Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 101-109, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-952 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Isara Patawang, Sarawut Kaewsri, Sitthisak Jantarat, Praween Supanuam, Sarun Jumrusthanasan, Alongklod Tanomtong (2021) Some molec- ular cytogenetic markers and classical chromosomal features of Spilopelia chinensis (Scopoli, 1786) and Tachy- baptus ruficollis (Pallas, 1764) in Thai- land. Caryologia 74(4): 101-109. doi: 10.36253/caryologia-952 Received: May 26, 2021 Accepted: December 17, 2021 Published: March 08, 2022 Copyright: © 2021 Isara Patawang, Sarawut Kaewsri, Sitthisak Jantarat, Praween Supanuam, Sarun Jum- rusthanasan, Alongklod Tanomtong. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, 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. Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis (Scopoli, 1786) and Tachybaptus ruficollis (Pallas, 1764) in Thailand Isara Patawang1,*, Sarawut Kaewsri2, Sitthisak Jantarat3, Praween Supanuam4, Sarun Jumrusthanasan2, Alongklod Tanomtong5 1 Department of Biology, Faculty of Science, Chiang Mai University, Muang, Chiang Mai, Thailand 2 Program of Biology, Department of Science, Faculty of Science, Buriram Rajabhat Uni- versity, Muang, Buriram, Thailand 3 Program of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University [Pattani Campus], Muang, Pattani, Thailand 4 Program of Biology, Faculty of Science, Ubon Ratchathani Rajabhat University, Muang, Ubon Ratchathani, Thailand 5 Program of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen, Thailand *Corresponding author. E-mail: isara.p@cmu.ac.th Abstract. This study analyzed the karyological features of two bird species – Spilope- lia chinensis and Tachybaptus ruficollis – from Northeastern Thailand. Mitotic chro- mosomes were indirectly prepared by fibroblast cell culture. The chromosomes were stained by conventional Giemsa staining and microsatellite repeat of fluorescence in situ hybridization techniques. Giemsa staining showed that the diploid chromosome number of S. chinensis was 2n=70 and T. ruficollis was 60. The types of chromosomes observed in S. chinensis were 4 large metacentric, 2 medium acrocentric, 2 small meta- centric, 2 small submetacentric, 2 sex chromosomes and 58 microchromosomes; the karyotype of T. ruficollis comprised 2 large metacentric, 2 large submetacentric, 2 large acrocentric, 8 small metacentric, 4 small submetacentric, ZW sex chromosomes and 40 microchromosomes. The molecular cytogenetical features that were exhibited only on the male T. ruficollis chromosome included two microsatellites and telomeric sequenc- es: two signals of d(CA)15 on two microchromosomes, one signal of d(GC)15 on one of the first pair, and signals of AGGGTTn sequences on each telomeric region of all macro- and microchromosomes. The karyotype formula was deduced as: 2n (70) = Lm4 + Ma2 + Sm2 + Ssm2 + 2 sex chromosomes (Sm1/Ssm1) + 58 microchromosomes for S. chinensis and 2n (60) = Lm2 +Lsm2 + La2 + Sm8 + Ssm4 + Z (Msm1) W (Ssm1) + 40 micro- chromosomes for T. ruficollis. Keywords: Spilopelia chinensis, Tachybaptus ruficollis, Bird chromosome, Bird karyo- type. 102 Isara Patawang et al. INTRODUCTION Birds, also known as avian dinosaurs, are a group of endothermic vertebrates, characterized by many fea- tures. Spilopelia chinensis (Figure 1a), or spotted dove, is a small pigeon that is a common local breeding bird throughout its native range on the Indian Subcontinent and in Southeast Asia. The species belongs to the