Research Article |
Corresponding author: Alisa O. Vershinina ( vershinina.alice@gmail.com ) Academic editor: Snejana Grozeva
© 2015 Alisa O. Vershinina, Boris A. Anokhin, Vladimir A. Lukhtanov.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Vershinina A, Anokhin B, Lukhtanov V (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. https://doi.org/10.3897/CompCytogen.v9i2.4715
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Ribosomal DNA clusters and telomeric repeats are important parts of eukaryotic genome. However, little is known about their organization and localization in karyotypes of organisms with holocentric chromosomes. Here we present first cytogenetic study of these molecular structures in seven blue butterflies of the genus Polyommatus Latreille, 1804 with low and high chromosome numbers (from n=10 to n=ca.108) using fluorescence in situ hybridization (FISH) with 18S rDNA and (TTAGG)n telomeric probes. FISH with the 18S rDNA probe showed the presence of two different variants of the location of major rDNA clusters in Polyommatus species: with one or two rDNA-carrying chromosomes in haploid karyotype. We discuss evolutionary trends and possible mechanisms of changes in the number of ribosomal clusters. We also demonstrate that Polyommatus species have the classical insect (TTAGG)n telomere organization. This chromosome end protection mechanism probably originated de novo in small chromosomes that evolved via fragmentations.
Lycanidae , ribosomal DNA, chromosome, taxonomy, karyotype evolution, telomeres
Most studied butterfly families and genera share the modal chromosome number of n=30 or n=31 (
These are the reasons explaining why the karyotype evolution is still poorly understood in Lepidoptera, though some data regarding karyotype organization and genome rearrangements are present for Bombyx mori (Linnaeus, 1758) (
A molecular hybridization technique, such as fluorescence in situ hybridization (FISH), is a very useful method for studying molecular organization of chromatin and for tracing individual chromosomes in different species (
The sparse data available have contributed to generalizations about the pattern and mode of the major rDNA cluster evolution in Lepidoptera. According to
Additionally, we analyzed molecular organization of telomeric repeats in Polyommatus (subgenus Agrodiaetus). In animals there are three main types of telomeric tandem repeats: TTAGGG, TTAGGC, and TTAGG. The TTAGGG motif is probably ancestral for all Metazoa and has been found in all multicellular animals, except round worms and arthropods (
Butterfly species were collected from 2005 to 2011 by V. Lukhtanov, A. Dantchenko and N. Shapoval in Iran (Table
List of Polyommatus (subgenus Agrodiaetus) populations used in the present study and their haploid chromosome numbers (n) according to original data.
Species | n | Province | Locality | altitude | date |
---|---|---|---|---|---|
P. (A.) caeruleus | 10 | Golestan | Shahkuh | 2700–3100 m | 2005.VII.22 |
P. (A.) hamadanensis | 19 | Lortestan | Sarvand, 33°22.38'N/ 49°10.25'E | 2070 m | 2009.VII.22 |
P. (A.) hamadanensis | 21 | Esfahan | Kuhe-Tamandar Mts, 33°12.72'N/ 49°56.43'E | 2336 m | 2011.VII.16 |
P. (A.) karindus | ca.68 | Kurdistan | 40 km SW Saqqez, 36°04.39'N/ 45°59.06'E | 1869 m | 2009.VII.29 |
P. (A.) morgani | 25 | Kurdistan | 14 km N of Chenareh, 35°42.12'N/ 46°22.35'E | 2025 m | 2009.VII.28 |
P. (A.) peilei | 39 | - | 14 km N of Chenareh, 35°42.127'N/ 46°22.35'E | 2025 m | 2009.VII.28 |
P. (A.) pfeifferi | ca.108 | Fars | Barm-i-Firuz Mts, 30°23'N/ 51°56'E | 2900 m | 2002.VII.19 |
P. (A.) sennanensis | 27 | Qom | Qom-Qamsar, 33°43.80'N/ 51°29.53'E | 1862 m | 2009.VII.16 |
18S rDNA and (TTAGG)n probe preparation and hybridization were carried out as described in
ca. (circa) approximately.
FISH fluorescence in situ hybridization.
MI meiotic metaphase I.
MII meiotic metaphase II.
NOR nucleolus organizer region.
In all karyotypes weak and strong telomeric signals were present (Figs
Localization of FISH signals on telomeres (red) and rDNA clusters (green) in squash chromosome preparations of seven species of Polyommatus (subgenus Agrodiaetus). Chromosomes are counterstained with DAPI. Note telomeric signals of different intensity. 1–7 one 18S rDNA cluster is found 8 two 18S rDNA clusters are found 1P. (A.) caeruleus, MII 2–3P. (A.) hamadanensis, MI cells from two different populations with different karyotypes (n=19 and n=21 accordingly) 4P. (A.) karindus, MII 5P. (A.) morgani, MI 6P. (A.) peilei, MII 7P. (A.) pfeifferi, MII 8P. (A.) sennanensis, MII. The inset in the upper right corner shows twice enlarged image of rDNA-carrying chromosomes.
P. (A.) caeruleus had n=10 with one rDNA cluster localized in one of the chromosome pairs (Table
In P. (A.) hamadanensis, the haploid chromosome number of n=19 was found in MI cells of one studied individual from Lorestan province. In the specimens from another population (Esfahan province) the number of n=21 was found in MI cells (Table
In P. (A.) karindus, the haploid chromosome number of n= ca.68 was found in MII cells (Table
In P. (A.) morgani, the haploid chromosome number of n=25 was found in MI cells of a single individual (Table
In P. (A.) peilei, the haploid chromosome number of n=39 was found in MII cells (Table
In P. (A.) pfeifferi, the chromosome number was only approximately established and was n=ca.108 (Table
In P. (A.) sennanensis, the haploid chromosome number of n=27 was found in MII cells (Table
Previous investigations by
Specifically for blue butterflies (Lycaenidae),
Chromosome fissions lead to strong decrease in size of fragmented chromosomes (
The third possible mechanism which can change the number of rDNA clusters is the formation of a hybrid lineage or a homoploid hybrid speciation (hybridization without a change in chromosome number,
On the basis of rDNA evolutionary dynamics and the repetitive structure of rDNA in Lepidoptera
In our study, FISH with telomeric (TTAGG)n probe conclusively demonstrate that Polyommatus (subgenus Agrodiaetus) blue butterflies have classical insect telomere organization. On small chromosomes of P. (A.) peilei, P. (A.) karindus and P. (A.) pfeifferi, originated by fragmentations, telomeric signals are also detected. Generally, fissions lead to breakdown in chromosome structure because after this reorganization the newly originated fragmented chromosomes lack telomeres and their chromosome ends need to be protected from degradation (
Appearance of a new telomere seems to be a highly important event in genome evolution, however its proximate and ultimate mechanisms are still unknown. Polyommatus (subgenus Agrodiaetus) butterflies with their diverse karyotypes represent a good model system for studying these processes.
We thank our colleagues Nazar Shapoval and Alexander Dantchenko for samples from Iran. We are also very grateful to Valentina Kuznetsova for laboratory facilities and valuable discussion. We are very grateful to Walther Traut, Francisco Panzera and František Marec for their suggestions during the preparation of this article. 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.