Carassius Ve Střední Evropě

MASARYKOVA UNIVERZITA Přírodovědecká fakulta

Ivo PAPOUŠEK

MOLEKULÁRNĚ-GENETICKÉ ANALÝZY DRUHŮ RODU CARASSIUS VE STŘEDNÍ EVROPĚ

Přílohy k disertační práci

Školitel: prof. RNDr. Jiřina Relichová, CSc. Školitel-specialista: RNDr. Věra Lusková, CSc. Brno, 2008

PŘÍLOHY – SOUBOR PUBLIKACÍ

Příloha I

Papoušek, I., Vetešník, L., Halačka, K., Lusková, V., Humpl, M. & Mendel, J. (2008). Identification of natural hybrids of gibel carp Carassius auratus gibelio (Bloch) and crucian carp Carassius carassius (L.) from lower Dyje River floodplain (Czech Republic). Journal of Fish Biology 72, 1230-1235.

Příloha II

Vetešník, L., Papoušek, I., Halačka, K., Lusková, V. & Mendel, J. (2007). Morphometric and genetic analysis of Carassius auratus complex from an artificial wetland in Morava River floodplain, Czech Republic. Fisheries Science 73, 817-822.

Příloha III

Papoušek, I., Lusková, V., Halačka, K., Koščo, J., Vetešník, L., Lusk, S. & Mendel, J. (2008). Genetic diversity of the Carassius auratus complex (Teleostei: Cyprinidae) in the central Europe. Journal of Fish Biology (submitted 10/2008).

Příloha I

Journal of Fish Biology (2008) 72, 1230–1235 doi:10.1111/j.1095-8649.2007.01783.x, available online at http://www.blackwell-synergy.com

Identiﬁcation of natural hybrids of gibel carp Carassius auratus gibelio (Bloch) and crucian carp Carassius carassius (L.) from lower Dyje River ﬂoodplain (Czech Republic)

I. PAPOUSˇEK*, L. VETESˇNI´K,K.HALACˇKA,V.LUSKOVA´,M.HUMPL AND J. MENDEL Department of Ichthyology, Institute of Vertebrate Biology, Czech Academy of Sciences, v.v.i., Kveˇtna´ 8, 603 65 Brno, Czech Republic

(Received 13 July 2007, Accepted 28 November 2007)

Morphometric and genetic variation were examined in parental Carassius auratus gibelio and Carassius carassius individuals, and their natural hybrids. Of meristic traits, only the number of gill rakers clearly distinguished hybrids (394 14) from parental C. a. gibelio (483 07) and C. carassius (286 11). MtDNA sequences showed that the hybrids were descendants of female C. a. gibelio. Microsatellite analysis conﬁrmed the presence in hybrids of variants typical of C. a. gibelio and C. carassius. # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles

Key words: crucian carp; gibel carp; gill rakers; hybrids; microsatellites; mtDNA.

The genus Carassius is represented in Europe by four taxa, native crucian carp Carassius carassius (L.), introduced goldfish Carassius auratus auratus (L.), gibel carp Carassius auratus gibelio (Bloch) and rare ‘ginbuna’ Carassius auratus langsdorfii Temminck & Schlegel (Szczerbowski, 2002; Kalous et al., 2007; Vetesˇnı´k et al., 2007). Even though C. a. gibelio and C. carassius are widely dis- tributed, published reports of hybrids between them are rare. Kux (1982) described the occurrence of such hybrids from the Danube Delta, and Goryunova & Skakun (2002) found hybrids of these taxa in natural lakes in Kazakhstan. In Polesi waters (Ukraine), several hybrids were found by Mezherin & Liseckij (2004). Ha¨nfling & Harley (2003) and Ha¨nfling et al. (2005) report the frequent occurrence of diploid hybrids arising from C. auratus and C. carassius in Great Britain. Despite the general prevalence of C. a. auratus and C. a. gibelio in Europe, information on the occurrence of natural hybrids with native C. carassius is scarce. This limited information may not reflect the

*Author to whom correspondence should be addressed. Tel.: þ420 543 422 529; email: [email protected] 1230 # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles IDENTIFICATION OF NATURAL CARASSIUS HYBRIDS 1231 extent of hybridization or may indicate that insufficient attention has been paid to hybrid identification. The aim of the present study was to identify with morphometric and genetic analyses natural hybrids of gibel carp and crucian carp in the lower Dyje River floodplain (Czech Republic). A total of 634 individuals attributed to genus Carassius [visually identified as C. a. gibelio (n ¼ 594), C. carassius (n ¼ 25) and possible hybrids (n ¼ 15)] were caught by electrofishing in the parapotamon side arm in the aluvium of the lower Dyje River (48°439250 N, 16°539200 E; in an area of 045 ha). Analyses of ploidy levels were performed by means of computer-assisted microscopic image cytometry according to Flajsˇhans (1997) and Halacˇka & Luskova´ (2000). Blood smears were stained by Harris hematoxyline (Sigma-Aldrich, Prague, Czech Republic) for 10 min. Ploidy diagnostic dimension (i.e. erythrocyte nuclear area) was determined by bright field microscopy, imaging by a charge-coupled device (CCD) camera and image processing in Olympus MicroImage 4.0 analyser. The ploidy status was determined in 75 fish, including C. a. gibelio [17 diploid males (m), 15 diploid and 43 triploid females (f)], 10 C. carassius (6 m and 4 f) and 15 hybrids (6 m and 9 f). Meristic traits were assessed according to Holcˇı´k (1989) in 12 individuals (6 m and 6 f) of diploid C. a. gibelio, eight individuals (4 m and 4 f) of C. carassius and 12 hybrids (5 m and 7 f). For the meristic analyses, individuals were killed by overdose of anaesthetic (2-phenoxy-ethanol) and preserved in 4% formaldehyde. Differences in values of meristic traits among C. a. gibelio, C. carassius and hybrids were tested using ANOVA in Statistica Cz 7 and detrended correspondence analysis (DCA) Canoco 3.10. All data were checked for normalcy before analyses. DNA analyses were performed on two C. carassius individuals (1 m and 1 f), two C. a. gibelio individuals (1 m and 1 f) and nine putative hybrids (4 m and 5 f). Tested fish were fin-clipped and the clips were stored in ethanol for genetic analyses. Total genomic DNA was isolated from fin clips by phenol-chloroform extraction according to Sambrook et al. (1989) with minor modifications. Frag- ments of mtDNA representing the hypervariable part of the control region (D-loop) were amplified using primers CarU32 (59-CCAAAGCCAGAATTCTAAAC-39) and CarL509 (59-CATGTGGGGTAATGA-39). Polymerase chain reaction (PCR) was performed in total volume of 50 ml, which contained 200 mMofeach primer, 200 mM dNTP, 15 mM MgCl2,1 reaction buffer, 100 ng of total genomic DNA and 15 U Taq polymerase. The following protocol for amplification was used: denaturation 95° C 1 min, 32 cycles 94° C 45 s/50° C30s/72° C 45 s, final extension 72° C for 7 min. PCR fragments were purified using poly- ethyleneglycol (PEG)/MgCl2/NaAc and sequenced on an automatic sequencer ABI PRISM 310 (Applied Biosystems, Foster City, CA, U.S.A.). Sequences (493 bp) were aligned by MEGA 3.1 (Kumar et al., 2004) and compared with GenBank sequences of C. a. gibelio (accession number AJ 388413.1), C. a. auratus (NC 006580), C. a. langsdorfii (AB 052327.1) and Carassius auratus cuvieri Temminck & Schlegel (AB 052334.1), and unpublished sequences of C. carassius. Microsatellite loci GF17, GF29, MFW2 and MFW7 were amplified with previously published primer sequences (Zheng et al., 1995; Crooijmans et al., 1997). PCR was performed according to Ha¨nfling et al. (2005). Resulting genotypes were compared with allele size ranges of C. carassius and C. a. gibelio as in Ha¨nfling et al. (2005).

