comparative karyotype investigations in the european

COMPARATIVE KARYOTYPE INVESTIGATIONS IN THE EUROPEAN CRAYFISH ASTACUS ASTACUS AND A. LEPTODACTYLUS (DECAPODA, ASTACIDAE) BY ´ 1 ), M. PAVLICA1 ), M. ŠRUT2 ), J. MLINAREC1,3 ), M. MUŽIC 2 ) and I. MAGUIRE2 ) ˇ G. KLOBUCAR 1 ) Faculty of Science, University of Zagreb, Division of Biology, Department of Molecular Biology, HR-10000 Zagreb, Croatia 2 ) Faculty of Science, University of Zagreb, Division of Biology, Department of Zoology, HR-10000 Zagreb, Croatia

ABSTRACT This study reports on the chromosome number and karyological characteristics of the endangered species of European crayfish, Astacus astacus and A. leptodactylus (Decapoda, Astacidae), both native to Croatian freshwater habitats. The karyotype of A. astacus and A. leptodactylus consists of 2n = 176 and 2n = 180 chromosomes, respectively. The haploid chromosome complement of A. astacus consists of 52 metacentric, 35 metacentric-submetacentric, and 1 acrocentric chromosomes. Fluorochrome staining with 4,6-diamino-2-phenylindole (DAPI) has revealed that the karyotypes of A. astacus and A. leptodactylus are characterized by large heterochromatic blocks located at centromeric and intercalary positions on the chromosomes. Interstitial heterochromatic blocks were more frequent in A. astacus than in A. leptodactylus. In both species pairing of chromosomes in meiosis was regular with the majority of bivalents in a ring- and a dumbbell-form. Fluorescence in situ hybridization (FISH) has revealed that two 45S rDNA loci were present in the investigated species. In A. astacus one of the two 45S rDNA-bearing chromosome pairs was highly heteromorphic, exhibiting a three-fold size difference between 45S rDNA sites on homologous chromosomes. Such a size difference was significantly less pronounced in A. leptodactylus. The karyotype differences between A. astacus and A. leptodactylus suggest changes in chromosome number as well as position of repetitive DNAs have played a role in the karyotype evolution of the species of Astacus.

RÉSUMÉ Cette étude rend compte du nombre de chromosomes et des caractéristiques caryologiques chez deux espèces d’écrevisse européennes menacées d’extinction, Astacus astacus et A. leptodactylus (Decapoda, Astacidae), toutes deux natives dans les habitats d’eau douce croates. Le caryotype de

3 ) e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2011 Also available online: www.brill.nl/cr

Crustaceana 84 (12-13): 1497-1510 DOI:10.1163/156854011X607015

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A. astacus et A. leptodactylus apparait constitué de 2n = 176 et 2n = 180 chromosomes, respectivement. Le complément de chromosomes haploïdes d’A. astacus consiste en 52 chromosomes métacentriques, 35 métacentrique-submétacentriques, et 1 acrocentrique. Une coloration fluorochrome par 4,6-diamino-2-phenylindole (DAPI) a révélé que les caryotypes de A. astacus et A. leptodactylus sont caractérisés par de larges portions hétérochromatiques localisées en position centromérique et intercalaires sur les chromosomes. Les portions hétérochromatiques interstitielles ont été plus fréquentes chez A. astacus que chez A. leptodactylus. Chez les deux espèces l’appariement des chromosomes à la méiose a été régulier avec une majorité de bivalents en forme d’anneau ou d’haltères. L’hybridisation in situ par fluorescence (FISH) a montré que deux loci 45S rDNA sont présents chez les espèces investiguées. Chez A. astacus une des deux paires de chromosomes porteurs de 45S rDNA est fortement hétéromorphique, montrant une différence de taille triple entre les sites 45S rDNA sur les chromosomes homologues. Une telle différence de taille a été significativement moins prononcée chez A. leptodactylus. Les différences de caryotype entre A. astacus and A. leptodactylus suggèrent que des changements dans le nombre de chromosomes ainsi que des positions des répétitions d’ADN ont joué un rôle dans l’évolution du caryotype des espèces d’Astacus.