genus Spilopelia, subfamily Columbinae, family Columbidae, order Columbiformes, clade Columbimorphae and class Aves (Gibbs et al. 2001). Tachybaptus ruficollis (Figure 1b) or little grebe, is native to Europe, Africa and Asia. Tachybaptus ruficollis is one of six grebe species in the genus Tachybaptus, family Podicipedidae, order Podici- pediformes, clade Phoenicopterimorphae and class Aves (BirdLife International 2020). Twenty-eight species of Columbidae and three species of Podicipedidae have been reported in Thailand, which the genus Spilopelia comprises three species (S. orientalis, S. chinensis and S. tranquebarica) and the genus Tachybaptus has only one species (T. ruficollis) (Pratumthong et al. 2011). Columbiformes, one of three orders in the Columbi- morphae clade, and Podicipediformes, one of two orders in the Phoenicopterimorphae clade, are both classified to the same Columbea group by genome analyses. Paleobi- ology and molecular biology suggest that neoavians and placental mammals originated about 66 million years ago during the late Cretaceous to early Paleogene period. The evolutionary lines of Columbimorphae, including mesites, sandgrouse and doves, and Phoenicopterimor- phae, comprising flamingos and grebes, divided about 70 million years ago during the late Cretaceous period (Pacheco et al. 2011; Ksepka and Boyd 2012; Yuri et al. 2013; Jarvis et al. 2014). Few avian chromosomal data studies have been reported, because of their difficulty compared to other vertebrates, as avian chromosomes are highly conserved compared to other vertebrate groups. At present, about 10% of total 10,857 bird species that have been report- ed karyotypic study. Approximately half the number of karyotyped birds (≈50.7%) have diploid number of 78 and 82 chromosomes, and about 21.7% have 2n=80. Extraordinary diversity of bird chromosome ranges from 2n=40 in Falco columbarius (Falconiformes) to 2n=142 in Corythaixoides concolor (Musophagiformes). The number of chromosomes, karyotypic features and sex chromosomes have been preserved in the avian genome on the chromosomal level and shared across all avian species (Degrandi et al. 2020). The diploid number of 2n=80 was proposed to the presumptive ancestral bird chromosome, which can be used to explain the chromo- somal evolution of birds well (Griffin et al. 2007). This classic chromosomal study of Thailand popula- tions of S. chinensis and T. ruficollis species is the new recorded; in addition, we are the first to report on the molecular cytogenetic features of the T. ruficollis species. MATERIALS AND METHODS Sample collection S. chinensis tissue samples were derived from whole embryo tissue from two eggs; T. ruficollis tissues sam- ples were derived from the feather coat. The S. chinen- sis eggs were collected from Ban Hauyrai (15°51’23.1”N, 102°50’06.1”E), Wang Muang Sub-district, Paui Noi Dis- trict, Khon Kaen Province, Thailand. The T. ruficollis samples were collected from a nesting area at the waste- water treatment plant of Khon Kaen University, Khon Kaen Province, Thailand. Chromosomes were prepared from the tissue samples using fibroblast cell culture. Figure 1. General characteristics of Spilopelia chinensis (a) and Tachybaptus ruficollis (b); scale bars = 5 centimeter. 103Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis and Tachybaptus ruficollis Fibroblast cell culture and chromosome preparation The chromosomes were prepared in three steps. First, the half-period old of eggs life cycle of S. chin- ensis and feather coat tissue of T. ruficollis used in this research were collected from bird nests as noted in the section above. Second, the embryos and feather coat tissue were isolated and washed three times with phos- phate buffered saline (PBS). The tissue samples were then chopped into pieces of 1 mm3 and placed onto the sur- face of a tissue culture flask at 41°C in a humidified air atmosphere containing 5% of CO2 for 3-4 h. Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum was added into the inverted flask and cul- tured overnight. The medium was refreshed after 2-3 d. Finally, colchicine was introduced and mixed for fur- ther incubation of 30 min. The cells were harvested at 80-90% confluence using 0.25% trypsin (m/v) solution; they were separated into culture flasks in ratios of 1:2 or 1:3. The cell mixtures were centrifuged at 3,000 rpm for 10 min. After discarding the supernatant, the cells were treated with 10 mL of hypotonic solution (0.075 M KCl) and incubated at room temperature for 30 min. The cells were centrifuged and the supernatant discarded. The cells were fixed by gradually adding fresh cool fixative (3 methanol: 1 acetic acid) up to 8 ml. After centrifuging, the cells were repeatedly fixed until the supernatant was clear. The cells were added to 1 ml fixative by dropping onto a clean cold slide and then air dried (Bai et al. 2011; Phimphan et al. 2015). Chromosome staining The chromosomes were conventionally stained using a 20%-Giemsa working solution for 30 minutes (Patawang et al. 2017). d(CA)15 and d(GC)15 microsat- ellites and telomeric (TTAGGG)n sequence were used as probes. These probes were generated by PCR (PCR DIG-Probe Synthesis Kit, Roche) in the absence of a DNA template. Fluorescence in situ hybridization (FISH) was performed under highly stringent conditions on mitotic chromosome spreads. Metaphase chromosomes and non-metaphase cells on slides were incubated with RNAse (40 lg/ml) for 1.5 h at 37 OC. After the chro- mosomal DNA was denatured for 4 min in 70 % for- mamide/29 SSC at pH 7.0 and 70 OC, the hybridization mixture (2.5 ng/ll probes, 2 lg/ll salmon sperm DNA, 50 % deionized formamide, and 10 % dextran sulphate) was dropped on the slides and the hybridization was performed for 14 h at 37 OC in a moist chamber con- taining 29 SSC. The first post-hybridization wash was performed with 29 SSC for 5 min at 65 OC, and a final washing was performed at room temperature in 19 SSC for 5 min. The microsatellite repeats and telomeric probe were detected using Anti-digoxigenin-FITC. Finally, the slides were counterstained with DAPI and mounted in an antifade solution (Getlekha et al. 2016). Chromosome checking and classifying The lengths of short arm (Ls) and long arm (Ll) chromosomes were measured to calculate the length of the total arm chromosome (LT, LT = Ls + Ll). Relative length (RL) and centromeric index (CI) were estimated. CI was also computed to classify the types of chromo- somes according to Chaiyasut (1989). All parameters were used in karyotyping and idiograming. RESULTS AND DISCUSSION Karyological characteristics of S. chinensis Both embryo samples of S. chinensis showed a dip- loid number of 70. The two embryos exhibited the same type of sex chromosome – Z and W, which lead to pre- sumed female embryo. The autosome comprised of 10 macrochromosomes –v4 large metacentric, 2 medium acrocentric, 2 small metacentric, and 2 small submeta- centric – and 58 microchromosomes (Table 1 and Fig- ures 2a-b). The diploid number found here differed from previ- ous reports in the genus Spilopelia: 2n=80 in S. chinensis (You-Sheng