# 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1230–1235 1232 I. PAPOUSˇEK ET AL.

Carassius carassius body colour was greenish with a golden tinged, luteous abdomen; C. a. gibelio had a dark grey back and silvery sides. Body colouring of hybrid individuals was similar to C. a. gibelio, with a tinge of dark grey and black on head, torso and fins. The upper line of the dorsal fin was convex and the upper edge was rounded in C. carassius, whereas in C. a. gibelio the upper line of the dorsal fin was ventrally slightly concave. The dorsal fin of hybrid individuals merged traits of both parental species: the cranial half of the dorsal fin was convex and corresponded to the fin of C. carassius; the caudal half of the dorsal fin was slightly concave as in C. a. gibelio. The inner side of the abdominal cavity (peritoneum) was light and unpigmented in C. carassius but was black with a pearly sheen in C. a. gibelio. Peritoneal colouring of the hybrids was variable – some individuals had darker and some had lighter colouring. The average number of gill rakers (Sp.br.) reached 483 in diploid C. a. gibelio, 286inC. carassius and 394 in hybrids (Table I). This trait clearly distinguished hybrids from parental species. Comparison of other meristic traits without Sp.br. discriminates C. a. gibelio from both C. carassius and hybrids, but C. carassius and hybrids could not be distinguished without Sp.br. (Table I). DCA of the number of gill rakers demonstrated clear differences between C. a. gibelio, C. carassius and their hybrids (Fig. 1). The number of gill rakers is useful for the identification of species and hybrids of the genus Carassius (Berg, 1932). The average number of gill rakers found in diploid C. a. gibelio individuals is consistent with data published by other authors (Vasileva, 1990; Vetesˇnı´k, 2005). Only Kux (1982) found 41 gill rakers in a single hybrid of these species. This number is close to the average number (394) Sp.br. of the hybrid individuals. The analysis of four microsatellite markers in control samples of C. a. gibelio and C. carassius produced alleles within or close to the size ranges described by Ha¨nfling et al. (2005); only MFW7 had an allele that was significantly different in size, but this allele was nevertheless consistent with alleles in C. Carassius, which occur with an even total length (LT). Microsatellite analysis of nine putative hybrid individuals revealed the presence of eight alleles in GF17 locus (range 180–210 bp), four alleles in GF29

TABLE I. Meristic traits – Carassius auratus gibelio, hybrids and Carassius carassius

C. a. gibelio (n ¼ 12) Hybrids (n ¼ 12) C. carassius (n ¼ 8)

Features Average S.D. Average S.D. Average S.D. ANOVA

Du 370528042902 P 0001 Db 170071570615505 P 0001 Au 30—27052405 P 001 Ab 60—510350—P 0001 l.l. 307073160531308 P 005 Sp.br. 483073941428611 P 0001

Du, number of unbranched rays in dorsal fin; Db, branched rays in dorsal fin; Au, number of unbranched rays in anal fin; Ab, branched rays in anal fin; l.l., number of scales in the lateral line; Sp.br., gill rakers.

# 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1230–1235 IDENTIFICATION OF NATURAL CARASSIUS HYBRIDS 1233

FIG. 1. Meristic traits – detrended correspondence analysis (DCA); , Carassius auratus gibelio, , hybrids and , Carassius carassius.

(199–218 bp), two alleles in MFW2 (157–160 bp), 11 alleles in MFW7 (158–199 bp; Table II). For each of the loci, GF17, GF29 and MFW2, one allele corresponded with C. carassius and the other with C. a. gibelio [either based on size ranges described by Ha¨nﬂing et al. (2005) or based on actually observed alleles present in control samples] for all putative hybrids, except for GF17 in the HY5 sample, where the allele with presumed origin from C. a. gibelio fell out of the expected size range, but only by a single repeat unit. In the most variable marker MFW7, one allele corresponding with C. a. gibelio (odd LT within expected size ranges) was found in all samples. The second allele corresponded with the allele found in the control samples of C. carassius in ﬁve

TABLE II. Microsatellite and mitochondrial genotypes in Carassius auratus gibelio (CAG), Carassius carassius (CC) and their respective hybrids (HY). CAG, CC – allele size ranges according to Ha¨nﬂing et al. (2005) for C. a. gibelio and C. carassius. Microsatellite alleles with presumed origin in C. a. gibelio typed in italics, alleles outside ranges CAG and CC are in bold

Specimen Microsatellite locus

CAG CC HYSex GF17 GF29 MFW2 MFW7 mtDNA

1 m 188/188 195/201 157/157 175/175 C. a. gibelio 2 f 188/188 199/199 157/157 175/175 C. a. gibelio 1 m 180/180 212/212 160/160 158/158 C. carassius 2 f 180/180 214/214 160/160 158/158 C. carassius 1 m 180/190 199/212 157/160 158/177 C. a. gibelio 2 m 180/192 199/218 157/160 180/195 C. a. gibelio 3 f 180/192 199/218 157/160 158/181 C. a. gibelio 4 f 180/188 199/212 157/160 158/179 C. a. gibelio 5 m 180/184 199/212 157/160 176/183 C. a. gibelio 6 f 180/204 199/214 157/160 180/199 C. a. gibelio 7 f 180/210 199/218 157/160 158/175 C. a. gibelio 8 m 180/194 199/212 157/160 179/196 C. a. gibelio 9 f 180/194 199/212 157/160 158/177 C. a. gibelio CAG 186–220 193–207 157 175–199 CC 182 210–228 160 178–196

# 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1230–1235 1234 I. PAPOUSˇEK ET AL. hybrid fish; in three other hybrid fish, the allele corresponding with C. carassius (even LT within the expected size ranges) was found. The final sample carried an allele slightly out of the expected range (by a single repeat unit). Based on these findings, all tested hybrid individuals appear to carry one allele from C. carassius and one allele from C. a. gibelio, which suggests that all of them were F1 hybrids rather than post-F1 hybrids. No suspected hybrid fish demonstrated a genotype with a locus homozygous for one species and a locus homozygous for the other species. Similarly, no genotype containing a locus with alleles of one species and a locus with alleles of both parental species was detected, which would suggest a possible backcross origin. Furthermore, relative frequencies of individuals possessing heterozygous genotypes in all four tested markers decrease rapidly from c. 6% in the first generation of backcrossing (BC-1) to <03% in the BC-2 and so on (Boecklen & Howard, 1997). Analysis of mtDNA haplotypes showed that all hybrid fish had the C. a. gibelio haplotype, indicating that hybrid offspring resulted from unidirectional crosses of C. carassius males and C. a. gibelio females. Hybrids between C. a. gibelio and C. carassius have been reported from other locations. The supposed relatively high abundances of C. a. gibelio and C. carassius hybrids, described by Goryunova & Skakun (2002) in the extreme conditions in Kazakhstan lakes, may not be correct because the identification of hybrids was based on peritoneal colouring in the absence of unambiguous discriminating factors. Goryunova & Skakun (2002) stated that hybrids mate with both other hybrids and parental species. Molecular evidence indicates that native C. carassius frequently hybridizes with the introduced species, C. auratus and Cyprinus carpio L., in Britain (Ha¨nfling et al., 2005). About 38% of investigated C. carassius populations contained hybrids with C. auratus or C. carpio, respectively. Hybrids between C. carpio and both crucian carp (C. carassius and C. a. gibelio) and C. auratus are produced artificially for commercial purposes (Ha¨nfling et al., 2005). However, hybrids between C. carassius, C. a. gibelio and C. carpio are not produced intentionally in the Czech Republic, and this study presents the first observation of these hybrids in this country. Hybrids between the two latter species, C. carpio and C. a. gibelio, have been previously recorded (Prokesˇ&Barusˇ, 1996; unpubl. data). The emergence of viable hybrids between C. a. gibelio and other cyprinid species is unlikely, as suggested by the results from experimental hybridization with Abramis brama (L.), Abramis bjoerkna (L.) and Rutilus rutilus (L.) (Flajsˇhans et al., 2004; unpubl. data). Non-native fish released into the wild is an increasing problem posing considerable ecolo- gical and genetic threats through direct competition and hybridization.

This study was supported by grant project of Grant Agency of Czech Republic no. 206/05/2159.

References Berg, L. S. (1932). U¨ber Carassius carassius und Carassius gibelio. Zoologischer Anzeiger 98, 15–18. Boecklen, W. J. & Howard, D. J. (1997). Genetic analysis of hybrid zones: numbers of markers and power of resolution. Ecology 78, 2611–2616.

