Introduction: Comparative cytogenetics is a vital approach for diagnosing chromosome abnormalities and identifying species-specific patterns. In this study, chromosomal analysis of three Anatolian endemic Cobitis species was performed: Cobitis bilseli, C. fahireae, and C. turcica. Methods: Conventional cytogenetic techniques such as Giemsa staining, C-banding, and Ag-NOR staining were applied, followed by measurements of chromosome arm lengths including analysis of the measured data. Results: The diploid chromosome number, 2n = 50, was determined for all three species. The karyotype formulas were as follows: four pairs of metacentric, 5 pairs of submetacentric, and 16 pairs of subtelo-telocentric chromosomes in C. bilseli; 11 pairs of metacentric, 7 pairs of submetacentric, and 7 pairs of subtelo-telocentric chromosomes in C. fahireae; and 4 pairs of metacentric, 4 pairs of submetacentric, and 17 pairs of subtelo-telocentric chromosomes in C. turcica. Dark C-bands were observed on the pericentromeres of nearly all chromosomes in C. bilseli and C. turcica, whereas light C-bands appeared on the pericentromeres of some chromosomes in C. fahireae. Silver-stained metaphases revealed signals on the short arm of a submetacentric chromosome pair in C. fahireae (each homologous chromosome carries one signal), while in C. bilseli and C. turcica, Ag-NOR signals were detected on the long arm of a single metacentric chromosome (only one homologous chromosome carries the signal, and the signal-carrying chromosome is the largest chromosome in the karyotype). Conclusion: This study provides new cytogenetic data consistent with the phylogenetic distances between the studied species, indicating that pericentric inversions and/or translocations govern the formation of Cobitis karyotypes.

The spined loaches Cobitidae are a species-rich group of small freshwater fishes [1]. Representatives of the genus Cobitis Linnaeus, 1758 show very little morphological variation, making it difficult to distinguish some species from others [2]. A comparative experimental taxonomy approach has been used in Cobitis loaches, applying genetic and molecular methods for identification of species [3]. Chromosome studies have shown that most Cobitis species have a diploid chromosome number of 2n = 50 (with the only exception being Cobitis taenia 2n = 48 [4]) but highly diversified karyotypes [3]. A karyotype is an arrangement of chromosomes into individual morphological categories based on the centromeric index (i). Morphological categories are generally divided into meta-, submeta-, subtelo-, acro-, and telocentric [5]. Differences that occurred during the karyotype evolution in Cobitidae play an important role in speciation [3]. Cytogenetics may provide a powerful tool in the complex systematics and taxonomy of spined loaches of the genus Cobitis [6]. In addition to the fish genus Cobitis, karyotype descriptions can reveal various chromosome rearrangements, such as pericentric or paracentric inversions, decay of an ancestral centromere followed by the establishment of a new one, fissions, and fusions. These rearrangements may be significant drivers of karyotype evolution [7‒9]. The presence of chromosome rearrangements can serve as an important indicator of diversity between species as well as inter-population diversity within a species, even when the morphological approach fails [10‒13].

The species diversity of the genus Cobitis in the Middle East was reviewed by Freyhof et al. [2], recognizing 25 species in Anatolia, Türkiye. Additionally, C. indus was recently recognized from Anatolia by Eagderi et al. [14]. Out of these 26 Cobitis species, most are endemic to Anatolia, Türkiye [2, 14]. Among these loaches, Cobitis bilseli Battalgil, 1942, is endemic to the Lake Beyşehir basin in Central Anatolia. Cobitis fahireae Erkakan, Atalay-Ekmekçi, and Nalbant, 1998, occurs as an endemic species in the eastern Aegean Sea basin in west Anatolia, while Cobitis turcica Hanko, 1924, is endemic to the wider Lake Tuz basin in Central Anatolia [2].

Contrary to the species richness of Cobitidae in Anatolia, karyological studies have been reported in only C. elazigensis [15], C. phrygica, C. simplicispina [16], and C. bilseli [17] from Türkiye. However, no chromosomal study has been performed on C. fahireae and C. turcica. The aim of this study was to process chromosomal measurement information and use conventional staining methods to provide detailed resolution of the karyotypes of three endemic Cobitis species (C. bilseli, C. fahireae, and C. turcica) from three different localities in Anatolia, Türkiye. Based on the measurement of chromosome arms and the number of cytogenetic markers, we discuss the possibilities of karyotype evolution.

