Short Communications
Short Communications
The blue butterfly Polyommatus (Plebicula) atlanticus (Lepidoptera, Lycaenidae) holds the record of the highest number of chromosomes in the non-polyploid eukaryotic organisms
expand article infoVladimir A Lukhtanov
‡ Zoological Institute of Russian Academy of Science, St. Petersburg, Russia
Open Access


The blue butterfly species Polyommatus (Plebicula) atlanticus (Elwes, 1906) (Lepidoptera, Lycaenidae) is known to have a very high haploid number of chromosomes (n= circa 223). However, this approximate count made by Hugo de Lesse 45 years ago was based on analysis of a single meiotic I metaphase plate, not confirmed by study of diploid chromosome set and not documented by microphotographs. Here I demonstrate that (1) P. atlanticus is a diploid (non-polyploid) species, (2) its meiotic I chromosome complement includes at least 224-226 countable chromosome bodies, and (3) all (or nearly all) chromosome elements in meiotic I karyotype are represented by bivalents. I also provide the first data on the diploid karyotype and estimate the diploid chromosome number as 2n=ca448-452. Thus, P. atlanticus is confirmed to possess the highest chromosome number among all the non-polyploid eukaryotic organisms.


Acipenser, Amoeba proteus, Astacus, Aulacantha scolymantha, chromosome number, karyotype evolution, linkage group, Lycaenidae, Ophioglossum, Pacifastacus, Plebicula, Polyommatus, vizcacha rat


Trends and mechanisms of chromosome number and chromosome structure changes are currently a matter of a sharp discussion (Qumslyeh 1994, Imai et al. 2002, Eichler and Sankoff 2003, Schubert 2007, Lukhtanov et al. 2005, 2011, 2015a, Vila et al. 2010, Dincă et al. 2011, Bureš and Zedek 2014, Fleischmann et al. 2014, Lukhtanov 2014, Vershinina et al. 2015). These changes are important in evolution of eukaryotic organisms since they can trigger speciation via hybrid-sterility or/and via suppressed-recombination mechanisms (Faria and Navarro 2010). Fixation of these changes plays a serious role in maintaining postzygotic isolation between well-established species and protects hybridizing lineages from merging (Kandul et al. 2007). Change of chromosome number results in change of linkage groups and thus affects rate of meiotic recombination (Dumont and Payseur 2011).

Comparative analysis of chromosomal data is a promising way for understanding the patterns of karyotype evolution (Vershinina and Lukhtanov 2013), and this analysis requires accurate and precise data on chromosome complements of species under study. The blue butterfly Polyommatus (Plebicula) atlanticus (Elwes, 1906) is mentioned in many publications devoted to chromosome number evolution since it is supposed to possess the highest chromosome number (n= circa 223) among all the non-polyploid metazoan animals (e.g. White 1973, Imai et al. 2002, Bureš and Zedek 2014). However, this approximate count made by Hugo de Lesse 45 years ago was based on analysis of a single meiotic I metaphase plate, not confirmed by studies of diploid chromosome set and not documented by microphotographs (de Lesse 1970).

The aim of this study is cytogenetic reinvestigation and documentation of P. atlanticus karyotype with a special consideration of diploid chromosome set of this species.

Material and methods

The studied species is often mentioned in the literature as a member of the genus Lysandra Hemming, 1933 (e.g. de Lesse 1970, White 1973). However, according to the last revision of the tribe Polyommatina, it should be transferred to the genus Polyommatus Latreille, 1804 (Talavera et al. 2013a). The adult male samples used for chromosomal analysis (NK02A032, NK02A033 and NK02A035) were collected in Morocco (Atlas range, Col du Zad pass, 2200 m alt., 27 June 2002) by Roger Vila, Santiago Ramirez and Nikolai Kandul. The methods of chromosomal analysis were described previously (Lukhtanov and Dantchenko 2002, Lukhtanov et al. 2008, 2014, Vershinina and Lukhtanov 2010, Talavera et al. 2013b, Przybyłowicz et al. 2014). Haploid (n) chromosome numbers were analyzed in meiotic I (MI) and meiotic II (MII) cells. Diploid (2n) chromosome numbers were analyzed in asynaptic meiotic cells that can be observed in so called atypical meiosis (see Lorković 1990 for more details on atypical meiosis in Lepidoptera).


