Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(4): 61-67, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-160 Citation: M. Alemdag, R.C. Ozturk, S.A. Sahin, I. Altinok (2019) Karyo- types of Danubian lineage brown trout and their hybrids. Caryologia 72(4): 61-67. doi: 10.13128/caryologia-160 Published: December 23, 2019 Copyright: © 2019 M. Alemdag, R.C. Ozturk, S.A. Sahin, I. Altinok. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Karyotypes of Danubian lineage brown trout and their hybrids Melike Alemdag, Rafet Cagri Ozturk, Sebnem Atasaral Sahin, Ilhan Altinok* Department of Fisheries Technology Engineering, Surmene Faculty of Marine Sciences, Karadeniz Technical University, 61530 Surmene, Trabzon, Turkey *Corresponding author: ialtinok@ktu.edu.tr Abstract. Cytogenetic analysis of brown trout, Salmo trutta, have been described for different populations and morphs; however, cytogenetic analysis of interspecific brown trout hybrids is unknown. Cultured kidney cells from four brown trout subspe- cies (Salmo trutta abanticus, S.t. caspius, S.t. fario and S.t. labrax) and their reciprocal hybrids were karyotyped using conventional staining, C-banding and Ag-NOR staining techniques. Chromosome number (2N) and chromosome arm number (NF) ranged from76 to 80 and 98 to 102, respectively. Silver staining revealed the presence of NOR sites on the short arm of the submetacentric chromosome. The size and number of NOR sites showed uniformity. The presence of heterochromatin on different chromo- some arms was confirmed by C-banding. The presence and position of constitutive heterochromatin showed variability among individuals. Chromosome structures of purebred brown trout subspecies belonging to the Danubian linage and their hybrids were similar, and no distinctive characteristics were observed in any of the species. The results of this study are applicable to the development of improved conservation and management strategies for brown trout. Keywords. Cytogenetic, Karyotype, Salmo trutta, Ag-NOR, C-banding. INTRODUCTION Brown trout, Salmo trutta (Linnaeus, 1758), is a polymorphic and wide- spread species. Its historic geographic range covers Europe, Western Asia and Northern Africa. During the past century, Salmo trutta have been introduced to different parts of the world, and the range of brown trout has been extended to all continents except Antarctica (Elliott, 1989). The systematic classification of Salmo trutta is plagued by many nomenclatu- ral issues. Salmo trutta was once recognized as a polymorphic species with three morphs based on life-history variation: resident trout, lake trout and river trout (Ferguson, 2004). Mitochondrial DNA (mtDNA) sequence varia- tion analysis revealed the existence of five major phylogenetic groups, which are believed to have been separated for some 500,000 to 2 million years (Ber- natchez, 1995). Over the years, distinct species or nominal subspecies have 62 Melike Alemdag et al. been described based on morphological and molecular analysis (Kottelat & Freyhof, 2007; Turan, Kottelat, & Engin, 2014). However, S. trutta subspecies such as S.t. abanticus, S.t. caspius, S.t. fario and S.t. labrax belong- ing to Danubian lineage have been proved to be a single biological species called Salmo trutta. Thus, it was rec- ommended that strains should be named according to location, such as Abant, Caspian, Anatolian and Black Sea (Kalayci et al., 2018). Inter- and intraspecific hybridization experiments in fish are often less concerned with identification of the genomic composition than with the