INTRODUCTION

Crayfish constitute a non-monophyletic group of some 600 described species, arranged into three families under the infraorder Astacidea: Astacidae, Cambaridae, and Parastacidae. These species occur on all continents except Antarctica and Africa (present on Madagascar), with centres of diversity in the south-eastern United States and in Victoria (Australia) (Crandall & Buhay, 2008). There are five species of crayfish native to European fresh waters, all from the family Astacidae (cf. Holdich et al., 2009). Recently, all five species have undergone a decline in number and size of their populations. They are threatened by the presence of non-native crayfish, water quality impoverishment, and climate changes (Holdich et al., 2009). The native species belong to two genera, Astacus and Austropotamobius. The genus Astacus is represented by two species: Astacus leptodactylus (Eschscholtz, 1823) and A. astacus (Linnaeus, 1758). Both species are naturally distributed in freshwater habitats belonging to the Black Sea drainage, with the latter being introduced into a few localities of the Adriatic Sea drainage (Maguire & Gottstein Matoˇcec, 2004). Recent research revealed that more than 36% of the A. astacus populations in Croatia have disappeared (Maguire et al., 2011). The same study showed that A. leptodactylus is spreading naturally to the west and south of Croatia, while at the same time the non-native species Orconectes limosus (Rafinesque, 1817) is displacing it from its habitats in the far eastern part of Croatia (Hudina et al., 2009). For years, crustaceans were considered to be problematic for karyological and cytogenetic studies, as the majority of species have relatively high diploid chromosome numbers and relatively small chromosomes. Indeed, the crayfish Pacifastacus leniusculus trowbridgii (Dana, 1852) has a diploid number 2n = 376

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(Niiyama, 1962) and holds the record in chromosome number for Metazoa (cf. Lécher et al., 1995). The majority of crustaceans have been poorly investigated at a chromosomal level, or not at all. Most cytogenetic studies date back to the early years of classical research, largely to reveal only chromosome numbers (reviewed in Lécher et al., 1995). Although the number of species examined still remains low (less than 2% of the Crustacea), the amount of variation observed is surprisingly high. Within the crayfish, chromosome numbers appear to be extremely variable as based on available data, displaying a broad range from 2n = 102 in Procambarus digueti (Bouvier, 1897) (cf. Diupotex Chong et al., 1997) to 2n = 376 in P. leniusculus trowbridgii (cf. Niiyama, 1962). Crayfish chromosomes are also typically small and of similar size. For A. astacus, there is only one study reporting on its chromosome number and that was published at the beginning of the 20th century by Prowazek (1902) (cited from Fetzner & Crandal, 2002). According to that report, A. astacus would have a diploid chromosome number 2n = ca. 116. Afterwards, Silver & Cukerzis (1964) (cited from Fetzner & Crandal, 2002) and Cukerzis (1988) have reported that the diploid number of A. leptodactylus was determined as 2n = 368. Fluorochrome banding as well as fluorescence in situ hybridization (FISH) to nuclear ribosomal RNA (rRNA) genes, have been used in evolutionary and taxonomic studies, and in population genetics research (Maluszyńska & HeslopHarrison, 1993; Zoldos et al., 1999; Adams et al., 2000; Lim et al., 2006; Mlinarec et al., 2011). In higher eukaryotes, 45S rRNA genes are organized as multiple tandem repeats of coding units and intergenic spacers. The number of nuclear ribosomal DNA (rDNA) loci as well as the chromosomal distribution of heterochromatin can be used to characterize species (Maluszyńska et al., 1998; Weiss-Schneeweiss et al., 2008; Parraguez et al., 2009; Kavalco et al., 2011). However, such modern cytogenetic approaches have not hitherto been used to investigate karyotype diversification and evolution in crustaceans. There is no information regarding 45S rDNA localization for any species within the family Astacidae. We show here that such analyses are valuable in distinguishing between the uniformly small size of chromosomes in the family Astacidae. Here we examine the European crayfish species A. astacus and A. leptodactylus with the aim to: (i) re-examine karyotypes and chromosome numbers, (ii) investigate male meiosis, and (iii) determine the chromosomal distribution of 45S rDNA and heterochromatin. This study thus contributes to understanding of patterns of evolution in the family Astacidae, including the first physical map of any DNA sequences to chromosomes of the genus Astacus.

