SC/65b/BRG04

Genome size of the bowhead whale (Balaena mysticetus)

Bickham1, J. W., C. W. Matson2, Elias M. Oziolor2, R. Suydam3, J. C. George3, and A. B. Baird4

1Battelle Memorial Institute, Houston, TX USA 2Department of Environmental Science, Center for Reservoir and Aquatic Systems Research (CRASR), and Institute for Biomedical Studies, Baylor University, Waco, TX USA 3Department of Wildlife Management, North Slope Borough, Barrow, AK USA 4University of Houston – Downtown, Department of Natural Sciences, 1 Main Street Houston, TX USA

Abstract The genome size of the bowhead whale (Balaena mysticetus) was measured from spleen cells of a male and a female using flow cytometry. The mean genome size (C value) was estimated to be 2.93 pg (2.87 Gb). There was a 3% difference in C value between the male and female bowhead which is consistent with the expected size difference between the X and Y chromosomes. This is the first direct genome size estimate for a baleen whale, and is the lowest value reported for any cetacean. It is near the low end of values reported for cetartiodactyls and is relatively low for mammals. Most mammals possessing lower genome sizes are small mammals, such as bats, shrews and rodents, with high metabolic rates. The total length of the reference genome sequence of this species is 2.3 Gb, which is approximately 21% less than our estimate based on flow cytometry. This discrepancy is likely the result of highly repetitive DNA which cannot be assembled effectively with current methods. Genome size correlates with cell size, and influences physiological processes such as metabolic rate and O2 exchange as small cells have higher surface area to volume ratios than large cells. It is also positively correlated with body size within some groups of mammals. The relatively small size of the genome of bowheads could be associated with metabolic rate, O2 exchange, or simply a plesiomorphic trait shared in common with other basal cetartiodactyls.

Introduction

Genome size evolution has received considerable attention due to a well-established relationship between nuclear DNA content, nuclear volume, cell volume and surface area. Specifically, smaller cells are associated with small genomes and have a higher surface area to volume ratio. These so called nucleotypic effects contribute towards improved gas exchange to meet metabolic demands (Szarski, 1983; Gregory, 2001).

Strong positive links between genome size and cell size and inverse relationships between genome size and metabolic rate have been shown in mammals (Vinogradov 1995; Gregory 2000). Additionally, relationships to various correlates have been identified within mammalian orders, such as a correlation between genome size and body size in rodents (Gregory 2002) and bats (Smith et al., 2013).

A link between genome size and rate of cell division is also well established, and this cellular trait leads to associations with other biological features. For example, it has been suggested that organ complexity, development, and ecological lifestyle have been important in the evolution of genome size among vertebrates (Gregory 2002, 2005a; Andrews and Gregory 2009). Roth et al. (1994) showed a negative correlation of genome size to brain complexity in amphibians. Fewer, less well differentiated neurons fit within the braincase when cells are large and divide slowly. That brain complexity and function influence aspects of animal behavior and ecology is a relationship known in mammals as well. For example, in bats, brain size has been linked to foraging ecology, feeding type, and habitat complexity (Eisenberg and Wilson 1978).

The negative relationship between genome size and cell division rate suggests that developmental traits may also be linked to genome size. This inverse relationship between genome size and developmental rate was demonstrated in amphibians (Bachmann 1972).

To date no direct estimates of genome size have been reported for any baleen whale. In this paper, we estimate the genome size of bowhead whales using flow cytometry and compare our findings to previous reports from studies of other Cetacea, Cetartiodactyla, and Mammalia. We also compare our estimated bowhead genome size to the length of the sequence scaffold constructed in the genome sequence project for this species. The purpose of the study was to gain insight into the genome size of the bowhead as a potentially adaptive trait that might be associated with their unique lifestyle including large body size, metabolic rate, diving capability, cold tolerance, and other features known or hypothesized to be influenced by nucleotypic effects.

Materials and Methods

Genome Size Determination

To determine a genome size estimate for bowhead whale, spleen tissues were acquired from one male (10B17) and one female (10B18). Both whales were harvested in 2010 as part of the Native subsistence hunt in Barrow, Alaska. Sample processing and staining followed the methods of Vindelov and Christiansen (1994). Instrument description and additional details provided in Oziolor et al. (2014). Briefly, flow cytometric genome size determination is based on propidium iodide fluorescent staining of nuclear DNA. Mean fluorescence is calculated for cells in the G0 and G1 phases of the cell cycle. This method requires direct comparison to known standards to convert measured fluorescence to pg of DNA. The primary standard used in this study is the domestic chicken (Gallus gallus domesticus). Chicken red blood cells are widely used as a genome size standard, with an accepted genome size of C = 1.25 pg. Chicken whole blood was purchased from Innovative Research (Novi, MI, USA). We also included mouse and rat as additional size standards and for internal checks for size estimates based on chicken. The house mouse, Mus musculus, has a genome size of C = 3.25 pg, and the genome size for Rattus norvegicus, is C = 3.05 pg (Vinogradov, 1998) . Spleen tissues from three male 129/SvEvTac laboratory mice and a single male Harlan SD Sprague-Dawley laboratory rat (Rattus norvegicus) were used.