evolution of perfor- mance and survival (Johnson & Wright, 1986). Morphol- ogy and variation in chromosome number have been proven useful in identifying fish populations (Phillips, 2005). Cytogenetically, the Salmo trutta complex is one of the best analyzed salmonid. The karyotype of Salmo trutta consists of 80 chromosomes with a fundamen- tal arm number (NF) ranging from 98 to 102 (Amaro, Abuin, & Sanchez, 1996; Woznicki, Jankun, & Luc- zynski, 1998; Woznicki, Sanchez, Martinez, Pardo, & Jankun, 2000). Although Salmo trutta have been sub- jected to numerous cytogenetic analyses, and karyo- types have been described for different populations and morphs, (Caputo, Giovannotti, Cerioni, Splendiani, & Olmo, 2009; Jankun, 2000; Kalbassi, Dorafshan, Tava- kolian, Khazab, & Abdolhay, 2006; Northland-Leppe, Lam, Jara-Seguel, & Capetillo-Arcos, 2009; Woznicki, Jankun, & Luczynski, 1997; Woznicki et al., 1998), the chromosome complement of interspecific brown trout hybrids seems to be comparatively less studied (Polonis, Fujimoto, Dobosz, Zalewski, & Ocalewicz, 2018; Ziomek, Debowska, Hliwa, & Ocalewicz, 2016). A cytogenetic characterization of hybrids and parental species would aid in a better understanding of their species status. Therefore, the aim of the present study was 1) to deter- mine the chromosomal characteristics of Abant trout (S.t. abanticus), Black Sea trout (S.t. labrax), Caspian trout (S.t. caspius), Anatolian trout (S.t. fario) and their reciprocal hybrids and 2) to determine if the NF of chro- mosomes varies among purebred and hybrid trout. MATERIALS AND METHODS Fish Abant, Anatolian, Black Sea and Caspian trout were crossed to each other to produce the F1 generation of all possible reciprocal crossing combinations (16 cross- types) (Table 1). After fertilization, each family was sep- arately incubated in a vertical incubator and transferred to a separate flow-through indoor tank after hatching. This study was approved by the Institutional Animal Care and Use Committee at Karadeniz Technical Uni- versity (approval #14/2013). Chromosome Preparation Five fish from each cross-type were used in chromo- some analysis (Table 1). Fish were anaesthetized with ice, and their anterior kidney tissue was sampled on ice. Tis- sue was cut into small pieces and incubated in 1.5 ml of RPMI media supplemented with penicillin G (75 U/ml), fungizone (1.5 μg/ml), gentamycin sulphate (30 μg/ml) and streptomycin sulphate (75 μg/ml) for 24 h at room temperature. Supplementing the culture media with antibiotics eliminated any growth of fungi, yeasts, myco- plasma and Gram-positive and Gram-negative bacteria. After incubation of the tissue with colchicine (0.1%) for 1 h, samples were centrifuged at 1000 x g for 10 min, and the supernatant was removed. Pellets were resuspended in 3 ml ice-cold 0.075 mol/l KCl solution, incubated at 4ºC for 30 min and then four drops of ice-cold Carnoy fixative (methanol: acetic acid, 3:1) were added. Samples were centrifuged at 1000 x g for 10 min, and the super- natant was removed. After that, 5 ml of fixative was add- ed to the sample, which was then centrifuged at 1000 x g for 10 min. This step was repeated three times to wash the cells. Tissues were transferred to a petri dish with one milliliter of fixative and then cut into small pieces with a surgery blade. Slides were placed over boiled Table 1. Cross-types of fish and their abbreviation, mean length and weight. Crosses (female X male) Family Abbreviation Mean Length (cm) Mean Weight (gr) S.t labrax X S.t. labrax LL 18.63±1.41 69.18±5.25 S.t. labrax