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MATERIAL AND METHODS

Two male adults of each species, Astacus astacus and A. leptodactylus, of 34.234.5 g and 63.1-87.7 g body weight, respectively, were used for the analyses. Specimens of A. astacus were sampled from the Trećak Stream (Slavonia, Croatia), and A. leptodactylus from the Mrežnica River (Duga Resa, Croatia). Chromosome preparation The mitotic inhibitor colchicine (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was used in a dose adjusted to the crayfish’s body weight (2 μg g−1 ) by slow injection into the peritoneal cavity at the base of the first pair of pleopods in the mature males. The testes were then excised after 5-6 h of pre-treatment, sliced into 1-2 mm segments, and immersed into a hypotonic solution of 0.1 M KCl for 45 minutes at 25◦ C. The tissue was fixed in Carnoy fixative (3 : 1 v/v, methanol : acetic acid) for 40 min. This was replaced with a fresh fixative for another 60 min. and then immersed in 1 : 1 (v/v), methanol : acetic acid Carnoy fixative for 24 h at 4◦ C. The fixed testis tissue was incubated in 50% glacial acetic acid for 1 h followed by maceration in 1 : 1 (v/v), methanol acetic acid Carnoy fixative. A coverslip was removed from the slide by freezing the slide on solid carbon dioxide (Sharma & Sharma, 1972). Then the slides were air-dried at room temperature for 24 h. Fluorochrome staining Fluorescent banding with 4,6-diamino-2-phenylindole (DAPI) allows localization of AT-rich blocks of heterochromatin. Chromosome preparations were first incubated in McIlvaine’s citric acid-Na2 HPO4 buffer (pH = 7) (Schweizer & Ambros, 1994) for 20 min. at room temperature (RT) followed by staining with (DAPI) (2 μg ml−1 ) for 20 min. at RT. After staining, slides were rinsed with McIlvaine buffer (pH = 7) and mounted in an anti-fade solution (Dako Corp.). The images were examined and photographs were taken with an Olympus BX51 microscope, equipped with a digital camera (Olympus DP70). Construction of karyotype Photographs of the chromosomes were taken at 600× magnification and the images processed using Adobe Photoshop 6.0. Chromosomes were measured on three well-spread metaphase plates using the Adobe Photoshop 6.0 measurement tools. Chromosome pairs were classified following the nomenclature of Levan et al. (1964) into: m = metacentric (arm ratio (r) = 1-1.7), sm = submetacentric (r = 1.7-3), t = telocentric (r = 3-7), and a = acrocentric (r = >7). The karyotype was constructed by grouping the chromosome pairs into classes based on their arm


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ratios and then arranging the chromosomes in each group in decreasing order of size. A heteromorphic chromosome pair was placed separately in the last row. Fluorescence in situ hybridization (FISH) FISH experiments were conducted according to Mlinarec et al. (2006) with minor modifications. The position and number of 45S rDNA sites were determined using the 2.4 kb HindIII fragment of the entire 18S rRNA-coding sequence, a component of 45S rRNA previously cloned into pUC19 as a probe (Torres-Ruiz & Hemleben, 1994). The probe was directly labelled with FITC-dUTP (Roche Diagnostics GmbH, Mannheim, Germany) by using a nick-translation kit according to the manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). The chromosome preparations were pre-treated with RNaseA (100 μg ml−1 ; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) followed by fixation with 4% (w/v) paraformaldehyde in water (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The hybridization mixture (40 μl) containing 50% (v/v) formamide, 10% (w/v) dextran sulphate, 0.6% (w/v) sodium dodecyl sulphate, in 2 × SSC and 2 ng μl−1 of labelled probe was denatured at 76◦ C for 15 min. Chromosome preparations were denatured at 73◦ C for 5 min. after applying the hybridization mix. Hybridization was carried out at 37◦ C overnight. Stringent washes were performed at 42◦ C in the following solutions: 2 × SSC, 0.1 × SSC, 2 × SSC and 4 × SSC/Tween20 (5 min. each). The preparations were mounted in antifade buffer Vectashield (Vector Laboratories, Peterborough, U.K.) containing DAPI (2 μg ml−1 ) and stored at 4◦ C. Images were examined and photographs were taken with an Olympus BX51 microscope, equipped with the digital camera Olympus DP70, again at a magnification of 600×. The images were merged and contrasted using Adobe Photoshop 6.0. An average of 35 well-spread metaphases was analysed for each species.

