Evolution of Genome Size in Drosophila. Is the Invader's Genome Being Invaded by Transposable Elements?

Cristina Vieira*, Christiane Nardon*, Christophe Arpin{dagger}, David Lepetit* and Christian Biémont*

*Laboratoire de Biométrie et Biologie Evolutive, UMR CNRS 5558, Université Lyon 1, Villeurbanne Cedex, France;
{dagger}INSERM-U503, CERVI, Lyon Cedex 07, France


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Genome size varies considerably between species, and transposable elements (TEs) are known to play an important role in this variability. However, it is far from clear whether TEs are involved in genome size differences between populations within a given species. We show here that in Drosophila melanogaster and Drosophila simulans the size of the genome varies among populations and is correlated with the TE copy number on the chromosome arms. The TEs embedded within the heterochromatin do not seem to be involved directly in this phenomenon, although they may contribute to differences in genome size. Furthermore, genome size and TE content variations parallel the worldwide colonization of D. melanogaster species. No such relationship exists for the more recently dispersed D. simulans species, which indicates that a quantitative increase in the TEs in local populations and fly migration are sufficient to account for the increase in genome size, with no need for an adaptation hypothesis.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Genome size (the C-value) is known to vary considerably among organisms but is relatively constant among individuals of the same species (Mirsky and Ris 1951Citation ), and its significant degree of variation among organisms, more than 200,000-fold among eukaryotes (Gregory 2001Citation ), is not linked to biological complexity (Petrov 2001Citation ). Several hypotheses have been proposed to explain this genome size variation. The "selfish DNA" hypothesis suggests that the accumulation of DNA over evolutionary time may result from the cell replication process. The increase in genome size thus ceases when it begins to compromise cell viability (Doolittle and Sapienza 1980Citation ; Orgel and Crick 1980Citation ; Pagel and Johnston 1992Citation ). According to this theory, larger cells can simply tolerate more DNA (Gregory 2000Citation ). An alternative hypothesis postulates that genome size may influence the fitness of the organism by a nucleoskeletal effect that is based on the amount of nuclear DNA and is independent of the nucleotide sequence. According to this hypothesis, genome size is considered to be a selective trait because larger cells will need larger nuclei to preserve a favorable metabolic balance (Cavalier-Smith 1978Citation , 1985Citation , pp. 253–265; Cavalier-Smith and Beaton 1999Citation ). This relationship implies an inverse correlation between genome size and some life history traits, such as the rate of cell division (Cavalier-Smith 1985Citation ), metabolism (Vinogradov 1995Citation , 1997Citation ), and development (Sessions and Larson 1987Citation ), with natural selection acting on the global phenotype of the organism. Both hypotheses relate the increase of genome size to cell volume, but their interpretations are strikingly different (see Gregory and Hebert 1999Citation for a review). In addition to these correlations, several studies have also identified relationships between genome size and climate. Hence, some fishes (Beaton and Hebert 1988Citation ), salamanders (Xia 1995Citation ; Jockush 1997Citation ), and several plants (Grime and Mowforth 1982Citation ; Kalendar et al. 2000Citation ; Tarchini et al. 2000Citation ) from high latitude and altitude exhibit larger genomes and more frequent polyploidy than species from low latitude and altitude.

Genome size variability is usually attributed to differences in the amount of repetitive DNA, which corresponds to a large fraction of the genome (Pagel and Johnston 1992Citation ). For example, the human genome consists of only 5% of coding sequences but more than 40% of transposable elements (TEs) (International Human Genome Sequencing Consortium 2001Citation ). A link between genome size and the amount of TEs is therefore to be expected. Recent data provide evidence that the TE copy number is related to environmental conditions and to the colonization process (Arnault and Dufournel 1994Citation ; Labrador and Fontdevila 1994Citation ; Wisotzkey, Felger, and Hunt 1997Citation ; Labrador et al. 1999Citation ). Kalendar et al. (2000)Citation have shown that the Bare-1 element in barley, is responsible for a large genome size variation, and its copy number increases in relation to the height and dryness of the environment. An association between genome size variation and TE content has also been found in rice, which contains 14% of LTR retrotransposons (Tarchini et al. 2000Citation ). An increase in the genome size of maize is the result of recent TE amplifications, which have occurred within the last 2 to 6 Myr (SanMiguel and Bennetzen 1998Citation ). TEs may also contribute to an increase in genome size after crosses between different species. This was observed when two wallaby species, Macropus eugenii and Wallabia bicolor, were crossed (O'Neill, O'Neill, and Graves 1998Citation ). The centromeric regions of the hybrids were amplified, and the amplified sequences presented homologies to human endogenous retroviral elements. In Drosophila, crosses between Drosophila buzzatti and D. koepferae mobilize the osvaldo element in hybrids (Labrador and Fontdevila 1994Citation ; Labrador et al. 1999Citation ).

