Abstract
Obligatory parthenogenesis in vertebrates is restricted to squamate reptiles and evolved through hybridisation. Parthenogens can hybridise with sexual species, resulting in individuals with increased ploidy levels. We describe two successive hybridisations of the parthenogenetic butterfly lizards (genus Leiolepis) in Vietnam with a parental sexual species. Contrary to previous proposals, we document that parthenogenetic L. guentherpetersi has mitochondrial DNA and two haploid sets from L. guttata and one from L. reevesii, suggesting that it is the result of a backcross of a parthenogenetic L. guttata × L. reevesii hybrid with a L. guttata male increasing ploidy from 2n to 3n. Within the range of L. guentherpetersi, we found an adult tetraploid male with three L. guttata and one L. reevesii haploid genomes. It probably originated from fertilisation of an unreduced triploid L. guentherpetersi egg by a L. guttata sperm. Although its external morphology resembles that of the maternal species, it possessed exceptionally large erythrocytes and was likely sterile. As increased ploidy level above triploidy or tetraploidy appears to be harmful for amniotes, all-female asexual lineages should evolve a strategy to prevent incorporation of other haploid genomes from a sexual species by avoiding fertilisation by sexual males.
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Introduction
Reproduction in vertebrates is mostly sexual, based on meiosis in both sexes and subsequent restoration of the diploid level by fusion of the egg and sperm genomes. However, several lineages have deviated from this canonical mode and developed different reproductive strategies: hybridogenesis, androgenesis and parthenogenesis1. Nearly all obligatory non-sexual lineages, including all-female parthenogenetic lineages, are of hybrid origin2, but see 3. As far as known, already the F1 generation of the hybrid lineages reproducing by parthenogenesis or sperm-dependent parthenogenesis (gynogenesis) is capable of producing non-reduced eggs, as documented in gynogenetic fishes4,5. The all-female offspring can spread rapidly due to their high reproductive rate, as they do not invest in males and thus do not pay the “double cost of sex”6. Moreover, due to their “clonal” genome inheritance, they do not suffer from inbreeding depression in small, isolated populations7. Environmental requirements of parthenogens are similar to these in sexual species8 and ecological niches of parthenogenetic and parental sexual species often overlap9. The distribution of the parthenogenetic lineage overlaps with one or both of the parental species immediately after its origin, but it can also have secondary overlap areas with related sexual lineages later in its evolution. In addition to the likely competition between the parthenogenetic and sexual forms and the risk of competitive exclusion10, the parthenogenetic lineage must deal with the presence of males of related sexual species that are very likely capable of fertilising their unreduced eggs11,12.
The evolutionary solution to this problem seems to vary greatly between groups, at least partly due to the constraints imposed by the need for sperm to initiate embryonic development. In teleost fishes and amphibians, all-female lineages possess gynogenesis, i.e. sperm-dependent parthenogenesis13,14,15. The gynogenetic lineages do not incorporate the sperm genome into the non-reduced egg but require a sperm to induce embryonic development. Gynogenetic vertebrates are therefore restricted to live only in places where the males of sexual species provide sperm capable of inducing egg development14. While the formation of unreduced eggs seems relatively easy even in F1 hybrids, the exclusion of sperm pronucleus after fertilisation remains challenging for some of those hybrids4,5,16.
It is known, that chondrichthyes and sauropsids (= reptiles and birds) do not require sperm to induce egg development, as evidenced by cases of facultative parthenogenesis and all-female parthenogenetic lineages allopatric with related sexuals15,17. Obligate parthenogenetic vertebrates occur exclusively in squamate reptiles1,15. They have evolved many times in the lizard families Gekkonidae18,19, Scincidae20, Teiidae21,22,23, Gymnophthalmidae24,25, Lacertidae26, Xantusiidae3, Liolaemidae27 and Agamidae28 and in the blind snake Indotyphlops braminus29. Backcrosses of an already parthenogenetic hybrid with a male of one of the parental species21 or even with other related sexual species30 can lead to polyploidisation12,18. However, mating with parthenogenetic rock lizards with sympatric parental species does not always lead to production of hybrids with elevated ploidy31. Depending on sex determination system, the triploids resulting from fertilisation of an unreduced diploid parthenogenetic egg by a haploid sperm can be sterile males or sterile females32, which should reduce the fitness of the parthenogenetic form in sympatry with sexual species. In fact, all polyploid reptile lineages are triploid parthenogens18,33,34,35 and most likely originated in this way. Further, after mating a triploid parthenogenetic female with a sexual male, a tetraploid can be formed, which might be a fertile parthenogenetic female. Tetraploid individuals were found in the field36, or they were produced by laboratory crosses of triploid parthenogens and a male of sexual species37,38,39. Single tetraploid hybrid was also found in sympatry of diploid parthenogenetic D. armeniaca with sexual D. valentini12. However, the fitness of tetraploid parthenogens is likely lower, as no tetraploid parthenogenetic lineage has been detected in the field to date. The reason why triploidy is well-tolerated by many parthenogenetic squamate reptiles, but tetraploidy and higher ploidy levels seem harmful for them is unclear. It may include problems with fertility, or could be mediated by the negative effect of polyploid, larger cells on metabolic rate due to relatively lower cell surface to cell volume40,41. In any case, sympatry with sexual relatives should be costly for parthenogenetic lineages due to competition and possible increase of ploidy level by hybridisation.