The haploid chromosome number n=ca 224–226 was found in MI cells of three studied individuals (Fig. 1a, b). This count was based on analysis of 12 selected MI plates with best quality of chromosome spreading. The meiotic karyotype included one large bivalent, one medium bivalent and 222–224 small chromosome bodies. Multiple MII cells were also observed. The MII cells demonstrated one large and one medium chromosome and multiple dot-like elements, however the precise count of these elements was impossible. The diploid chromosome set was observed in male atypical (asynaptic) meiosis (Fig. 1c, d) in three studied individuals (20 cells were analysed). At this stage at least 434 chromosome entities could be observed: one pair of large chromosomes, one pair of medium chromosomes and at least 430 (most likely more) very small, dot-like chromosomes. Combination of chromosome number count at MI and diploid stages results in conclusion that all (or nearly all) chromosome elements in MI karyotype are represented by bivalents. This assumption results in diploid chromosome number estimation of 2n=ca 448–452.

Figure 1.

Male karyotype of Polyommatus (Plebicula) atlanticus, sample NK02A032. a MI plate b chromosome count in MI plate: red dots indicate distinct separate entities, blue dots indicate doubtful entities, n=224 red dots + 2 blue dots c diploid chromosome set observed in male asynaptic meiosis d chromosome number count in diploid chromosome set; at least 434 entities can be distinguished. Bar = 10 μm.


Previously, the chromosome number was estimated in P. atlanticus as n=ca217-223 (de Lesse 1970). This number has later been interpreted as 2n=446 (e.g. see Bureš and Zedek 2014). However, interpretation of all chromosome bodies visible at MI stage as bivalents should be considered with caution. As it was mentioned by White (1973), “there seems to be no means of distinguishing between univalents, bivalents and multivalents in lepidopteran spermatogenesis – they all look like small spheres or isodiametric bodies in which no structure is observable”. For example, multiple B-chromosomes (which can be often represented by univalents in meiosis) can sometimes accumulate through processes of mitotic or meiotic drive (Jones 2008). Therefore, I believe that analysis of diploid karyotype is indispensable prerequisite for inferring the diploid chromosome number. In my research the combination of chromosome number counts at MI and diploid stages results in conclusion that all (or nearly all) chromosome elements in MI karyotype are represented by bivalents. This assumption leads to conclusion that diploid chromosome number can be estimated in P. atlanticus as 2n=ca 448–452, and the haploid number can be estimated as n=ca 224–226.

In eukaryotic organisms the highest number of chromosomes has been so far reported in radiolarian species, e.g. in Aulacantha scolymantha Haeckel, 1862 (Cercozoa, Aulacanthidae) there are more than 2000 chromosomes (Lecher 1978). This high number is an output of polyploidization (Lecher 1978, Parfrey et al. 2008), which includes 7 or 8 cycles of endomitosis resulting in each chromosome represented by 128 or 256 copies (Lecher 1978).

500 chromosomes were reported for asexual lobose amoebae, Amoeba proteus (Pallas, 1766) (Amoebozoa, Amoebidae) (Parfrey et al. 2008). This high number is also considered to be polyploid although the questions about the precise number of chromosomes and the ploidy level are still unanswered despite the fact that cytology of this well-known species has been under study for about 200 years (Podlipaeva et al. 2013).

Very high chromosome numbers are known in some plants, e.g. in ferns of the genus Ophioglossum Linnaeus, 1753 (Pteridophyta, Ophioglossaceae) n=120–720 (Shinohara et al. 2013). However, this genus is also characterized by a high degree of polyploidization with x=120 as a basic chromosome number and with the highest n=720 in hexaploid species Ophioglossum reticulatum Linnaeus, 1753 (Khandelwal 1990, Barker 2013, Shinohara et al. 2013).

In vertebrate animals the highest chromosome number (372 elements in mitotic cell divisions) is known in sturgeon Acipenser brevirostrum Lesueur, 1818 (Acipenseriformes, Acipenseridae) (Kim et al. 2005), however this species is hexaploid one, too (Kim et al. 2005). In mammals the highest chromosome number 2n=102 is found in vizcacha rat Tympanoctomys barrerae (B. Lawrence, 1941) (Rodentia, Octodontidae) (Suárez-Villota et al. 2012).