Most of the work done on genome size is based on comparisons between different species. However, the few studies which have been done at the population level reveal differences in genome size between populations in mammals, such as the pocket Gophers (Sherwod and Patton 1982Citation ), fishes (Gold and Amemiya 1987Citation ; Gold, Ragland, and Schliesling 1990Citation ; Lockwood and Derr 1990Citation ), and insects, such as mosquitoes (Rao and Rai 1987Citation ; Black and Rai 1988Citation ; Warren and Crampton 1991Citation ) and Drosophila (Dawley 1997Citation , pp. 143–184). One way to explain these intraspecific differences is to view genome size as a selective trait (Gregory 2001Citation ). An alternative hypothesis, however, is that TEs may be mobilized under stressful environmental conditions (Arnault and Dufournel 1994Citation ) or genetic conditions (Wisotzkey, Felger, and Hunt 1997Citation ; Biémont et al. 1999Citation ; Labrador et al. 1999Citation ), in some cases after horizontal transfer (Kidwell and Lish 1997Citation ). As a consequence, the colonization of new habitats could lead to the mobilization of certain TEs in the genome of invasive species, leading to an increase in genome size. In the present study we used flow cytometry to estimate genome size of individuals from various geographically distinct populations of D. melanogaster and D. simulans, and we correlated genome size with the total amount of TEs previously estimated by in situ hybridization (Biémont et al. 1999Citation ; Vieira et al. 1999Citation ). We show that differences in TE amount in the euchromatic part of the chromosomes do indeed account for some of the differences in genome size between species and between populations within a given species. The amount of TEs in centromeric heterochromatin does not seem to be involved in this correlation in a simple way.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Natural Populations
We worked on fly samples collected from several geographically distinct natural populations. The D. melanogaster populations considered were from Arabia, Argentina (Virasoro), Bolivia, China (Canton), Congo (Brazzaville), France (St Cyprien), Portugal (Chicharo), Réunion Island, Senegal, and USA (Seattle). The populations of D. simulans were from Australia (Canberra, Cann River, Eden), France (Valence), Kenya (Makindu), Madagascar, New Caledonia (Amieu), Polynesia (Noumea, Papeete), Portugal (Madeira), Réunion Island, Russia (Moscow), and Zimbabwe. These populations were maintained in the laboratory at 18°C as isofemale lines or small mass cultures with around 50 pairs in each generation.

Estimation of the Euchromatic TE Copy Number
The TE insertion site number of polytene chromosomes had previously been determined for 31 elements in samples of the populations described above (Biémont et al. 1999Citation ; Vieira et al. 1999Citation ).

Estimation of the Total Amount of TEs
We estimated the relative total amount of each TE individually by an inverse dot blot technique, which was developed by D. Lepetit. One microgram of each plasmidic DNA containing a TE sequence and 1 µg of actin DNA used as control were blotted on a nylon N+ membrane (Amersham). The membrane was hybridized with 100 ng of DNA extracted by classical technique from 20 adult female flies from a given population. This DNA was sonicated, and the resulting fragments were labeled with 32P dCTP by random priming. The same blot was thus hybridized successively with the DNA from four different populations, Canton and Senegal for D. melanogaster and Canberra and Madagascar for D. simulans. The list of the TEs used in the dot blot experiment and their euchromatic copy number determined by in situ hybridization are given in table 1 . Autoradiographic signal intensity was acquired with the Molecular Imager System, analyzed with Biorad Molecular Analyst R System and Biorad software, and adjusted as a function of the actin activity. Because the labeling of the probe by random priming is not independent of the base composition, we considered the ratio between populations for each TE. This hybridization with whole fly DNA made it possible to estimate the amount of heterochromatic and euchromatic copies for each TE. In comparison, in situ hybridization gave only the number of TE insertion sites distributed along the euchromatic part of the chromosomes; therefore, it was an underestimation of the real TE copy number because each site labeled could in fact bear more than one TE copy.


