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Karyological analysis of five tooth-carps (Actinopterygii:

Cyprinodontidae) from Iran

Hamid Reza Esmaeili a,*, Mehrgan Ebrahimi b, Mahvash Saifali c

a Department of Biology, College of Sciences, Shiraz University, Shiraz, Iranb Department of Biodiversity and Ecosystem Management, Environmental Sciences Research Institute, Shehid Beheshti University, Evin, Tehran, Iran

c Department of Biology, College of Sciences, Alzahra University, Tehran, Iran

Received 27 August 2006; received in revised form 30 December 2006; accepted 31 December 2006

Abstract

The karyotypes of five tooth-carps of Iran, Aphanius persicus, Aphanius sophiae, Aphanius dispar, and Aphanius sp. have been investigated by

examining metaphase chromosomes spreads obtained from gill epithelial and kidney cells. The diploid chromosome numbers of all five species

were 2n = 48. The karyotypes consisted of 8 pairs of submetacentric and 16 pairs of subtelocentric chromosomes in A. persicus; 4 submetacentric

and 20 subtelocentric in A. sophiae; 7 submetacentric and 17 subtelocentric in Aphanius ginaonis and 8 submetacentric and 16 subtelocentric in A.

dispar. It was 16 metacentric, 1 submetacentric and 7 telocentric chromosome pairs in Aphanius sp. specimen. The arm numbers were 32, 28, 31

and 32 in A. persicus, A. sophiae, A. ginaonis and A. dispar, respectively. It was 41 in Aphanius sp. Sex chromosomes were cytologically

indistinguishable in these tooth-carps. Cluster analysis revealed the closeness of A. sophiae and A. persicus. According to our data A. ginaonis is

related to a group of A. sophiae + A. persicus + A. dispar.

# 2007 Published by Elsevier Ltd.

Keywords: Aphanius; Karyotype; Chromosome; Idiogram; Iran

1. Introduction

The genus Aphanius is the only genus of Cyprinodontidae

available in Iran which is represented by seven named species

(Scheel, 1990; Coad, 1988, 1995, 1996; Hrbek et al., 2006):

Aphanius ginaonis (Holly, 1929), Aphanius mento (Heckel,

1843), Aphanius dispar (Ruppell, 1828), Aphanius vladykovi

(Coad, 1988), Aphanius sophiae (Heckel, 1849), Aphanius

persicus (Jenkins, 1910) and Aphanius isfahanensis (Hrbek

et al., 2006). They are very colorful fish and can be kept in

aquarium, hence they may enter the aquarium trade. Aphanius

ginaonis (Geno tooth-carp) is an endemic species restricted to a

hot spring in south of Iran. A. dispar is a euryhaline fish which

apparently prefers brackish waters of costal areas of Iran. A.

vladykovi or Zagros tooth-carp is an endemic species found in a

restricted area of the central Zagros Mountains (Coad and

Keivany, 2000). A. persicus or Persian tooth-carp is an endemic

species found in lake Maharlu basin. This lake is located in a

basin of Shiraz valley in Fars province at an altitude of about

1460 m. This is a chloride lake and is fishless except for

inflowing streams and springs. A. sophiae is found in the

endorheic Kor River basin of this province. A. isfahanensis

(Isfahan tooth-carp) is a new species described from Isfahan

basin of Iran in 2006 (Hrbek et al., 2006). Obvious sex

determination, beautiful coloration, small size, high tolerance,

and easy adaptation to aquarium conditions, make these tooth-

carps very suitable species for keeping in aquaria.

Little work has been done on the Iranian populations of

Aphanius (Hrbek et al., 2006); however, basic data on

reproduction, alimentation and habitat of A. vladykovi (Keivany

and Sofiani, 2004) and reproductive biology of A. persicus are

available (Esmaeili and Shiva, 2006). Tooth-carps of Iran have

been studied mainly based on their morphology but their

morphology is conservative and species identification on this

basis is not always possible. The application of non-

morphological methods such as cytogenetic studies may

provide a complementary data source for more accurate and

precise identification of these fishes. Application of this type of

studies has received considerable attention in recent years

(Galetti et al., 2000; Ozouf-Costaz and Foresti, 1992). Fish

www.elsevier.com/locate/micron

Micron 39 (2008) 95–100

* Corresponding author. Tel.: +98 711 2280916; fax: +98 711 2280926.