Data on sympatry of reptile parthenogenetic and sexual forms and their consequences are still rare. The coexistence of parthenogenetic and sexual lizards has been described in the lacertid rock lizards of the genus Darevskia11,12,42,43, the teiid lizards of the genus Aspidoscelis44,45,46 and Teius47 as well as members of the butterfly lizards of the agamid genus Leiolepis48,49. The Indo-Malayan lizard genus Leiolepis contains ten species, six sexual and four obligatory parthenogenetic species50. To find if the hybridisation between parthenogenetic and sexual butterfly lizards is possible, we examined several populations of triploid parthenogenetic Peters’ butterfly lizards (L. guentherpetersi), looking for sympatry with different species and for hybrids between this parthenogen and the parental species L. guttata or L. reevesii33. We also evaluated phenotypic consequences of such possible hybridisation and changes in ploidy level and cell size. Our study describes relationships between the sexual and parthenogenetic lineages in members of the genus Leiolepis and tests the effect of their sympatry and backcrossing with sexual relatives on obligatory parthenogenetic lineages of a hybrid origin.
Materials and methods
Study area and sampled specimens
We inspected 13 localities of L. guentherpetersi in the distribution range described in Darevsky and Kupriyanova28, Grismer and Grismer49 and Grismer et al.33. The research was established from 10 to 27 March and from 15 to 30 June 2023 (Table S1). Peters’ butterfly lizards occupy sandy dunes covered with grassland or bushes along the coast and several km deep into the mainland. In addition to the sites described in the previous studies, we searched for the lizards in the suitable biotopes in the vicinity of Da Nang and Hue. The lizards were captured by hand, by noose or by digging the burrow.
The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Total list of studied individuals with sampled localities and analysis is present in Table S1. Some specimens are stored in the Zoological Museum of Moscow State University (ZMMU) with given museum numbers (Table S1). One aberrant individual N34_H (ZMMU R-17884) was captured on sandy dunes in the vicinity of Lập An (N16.258022; E108.066678), where L. guentherpetersi is also present (Table S1). We noted that this lizard (Fig. 1) had a yellowish dorsum and bright yellow flank stripe like L. guentherpetersi, but it was a male with well-developed hemipenes. We speculated that this lizard arised through a hybridisation and explored it in detail.
Photo of the putative hybrid male N34_H (ZMMU R-17884) in situ.
DNA barcoding
Tail tips of 37 lizards captured in the study areas (Table S1) were collected for barcoding. We isolated genomic DNA from the 96% ethanol fixed tissues using Evrogen ExtractDNA Blood & Cells kits based on spin-column purification method. We amplified 1088 bp of the mitochondrial gene cytochrome b (cyt b) using primers L1491051 and H1606452 according to Lin et al.53. PCR products were purified using the Evrogen Cleanup Mini kit. Prepared samples were Sanger sequenced in Evrogen. The obtained sequences with GenBank accession numbers (PQ162479 – PQ162515) are presented in Table S1.
The sequences were aligned in Unipro UGENE 49.154. and adjusted manually. We used sequences of L. guttata from complete mitochondrion55 and L. reevesii53 for our results validation; and Uromastyx geyri (AB474755) as an outgroup.
Phylogenetic trees were reconstructed under Bayesian inference (BI) and using the maximum likelihood (ML) method. The ML trees were generated using IQ-tree software56 with ultrafast bootstrap = 10,000 (UFBoot)57, partitioning scheme and model were selected using ModelFinder software58. Best-fit model according to BIC was GTR + F. The optimal partitioning schemes and models for Bayesian Inference analysis were identified with PartitionFinder software59 using greedy search algorithm under AIC criterion: GTR for 1st position and GTR + I for 2nd position, GTR + I for 3rd position. BI was performed using MrBayes v3.2.660 with two simultaneous runs, each with four chains, for 5 million generations. We checked the convergence of the runs and that the effective sample sizes (ESS) were all above 200 by exploring the likelihood plots using TRACER v1.7.161. The initial 10% of trees were discarded as burnin. Confidence in tree topology was assessed by posterior probability (PP)62. Based on the clade distribution in dendrogram we calculated the uncorrected inter- and intragroup pairwise distances (p-distances) using MEGA 1163 with 1000 bootstrap replications (Table S2).
External morphology
A morphological study was performed on the live and preserved individuals captured during field work (Tables S1, S3, S4). Additional comparative material was examined from the collections of the Zoological Institute, Russian Academy of Science (ZISP), St. Petersburg, Russia and Zoological Museum of the M. V. Lomonosov Moscow State University (ZMMU), Moscow, Russia. In total, besides the putative hybrid, we studied 28 specimens of L. guentherpetersi, seven L. reevesii and 18 L. guttata.