According to White (1973), the highest haploid chromosome number recorded in invertebrate animals (except for P. atlanticus) is n=191 in the butterfly Polyommatus nivescens (Keferstein, 1851) (Lepidoptera, Lycaenidae) (de Lesse 1970, White 1973). The next highest haploid numbers were reported in crayfish, Pacifastacus leniusculus trowbridgii (Stimpson, 1857) (Crustacea, Astacidae) (n=188, Niiyama 1962) and Astacus leptodactylus (Eschscholtz, 1823) (Crustacea, Astacidae) (n=184, Silver and Tsukersis 1964). The last two counts were even erroneously cited as the records for the highest chromosome numbers in the animal kingdom (Fetzner and Crandall 2002). However, the numbers in crayfish are, first, lower than the numbers discovered in the blue butterflies. Second, they were disputed in the more recent publications (e.g. n=93 was mentioned in P. l. trowbridgii, Murofushi 1999, Imai et al. 2002 and n=90 was mentioned in A. leptodactylus, Mlinarec et al. 2011). All these haploid numbers are essentially lower than numbers found in P. atlanticus.

The data obtained indicate that P. atlanticus is a diploid (not polyploid) species since it possesses double (not multiple) number of chromosomes that can be individually recognized: one pair of large and one pair of medium chromosomes. Thus, P. atlanticus is confirmed to have the highest chromosome number among all the non-polyploid eukaryotic organisms.


I thank Roger Vila, Santiago Ramirez and Nikolai Kandul for providing collected specimens. I thank Valentina Kuznetsova, Vladimir Gokhman, Nikolai Kandul and Vladimir Trifonov for valuable comments. The financial support for this study was provided by the grant from the Russian Science Foundation N 14-14-00541 to Zoological Institute of the Russian Academy of Sciences.