E-mail address: esmaeili@susc.ac.ir (H.R. Esmaeili).

0968-4328/$ – see front matter # 2007 Published by Elsevier Ltd.

doi:10.1016/j.micron.2006.12.007

Author's personal copy

chromosome data have great importance in studies concerning

evolutionary systematics, aquaculture, mutagenesis, genetic

control and the rapid production of inbred lines (Al-Sabti,

1991). About 1300 freshwater and saltwater fish species have

been reviewed in this respect (Demirok and Unlu, 2001) which

is less than 5% of the 27,000 described fish species. The

increasing importance of chromosomal studies on fish and lack

of data on karyotypy of Iranian fish encouraged us to do this

first cytogenetical analysis (i.e., diploid chromosome numbers,

description of karyotypes and idiograms) of the five tooth-carps

of Iran.

2. Materials and methods

Specimens of A. persicus were collected from Barm-e-Shoor

spring stream system (2982709N–5284200E, Alt. 1465 m) in

lake Maharlu basin, A. sophiae from Ghadamgah spring stream

system in Kor basin (308150N–5282500E, Alt. 1660 m) in Fars

province Southwest of Iran, A. ginaonis from hot spring of

Geno (27828029.8600N and 5681702.6700E, Alt. 197 m), A. dispar

from Khorgo hot spring (Hormuz basin), in Hormuzgan

province, southern Iran (278310N; 568280E, Alt. 154 m) and

Aphanius sp. from Cheshmeh-e-Ali spring located at Northwest

of Damghan city (368170N and 548050E), Semnan province

(Fig. 1). The fish were transported live to the laboratory and

kept in a well-aerated aquarium at 20–25 8C before analysis.

For karyological studies the modified method of Uwa (1986)

was followed. Colchicine solution was prepared with 0.005 g in

20 ml physiological serum. The fish were injected intraper-

itoneally with 0.02 ml of colchicine per gram of body weight

using an insulin syringe and then were taken back to the

aquarium for 4–5 h. The gill filaments and kidneys of the

specimens were then removed and placed in hypotonic 0.36%

KCl solution for 45 min in room temperature (25 8C).

Thereafter, the solutions were centrifuged for 10 min at

1000 rpm, adding 2–3 drops of fresh and cold Carnoy fixative

(1:3, acetic acid:methanol) before centrifugation. The super-

natants were then discarded and 5 ml fresh and cold fixative was

Fig. 1. Map of collection sites of Iranian Aphanius made for this study.

Fig. 2. Giemsa stained chromosome spreads of five tooth-carps from Iran.

H.R. Esmaeili et al. / Micron 39 (2008) 95–10096

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added to sediments, mixed thoroughly and then were left for

1 h. The fixation and centrifugation stages were repeated twice.

The suspensions now were trickled to cold slides from almost

2 m height. These slides were stained with 10% Giemsa for

20 min. Chromosomes were observed, selected and photo-

graphed by Olympus light microscope mounted with a camera.

Karyotypes were prepared by arranging chromosomes in pairs

by size. For each chromosome, the average lengths of the short

and long arms and arm ratio (the ratio of the long arm length to

the short arm length of chromosomes or r value) were

calculated and then the chromosomes were classified according

to the criteria of Levan et al. (1964). For numerical karyotype

analysis karyotype total form (% TF or the ratio of the sum of

short arm length to sum of total arm length), % S (the ratio of

the length of smallest chromosome to the length of longest

chromosome), % F (the ratio of the short arm length to the total

length of chromosome) and also d-value (difference of long and

short length of chromosome) were calculated. Fundamental

number (NF) was expressed as twice the number of atelocentric

plus the number of telocentric chromosomes. Grouping of the

chromosomal parameters was performed using the average

linkage group (UPGMA) method. The idiograms were prepared

in Excel 2003 software (Microsoft). Due to smaller number of

live specimens of Aphanius sp. only the metaphase spread and

the karyotype formula were presented for this tooth-carp.