et al. 2008), 2n=66 in S. risoria (Tange and Nakahara 1938-1939), 2n=78; 10 macrochromosomes + two sex-chromosomes (ZZ/ZW) + 66 microchromo- somes and 2n=76; 16 macrochromosomes + 60 micro- chromosomes in S. decaocto (Srivastava and Misra 1971), and 2n=76; 16 macrochromosomes + 60 microchromo- somes in S. orientalis orientalis (Makino et al. 1956). Chromosomal features of T. ruficollis T. ruficollis had a diploid number of 60 and funda- mental number of 80 in both male and female (Figures 3a-b). The karyotype comprised of 20 macrochromo- somes –2 large metacentric, 2 large submetacentric, 2 large acrocentric, 8 small metacentric, 4 small submeta- centric and two sex chromosomes – and 40 microchro- mosomes. The sex chromosomes of T. ruficollis were classified to the ZZ/ZW system; Z was a medium sub- metacentric chromosome and W was a small submeta- centric chromosome (Table 2 and Figures 3a-b). Also, 104 Isara Patawang et al. the karyotype showed the gradually series size of the 11th to 30th pairs of microchromosomes. Our result differed from Ebied et al. (2005), who found a diploid number of 58 in T. ruficollis from an Egyptian population. Howev- er, many of the karyotypic features of these two popula- tions of T. ruficollis were the same, including the num- ber of macrochromosomes (18) and sex chromosomes (2), and the type and size of each. The molecular cytogenetical features in this report that exhibited only on the male T. ruficollis chromosome included two microsatellites and telomeric sequences. First, signals of d(CA)15 microsatellites showed two sig- nals on two microchromosomes; these presented alike in interphase (Figure 4a), prophase (Figure 4b) and metaphase (Figure 4c) cells. Next, microsatellite d(GC)15 appeared on the sub-centromeric region of the long arm of one chromosome of the first pair macrochromo- some (Figure 4d), shown in the idiogram as pair 1a and 1b (Figure 4e), which is same only one signal of both non-metaphase and metaphase cells. Finally, AGGGTTn sequence signals showed on each telomeric region of all macro- and microchromosomes, which appeared as green signals on interphase, prophase and metaphase cells as shown in Figures 4(f-h). Ours is the first study of these markers in this species, and is one of only a few avian chromosomal reports. Microsatellites, simple sequence repeats (SSR), short tandem repeats (STR) and simple sequence length poly- morphisms (SSLP) are found in prokaryotes and eukary- otes. They are widely dispersed in the genome, especially in the euchromatin of eukaryotes, and coding and non- coding nuclear and organellar DNA (Vieira et al. 2016; Kumar 2018). The signals of d(CA)15 microsatellites on two microchromosomes of male T. ruficollis showed the one functional that was needed to find the answer in the future study. The signal of the d(GC)15 micro- satellite that exhibited on only one chromosome of the 1st pair is another issue that needs to be addressed. We used AGGGTTn sequence probes to investigate the fea- ture of the male T. ruficollis chromosome. AGGGTTn are repeated sequences on the terminal end of the chromo- some arm of general vertebrates, for example humans, mice and the Xenopus frog (Ichikawa et al. 2015). The AGGGTTn signals that appeared on the interphase, prophase and metaphase cells of the male T. ruficol- lis showed the existence of this sequence in this species (Figures 4f-h). Overview of avian chromosome In birds, females are the heterogametic sex with Z and W sex chromosomes; males are the homogametic sex, with ZZ sex chromosomes. Studies