Research Article |
Corresponding author: Artem P. Lisachov ( a.p.lisachev@utmn.ru ) Academic editor: Larissa Kupriyanova
© 2016 Artem P. Lisachov, Pavel M. Borodin.
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:
Lisachov AP, Borodin PM (2016) Microchromosome polymorphism in the sand lizard, Lacerta agilis Linnaeus, 1758 (Reptilia, Squamata). Comparative Cytogenetics 10(3): 387-399. https://doi.org/10.3897/CompCytogen.v10i3.7655
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Most true lizards (Lacertidae) share a conservative karyotype, consisting of 18 pairs of macrochromosomes and one microchromosome pair. Homeologues of the microchromosome are present in other squamates and even in chickens. No structural autosomal microchromosome polymorphisms have been described previously in lizards. We found homozygous and heterozygous carriers of a microchromosome variant in a Siberian population of the sand lizard, Lacerta agilis Linnaeus, 1758. The variant microchromosome was almost twice as long as the standard one. In heterozygotes at pachytene, the microchromosomes firstly pair in proximal regions and the central part of the longer axial element undergoes foldback synapsis, then its distal region pairs with the distal region of the standard partner. At metaphase-I, the heteromorphic microchromosome bivalents have a proximal chiasma. The content of the additional segment was Ag-NOR, C-like DAPI, CMA3 negative. FISH with telomere PNA probe did not detect interstitial (TTAGGG)n sequences in the heteromorphic and any other bivalents. Both homo- and heterozygous carriers were phenotypically normal. The presence of homozygotes shows that heterozygotes are fertile. Reduction in the number of microchromosomes is a clear trend in squamate evolution, as a result of microchromosomes fusing together or with macrochromosomes. Our findings indicate that gaining additional DNA may lead to a transformation of microchromosomes into small macrochromosomes without fusion.
Synaptonemal complex, Lacertidae , chromosome evolution
Microchromosomes are considered as the part of the ancestral tetrapod genome (
Chromosome polymorphism in the lacertids is poorly known. We are aware of only one case of chromosome polytypism: variation in C-band distribution between subspecies and populations of Italian wall lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) (
In this paper we describe a long microchromosome variant which covers the gap between micro- and macrochromosomes. We found this variant in Siberian population of the sand lizard, Lacerta agilis Linnaeus, 1758, and examined its meiotic behavior in homo- and heterozygotes by fluorescent microscopy of synaptonemal complexes (SCs) and metaphase-I spreads using immunolocalization of SYCP3 (the major protein of the SC axial elements) and centromeres, and electron microscopy using Ag-NOR staining.
The SC analysis via electron microscopy and immunofluorescent staining is widely used in vertebrate cytogenetics (
The lizards were caught near Berdsk (54°46.37'N, 83°5.77'E) (ten specimens) and Novosibirsk (54°50.78'N, 82°57.92'E) (four specimens), Novosibirsk region, Russia. Trapping, handling, and euthanasia of animals were performed according to the protocols approved by the Animal Care and Use Committee at the Institute of Cytology and Genetics of the Russian Academy of Sciences. All institutional and national guidelines for the care and use of laboratory animals were followed. No additional permits are required for research on this non-listed species in Russia. The specimens were deposited in the research collections of the Institute of Cytology and Genetics of the Russian Academy of Sciences.
The spreads of spermatocytes were prepared according to the protocol of
For electron microscopic examination the spreads were stained with silver nitrate (
Immunostaining was performed according to the protocol described by
The heterochromatic regions were visualized by a previously described C-like DAPI staining technique (
For chromomycin A3 (CMA3) staining, we used the solution of 0.4 mg/ml CMA3 and 0.01 M MgCl2 in PBS. After preparing, the solution was left to stabilize at +4 °C for two days. Then 25 µl of the solution was put onto the slide already subjected to immunostaining, and covered by the coverslip. After 1 h, the slide was washed in PBS for 5 min and then mounted in the antifade solution with DAPI. After staining, the slide was again left to stabilize at the room temperature in the dark for three days.
The telomeric (TTAGGG)n sequences were detected with a commercial FITC-conjugated PNA probe (LifeTechnologies) according to the manufacturer’s protocol.
We examined 14 male lizards, ten from Berdsk and four from Novosibirsk. In all of them, 19 acrocentric bivalents were seen at synaptonemal complex spreads and at metaphase I plates (2n=38). The mean total length of the SCs was 178±21 µm. The macroSCs formed a gradually decreasing set. In five individuals from Berdsk and the four from Novosibirsk, the microchromosome (SC 19) was significantly smaller than the smallest macrochromosome (SC 18). Their mean sizes, relative to the total SC length, were 1.68±0.14% and 3.14±0.31% respectively (P <0.001)). The microchromosome was thus easily identifiable at SC spreads, as well as at meiotic metaphase I plates (Figs
In one Berdsk individual (#3) the difference between SC 18 and SC 19 was less pronounced although still significant, 3.24±0.24% and 2.78±0.14% respectively (P <0.001) (Figs
SC spreads of sand lizards. a standard karyotype b homozygote for the long variant of SC 19 c heterozygote for the long variant of SC 19. Arrowheads indicate SC 19. Red: SYCP3. Green: ACA. Scale bar: 10 µm.