X S.t. abanticus LA 19.70±1.50 71.51±5.31 S.t. labrax X S.t. caspius LC 24.36±1.81 156.0±10.12 S.t. abanticus X S.t. abanticus AA 17.37±1.28 38.84±3.00 S.t. abanticus X S.t. labrax LL 16.45±1.11 48.58±3.41 S.t. abanticus X S.t. caspius LA 15.20±1.08 34.78±2.04 S.t. caspius X S.t. labrax LC 15.88±1.12 41.70±3.06 S.t. caspius X S.t. abanticus AA 11.62±0.84 13.67±0.07 S.t. caspius X S.t. caspius LL 12.54±0.92 18.30±1.025 S.t. fario X S.t. fario FF 7.15±0.41 5.11±1.01 S.t. fario X S.t. abanticus FA 6.01±0.28 5.09±0.09 S.t. fario X S.t. caspius FC 5.12±0.17 4.81±0.41 S.t. fario X S.t. labrax FL 6.57±0.65 4.51±0.46 S.t. abanticus X S.t. fario AF 7.24±0.47 5.11±1.06 S.t. caspius X S.t. fario CF 5.03±0.21 4.24±0.38 S.t. labrax X S.t. fario LF 7.31±0.58 5.19±0.91 63Karyotypes of Danubian lineage brown trout and their hybrids water steam, and three drops of cell suspension were dropped onto slides from a height of 30–40 cm. For each fish species, a total of 15 slides were prepared and air dried, and 5 of them were stained with 10% Giemsa. The remaining 10 were used for C-banding (5 slides) and Ag- NORs analysis as explained below. C-banding was performed according to the method described by Sumner (1972), with slight modifications. Slides containing the chromosome preparation were treated with 0.2 mol/l HCl solution at 37ºC for 1 h and rinsed with distilled water. Washed slides were incubat- ed in 2X SSC (pH 7.0) at 60ºC for 1 h, rinsed with dis- tilled water and finally stained with 10% Giemsa for 20 min. Silver staining of nucleus organizer regions (Ag- NORs) were performed according to the method described by Howell and Black (1980). Two drops of colloidal developer and a single drop of aqueous silver nitrate were dropped onto a slide on which the chromo- some preparation was mounted and covered with a cover glass. The slide was incubated at 70ºC until the silver- staining mixture turned a golden-brownish color. The slides were then rinsed with distilled water, air dried and stained with 10% Giemsa. Metaphase cells were screened with a fully automat- ed karyotyping software system (CytoVision ver. 3.92) connected to an Olympus light microscope. Metaphase cell photos were captured at 100x magnification for fur- ther analysis. Ten high-quality metaphase spreads from each slide were used in chromosome analysis. Image-Pro Premier (Media Cybernetics), SmartType 3.1.0.43 (Digi- tal Scientific, Cambridge, UK) and tpsDig2 v2.26 (New York State University, Stony Brook, USA) were used in karyotyping. The NF value was estimated by counting biarmed (metacentric and submetacentric) and unarmed (acrocentric and subtelocentric) chromosomes and cal- culated according to the formula given by Naran (1997). RESULTS The chromosome numbers and structures of four subspecies of brown trout and their cross-types (n = 16) were successfully determined. Furthermore, karyogram and chromosome measurement tables were generated. About 500 metaphase plates from 80 individuals were examined. Cross-types were karyotyped based on the representative chromosome image (Fig. 1) and chromo- some arm scale (Table 2). Diploid chromosome numbers (2N) of all examined cross-types ranged from 76 to 80, but the majority of cross-types had 2N = 80 chromo- somes (Table 3). The pure breed LL (see Table 1 for abbre- viation) and the hybrid CA had 76 chromosomes, while CL had 78 chromosomes The NF varied from 96 to 102, the lowest being obtained from CL (96) followed by CC, LL and CA (98) (Table 3). Metacentric (M), submetacen- tric (SM) and acrocentric/telocentric (A/T) chromosome