Polyploidy in Mammals: The First Tetraploid Rodent.
Milton H. Gallardo1, Ricardo A. Ojeda2, Claudio Bidau3, and John A. Kirsch4..

1Instituto Ecología y Evolución, Universidad Austral de Chile. 2 Grupo de Investigaciones de la Biodiversidad, IADIZA, CONICET,  Cricyt, 5500 Mendoza, Argentina. 3Laboratorio de Genética, Universidad Nacional de Misiones, 3300 Posadas, Argentina. 4University of Wisconsin Zoological Museum, Madison, Wisconsin 53706 USA.

The red vizcacha rat, Tympanoctomy barrerae (Rodentia, Octodontidae) is an endemic desert specialist distributed in the Monte Desert in west-central Argentina. Being remarkably specialized in morphological, physiological, and behavioural traits, Tympanoctomys has a totally biarmed karyotype with the highest chromosome number recorded in any mammal (2n = 102). Associated to this perplexing karyotype, unusually large sperms are found in Tympanoctomys. These peculiarities prompted further study since we aimed to determine whether these features could be derived from whole-genome duplication (= tetraploidy)

Karyotypes: C-bands were induced by the barium hydroxide technique. Sixteen C-banded karyotypes were prepared from selected material. Chromosomes were captured using a Zeiss Axioplan equiped with a digital camera. Images were contrast-enhanced and black-and-white images were obtained using Macktype 3.0.

Flow cytometry: Nuclei of disrupted kidney cells were stained with propiduim iodide. Cells were run in the stain solution and the G1 peak was adjusted to channel 25 using chicken cells as internal standard. Flow analysis of 10,000 cells in the G1 (2C) peak was performed using a Leitz MPV flow cytometer. DNA content was calculated by the ratio of X divided by the channel number for the animal tested. .

Sperm morphology: Semen was extruded from the epididymis and placed in Hanks balanced salt solution. Smears were fixed in Carnoy and stained in Giemsa. Images of sperms were digitally captured and processed with Adobe Photoshop 5.0.

RESULTS

The karyotype of Tympanoctomys barrerae (2n = 102, FN = 200) is totally biarmed, and includes 36 pairs of metacentric to submetacentric chromosomes and 14 pairs of subtelocentric autosomes. All chromosomes showed conspicuous, pericentromeric blocks of heterochromatin. No whole-heterochromatic arms and no insterstitial or telomeric C-bands were recorded. Pair 43 has secondary constriction in the long arm. The X chromosome is large-sized and the Y is the only acrocentrics in the karyotype.

Genome size estimates and karyotypic data does not support a gradual trend towards increasing genome size in the octodontids and allied taxa. On the contrary, the discontinuity separating the 7-9 pg species from T. barrerae (16.8 pg) contradicts a gradual explanation and supports its tetraploid condition.

Tympanoctomys has a spatulated sperm head with a broad lateral face and a relatively short tail. Although its more square-shaped head differs from the pyriform or oval shape of the other octodontids, its abnormally large size (13 x 13m ) contrasts markedly with the dimensions of all the octodontid genera and allied families. As expected in polyploid organisms, sperm head dimensions are related to DNA content and T. barrerae fitted that expectation by having 9.2 pg DNA that is in the range of diploid values (Fig 5).

Larger somatic cell diameter were also recorded as expected intetraploid organisms. A highly significant paired t-test supported the difference in maximum diameter of liver cells (N = 150) between Abrocoma bennetti (22.3 m ± 3.5) or Octodon lunatus (21.3 m ± 2.7) and T. barrerae (26.1m ± 3.8).

Discussion

Nuclear DNA content is relatively constant in several mammal orders. An average genome size of 10 pg DNA is found in megachiropterans and marsupials whereas only 6 pg DNA characterize the microchiropterans. A similar average value is found in some artiodactyls, and 7.3 pg DNA have been reported for humans. Genome size in the hystricognath rodents fluctuates from 6 to 9 pg DNA.

The sperm head of Tympanoctomys is is by far the largest naturally occurring in mammals, and its size is causally related to the double genome size it has to accomodate.

The allozymic relationships of the Octodontoidea and sequencing data from the 12S mitochondrial gene indicated sister relationships between Tympanoctomys barrerae (2n = 102) Octomys mimax (2n = 56).DNA-DNA hibridization, and sequence data from the 12S rDNA suggests that the lineage conducting to Octomys mimax may represent the ancestral species from which tetraploidy developed.

Regardless the origin of this tetraploid, its origin cannot be traced to any recent karyotype if a perfect duplication is expected. The difference between observed (2n=102) and expected numbers (2n=112) involves the secondary constriction bearing pair, and the sex chromosomes, not found in four copies. This numerical difference probably reflects underlying physiological mechanisms in which local disomy is needed to avoid the combined lethal effects of chromosome imbalance. By the same token, disomy for the X-chromosome in Tympanoctomys is either sufficient or the only possibility for functional tetraploid female eutherians to develop.

Regardless of its origin, this tetraploid lineage probably was reproductively isolated by chromosome barriers. Apparently instantaneous speciation then is also possible in mammals, and the role of genome duplications in triggering evolutionary novelties is stressed by this unique rodent.