View this table:
[in this window]
[in a new window]
 
Table 1 Average Copy Numbers of TEs Per Diploid Genome on Euchromatin Determined by In Situ Hybridization

 
Estimation of the Genome Size
Nuclei were extracted from five heads of 5- to 10-day-old males or females. The heads were crushed in a small, siliconized Eppendorf vial containing 200 µl labeling solution (0.1 g trisodium citrate, 0.01 ml Triton X-100, 0.05 mg RNAse-A, water UHQ 100 ml, following R. Dawley, personal communication) with 1 µg/ml propidium iodide (Aldrich). Tetraodon (Tetraodon nigroviridis) blood was used as the internal standard for diploid genome size (0.8 pg). In order to distinguish between this standard and the fly head nuclei, the tetraodon blood was labeled with the fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) at 2 µg/ml in PBS for 15 min, at 37°C. The reaction was blocked by adding an excess of proteins in the form of an equal volume of cold fetal calf serum. Twenty microlitres of stained tetraodon blood was then added to the solution of fly head nuclei. The final mixture thus contained similar amounts of blood cells and fly nuclei, as estimated using a Thoma nucleus-counting cell. The mixture was incubated for 10 min in ice and filtered across 140 µm and then 30 µm nylon meshes. Six-hundred microlitres of initial labeling solution was then added to achieve an appropriate dilution. The resulting solution was analyzed on a FACScalibur flow cytometer (Becton Dickinson Instruments) fitted with an argon laser at 488 nm wavelength. About 20,000 nuclei were analyzed, with an average rate of 300 events/s for each determination of the genome size of the flies. Genome sizes were estimated for the 10 populations of D. melanogaster or the 13 populations of D. simulans simultaneously, and the experiment was replicated four times for males and four times for females, independently. Flow cytometry, which is commonly used in the medical field and in plant biology, provides an accurate determination of differences in genome size (Ulrich 1990Citation ; Michaelson et al. 1991Citation ; Lauzon et al. 2000Citation ) and is considered to be highly reliable for detecting tiny differences in genome size, such as a difference of 1.5% (Kent, Chandler, and Wachtel 1988Citation ). An example of a flow cytometry analysis of one sample of fly head nuclei and tetraodon blood is shown in figure 1 .



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.—Example of flow cytometry analysis of nuclei samples. (A) Dual FL3-width/FL-3-area plot was used to eliminate nuclei doublets from the analysis (R1). (B) Dual FL1-H/FL-3H plot analysis was used to separate fly nuclei (R2) and the tetraodon blood internal standard (R3). A threshold was set on FL3-H to eliminate cell debris from the analysis. (C) FL3-H fluorescence distribution of propidium iodide–stained DNA samples from tetraodon internal control (R1 and R2) and fly head nuclei (R2 and R3). The two peaks observed in the fluorescence profile of fly nuclei DNA correspond to the diploid (M1) and tetraploid (M2) nuclei. The mean FL3-H fluorescence from diploid fly nuclei and from tetraodon blood nuclei was used to calculate the Drosophila genome size of each sample

 

    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Genome Size Variation
Two-way analysis of variance was performed to detect the effects of sex and population in each species (see tables 2 and 3 ). There was no significant difference in mean genome size between males (0.395 ± 3.8 x 10-5 pg) and females (0.394 ± 7.4 x 10-5 pg) for D. melanogaster. However, in D. simulans, the males had a bigger genome (0.356 ± 3.4 x 10-5 pg) than the females (0.348 ± 3.3 x 10-5 pg). Significant differences were also detected between populations within each species (see tables 2 and 3 ).


View this table:
[in this window]
[in a new window]
 
Table 2 ANOVA of Genome Size in Populations of D. melanogaster

 

View this table:
[in this window]
[in a new window]
 
Table 3 ANOVA of Genome Size in Populations of D. simulans

 
Euchromatic TE Amount and Genome Size
As the genome size was significantly different in different populations, we investigated whether there was any correlation between genome size and the total amount of TEs on the polytene chromosomes. Figure 2 shows significant linear correlations and regressions between genome size and the TE insertion site numbers of males and females for the 10 populations of D. melanogaster and the 13 populations of D. simulans. Drosophila melanogaster had a larger genome than D. simulans and also displayed a stronger correlation between genome size and TE copy number.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.—Genome size (pg) versus number of insertion sites of TEs along the chromosomes. (a) males of D. simulans (black circles) and D. melanogaster (white circles), (b) females of D. simulans (black circles) and D. melanogaster (white circles). Linear correlation coefficients: Drosophila melanogaster, r = 0.55, P < 0.05 for males; r = 0.67, P < 0.001 for females; D. simulans, r = 0.40, P < 0.01 for males; r = 0.42, P < 0.01 for females. The regression lines for males correspond to Y = 0.339 + 0.000043 X for D. simulans, and Y = 0.358 + 0.000036 X for D. melanogaster. The regression lines for females correspond to Y = 0.330 + 0.000045 X for D. simulans and Y = 0.331 + 0.000061 X for D. melanogaster. The variable Y is genome size (pg) and X the TE insertion site number