numbers varied from 14 to 18, 4 to 8, 2 to 14 and 46 to 56, respectively, among cross-types (Table 3). Ag-NOR staining revealed the presence of one pair of NOR sites on the short arm of the SM chromosome in all the analyzed specimens (Fig 2). C-banding showed constitutive heterochromatin at the centromeres and arms of most of the chromosomes (Fig. 3) and the pres- ence and position of constitutive heterochromatin with- in cross-types were variable even in pure breeds (Fig. 3). C-banding was not discriminative for brown trout sub- species. DISCUSSION Several cytogenetic methods of chromosome isola- tion have been developed. The main objective of all such methods is to obtain cells at the metaphase stage by dis- rupting the cell spindle (Pack, 2002). Solid tissues and cultured cells, together with colchicine treatment, are the most common sources of samples for the preparation of slides of fish chromosomes. Spleen, kidney, liver, gills and scales are the preferred sources of chromosomes. To prepare chromosomes, we first used the solid-tissue technique by harvesting various fish tissues and then empirically tested the colchicine concentration, expo- sure method (injection and bath) and fixation duration to obtain the most efficient means of chromosome prep- aration. Despite our efforts, we were unable to prepare metaphase plates for all but a couple of samples. With Figure 1. Karyotype of Abant trout Salmo t. abanticus (2N=80) stained conventionally with Giemsa. Metacentric (M), submetacen- tric (SM), subtelocentric (ST), acrocentric and telocentric chromo- some (A/T) of cross-types. 64 Melike Alemdag et al. the cell culture technique as described in the Materials and Methods section, we were able to obtain numerous well-spread metaphase chromosomes. The solid-tissue technique is applicable to various eukaryotic organisms (Kligerman & Bloom, 1977), but we favor the culture technique when working with salmonid fish, especially Salmo trutta. The ty pical kar yoty pes of all three ecological forms of Salmo trutta (2N = 80 and NF = 100 – 102) were found, in agreement with numerous other studies Table 2. Relative arm lenght (µ), total lenght (µ), arm ratio (p/q) and chromosome type of Abant trout. Chromosome number (2n) Short arm length (p) Long arm length(q) Total Lenght Arm ratio (q/p) Chromosome Type 1 0.12 0.12 0.24 1.00 M 2 0.12 0.12 0.24 1.00 M 3 0.12 0.12 0.24 1.00 M 4 0.12 0.12 0.24 1.00 M 5 0.90 0.90 1.80 1.00 M 6 0.10 0.10 0.20 1.00 M 7 0.80 0.80 1.70 0.89 M 8 0.05 0.12 0.17 2.40 SM 9 0.07 0.13 0.20 1.86 SM 10 0.05 0.10 0.15 2.00 SM 11 0.03 0.12 0.15 4.00 ST 12 0.06 0.19 0.25 3.17 ST 13 0.02 0.13 0.15 6.50 ST 14 0.00 0.22 0.22 ∞ A 15 0.00 0.09 0.09 ∞ A 16 0.00 0.14 0.14 ∞ A 17 0.00 0.12 0.12 ∞ A 18 0.00 0.14 0.14 ∞ A 19 0.00 0.15 0.15 ∞ A 20 0.00 0.11 0.11 ∞ A 21 0.00 0.11 0.11 ∞ A 22 0.00 0.11 0.11 ∞ A 23 0.00 0.11 0.11 ∞ A 24 0.00 0.11 0.11 ∞ A 25 0.00 0.12 0.12 ∞ A 26 0.00 0.10 0.10 ∞ A 27 0.00 0.11 0.11 ∞ A 28 0.00 0.10 0.10 ∞ A 29 0.00 0.08 0.08 ∞ A 30 0.00 0.10 0.10 ∞ A 31 0.00 0.12 0.12 ∞ A 32 0.00 0.12 0.12 ∞ A 33 0.00 0.11 0.11 ∞ A 34 0.00 0.11 0.11 ∞ A 35 0.00 0.07 0.07 ∞ A 36 0.00 0.08 0.08 ∞ A 37 0.00 0.08 0.08 ∞ A 38 0.00 0.10 0.10 ∞ A 39 0.00 0.08 0.08 ∞ A 40 0.00 0.13 0.13 ∞ A Table 3. Chromosome number (N) fundamental number (NF) and structure [metacentric (M), submetacentric (SM), subtelocentric (ST), acrocentric and telocentric chromosome (A/T)] of cross-types. Cross- type M SM ST A/T N NF AA 14 8 2 56 80 102 CC 14 4 4 58 80 98 LL 16 6 4 50 76 98 FF 14 6 4 56 80 100 AC 16 4 8 52 80 100 AL 16 6 2 56 80 102 CA 