# 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1230–1235 IDENTIFICATION OF NATURAL CARASSIUS HYBRIDS 1235

Crooijmans, R. P. M. A., Bierbooms, V. A. F., Komen, J., VanderPoel, J. J. & Groenen, M. A. M. (1997). Microsatellite markers in common carp (Cyprinus carpio L.). Animal Genetics 28, 129–134. Flajsˇhans, M. (1997). A model approach to distinguish diploid and triploid fish by means of computer - assisted image analysis. Acta Veterinaria Brno 66, 101–110. Flajsˇhans, M., Luskova,´ V., Vetesˇnı´k, L., Halacˇka, K., Rodina, M., Lusk, S. & Gela, D. (2004). Diploid, triploid and tetraploid silver crucian carp (Carassius auratus) from the lower reach of Dyje River: the first results of reproductive characteristics and experimental hybridization. Biodiverzita ichtyofauny CˇR V, 35–43 (Czech with English summary). Goryunova, A. I. & Skakun, D. (2002). Biological characterization on crucian carps. Tethys Aqua Zoological Research 1, 33–48. Halacˇka, K. & Luskova,´ V. (2000). Polyploidy in Carassius auratus in the lower reaches of the River Dyje – determination using the size of erythrocyte nuclei. Proceedings of the 4th Czech Conference of Ichthyology, pp. 110–113. (Czech with English summary). Ha¨nfling, B. & Harley, M. (2003). A molecular approach to detect hybridization between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius auratus and Cyprinus carpio) in UK waters, including a consideration of the taxonomic status of the giebel carp (Carassius spp.). Environment Agency R&D Technical Report W2–077/TR. Ha¨nfling, B., Bolton, P., Harley, M. & Carvalho, G. R. (2005). A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio). Freshwater Biology 50, 403–417. Holcˇı´k, J. (1989). The Freshwater Fishes of Europe. Vol. 1, Part II. General Introduction to Fishes, Acipenseriformes. Wiesbaden: AULA-Verlag. Kalous, L., Sˇlechtova,´ V., Bohlen, J., Petrty´l, M. & Sˇvatora,´ M. (2007). First European record of Carassius langsdorfii from the Elbe basin. Journal of Fish Biology 70 (Suppl. A), 132–138. doi: 10.1111/j.1095-8649.2006.01290.x Kux, Z. (1982). Prˇı´speˇvek k problematice krˇı´zˇencu˚rodu Carassius. Acta Musei Moraviae 67, 181–188. (in Czech). Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5, 150–163. Mezherin, S. V. & Liseckij, I. L. (2004). Estestvennaja gibridizacija serebrjanogo karasja (Carassius auratus) i zolotogo (Carassius carassius) karasej: evoljucionnyj fenomen ili poglosˇcˇenie odnogo vida drugim? Reports of the NASU 9, 162–166 (in Russian). Prokesˇ, M. & Barusˇ, V. (1996). On the natural hybrid between common carp (Cyprinus carpio) and Prussian carp (Carassius auratus gibelio) in the Czech Republic. Folia Zoologica 45, 277–282. Sambrook, J., Fritsch, E. & Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Szczerbowski, J. A. (2002). Carassius auratus (Linnaeus, 1758). In The Freshwater Fishes of Europe, Vol. 5/III, Cyprinidae 2 (Ba˘na˘rescu, P. & Paepke, H. J., eds), pp. 5–41. Wiesbaden: AULA-Verlag. Vasileva, E. D. (1990). On morphological divergence of gynogenetical and bisexual forms of Carassius auratus (Cyprinidae, Pisces). Zoologicˇeskij Zˇurnal 11, 97–110. Vetesˇnı´k, L. (2005). Biological characteristic of silver crucian carp (Carassius auratus) under the aspect of different ploidy level between individuals. Mendel University of Agriculture and Forestry, Dissertation, Brno (Czech with English summary). Vetesˇnı´k, L., Papousˇek, I., Halacˇka, K., Luskova,´ V. & Mendel, J. (2007). Morphometric and genetic analysis of the Carassius auratus complex from an artificial wetland in floodplain of the Morava River (Czech Republic). Fisheries Science 73, 817–822. Zheng, W., Stacey, N. E., Coffin, J. & Strobeck, C. (1995). Isolation and characterization of microsatellite loci in the goldfish Carassius auratus. Molecular Ecology 4, 791–792.

# 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 1230–1235

Příloha II

FISHERIES SCIENCE 2007; 73: 817–822

Morphometric and genetic analysis of Carassius auratus complex from an artiﬁcial wetland in Morava River ﬂoodplain, Czech Republic

Lukáš VETEŠNÍK, Ivo PAPOUŠEK,* Karel HALACˇ KA, Veˇra LUSKOVÁ AND Jan MENDEL

Institute of Vertebrate Biology of the Academy of Sciences of Czech Republic, v.v.i., Kveˇtná 8, 603 65 Brno, Czech Republic

ABSTRACT: A Carassius auratus complex from an artificial wetland in the Morava River basin is composed of triploid females. Based on body depth, sampled females could be divided into two groups: (i) high-dorsal (42.5% of standard length); and (ii) low-dorsal (36.1% of standard length). Both groups differed also in number of gill rakers (50.2 versus 45.4, respectively). In concordance with morphological differences, genetic analysis proved the existence of two haplotypes in examined individuals. The first haplotype is bound to the high-dorsal form with higher number of gill rakers. This is the most frequent haplotype in populations of the C. a. gibelio form in the Czech Republic. The second haplotype is characteristic of the low-dorsal form with a lower number of gill rakers. This haplotype is close to haplotypes described in the C. a. langsdorfii form, which is known from Japan, and its occurrence within haplotypes specified in European territory is sporadic.

KEY WORDS: artiﬁcial wetland, Carassius auratus gibelio, Carassius auratus langsdorﬁi, Czech Republic, microsatellites, morphometry.

INTRODUCTION with different ploidy, with both gynogenetic and sexual reproduction.5–8 The waters of the Czech Republic are inhabited Within the scope of C. auratus, five subspecies by a native species Carassius carassius and (or forms) are distinguished in Japan: C. a. langs- non-indigenous species considered as Carassius dorfii (ginbuna), C. a. grandoculis (nigorobuna), auratus complex. The taxonomic status of this C. a. ssp. (kinbuna), C. a. buergeri (nagabuna), and complex is unclear; therefore, we have used a term C. a. cuvieri (gengorobuna).9 In a revision of the ‘form’ for its individual components. In Western Carassius complex in Japan based on meristic European countries, a diploid form of C. a. aura- traits and ploidy, only two ‘taxons’ or ‘species’ tus, originating from decorative goldfish, is most were specified: C. cuvieri and C. auratus complex common.1–3 In Central and Eastern Europe, the encompassing all others. Carassius cuvieri is con- form C. a. gibelio, which is very similar to the form sidered a valid endemic species to Lake Biwa.10 In described in the Amur basin,4 prevails. Variable another study from Japan, C. a. cuvieri was also proportions of males and females with different interpreted as a separate species of C. cuvieri by ploidy levels and ways of reproduction are charac- genetic analysis.11 All other traditionally distin- teristic of this form. The invasive C. a. gibelio form guished morphotypes overlap both genetically and consisting of triploid gynogenetically reproducing morphologically and it is difficult to identify them females permeated into Czech waters from the by morphometric means.11 Danube River in 1976, and with human help colo- An incidental occurrence of two individuals of nized all suitable aquatic biotopes in 20 years. This the C. a. langsdorfii form in the Chrudimka River form gradually transformed into the present-day (Elbe basin, Czech Republic) is the first published12 type of mixed populations of males and females discovery of this form in Europe. The aim of the present study was to use morphometry (meristic and plastic traits) and genetic *Corresponding author: Tel: 420-543422529. analyses (mtDNA and microsatellites) to distin- Fax: 420-543211346. Email: [email protected] guish between two forms of C. auratus (C. a. gibe- Received 29 May 2006. Accepted 16 March 2007. lio and C. a. langsdorfii). doi:10.1111/j.1444-2906.2007.01401.x © 2007 Japanese Society of Fisheries Science 818 FISHERIES SCIENCE L Vetešník et al.

MATERIALS AND METHODS was amplified using primers CarU32 (5′- CCAAAGCCAGAATTCTAAAC-3′) and CarL509 Description of monitored area and (5′-GCATGTGGGGTAATGA-3′). Polymerase chain collection of material reaction (PCR) was performed in a T-Gradient thermocycler (Biometra, Göttingen, Germany), An artificial wetland was created in the Morava in a total volume of 50 mL, containing 1¥ reaction River floodplain in 1996. It consists of a closed buffer, 1.5 mM MgCl2, 200 mM each of dNTPs, system of channels and a larger pond with total 2 mM of each primer, 100 ng genomic DNA, and area of 0.13 ha. In 1997, during an extreme flood, it 1.5 U Taq polymerase. For amplification, the fol- was submerged and colonized by fishes brought by lowing protocol was used: denaturation at 95°C the flood. The occurrence of 15 species was docu- for 1 min, 32 cycles at 94°C for 45 s, 50°C for 30 s, mented; the dominant species included Rutilus 72°C for 45 s, and final extension at 72°C for 7 min. rutilus, Scardinius erythrophthalmus, C. auratus, PCR fragments were subsequently purified using 13 and Perca fluviatilis. Collection of material was PEG–MgCl2–NaAc and sequenced on an automatic performed by electrofishing in August 2005. In sequencer (Abiprism 310, Applied Biosystems, total, 91 individuals of C. auratus were caught. Foster City, CA, USA). Blood was taken for determination of ploidy status, Acquired sequences of total length approxi- and parts of fins were clipped into ethanol for mately 493 bp were aligned using MEGA 3.1 DNA analysis. For morphometric examinations, 20 software,19 and compared with sequences of individuals were selected, killed by overdose of C. a. gibelio (GenBank accession number anesthetic (2-phenoxy-ethanol), and stored in AJ388413), C. a. auratus (NC_006580), C. a. langs- formaldehyde. The fishes were divided visually into dorfii (AB052327), C. cuvieri (AB052334), and two groups based on body depth. High-dorsal C. carassius (Papoušek I., unpubl. data, 2005). A females were entitled C1 and low-dorsal, C2. newly reported sequence of C. a langsdorfii (haplotype HT 2) appears in GenBank under accession number AY940118. Ploidy determination and morphometry For comparison with other C. a. langsdorfii,previously published sequences (GenBank accession Ploidy level of examined individuals of C. auratus numbers AB052299–AB052335) were used.11 was determined by computer analysis of micro- All phylogenetic analyses were carried out by scopic cell image (image cytometry, ICM). Blood MEGA 3.1 software.19 Phylogenetic trees were smears stained by Harris hematoxyline (Sigma- constructed using the neighbor-joining (NJ) Aldrich, Prague, Czech Republic) for 10 min, were algorithm.20 used for ICM. The ploidy-diagnostic dimension (erythrocyte nuclear area) was determined by bright field microscopy, imaging by a CCD camera Analysis of microsatellites and image processing in a MicroImage 4.0 analyzer (Olympus, Tokyo, Japan).14–16 Classic methodolo- Analysis of microsatellites was performed on ten gies were used for assessment of plastic and mer- fishes each from C1 and C2 groups. Six microsatel- istic traits (gill rakers, and spinae branchiales, lite loci (GF17, GF29, MFW7, MFW17, J1, and J62) Sp.br.).17 After determination of age, scales were were amplified under previously described taken from the left side of body between the anal conditions.21–23 fin and lateral line. The differences in values of plastic and meristic traits between C1 and C2 were tested by a Mann–Whitney test (MW-test) using RESULTS Statistica v6.0 software (Statsoft, Tulsa, OK, USA). Ploidy, sex, length, and age structure