 
The difference observed between the two species for their TE amount, the correlation between genome size and TE copy number between populations within the same species, and our experimental protocol, makes it unlikely that differences in DNA stainability could account for differences in genome size between populations. Changes in chromatin structure or chromatin condensation could have indeed lead to a wrong estimation of genome size because the DNA fluorescence would be incorrect (Noirot et al. 2000Citation ). However, this was unlikely in the present study because there is no reason to suppose that the different populations would have different levels of chromatin condensation associated with their TE copy number.

Contribution of TEs to Genome Size Variation
To determine the contribution of the TEs to genome size variation, we estimated the amount of TEs in picograms for the populations of extreme genome size for both species (Canton and Senegal for D. melanogaster; Canberra and Madagascar for D. simulans). The number of copies of each TE, estimated by in situ hybridization (table 1 ), was multiplied by the TE length in pg (1 pg equals 0.9869 x 109 bp). The difference in TE amount between the two extreme genome size populations within each species was then expressed as a percent of the genome size difference. It appears that variations in TE copy number in euchromatin account for only 5% to 9% of the observed difference in genome size. Other factors, such as the proportion of repetitive sequences in heterochromatin or the interspersion of highly repetitive DNA in the genome, must be involved to account for the difference in genome size, for which the TE copy number seems to be a distinctive label. We thus determined by dot blot the total amount of TEs in the heterochromatic and euchromatic parts of the genome of whole flies from the two populations with extreme size in each species (see an example of dot blot in fig. 3 ). In D. simulans, the ratio of the blot intensities from whole DNA between Canberra and Madagascar was equal to 1.10, whereas the ratio of the copy number estimated by the in situ technique was equal to 1.72 (the TEs with no insertion sites were excluded from the calculus). If we removed the 412 element from the calculation (the element that is overrepresented in the Canberra population), the dot blot and the in situ techniques gave copy number ratios between Canberra and Madagascar equal to 1.03 and 1.34, respectively. This shows that the Canberra and Madagascar populations did not differ significantly when the total genomic amount of TEs was considered. The number of insertion sites on the chromosome arms was thus not associated with the amount of TEs within the heterochromatin. It is thus the variation in the number of insertion sites along the chromosomes which was responsible for the main variation in total amount of TEs in D. simulans. This conclusion is sustained by the strong correlation between the ratios of the dot intensity and the in situ insertion site number of Canberra over Madagascar (r = 0.895, P < 0.0001). Again, when we excluded the 412 element from the analysis, the correlation dropped to r = 0.693 but was still significant (P < 0.01) (fig. 4 ). Drosophila melanogaster presented a different picture. The ratios of the most extreme populations for genome size, Canton and Senegal, were equal to 1.24 and 1.36 for the dot blot and the in situ hybridization techniques, respectively. This suggests that (1) the TE insertions on the euchromatin, to a great extent, determine the total amount of TEs, (2) the amount of TEs in heterochromatin parallels the amount on euchromatin, or (3) in Canton, the TE amount on heterochromatin increased, whereas it varied independently of the insertion site number in euchromatin. The first hypothesis can be discounted because the ratio of the total genomic amount of eight TEs with very low insertion sites determined by in situ was equal to 1.47 and thus did not differ from the value obtained when all TEs were considered. The second hypothesis can be eliminated because correlation between the ratios of dot blot intensity and the in situ insertion site number of Canton and Senegal (r = -0.13) (fig. 4 ) was low and nonsignificant, in contrast with that in D. simulans. This suggests that the TE amount within heterochromatin was higher in Canton than in Senegal and that this TE amount in heterochromatin increased from Senegal to Canton independent of the increase in insertion sites on euchromatin, as proposed by the third hypothesis.