16 6 6 48 76 98 CL 14 4 8 52 78 96 LA 16 4 14 46 80 100 LC 18 4 2 56 80 102 AF 18 4 2 56 80 102 FA 16 4 4 56 80 100 FC 18 4 4 54 80 102 CF 16 4 6 54 80 100 LF 16 6 4 54 80 102 FL 16 4 6 54 80 100 Figure 2. Karyotype of Abant trout Salmo t. abanticus with silver staining. Presence of NOR sites on the short arm of the submeta- centric chromosome indicated with red ring. 65Karyotypes of Danubian lineage brown trout and their hybrids (Woznicki et al., 1998). This study documented slight karyotype variation among cross-types, with a diploid chromosome number and NF ranging from 76 to 80 and 98 to 102, respectively, while the majority of the cross- types exhibited 2N = 80, in agreement with previous reports (Woznicki et al., 1998). Intra-specific variation in both chromosome number and NF was previously docu- mented among different fish species, including brown trout (Gjedrem, Eggum, & Refstie, 1977). Intra-specific variation in chromosome numbers in these trout forms and their hybrids suggest centric fusion between acro- centric chromosome pairs during the karyotype evolu- tion of Robertsonian translocation. Loss of chromosome number due to counting errors and chromosome loss during preparation of slides is within the bounds of pos- sibility (Gold & Gall, 1975; Zenzes & Voiculescu, 1975). Allopolyploids have genomes from different species; therefore, it is associated with hybridization. Allopoly- ploidy can be occurred in the nature as a results of interspecific or intergeneric hybridizations and offspring holds two different diploid chromosome sets (Zhou & Gui, 2017). Consequence of interhomolog recombination in genomic rearrangements can cause gene losses, and gametic aneuploidy (Hollister, 2015). Polymorphic NOR size is common in fish and par- ticularly in salmonids (Gold, 1984; Woznicki & Jankun, 1994). The NORs are commonly located on chromosome pair number 11 in Salmo trutta, but multichromosomal NOR-site polymorphism and variation in NOR size has also been reported (Sanchez, Martinez, Vinas, & Bouza, 1990; Schmid et al., 1995; Zhuo, Reed, & Phillips, 1995). In our study, the positions of NORs showed remarkable uniformity among individuals and cross-types. We could not detect any variation in the size and number of NORs. Chromosomal characteristics of brown trout hybrids were studied for the first time in the present study. Chromosome structures of purebred brown trout sub- species (S.t. abanticus, S.t. caspius, S.t. fario and S.t. lab- rax) belonging to the Danubian linage and their hybrids were similar, and no distinctive characteristic was observed in any of the species. Therefore, they should be the same species but different strains. This statement was confirmed by Kalayci et al. (2018). They found that S.t. abanticus, S.t. caspius, S.t. fario and S.t. labrax are single biological species which should be called Salmo trutta. The results of this study are applicable to the develop- ment of improved conservation and management strat- egies for brown trout. Brown trout population in the nature is very low and governmental fisheries agencies are releasing hatchery reared brown trout to the stream or rivers to restore the population. Therefore, extra pre- caution should be should be taken in order to protect local brown trout population genetics ACKNOWLEDGEMENTS This study was funded by the Scientific and Tech- nological Research Council of Turkey (TUBITAK: 214O595). DISCLOSURE STATEMENT The authors declare that they have no conflict of interest REFERENCES Amaro, R., Abuin, M., & Sanchez, L. (1996). Chromo- somal evolution in