Corresponding author: Artem P. Lisachov ( email@example.com )
Academic editor: Andrei Barabanov
© 2017 Artem P. Lisachov, Vladimir A. Trifonov, Massimo Giovannotti, Malcolm A. Ferguson-Smith, 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, Trifonov VA, Giovannotti M, Ferguson-Smith MA, Borodin PM (2017) Immunocytological analysis of meiotic recombination in two anole lizards (Squamata, Dactyloidae). Comparative Cytogenetics 11(1): 129-141. https://doi.org/10.3897/CompCytogen.v11i1.10916
Although the evolutionary importance of meiotic recombination is not disputed, the significance of interspecies differences in the recombination rates and recombination landscapes remains under-appreciated. Recombination rates and distribution of chiasmata have been examined cytologically in many mammalian species, whereas data on other vertebrates are scarce. Immunolocalization of the protein of the synaptonemal complex (SYCP3), centromere proteins and the mismatch-repair protein MLH1 was used, which is associated with the most common type of recombination nodules, to analyze the pattern of meiotic recombination in the male of two species of iguanian lizards, Anolis carolinensis Voigt, 1832 and Deiroptyx coelestinus (Cope, 1862). These species are separated by a relatively long evolutionary history although they retain the ancestral iguanian karyotype. In both species similar and extremely uneven distributions of MLH1 foci along the macrochromosome bivalents were detected: approximately 90% of crossovers were located at the distal 20% of the chromosome arm length. Almost total suppression of recombination in the intermediate and proximal regions of the chromosome arms contradicts the hypothesis that “homogenous recombination” is responsible for the low variation in GC content across the anole genome. It also leads to strong linkage disequilibrium between the genes located in these regions, which may benefit conservation of co-adaptive gene arrays responsible for the ecological adaptations of the anoles.
Synaptonemal complex, chromosomes, crossing over, Anolis, Deiroptyx, lizard, Reptilia
Meiotic recombination (crossing over) plays a dual role in sexually reproducing organisms. At least one crossover per chromosome is necessary and sufficient to secure orderly segregation of homologous chromosomes during the first meiotic division. Crossing over shuffles allele combinations between homologous chromosomes, increasing the genetic variation in the progeny, on the one hand, and shaping local patterns of GC-content (i.e., creating or modifying isochores) along the chromosome length, on the other hand (
The number and distribution of the crossovers along a chromosome depends on its length, chromatin composition, genetic content and crossover interference (
The patterns of crossover distribution have been studied across a variety of vertebrates such as fish (
There are several hypotheses which connect the recombination landscape with species’ ecology and speciation (
Reptiles are particularly interesting organisms in which to study the evolution of recombination because they show a wide array of karyotypes and ecological specializations, and extensive homology and synteny between reptilian and avian chromosomes has been demonstrated (
The most widespread technique for studying recombination rate and localization is by immunofluorescent mapping of MLH1 (the mismatch repair protein associated with mature recombination nodules) along the synaptonemal complexes (SCs) at prophase (
One of the most species-rich and diverse reptilian clades are iguanians (infraorder Iguania), which include nearly 30% of all lizard species (Uetz and Hošek 2005). Iguanians are further subdivided into pleurodonts (Pleurodonta) and acrodonts (Acrodonta). The former clade includes New World and Madagascan species (former family Iguanidae sensu lato), and the latter includes chameleons (Chamaeleonidae Rafinesque, 1815) and Old World and Australian dragon lizards (Agamidae Gray, 1827). Many of them have a conservative karyotype with 2n = 36, including 12 submetacentric and metacentric macrochromosomes and 24 microchromosomes. This karyotype is presumed to be ancestral for Iguania (
Among iguanians, anoles (Dactyloidae Fitzinger, 1843, Pleurodonta) are one of the best studied lineages. They are the classical model organisms in studies of reptilian ecology, evolution, biogeography, karyology and genetics (
In this study, we assessed the pattern of meiotic recombination in two anole species, A. carolinensis and Deiroptyx coelestinus (Cope, 1862). Although these species are separated by a relatively long evolutionary history (
The specimens, two male A. carolinensis and one male D. coelestinus, were purchased from commercial breeders. Handling and euthanasia of the animals were performed according to the protocols approved by the Animal Care and Use Committee at the Institute of Cytology and Genetics. The specimens were deposited in the research collections of the institute.
The spreads of meiotic cells were prepared according to the protocol of
The preparations were visualized with an Axioplan 2 Imaging microscope (Carl Zeiss) equipped with a CCD camera (CV M300, JAI), CHROMA filter sets, and ISIS4 image processing package (MetaSystems GmbH).