Analysis of mitochondrial DNA All 91 analyzed C. auratus individuals were triploid females. Length structure of a sample caught in Molecular-genetic analysis of mitochondrial DNA August 2005 is shown in Figure 1. Most frequent was performed on 16 fishes from the C1 group were females of standard length of 160–190 mm. and 22 fishes from the C2 group. Whole genomic The age of fishes ranged from 3+ to 8+, the most DNA was isolated by classic phenol–chloroform frequent were categories of females aged 4+ and 5+. extraction with minor modifications.18 A frag- No fishes aged 1+ and 2+ were found. The C. aura- ment of mtDNA, which represented a hyper- tus form of the C1 group grows faster than the variable segment of the control region (D-loop), C. auratus form of C2 group; annually, e.g. 5+

18 Analysis of mitochondrial DNA 16 14 Analysis of the control region of mitochondrial 12 DNA (D-loop) revealed the presence of two haplo- 10 types of C. auratus in the wetland: HT 1 (in C1 n 8 females) and HT 2 (in C2 females). HT 1 is identical 6 to the haplotype of the C. a. gibelio form (GenBank 4 accession number AJ388413). This haplotype is 2 very common in Czech territory (Papoušek I., 0 unpubl. data, 2005). HT 2 is novel, but phyloge- 141-150 151-160 161-170 171-180 181-190 191-200 211-220 241-250 261-270 271-280 281-290 291-300 131-140 201-210 221-230 231-240 251-260 301-310 netically close to haplotypes of C. a. langsdorﬁi (ginbuna). The phylogenetic tree inferred from SL (mm) NJ analysis is illustrated in Figure 2. In addition to the NJ algorithm, minimum evolution (ME) and Fig. 1 Length distribution of Carassius auratus caught maximum parsimony (MP) algorithms were used. in August 2005. SL, standard length range; n, number of Resulting phylogenetic trees had identical topolo- individuals. gies, and therefore, are not shown. female C1 average standard length was 215.5 mm, Analysis of microsatellites and 5+ female C2 average standard length was 198.2 mm. A similar trend was found in other age < Analysis of six microsatellite loci has conﬁrmed the categories (MW-test, P 0.01). presence of two separate clonal lines corresponding to groups C1 and C2. Microsatellite genotypes of clonal lines are summarized in Table 2. Morphometry

Meristic characteristics DISCUSSION

A statistically significant difference (P < 0.01) bet- Morphometric and genetic analyses of C. auratus ween samples C1 and C2 was found only in number individuals from an artificial wetland have clearly of gill rakers (Sp.br.). No statistical difference was confirmed the existence of a non-hybrid complex found between C1 and C2 for other meristic traits. of C. auratus constituted by two independent The average number of gill rakers in the C1 sample populations of two forms of C. auratus. Popula- was 50.17 (min–max, 49–51), and 45.42 (44–46) in tions of both forms in the wetland reproduce in a C2. Of meristic traits, the number of gill rakers dis- natural gynogenetic way, which is proved by the tinguishes groups C1 and C2 unambiguously. existence of multi-age structure of the examined sample (age 3+ to 8+). The average number of gill rakers in sample C1 Plastic characteristics (50.17) was very close to values previously found in triploid individuals of the C. a. gibelio form: 51.96 Comparison of plastic characteristics of samples (50–53)24 and 51.50 (50–53).25 The average number C1 and C2 (Table 1) shows differences in several of Sp.br. in sample C2 (45.42) was similar to values parameters. The most significant difference (44.60 and 45.90) found in the triploid C. a. langs- (P < 0.01) was found in body depth, which clearly dorfii form in Japan.26,27 In two individuals of the divides examined fishes into two groups. The body C. a. langsdorfii form previously recorded in the depth of the C1 sample was 6.47% higher on Czech Republic, 47 and 46 gill rakers were found.12 average. A statistically significant difference In another study, individuals of ginbuna from Japa- (P < 0.01) was also found in the predorsal distance nese lakes and rivers were also described, and (3.46%), preanal distance (3.10%), and length of number of Sp.br. varied from 41 to 51.28 These rela- the caudal peduncle (3.52%) between C1 and C2 tively large differences could be explained either (Table 1). Although the MW-test shows differences by possibly different ploidy levels in tested indi- at significance level P < 0.05 in some other viduals, or by possibly different body length; with assessed parameters (Table 1), they should not be increasing body length in the C. a. gibelio form overrated, considering possible influence of the and C. carassius, the number of gill rakers also environment. Of plastic traits, body depth distin- increases.29,30 We presume that an identical situ- guishes groups C1 and C2 unambiguously. ation arises in C. a. langsdorfii; therefore, it is

Table 1 Plastic characteristics of C1 and C2 Carassius auratus samples Parameters C1 C2 P n 10 10 Features¯ X Ϯ SD X¯ Ϯ SD Standard length (mm) in percent standard length: 143.00 Ϯ 7.85 162.60 Ϯ 11.18 * Head length 30.25 Ϯ 1.27 28.81 Ϯ 0.89 * Head depth 24.48 Ϯ 0.39 23.61 Ϯ 0.54 * Head width 17.65 Ϯ 0.57 18.68 Ϯ 0.33 * Preorbital distance 9.52 Ϯ 0.49 8.86 Ϯ 0.13 Postorbital distance 14.98 Ϯ 0.29 15.36 Ϯ 0.51 Diameter of the eye 6.12 Ϯ 0.52 5.15 Ϯ 0.19 * Distance between nostrils 5.31 Ϯ 0.45 5.91 Ϯ 0.54 Interorbital distance 11.34 Ϯ 0.37 11.80 Ϯ 0.40 Prepectoral distance 29.84 Ϯ 1.40 28.80 Ϯ 0.62 Preventral distance 48.28 Ϯ 1.04 48.23 Ϯ 0.77 Preanal distance 77.65 Ϯ 0.74 74.55 Ϯ 0.42 ** Predorsal distance 52.90 Ϯ 1.31 49.44 Ϯ 0.90 ** Body depth 42.52 Ϯ 1.78 36.05 Ϯ 0.47 ** Length of caudal peduncle 19.02 Ϯ 0.77 22.54 Ϯ 0.39 ** Caudal peduncle width 5.74 Ϯ 0.63 6.42 Ϯ 0.60 Caudal peduncle depth 18.18 Ϯ 0.58 17.00 Ϯ 1.19 Minimum body depth 15.68 Ϯ 0.33 14.78 Ϯ 0.69 Distance between pectoral and ventral fin 19.97 Ϯ 0.49 20.84 Ϯ 1.02 Distance between ventral and anal fin 30.94 Ϯ 1.30 28.20 Ϯ 1.49 * Length of dorsal fin 34.40 Ϯ 0.73 32.18 Ϯ 0.85 * Length of anal fin 9.78 Ϯ 0.55 9.26 Ϯ 0.65 Length of pectoral fin 19.42 Ϯ 0.80 19.90 Ϯ 0.76 Length of ventral fin 22.54 Ϯ 0.66 21.42 Ϯ 0.81 *

*P < 0.05; **P < 0.01.