of sex chromosome evolu- tion in birds and other systems with female heterogamety are important, because they offer independent replication of observations from X–Y species. We observed the het- erogametic ZW sex chromosomes in female T. ruficollis (Figure 5a) and found heterogametic chromosomes in two embryonic S. chinensis samples (Figure 5b) in this study; this agreed with other avian sex chromosome studies (Ellegren 2000; Shibusawa et al. 2004). Most avian chromosome studies have shown con- served characteristics on three macrochromosome pairs, including the 1st (metacentric, m), 2nd (submetacentric, sm) and 3rd (acrocentric, a) pairs. In addition, the 4th pair (metacentric or submetacentric) have been shown to exhibit the semi-conserved characteristic typical of many avian species. These characteristics have been Table 1. Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL), centro- meric index (CI), and standard deviation (SD) of RL, CI from 20 metaphase cells of two female individuals spotted dove (Spilopelia chinen- sis), 2n=70. Ch.p Ls Ll LT RL±SD CL±SD Ch.s Ch.t 1 3.630 5.190 8.820 0.242±0.012 0.588±0.024 L m 2 2.690 3.920 6.610 0.181±0.010 0.593±0.030 L m 3 1.180 4.320 5.500 0.151±0.008 0.785±0.026 M a 4 1.770 2.260 4.030 0.110±0.008 0.561±0.032 S m 5 1.450 2.210 3.660 0.100±0.009 0.604±0.028 S sm 1st Sex chro. 1.850 2.220 4.070 0.111±0.008 0.545±0.024 S m 2nd Sex chro. 1.340 2.490 3.830 0.105±0.010 0.650±0.030 S sm 7-35 - - - - - Microchromosomes Abbreviations: Ch.p, chromosome pair; Ch.s, chromosome size; Ch.t, chromosome type; L, large size; M, medium size; S, small size; m, metacentric; sm, submetacentric; a, acrocentric. 105Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis and Tachybaptus ruficollis observed in many species, for example Agelaius phoeni- ceus [2n=76] (Cox and James 1984), Anas platyrhynchos [2n=80] (Skinner et al. 2009), Rupornis magnirostris [2n=68], Buteogallus meridionallis [2n=68], and Asturina nitida [2n=68] (de Oliveira et al. 2013), Lonchura punct- ulata [2n=72] (Kaewmad et al. 2013), Ara macao [2n=62- 64] (Seabury et al. 2013), Turdus rufiventris [2n=78], T. albicollis [2n=78] (Kretschmer et al. 2014), Gallus gallus [2n=78] (Phimphan et al. 2015); with the 1st pair m, 2nd sm, 3rd a and 4th m/sm. We found the same conserved chromosome pair characteristics in the two species in our study as in these other avian reports. In addition, the microchromosome is one of many characteristics that has been conserved in the genome of all avian and many reptilian species. The archetypal avi- an chromosome comprises about 40 chromosome pairs Figure 2. Metaphase chromosome plates and standardized karyotypes of embryonic individual 1 (a) and embryonic individual 2 (b) Spilope- lia chinensis, 2n=70 by conventional staining. 106 Isara Patawang et al. and usually 30 small to tiny microchromosome pairs. This karyotypic feature perhaps evolved 100-250 million years ago (Burt 2002). The S. chinensis and T. ruficollis in this study had a microchromosome number of 58 and 40, respectively, indicating the close evolutionary lines between these two species and other avian species. ACKNOWLEDGEMENTS This research was financially supported by the Chi- ang Mai University, Thailand. We would like to thank the Cytogenetics and Cytosystematics Research labo- ratory of the Department of Biology, Faculty of Sci- ence, Chiang Mai University for their help. The Insti- tute of Animals for Scientific Purpose Development of the National Research Council of Thailand (Resolution U1-04491-2559) approved this project. REFERENCES Bai C, Wang D, Li C, Jin D, Li C, Guan W, Ma Y. 2011. Establishment and biological characteristics of a Jin- gning chicken embryonic fibroblast bank. Eur J His- tochem. 