One male of C. bilseli was caught from Sarıöz Stream, Beyşehir Lake, Konya prov., Türkiye (38°42′N, 31°41′E). Two females and five males of C. fahireae were captured in Gediz River, Salihli, Manisa prov., Türkiye (38°31′N, 28°08′E). Two females of C. turcica were caught from Selime Village, Melendiz Creek, Aksaray prov., Türkiye (38°18′N, 34°15′E). The collection locations of all three species studied are depicted in Figure 1a. The live specimens were carried to the laboratory. The process was approved by the Local Animal Ethics Committee of Türkiye (Protocol Number: 68429034/15/17). Species were identified based on morphological characteristics according to Freyhof et al. [2]. Chromosome slides were prepared according to the method of Bertollo et al. [18]. Each fish was injected intra-peritoneally with a colchicine solution (0.1%; 1 mL/100 g body weight). The fish were kept in a well-aerated aquarium and after 2 h, the cephalic kidney tissue was dissected out and placed in a hypotonic 0.56% KCl solution. After this step, the cellular suspension was centrifuged at 1,200 rpm for 10 min. The hypotonic solution was removed and the pellet was suspended and washed three times in methanol:glacial acetic acid (3:1). After centrifugation at 1,200 rpm for 10 min, the drops of cellular suspension were put on a clean slide. The slides were air-dried. Some of them were stained with 10% Giemsa solution in distilled water for 20 min. At least 10 slides were prepared from each specimen. Constitutive heterochromatin regions and Ag-NORs were detected by the following methods: Sumner [19] and Howel and Black [20], respectively.

Fig. 1.

a Geographical map of collection sites of Cobitidae specimens. Each location of the species has a different color and mark, i.e., C. bilseli square in blue, C. fahireae star in orange, C. turcica triangle in green. b-d Male of C. bilseli with one lamina circularis. e-g Male of C. fahireae with one lamina circularis.

Fig. 1.

a Geographical map of collection sites of Cobitidae specimens. Each location of the species has a different color and mark, i.e., C. bilseli square in blue, C. fahireae star in orange, C. turcica triangle in green. b-d Male of C. bilseli with one lamina circularis. e-g Male of C. fahireae with one lamina circularis.

Close modal

Metaphases were photographed under a Leica DM 3000 microscope and photographs were taken using AKAS software. Both arms of each chromatid were measured in pixels using ImageJ, v 1.54f [21]. The short (p) and long (q) arm lengths were quantified as described in Knytl and Fornaini [22]. To identify each chromosome, we analyzed chromosomal length (l), centromeric index (i), and q/p arm ratio (r2) [5]; for formulas, see Knytl et al. [23]. Data were analyzed in R software for statistical computing, R 4.3.1 [24], using ggplot2 and ggpubr packages. A one-way analysis of variance was performed to compare the mean values of l and i between the karyotypes of all three species. The analysis aimed to test the null hypothesis, which states that there is no difference in the means, against the alternative hypothesis, which suggests that the mean values of individual orthologous chromosomes significantly differ from those of the orthologous chromosomes in the other species. We used and modified R scripts developed by Knytl and Fornaini [22]. Chromosome pairs were arranged according to their l and i values into individual morphological categories in the karyotypes. Chromosome categories and identical chromosome classification were taken from Levan et al. [5]. To determine the fundamental arm number (FN), meta- and submetacentrics were considered as biarmed and subtelo-, acro-, and telocentrics as uniarmed.

The diploid chromosome number for each of the three Cobitis species was determined to be 2n = 50 (Figs. 2a, 3a, 4a). The karyotypes consist of 4 pairs of metacentric, 5 pairs of submetacentric, and 16 pairs of subtelo-telocentric chromosomes in C. bilseli (Fig. 2b); 11 pairs of metacentric, 7 pairs of submetacentric, and 7 pairs of subtelo-telocentric chromosomes in C. fahireae (Fig. 3b); and 4 pairs of metacentric, 4 pairs of submetacentric, and 17 pairs of subtelo-telocentric chromosomes in C. turcica (Fig. 4b). FN was calculated as 72 in C. bilseli; as 86 in C. fahireae; and as 66 in C. turcica. No heteromorphic sex chromosomes were found in any of the three karyotypes. Dark C-bands were found on the centromeres of almost all chromosomes in C. bilseli (Fig. 2c, d) and C. turcica (Fig. 4c, d). Otherwise, slightly stained C-bands were observed on the centromeres of most chromosomes in C. fahireae, for instance, metacentric pairs 1–6, 8, 9, and 11 (Fig. 3c, d). The Ag-NOR signals were detected in the terminal region of the p arm of the 6th submetacentric chromosome pair in C. fahireae (Fig. 3e, f) and in the terminal region of the q arm of only one chromosome from the 1st metacentric pair in C. bilseli (Fig. 2e, f) and C. turcica (Fig. 4e, f). In the latter two species, the chromosome carrying the signal is the largest chromosome of the karyotype. Regarding the exceptional pattern of single-chromosome Ag-NOR signal, no polymorphisms in the number of Ag-NORs were observed within a species or an individual.

