Laboratory synthesis of an independently reproducing vertebrate species

Abstract

Speciation in animals commonly involves an extrinsic barrier to genetic exchange followed by the accumulation of sufficient genetic variation to impede subsequent productive interbreeding. All-female species of whiptail lizards, which originated by interspecific hybridization between sexual progenitors, are an exception to this rule. Here, the arising species instantaneously acquires a novel genotype combining distinctive alleles from two different species, and reproduction by parthenogenesis constitutes an effective intrinsic barrier to genetic exchange. Fertilization of diploid parthenogenetic females by males of sexual species has produced several triploid species, but these instantaneous speciation events have neither been observed in nature nor have they been reconstituted in the laboratory. Here we report the generation of four self-sustaining clonal lineages of a tetraploid species resulting from fertilization of triploid oocytes from a parthenogenetic Aspidoscelis exsanguis with haploid sperm from Aspidoscelis inornata. Molecular and cytological analysis confirmed the genetic identity of the hybrids and revealed that the females retain the capability of parthenogenetic reproduction characteristic of their triploid mothers. The tetraploid females have established self-perpetuating clonal lineages which are now in the third generation. Our results confirm the hypothesis that secondary hybridization events can lead to asexual lineages of increased ploidy when favorable combinations of parental genomes are assembled. We anticipate that these animals will be a critical tool in understanding the mechanisms underlying the origin and subsequent evolution of asexual amniotes.

Fig. 1.

Morphology of parental species and tetraploid hybrid animals. (A) Dorsal view of A. inornata (Left), A. exsanguis (Right), and the A. exsanguis/A. inornata hybrid (Center). (Scale bar, 10 mm.) (B) Individuals representing the first (H1, Left), second (H2, Center), and third (H3, Right) hybrid generation of the tetraploid species. The H1 and H2 individuals are adults photographed on day 1,168 and 645 after hatching, respectively. The H3 individual is shown at an age of 44 d and displays the color and pattern typical for juveniles.

Fig. 2.

DNA and chromosome analysis. (A) Determination of DNA content of whole blood nuclei by propidium-iodide staining followed by FACS analysis. A comparison between diploid A. inornata (red), triploid A. exsanguis (green) and a putative hybrid (blue) indicates a tetraploid DNA content for the hybrid. (B) The karyotype of the A. exsanguis/A. inornata hybrid was determined from metaphase chromosomes of cultured cells. Each row shows a haploid chromosome set, with chromosomes arranged by decreasing size. The centric fission of one of the three large chromosomes is indicated with an arrow. (C) Microsatellite analysis at nine loci in the A. exsanguis and A. inornata parents and the six hybrid progeny. The tree depicts the relationships between the eight animals. Unique alleles of A. exsanguis and A. inornata are highlighted in red and blue, respectively. In some experiments, an additional peak at 261 was observed for MS15, but was not reproducible in repeat runs and is considered a technical artifact.

Fig. 3.

Ovaries and mechanism of oogenesis. (A) A large yolked and shelled egg was found in the body cavity of a deceased tetraploid hybrid. (B) One of two fully developed ovaries from the same lizard shown in A. (Scale bar, ∼0.5 mm.) (C) Three-dimensional projections of DAPI-stained germinal vesicles (GV) in prophase I of meiosis from a diploid parthenogenetic A. tesselata. (Scale bar, 20 μm.) (D) GVs from the tetraploid hybrid.

Fig. 4.

Maintenance of heterozygosity over three generations. Microsatellite analysis at nine loci for a first-generation hybrid (H2), two of its daughters (H2), and four granddaughters (H3). Alleles originally inherited from A. inornata are highlighted in blue, those from A. exsanguis in red. A single novel allele of MS14 is highlighted in pink.