Fifteen meristic traits were scored in the analysis28,49, our data]: SL – number of supralabials; IL – number of infralabials; FMP – total number of femoral pores; NPBS – number of non-pore-bearing scales between pore-bearing-femoral scales; FB – number of scales across the frontal bone between the midpoint of the supraorbital regions, excluding supraorbital scales; KSFA – number of enlarged, keeled scale rows across the forearm midway between the elbow and wrist; TS – number of enlarged scales across the lower part of tibia; DSS – number of dorsal scales between inner margins of the dorsolateral stripes at the widest part of the body; DLS1 – number of scale across the dorsolateral stripe in the widest part; L1T – number of enlarged, plate-like scales along the dorsal surface of the first toe; ESL3T – number of enlarged, subdigital lamellae on the third toe; SL4T (SDS) – number of enlarged, subdigital lamellae on the fourth toe; L4T – number of enlarged, plate-like scales on the dorsal surface of the fourth toe; ESS – number of eye spots between dorsolateral stripes; ROS – number of scales in a straight line between the rostral shield and a perpendicular line between the middle of the eyes. We also noted colouration characters: VNS – light ventrolateral stripe – absence (0) or presence (1); DLS2 – presence, shape and colour of dorsolateral stripe – dorsolateral stripes absence (or very thin and fragmented) (0), dorsolateral stripe yellow, same width along the entire length with clearly defined edges, contacting occipital region, dark dorsolateral stripe under light dorsolateral stripe absence (1), dorsolateral stripe light grey, different width along the entire length with unclearly defined edges, not contacting occipital region, dark dorsolateral stripe under light dorsolateral stripe (some time with reticular elements) presence (2); LFS – light femur stripe – light stripe on the back of femur absence (or lightly visible fragmented) (0) or presence (1); TCC – throat and chest colour – round light blotch on throat and chest (0), light marks and stripes on the throat and chest (1).
Statistical analysis and visualisation were performed in RStudio (v.2022.12.0 + 353). We applied Factor Analysis of Mixed Data (FAMD) to analyse morphological variability. (FAMD) allows the use of numerical and factorial data. The FactoMineR package and packages vcd64, factoextra65 and ggplot266 were applied. We did not include DSS, DLS1, and ESS in this analysis, as they are absent in L. reevesii. However, we provide them in the table (Table S3) because they effectively distinguish L. guttata from L. guentherpetersi.
Flow cytometry
As an additional test of the ploidy of the studied individuals, we measured the DNA content of blood cell nuclei of one L. guttata (N40), one L. reevesii (N41), five L. guentherpetersi (N11, N21, N22, N24, N44), and the putative hybrid. Full blood was collected from the tail tip to 70% ethanol. To release and stain the cell nuclei and to discard the total RNA, the samples were incubated with 10 µg/ml propidium iodide (Sigma-Aldrich) and 0.15 mg/ml RNase A (Sigma-Aldrich) in 0.1% Triton X-100 / 1× phosphate buffered saline (PBS, Sigma-Aldrich) at 4 °C overnight. DNA content was measured by using a BD FACSAria™ flow cytometer and at least 10,000 nuclei in total were analysed per individual. The chicken full blood was used as an internal technical control for measurements. Data was further analysed by BD FACSDiva software (version 6.1.3).
Erythrocyte size
We prepared blood smears from the tail tips of seven L. guentherpetersi, six L. guttata and the putative hybrid (Table S6). They were air dried and stained by Giemsa according to the protocol published in Wright67. Erythrocytes were then photographed using Axiostar Plus microscope (Carl Zeiss) with SCMOS05100KPA (ToupCam) camera at 400 magnification. We measured maximum cell length, maximum nucleus length, cell projection area and nucleus projection area of 50 randomly chosen erythrocytes with ImageJ 1.54i software. Prior to measuring parameters of erythrocytes, we calibrated ImageJ measuring tools with a Micromed calibration slide.
We made mixed linear models for prediction of each erythrocyte size parameter. Species was used as a fixed effect and ID was a random intercept. We then performed Kenward-Roger test with Holm’s adjustment for pairwise comparisons of maximum cell length, maximum nucleus length, cell projection area and nucleus projection area between species. Statistical analysis was performed with lmerTest68 and emmeans69 R packages. Plotting was performed with ggplot266 and ggpubr70 R packages. Means are given with standard deviation (SD) and significance P-value of < 0.05.
Mitotic chromosome preparation
To determine the ploidy level by counting chromosomes, we examined the karyotypes of three L. guttata (N100, N203, N140), one L. reevesii (N51), eight L. guentherpetersi(N3, N10, N12, N13, N14, N21, N33, N34_G); and the putative hybrid. Lizards were treated intraperitoneally with 0.15% colchicine (0.04 ml per 1 g of body weight) for 19–24 h and euthanized by blunt force trauma to the head. Cell suspensions were obtained from the small intestine and bone marrow from epiphysis of the tibia. The cells were first hypotonised in 0.075 M potassium chloride for 40–60 min at room temperature (RT). The samples were then centrifuged for 15 min at 3000 rpm, and the supernatant was discarded. The concentrated cell suspension was fixed in a refrigerated 25% solution of glacial acetic acid in methanol, following the protocol of Ford and Hamerton71. The chromosome spreads were stained with 4’,6-diamidino-2-phenylindole (DAPI, Elabscience) and analysed using a Leica DM2500 Imaging microscope equipped with a Leica Microsystems DFC420 C camera and Leica Application Suite 4.13.0 software.