Fig. 2.

Metaphases and karyotypes of C. bilseli. a Giemsa-stained metaphase. b Arranged karyotype of the Giemsa-stained metaphase. c C-banded metaphase. d Arranged karyotype of the C-banded metaphase. e Silver-stained metaphase (arrow indicates the Ag-NOR). f Arranged karyotype of the silver-stained metaphase. Scale bar = 3 μm. m, metacentric; sm, submetacentric; st-T, subtelo-telocentric chromosomes.

Fig. 2.

Metaphases and karyotypes of C. bilseli. a Giemsa-stained metaphase. b Arranged karyotype of the Giemsa-stained metaphase. c C-banded metaphase. d Arranged karyotype of the C-banded metaphase. e Silver-stained metaphase (arrow indicates the Ag-NOR). f Arranged karyotype of the silver-stained metaphase. Scale bar = 3 μm. m, metacentric; sm, submetacentric; st-T, subtelo-telocentric chromosomes.

Close modal
Fig. 3.

Metaphases and karyotypes of C. fahireae. a Giemsa-stained metaphase. b Arranged karyotype of the Giemsa-stained metaphase. c C-banded metaphase. d Arranged karyotype of the C-banded metaphase. e Silver-stained metaphase (arrows indicate the Ag-NORs). f Arranged karyotype of the silver-stained metaphase. Scale bar = 5 μm. m, metacentric; sm, submetacentric; st-T, subtelo-telocentric chromosomes.

Fig. 3.

Metaphases and karyotypes of C. fahireae. a Giemsa-stained metaphase. b Arranged karyotype of the Giemsa-stained metaphase. c C-banded metaphase. d Arranged karyotype of the C-banded metaphase. e Silver-stained metaphase (arrows indicate the Ag-NORs). f Arranged karyotype of the silver-stained metaphase. Scale bar = 5 μm. m, metacentric; sm, submetacentric; st-T, subtelo-telocentric chromosomes.

Close modal
Fig. 4.

Metaphases and karyotypes of C. turcica. a Giemsa-stained metaphase. b Arranged karyotype of the Giemsa-stained metaphase. c C-banded metaphase. d Arranged karyotype of the C-banded metaphase. e Silver-stained metaphase (arrow indicates the Ag-NOR). f Arranged karyotype of the silver-stained metaphase. Scale bar = 3 μm. m, metacentric; sm, submetacentric; st-T, subtelo-telocentric chromosomes.

Fig. 4.

Metaphases and karyotypes of C. turcica. a Giemsa-stained metaphase. b Arranged karyotype of the Giemsa-stained metaphase. c C-banded metaphase. d Arranged karyotype of the C-banded metaphase. e Silver-stained metaphase (arrow indicates the Ag-NOR). f Arranged karyotype of the silver-stained metaphase. Scale bar = 3 μm. m, metacentric; sm, submetacentric; st-T, subtelo-telocentric chromosomes.

Close modal

Both p and q arms of each chromatid were measured from 10, 9, and 7 metaphase spreads for C. bilseli, C. fahireae, and C. turcica, respectively. Chromosomes were identified based on the calculated median values of l and i. The l value was quantified as a percentage of the sum of l for all chromosomes to account for variation in resolution and pixel size of our images. Each individual chromosome was also assigned a chromosomal category based on the i value. If the i value was equal to or greater than 37.5, the chromosome was categorized to be metacentric (m). If the i value was equal to or higher than 25 and lower than 37.5, the chromosome was categorized as submetacentric (sm). If the i value was equal to or greater than 12.5 and lower than 25, the chromosome was categorized as subtelocentric (st). If the i value was higher than 0 and lower than 12.5, the chromosome was classified as acrocentric (a). If the i value was equal to 0, the chromosome was identified as telocentric sensu stricto (T), i.e., without the p arm (Table 1). The m, sm, and T categories were present in all three species. In addition, we identified one pair of st and one pair of a chromosomes in C. bilseli.

Table 1.