salmonids: A comparison of Atlantic salmon, brown trout, and rainbow trout R-band chromosomes. Genetica, 98(3), 297-302. doi:10.1007/Bf00057594 Bernatchez, L. (1995). A role for molecular systematics in defining evolutionarily significant units in fishes. Evolution and the Aquatic Ecosystem: Defining Unique Units in Population Conservation, 17, 114-132. Caputo, V., Giovannotti, M., Cerioni, P. N., Splendiani, A., & Olmo, E. (2009). Chromosomal study of native and hatchery trouts from Italy (Salmo trutta com- plex, Salmonidae): conventional and FISH analysis. Cytogenetic and Genome Research, 124(1), 51-62. doi: 10.1159/000200088 Elliott, J. M. (1989). Wild Brown Trout Salmo-Trutta - an Important National and International Resource. Freshwater Biology, 21(1), 1-5. doi: 10.1111/j.1365- 2427.1989.tb01343.x Figure 3. C-banded karyotype of Abant trout Salmo t. abanticus. Constitutive heterochromatin at the centromeres and arms of most of the chromosomes. 66 Melike Alemdag et al. Ferguson, A. (2004). Brown trout genetic diversity: ori- gins, importance and impacts of supplemental stocking. Paper presented at the Proceedings of the Institute of Fisheries Management 34th Annual Study Course. Gjedrem, T., Eggum, A., & Refstie, T. (1977). Chromosomes of Some Salmonids and Salmonid Hybrids. Aquaculture, 11(4), 335-348. doi: 10.1016/0044-8486(77)90083-7 Gold, J. R. (1984). Silver-Staining and Heteromorphism of Chromosomal Nucleolus Organizer Regions in North-American Cyprinid Fishes. Copeia(1), 133- 139. doi: 10.2307/1445043 Gold, J. R., & Gall, A. E. (1975). Chromosome cytol- ogy and polymorphism in the Californian high sierra golden trout (Salmo aguabonita). Canadian Journal of Genetics and Cytology, 17, 41-53. Hollister, J.D. 2015. Polyploidy: adaptation to the genom- ic environment. New Phytologist (2015) 205: 1034– 1039. doi: 10.1111/nph.12939 Howell, W. M., & Black, D. A. (1980). Controlled Silver- Staining of Nucleolus Organizer Regions with a Pro- tective Colloidal Developer - a 1-Step Method. Expe- rientia, 36(8), 1014-1015. doi: 10.1007/Bf01953855 Jankun, M. (2000). Standard karyotype of sea trout (Sal- mo trutta morpha trutta) based on replication band- ing patterns. Cytobios, 103(403), 79-89. Johnson, K. R., & Wright, J. E. (1986). Female Brown Trout X Atlantic Salmon Hybrids Produce Gyno- gens and Triploids When Backcrossed to Male Atlantic Salmon. Aquaculture, 57(1-4), 345-358. doi: 10.1016/0044-8486(86)90213-9 Kalayci, G., Ozturk, R. C., Capkin, E., & Altinok, I. 2018. Genetic and molecular evidence that brown trout Salmo trutta belonging to the Danubian lineage are a single biological species. Journal of Fish Biology, 93, 792-804. doi: 10.1111/jfb.13777 Kalbassi, M. R., Dorafshan, S., Tavakolian, T., Khazab, M., & Abdolhay, H. (2006). Karyological analysis of endangered Caspian salmon, Salmo trutta caspius (Kessler, 1877). Aquaculture Research, 37(13), 1341- 1347. doi: 10.1111/j.1365-2109.2006.01560.x Kligerman, A. D., & Bloom, S. E. (1977). Rapid Chromo- some Preparations from Solid Tissues of Fishes. Jour- nal of the Fisheries Research Board of Canada, 34(2), 266-269. doi: 10.1139/f77-039 Kottelat, M., & Freyhof, J. (2007). Handbook of European Freshwater Fishes. Cornol, Switzerland Naran, D. (1997). Cytogenetic studies of Pseudobarbus and selected Barbus (Pısces: Cyprinidae) of Southern Afrıca. (Master of Science), Rhodes University, Gra- hamstown, South Africa. Northland-Leppe, I., Lam, N., Jara-Seguel, P., & Capetil- lo-Arcos, J. (2009). Chromosomes And Ag-Nor Loca- tion in Fluviate Populations of Salmo trutta fario L. 