Relative lengths of the SCs in three sand lizards. a standard karyotype (21 spreads) b homozygote for the long variant of SC 19 (22 spreads) c heterozygote for the long variant of SC 19 (18 spreads). Red column: the long variant in the heterozygote. Bars show standard deviation.
Meiotic metaphase I in sand lizards. a standard karyotype b heterozygote for the long variant of SC 19. Arrowheads and schematic inserts show bivalent 19. In schematic inserts red and yellow colors show the homologues. Red: SYCP3. Blue: DAPI. Scale bar: 10 µm.
In four Berdsk individuals ##1, 7, 9, 10 we detected a heteromorphic SC 19 with one axial element element significantly longer than the other (P=0.03). The relative length of the longer element was 3.08±0.53% of the total macrochromosomal SC length plus the long element 19. The shorter element was 1.77±0.33% of the total macrochromosomal SC length plus the short element 19 (Figs
Synaptic configurations of the heteromorphic SC 19. a synapsis in the proximal regions b the longer element showing foldback self-synapsis c completely paired forming a T-shaped configuration. Scale bar: 5 µm.
Fig.
An interesting feature of the metaphase I bivalents of the sand lizard is that they retain some traces of SYCP3 (Fig.
Insertions or/and amplifications of C-positive chromatin have been suggested as common causes of an increase in chromosome size (
SC spreads of the sand lizards after C-like DAPI staining. a–c standard homozygote d–f homozygote for the long variant of chromosome 19 a, d merged images b,e SYCP3 c, f DAPI. Arrowheads show SC 19. Scale bar: 10 µm.
To test whether there is an accumulation of GC-rich sequences in the long variant of the chromosome 19, we used CMA3 staining. This fluorochrome mostly gave a uniform fluorescence along all the chromosomes, including the enlarged chromosome 19 (Fig.
The telomeric sequences are known to extensively accumulate at the W chromosome of L. agilis (
CMA3 and DAPI staining of the SC spread from a heterozygous individual. a merged image b SYCP3 c CMA3 d DAPI. Arrowhead shows SC 19. Scale bar: 10 µm.
We have concluded that the carriers of the heteromorphic SC 19 (specimens ##1, 7, 9, 10) were heterozygotes for the long variant of the chromosome 19, while the specimen #3 was homozygous for this variant, and all other individuals were homozygous for the standard variant described for this species previously. Since similar karyotypes consisting of 18 macrochromosome pairs and 1 microchromosome pair are characteristic for most other lacertid species (
Based on the synaptic configurations observed in the heterozygote, we suggest that the long variant of the microchromosome probably contains a palindromic sequence in its median region. This sequence shows foldback self-synapsis (Figs
The polymorphic variant of the microchromosome seems not to affect the fitness of the carriers. Homozygous and heterozygous carriers of the variant were phenotypically normal compared with the specimens having the normal karyotype. The occurrence of the homozygote and the presence of the mature spermatids on the preparations from the heterozygotes (not shown) indicate that heterozygotes are fertile.
The five carriers of the long microchromosome were found in the same area of several thousand square meters of grassy river terrace slope, between a motorway and a river. This variant is possibly local and shared by a group of related animals. If this is the case, a homozygote can be produced in the third generation after the origin of the variant chromosome.
Fusion of microchromosomes with each other and with macrochromosomes is considered as the main mechanism of the reduction of the number of the microchromosomes (
We found a polymorphic variant of the 19th chromosome in one population of the sand lizard, Lacerta agilis. It is presented in both heterozygous and homozygous states, and the carriers seem to be phenotypically normal and fertile. The polymorphic variant is two-fold larger than the normal one. Its exact content is unknown. We suggest that enlargement of an individual microchromosome by accumulating repetitive and other sequences may serve as alternative way in the process of the disappearance of the microchromosomes, along with the fusion events.
We thank Mrs. Marina Rodionova for her help in SC spreading, Prof John Parker for English editing and important suggestions and the Microscopic Center of the Siberian Department of the Russian Academy of Sciences for granting access to microscopic equipment. This work was supported by the Federal Agency of Scientific Organizations via the Institute of Cytology and Genetics (project # 0324-2015-0003) and Russian Foundation for Basic Research (grant #16-04-00087).
The colocalization of dense SYCP3 signal with the centromeres at meiotic metaphase I in the sand lizard
Data type: Tif file
Explanation note: The colocalization of dense SYCP3 signal with the centromeres at meiotic metaphase I in the sand lizard. Blue: DAPI, red: SYCP3, green: centromere. Scale bar: 5 µm.
FISH with the telomeric probe on the SC spread of the sand lizard heterozygous for the long variant of chromosome 19
Data type: Tif file
Explanation note: FISH with the telomeric probe on the SC spread of the sand lizard heterozygous for the long variant of chromosome 19. Blue: DAPI, red: SYCP3, green: telomeric probe. Insert and arrowhead shows SC 19. Scale bar: 10 µm.