Results

Simple ratios, assuming a chicken genome size of C = 1.25 pg, were used to convert mean fluorescence to pg of DNA. Mouse and rat tissues, which were included as an additional confirmation of genome size estimation accuracy, were within 2% and 3%, respectively, of published values (data not shown). Bowhead whale genome sizes were estimated using both chicken as a size standard, and by averaging the estimates produced from all three size standards (chicken, mouse, and rat) independently. The results from these two methods yielded estimates of 2.93 and 2.92 pg, respectively. Of particular interest was the variability in individual bowhead whale genome size estimates, an approximately 3% difference between our two samples (Figure 1). While not known during sample processing and initial analysis, bowhead #10B17, the individual with the smaller genome (2.88 pg), was a male, whereas bowhead #10B18, the individual with the larger genome (2.98 pg) was a female. This difference in genome size is entirely accounted for by the expected differences in masses of X and Y chromosomes. As is customary, the final bowhead whale genome size estimate was calculated as the average of the male and female genome sizes, 2.93 pg or 2.87 Gb (Figure 1).

Discussion

This is the first estimate of genome size for a baleen whale. The value C = 2.93 pg is the lowest value yet for a cetacean (Figure 2) and is on the low end of values for Cetartiodactyla (artiodactyls and cetaceans). The average of all mammals is C = 3.5 pg, and most mammalian species with lower genome sizes are animal with small body size and high metabolic rates including bats, shrews and some rodents. Only toothed whales are available for comparison and thus it is not known if bowheads are atypical for baleen whales. Nevertheless it is apparent from these results that bowheads are at the low end of the scale for mammals in general.

There are two possible explanations for the relatively small genome of the bowhead whale. The first is that it could be a plesiomorphic character unchanged during the evolution and diversification of cetartiodactyls. This is possible given the fact that low genome sizes are also found in suids, camelids, giraffids, cervids and bovids, notwithstanding the fact that most cetartiodactyls have higher values (http://www.genomesize.com/) and the ancestral character state is not known.

Figure 1.—DNA flow histograms of a male (A) and female (B) bowhead whale showing an approximately 3% difference in estimated genome sizes. The mean estimated genome size is C = 2.93 pg.

The second possible explanation is that the low genome size of the bowhead is a derived, adaptive, character state that has evolved as a result of nucleotypic effects. A correlate to small genome size is not obvious but could be related to metabolic rate or gas exchange in this highly specialized diving mammal.

Significance of the genome size estimate of bowheads also relates to its genome sequence. The genome size estimated from flow cytometry gives us an independent estimate of the amount of sequence not represented in the assembled genome sequence. The genome and transcriptome sequences of this species will be published in the near future and have been reported on at these meetings. However, there is a discrepancy in the genome size as measured in base pairs (one picogram = 978 megabases) with flow cytometry compared to the total sequence length in the genome sequence. The flow cytometric estimate is higher than the sequence total (2.87 Gb versus 2.3 Gb), likely due to the inability of the bioinformatics methods to assemble repetitive DNA sequences.

The total sequence length of the bowhead (2.3 Gb) is comparable to 2.44 Gb reported for the sequence of the minke whale (Balaenoptera acutorostrata; Yim et al., 2014). However, it cannot be inferred from this that their genome sizes are similar as the repetitive fraction of the two genomes could be quite different. Therefore, additional studies of genome size are needed for baleen whales in order to determine if the bowhead is an outlier or if this group of mammals is characterized by unexpectedly small genome sizes. In this way perhaps the adaptive correlates, if any exist, can be determined. In addition, it is anticipated that other baleen whales will be the subjects of genome sequencing efforts and a better understanding of the amount of DNA sequence not assembled is useful for determining the overall percent coverage of the genome sequence.

Figure 2.—Distribution of genome sizes of Mammalia, Cetartiodactyla, and Cetacea. Bowhead whales have an estimated genome size (2.93 pg) well below the mammalian mean (3.5 pg). This is the first species of baleen whale to be reported and has the lowest C-value of any cetacean. Some cetartiodactyls have lower genome sizes but most are higher than bowheads.

Acknowledgments

We thank the Alaska Eskimo Whaling Commission and the whaling captains and their crews for their support of this research.

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