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 3.—Dot blot obtained for the two populations with extreme genome size for D. simulans (see Materials and Methods for the technical procedure). Spots from left to right: 17.6, 297, 412, 1731, Bari-1, Bell, blood, burdock, copia, coral, Doc, F, Flea, gypsy, HMS Beagle, hobo, I, Idefixe, jockey, mdg1, mdg3, mariner, nomade, opus, pogo, roo/B104, springer, stalker, tirant, Zam, actine. Mk—Madagascar, Cb—Canberra

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.—Dot blot versus in situ hybridization ratios of TE amount in populations with extreme genome size for (a) D. melanogaster and (b) D. simulans (see text for the correlation values)

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The fact that the genome of D. melanogaster is larger than that of D. simulans concurs with the usual observation that the genome of D. melanogaster contains more middle repetitive DNA than that of D. simulans (Dowsett and Young 1982Citation ; Vieira et al. 1999Citation ). We have previously shown that D. melanogaster populations from Africa (the cradle of this species) harbor a low total amount of TEs but that this amount increases in populations from other continents. We therefore suggested that TE acquisition and accumulation may parallel the D. melanogaster colonization process (Vieira et al. 1999Citation ). We show here that genome size variation in the D. melanogaster populations is correlated with the TE amount; thus, it also parallels the species colonization. No global association between geographic distribution of populations and the overall TE amount was observed in D. simulans populations (Vieira et al. 1999Citation ), and the correlation between TE amount and genome size is lower than that in D. melanogaster. This lower level of correlation is attributed to the fact that certain populations of D. simulans have a high copy number of some TEs but not of others, whereas populations of D. melanogaster have high copy number for most TEs (Vieira et al. 1999Citation ). Indeed, for most populations of D. simulans, any increase in the copy number for one or only a few TEs is not sufficient to influence genome size significantly. However, the Canberra population of D. simulans has the largest genome in the species and has a significantly larger number of copies of most TEs, especially when compared with the Madagascar population, which has small genome size and a low TE copy number (Vieira, Piganeau, and Biémont 2000Citation ). The main point that emerged from these findings is that the TE insertion site number on chromosome arms (the euchromatic copies) only contributes to about 5% to 9% of the observed genome size variations. This suggests that genome size variation is caused by other factors as well as by the TEs in euchromatin. TE copies and blocks of complex DNA embedded in the heterochromatic regions of the chromocenter are good candidates for explaining such differences in genome size (Vaury, Bucheton, and Pélisson 1989Citation ; Miklos and Cotsell 1990Citation ; Nurminsky et al. 1994Citation ; Le, Duricka, and Karpen 1995Citation ). We have shown in D. melanogaster that the variation in the TE amount within heterochromatin does not follow the variation in TE number in euchromatin when TEs are considered individually, although the Canton population with a high genome size has both a high amount of TEs within heterochromatin and high TE insertion site numbers in euchromatin. We must therefore envisage that the amplification of other repetitive sequences in heterochromatin or euchromatin may be associated in one way or another with the TE copy number on chromosome arms. For example, in plants it is the size of repetitive blocks between genes that correlates with genome size (Chen et al. 1997Citation ), and amplification and deletion of satellite DNA has been observed in many species (Csink and Henikoff 1998Citation ; Slamovits et al. 2001Citation ), all phenomena which could have involved chromatin remodeling (Henikoff, Ahmad, and Malik 2001Citation ). The fact that D. simulans males have bigger genomes than females may indicate a specific increase of repeated sequences in the heterochromatin of the Y chromosome of that species.

Historical processes have led to the invasion of the D. melanogaster genome by many TEs a long time ago, apart from the P and I elements, which only invaded this species at the beginning of the 1900s. In contrast, populations of the D. simulans species still seem to be in the process of being invaded by TEs (Vieira et al. 1999Citation ). Increases in genome size could therefore result from an increase in TE copy number, which could result from mobilization of TEs induced by environmental stresses, as has been reported in bacteria, yeast, and plants (Newton, Wakamiya, and Price 1993Citation , pp. 321–345; Arnault and Dufournel 1994Citation ; Kidwell and Lish 1997Citation ). When populations are subjected to new environmental conditions, as may happen in populations that have invaded novel habitats, some of their TEs may be mobilized (Capy et al. 2000Citation ; Kalendar et al. 2000Citation ) and their genome size increased, leading to the observation that genome size increases during geographical colonization (Cros et al. 1995Citation ). Drosophila melanogaster and D. simulans both originated in East Africa (Lachaise et al. 1988Citation ), and the relationship between genome size and geography in D. melanogaster could therefore mainly reflect the geographic distance from the African birthplace, rather than any adaptive process. The high copy number of the uhu and LOA elements in Hawaiian Drosophila suggests that the colonization of new islands from older islands may have been associated with a significant increase in TE copy number (Wisotzkey, Felger, and Hunt 1997Citation ). The unusual distribution of the osvaldo element in populations from the Iberian Peninsula compared with the original populations in Argentina (Labrador et al. 1999Citation ) also implies an association between an increase in TE copies and colonization processes. TE transposition has also been seen to increase in both Drosophila hybrids (Evgen'ev et al. 1982Citation ; Labrador et al. 1999Citation ) and in the hybrid Australian wallaby (O'Neill, O'Neill, and Graves 1998Citation ), resulting from confronting populations or species that did not previously overlap.