99 HT 1 (ii) cluster II contained 2 haplotypes of polyploid 93 AJ388413.1 C.auratus gibelio ginbunas and all goldﬁsh haplotypes; and (iii) the 75 NC 006580.1 C.auratus auratus HT 2 most heterogenous cluster III was formed by hap-

89 AB052327.1 C.auratus langsdorfii lotypes of diploid and triploid ginbuna, nigor- C.carassius obuna, and nagabuna. In comparison with this AB052334.1 C.cuvieri study our novel haplotype HT 2 falls into cluster III,

0.01 sharing a subcluster with haplotypes of diploid and triploid ginbunas from Lake Shiwa and Lake Imba Fig. 2 Consensual phylogenetic tree based on analysis (Fig. 3). of mitochondrial control region haplotypes with Analysis of microsatellites clearly proved that C1 neighbor-joining algorithm. Numbers at nodes indicate and C2 lines are separate and do not hybridize: this percent bootstrap support (1000 pseudoreplications). separation is illustrated by finding that they do not Scale bar for branch lengths is shown. share any alleles in microsatellite loci MFW17 and J1, and differ by at least one allele in remaining loci (Table 2). necessary to compare fish of similar body length. Considering these findings, it can be assumed Differences can be also caused by human factors – that further application of molecular-genetic each scientist could determine the number of gill analysis should strongly contribute to identifica- rakers differently (i.e. difficulty in computing Sp.br. tion of the structure of the C. auratus complex in in juvenile fishes, where the edge of the branchial Central Europe. arch breaks easily, and therefore, all Sp.br. can not The usual development in situations when there be computed). is no isolation between populations is that changes Genetic analysis of 169 Carassius auratus from towards mixed hybrid populations are expected, Japan, which used D-loop sequencing as genetic and morphometric differences of original forms are marker,11 revealed the existence of 37 haplotypes suppressed. On the contrary,we observed preserva- divided into three major phylogenetic clusters: (i) tion of morphometric traits, which is facilitated by cluster I consisted exclusively of the gengorobuna; gynogenetic reproduction of two isolated unisexual

Table 2 Microsatellite genotypes in two clonal lines of Carassius auratus Microsatellite marker C1 C2 GF17 188/190/192 190/190 or 192/192 GF29 199/199 or 207/207 191/191 or 199/199 MFW7 179/179 or 199/199 175/175 or 199/199 MFW17 222/222 or 226/226 212/212 or 216/216 J1 147/147 or 163/163 127/127 or 135/135 J62 176/176 or 178/178 124/124 or 178/178

Ten samples were used for each of the two clonal lines.

langsdorfii).31,32 A possible explanation is that the C. a. langsdorfii was present in limited numbers in the first invasion wave, which consisted mostly of individuals of the C. a. gibelio form. This formation intruded into the hydrologic system of the Czech Republic by automigration from the Danube River in 1976.5 This hypothesis is supported by findings of more individuals of the C. a. langsdorfii form, which were sporadically found in several other localities together with numerically dominant C. a. gibelio form (Papoušek I., unpubl. data 2005). Another possible way would be importation in conjunction with commercially introduced fish species from Asia.12 We found no other information on the occurrence of C. a. langsdorfii in Europe.

ACKNOWLEDGMENT

This study was supported by grant project of the Grant Agency of Czech Republic No. 206/05/2159.

REFERENCES

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Příloha III

Genetic diversity of the Carassius auratus complex (Teleostei: Cyprinidae) in the central Europe I. Papoušek*‡, V. Lusková*, K. Halačka*, J. Koščo†, L. Vetešník*, S. Lusk* and J. Mendel*

*Department of Ichthyology, Institute of Vertebrate Biology, Czech Academy of Sciences, v.v.i., Květná 8, 603 65 Brno, Czech Republic, †Department of Ecology, University of Prešov, 17. novembra 1, 081 16 Prešov, Slovakia

Running headline: Diversity of Carassius in central Europe

‡ Author to whom correspondence should be addressed. Tel.: +420 543 422 529; e-mail: [email protected]

Genetic diversity of the Carassius auratus complex, an invasive cyprinid fish, was evaluated based on the sequencing of the mitochondrial control region and the cytochrome b gene in samples from central Europe. Analyses have revealed presence of four distinctive genetic lineages in the waters of the Czech Republic and Slovakia. These lineages are related to four forms of the Carassius auratus complex – C. a. gibelio, C. a. auratus, C. a. langsdorfii and a fourth form previously unknown in Europe.

Key words: gibel carp, crucian carp, mtDNA, Czech Republic, Slovakia

INTRODUCTION From the point of view of distribution, the silver crucian carp (Carassius auratus) is one of the most successful non-indigenous invasive taxa in Europe (Szczerbowski, 2002). Although some authors admit the indigenousness of this fish for Europe, specifically for the Danube River basin (Holčík, 1980), a clear evidence is not available (Hensel, 1971). In a number of cases C. auratus had been mistaken for the indigenous species Carassius carassius (L.) (Pelz, 1987) and until secure distinction of C. auratus from C. carassius on the basis of the number of gill rakers (Berg, 1932) was made, variable forms of C. carassius were frequently labelled as C. gibelio (Jeitteles, 1863 and others). At present it is possible to be satisfied that the fish comes from East Asia (the Amur River basin, China), where it occurs in several forms (e.g. Raicu et al., 1981, Zhou et al., 2001). After casting doubts on the species status of C. gibelio (Bloch 1782) and abandoning the category of subspecies in the species taxonomy, the taxonomic identification of the silver crucian carp is unclear. The situation is also complicated by occurrence of hybrids between C. carassius and some forms of C. auratus, especially the hybrids with the form C. a. auratus, with which hybridization is common due to its exclusive diploidy (Hänfling et al., 2005); on the contrary, findings of hybrids with the form C. a. gibelio are sporadic (Kux, 1982; Papoušek et al., 2008). Other complications are caused by uncertain developmental relations of the individual forms, variable biological characteristics within populations in the course of time (diploidy-polyploidy, sexual-asexual form of reproduction, migrability) and low level of morphometric and anatomical distinguishability of the individual forms of this fish. Therefore we meet with approaches that some authors use subspecific names C. a. gibelio and C. a. auratus in the complex species name Carassius auratus (e.g., Berg, 1949; Banarescu, 1964; Pelz, 1987; Baruš & Oliva, 1995; Szczerbowski, 2002), other authors use the species names C. auratus, C. gibelio (Kottelat, 1997; Kottelat & Freyhof, 2007). In connection with the use of the common name, especially in English

(prussian carp, crucian carp, goldfish, silver crucian carp, gibel carp) and in other national languages (e.g., Russian) additional ambiguities arose. In this study we regard the taxon “Carassius auratus“ as a complex composed of several forms. In the first place it is the ornamental form C. a. auratus, characterized by exclusive sexual reproduction and diploid individuals of both sexes. Another form C. a. gibelio and other known forms originating from Japan – C. a. langsdorfii and C. a. buergeri – are characterized by polyploidy and both sexual and asexual form of reproduction (gynogenesis) (Murakami et al., 2001; Lusková et al., 2002; Halačka et al., 2003). Another Japanese form, C. a. grandoculis, is diploid and together with C. a. buergeri it is sometimes viewed as a morphological variation of C. a. langsdorfii (Murakami et al., 2001). The possibility that C. auratus is a complex of forms or species is admitted also by Vasil’eva & Vasil’ev (2005). In connection with human activities (aquaristics, decoration) the diploid decorative form C. a. auratus was imported into Western Europe in the seventeenth century and gradually expanded to other countries (England, France, Portugal, the Netherlands, Germany, Italy, etc.). The form C. a. auratus is able to produce stable natural populations of feral individuals (Pelz, 1987; Kottelat, 1997; Balon, 2004; Hänfling et al., 2005). The eastern half of Europe was extensively populated as late as the latter half of the twentieth century, prevailingly by the form C. a. gibelio (Holčík, 1980; Lusková et al., 2004). Originally it was the case of sporadic patchy occurrence (Gasowska, 1936; Libosvárský, 1962), not always evidentiary as the form C. a. gibelio. The form C. a. gibelio started to invade the Central European space of the above mentioned countries gradually after 1960. In Slovakia, the first finds in the Danube River basin were in 1961 (Balon, 1962), in the Tisa River basin in 1963 (Žitňan, 1965). In the Czech Republic, the first finds were in the area of the confluence of the rivers Morava and Dyje in 1976 (Lusk et al., 1977). In the course of 10 - 15 years the silver crucian carp expanded in the waters of both countries, when it (with human help) got over migration barriers and geographic boundaries between river and sea basins (Lusk et al., 1980, 1998; Lusk 1986; Lusková et al. 2004). During the last fifteen years a gradual transformation of originally monosexual female populations, reproducing asexually, into mixed populations took place. These populations are composed of males and females of various ploidy (2n, 3n and rarely 4n). Reproduction is ensured both by asexual (females 3n, gynogenesis) and sexual form (males 2n, rarely 3n, females 2n) (Halačka et al., 2003; Lusková et al., 2004). The biological and morphological variability and differences within the form C. a. gibelio are pointed out by several authors. The diversity of the ploidy status, i.e., the occurrence of