3. Results

Metaphase spreads of five species are given in Fig. 2. The

diploid chromosome numbers of all five species were 2n = 48

(Fig. 3). The quantitative data of the different measurements

used to classify chromosomes and idiograms are given in

Table 1 and Figs. 4 and 5. The karyotypes consisted of 8 pairs of

submetacentric and 16 pairs of subtelocentric chromosomes in

A. persicus (16Sm + 32St), 4 submetacentric and 20 subtelo-

centric chromosome pairs in A. sophiae (8Sm + 40St), 7

submetacentric and 17 subtelocentric chromosome pairs in A.

ginaonis (14Sm + 34St) and 8 submetacentric and 16 subtelo-

centric pairs in A. dispar (16Sm + 32St). It was 16 metacentric,

1 submetacentric and 7 telocentric chromosome pairs in

Aphanius sp. (32M + 2Sm + 14t). The arm numbers were 32,

28, 31 and 32 in A. persicus, A. sophiae, A. ginaonis and A.

dispar, respectively. It was 41 in Aphanius sp. Sex chromo-

somes were cytologically indistinguishable in these tooth-

carps. TF was 27.29 in A. dispar and 25.47, 24.31 and 23.14 in

A. ginaonis, A.persicus and A. sophiae, respectively, indicating

asymmetry in chromosome shape. Cluster analysis of

karyological data are represented in Fig. 6.

4. Discussion

Karyotypes are descriptions of the number and morphology

of chromosomes. The number of chromosomes per cell seems

to be a rather conservative characteristic and so may be used as

an indicator of the closeness of species interrelationships within

families (Moyle and Cech, 2004).

According to our observations, the diploid chromosome

numbers of all Aphanius species were 2n = 48 and it is in

conformation with the chromosome number of other species of

this genus. Klinkhardt et al. (1995) and Arkhipchuk (1999)

reported the chromosome number of A. sophiae to be 2n = 48

and that of A. mento 2n = 48 (Arkhipchuk, 1999; Klinkhardt

et al., 1995; Vasil’ev, 1980). The chromosome numbers of A.

dispar, A. asquamatus, A. iberus and A. fasciatus were also

2n = 48 (Arkhipchuk, 1999; Gyldenholm and Scheel, 1974;

Karbe, 1961). It can be concluded that the chromosome number

Fig. 3. Karyotypes of four tooth-carps from Iran.

H.R. Esmaeili et al. / Micron 39 (2008) 95–100 97

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Table 1

Relative length, arm ratio and chromosome type of four Iranian tooth-carps

No. Aphanius dispar Aphanius ginaonis Aphanius sophiae Aphanius persicus

Relative

length

Arm ratio Type Relative

length

Arm ratio Type Relative

length

Arm ratio Type Relative

length

Arm ratio Type

1 5.59 � 0.54 2.95 � 1.45 Sm 5.34 � 0.77 3.51 � 0.83 St 5.26 � 0.82 3.90 � 1.08 St 5.51 � 0.81 3.39 � 1.32 St

2 5.13 � 0.65 2.13 � 1.16 Sm 4.97 � 0.81 3.34 � 1.38 St 5.12 � 0.82 3.74 � 0.74 St 5.07 � 0.67 3.52 � 0.67 St

3 4.97 � 0.72 3.17 � 0.64 St 4.85 � 0.78 3.20 � 0.30 St 4.85 � 0.68 3.94 � 1.11 St 5.02 � 0.67 3.63 � 0.39 St

4 4.92 � 0.72 3.42 � 0.45 St 4.76 � 0.76 3.24 � 0.62 st 4.75 � 0.67 3.52 � 0.59 st 4.84 � 0.34 3.36 � 0.67 st

5 4.85 � 0.77 3.21 � 0.71 St 4.68 � 0.74 3.65 � 1.16 st 4.69 � 0.64 3.19 � 0.36 st 4.77 � 0.33 3.61 � 1.09 st

6 4.77 � 0.69 3.25 � 1.17 St 4.57 � 0.74 2.86 � 1.29 Sm 4.62 � 0.65 3.42 � 0.34 st 4.66 � 0.40 3.34 � 0.60 st

7 4.70 � 0.67 2.89 � 1.37 Sm 4.55 � 0.74 3.03 � 0.56 St 4.55 � 0.64 3.08 � 0.23 st 4.58 � 0.42 3.06 � 0.35 st