We still have much to learn about the genome. This paper and other recent studies on Drosophila, plants, fishes, and yeast, should trigger a fresh debate on genome changes associated with species invasion of new habitats, geographical locations, and evolution. Progress in this area of research can benefit from the comparative analysis of genomes of native and newly derived populations of the numerous pest species that are continually invading new areas of our world. The Zebra mussel, cheatgrass, European house sparrow, Argentine ant (Orr and Smith 1998Citation ; Duke and Mooney 1999Citation ; Duvernell and Turner 1999Citation ), and many other invaders, in addition to the more common Drosophila species, should help us to understand the link between chromatin structure, TE mobilization, genome size, success of colonization, and various adaptive characteristics.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank J. Marvel, R. Dawley, and J. S. Johnston for their advice on the flow cytometry analysis of fly nuclei, C. Fisher for her gift of Tetraodon blood, and R. Grantham and C. Loevenbruck for their help. This work was funded by the Centre National de la Recherche Scientifique and the Association pour la Recherche sur le Cancer. We thank M. Ghosh for reviewing the English text.


    Footnotes
 
Pierre Capy, Reviewing Editor

Keywords: genome size Drosophila transposable elements Back

Address for correspondence and reprints: Cristina Vieira, Laboratoire de Biométrie et Biologie Evolutive, UMR CNRS 5558, 43 bd 11 novembre, Université Lyon 1, 69622 Villeurbanne Cedex, France. vieira{at}biomserv.univ-lyon1.fr Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Arnault C., I. Dufournel, 1994 Genome and stresses: reactions against aggressions, behavior of transposable elements Genetica 93:149-160[ISI][Medline]

    Beaton M. J., P. D. N. Hebert, 1988 Geographical parthenogenesis and polyploidy in Daphnia pulex Am. Nat 132:157-163

    Biémont C., C. Vieira, N. Borie, D. Lepetit, 1999 Transposable elements and genome evolution: the case of Drosophila simulans Genetica 107:113-120[ISI][Medline]

    Black W. C., K. S. Rai, 1988 Genome evolution in mosquitoes: intraspecific and interspecific variation in repetitive DNA amounts and organization Genet. Res 51:185-196[ISI][Medline]

    Capy P., G. Gasperi, C. Biémont, C. Bazin, 2000 Stress and transposable elements: co-evolution or useful parasites? Heredity 85:101-106[ISI][Medline]

    Cavalier-Smith T., 1978 Nuclear volume control by nucleoskeletal DNA selection for cell volume and cell growth rate, and the solution of the DNA C-value paradox J. Cell. Sci 34:247-278[Abstract]

    ———. 1985 The evolution of genome size John Wiley & Sons, Chichester, UK

    Cavalier-Smith T., M. J. Beaton, 1999 The skeletal function of non-genic nuclear DNA: new evidence from ancient cell chimaeras Genetica 106:3-13[ISI][Medline]

    Chen M., P. San Miguel, A. C. de Oliveira, S. S. Woo, H. Zhang, R. A. Wing, J. L. Bennetzen, 1997 Microcolinearity in sh2-homologous regions of the maize, rice, and sorghum genomes Proc. Natl. Acad. Sci. USA 94:3431-3435[Abstract/Free Full Text]

    Cros J., et al 1995 Nuclear DNA content in the subgenus Coffea (Rubiaceae): inter- and intra-specific variation in African species Can. J. Bot 73:14-20[ISI]

    Csink A. K., S. Henikoff, 1998 Something from nothing: the evolution and utility of satellite repeats Trends Genet 14:200-204[ISI][Medline]

    Dawley R., 1997 Progress and prospects in evolutionary biology The Drosophila model. J. P. Powell, New Oxford University Press, New York