RESULTS

Scoring 35 well-spread male metaphase I cells revealed that the number of bivalents in Astacus astacus varied from 82 to 92, with a mode at 88 bivalents in 40% of the cells (fig. 1). In A. leptodactylus, the number of bivalents varied from 82 to 94 with a mode at 90 bivalents in 35% of the cells (fig. 2). In A. astacus, analyses of three well-spread spermatogonial mitotic cells are consistent with a haploid chromosome number n = 88, consisting of 52 metacentric, 35 submetacentric, and 1 acrocentric chromosomes (fig. 3, n = 88, 52 m + 35 sm + 1 a), with chromosome lengths varying from 1.7 to 6.2 μm. It was not

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Fig. 1. Distribution of chromosome numbers recorded in 35 haploid metaphases of male Astacus astacus (Linnaeus, 1758).

possible to analyse the mitotic chromosomes of A. leptodactylus because of the quality of the spreads and the scarcity of material. In A. astacus, centromeric AT-rich blocks were observed on all chromosomes, while interstitial AT-rich blocks were observed primarily on on the larger chromosomes (figs. 3, 4a). Six out of 52 pairs of metacentric chromosomes and 18 of 35 pairs of submetacentric chromosomes exhibited an interstitial AT-rich block on their longer arm (fig. 3, arrows). AT-rich blocks were of a different size and intensity, allowing us to distinguish individual chromosomes. In A. astacus, one

Fig. 2. Distribution of chromosome numbers recorded in 35 haploid metaphases of male Astacus leptodactylus (Eschscholtz, 1823).


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Fig. 3. Karyotype of Astacus astacus (Linnaeus, 1758) with 2n = 176 after DAPI staining. m, metacentric chromosomes; sm, submetacentric; and a, acrocentric chromosomes. Arrows show DAPI blocks on the long arms of chromosomes. Scale bar = 10 μm.

pair of homologous chromosomes was almost entirely heterochromatic (fig. 4a, arrows) and in meiosis they formed a bivalent in the form of a dumbbell that segregated regularly (fig. 4c, arrow). We were not able to get good-quality images of spermatogonial mitosis of A. leptodactylus because of the scarcity of material. Nevertheless, from the meiotic I metaphases and mitotic cells (data not shown) it was clear that A. leptodactylus has distinctive centromeric AT-rich blocks positioned on all chromosomes and interstitial AT-rich blocks on some chromosomes, as observed in A. astacus. However, in contrast to A. astacus, which only has inter-

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Fig. 4. a, Mitotic metaphase; and, c, meiotic metaphase I of Astacus astacus (Linnaeus, 1758); and, e, meiotic metaphase I of A. leptodactylus (Eschscholtz, 1823) after staining with DAPI; heterochromatic chromosome pair of A. astacus and corresponding bivalent marked with arrows (a, c); bivalents of A. leptodactylus displaying interstitial AT-rich blocks are marked with arrows (e). FISH localization of 45S rDNA (green signals) on, b, mitotic metaphase; and, d, meiotic metaphase I of A. astacus and, f, meiotic metaphase I of A. leptodactylus; additional 45S rDNA signal in A. leptodactylus is marked with arrow (f). g, 45S rDNA bearing chromosomes and 45S rDNA bearing bivalents of A. astacus after staining with DAPI (left) and after FISH with 45S rDNA as a probe (right). h, 45S rDNA bearing bivalents of A. leptodactylus after staining with DAPI (left) and after FISH with 45S rDNA as a probe (right); 45S rDNA bearing heteromorphic chromosome pair and corresponding bivalent of A. astacus and A. leptodactylus are framed. Scale bars = 10 μm in every picture.

stitial AT-rich blocks on most large chromosomes (fig. 3, arrows), in A. leptodactylus these sites were observed on only six pairs of chromosomes (fig. 4e, arrows). Telomeric AT-rich heterochromatic blocks were not observed either in A. astacus or A. leptodactylus.