8 4.54 � 0.67 3.77 � 1.36 St 4.47 � 0.69 2.96 � 1.13 Sm 4.47 � 0.65 3.60 � 0.45 st 4.54 � 0.40 3.17 � 1.00 st

9 4.47 � 0.64 4.53 � 0.93 St 4.41 � 0.73 3.07 � 0.47 St 4.41 � 0.62 3.71 � 0.53 st 4.40 � 0.44 3.25 � 0.42 st

10 4.34 � 0.58 3.01 � 0.84 St 4.29 � 0.69 3.08 � 0.90 St 4.37 � 0.61 3.27 � 0.44 st 4.34 � 0.45 3.11 � 0.61 st

11 4.27 � 0.56 3.72 � 0.62 St 4.28 � 0.68 3.20 � 0.51 St 4.34 � 0.61 3.34 � 0.54 st 4.31 � 0.45 3.25 � 0.90 st

12 4.19 � 0.61 3.15 � 0.71 St 4.24 � 0.65 3.55 � 0.71 St 4.31 � 0.61 3.59 � 0.69 st 4.17 � 0.50 3.22 � 0.62 st

13 4.14 � 0.61 3.24 � 0.59 St 4.15 � 0.56 2.06 � 0.79 Sm 4.23 � 0.61 3.46 � 0.66 st 4.12 � 0.52 2.95 � 0.69 Sm

14 4.07 � 0.67 2.91 � 1.22 Sm 4.07 � 0.51 2.93 � 1.49 Sm 4.13 � 0.56 3.70 � 0.69 st 4.09 � 0.51 3.00 � 0.28 St

15 3.98 � 0.66 2.79 � 1.08 Sm 4.00 � 0.52 2.85 � 0.72 Sm 4.03 � 0.53 3.30 � 0.56 st 4.03 � 0.52 2.78 � 0.52 Sm

16 3.89 � 0.60 3.34 � 0.78 St 3.90 � 0.46 3.31 � 0.67 St 3.91 � 0.50 2.60 � 0.50 Sm 3.97 � 0.56 2.95 � 0.46 Sm

17 3.77 � 0.61 2.98 � 1.06 Sm 3.83 � 0.46 2.54 � 0.34 Sm 3.83 � 0.54 3.37 � 0.99 St 3.88 � 0.58 3.22 � 0.36 St

18 3.75 � 0.63 3.73 � 0.95 St 3.77 � 0.43 3.06 � 0.94 St 3.76 � 0.50 3.26 � 0.78 St 3.82 � 0.62 2.69 � 0.48 Sm

19 3.70 � 0.58 3.67 � 1.42 St 3.71 � 0.43 3.23 � 0.68 St 3.65 � 0.44 3.25 � 0.53 St 3.68 � 0.52 3.01 � 0.36 St

20 3.6 � 0.53 3.24 � 0.74 St 3.61 � 0.40 2.57 � 0.56 Sm 3.60 � 0.46 3.08 � 0.82 St 3.61 � 0.53 2.61 � 0.46 Sm

21 3.44 � 0.39 3.81 � 0.92 St 3.53 � 0.38 3.89 � 1.85 St 3.53 � 0.49 3.23 � 0.67 st 3.56 � 0.54 2.68 � 0.94 Sm

22 3.23 � 0.34 2.22 � 0.80 Sm 3.48 � 0.39 3.38 � 0.35 St 3.44 � 0.47 2.79 � 0.50 Sm 3.24 � 0.70 3.02 � 0.71 St

23 3.02 � 0.27 3.04 � 0.92 St 3.31 � 0.32 3.17 � 0.55 St 3.31 � 0.46 2.90 � 0.35 Sm 3.05 � 0.54 2.86 � 0.61 Sm

24 2.65 � 0.20 2.04 � 0.61 Sm 3.22 � 0.25 3.51 � 0.28 St 2.86 � 0.46 2.28 � 0.65 Sm 2.74 � 0.28 2.68 � 0.56 Sm

Fig. 4. Haploid idiograms of four species of Aphanius from Iran.