    Doolitle W. F., C. Sapienza, 1980 Selfish genes, the phenotype paradigm and genome evolution Nature 284:601-603[ISI][Medline]

    Dowsett A. P., M. W. Young, 1982 Differing levels of dispersed repetitive DNA among closely related species of Drosophila Proc. Natl. Acad. Sci. USA 79:4570-4574[Abstract]

    Duke J. S., H. A. Mooney, 1999 Does global change increase the success of biological invaders? Trends Ecol. Evol 14:135-139[ISI][Medline]

    Duvernell D. D., B. J. Turner, 1999 Variation and divergence of Death Valley pupfish populations at retrotransposon-defined loci Mol. Biol. Evol 16:363-371[Free Full Text]

    Evgen'ev M. B., G. N. Yenikolopov, N. I. Peunova, Y. V. Ilyin, 1982 Transposition of mobile genetic elements in interspecific hybrids of Drosophila Chromosoma 85:375-386[ISI][Medline]

    Gold J. R., C. T. Amemiya, 1987 Genome size variation in North American minnows (Cyprinidae). II. Variation among 20 species Genome 29:481-489[ISI][Medline]

    Gold J. R., C. J. Ragland, L. J. Schliesling, 1990 Genome size variation and evolution in North American cyprinid fishes Genet. Sel. Evol 22:11-29[ISI]

    Gregory T. R., 2000 Nucleotypic effects without nuclei: genome size and erythrocyte size in mammals Genome 43:895-901[ISI][Medline]

    ———. 2001 Coincidence, coevolution, or causation? DNA content, cell size, and the C-value enigma Biol. Rev. Camb. Philos. Soc 76:65-101[Medline]

    Gregory T. R., P. D. Hebert, 1999 The modulation of DNA content: proximate causes and ultimate consequences Genome Res 9:317-324[Abstract/Free Full Text]

    Grime J. P., M. A. Mowforth, 1982 Variation in genome size—an ecological interpretation Nature 299:151-153[ISI]

    Henikoff S., K. Ahmad, H. S. Malik, 2001 The centromere paradox: stable inheritance with rapidly evolving DNA Science 293:1098-1102[Abstract/Free Full Text]

    International Human Genome Sequencing Consortium. 2001 Initial sequencing an analysis of the human genome Nature 409:860-921[ISI][Medline]

    Jockush E. J., 1997 An evolutionary correlate of genome size change in plethondontid salamanders Proc. R. Soc. Lond. B Biol. Sci 264:597-605[ISI]

    Kalendar R., J. Tanskanen, S. Immonen, E. Nevo, A. H. Schulman, 2000 Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence Proc. Natl. Acad. Sci. USA 97:6603-6607[Abstract/Free Full Text]

    Kent M., R. Chandler, S. Wachtel, 1988 DNA analysis by flow cytometry Cytogenet. Cell Genet 47:88-89[ISI][Medline]

    Kidwell M. G., J. F. Lish, 1997 Transposable elements as source of variation in animals and plants Proc. Natl. Acad. Sci. USA 94:7704-7711[Abstract/Free Full Text]

    Labrador M., M. Farré, F. Utzet, A. Fontdevilla, 1999 Interspecific hybridization increase tranposition rates of Osvaldo Mol. Biol. Evol 16:931-937[Abstract]

    Labrador M., A. Fontdevila, 1994 High transposition rate of Osvaldo, a new Drosophila buzzatti retrotransposon Mol. Gen. Genet 245:661-674[ISI][Medline]

    Lachaise D., et al 1988 Historical biogeography of the Drosophila melanogaster species subgroup Evol. Biol 22:159-225[ISI]

    Lauzon W., J. S. Dardon, D. W. Cameron, A. D. Badley, 2000 Flow cytometry measurements of telomere length Cytometry 42:159-164[ISI][Medline]

    Le M.-H., D. Duricka, G. H. Karpen, 1995 Islands of complex DNA are widespread in Drosophila centric heterochromatin Genetics 141:283-303[Abstract/Free Full Text]

    Lockwood S. F., J. N. Derr, 1990 Intra and interspecific genome size variation in the salmonidae Cytogenet. Cell Genet 59:303-306

    Michaelson M. J., H. J. Price, J. R. Ellison, J. S. Johnston, 1991 Comparison of plant DNA contents determined by feulgen microspectrophotometry and laser flow cytometry Am. J. Bot 78:183-188[ISI]

