Natural repeated backcrosses lead to triploidy and tetraploidy in parthenogenetic butterfly lizards (Leiolepis: Agamidae)

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.

Keywords: Leiolepis; Hybridisation; Meiosis; Parthenogenesis; Tetraploidy; Vietnam.

Fig. 1

Photo of the putative hybrid male N34_H (ZMMU R-17884) in situ.

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.

Fig. 3

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.

Fig. 4

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).

Fig. 5

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.

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.

Fig. 7

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.. 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.