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At metaphases I of both A. astacus and A. leptodactilus, bivalents formed different configurations: ring-, cross-, v-, and dumbbell forms. The dumbbell configuration is typical of crustaceans (Niiyama, 1959) and is derived from endby-end pairing. In A. astacus and A. leptodactylus, the majority of bivalents were in dumbbell- and ring-configurations, while cross- and v-configurations were significantly less prevalent. Detailed analyses of seven metaphases I of A. astacus and A. leptodactylus showed that in A. astacus out of 88 bivalents, 41 formed dumbbell-, 38 ring-, 7 v-, and 2 cross-configurations (fig. 4c), while in A. leptodactylus out of 90 bivalents 55 formed dumbbell-, 30 ring-, 3 v-, and 2 cross-configurations (fig. 4e). FISH experiments with the 45S rDNA probe on mitotic chromosomes of A. astacus revealed four sites of probe hybridization (fig. 4b). One 45S rDNAbearing homologous pair was highly heteromorphic in size, morphology, and size and intensity of FISH signal: one of the pair was amongst the largest of the complement, while the other was amongst the smallest, being only one-third the size of its homologue (fig. 4g, upper row, framed). The long arm of the larger of these acrocentric chromosomes was entirely labelled with the 45S rDNA probe. Likewise, the long arm of its smaller homologue was similarly labelled, although the total size of the locus was one-third of its homologous locus. An additional 45S rDNA locus was positioned on the short arms of a metacentric chromosome pair, where the entire chromosome arm was decorated with probe signal (fig. 4g, upper row, out of frame). This distribution of 45S rDNA was observed also at metaphase I (fig. 4d), where probe signals to the four 45S rDNA sites were observed on two bivalents that had a dumbbell morphology. The heteromorphic chromosome pair formed a bivalent with 45S rDNA signal on one end, being three times the intensity and size of the signal on the opposite end (fig. 4g, lower row, framed). Such pattern was constantly observed in 35 spermatogonial mitoses and meiotic metaphases I of two A. astacus males. FISH experiments with the 45S rDNA probe on meiotic metaphases I of A. leptodactylus revealed four major FITC-green signals, corresponding to four sites of 45S rDNA (fig. 4f); and one additional 45S rDNA signal of significantly smaller size and intensity (fig. 4f, arrow). The four major 45S rDNA sites were positioned on bivalents of a dumbbell-form, as in A. astacus, while the fifth 45S rDNA site was positioned on one end of another bivalent (fig. 4h). In A. leptodactylus, the heteromorphic 45S rDNA-bearing chromosome pair was less heteromorphic than observed in A. astacus, displaying only a two-fold difference in the size of the 45S rDNA sites between homologues (fig. 4h, framed). This pattern was observed in 35 spermatogonial mitoses and metaphase I cells of the two A. leptodactylus males.

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DISCUSSION

Karyotype divergence The diploid chromosome numbers obtained in this study for Astacus astacus (2n = 176) and A. leptodactylus (2n = 180) are inconsistent with previous counts, being one-and-half times higher than previously reported for A. astacus (cf. Prowazek, 1902) and less than half the number previously reported for A. leptodactylus (cf. Silver & Cukerzis, 1964). Several hypotheses could explain the discrepancies. Microscopy has developed over the last century, and today chromosome counts can be done faster and more accurately. Furthermore, the gonad tissues consist of cells of different ploidy levels, potentially causing confusion. To further test these hypotheses, more studies are needed, including from a larger number of individuals. However, the chromosome numbers reported here fall within the range of published chromosome counts in members of the other two families of Astacidea (Cambaridae and Parastacidae) such as Orconectes virilis (Hagen, 1870) (2n = 200; Fasten, 1914), O. immunis (Hagen, 1980) (2n = 208; Fasten, 1914), Cambaroides japonicus (De Haan, 1841) (2n = 196; Niiyama, 1934), Procambarus clarkii (Girard, 1852) (2n = 188; Murofushi et al., 1984), P. digueti (Bouvier, 1897) (2n = 102; Diupotex Chong et al., 1997), Cherax quadricarinatus (Von Martens, 1868) (2n = 200; Tan et al., 2004), and C. destructor (Clark, 1936) (2n = 188; Scalici et al., 2010). The variations in chromosome numbers indicate that karyotype divergence has played a role in the evolution of crayfish. The diploid chromosome number of Pacifastacus leniusculus trowbridgii (Dana, 1852) (2n = 376; Niiyama, 1962), in the family Astacidae, is approximately twice the diploid chromosome number in the sister genus Astacus. Potentially, polyploidy has played a role, as previously suggested by Lécher et al. (1995). Further studies involving molecular cytogenetic methods across a wide range of species are needed to test this hypothesis. Divergence in distribution of 45S rDNA In A. astacus, one of the two 45S rDNA-bearing chromosome pairs was highly heteromorphic in length and size of the 45S rDNA locus: one of the homologues had a locus three times as large as that of the other. The variation in the size of the 45S rDNA locus probably occurred as a result of unequal crossing over between homologues. The polymorphism did not, however, seem to influence chromosome pairing and segregation in meiosis. Polymorphism in 45S rDNA locus size has been reported for many species, including in amphibians (Schmidt, 1982), salmonid fishes (Reed & Philips, 1997), mammals (Hirai et al., 1998), and