55(1):e4. BirdLife International, Species factsheet: Tachybaptus ruficollis. 2020. Cambridge: BirdLife International; [accessed 2020 May 25]. http://www.birdlife.org/. Burt DW. 2002. Origin and evolution of avian microchro- mosomes. Cytogenet Genome Res. 96(1-4):97–112. Chaiyasut K. 1989. Cytogenetics and cytotaxonomy of the genus Zephyranthes. Bangkok: Department of Botany, Faculty of Science, Chulalongkorn University. Thai. Cox J, James FC. 1984. Karyotypic uniformity in the red- winged blackbird. Condor. 86:416–422. Degrandi TM, Barcellos SA, Costa AL, Garnero ADV, Hass I, Gunski RJ. 2020. Introducing the bird chro- mosome database: An overview of cytogenetic stud- ies in birds. Cytogenet Genome Res. 160(4):199–205. de Oliveira EH, Tagliarini MM, dos Santos MS, O’Brien PC, Ferguson-Smith MA. 2013. Chromosome paint- ing in three species of Buteoninae: a cytogenetic sig- nature reinforces the monophyly of South American species. PLoS One. 8(7):e70071. Ebied AM, Hassan HA, Abu Almaaty AH, Yaseen AE. 2005. Karyotypic characterization of ten species of birds. Cytologia. 70(2):181–194. Ellegren H. 2000. Evolution of the avian sex chromo- somes and their role in sex determination. Trends Ecol Evol. 15(5):188–192. Getlekha N, Molina WF, Cioffi MB, Yano CF, Manee- chot N, Bertollo LNC, Supiwong W, Tanomtong A. 2016. Repetitive DNAs highlight the role of chromo- somal fusions in the karyotype evolution of Dascyl- lus species (Pomacentridae, Perciformes). Genetica. 144(2):203–211. Gibbs D, Barnes E, Cox J. 2001. Pigeons and doves: a guide to the pigeons and doves of the world. Con- necticut: Yale University Press. Table 2. Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL), cen- tromeric index (CI), and standard deviation (SD) of RL, CI from 20 metaphase cells of male and female little grebe (Tachybaptus ruficollis), 2n=60. Ch.p Ls Ll LT RL±SD CL±SD Ch.s Ch.t 1 4.410 5.920 10.330 0.185±0.004 0.573±0.020 L m 2 3.150 5.750 8.900 0.159±0.005 0.646±0.032 L sm 3 0.900 5.900 6.800 0.122±0.004 0.868±0.015 L a 4 1.800 2.530 4.330 0.078±0.006 0.584±0.025 S m 5 1.450 2.560 4.010 0.072±0.005 0.638±0.040 S sm 6 1.500 2.340 3.840 0.069±0.004 0.609±0.035 S sm 7 1.340 1.760 3.100 0.056±0.005 0.568±0.042 S m 8 1.300 1.450 2.750 0.049±0.007 0.527±0.045 S m 9 1.250 1.400 2.650 0.047±0.003 0.528±0.038 S m Z 2.030 3.450 5.480 0.098±0.004 0.630±0.042 M sm W 1.320 2.290 3.610 0.065±0.003 0.634±0.036 S sm 11-30 - - - - - Microchromosomes Abbreviations: Ch.p, chromosome pair; Ch.s, chromosome size; Ch.t, chromosome type; L, large size; M, medium size; S, small size; m, metacentric; sm, submetacentric; a, acrocentric. 107Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis and Tachybaptus ruficollis Figure 3. Metaphase chromosome plates and standardized karyotypes of male (a) and female (b) Tachybaptus ruficollis, 2n=60 by conven- tional staining. 108 Isara Patawang et al. Griffin DK, Robertson LBW, Tempest HG, Skinner BM. 2007. The evolution of the avian genome as revealed by comparative molecular cytogenetics. Cytogenet Genome Res. 117(1–4):64–77. Ichikawa Y, Nishimura Y, Kurumizaka H, Shimizu M. 2015. Nucleosome organization and chromatin dynamics in telomeres. Biomol Concepts. 6(1):67–75. Jarvis ED, Mirarab S, Aberer AJ, Li B, Houde P, Li C, Ho SYW, Faircloth BC, Nabholz B, Howard JT, et al. 2014. Whole-genome analyses resolve early branches in the tree of life of modern birds. Science. 