H.R. Esmaeili et al. / Micron 39 (2008) 95–10098

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in this genus is conservative. The number of chromosomes in

these tooth-carps is also similar to that of other species of

Cyprinodontidae such as Orestias agassizii, Cyprinodon

bovines and Cyprinodon macularius (Arkhipchuk, 1999;

Klinkhardt et al., 1995). In the order Cyrinodontiformes, the

most common fish species which have so far been cytologically

investigated, such as Gambusia holbrooki, G. affinis, G.

hurtadoi, Girardinus metallicus; Poecilia latipinna (Poecilli-

dae); Fundulus majalis (Fundulidae); Allotoca diazi, Ameca

splendens, Goodea atripinnis, Goodea gracilis, Hubbsina

turneri, Hyodon furcidenes, Skiffia bilineata, Xenotaenia

resolanae, Xenotoca eiseni, X. melanosoma, X. variata

(Goodeidae), have the diploid chromosome number 2n = 48

(Sola et al., 1990; Arkhipchuk, 1999; Klinkhardt et al., 1995).

Yet in few species of Cyprinodontiformes such as Aphyosemion

viride, Fundulopanchax sjostedti (Aplocheilidae); Allodon-

tichthys hubbsi and Ameca splendens (Goodeidae) the diploid

chromosome number reported to vary from 2n = 26 to 42

(Arkhipchuk, 1999; Klinkhardt et al., 1995). It could be

suggested that the diploid chromosome number of 2n = 48 is

the modal number in cyprinodont fishes. In interpretation of

karyotypic evolution it is often assumed that the primitive fish

karyotype consists of 48 rods from which the kayotypes of all

existing fish forms have been derived (Khuda-Bukhsh et al.,

1986) but the issue seems yet to be resolved. The discovery of

48 rather large acrocentric chromosomes in the Pacific hag fish,

Eptatretus stoutii, belonging to the order Myxiniformes

(Taylor, 1967; Vasil’ev, 1980) and the occurrence of 48 rods

in the majority of fishes studied prior to 1967 led to the idea that

the primitive karyotype of ancestral vertebrate freshly evolved

from chordate might consist of 48 rods (Khuda-Bukhsh et al.,

1986). Therefore, most of the subsequent workers assumed the

karyotypic evolution in different groups of fishes based on this

basic assumption of 48 rods as the primitive number (Khuda-

Bukhsh et al., 1986). But the discovery of 2n = 24 rods in two

species of freshwater eels (Rishi and Haobam, 1984; Kitada and

Tagawa, 1973), 2n = 36 rods in two species of Myxine, low

diploid numbers ranging between 14 and 42 in a large number

of fish families showing NF less than 36 in some cases (Khuda-

Bukhsh et al., 1986) would possibly call for a more cautious

prediction on the primitive karyotype of fish.

The karyotypes formula of these tooth-carps were found

to be different, being 16Sm + 32St in A. dispar and

14Sm + 34St in A. ginaonis. It was 8Sm + 40St in A.

sophiae, 16Sm + 32St in A. persicus and 32M + 2Sm + 14T

in Aphanius sp. specimens. The chromosome arm number of

A. dispar and A. persicus (32) was higher than A. ginaonis

(31) and A. sophiae (28). The chromosome arm number of

Aphanius sp. (41) was higher than all the other four Aphanius

species. It may be due to less number of Aphanius sp.

specimens. In the present study, no cytological evidence was

found for sex chromosome dimorphism in any of tooth-carps

which agrees with reports on many fish species. Cluster

analysis (Fig. 6) revealed the closeness of A. sophiae and A.

persicus which is in agreement with Coad (1996) based on

morphological characters and with Hrbek et al. (2006) based

on molecular sequence data. According to our data A.

ginaonis is related to a group of A. sophiae + A. persicus + A.

dispar. However, further molecular, cytological, anatomical,

morphological and biological investigations towards better

recognition and understanding of the genus Aphanius in Iran

need to be made.

Acknowledgments

The authors thank B.W. Coad from Canadian Museum of

Nature, Sh. Hosseini and F. Hosseini from Shiraz University

for their invaluable comments and suggestions on the

manuscript, A. Teimory, Z. Gholami and T. Hojat Ansari for

Fig. 5. Relative length of the chromosomes of four species of Aphanius form Iran.

Fig. 6. Dendrogram obtained from cluster analysis of four tooth-carps from

Iran.

H.R. Esmaeili et al. / Micron 39 (2008) 95–100 99

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helping in fish collection and Shiraz University for financial

support.

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