Interspecific comparative genomic hybridisation
In order to reveal the origin of a tetraploid hybrid, we used comparative genomic hybridisation (CGH), which can detect species-specific repetitive DNA and thus might distinguish the chromosome sets originated from different species. Only L. gutatta, L. reevesii and their asexual triploid hybrid L. guentherpetersi33 occur in the region, therefore we used whole genomic DNA (gDNA) of L. guttata and L. reevesii for the probe preparation and applied it on chromosome spreads of three L. guentherpetersi (N10, N13, N34_G; 31 metaphases) and the putative hybrid (90 metaphases). Chromosome spreads of one L. guttata and one L. reevesii were used as controls. In total, 3 µg of L. guttata gDNA and 3 µg of L. reevesii gDNA were directly labelled through nick translation using a commercial kit: L. guttata DNA was labelled with a Fluorescein NT Labelling Kit, and L. reevesii DNA was labelled with Cy3 NT Labelling Kit (both Jena Bioscience). After nick translation, for each experiment, a probe containing 500 ng of L. guttata and 500 ng of L. reevesii labelled DNA was co-precipitated together with 2.5 µl of sonicated salmon sperm DNA (Sigma-Aldrich), 0.1% (v/v) 3 M sodium acetate (ThermoFisher Scientific) and 2.5% (v/v) cold 96% ethanol overnight at −20°C. Following precipitation, it was washed in 70% ethanol, air-dried and re-dissolved in 15 µl of hybridisation buffer containing 50% formamide, 10% dextran sulphate, 10% sodium dodecyl sulphate, 2× saline-sodium citrate buffer (SSC) and 1× Denhardt’s solution (ThermoFisher Scientific), pH 7.0. The probe was then denatured for 6 min at 86 °C and chilled on ice prior to hybridization. Slides with chromosomal spreads were prepared for hybridization following the protocol72. Hybridization was carried out over 3 days at 37 °C. Post-hybridization washes were performed in 1× SSC for 2 × 5 min at 65 °C, in 4× SSC/ 0.1% Tween 20 for 5 min at 44 °C, soaked in 1× PBS for 1 min at RT and rinsed in dH2O. Finally, the chromosomes were counterstained by Vectashield Antifade containing DAPI (1.5 mg/ml) (Vector Laboratories). Metaphases were captured by an Axio Imager Z2 microscope equipped with a CoolCube 1 b/w digital camera (MetaSystems) by the MetaSystems platform for automatic search, capture, and image processing. For full protocol see Altmanová et al.72.
Preparation of synaptonemal complexes and immunostaining
We obtained spreads of synaptonemal complexes (SCs) from one L. reevesii (N50), one L. guttata (N301), and the putative hybrid (N34_H) male following the protocol described by Peters et al.73. After dissection, testes were cut into several pieces and placed in hypotonic solution (30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, pH 8.2). After incubation for 40 min at RT in the hypotonic solution, small pieces of gonads were transferred into 80 µl of 100 mM sucrose for 10 min at RT, and minced to prepare homogenous cell suspension. The suspension was dropped onto SuperFrost® slides with polylysine (Menzel Gläser), which had been preliminary treated with fresh 1% paraformaldehyde (PFA) solution containing 0.15% Triton X-100 (pH of 8.5). After spreading cells by the inclination of the slide, slides were incubated in a humidity chamber for 1 h at RT, dried, washed in 1× PBS or 0.4% Kodak Photo-Flo solution (Kodak, Rochester, NY, USA), and used for immunofluorescent staining.
To visualise SCs, we applied a rabbit polyclonal antibody against its lateral component, the SYCP3 protein (1:200, ab15093, Abcam) and against its transverse filaments of central space, SYCP1 protein (1:250, ab15087, Abcam). Additionally, we applied mouse monoclonal antibody (1:50, ab14206, Abcam) against MLH1 to visualise crossing-overs. Centromeres were detected by human anti-centromere serum (1:50, 15–235, Antibodies Incorporated or 1:250-1:500, CREST, 90 C-CS1058, Fitzgerald Industries International). Primary antibodies were diluted in 1% blocking reagent (Roche) in 1× PBS and 0.01% Tween-20 and applied for 3 h at RT. Afterward, slides were washed three times for 5 min in 1× PBS at RT. We applied the following secondary antibodies: Alexa-595-conjugated goat anti-rabbit IgG (H + L) (1:200, A-11012, Invitrogen), Alexa-488-conjugated goat anti-mouse IgG (H + L) (1:200, A-11001, Invitrogen) and Alexa-488-conjugated goat anti-human IgG (H + L) (1:200, 11013, Invitrogen) diluted in 1% blocking reagent (Roche) in 1× PBS for 1 h at RT. After three times for 5 min in 1× PBS at RT slides were dehydrated in ethanol series (50%, 70% and 96%), dried and mounted in Vectashield/DAPI (1.5 mg/ml) (Vector Laboratories).
