Widespread failure to complete meiosis does not impair fecundity in parthenogenetic whiptail lizards

Abstract

Parthenogenetic species of whiptail lizards in the genus Aspidoscelis constitute a striking example of speciation by hybridization, in which first-generation hybrids instantly attain reproductive isolation and procreate as clonal all-female lineages. Production of eggs containing a full complement of chromosomes in the absence of fertilization involves genome duplication prior to the meiotic divisions. In these pseudo-tetraploid oocytes, pairing and recombination occur exclusively between identical chromosomes instead of homologs; a deviation from the normal meiotic program that maintains heterozygosity. Whether pseudo-tetraploid cells arise early in germ cell development or just prior to meiosis has remained unclear. We now show that in the obligate parthenogenetic species A. neomexicana the vast majority of oocytes enter meiosis as diploid cells. Telomere bouquet formation is normal, but synapsis fails and oocytes accumulate in large numbers at the pairing stage. Pseudo-tetraploid cells are exceedingly rare in early meiotic prophase, but they are the only cells that progress into diplotene. Despite the widespread failure to increase ploidy prior to entering meiosis, the fecundity of parthenogenetic A. neomexicana is similar to that of A. inornata, one of its bisexual ancestors.

Keywords: Aspidoscelis; Endoreplication; Meiosis; Parthenogenesis; Synaptonemal complex.

Fig. 1.

DNA content in oocytes and somatic cells in the germinal bed. (A) Schematic of meiosis in parthenogenetic whiptail lizards. A single pair of homologous chromosomes are shown in light and dark blue. Following premeiotic S-phase and an additional doubling, identical chromosomes – not homologs – pair and recombine. The two meiotic divisions give rise to a diploid oocyte and three polar bodies. (B) Schematic of ovaries comprised of previtellogenic follicles and the germinal bed, which contains the earlier stages of germ cell development including primordial and early primary follicles embedded in connective tissue within its cortex. (C) DNA content analysis for germinal bed cells from the bisexual species A. inornata (n=292). Tissues were DAPI-stained and DNA content measured based on fluorescence intensity of individual nuclei. (D) DNA content analysis as in C for germinal bed from parthenogenetic A. neomexicana (n=351). (E) Germinal beds of A. inornata (top) and A. neomexicana (bottom) stained with DAPI (blue) and anti-SYCP3 serum (green). The boxed area in the left images is enlarged on the right. Scale bars: 20 µm, left; 5 µm, right. (F) DNA content for oocytes from germinal beds of newly hatched A. inornata (left, n=167), newly hatched A. neomexicana (middle, n=148), and adult A. neomexicana ranging in age from 16-22 months (right, n=179).

Fig. 2.

Stages of meiotic prophase in A. inornata. Germinal bed stained with DAPI (blue), anti-SYCP3 (magenta) and telomere FISH (yellow). Stages were assigned based on SYCP3 staining pattern, telomere position and cell size. Representative examples for (A) pre-leptotene, (B) late leptotene/early zygotene, (C) zygotene, (D) pachytene, and (E) early diplotene. The diplotene nucleus is outlined with a dashed white line in the DAPI image. Scale bars: 2 µm. Schematics of prophase nuclei at the respective stages are shown to the right of the microscopic images showing DNA in blue, telomeres in yellow and SYCP3 in magenta.

Fig. 3.

Distribution of oocytes across prophase I in hatchlings and adults. Meiotic stages were assigned based on anti-SYCP3 staining and telomere localization in newly hatched and adult A. inornata (solid blue) and A. neomexicana (striped). Number of oocytes assigned to specific stages: 211 (hatchling A. inornata), 480 (hatchling A. neomexicana), 26 (adult A. inornata), 332 (adult A. neomexicana). Absolute numbers of oocytes in each class are shown in parentheses.

Fig. 4.

Rad51 localization in oocytes. Germinal beds from bisexual A. inornata (A) and parthenogenetic A. neomexicana (B) were stained with DAPI (blue), anti-Rad51 (yellow) and telomere FISH (magenta). Telomere bouquets are indicated with white arrows. The chromosome internal arrays of telomeric sequence found on 13 chromosomes in A. neomexicana do not contribute to the meiotic bouquet. Scale bars: 2 µm.

Fig. 5.

Structured illumination microscopy (SIM) of SYCP3 threads resolves lateral elements on paired chromosomes. (A) Germinal beds were stained with anti-SYCP3 and the same oocytes were imaged by confocal microscopy (left) and SIM (right). The boxed areas in each image are shown enlarged below the main images. (B) Examples of oocytes with unpaired, partially paired and fully paired chromosomes from A. inornata hatchling. (C) Unpaired and partially paired chromosomes from A. neomexicana hatchling. (D) Example of partially paired chromosomes from an adult A. neomexicana. (E) Quantification of pairing in hatchling A. inornata (solid blue bar; n=85), hatchling (light blue stripes; n=115) and adult (dark blue stripes; n=33) A. neomexicana. Scale bars: 2 µm.

Fig. 6.

DNA content analysis for pachytene-like and diplotene oocytes. (A) DNA content analysis of pachytene-like oocytes based on SYCP3 and telomere localization. DNA content was measured based on nuclear DAPI fluorescence for each oocyte relative to at least three surrounding stromal cells assumed to be in G1/G0. DNA content differences are not significant [P=0.585 one-way analysis of variance (ANOVA) test]. Germinal beds were isolated from hatchling of A. inornata (bisexual) and A. neomexicana (parthenogenetic). (B) Image cytometry as in A for early diplotene oocytes identified based on cell morphology and size, as well as absence of anti-SYCP3 staining and telomere clustering. DNA content differences between the two species are highly significant [P<2×10⁻¹⁶ one-way analysis of variance (ANOVA) test].

Fig. 7.

Fecundity and hatch rates for A. inornata (bisexual) and A. neomexicana (parthenogenetic). (A) Fecundity based on the number of eggs recovered from pens collectively housing 37 female and 24 male A. inornata and 57 A. neomexicana, respectively. As not all animals were present for the full 365 day collection period, the number of eggs recovered was divided by the sum of days for all females in the experiment (10169, A. inornata; 17490, A. neomexicana) and normalized to one year. Mean and standard errors were calculated by treating animals housed in separate pens as biological replicates. (B) Hatch rate for eggs collected in A after incubation at 28°C for ∼2 months. Some late-stage embryos (28 for A. neomexicana and 1 for A. inornata) were used in research and those eggs were excluded from the analysis.