346(6215):1320–1331. Kaewmad P, Tanomtong A, Gomontean B, Wonkaonoi W, Khunsook S, Sanoamuang L. 2013. First karyo- Figure 4. The molecular cytogenetical features of male Tachybaptus ruficollis, including: d(CA)15 microsatellite signals on two microchromo- somes of interphase (a) prophase (b) and metaphase (c); d(GC)15 microsatellite signals on only one chromosome of the 1st pair of metaphase (d, red arrow), interphase (d, yellow arrow) and the position of this signal on idiogram (e); and AGGGTTn telomeric sequences on inter- phase (f ), prophase (g) and metaphase (h). 109Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis and Tachybaptus ruficollis logical analysis of black crowned crane (Balearica pavonina) and scaly breasted munia (Lonchura punct- ulata) by conventional staining technique. Cytologia. 78(3):205–211. Kretschmer R, Gunski RJ, Del Valle Garnero A, de Oliveira Furo I, O’Brien PCM, Ferguson-Smith MA, de Oliveira EHC. 2014. Molecular cytogenetic char- acterization of multiple intrachromosomal rearrange- ments in two representatives of the genus Turdus (Turdidae, Passeriformes). PLoS One. 9(7):e103338. Ksepka DT, Boyd CA. 2012. Quantifying historical trends in the completeness of the fossil record and the con- tributing factors: an example using Aves. Paleobiol- ogy. 38(1):112–125. Kumar R. 2018. Microsatellite marker. In: Vonk J, Shack- elford T. (eds) Encyclopedia of Animal Cognition and Behavior. Cham: Springer. Makino S, Udagama T, Yamashina Y. 1956. Karyotype studies in birds. 2: a comparative study of chromo- somes in the Columbidae. Caryologia. 8(2):275– 293. Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S, Escalante AA. 2011. Evolution of mod- ern birds revealed by mitogenomics: timing the radiation and origin of major orders. Mol Biol Evol. 28(6):1927–1942. Patawang I, Tanomtong A, Getlekha N, Phimphan S, Pin- thong K, Neeratanaphan L. 2017. Standardized kary- otype and idiogram of Bengal monitor lizard, Vara- nus bengalensis (Squamata, Varanidae). Cytologia. 82(1):75–82. Phimphan S, Tanomtong A, Chuaynkern Y, Pratumtong D. 2015. Karyological analysis of red jungle fowl (Gallus gallus gallus Linnaeus, 1758) using egg fibro- blastic cell culture. KKU Sci J. 43(1):39–48. Thai. Pratumthong D, Thunhikorn S, Duengkae P. 2011. A checklist of the birds in Thailand. Journal of Wildlife in Thailand. 18(1):152–319. Thai. Seabury CM, Dowd SE, Seabury PM, Raudsepp T, Brightsmith DJ, Liboriussen P, Halley Y, Fisher CA, Owens E, Viswanathan G, et al. 2013. A multi-plat- form draft de novo genome assembly and compara- tive analysis for the Scarlet Macaw (Ara macao). PLoS One. 8(5):e62415. Shibusawa M, Nishibori M, Nishida-Umehara C, Tsud- zuki M, Masabanda J, Griffin DK, Matsuda Y. 2004. Karyotypic evolution in the Galliformes: an examina- tion of the process of karyotypic evolution by com- parison of the molecular cytogenetic findings with the molecular phylogeny. Cytogenet Genome Res. 106(1):111–9. Skinner BM, Robertson L BW, Tempest HG, Lang- ley EJ, Ioannou D, Fowler KE, Crooijmans R PMA, Hall AD, Griffin DK, Völker M. 2009. Comparative genomics in chicken and Pekin duck using FISH mapping and microarray analysis. BMC Genomics. 10:357. Srivastava MDL, Misra M. 1971. Somatic chromosomes of Streptopdia decaocto (Golumbiformes). J Hered. 