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marine catfish (Sczepanski et al., 2010). Foresti et al. (1981) reported a six-fold size difference between 45S rDNA sites of homologous chromosomes in some species of Eigenmannia (Teleostei, Sternopygidae). However, this is the first report on polymorphism in a 45S rDNA locus size in Crustacea Decapoda. Divergence in distribution of AT-rich heterochromatin At cytogenetic level, A. astacus and A. leptodactylus show considerable differences in diploid chromosome numbers and distribution of AT-rich heterochromatin. However, both species have substantial blocks of AT-rich chromatin, suggesting that their genomes are composed of a large amount of repetitive, satellite DNA. Satellite DNA is considered an important source of genetic variability between species (Lim et al., 2000; Mravinac & Plohl, 2010; Sarri et al., 2011). Given the likely role of satellite DNA in maintaining chromosome structure, pairing of chromosomes at meiosis and the organization of chromosomes at interphase, it would be valuable to isolate and characterize the repetitive sequences from the genomes of A. astacus and A. leptodactylus and use them for evolutionary studies in Astacidae.

CONCLUSION

The divergence of chromosomes and karyotypes observed between Astacus astacus and A. leptodactylus probably contributes to a lack of success in crossing experiments. Furrer et al. (1998) revealed that hybrid offspring between A. astacus males and A. leptodactylus females die two months after hatching and so never reach reproductive age. In the reciprocal cross, A. astacus females discard eggs after breeding with A. leptodactylus males. As far as we know, there are no valid reports of the existence of natural hybrid populations between A. astacus and A. leptodactylus.

ACKNOWLEDGEMENTS

We thank A. R. Leitch, V. Zoldoš, V. Besendorfer, and anonymous reviewers for valuable suggestions during the preparation of the manuscript. This work was funded by the Ministry of Science, Education and Sport of the Republic of Croatia, grants no. 119-0000000-3167, 119-0982934-3110, and 119-1191196-1201.