Characteristics of chromosome measurements

Cobitis bilseliCobitis fahireaeCobitis turcicaANOVA test
chromosomel (%)icategoryl (%)icategoryl (%)icategorycbi × cfacbi × ctucfa × ctu
lilili
2.46 44.71 2.32 37.79 2.38 42.68   a  
2.27 46.61 2.30 49.45 2.36 48.61 a   ** 
2.25 41.37 2.26 38.58 2.27 45.66 **   *** 
2.14 49.44 2.26 39.55 2.22 41.01  ***    *** 
2.59 32.51 sm 2.25 46.86 2.43 34.63 sm ***   *** 
2.41 36.59 sm 2.18 41.21 2.40 33.54 sm  ***    *** 
2.23 35.01 sm 2.17 42.55 2.31 30.41 sm ** ***    *** 
2.08 28.32 sm 2.11 41.72 2.24 36.68 sm  ***    *** 
2.02 25.16 sm 2.11 47.74 2.36 *** *** ***  *** 
10 1.88 15.38 st 2.03 45.14 2.15 *** *** ** ***  *** 
11 2.08 9.20 1.97 44.45 2.10 ** *** a *** a *** 
12 2.08 2.36 37.10 sm 2.02 a ***  **  *** 
13 2.04 2.23 33.23 sm 2.00  ***    *** 
14 1.99 2.07 31.88 sm 1.98  ***    *** 
15 1.95 2.06 30.39 sm 1.93 ***  ** *** 
16 1.90 2.01 36.01 sm 1.91 ***   *** 
17 1.87 1.95 34.83 sm 1.87 ***   *** *** 
18 1.82 1.71 25.31 sm 1.81  ***   *** *** 
19 1.79 1.92 1.75  ***  ** 
20 1.77 1.84 1.69  **  
21 1.72 1.75 1.64   ***  a  
22 1.70 1.68 1.52   ***    
23 1.65 1.63 1.40 a  ***    
24 1.60 1.51 1.34 **  ***    
25 1.52 1.19 1.29 ***  ***    
Cobitis bilseliCobitis fahireaeCobitis turcicaANOVA test
chromosomel (%)icategoryl (%)icategoryl (%)icategorycbi × cfacbi × ctucfa × ctu
lilili
2.46 44.71 2.32 37.79 2.38 42.68   a  
2.27 46.61 2.30 49.45 2.36 48.61 a   ** 
2.25 41.37 2.26 38.58 2.27 45.66 **   *** 
2.14 49.44 2.26 39.55 2.22 41.01  ***    *** 
2.59 32.51 sm 2.25 46.86 2.43 34.63 sm ***   *** 
2.41 36.59 sm 2.18 41.21 2.40 33.54 sm  ***    *** 
2.23 35.01 sm 2.17 42.55 2.31 30.41 sm ** ***    *** 
2.08 28.32 sm 2.11 41.72 2.24 36.68 sm  ***    *** 
2.02 25.16 sm 2.11 47.74 2.36 *** *** ***  *** 
10 1.88 15.38 st 2.03 45.14 2.15 *** *** ** ***  *** 
11 2.08 9.20 1.97 44.45 2.10 ** *** a *** a *** 
12 2.08 2.36 37.10 sm 2.02 a ***  **  *** 
13 2.04 2.23 33.23 sm 2.00  ***    *** 
14 1.99 2.07 31.88 sm 1.98  ***    *** 
15 1.95 2.06 30.39 sm 1.93 ***  ** *** 
16 1.90 2.01 36.01 sm 1.91 ***   *** 
17 1.87 1.95 34.83 sm 1.87 ***   *** *** 
18 1.82 1.71 25.31 sm 1.81  ***   *** *** 
19 1.79 1.92 1.75  ***  ** 
20 1.77 1.84 1.69  **  
21 1.72 1.75 1.64   ***  a  
22 1.70 1.68 1.52   ***    
23 1.65 1.63 1.40 a  ***    
24 1.60 1.51 1.34 **  ***    
25 1.52 1.19 1.29 ***  ***    

Chromosome measurements of C. bilseli, C. fahireae, and C. turcica are shown in median length (l, in %) and centromeric index (i).

Chromosomal categories correspond to m, metacentric; sm, submetacentric; st, subtelocentric; a, acrocentric; and T, telocentric chromosomes. ANOVA test cbi x cfa, ANOVA test for l and i values of orthologous chromosomes in C. bilseli compared to those in C. fahireae. Similarly, C. bilseli and C.fahireae orthologs were compared to those in C. turcica, respectively (cbi x ctu, cfa x ctu).

