ARTICLE IN PRESS Organisms, Diversity & Evolution 8 (2008) 337–345 www.elsevier.de/ode A cytogenetical study of Ischyroceridae (Amphipoda) allows the identification of a new species, Jassa cadetta sp. n., in the Lagoon of Venice Traudl Krappa, Massimiliano Rampinb, Angelo Libertinib,Ã aForschungsinstitut Museum A. Koenig, Adenauerallee 160, 53113 Bonn, Germany bCNR-Institute of Marine Science, Riva 7 Martiri 1364/A, 30122 Venezia, Italy Received 20 November 2007; accepted 16 June 2008 Abstract Jassa cadetta sp. n. (Amphipoda: Ischyroceridae) is described from the Venice Lagoon, northern Adriatic Sea, and a key to Mediterranean members of the genus Jassa Leach is provided. The new species is separated from J. marmorata Holmes primarily by cytogenetics, differing in chromosome number (2n ¼ 10 in J. cadetta vs. 2n ¼ 12 in J. marmorata), karyotype morphology (FN ¼ 20 vs. FN ¼ 22), and chromosome location of 18S-5.8S-28S ribosomal cistrons. Cytogenetic analysis of Ischyrocerus anguipes Krøyer (2n ¼ 10, FN ¼ 18) gives a first insight into karyological diversity among Ischyroceridae. Analysis of random amplified polymorphic DNA markers confirms the distinction between J. cadetta sp. n. and J. marmorata. r 2008 Gesellschaft fu¨ r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. Keywords: Amphipoda; Northern Adriatic Sea; Cytogenetics; PCR-RAPD; New species Introduction examines early embryos as material for karyotyping (Libertini et al. 2000); the body of the corresponding Libertini et al. (2000) published a monograph on the ovigerous female is usually preserved for species cytogenetics of Jassa marmorata Holmes, 1903 from the identification. One female from the Lido mouth, Venice Lagoon. The population was characterized by a identified as J. marmorata, possessed a karyotype clearly karyotype of 2n ¼ 12 chromosomes, with 5 pairs of different from that outlined above. This discrepancy was metacentrics and 1 pair of subtelocentrics. These data initially explained as a confusion in labeling of the were consistent with those from an Atlantic population female, because many other J. marmorata specimens of the species in which haploid and diploid chromosome with a ‘regular’ karyotype were found in the same numbers had been found to be 6 and 12, respectively sample. (Coleman 1994). In subsequent years, other fouling samples from the A fouling sample was collected from wooden piles at deepest part of blue mussel cultivation socks in the the northern mouth of the Venice Lagoon (Lido mouth) central mouth of the Venice Lagoon (Malamocco in 2000 and studied cytogenetically. Our lab regularly mouth) contained many Jassa specimens with a karyo- type different from that of J. marmorata, compelling ÃCorresponding author. Tel.: +39 041 2404711; the authors to propose the presence of another fax: +39 041 5204126. species, recently recognized as new, and described E-mail address: [email protected] (A. Libertini). below. 1439-6092/$ - see front matter r 2008 Gesellschaft fu¨ r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ode.2008.06.001 ARTICLE IN PRESS 338 T. Krapp et al. / Organisms, Diversity & Evolution 8 (2008) 337–345 The present paper reports on a genetic comparison room temperature (RT), once (5 min) in 2 Â SSC/0.1% between the two Jassa species from the Venice Lagoon, Tween20 at RT, and once (5 min) in PBS/0.1% based on karyotypes and random amplified poly- Tween/0.5% skimmed milk powder at RT. Hybridi- morphic DNA (PCR-RAPD), and provides the descrip- zation signals were detected with FITC-conjugated tion of Jassa cadetta sp. n. The cytogenetic comparison antidigoxigenin (Roche Molecular Biochemicals) or Cy3- is extended to Ischyrocerus anguipes Krøyer, 1838, conjugated extravidin (Sigma). Slides were mounted in considered here as an outgroup belonging to the same AF1 antifade solution (Citifluor) containing 2 mg/ml tribe, Ischyrocerini, according to the classification DAPI (40,6-diamidino-2-phenylindole). scheme by Myers and Lowry (2003). Observations were made with a JenaMed 2-fluores- cence microscope (Carl Zeiss Jena, Germany) equipped with the 410/450 and the 510/570 filter sets. Normal light and fluorescence images were taken with a Canon Material and methods EOS 10D digital camera, processed and merged with Adobe Photoshop Elements 2.0. The Jassa specimens used in this study were collected For PCR-RAPD analysis, genomic DNA was ex- at different sites in the Venice Lagoon (NE Italy). tracted and purified from 10 males of J. marmorata and Ischyrocerus anguipes was collected along the shores 6 males of J. cadetta sp. n. with the GenElute near the Sandgerji Marine Centre of the University of Mammalian Genomic Miniprep kit (SIGMA). The Iceland (SW Iceland), during low tide. PCR-RAPD protocol was performed following Costa Chromosome preparations were made with the et al. (2004) with slight