Synaptonemal complexes were analysed using Olympus BX53 equipped with standard fluorescence filter sets and captured by a CCD camera (DP30W Olympus) using Olympus Acquisition Software. Final images were processed using Gimp software (GIMP Development Team, Open Source).
Results
Barcoding
Mitochondrial DNA of the sampled individuals represented two clades. The first included all L. reevesii, while the second comprises northern clade of L. guttata with parthenogenetic L. guentherpetersi and southern clade of L. guttata. The position of our putative hybrid individual was within L. guentherpetersi, documenting close relatedness between the female ancestor of the hybrid and L. guentherpetersi (Fig. 2).
Phylogenetic tree of sampled Leiolepis with field numbers based on a fragment of cyt b. Values over and under the nodes represent posterior probabilities by BI/bootstrap values by ML. Accession numbers from GenBank are shown in blue colour.
External morphology
The first two axes of FAMD account for a considerable amount of the total variance (dimension 1: eigenvalue 6.69, 41.2% of the variance; dimension 2: eigenvalues 2.48, 23.7% of the variance). The variables DLS2, VNS and TCC contributed most to the first axis, while KSFA, IL and LFS to the second axis (Table S5). All three studied species are well-separated on the FAMD plot (Fig. 3). The similarity of the putative hybrid male to L. guentherpetersi is most noticeable in its colouration, while the meristic characters present a more ambiguous picture (Tables S3, S4).
FAMD scatterplot of the first two dimensions based on the scalation and colouration characters of parthenogenetic females L. guentherpetersi (n = 28); 12 females and 6 males of L. guttata, and 3 females and 4 males of L. reevesii. The putative hybrid male N34_H (R-17884) is morphologically close to L. guentherpetersi.
Genome and erythrocyte size
The relative content of DNA per nucleus of blood cells measured by flow cytometry for each measured individual showed approximately 1.5× higher DNA content in triploid L. guentherpetersi (n = 5) in comparison to diploid L. guttata. The putative male hybrid had 2× higher DNA content per nucleus than L. guttata, pointing to its tetraploidy (Fig. 4a-c).
Relative DNA content of blood cell nuclei measured by flow cytometry and cell parameters of erythrocytes. All histograms depict the measurements of samples mixed with the internal control of diploid standard (Gallus domesticus). The measurements support diploidy in L. guttata (a), triploidy in L. guentherpetersi (b) and tetraploidy in the male hybrid (c). Boxplots show maximum cell length MCL (d), maximum nuclei length MNL (e) and cell projection area CPA (f) of erythrocytes in L. guttata (green), L. guentherpetersi (yellow) and 4n-hybrid (blue).
We expected that erythrocyte size as well as the size of their nuclei will correspond with the ploidy level. However, the results differ among variables. Mean CPA was 2.22 times higher in the tetraploid hybrid (242.10 ± 46.68 µm2) than in L. guttata and 1.84 times higher than in L. guentherpetersi (Kenward-Roger test, p < 0.001 in both cases). This measure differed significantly between L. guttata and L. guentherpetersi (Kenward-Roger test, p = 0.023). Maximum cell length (MCL) differed significantly between all three groups (Kenward-Roger test, p < 0.01 in all cases; Fig. 4d-f) and increased with ploidy level. The mean MCL in the tetraploid L. guentherpetersi × L. guttata hybrid was the largest (21.43 ± 2.55 μm), 1.23 times longer than in triploid L. guentherpetersi (18.59 ± 1.24 μm) and 1.42 times longer than in diploid L. guttata (16.26 ± 0.96 μm) (Table S6). Maximum nucleus length did not differ significantly between neither group (Kenward-Roger test, p > 0.056 in all comparisons, Table S6). Mean nucleus projection area differed marginally significantly between L. guentherpetersi × L. guttata hybrid and L. guttata (Kenward-Roger test, p = 0.043), but not between L. guentherpetersi × L. guttata hybrid and L. guentherpetersi (Kenward-Roger test, p = 0.057) and L. guentherpetersi and L. guttata (Kenward-Roger test, p = 0.436).
Mitotic chromosomes and comparative genomic hybridisation
The haploid set of chromosomes comprised six macrochromosomes in all examined Leiolepis individuals (Fig. 5). Accurate counting of microchromosomes in the triploids and the tetraploid hybrid was challenging due to their small size and frequent overlap. Karyotypes with 2n = 36 chromosomes (12 macro- and 24 microchromosomes) in L. guttata and L. reevesii, and 3n ~ 54 (18 macro- and likely 36 microchromosomes) in L. guentherpetersi were consistent with the previous report28. The putative hybrid male possessed 4n ~ 72 chromosomes, with 24 macro- and about 48 microchromosomes.