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REFERENCES A DAMS , S. P., I. J. L EITCH , M. D. B ENNETT, M. W. C HASE & A. R. L EITCH, 2000. Ribosomal DNA evolution and phylogeny in Aloe (Asphodelaceae). American Journ. Bot., 87(11): 15781583. C RANDALL, K. A. & J. E. B UHAY, 2008. Global diversity of crayfish (Astacidae, Cambaridae, and Parastacidae — Decapoda) in freshwater. Hydrobiologia, 595: 295-301. C UKERZIS, J. M., 1988. Astacus astacus in Europe. In: D. M. H OLDICH & R. S. L OWERY (eds.), Freshwater crayfish: biology, management and exploitation: 309-340. (Cambridge University Press, Cambridge). D IUPOTEX C HONG, M. E., N. R. F OSTER & C. A. Z ARATE, 1997. A cytogenetic study of the crayfish Procambarus digueti (Bouvier, 1897) (Decapoda Cambaridae) from lake Camecuaro, Michoacan, Mexico. Crustaceana, 70: 875-885. FASTEN, N., 1914. Spermatogenesis of the American crayfish, Cambarus virilis and C. immunis (?), with special reference to synapsis and the chromatoid bodies. Journ. Morphol., 25: 587-694. F ETZNER, J. W., J R . & K. A. C RANDALL, 2002. Genetic variation. In: D. M. H OLDICH (ed.), Biology of freshwater crayfish: 291-326. (Blackwell Science Ltd., Oxford). F ORESTI, F., L. F. A LMEIDA T OLEDO & S. A. F. T OLEDO, 1981. Polymorphic nature of nucleolus organizer regions in fishes. Cytogenet. Cell Genet., 31: 137-144. F URRER, S. C., M. C ANTIENI & N. D UVOISIN, 1998. Freshly hatched hybrids between Astacus astacus and Astacus leptodactylus differ in chela shape from purebred offspring. Freshwater Crayfish, 12: 90-97. H IRAI, H., Y. H ASEGAWA, Y. K AWAMOTO & E. T OKITA, 1998. Tandem duplication of nucleolus organizer region (NOR) in the Japanese macaque, Macaca fuscata fuscata. Chromosome Res., 6: 191-197. H OLDICH, D. M., J. D. R EYNOLDS, C. S OUTY-G ROSSET & P. J. S IBLEY, 2009. A review of the ever increasing threat to European crayfish from non-indigenous crayfish species. Knowl. Managt. Aquatic Ecosyst., 11: 394-395. ˇ H UDINA, S., M. FALLER, A. L UCI C´ , G. K LOBU CAR & I. M AGUIRE, 2009. Distribution and dispersal of two invasive crayfish species in the Drava River basin, Croatia. Knowl. Managt. Aquatic Ecosyst., 9: 394-395. K AVALCO, K. F., R. PAZZA, K. D. B RANDAO, C. G ARCIA & L. F. A LMEIDA -T OLEDO, 2011. Comparative cytogenetics and molecular phylogeography in the group Astyanax Altiparanae — Astyanax aff. bimaculatus (Teleostei, Characidae). Cytogenet Genome Res., 134(2): 108119. L ÉCHER, P., D. D EFAYE & P. N OEL, 1995. Chromosomes and nuclear DNA of Crustacea. Invertebr. Reprod. Dev., 27: 85-114. L EVAN, A., K. F REDGA & A. S ANDBERG, 1964. Nomenclature for centromeric position of chromosomes. Hereditas, 52: 201-220. L IM, K. Y., A. KOVARIK, R. M ATYASEK, M. W. C HASE, S. K NAPP, E. M C C ARTHY, J. J. C LARK SON & A. R. L EITCH , 2006. Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae. Plant Journ., 48: 907-919. L IM, K. Y., R. M ATYÁŠEK, C. P. L ICHTENSTEIN & A. R. L EITCH, 2000. Molecular cytogenetic analyses and phylogenetic studies in the Nicotiana section Tomentosae. Chromosoma, 109: 245-258. ˇ M AGUIRE, I. & S. G OTTSTEIN M ATO CEC , 2004. The distribution pattern of freshwater crayfish in Croatia. Crustaceana, 77(1): 25-49. ˇ M AGUIRE, I., M. J ELI C´ & G. K LOBU CAR , 2011. Update on the distribution of freshwater crayfish in Croatia. Knowl. Managt. Aquatic Ecosyst., 31: 401. ´ M ALUSZY NSKA , J., R. H ASTEROK & J. S. H ESLOP -H ARRISON, 1993. Physical mapping of rDNA loci in Brassica species. Genome, 36: 774-781.