  • Barker MS (2013) Karyotype and genome evolution in Pteridophytes. In: Leitch IJ, Greilhuber J, Doležel J, Wendel JF (Eds) Plant Genome Diversity 2: Physical Structure, Behaviour and Evolution of Plant Genomes. Springer, Wien, 245–253. doi: 10.1007/978-3-7091-1160-4_15
  • Bureš P, Zedek F (2014) Holokinetic drive: centromere drive in chromosomes without centromeres. Evolution 68(8): 2412–2420. doi: 10.1111/evo.12437
  • de Lesse H (1970) Les nombres de chromosomes dans le groupe de Lysandra argester et leur incidence sur sa taxonomie [Lep. Lycaenidae]. Bulletin de la Société entomologique de France 75: 64–68.
  • Dincă V, Lukhtanov VA, Talavera G, Vila R (2011) Unexpected layers of cryptic diversity in Wood White Leptidea butterflies. Nature Communications 2: 234. doi: 10.1038/ncomms1329
  • Dumont BL, Payseur BA (2011) Genetic analysis of genome-scale recombination rate evolution in house mice. PLoS Genetics 7: e1002116. doi: 10.1371/journal.pgen.1002116
  • Eichler EE, Sankoff D (2003) Structural dynamics of eukaryotic chromosome evolution. Science 301: 793–797. doi: 10.1126/science.1086132
  • Faria R, Navarro A (2010) Chromosomal speciation revisited: rearranging theory with pieces of evidence. Trends in Ecology and Evolution 25: 660–669. doi: 10.1016/j.tree.2010.07.008
  • Fetzner JW, Crandall KA (2002) Genetic Variation In: Holdich DM (Ed.) Biology of freshwater crayfish. Blackwell, Oxford, 291–326.
  • Fleischmann A, Michael TP, Rivadavia F, Sousa A, Wang W, Temsch EM4, Greilhuber J, Müller KF, Heubl G (2014) Evolution of genome size and chromosome number in the carnivorous plant genus Genlisea (Lentibulariaceae), with a new estimate of the minimum genome size in angiosperms. Annals of Botany 114: 1651–1663. doi: 10.1093/aob/mcu189
  • Imai HT, Satta Y, Wada M, Takahata N (2002) Estimation of the highest chromosome number of eukaryotes based on the minimum interaction theory. Journal of Theoretical Biology 217: 61–74. doi: 10.1006/jtbi.2002.3016
  • Jones RN, Gonzalez-Sanchez M, Gonzalez-Garcia M, Vega JM, Puertas MJ (2008) Chromosomes with a life of their own. Cytogenetic and Genome Research 120: 265–280. doi: 10.1159/000121076
  • Kandul NP, Lukhtanov VA, Pierce NE (2007) Karyotypic diversity and speciation in Agrodiaetus butterflies. Evolution 61(3): 546–559. doi: 10.1111/j.1558-5646.2007.00046.x
  • Kim DS, Nam YK, Noh JK, Park CH, Chapman FA (2005) Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the Acipenseriformes. Ichthyological Research 52(1): 94–97. doi: 10.1007/s10228-004-0257-z
  • Lecher P (1978) The synaptonemal complex in the bipartition division of the radiolaria Aulacantha scolymantha. Canadian Journal of Genetics and Cytology 20(1): 85–95. doi: 10.1139/g78-010
  • Lorkoviç Z (1990) The butterfly chromosomes and their application in systematics and phylogeny. In: Kudrna O (Ed.) Butterflies of Europe. Volume 2. Aula-Verlag, Wiesbaden, 332–396.
  • Lukhtanov V (2014) Chromosome number evolution in skippers (Lepidoptera, Hesperiidae). Comparative Cytogenetics 8(4): 275–291. doi: 10.3897/CompCytogen.v8i4.8789
  • Lukhtanov VA, Dantchenko AV (2002) Principles of highly ordered metaphase I bivalent arrangement in spermatocytes of Agrodiaetus (Lepidoptera). Chromosome Research 10(1): 5–20. doi: 10.1023/A:1014249607796
  • Lukhtanov VA, Kandul NP, Plotkin JB, Dantchenko AV, Haig D, Pierce NE (2005) Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies. Nature 436(7049): 385–389. doi: 10.1038/nature03704
  • Lukhtanov VA, Shapoval NA, Dantchenko AV (2008) Agrodiaetus shahkuhensis sp. n. (Lepidoptera, Lycaenidae), a cryptic species from Iran discovered by using molecular and chromosomal markers. Comparative Cytogenetics 2(2): 99–114.
  • Lukhtanov VA, Dincă V, Talavera G, Vila R (2011) Unprecedented within-species chromosome number cline in the Wood White butterfly Leptidea sinapis and its significance for karyotype evolution and speciation. BMC Evolutionary Biology 11: 109. doi: 10.1186/1471-2148-11-109
  • Lukhtanov VA, Shapoval NA, Dantchenko AV (2014) Taxonomic position of several enigmatic Polyommatus (Agrodiaetus) species (Lepidoptera, Lycaenidae) from Central and Eastern Iran: insights from molecular and chromosomal data. Comparative Cytogenetics 8(4): 313–322. doi: 10.3897/CompCytogen.v8i4.8939