*p < 0.05.

**p < 0.01.

***p < 0.001.

ap < 0.1.

The extreme T category was represented by 14, 7, and 17 chromosomes (homologous pairs) of C. bilseli, C. fahireae, and C. turcica, respectively (i.e., 14, 7, and 17 points lie on the dashed vertical line of interval 0 in Fig. 5). The T chromosomes had a relatively wide range of l values in all three species (most of the chromosomes from 1.5 to 2.1%). Another extreme M category (metacentric sensu stricto, i = 50, sensu; Levan et al. [5]) was not present in any of the species studied, but chromosome 1 in C. bilseli and C. fahireae had the i value close to 50, i.e., 49.44 and 49.45, respectively (see two points close to the dashed vertical line of interval 50) (Fig. 5). Four chromosomes belonged to the m category in C. bilseli and C. turcica, with l values ranging from 2.14 and 2.22 to 2.46 and 2.38%, respectively. Cobitis fahireae possessed 11 m chromosomes with the l values ranging from 1.97 to 2.23%. Apart from the four m chromosomes in C. bilseli and C. turcica, the number of other categories differed, e.g., five, seven, and four sm chromosomes in C. bilseli, C. fahireae, and C. turcica, respectively. The st and a chromosomes were present solely in C. bilseli, each of these two categories being represented by one chromosome.

Fig. 5.

Relationship between centromeric index (i), x axis, and chromosome length (l), y axis. Chromosome complements of C. bilseli, C. fahireae, and C. turcica are depicted in green, red, and dark blue, respectively. Black dashed vertical lines define intervals 0–12.5, 12.5–25, 25–37.5, and 37.5–50, which correspond to acrocentric, subtelocentric, submetacentric, and metacentric chromosomes, respectively. The dashed vertical line at i = 0 defines telocentric chromosomes. Plotted values of i and l are medians for each chromosome. The relative l value is given as a percentage of the overall length of all chromosomes.

Fig. 5.

Relationship between centromeric index (i), x axis, and chromosome length (l), y axis. Chromosome complements of C. bilseli, C. fahireae, and C. turcica are depicted in green, red, and dark blue, respectively. Black dashed vertical lines define intervals 0–12.5, 12.5–25, 25–37.5, and 37.5–50, which correspond to acrocentric, subtelocentric, submetacentric, and metacentric chromosomes, respectively. The dashed vertical line at i = 0 defines telocentric chromosomes. Plotted values of i and l are medians for each chromosome. The relative l value is given as a percentage of the overall length of all chromosomes.

Close modal

Another analysis we performed concerned the intrachromosomal variation of l and i values. The l and i values were assigned to each chromosome 1–25 and plotted (Fig. 6, 7). A measure of statistical dispersion, the interquartile range (Q1-Q3), was used to evaluate the extent of morphological divergence of each orthologous pair between Cobitis species. Each box depicts a group of measured l (Fig. 6) and i (Fig. 7) values. Chromosomes were ordered according to the morphological categories from metacentric chromosomes on the left side of plots to telocentric chromosomes on the right side of plots. If the i value was equal, chromosomes were ordered decreasingly according to the l value (T chromosomes with i = 0). Within each category, chromosomes were ordered decreasingly according to the l value from the largest to the shortest chromosome. For instance, the largest metacentric chromosome for all three species was chromosome 1 (hereafter chr1). The largest submetacentric chromosome was chr5 in C. bilseli and C. turcica. We compared both l and i values of each orthologous chromosome within three Cobitis species (between C. bilseli and C. fahireae, C. bilseli and C. turcica, and C. fahireae and C. turcica, Table 1) with the aim to determine whether significant variation occurs or whether chromosomes are morphologically similar. ANOVA test revealed significant variation in the i value in the most chromosomes of C. fahireae even when grouped within the same morphological categories (Table 1). The highest variation in i values is clearly visible for chrs10–12 in C. bilseli, chrs19–20 in C. fahireae, and chr9 in C. turcica. The Q1-Q3 interval within these variable chromosomes covers i values from 0 to 15 (Fig. 7). The order of chromosomes on karyotypes (Figs. 2-4) corresponds exactly to the order of chromosomes on box plots.

Fig. 6.