Metaphase of diploid L. guttata N140 with 12 macrochromosomes (a), triploid L. guentherpetersi N3 with 18 macrochromosomes (b), and tetraploid hybrid N34_H with 24 macrochromosomes (c). In line with the consistent pattern in diploid species L. guttata (d), comparative genomic hybridisation identified six macrochromosomes inherited from L. reevesii (labelled by Cy3, red) and 12 chromosomes from L. guttata (labelled by Fluorescein, green) in triploid L. guentherpetersi (e), and the tetraploid hybrid male N34_H (f) possessed six chromosomes from L. reevesii and 18 chromosomes from L. guttata. Scale bar = 10 μm.
CGH identified two chromosome sets from L. guttata and one set from L. reevesii in all analysed metaphases of L. guentherpetersi (Fig. 5e). In contrast, three chromosomal sets from L. guttata and one set from L. reevesii were identified in the tetraploid male N_34H (Fig. 5f). In control experiments, no colour difference between the chromosome sets has been detected, confirming that L. guttata chromosomes preferentially hybridised with L. guttata-labelled DNA (the entire karyotype appeared greenish; Fig. 5d) and the same occurred in L. reevesii, where the karyotype appeared reddish (results not shown).
Synaptonemal complexes
Detection of lateral components of the synaptonemal complex (SYCP3), and recombination foci (MLH1) allowed the discrimination of bivalents from univalents based on the thickness of SYCP3 and the presence of recombination foci74. We found that SCs of L. reevesii (in 152 SC spreads analyzed) and L. guttata (in 48 SC spreads) males had 18 bivalents including six bivalents formed by macrochromosomes and 12 bivalents formed by microchromosomes. Each bivalent carried at least one crossing-over locus (Fig. 6).
The testes of the tetraploid L. guentherpetersi × L. guttata hybrid were very small and the testicular cell suspension was almost transparent (cell poor). Only 2–4 spermatocytes per slide were found, with a total of 18 spermatocytes were analysed. Twelve SC bivalents formed by 24 macrochromosomes and the remaining SC bivalents formed by microchromosomes were identified in pachytene spermatocytes (Figs. 6, S1). The centromeric regions were labelled with CREST antibodies as dots or as small diffuse clouds (Fig. S1). SYCP1-positive central elements were formed along all SCs (Fig. 6). Only a part of the macrobivalents had MLH1 dots and most bivalents did not have these signals (Fig. 6).
Synaptonemal complex (SC) spreads of L. reevesii (a), L. guttata (b) and Leiolepis tetraploid hybrid (c,d) males. Lateral components of the synaptonemal complex were detected by antibodies against SYCP3 protein (green); crossing over loci were visualised by antibodies against MLH1 protein (indicated by arrows, red); transverse filaments of central space of the SCs were identified by antibodies against SYCP1 protein (magenta); chromatin stained by DAPI (blue). SC spreads of L. reevesii (a) and L. guttata (b) are represented by 18 bivalents. In a hybrid, 12 bivalents are formed by 24 macrochromosomes and the remaining bivalents are formed by microchromosomes (c,d). Black and white (c) and colour (d) images are presented on the same nucleus. Scale bar = 10 μm.
Discussion
We reconstructed two subsequent hybridizations of the parthenogenetic form with its parental species, each of which led to an increased ploidy level (Fig. 7). The first resulted in the formation of triploid L. guentherpetersi, and the second in the expansion of the genome to tetraploidy.
The reconstructed hybridisation events led to the emergence of triploid L. guentherpetersi and its tetraploid hybrid. Distribution map of L. reevesii, L. guttata and L. guentherpetersi is modified after Grismer et al.33. The map of Vietnam from https://data.humdata.org/dataset/cod-ab-vnm edited using QGIS 3.32 Software. Figures represent specific coloration of each species according to sex.
Grismer et al.33 suggested that L. guentherpetersi is a triploid parthenogenetic hybrid of a female of northern clade of L. guttata with a male of L. reevesii, which hybridised back with a male of L. reevesii. In agreement with their study, our data show that L. guentherpetersi is triploid in all studied populations (Table S1) and that according to mitochondrial DNA, the northern lineage of L. guttata was the maternal species of this parthenogen (Fig. 2). However, our results clearly show that L. guentherpetersi contains two haploid genomes of L. guttata and only one of L. reevesii (Fig. 5e), correcting the earlier suggestion about the origin of this triploid parthenogenetic lineage by a backcross. Triploid lineages assumed to be formed by a backcross between a diploid parthenogenetic hybrid with a male of a related form are known in nearly all reptile parthenogenetic complexes. Next to the here studied agamid genus Leiolepis28,33,48, triploid obligatory parthenogenetic lineages were reported in the geckos of the genera Lepidodactylus, Hemiphyllodactylus, Heteronotia, Hemidactylus and Nactus18,19,75,76,77, in the scincid genus Menetia20, members of the teiid genera Aspidoscelis and Cnemidophorus (Teiidae)44,78, the gymnophthalmid genus Loxopholis79, the liolaemid genus Liolaemus27 and the blind snake Indotyphlops braminus80.