diploid and polyploid individuals, different forms of reproduction (sexual reproduction or gynogenesis) and time shifts in these characteristics are probatively evidenced (Halačka et al., 2003; Lusková et al., 2004). Some studies mention also other differences. According to Nikolskij (1956) both the rapidly growing populations and the form with slow growth occur in the Amur River basin, in addition they differ in the number of scales in the lateral line, number of beams in the dorsal fin and number of gill rakers. In the Danube River, Bušnica & Kristian (1959) distinguish two forms on the basis of a different shape of the head. In the lower Danube River, Kukuradze & Mariaš (1975) distinguished a form with slow growth and smaller scales and a form with faster growth which is composed exclusively of females. On the basis of biochemical genetic analyses of triploid individuals of C. gibelio, Mezhzherin & Kokodij (2006) discovered occurrence of several forms which they labelled as C. gibelio 1, 2 and 3. The aim of this study was to evaluate the genetic diversity of the silver crucian carp from the natural waters of the Czech Republic and Slovakia. We already documented the possibility of occurrence of multiple forms by a detailed genetic analysis of the population of triploid females in an artificial wetland, where the forms C. a. gibelio and C. a. langsdorfii occurred concurrently (Vetešník et al., 2007).

MATERIALS AND METHODS

SAMPLING 338 specimens of the silver crucian carp from 23 localities in the Czech Republic and Slovakia were collected. Numbers of specimens collected from the individual localities are summarized in Table I. The map of the geographic position of the localities is in Fig. 1. The specimens were caught by electrofishing between 2004 and 2007. Fin clips of the studied fish were put into ethanol and the fish were subsequently released back into water or transported alive into laboratory where further analyses were performed. Furthermore, 12 ornamental crucian carps were analyzed (8 shubunkin, 2 demekin, 1 ryukin, 1 oranda). They were obtained from commercial aquarium breeds.

PLOIDY DETECTION Ploidy level of the studied specimens was determined using one of two methods: a) a method of computer-assisted analysis of the microscopic image of cells (image cytometry, ICM): blood smears for ICM were stained 10 min in Harris haematoxylin (Sigma - Aldrich, Czech

Republic). Ploidy-diagnostic dimensions of the erythrocyte nuclei were determined by means of bright field microscopy and image capture with the CCD camera and subsequent image information processing using Olympus MicroImage 4.0 analyzer (Flajšhans, 1997; Halačka & Lusková, 2000; Flajšhans & Ráb, 2003), b) using flow cytometry: Flow cytometry was done on Partec CCA 1 cytometer (Partec GmBH, Germany) to determine the relative DNA content in nuclei of peripheral blood cells according to Vindeløv & Christensen (1994), using High Resolution DNA kit T (Partec GmBH, Germany) which contained 4´,6-diamidino-2- phenylindol (DAPI; excitation/emission maxima 358/461 nm) to stain nuclear DNA (Otto, 1994). Diploid form of C. a. gibelio was used as a reference standard.

DNA ISOLATION, PCR AMPLIFICATION AND SEQUENCING The complete genome DNA was isolated from fin clips using a standard method of extraction with the mixture of fenol-chloroform-isoamylalcohol (Sambrook et al., 1989) with minor modifications. Primary mitochondrial DNA analysis was based on amplification and sequencing of a portion of the of the hypervariable part of the control region (ca. 493 bp in length) with primers CarU32 (5‘-CCA AAG CCA GAA TTC TAA AC-3‘) and CarL509 (5‘- GCA TGT GGG GTA ATG A-3‘). Polymerase chain reaction was performed in T-GRADIENT thermocycler (Biometra, Germany), in total volume of 50 µl, containing 1x reaction buffer, 1.5 mM MgCl2, 200 µM each of dNTPs, 2 µM of each primer, 100 ng genomic DNA, 1.5 U Taq polymerase. For amplification, the following protocol was used: denaturation 95 °C 1 min, 32 cycles 94 °C 45 s/50 °C 30 s/72 °C 45 s, final extension 72 °C 7 min. In selected specimens also the cytochrome b gene (1141 bp) was amplified using primers GluDG.L (Palumbi, 1996) and H16460 (Perdices & Doadrio, 2001) and the following protocol: denaturation 94 °C 2 min, 34 cycles 94 °C 45 s/58 °C 45 s/72 °C 90 s, final extension 72 °C 7 min. Resulting PCR fragments of control region and cytochrome b gene were subsequently purified using mixture of 26% polyethylenglycol, 6.5 mM MgCl2.6H2O and 0.6 M

NaAc.3H2O, and sequenced on automatic sequencer ABIPRISM 310 (Applied Biosystems, United States).

ANALYSIS OF THE CONTROL REGION Obtained sequences (475-493 bp in length) were aligned in MEGA 4 (Tamura et al., 2007). Found haplotypes were stored in the GenBank database under accession numbers FJ167410-

FJ167425. Chi-square homogeneity test of base frequencies was performed in PAUP ver 4.0b10 (Swofford, 2003). ModelTest web site (http://darwin.uvigo.es/software/modeltest_server.html) (Posada, 1998) was employed in all cases to determine the best model of evolution that fits to our data. Phylogenetic trees were constructed in MEGA 4 programme with application of neighbour- joining (NJ) (Saitou & Nei, 1987) and maximum parsimony (MP) algorithms. As outgroup the sequence of C. carassius from locality no. 7 “Soutok“ was used (GenBank accession number FJ167426). Consequently all genetic distances were computed and NJ phylogenetic trees constructed using TN93 model with homogenous pattern among lineages and uniform rates among sites (Tamura & Nei, 1993). In MP analysis, heuristic approach was employed and only minimal trees were retained. Bootstrap analysis (1000 replicates) was used to evaluate robustness of topology. Bayesian analysis was performed using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2005). Starting from a random tree, four Markov chains were run for 1 x 106 generations with a sampling frequency of 100. The trees generated prior to reaching stationarity were discarded as burn-in. Resulting 50% majority rule consensus tree was taken. Data generated by MrBayes vere visualised with FigTree 1.1.2 (Rambaut, 2008). Selected sequences were further compared with sequences of C. a. gibelio (accession numbers EF633642-EF633680), C. a. auratus (NC006580) and Carassius spp. (AB052299- AB052335) obtained from the GenBank database. Phylogenetic tree for comparison of haplotypes with the representatives of Japanese haplotypes of the C. auratus complex was constructed on an alignment of 331 bp length using NJ algorithm, with model TN93+G with homogenous pattern among lineages and gamma equal 0.6457. As outgroup D-loop sequence of carp (FJ167427) was used. Bootstrap analysis (1000 replicates) was used to evaluate robustness of topology. In order to identify ancestral haplotype and genealogical relationships among D-loop haplotypes, haplotype network was constructed using Network 4.2.0.1 programme (Bandelt et al., 1999), using median joining algorithm.

ANALYSIS OF THE CYTOCHROME b GENE Obtained sequences (1141 bp in length) were aligned in MEGA 4 (Tamura et al., 2007) and stored in GenBank under accession numbers FJ169951-FJ169954. They were further compared with sequences of C. a. gibelio (accession number EF055472), C. a. auratus (AB111951), C. a. langsdorfii (AB006953) and C. cuvieri (AB045144) from the GenBank. As outgroup sequence of C. carassius from locality no. 7 was used (GenBank accession

number FJ167428). Phylogenetic trees were constructed using NJ and MP algorithms. ModelTest web site (http://darwin.uvigo.es/software/modeltest_server.html) was employed to determine the best model of evolution that fits to our data. Consequently, genetic distances were computed, and NJ phylogenetic tree was constructed using the TN93 model with homogenous pattern among lineages and uniform rates among sites. In MP analysis, only minimal trees were retained. Bootstrap analysis (1000 replicates) was used to evaluate robustness of topology.

RESULTS

ANALYSIS OF THE CONTROL REGION Molecular characteristics and composition Nucleotide sequences (475-493 bp in length) were obtained in 350 specimens of C. auratus. Alignment of these sequences revealed the presence of 60 variable sites (12,2 %), including 23 indels. 59 variable sites were parsimony informative. Base frequencies were homogenous across variable sites (chi square = 0.717, df = 45, P = 1.0). The variable sites constituted altogether 16 different haplotypes, which were labelled as G01-G13, A01, L01 a M01 (Table II). Sequence variability was mainly due to transitions (Ti/Tv ratio = 4.1). Genetic distances between haplotypes ranged from 0.0000 (insertions/deletions only) to 0.0516 (0.0167 on average). The most frequent haplotype was G02 (163 specimens). On the contrary, haplotypes G08 and G10 were found in a single specimen only (Table II). In specimens with the silver crucian carp fenotype all identified haplotypes were recorded. In fish from the locality no. 7 „Soutok“ with goldfish phenotype, haplotypes G04 and A01 were recorded. In the commercially available ornamental forms of goldfish, haplotypes G02 and A01 were recorded.