Intrachromosomal variability of l value (y axis) is displayed for the haploid complement of 25 chromosomes of C. bilseli (a), C. fahireae (b), and C. turcica (c) (x axis). Each box defines interquartile range (Q1-Q3) with the median value indicated by black lines. Upper and lower whiskers show the maximum and minimum values of the data set, respectively. Outliers are drawn as black points. Black dashed vertical lines delineate the chromosome categories as follows: in C. bilseli, from the left to right: metacentric, submetacentric, subtelocentric, acrocentric, and telocentric chromosomes; in C. fahireae and C. turcica: metacentric, submetacentric, and telocentric chromosomes.

Fig. 6.

Intrachromosomal variability of l value (y axis) is displayed for the haploid complement of 25 chromosomes of C. bilseli (a), C. fahireae (b), and C. turcica (c) (x axis). Each box defines interquartile range (Q1-Q3) with the median value indicated by black lines. Upper and lower whiskers show the maximum and minimum values of the data set, respectively. Outliers are drawn as black points. Black dashed vertical lines delineate the chromosome categories as follows: in C. bilseli, from the left to right: metacentric, submetacentric, subtelocentric, acrocentric, and telocentric chromosomes; in C. fahireae and C. turcica: metacentric, submetacentric, and telocentric chromosomes.

Close modal
Fig. 7.

Intrachromosomal variability of i value (y axis) is displayed for the haploid complement of 25 chromosomes of C. bilseli (a), C. fahireae (b), and C. turcica (c) (x axis). Each box defines interquartile range (Q1-Q3) with the median value indicated by black lines. Upper and lower whiskers show the maximum and minimum values of the data set, respectively. Outliers are drawn as black points. Black dashed vertical lines delineate the chromosome categories as follows: in C. bilseli, from the left to right: metacentric, submetacentric, subtelocentric, acrocentric, and telocentric chromosomes; in C. fahireae and C. turcica: metacentric, submetacentric, and telocentric chromosomes.

Fig. 7.

Intrachromosomal variability of i value (y axis) is displayed for the haploid complement of 25 chromosomes of C. bilseli (a), C. fahireae (b), and C. turcica (c) (x axis). Each box defines interquartile range (Q1-Q3) with the median value indicated by black lines. Upper and lower whiskers show the maximum and minimum values of the data set, respectively. Outliers are drawn as black points. Black dashed vertical lines delineate the chromosome categories as follows: in C. bilseli, from the left to right: metacentric, submetacentric, subtelocentric, acrocentric, and telocentric chromosomes; in C. fahireae and C. turcica: metacentric, submetacentric, and telocentric chromosomes.

Close modal

Taken together, we determined standardized karyotypes for C. bilseli (Fig. 2), C. fahireae (Fig. 3), and C. turcica (Fig. 4). Although we found all three karyotypes to be identical in terms of chromosome number, differences between karyotypes were found in NOR and C-band localization and chromosome structure (morphology, l and i values).

Until recently, cytogenetic data were available for only three Cobitis species that have been distributed in Türkiye – C. elazigensis, C. phrygica, and C. simplicispina [15, 16]. Here we describe for the first time the karyotypic properties of two other Anatolian endemic Cobitis species – C. fahireae and C. turcica. The C. bilseli karyotype [17] was published during the review process of our study. Accordingly, we now also report the karyotype of C. bilseli. The present study reveals conserved diploid (2n) chromosome numbers but variable karyotype formulas. Pericentric inversions, the extinction of an old centromere and the origin of a new one, and/or translocations are likely to play an important role in the karyotype evolution of this species, as has already been stated for the other Cobitis species C. elazigensis, C. phrygica, and C. simplicispina [16]. Based on a detailed analysis of the i values, the karyotype composition of C. bilseli and C. turcica appeared to be more similar to each other than to the karyotype of either of these two species to that of C. fahireae (Table 1). All three species in this study share the 2n number with all the Cobitis species karyotyped so far from Türkiye (Table 2). Given the equal number of chromosomes in our studied species, we can exclude fissions and fusions that could have shaped the karyotype evolution because these rearrangements affect the number of chromosomes (e.g., Sember et al. [25]). In the Anatolian spined loaches, intrachromosomal rearrangements likely occurred, leading to spatial shifts in the position of the centromere without changes in the chromosome number. The most probable responsible mechanisms should be pericentric inversions, as reported recently in the other groups of fish [26, 27]. Some representatives of Anatolian Cobitidae species possess a karyotype composed of more uniarmed chromosomes than biarmed chromosomes, e.g., C. elazigensis, C. phrygica, C. bilseli, and C. turcica (Table 2). Contrary to this phenomenon, C. simplicispina and C. fahireae have more biarmed chromosomes in their karyotypes than uniarmed ones (Table 2). According to these differences, FNs in C. simplicispina and C. fahireae are higher than FNs in C. elazigensis, C. phrygica, C. bilseli, and C. turcica.