Parthenogenesis has to be connected with a production of unreduced eggs. Up to now, three mechanisms of this process has been documented in vertebrates: a normal meiosis followed up by a restoration of ploidy by a fusion of egg and polar body nucleus, genome doubling of haploid egg genome after meiosis, and premiotic endoreplication81,82. The first two mechanisms were reported only in facultative parthenogenesis82,83. As far as known, obligatory parthenogens rely during the production of unreduced eggs on premeiotic endoreplication, i.e. by genome doubling in gonial cells before meiosis, which proceed in a canonical way just with a pairing and recombination of identical (duplicated) chromosomal copies during the first meiotic division16,38,44,77,84. Dedukh et al.77 suggested that premeiotic genome endoreplication might be an important preadaptation for parthenogen polyploidisation, as at least some triploid females emerged from a fertilisation of unreduced diploid eggs by a sperm may exploit the similar alteration as their diploid parental parthenogenetic lineages. On the other hand, a failure of a proper bivalent formation in a triploid oocyte would likely prevent the parthenogenetic reproduction of a triploid emerged from a diploid ancestor using the fusion or genome doubling. As the parthenogenetic butterfly lizards include triploid lineages, we predict that they produce unreduced eggs by premeiotic endoreplication, as well as parthenogens arising from nine independent origins of obligate parthenogenesis studied to date16.
CGH revealed that the tetraploid male has three haploid sets of L. guttata and one set of L. reevesii chromosomes (Fig. 5f). The chromosomal constitutions as well as its mitochondrial haplotype (Fig. 2) shows that the individual was a hybrid between L. guentherpetersi and L. guttata. Spermatocytes of this tetraploid male exhibit partial pairing and recombination during meiotic prophase (Fig. 6). Most likely, such spermatocytes cannot accomplish meiosis, and in agreement, no sperm was found in epididymis. The reproductive failure of the hybrid male is not very surprising, as male and female meiosis differ substantially and in contrast to certain hybrid females, hybrid males are usually not able to produce clonal gametes through endoreplication or other gametogenetic alterations5,85.
The hybrids between parthenogens and males of sexual species were documented previously also in other lineages. In the lacertid genus Darevskia, the hybridisation between different combinations of parthenogenetic lineages with males of their paternal species leads to triploid and even tetraploid males and females, some of them with reduced reproductive organs11,12,32. A triploid male from the cross of D. unisexualis × D. valentini studied in detail was able to pass meiosis, but produced abnormal spermatids85. The triploid hybrids between different lineages of parthenogenetic and sexual whiptail lizards of the genus Aspidoscelis were of both sexes and often sterile86,87,88,89,90. Tetraploid individuals were reported in nature as well36,91, but no tetraploid parthenogenetic all-female lineage of this genus have been found in the field, although they were repeatedly produced by experimental crosses38,39,92. All this evidence suggests that fertilisation by a male of sexual species is likely costly for females from a parthenogenetic lineage, as it may lead to often inviable or infertile progeny with an increased ploidy level.
Polyploid lineages, with extremes up to ploidy 12×93 are common in amphibians and actinopterygian fishes and two allotetraploid species were reported in mammals94,95. Many of these polyploids reproduce sexually. In contrast, with the exception of a few spontaneous triploid individuals96,97,98 and various combinations of diploids, triploids and diploid-triploid mosaicism in two species99,100, polyploidy is in sauropsids restricted to obligatory parthenogenetic lineages and to our knowledge, only triploid stable lineages are known in nature. Reported viable tetraploids in reptiles are either sterile individuals18,101, this study] or parthenogenetic lineages produced by hybridisations in the lab37,38,39,92. As far as we know, no viable individuals of higher ploidy than 4n have been reported in amniotes, suggesting that in contrast to some other vertebrate lineages, it is their highest possible viable level. The reasons why amniotes do not form lineages with ploidy level above tetraploidy are unknown. It can reflect problems with fertility or viability, possibly connected with an effect of polyploidisation on cell size and in turn on development and metabolism.
The tetraploid L. guentherpetersi × L. guttata hybrid examined by us had much larger erythrocytes than triploid L. guentherpetersi and diploid L. guttata in each size measurement (Fig. 4). Cell size reflects differences in ploidy better than nucleus parameters (Fig. 4d–f; Table S6), suggesting that nucleus can be differently dense102. Cell projection area differed between diploids and triploids versus the tetraploid, but diploid and triploid individuals had more similar cell size than this in tetraploid (Fig. 4d-f). We speculate that generally in amniotes, triploidy does not affect cell phenotype in the same scale that much as tetraploidy, which would explain why triploid lineages are common, while tetraploid lineages have never been recorded in the field in squamates. However, the effect of polyploidy on cell size might be lineage-specific. In contrast to the Leiolepis lizards, triploids in the rock lizards of the genus Darevskia have significantly larger erythrocytes than diploids, but there is no significant difference in cell size between triploids and tetraploids12. This could explain why there are no triploid parthenogenetic lineages in the genus Darevskia, although triploid individuals emerged from hybridisation are quite frequent there.