Brightness and contrast of all images were enhanced using Corel PaintShop Photo Pro X6 (Corel Corp). The centromeres were identified by the ACA foci. The MLH1 signals were scored only if they were localized on SCs. The length of the SC of each chromosome arm was measured in micrometers and the positions of centromeres and MLH1 foci in relation to the centromeres were recorded using MicroMeasure 3.3 software (
To map the MLH1 foci distribution along the macroSCs we calculated the absolute position of each MLH1 focus multiplying the relative position of each focus by the average absolute length for the corresponding chromosome arm. These data were pooled for each arm and plotted to represent a recombination map.
Statistica 6.0 software package (StatSoft) was used for descriptive statistics. MLH1 foci distribution along the SCs was analyzed using CODA v.1.1 software (
The SC spreads of A. carolinensis (a, b) and D. coelestinus (c, d). a, c immunofluorescence and DAPI. Red: SYCP3, green: centromere and MLH1, blue: DAPI b, d DAPI channel separately. Arrowheads show the XY bivalent (
All the macroSCs of A. carolinensis show DAPI-positive bands in their pericentromeric regions. In D. coelestinus such bands were detected only at SC2 and at one microSC. Such bands are observed in many species and generally correspond to C-heterochromatin. They mainly contain satellite repeats (
The mean number of MLH1 foci on each of the macrochromosomal bivalents was calculated (Table
Average SC length (µm) and of MLH1 foci number (±S.D.) in macroSCs in two anole species.
|A. carolinensis||D. coelestinus|
|SC rank||SC length (µm)||No. of MLH1 foci||SC length (µm)||No. of MLH1 foci|
The distribution of MLH1 foci along all the macroSCs in both species was extremely uneven (Fig.
The distribution of MLH1 foci along the macrochromosomes of A. carolinensis and D. coelestinus. The X-axis shows the position of MLH1 foci, the marks on this axis are separated by 1 μm. Black dots indicate centromeres. The Y-axis indicates the frequency of MLH1 foci in each 1 μm – interval. Stacked columns show the frequency for the SCs containing MLH1 foci at each interval.
The most interesting feature of the recombination pattern of A. carolinensis and D. coelestinus macrochromosomes is an extreme polarization of the recombination events. Similar terminal localization of crossovers was previously observed in several anole species, including A. carolinensis, using chiasmata analysis at diakinesis-metaphase I (
Subtelomeric peaks in the distribution of crossovers are common for most vertebrates. This phenomenon is explained by the fact that meiotic pairing of the homologs is usually initiated at the telomeres (
Crossover interference is unlikely to be the cause of almost complete suppression of recombination beyond the subtelomeric regions, because the macrochromosomes of both species demonstrate a rather moderate degree of interference. Our estimate of the ν-value (approximately 5.0) is the first estimate of this parameter in reptiles, so we can only compare it with estimates obtained for mammalian chromosomes of similar size. It was substantially lower than the values detected in the largest chromosomes of common shrews (11.1:
Additional evidence against crossover interference as the cause of the extreme distal location of the crossovers is the fact that single crossovers are also located distally. Single crossovers tend to be located in the middle of mammalian chromosomes, because they suppress the occurrence of other crossovers at both chromosome ends (
Almost total suppression of recombination in the intermediate and proximal regions of chromosome arms would lead to strong linkage disequilibrium between the genes located in these regions. This may benefit the conservation of co-adaptive gene arrays or “supergenes” (
The divergence between Anolis and Deiroptyx is one of the basal radiations among Dactyloidae (
The results of our analysis of crossover distribution along anole macrochromosomes might shed light on a peculiarity of their genome organization. One of the specific characters of the genomes of cold-blooded vertebrates is weak regional variation in GC-content (i.e. less prominent isochore structure) in comparison with birds and mammals (
The extremely distal localization of crossovers in the males of both anole species here analyzed might be considered as evidence against this hypothesis. The weak prominence of isochores in reptiles is apparently produced by some forces other than gBGC and does not reflect the distribution of recombination hotspots. Moreover, intense and homogenous recombination, which is known for example for birds, is apparently not enough to drive intense karyotypic evolution, since bird karyotypes are the most conservative and archaic among all vertebrates (
There are two additional important points to be considered in the discussion. In some reptile species chiasma number and localization depend on environmental conditions (
Sex difference in recombination rate and distribution should also be taken into account. Females tend to have higher recombination rates than males and more even distribution of crossovers along the chromosomes (
For the first time we directly assessed meiotic recombination in reptilian species using MLH1 mapping in SCs. We found that, in male anole lizards Anolis carolinensis and Deiroptyx coelestinus, MLH1 foci are mainly located in the terminal parts of the chromosome arms, whereas recombination intensity in the median parts of the chromosomes is extremely low. This result disagrees with the hypothesis of “homogenous recombination” as the cause of low isochore prominence in the genome of anoles. However, recombination in females has to be studied before drawing any final conclusions about overall recombination rate and distribution in anoles.
The authors declare that they have no competing interests.
We thank Mrs. Marina Rodionova for her help in SC spreading and the Microscopic Center of the Siberian Branch 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 # 0324-2016-0003) and research grant from the Russian Foundation for Basic Research # 16-04-00087.