Geographic distribution of the haplotypes Numbers of individuals possessing respective haplotypes from particular drainages are summarized in Table III. Haplotype G02 was the only haplotype found in all tested basins and localities, except for the locality no. 21 „Sv. Mária“. Other haplotypes (G01, G06, G07, G09, G11, G13) were found in various basins of both countries. On the other hand, haplotypes G03, G05, G08, G10 a L01 were detected only at one locality.. Haplotype G04 was found only in the Czech Republic (in all basins), haplotype G06 was found in all basins except the Czech and Slovak basin of the Danube River. Haplotype M01 was detected both in Czech and

Slovak parts of the Black Sea basin. Highest lineage diversity was found in the Danube and its tributary, Tisza.

Relationship of haplotypes and ploidy levels Most tested specimens were diploids or triploids. Haplotypes G01, G02, G04, G07 and G09 were detected in individuals of either ploidy. Fish with haplotypes A01 and M01 were exclusively diploid, fish with haplotypes G06 and L01 were exclusively triploid. Occurrence of remaining haplotypes was too rare for assessment of their eventual connection with the ploidy level. A single tetraploid individual possessed haplotype G01.

Phylogenetic analysis Phylogenetic analysis of 16 detected haplotypes of the mitochondrial DNA control region was performed using the „neighbour-joining“ (NJ) and maximum parsimony (MP) algorithms, and the Bayesian analysis (BA). Although the resulting phylogenetic trees slightly differ in topology, analysis in all three cases with high bootstrap support (NJ, MP) or posterior probability demonstrated existence of four clearly distinguished monophyletic haplotype groups or lineages (Fig. 2, 3 and 4). The largest group G consists of 13 haplotypes (G01-13), the other lineages A, L and M consist of a single haplotype each (A01, L01 and M01, respectively). Genetic distances among such defined lineages ranged from 3,16 % (M-L distance) to 4,94 % (L-A distance). Genetic distances of the lineages with regard to the C.carassius amount to 5,31-6,65 % (Table IV). The average intralineage diversity among the 13 haplotypes of the lineage G equalled to 0,44 %.

Haplotype network 16 haplotypes of the control region, representing 350 sequences, were used for construction of a haplotype network (Fig. 5). This network can be divided into four parts corresponding to discovered lineages G, A, L and M. The network demonstrates that within the scope of the lineage G, consisting of 13 haplotypes, the most frequent haplotype G02 is the ancestral haplotype.

ANALYSIS OF THE CYTOCHROME b GENE In order to confirm results of the control region analysis, the cytochrome b gene was sequenced in 12 C. auratus individuals with control region haplotypespossessing G02, L01,

M01 and A01 (three individuals of each haplotype), and in one C. carassius individual. In every three specimens from the same D-loop lineage an identical cytochrome b gene sequence was identified, therefore representative for each respective lineage. Alignment of sequences (1141 bp in length) showed presence of 106 variable sites (9,3 %), including. 25 parsimony informative. Genetic distances between haplotypes of the cytochrome b gene ranged from 1,96 to 7,23 %. Genetic distances of these lineages from C. carassius reached 10,55 to 10,86 % (Table IV).

DISCUSSION

ANALYSIS OF THE CONTROL REGION Analysis of the mitochondrial control region in 350 fish belonging to the Carassius auratus complex has proven presence of four distinct lineages on the territory of the Czech Republic and Slovakia. Those lineages were labelled A, G, L and M. The lineage G manifests substantial haplotype richness, while the other lineages are represented by a single haplotype each. In order to compare those lineages with existing taxonomic structure, we compared those haplotypes (the most frequent haplotype G02 was selected as a representative of the lineage G) with sequences available from the GenBank databases and also with additional publications regarding with the D-loop variability in the C. auratus. The haplotypes of the lineage G were found in most individuals with the silver crucian carp phenotype, diploids, triploids and a single tetraploid. The G02 haplotype is identical with sequences typical for the C. a. gibelio form in China (Li & Gui, 2008; GenBank accession numbers EF633642-EF633680). From the fact, that an identical haplotype occurs not only in Czech Republic and Slovakia, but also in Ukraine and Russia (Papoušek et al., unpublished) and in China, it can be deduced that this haplotype could be the basic (ancestral) haplotype typical for the C. a. gibelio not only in the Central Europe, but in other parts of its distribution area as well. The rest of the haplotypes belonging to the group G are most probably derivatives of this ancestral haplotype. Apart of the fish of the silver crucian carp phenotype, haplotypes of the G group were detected also in two individuals of shubunkin goldfish from commercial cultures and in a single shubunkin goldfish living at the locality no. 7 „Soutok“. Because of maternal inheritance of the mitochondrial DNA, those individuals could possibly represent hybrids originally descending from a cross of a C. a. gibelio female and a goldfish male, which had escaped (or had been released) into free waters. In artificial breeding of those

ornamental forms often occur unattractive discoloured specimens, which are often released into the wild, where they can hybridize with the C. a. gibelio. The L01 haplotype (lineage L) was detected in individuals from a single locality, an artificial wetland in the floodplain of the Morava River (locality no. 8 „Chomoutov“). Two clonal populations of triploid silver crucian carp females coexist in this wetland (Vetešník et al., 2007), whereas one population carries the G02 haplotype and the other carries the L01 haplotype. Aside from mitochondrial DNA, these populations are distinguishable also based on morphometric characters – body depth and number of gill rakers (Vetešník et al., 2007). In comparison with the GenBank database, the L01 haplotype is most similar to haplotypes of Japanese forms of Carassius auratus described by Murakami et al. (2001). This study divided Japanese Carassius into three main phylogenetic clusters: (i) cluster I consists exclusively of C. cuvieri (gengorobuna); (ii) cluster II included two haplotypes of polyploid C. a. langsdorfii and all haplotypes of the ornamental goldfish; and (iii) the most heterogenous cluster III, consisting of haplotypes of diploid and triploid C. a. langsdorfii (ginbuna), C. a. grandoculis (nigorobuna) and C. a. buergeri (nagabuna). Our haplotype L01 falls into the cluster III, sharing subcluster with haplotypes of diploid and triploid ginbunas from lakes Shiwa and Imba (Fig. 6). The haplotype lineage L can therefore be considered a representative of the Carassius auratus langsdorfii form. This form is genetically heterogenous and probably polyphyletic; Murakami et al. (2001) even states that the C. a. grandoculis (nigorobuna) and C. a. buergeri (nagabuna) forms could be simply morphological variants of the C. a. langsdorfii (ginbuna). With the finding of the lineage L, the C. a. langsdorfii has been detected on the territory of the Czech Republic for the second time, this time in form of numerous self-sustaining population. The first record by Kalous et al. (2007) consisted of two soliters. The M01 haplotype (lineage M) was detected in diploid fish (both males and females) wi the silver crucian carp phenotype at four localities in the Danube and Tisza basins (localities no. 7 „Soutok“, no. 10 „Trnava“, no. 12 „Duchonka“ and no. 19 „Zemplínska Šírava“). At the last mentioned locality the M01 haplotype even prevailed (22 out of 43 specimens). No close sequence has been found in comparison with the GenBank. Compared with Murakami et al. (2001), this haplotype does not fall into either of three clusters, forming an isolated lineage splitting from cluster II, which can be labelled as an independent cluster. With regards to relatively high genetic distance separating the lineage M from other lineages, it can be concluded that this lineage represents an independent form on the same taxonomic level

with C. a. gibelio, C. a. auratus a C. a. langsdorfii. Identity of these lineage is still unclear and will necessitate further, particularly morphological studies. The A01 haplotype (lineage A) was found in almost all examined specimens of commercially available goldfish strains and in almost all shubunkin goldfish living in free waters in the locality no. 7 „Soutok“, which supports evidence of monophyletic origin of the domesticated forms. In comparison with the GenBank database, this haplotype is identical in full length with sequences of C. a. auratus (i. e. complete mitochondrial genome of the ornamental wakin form from Japan z Japonska, accession number AB111951). Also when compared with Murakami et al. (2001), the A01 haplotype falls into cluster II containing haplotypes of ornamental goldfish from Japan. The lineage A can therefore be identified with the C. a. auratus form. Apart of specimens with characteristical goldfish phenotype, this haplotype was also detected in a single individual with the silver crucian carp phenotype from the Odra River basin. Analogically to „goldfish“ with the „G“ haplotype, it was most probably a hybrid resulting from mating of a silver crucian carp male with a goldfish female..