When mated, diploid parthenogens can sometimes produce parthenogenetic triploid lineages with a high fitness7, however, triploids would produce tetraploids with a lower fitness. Therefore, we suggest that an all-female asexual lineage formed by hybridization should evolve a strategy to solve the problem of non-incorporation of additional haploid genome from a sexual species to prevent further increase in ploidy level and production of individuals with low fitness. Gynogenetic (sperm-dependent parthenogenetic) fish and amphibians are restricted to sympatry with related sexual species and need to evolve a strategy to eliminate sperm DNA. Unlike them, parthenogenetic reptiles should prevent mating and fertilisation by sexual males, which can be ensured by several ways. Parthenogenetic reptile females can be non-receptive or non-attractive for males, evolve post-copulatory barriers to fertilisation31, or adopt a strategy to eliminate sperm DNA, which was up to now not reported in reptiles. Analogous situations were studied in parthenogenetic stick insects. Post-mating barrier to fertilisation were found in some lineages103, while parallel decay of female pheromones and contact signals used in communication with males, sperm storage organs, and lost the ability to fertilise eggs was found in other lineages104. Nevertheless, the easiest strategy seems to be an allotopy with respect to related sexual forms, for example, by expanding their range beyond the boundaries of parental species distribution.
It seems that only those parthenogenetic hybrids that would manage to avoid frequent fertilisation of their unreduced eggs by males of their sexual relatives can succeed. We speculate that parthenogenetic lineages might be produced by hybridization in nature more often than assumed (already F1 generation of species with chromosomal incompatibilities might be parthenogenetic, adopting premeiotic endoreplication as a mechanism to produce unreduced eggs77; however, that females of most of them are fertilised by males of a parental species and are effectively turned to unfit polyploids. We assume that most true parthenogens had to escape to allopatry and maybe this explains why asexual lineages were claimed to be so common in harsh environments where no sexuals are present105. We predict that successful parthenogens evolve in fragmented populations or under low densities, where it is easier to escape from harassing and polyploidy-inducing males of sexual species.
Data availability
All sequences generated in the present work have been submitted to the National Centre for Biotechnology Information (NCBI) under the following accession numbers: PQ162488, PQ162482, PQ162479, PQ162513, PQ162512, PQ162511, PQ162495, PQ162510, PQ162508, PQ162503, PQ162502, PQ162501, PQ162500, PQ162499, PQ162510, PQ162508, PQ162503, PQ162502, PQ162501, PQ162500, PQ162499, PQ162508, PQ162503, PQ162502, PQ162501, PQ162500, PQ162499, PQ162493, PQ162492, PQ162490, PQ162491, PQ162487, PQ162486, PQ162485, PQ162484, PQ162481, PQ162480, PQ162515, PQ162509, PQ162504. Please, contact the corresponding author for any queries.
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Funding
This work was supported by the Russian Science Foundation (grant number RSF 22-14-00227), the involvement of DD, MAl and LK by the Czech Science Foundation (project no. 23–07665 S). DD and MAl were supported by the Institutional Research Concept (RVO67985904), MAl also by the Charles University Research Centre program no. UNCE/24/SCI/006. SM was supported by Vavilov Institute of General Genetics state assignment contracts No. 0092-2022-0002.
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E.G. – project managing, field trip organisation, data processing and manuscript writing; R.N., M.Ar., I.K. – field work, catching the hybrid; D.D., S.M. – meiotic analysis; E.I. – DNA barcoding and preparation of sequences for the phylogenetic analysis; I.K., M.P., M.Al. – preparation of metaphases and CGH; J.K., M.Al. – flow cytometry; N.S. – figure preparation, data processing; N.O. - erythrocyte size measuring and paper writing; O.N. – text editing and supervising of the field survey; E.I., E.S. – phylogenetic analysis and phylogenetic tree preparation; N.T. – field work support and permissions; L.K. – results interpretation and supervision, draft of the first version of the manuscript. All authors edited and approved the final version.
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The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the Zoological Institute Russian Academy of Sciences (protocol No.1-3-15-06-2021, 15 June 2021); by the Experimental Animal Ethics Committee of A.N. Severtsov Institution of Ecology and Evolution, N48 27 May, 2021 and by Vietnam Academy of Science and Technology – Institute of Genome research. (protocol No.115/QĐ-NCHG, 4th July 2021, based on the Vietnam Museum of Nature - Academy of Sciences and Vietnam Technology i (No. 639/BTTNVN dated September 12, 2019). All applicable international, national and institutional guidelines for the care and use of animals were followed during this research.
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Galoyan, E., Nazarov, R., Altmanová, M. et al. Natural repeated backcrosses lead to triploidy and tetraploidy in parthenogenetic butterfly lizards (Leiolepis: Agamidae). Sci Rep 15, 3094 (2025). https://doi.org/10.1038/s41598-024-83300-y
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DOI: https://doi.org/10.1038/s41598-024-83300-y