  • Lukhtanov VA, Shapoval NA, Anokhin BA, Saifitdinova AF, Kuznetsova VG (2015a) Homoploid hybrid speciation and genome evolution via chromosome sorting. Proceedings of the Royal Society B: Biological Sciences 282(1807): 20150157. doi: 10.1098/rspb.2015.0157
  • Lukhtanov VA, Dantchenko AV, Vishnevskaya MS, Saifitdinova AF (2015b) Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae). Biological Journal of the Linnean Society 116(2): 468–485. doi: 10.1111/bij.12596
  • Mlinarec J, Mužić M, Pavlica M, Šrut M, Klobuč G, Maguire I (2011) Comparative karyotype investigations in the European crayfish Astacus astacus and A. leptodactylus (Decapoda, Astacidae). Crustaceana 84(12-13): 1497–1510. doi: 10.1163/156854011X607015
  • Murofushi M (1999) Cytogenetical study of species specificity in freshwater crawfish. In: Okutani T, Ohta S, Ueshima R (Eds) Updated progress in aquatic invertebrate zoology. Tokyo University Press, Tokyo, 249–260.
  • Niiyama H (1962) On the unprecedentedly large number of chromosomes of the crayfish, Astacus trowbridgii Stimpson. Annotationes Zoologicae Japonenses 35(4): 229–233.
  • Parfrey LW, Lahr DJG, Katz LA (2008) The dynamic nature of eukaryotic genomes. Molecular Biology and Evolution 25(4): 787–794. doi: 10.1093/molbev/msn032
  • Podlipaeva Y, Demin S, Goodkov A (2013) New method for cell cycle synchronization in Amoeba proteus culture. Protistology 8(1): 3–7.
  • Przybyłowicz Ł, Lukhtanov V, Lachowska-Cierlik D (2014) Towards the understanding of the origin of the Polish remote population of Polyommatus (Agrodiaetus) ripartii (Lepidoptera: Lycaenidae) based on karyology and molecular phylogeny. Journal of Zoological Systematics and Evolutionary Research 52(1): 44–51. doi: 10.1111/jzs.12040
  • Qumslyeh MB (1994) Evolution of number and morphology of mammalian chromosomes. Journal of Heredity 35: 455–465.
  • Shinohara W, Nakato N, Yatabe-Kakugawa Y, Oka T, Kim JK, Murakami N, Noda H, Sahashi N (2013) The use of matK in Ophioglossaceae phylogeny and the determination of Mankyua chromosome number shed light on chromosome number evolution in Ophioglossaceae. Systematic Botany 38(3): 564–570. doi: 10.1600/036364413X670232
  • Silver DS, Tsukersis YM (1964) The chromosome number of the crayfish Astacus leptodactylus Esch. Tsitologiya 6: 631–633.
  • Suárez-Villota EY, Vargas RA, Marchant CL, Torres JE, Köhler N, Núñez JJ, de la Fuente R, Page J, Gallardo MH (2012) Distribution of repetitive DNAs and the hybrid origin of the red vizcacha rat (Octodontidae). Genome 55(2): 105–117. doi: 10.1139/g11-084
  • Talavera G, Lukhtanov VA, Pierce NE, Vila R (2013a) Establishing criteria for higher-level classification using molecular data: the systematics of Polyommatus blue butterflies (Lepidoptera, Lycaenidae). Cladistics 29: 166–192. doi: 10.1111/j.1096-0031.2012.00421.x
  • Talavera G, Lukhtanov V, Rieppel L, Pierce NE, Vila R (2013b) In the shadow of phylogenetic uncertainty: the recent diversification of Lysandra butterflies through chromosomal change. Molecular Phylogenetics and Evolution 69: 469–478. doi: 10.1016/j.ympev.2013.08.004
  • Vershinina AO, Lukhtanov VA (2010) Geographical distribution of the cryptic species Agrodiaetus alcestis alcestis, A. alcestis karacetinae and A. demavendi (Lepidoptera, Lycaenidae) revealed by cytogenetic analysis. Comparative Cytogenetics 4(1): 1–11. doi: 10.3897/compcytogen.v4i1.21
  • Vershinina AO, Lukhtanov VA (2013) Dynamics of chromosome number evolution in the Agrodiaetus phyllis species complex (Insecta: Lepidoptera). Cell and Tissue Biology 7(4): 379–381. doi: 10.1134/S1990519X13040159
  • Vershinina AO, Anokhin BA, Lukhtanov VA (2015) Ribosomal DNA clusters and telomeric (TTAGG)n repeats in blue butterflies (Lepidoptera, Lycaenidae) with low and high chromosome numbers. Comparative Cytogenetics 9(2): 161–171. doi: 10.3897/CompCytogen.v9i2.4715
  • Vila R, Lukhtanov VA, Talavera G, Gil-T F, Pierce NE (2010) How common are dot-like distribution ranges? Taxonomical oversplitting in Western European Agrodiaetus (Lepidoptera, Lycaenidae) revealed by chromosomal and molecular markers. Biological Journal of the Linnean Society 101: 130–154. doi: 10.1111/j.1095-8312.2010.01481.x
  • White MJD (1973) Animal cytology and evolution. Cambridge University Press, 961 pp.