Table 2.

Karyological data of the genus Cobitis from Türkiye

Species studied2nKaryotype formulaFNReferences
C. elazigensis 50 18m-sm + 32a 68 [15
C. phrygica 50 8m + 8sm + 34st-a 66 [16
C. simplicispina 50 16m + 16m + 18st-a 82 [16
C. bilseli 50 8m + 6sm + 6st + 30a 70 [17
C. bilseli 50 8m + 10sm + 32st-T 72 This study 
C. fahireae 50 22m + 14sm + 14st-T 86 This study 
C. turcica 50 8m + 8sm + 34st-T 66 This study 
Species studied2nKaryotype formulaFNReferences
C. elazigensis 50 18m-sm + 32a 68 [15
C. phrygica 50 8m + 8sm + 34st-a 66 [16
C. simplicispina 50 16m + 16m + 18st-a 82 [16
C. bilseli 50 8m + 6sm + 6st + 30a 70 [17
C. bilseli 50 8m + 10sm + 32st-T 72 This study 
C. fahireae 50 22m + 14sm + 14st-T 86 This study 
C. turcica 50 8m + 8sm + 34st-T 66 This study 

2n, diploid chromosome number; FN, fundamental number; m, metacentric; sm, submetacentric; st-a, subtelo-acrocentric; st-T, subtelo-telocentric chromosomes (sensu stricto).

Otherwise, C. taenia from Europe [4] has a different 2n number from C. bilseli, C. fahireae, and C. turcica. Cobitis taenia’s karyotype contains a remarkable large metacentric chromosome, which is considered to be the result of a centric fusion of two acrocentric chromosomes [28]. Moreover, spined loaches from different countries named C. calderoni, C. maroccana [29], C. vardarensis [6], C. taurica [30], C. lutheri, C. choii, C. melanoleuca [31], C. linea [32], C. strumicae [33], C. derzhavini, C. saniae [34], and C. satunini [1] have the same diploid chromosome number, which is consistent with this study. However, the karyotype composition of the abovementioned species differs from C. bilseli, C. fahireae, and C. turcica. Majtánová et al. [3] observed a large variability in the karyotype structure and chromosomal markers in European C. elongatoides, C. taenia, and C. tanaitica and their hybrids. Most Cobitis species differ in their karyotype, which is routinely defined by variation in number, size, and morphology of chromosomes, as revealed in this study. Homomorphic, i.e., morphologically undifferentiated, sex chromosomes are a common feature among Cobitis loaches [15, 16, 32], and we also did not find any structural differences in male and female karyotypes.

Another significant difference between the karyotypes of C. bilseli, C. fahireae, and C. turcica is the variation in the number and location of Ag-NOR signals and C-banding patterns. These differences may be the result of specific chromosomal rearrangements such as inversions and translocations, which have occurred during their evolutionary history, as reported in the other Cobitis species [35]. The number and location of ribosomal DNA (rDNA) sites are essentially species specific and provide a useful karyotypic marker in fish cytotaxonomy [22, 26, 36]. The most of rDNAs are associated with nucleolus [37]; thus, their location on chromosomes is identical to that of NORs and is consistent with our analysis of silver staining. Cobitidae karyotypes have variable numbers and locations of NORs, ranging from single to complex multiple NOR phenotypes [6]. These variations do not consistently correlate with phylogenetic distance between Cobitidae species [38]. The NOR phenotypes of C. bilseli and C. turcica are the same. However, C. fahireae has a different NOR phenotype than the former two species. The number and location of Ag-NORs on submetacentric chromosomes in C. fahireae show similarity to C. elazigensis [15], C. phrygica, C. simplicispina [16] from Türkiye and C. strumicae from Europe [33]. The Ag-NORs on metacentric chromosomes have been reported in C. vardarensis from Europe [6], as well as in C. bilseli and C. turcica from Türkiye (this study, [16]). Cobitis taenia was characterized by one pair of Ag-NOR located on subtelocentric chromosomes [4]. Intraindividual polymorphism in the number of Ag-NORs that was reported in C. simplicispina [16] is not observed in any of the species in this study.

