as long) in A. a. cavernicolus is also prominent, and the corresponding exopodite having a

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symmetric but well-expressed concavity with more than 5 setae (mainly 7–15), while in A. kosswigi the protopodite is wider and exopodite has hardly visible concavity (extremely shallow or non-existent) with only up to 5 simple setae (mainly 0–4). A. kosswigi has on an average less numerous and shorter simple setae on the appendages than A. a. cavernicolus (values in parenthesis): pleopod II exopodite 5–9 (8–14), uropod endopodite 15–25 (28–66), propodus IV superior margin and submarginal surface 6–12 (13–34), propodus VII inferior margin 7–13 (14–23) and superior margin and submarginal surface10–17 (21–56) simple setae, the longest robust seta on the inferior propodus VII margin reaching only to 8–13% (12–20%) propodus length. Etymology. The species is named after Curt Kosswig who first discovered asellids in the subterranean Reka/ Timavo and together with Leonore Kosswig used A. aquaticus as a model organism for the study of the evolution of cave animals (Kosswig and Kosswig 1940).

4. Phylogeny The Bayesian tree of the COI sequences (Fig. 2) displayed high structuring of haplotypes with several well-supported clades generally in agreement with their geographic distribution. The haplotypes of the neotype population from Uppsala (UP) were imbedded in the western and northern European clade. A. kosswigi was positioned sister to a clade from isolated karst basins in the northwestern part of the Dinaric Mountains. The support for this relationship, however, was low (0.83 posterior probability). These populations are morphologically highly diverse and include several troglomorphic cave populations (Fig. 1). The only major geographic disagreement was in the Reka/Timavo River where specimens belonged to two different clades in accordance with their morphological classification. In the resurgence of the Reka/Timavo River (S. Giovanni al Timavo/Sˇtivan – TM), where both troglomorphic and non-troglomorphic individuals occurred syntopically, two troglomorphic specimens shared the same haplotype (TM10), which belonged to the clade of the upstream troglomorphic population from the Trebiciano Cave (LB). The haplotypes of the non-troglomorphic specimens (TM1, TM4) belonged to the clade geographically covering the Adriatic drainages. As the nontroglomorphic and troglomorphic populations from Reka River do not share common haplotypes, and are in fact highly differentiated (D ¼ 0.11770.014), this could only be explained by lack of gene flow due to a reproductive barrier. The resulting tree of the nuclear 28S rDNA sequences (Fig. 3) agrees well with the COI phylogeny, again joining A. kosswigi with populations form Dinaric karst

river systems. Other populations of A. aquaticus have highly uniform 28S rDNA throughout the range (see also Verovnik et al. 2005 for explanation). In the sample from the resurgence of the Reka/Timavo River (S. Giovanni al Timavo/Sˇtivan – TM) one of the two troglomorphic specimens shared the same sequence (TM10) with upstream troglomorphic population from the Trebiciano Cave (LB) and the other (TM11) differed only in a single nucleotide position from them. The non-troglomorphic specimens form the same locality had sequences equal to the widely distributed EUR-CP sequence. Again, as the non-troglomorphic and troglomorphic populations from Reka River do not share common 28S rDNA sequences this can be seen as independent support for their status as distinct species.

5. Discussion As one of the most common and widespread freshwater crustaceans in Europe, A. aquaticus has been attracting the attention of the scientist for some time. Initial taxonomic (summarized in Gruner 1965) and reproductive strategy studies (van Emden 1922; Vandel 1926) were followed by chemical toxicity (de Nicola Giudici et al. 1987; Le Bras 1990), ecological (Rossi et al. 1983; Petridis 1990) and molecular studies (Di Castro et al., 1979; Pelliccia et al. 1991). Nowadays, two roles of this expansive macroinvertebrate are mainly stressed. A. aquaticus was found to be an important food item in the diet of birds and different fish species (Pliszka 1953; Rask and Hiisivuori 1985; Petridis 1990). In addition, due to its relative tolerance of a range of pollutants (primary scientific literature summarized in PAN Pesticides Database– Chemical Toxicity Studies on Aquatic Organisms: web address cited in the literature) it has been used as a bioindicator of water quality (Maltby 1991; Mulliss et al. 1994, 1996). Especially the co-occurrence of the amphipod Gammarus pulex and the isopod A. aquaticus has been shown to be a useful tool in the assessment of water quality (Whitehurst 1991). Speciation in subterranean aquatic crustaceans is well exemplified in the genus Asellus in Japan (Matsumoto 1963), and even more pronounced in other asellids like Proasellus and Caecidotea, and the amphipod genus Niphargus (Botosaneanu 1986). Apparently, the same process has only just started in Europe, which was invaded by A. aquaticus approximately 8–12 Myr ago (Verovnik et al. 2005). As the karstification and therefore the availability of the subtteranean aquatic habitats in the range of A. kosswigi sp. nov., started only 1–5 Myr ago (Mihevc 2001), the process may be regarded as relatively young. This is in accordance with the coalescence time estimate of 2.7–4.3 Myr (Verovnik

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et al. 2005) for the monophyletic group of haplotypes from isolated Dinaric karst basins which includes A. kosswigi. This explains the paraphyly of the COI haplotypes of A. aquaticus caused by erecting the new species. The paraphyly could be explained by incomplete lineage sorting (Avise 2000) or the presence of additional species in the isolated Dinaric karst basins. Although the latter idea seems tempting, we have not been able to convincingly exclude recent gene flow between these populations and populations from other parts of Europe in previous studies (Verovnik et al. 2003, 2004, 2005). On the other hand, evidence of the existence of a separate species of Asellus inhabiting the subterranean part of the Reka River, has accumulated continually with morphological (Prevorcˇnik et al. 2004), as well as molecular studies (Verovnik et al. 2003, 2004, 2005). The best possible and conclusive evidence confirming the species status of A. kosswigi, under any species concept, is the fact that the mixed sample from the springs of Timavo shows no traces of genetic exchange between the cave and the surface population, both for mitochondrial and nuclear genes. According to these genetic differences, a strong isolating mechanism has obviously been preventing gene flow between these two syntopic taxa. Caves have once again proved to be evolutionary laboratories which facilitate speciation. The key factor in speciation in caves is the sharp ecological boundary between subterranean and surface habitats that in most cases results in a fast differentiation of populations due to strong directional selection in the cave environment (Culver et al. 1995). The resulting troglomorphoses could act as the pre-mating barriers that prevent gene exchange in areas of secondary contact. The second reason is fragmentation of suitable subterranean aquatic habitats that act as islands (Culver et al. 1995). As there are currently no known pigmented surface populations upstream from the cave system in the Reka/Timavo River, we consider the syntopic occurrence of both species in the resurgence as a secondary contact, following a long period of isolation.

Acknowledgements We are particularly grateful to Boris Sket for the idea and proposition of describing the neotype for this important species, providing samples and for helpful comments on the manuscript. Peter Trontelj kindly contributed many valuable comments on the manuscript. We are thankful to Jozˇica Murko-Bulic´ for her help in the laboratory. We also thank Cene Fisˇer, Morten S. My´lgaard, Fabio Stoch, G. Magniez, B. Frajman, K. Sindemark, K. Larsson and P. Kloss for providing samples.

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