modifications, using the PCR hot-dry method applied to early embryos as described ReadyMix kit (Sigma). PCR reactions were performed by Libertini et al. (2000). The body of the mother in a final volume of 25 ml, containing 0.2 mM each of carrying the embryos was preserved in 70% ethanol for dNTP, 0.75 U of Taq DNA polymerase, 1 Â PCR buffer identification. (20 mM Tris–HCl, pH 8.3; 50 mM KCl), 2.5 mM MgCl2, For conventional karyotyping, slides were stained 0.8 mM primer, and 20 ng template DNA. Primers were for 10 min in 5% Giemsa solution in phosphate buffer chosen from those successfully used by Costa et al. (pH 6.8). At least 100 chromosome plates for each (2004) for Gammarus locusta; their respective names species were counted to determine the diploid chromo- and sequences were (50-to-30): A2 (TGCCGAGCTG), some number. Karyotypes were arranged according to A9 (GGGTAACGCC), A10 (GTGATCGCAG), A16 chromosome size and shape using digitized pictures of (AGCCAGCGAA), D2 (GGACCCAACC), D3 (GTC metaphase figures edited using Corel PhotoPaint. GCCGTCA), D5 (TGAGCGGACA), D7 (TTGGCA Chromosome classification is according to Levan et al. CGGG). All primers were purchased from MWG (1964); the centromeric index was evaluated following Biotech. Cycling conditions consisted of initial dena- Naranjo et al. (1983). At least 20 karyotypes were turation at 95 1C for 5 min, followed by 40 amplification examined for each species. cycles (denaturation at 93 1C for 40 s, annealing at 37 1C The chromosomal localization of major (18S-5.8S- for 60 s, extension at 72 1C for 60 s), and by a final 28S) and minor (5S) rDNA sequences was performed by extension for 6 min at 72 1C. DNA was then precipitated means of fluorescent in situ hybridization (FISH). from the aqueous phase with ethanol and stored at A probe containing 18S-5.8S-28S genes plus intergenic À20 1C for at least 2 h or overnight. The samples spacer of the fruit fly Drosophila melanogaster (pDm238; were then washed in 70% ethanol. The amplification Roiha et al. 1981) was used for major rDNA FISH. As products were maintained at 4 1C until being loaded the probe for 5S rDNA FISH, genomic DNA of onto the gels. Electrophoresis was conducted on J. marmorata was amplified by PCR, using two primers 2.5% agarose gels in TAE buffer (40 mM Tris, (50-GAAAGCACCACTTCTCGTCC-30 and 50-AACG pH 7.6; 20 mM acetic acid, 1 mM EDTA), run at 90 V TGGTATGGCCGTTGAC-30) obtained from the 5S for 2.5 h or for 7 cm. The gels were stained in an rRNA sequence of the isopod Proasellus coxalis ethidium bromide solution (30 mg/l), then photo- (Dollfus, 1892) (Pelliccia et al. 1998). Probes were graphed under UV light with a Canon EOS 10D digital labeled by nick translation with digoxigenin-11-dUTP camera. A molecular size standard consisting of or biotin-14-dATP (Roche Molecular Biochemicals or 50–3000 bp ‘‘direct load’’ ladder (Sigma) was run in Invitrogen). lanes flanking the samples, and a negative control in Chromosomes were denatured for 4 min in 70% each gel. formamide/2 Â SSC at 69 1C. Denaturation of the For the morphological description of Jassa cadetta probes was performed for 10 min at 75 1C. Hybridiza- sp. n., the typical procedure for amphipods was used: direct tion was allowed to proceed overnight at 37 1C. Slides observation of specimens in alcohol and glycerine under were washed three times in 50% formamide/2 Â SSC Reichert and Wild M5 dissecting microscopes; dissec- at 40 1C (5 min each), twice (5 min) in 2 Â SSC at tion, slide preparation (and storage) in glycerine or ARTICLE IN PRESS T. Krapp et al. / Organisms, Diversity & Evolution 8 (2008) 337–345 339 Faure’s medium; then drawing under a Wild M20 et al. 2000; shown in Fig. 2A, for colour version see the microscope. online edition of this paper) and in I. anguipes (Fig. 2C). In J. cadetta with 2n ¼ 10, 18S-5.8S-28S rDNA cistrons were located at the end of the shorter arm in the second- Results and discussion longest metacentric pair (Fig. 2B). Therefore, in I. anguipes and J. cadetta nucleolus organizer regions (NORs) correspond to the secondary constrictions Genetics previously observed in Giemsa-stained chromosomes. The 5S rDNA probes hybridized in an intercalate According to Libertini et al. (2000), J. marmorata region of the longest arm of an average-sized metacentric from the Venice Lagoon has a karyotype (Fig. 1A) pair in all three species studied (Fig. 2D–F). In 6 of 7 composed of 5 pairs of metacentric chromosomes examined metaphase figures of I. anguipes an additional (pairs 1–5) and 1 pair of subtelocentrics (pair 6) much metacentric chromosome had positive intercalate FISH shorter than the other elements. The diploid chromo- signals (Fig. 2D–F). 18S-5.8S-28S rDNA and 5S rDNA some number (2n) is 12, the fundamental number of cistrons are localized in two different chromosome pairs in chromosome arms (FN) is 22.