ANALYSIS OF THE CYTOCHROME b GENE Comparison of the cytochrome b sequences in selected individuals possessing G02, L01, M01, A01 and Carassius carassius with sequences acquired from GenBank (sequences of the C. a. buergeri and C. a. grandoculis have not been available) resulted in phylogenetic trees of identical topology (Fig. 7), confirming that the lineage G corresponds to C. a. gibelio, the lineage A corresponds to C. a. auratus, the lineage L is close to C. a. langsdorfii and the lineage M is relatively isolated part of the C. auratus complex.

CONCLUSIONS A hypothesis, that area of Danube is inhabited by several forms or species named „C. auratus“, has been proposed by Holčík & Žitňan (1978). Authors deduced this from variable biological properties (growth rate, sex ratio) and speculated diverse geographical origin of those forms. They assume that the expansion of the C. a. gibelio in the Danube basin proceeded from three source centres: 1) Ponds in the Körös River drainage area (Hungary), where the C. a. gibelio had been imported in 1954 from Bulgaria, 2) Bulgaria itself, particularly tributaries of the Danube (those fish probably originated from imports from the USSR), 3) delta and lower reaches of the Danube on the territory of Romania (Holčík & Žitňan, 1978). In the opinion of Holčík & Žitňan (1978), Czechoslovakian populations originated from the Hungarian import.

Apart from native crucian carp (C. carassius), Kalous et al. (2007) distinguished in the territory of the Central Europe three more species: the goldfish C. auratus, the Prussian carp C. gibelio, and C. langsdorfii. Our present data suggest presence of four forms, which we classify as forms of the Carassius auratus complex. The origin of the „Japanese“ forms of the silver crucian carp (C. a. langsdorfii and a close form represented by our lineage M) in Europe has not been reliably proved yet. One of possible theories states that these forms have been accidentally introduced as an admixture together with imports of the „koi“ carps (Kalous et al., 2007). However, dispersion of these forms across various drainages (Vltava, Morava, Danube-Nitra, Tisa) is surprising. It can not be ruled out that these forms have been imported from China to Romania together with other species (Holčík, 1980) and such could have become a minor part of occupational populations otherwise consisting mostly of the C. a. gibelio form. Those „Japanese“ forms could have preserved their limited dispersive occurrence, and locally they can constitute a self-sustaining population, like the population of the Chomoutov wetland (Vetešník et al., 2007).

Acknowledgements

This study was supported by grant project of Grant Agency of Czech Republic no. 206/05/2159 and by grant project of the the Research Programme of the Ministry of Environment of the Czech Republic No. SP/2d4/55/07. Authors are thankful to Ladislav Pekárik for providing samples of the Carassius auratus from the Poprad River..

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Table I. Carassius auratus sampling localities and numbers of samples. No. locality water course basin sea country No. of samples 1 Střekov Labe 15 2 Novořecká bašta Lužnice 3 3 Soběslav Lužnice Labe North 5 4 Vodňany Blanice 11 Czech 5 Blatná Blatná ponds 7 Republic 6 CHKO Poodří Odra Odra Baltic 17 7 Soutok Dyje/Morava 103 (11)* 8 Chomoutov artificial wetland Danube Black 41 9 Hrdibořice Hrdibořice ponds 2 10 Trnava artificial pond 7 11 reservoir Kolíňany Bocegaj 4 12 reservoir Duchonka Železnica Danube 9 13 Kalinovo Ipeľ 5 14 Papradno Papradnianka 6 15 Seňa Belžanský creek 1 16 Perín Perín ponds Black 19 Slovakia 17 Veľké Trakany Tisa 6 18 Hraň Trnávka 10 Tisa 19 Zemplínská Šírava Čierna voda 43 20 Oborín Starý Laborec 12 21 Sv.Mária channel „Radský“ 2 22 Státní hranice channel „Panelový“ 4 23 Kežmarok Poprad Visla Baltic 6

* - Eleven fish from the locality no. 7 „Soutok“ possessed goldfish phenotype

Table II. Haplotypes and sequence variation of the control region in examined Carassius auratus.

Variable site 111 1112222222 2333333333 3334444444 Haplotype silver "wild" goldfish goldfish total 3445566777 8888888888 9999999244 5560223477 9001112345 5670122677 7130707789 0123456789 0123467618 1323599557 9890162962 6649634669 G01 17 17 GACATA-TAT TACATTAATG CATTATATAG ATAATGTCTC ACTAATGAGT TTGTCATAGC G02 161 2 163 ...... A...... GC...... T..... G03 3 3 ...... A...... A...A ....C...... T..... G04 35 1 36 ...... A------...... GC...... T..... G05 2 2 ...... A...... GC...C...... T..... G06 17 17 ...... A...... GC...... G07 11 11 ...... -...... GC...... T..... G08 1 1 ...... -...... GC...... GT....A G09 7 7 ...... A...... GC...... GT..... G10 1 1 ...... -...... GC...... GT..... G11 5 5 ....C.A...... GC...... T..... G12 3 3 ...... A... ..A...... GC...... T..... G13 8 8 ...... -...... C...... A01 1 10 10 21 ...G..A...... G.. T...... A.A .CG.C.CT.. G.C-...... CA...C... L01 25 25 -G-..GA...... T...... GA TC..CA.T.T .TC-G.TGAC C.AG...G.. M01 30 30 ...... A...... T.....G..A TC..C..T.T .....C.GA- ..AG.TC.T.

total 327 11 12

Fish origin: silver – silver crucian carp from from free waters; „wild“ goldfish – fish from the locality no. 7 „Soutok“ with goldfish phenotype; goldfish – comercially available goldfish from local fish dealers. Values show number of tested individuals. Dots indicate identical nucleotides and dashes indicate gaps in comparison with the haplotype G01, respectively. All haplotypes reported here will appear in the GenBank database with accession numbers FJ167410-FJ167425.

Table III. Observed numbers and ploidy characteristics of individual haplotypes in the waters of the Czech Republic and Slovakia. Czech Republic Slovakia fish Ploidy Haplotype total Labe Odra Danube Danube Tisa Visla dealers 2n 3n 4n G01 6 3 7 1 17 x x x G02 32 5 67 25 31 1 2 163 x x G03 3 3 x G04 7 3 26 36 x x G05 2 2 x G06 1 4 8 4 17 x G07 1 10 11 x x G08 1 1 x G09 4 1 2 7 x x G10 1 1 x G11 3 2 5 x G12 3 3 x G13 1 7 8 x A01 1 10 10 21 x L01 25 25 x M01 5 3 22 30 x total 350

Table IV. Average pairwise genetic distances among four lineages of the Carassius auratus complex and Carassius carassius in the Czech Republic and Slovakia. G A L M C.carassius G x 0,0196 0,0703 0,0420 0,1076 A 0,0338 x 0,0723 0,0468 0,1055 L 0,0487 0,0494 x 0,0693 0,1076 M 0,0372 0,0379 0,0316 x 0,1086 C.carassius 0,0612 0,0665 0,0602 0,0531 x

Over diagonal: distances in the cytochrome b gene, under diagonal: distances in the control region.

Fig. 1. Carassius auratus sampling locations in the Czech Republic and Slovakia. More information about localities can be found in the Table I.

Fig. 2. Phylogenetic tree for sixteen D-loop haplotypes of Carassius auratus complex in the Czech Republic and Slovakia computed by neighbour-joining algorithm. Numbers at the nodes represent estimated per cent bootstrap support. Branch lengths are in scale.

Fig. 3. Phylogenetic tree for sixteen D-loop haplotypes of Carassius auratus complex in the Czech Republic and Slovakia computed by maximum parsimony algorithm (CI = 0,760, RI = 0,519).

Fig. 4. Consensual phylogenetic tree for sixteen D-loop haplotypes of Carassius auratus complex in the Czech Republic and Slovakia inferred by Bayesian analysis. Numbers at the nodes represent posterior probabilities of topology. Branch lengths are in scale.

Fig. 5. Unrooted haplotype network for sixteen D-loop haplotypes of Carassius auratus complex. Branch lengths are in approximate scale to number of mutation changes between haplotypes, except for G04 (deletion of 17 bp shown as a single mutational change). Mv1-8 – hypotetical intermediate haplotypes.

Fig. 6. Neighbour-joining tree for comparison of L01, M01 and A01 haplotypes (black dots) with 37 haplotypes of Carassius spp. from Japan (1-37). Numbers at the nodes represent estimated per cent bootstrap support. Only values greater than 50 per cent are shown. Branch lengths are in scale.

Fig. 7. Relationships among found phylogenetic groups (black dots) and forms of the C. auratus complex based on the cytochrome b gene. Numbers at the nodes represent estimated bootstrap support (NJ/MP). Branch lengths are in scale.