Interestingly, we found Ag-NORs on only one chromosome of the largest homologous metacentric pair in both C. bilseli and C. turcica. This heterozygosity in both species confirms their close evolutionary relationship and more divergent relationship with C. fahireae, which has Ag-NOR on both homologous chromosomes of the submetacentric category. Contrary to our finding, Doori and Arslan [17] identified Ag-NOR on the p arm (not on the q arm as we did) of both chromosomes of the largest metacentric pair in C. bilseli. They did not measure the length of chromosome arms, and thus the NOR position may indeed be on the q arm, as we designated by accurate measurements. Also, slight differences in the karyotype formulas of C. bilseli in our study and the study of Doori and Arslan [17] were found. An explanation for the presence of the heterozygous Ag-NOR position may be the inactivity of NOR in the previous interphase since Ag binds NOR-associated proteins. If NORs were not active, rRNA synthesis would not occur and therefore Ag-NORs would not be detectable by silver staining technique [23, 39]. One future direction could be FISH with an rDNA probe (e.g., [40]) that hybridizes with a specific genome sequence associated with the NOR (e.g., 28S rDNA), thereby revealing the true NORs [41]. Even though the geographical distribution of C. bilseli and C. turcica is close (Fig. 1a), we assume that the similarity in their karyotype structure is a consequence of evolutionary history and timing of divergence rather than hybridization or gene introgression. Due to the polymorphism in number and location of NOR [4, 15, 16], interspecies hybridizations [3], and incomplete NOR mapping in Cobitis species, it is difficult to estimate the ancestral position of NOR in the entire Cobitis genus.

C-bands indicate the presence of constitutive heterochromatin regions [19]. These regions contain transcriptionally inactive and highly repetitive DNA sequences. The distribution of C-bands could be applied to identify homologous chromosomes within and orthologous chromosomes between species and improve chromosome classification [4, 42]. Numbers and positions of C-bands in C. bilseli and C. turcica are similar to C. elazigensis [15], C. phrygica, C. simplicispina [16] from Türkiye and C. vardarensis [6] and C. taenia [4] from Europe. A low C-banding content distinguishes C. fahireae from the above species, while this pattern is similar to that of C. strumicae from Europe [33]. Another explanation for the low intensity of C-bands in C. fahireae could be the low efficacy and thus non-specificity of the technique. Notably, relatively close phylogenetic relationships between C. fahireae and C. strumicae, as well as among C. bilseli, C. turcica, C. elazigensis, C. phrygica, and C. simplicispina, reflect their similar C-banding patterns and indicate a true specificity of C-bands.

Overall, the obtained results contributed to a better understanding of the karyotype structure and chromosomal evolution of Anatolian spined loaches. The cytogenetic findings, which showed a higher degree of similarity in C. bilseli and C. turcica and a higher degree of divergence in C. fahireae, are consistent with phylogenetic relationships of the genus Cobitis [38]. Also, our results indicate that main drivers of karyotype evolution are pericentric inversions, translocations, and the decay of old centromeres followed by the establishment of new ones, resulting in centromere repositioning. However, more detailed cytogenetic studies in other Anatolian Cobitis species are needed to solve the puzzle of chromosomal evolution and to discover the precise mechanisms driving changes in karyotype structure. In addition, we improved the R scripts previously developed for the determination of a standardized karyotype [22] and applied them in the Cobitis species.

We thank two anonymous reviewers for their constructive comments on early versions of the manuscript. The karyotype of C. bilseli was presented as an oral presentation in II. International Turkish World Engineering and Science Congress (2019) and the summary of the work was included in the abstract book of the congress.

The process was approved by the Local Animal Ethics Committee of Türkiye (Protocol Number: 68429034/15/17).

The authors have no conflicts of interest to declare.

This study was supported by Bartın University Scientific Research Projects, project code: 2019-FEN-B-006 (S.U.K.), Kırşehir Ahi Evran University Scientific Research Projects, project code: SYO.A4.21.006 (M.K.A.), and the P JAC project MSCA Fellowships CZ-UK, CZ.02.01.01/00/22_010/0002902 (M.K.).

All authors contributed to the study conception and design and project administration. Material preparation, data collection, and investigation were performed by S.U.K., M.G., and M.K.A. Funding was secured by S.U.K., M.K.A., and M.K. Formal analysis, software, and supervision were provided by M.K. The first draft of the manuscript was written by S.U.K. and M.K.A. All authors commented on previous versions of the manuscript, and read and approved the final manuscript.

All metaphase images and scripts within exported R files, including steps outlining how the measured values were calculated and processed into data frames and plots, are available at https://github.com/martinknytl/2023_Cobitis_karyotype.

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