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´ M ALUSZY NSKA , J., R. H ASTEROK & H. W EISS, 1998. rRNA genes — their distribution and activity ´ (ed.), Plant cytogenetics: 75-95. (Silesian University Press, in plants. In: J. M ALUSZY NSKA Katowice). M LINAREC, J., D. PAPEŠ & V. B ESENDORFER, 2006. Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis. Bot. Journ. Linn. Soc., London, 150: 177-186. M LINAREC , J., Z. Š ATOVI C´ , D. M IHELJ , N. M ALENICA & V. B ESENDORFER, 2011. Cytogenetic and phylogenetic studies of diploid and polyploid members of tribe Anemoninae (Ranunculaceae). Plant Biol., doi: 10.1111/j.1438-8677.2011.00519.x M RAVINAC, B. & M. P LOHL, 2010. Parallelism in evolution of highly repetitive DNAs in sibling species. Mol. Biol. Evol., 27(8): 1857-1867. M UROFUSHI, M., Y. D EGUCHI & H. YOSHIDA, 1984. Karyological study of the red swamp crayfish and the Japanese lobster by air-drying method. Proc. Japanese Acad., (B) 60: 306-309. N IIYAMA, H., 1934. The chromosomes of crayfish Cambarus japonicus (de Haan). Journ. Fac. Sci., Hokkaido Univ., (0) 3: 41-53. — —, 1937. The problem of male heterogamety in the decapod Crustacea, with special reference to the sex chromosomes in Plagusia dentipes De Haan. Journ. Fac. Sci. Hokkaido Univ., (0) 6: 283-295. — —, 1959. A comparative study of the chromosomes in decapods, isopods and amphipods, with some remarks on cytotaxonomy and sex-determination in the Crustacea. Mem. Fac. Fish. Hokkaido Univ., 7: 1-60. — —, 1962. On the unprecedently large number of chromosomes of the crayfish Astacus trowbridgii Stimpson. Annot. zool. Japonenses, 35: 229-233. PARRAGUEZ, M., G. G AJARDO & J. A. B EARDMORE, 2009. The New World Artemia species A. franciscana and A. persimilis are highly differentiated for chromosome size and heterochromatin content. Hereditas, 146(2): 93-103. P ROWAZEK, S., 1902. Ein Beitrag zur Krebsspermatogenese. Zeitschr. wiss. Zool., 71: 445-456. R EED, K. M. & R. B. P HILIPS, 1997. Polymorphism of the nucleolus organizer region (NOR) on the putative sex chromosomes of Arctic char (Salvelinus alpinus) is not sex related. Chromosome Res., 5: 221-227. S ARRI, V., M. C ECCARELLI & P. G. C IONINI, 2011. Quantitative evolution of transposable and satellite DNA sequences in Picea species. Genome, 54(5): 431-435. S CALICI, M., E. S OLANO & G. G IBERTINI, 2010. Karyological analyses on the Australian crayfish Cherax destructor (Decapoda: Parastacidae). Journ. Crust. Biol., 30: 528-530. S CHMIDT, M., 1982. Chromosome banding in Amphibia. VII. Analysis of the structure and variability of NORs in Anura. Chromosoma, 87: 327-344. S CHWEIZER , D. & P. F. A MBROS, 1994. Chromosome banding. In: J. R. G OSDEN (ed.), Methods in molecular biology, 29, Chromosome analysis rotocols. (Humana Press Inc., Totowa, NJ). S CZEPANSKI, T. S., R. B. N OLETO, M. M. C ESTARI & R. F. A RTONI, 2010. A comparative study of two marine catfish (Siluriformes, Ariidae): cytogenetic tools for determining cytotaxonomy and karyotype evolution. Micron, 41: 193-197. S HARMA, A. K. & A. S HARMA, 1972. Chromosome techniques — theory and practice: 1-000. (Butterworth Publishers Ltd., London). S ILVER, D. & J. M. C UKERZIS, 1964. The number of chromosomes in Astacus leptodyctylus. Tsitologiya, 6: 631-633. TAN, X., J. G. Q IN, B. C HEN, L. C HEN & X. L I, 2004. Karyological analyses on redclaw crayfish Cherax quadricarinatus (Decapoda: Parastacidae). Aquaculture, 234: 65-76. T ORRES -RUIZ, R. A. & V. H EMLEBEN, 1994. Pattern and degree of methylation in ribosomal RNA genes of Cucurbita pepo L. Plant mol. Biol., 26: 1167-1179.

1510

J. MLINAREC ET AL.

W EISS -S CHNEEWEISS, H., K. T REMETSBERGER, G. M. S CHNEEWIESS, J. S. PARKER & T. F. S TUESSY, 2008. Karyotype diversification and evolution in diploid and polyploid South American Hypochaeris (Asteraceae) inferred from rDNA localization and genetic fingerprint data. Ann. Bot., 101: 909-918. Z OLDOS, V., D. PAPES, M. C ERBAH, O. PANAUD, V. B ESENDORFER & S. S ILJAK -YAKOVLEV, 1999. Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species. Theor. appl. Genet., 99: 969-977.

First received 8 January 2011. Final version accepted 26 August 2011.