62(6):373–374. Tange M, Nakahara K. 1938-1939. On the chromosomes of the Ring Dove, Streptopelia risoria. Okajimas Folia Anat Jpn. 17(5):477–478. Vieira MLC, Santini L, Diniz AL, de Freitas Munhoz C. 2016. Microsatellite markers: what they mean and why they are so useful. Genet Mol Biol. 39(3):312– 328. You-Sheng R, Zhang-Feng W, Xue-Wen C, Huai-Yu Z. 2008. Study on karyotype of Streptopelia chinensis and Columba livia domestica. J Nanchang Normal Univ. 3:31–33. Yuri T, Kimball RT, Harshman J, Bowie RCK, Braun MJ, Chojnowski JL, Han KL, Hackett SJ, Huddleston CJ, Moore WS, et al. 2013. Parsimony and model- based analyses of indels in avian nuclear genes reveal congruent and incongruent phylogenetic signals. Biol(Basel). 2:419–444. Figure 5. Standardized macro-chromosomal idiogram of Tachybap- tus ruficollis, 2n=60 (a) and Spilopelia chinensis, 2n=70 (b) by con- ventional staining. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 4 - 2021 Firenze University Press Cytogenetic analyses in three species of Moenkhausia Eigenmann, 1903 (Characiformes, Characidae) from Upper Paraná River (Misiones, Argentina) Kevin I. Sánchez1,*, Fabio H. Takagui2, Alberto S. Fenocchio3 Genetic variations and interspesific relationships in Lonicera L. (Caprifoliaceae), using SCoT molecular markers Fengzhen Chen1, Dongmei Li2,* , Mohsen Farshadfar3 The new chromosomal data and karyotypic variations in genus Salvia L. (Lamiaceae): dysploidy, polyploidy and symmetrical karyotypes Halil Erhan Eroğlu1,*, Esra Martin2, Ahmet Kahraman3, Elif Gezer Aslan4 Cytogenetic survey of eight ant species from the Amazon rainforest Luísa Antônia Campos Barros1, Gisele Amaro Teixeira2, Paulo Castro Ferreira1, Rodrigo Batista Lod1, Linda Inês Silveira3, Frédéric Petitclerc4, Jérôme Orivel4, Hilton Jeferson Alves Cardoso de Aguiar1,5,* Molecular phylogeny and morphometric analyses in the genus Cousinia Cass. (Family Asteraceae), sections Cynaroideae Bunge and Platyacanthae Rech. f. Neda Atazadeh1,*, Masoud Sheidai1, Farideh Attar2, Fahimeh Koohdar1 A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.) Melika Tabasi1, Masoud Sheidai1,*, Fahimeh Koohdar1, Darab Hassani2 Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers: A high value medicinal plant Lu Feng1,*, Fariba Noedoost2 Morphometric analysis and genetic diversity in Rindera (Boraginaceae-Cynoglosseae) using sequence related amplified polymorphism Xixi Yao1, Haodong Liu2,*, Maede Shahiri Tabarestani3 Biosystematics, fingerprinting and DNA barcoding study of the genus Lallemantia based on SCoT and REMAP markers Fahimeh Koohdar*, Neda Aram, Masoud Sheidai Karyotype analysis in 21 plant families from the Qinghai–Tibetan Plateau and its evolutionary implications Ning Zhou1,2, Ai-Gen Fu3, Guang-Yan Wang1,2,*, Yong-Ping Yang1,2,* Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis (Scopoli, 1786) and Tachybaptus ruficollis (Pallas, 1764) in Thailand Isara Patawang1,*, Sarawut Kaewsri2, Sitthisak Jantarat3, Praween Supanuam4, Sarun Jumrusthanasan2, Alongklod Tanomtong5 Centromeric enrichment of LINE-1 retrotransposon in two species of South American monkeys Alouatta belzebul and Ateles nancymaae (Platyrrhini, Primates) Simona Ceraulo, Vanessa Milioto, Francesca Dumas* Repetitive DNA mapping on Oligosarcus acutirostris (Teleostei, Characidae) from the Paraíba do Sul River Basin in southeastern Brazil Marina Souza Cunha1,2,*,#, Silvana Melo1,3,#, Filipe Schitini Salgado1,2, Cidimar Estevam Assis1, Jorge Abdala Dergam1,* Karyomorphology of some Crocus L. taxa from Uşak province in Turkey Aykut Yilmaz*, Yudum Yeltekin Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis L. Egizia Falistocco1,*,+, Gianpiero Marconi1,+, Lorenzo Raggi1, Daniele Rosellini1, Marilena Ceccarelli2, Emidio Albertini1