1758 (Salmoniformes: Salmonidae) From Atacama Desert, Chile. Gayana, 73(1), 45-48. Pack, S. D., Stratakis, C.A. (2002). Chromosomes: MEth- od for preperation Encyclopedia of life sciences. New Jersey: John Wiley and Sons. Phillips, R. (2005). Chromosome morphology. In F. K. Cadrin SX, Waldman JR (Ed.), Stock Identification Methods (pp. 273-294). Amsterdam: Elsevier Aca- demic Press. Polonis, M., Fujimoto, T., Dobosz, S., Zalewski, T., & Ocalewicz, K. (2018). Genome incompatibility between rainbow trout (Oncorhynchus mykiss) and sea trout (Salmo trutta) and induction of the inter- species gynogenesis. Journal of Applied Genetics, 59(1), 91-97. doi: 10.1007/s13353-017-0425-2 Sanchez, L., Martinez, P., Vinas, A., & Bouza, C. (1990). Analysis of the Structure and Variability of Nucleolar Organizer Regions of Salmo-Trutta by C-, Ag-, and Restriction Endonuclease Banding. Cytogenetics and Cell Genetics, 54(1-2), 6-9. doi: 10.1159/000132944 Schmid, M., Feichtinger, W., Weimer, R., Mais, C., Bola- nos, F., & Leon, P. (1995). Chromosome-Banding in Amphibia .21. Inversion Polymorphism and Multi- ple Nucleolus Organizer Regions in Agalychnis-Cal- lidryas (Anura, Hylidae). Cytogenetics and Cell Genet- ics, 69(1-2), 18-26. doi: 10.1159/000133929 Sumner, A. T. (1972). Simple Technique for Demon- strating Centromeric Heterochromatin. Experimen- tal Cell Research, 75(1), 304-&. doi: 10.1016/0014- 4827(72)90558-7 Turan, D., Kottelat, M., & Engin, S. (2014). Two new spe- cies of trouts from the Euphrates drainage, Turkey (Teleostei: Salmonidae). Ichthyological Exploration of Freshwaters, 24(3), 275-287. Woznicki, P., & Jankun, M. (1994). Chromosome Poly- morphism of Atlantic Salmon (Salmo-Salar) from the River Dzwina, Baltic Sea Basin - Arm Length and nor Location Variation of the 8th Chromosome. Canadian Journal of Zoology-Revue Canadienne De Zoologie, 72(2), 364-367. doi: 10.1139/z94-050 Woznicki, P., Jankun, M., & Luczynski, M. (1997). Chro- mosome studies in brown trout (Salmo trutta m. far- io) from Poland: hypothetical evolution of the 11th, 12th and 14th chromosome pairs in the Salmo kary- otype. Cytobios, 91(366-67), 207-214. Woznicki, P., Jankun, M., & Luczynski, M. (1998). Chro- mosome polymorphism in Salmo trutta morpha lacustris from Poland, Wdzydze Lake population: Variation in the short arm length of chromosome eleven. Aquatic Sciences, 60(4), 367-375. doi: 10.1007/ s000270050047 67Karyotypes of Danubian lineage brown trout and their hybrids Woznicki, P., Sanchez, L., Martinez, P., Pardo, B. G., & Jankun, M. (2000). A population analysis of the structure and variability of NOR in Salmo trutta by Ag, CMA(3) and ISH. Genetica, 108(2), 113-118. doi: 10.1023/A:1004055125295 Zenzes, M. T., & Voiculescu, I. (1975). C-Banding Pat- terns in Salmo-Trutta, a Species of Tetraploid Origin. Genetica, 45(4), 531-536. doi: 10.1007/Bf01772875 Zhuo, L., Reed, K. M., & Phillips, R. B. (1995). Hypervar- iability of Ribosomal DNA at Multiple Chromosomal Sites in Lake Trout (Salvelinus-Namaycush). Genome, 38(3), 487-496. doi: 10.1139/g95-064 Zhou, L., & Gui, J. 2017. Natural and artificial polyploids in aquaculture. Aquaculture and Fisheries 2, 103-111. doi: 10.1016/j.aaf.2017.04.003 Ziomek, E., Debowska, M., Hliwa, P., & Ocalewicz, K. (2016). Impaired gonadal development in the sea trout (Salmo trutta) x Atlantic salmon (Salmo salar) F1 hybrid females. Oceanological and Hydrobiological Studies, 45(3), 337-343. doi: 10.1515/ohs-2016-0028 Substantia An International Journal of the History of Chemistry Vol. 2, n. 1 - March 2018 Firenze University Press