    Miklos G. L., J. N. Cotsell, 1990 Chromosome structure at interfaces between major chromatin types: alpha- and beta-heterochromatin Bioessays 12:1-6[ISI][Medline]

    Mirsky A. E., H. Ris, 1951 The deoxyribonuclei acid content of animal cells and its evolutionary significance J. Gen. Physiol 34:451-462[ISI][Medline]

    Newton E. J., I. Wakamiya, H. J. Price, 1993 Handbook of Plant and crop stress Marcel Dekker, Inc. New York

    Noirot M., P. Barre, J. Louarn, C. Duperray, S. Hamon, 2000 Nucleus-cytosol interactions—a source of stoichiometric error in flow cytometric estimation of nuclear DNA content in plants Ann. Bot 86:309-316[Abstract/Free Full Text]

    Nurminsky D. I., Y. Y. Shevelyov, S. V. Nuzhdin, V. A. Gvozdev, 1994 Structure, molecular evolution and maintenance of copy number of extended repeated structures in the X-heterochromatin of Drosophila melanogaster Mol. Gen. Genet 103:277-285

    O'Neill R. J. W., M. J. O'Neill, J. A. M. Graves, 1998 Undermethylation associated with retroelement activation and chromosome remodeling in an interspecific mammalian hybrid Nature 393:68-72[ISI][Medline]

    Orgel L. E., F. H. C. Crick, 1980 Selfish DNA: the ultimate parasite Nature 284:604-607[ISI][Medline]

    Orr M. R., T. B. Smith, 1998 Ecology and speciation Trends Ecol. Evol 13:502-506[ISI]

    Pagel M., R. A. Johnston, 1992 Variation across species in the size of nuclear genome supports the junk DNA explanation for the C-value paradox Proc. R. Soc. Lond. B Biol. Sci 249:119-124[ISI][Medline]

    Petrov D. A., 2001 Evolution of genome size: new approaches to an old problem Trends Genet 17:23-28[ISI][Medline]

    Rao P. N., K. S. Rai, 1987 Inter and intraspecific variation in nuclear DNA content in Aedes mosquitoes Heredity 59:253-258[ISI][Medline]

    SanMiguel P., J. L. Bennetzen, 1998 Evidence that a recent increase in maize genome size was caused by the massive amplification of intergene retrotransposons Ann. Bot 81:37-44

    Sessions S. K., A. Larson, 1987 Developmental correlates in genome size in plethodontid salamanders and their implications for the genome evolution Evolution 41:1239-1251[ISI]

    Sherwod S. W., S. L. Patton, 1982 Genome evolution in Pocket Gophers (Genus Thomomys). II. Variation in cellular DNA content Chromosoma 85:163-179[ISI][Medline]

    Slamovits C. H., J. A. Cook, E. P. Lessa, M. S. Rossi, 2001 Recurrent amplifications and deletions of satellite DNA accompanied chromosomal diversification in South American Tucos-tucos (genus Ctenomys, rodentia: octodontidae): a phylogenetic approach Mol. Biol. Evol 18:1708-1719[Abstract/Free Full Text]

    Tarchini R., P. Biddle, R. Wineland, S. Tingey, A. Rafalski, 2000 The complete sequence of 340 kb of DNA around the rice Adh1-adh2 region reveals interrupted colinearity with maize chromosome 4 Plant Cell 12:381-391[Abstract/Free Full Text]

    Ulrich W., 1990 Aneuploid and polyploid cellular DNA heterogeneity in insect cell material of diptera species analyzed by flow cytometry Z. Naturforsh 45:1027-1030

    Vaury C., A. Bucheton, A. Pélisson, 1989 The ß-heterochromatic sequences flanking the I elements are themselves defective transposable elements Chromosoma 98:215-224.[ISI][Medline]

    Vieira C., D. Lepetit, S. Dumont, C. Biémont, 1999 Wake up of transposable elements following Drosophila simulans worldwide colonization Mol. Biol. Evol 16:1251-1255[Abstract]

    Vieira C., G. Piganeau, C. Biémont, 2000 Mobilization of various transposable elements in an Australian population of D. simulans Genet. Res 76:117-119[ISI][Medline]

    Vinogradov A. E., 1995 Nucleotypic effect in homeotherms: Body mass-corrected basal metabolic rate of mammals is related to genome size Evolution 49:1249-1259[ISI]

    ———. 1997 Nucleotypic effect in homeotherms: body-mass independent metabolic rate of passerine birds is related to genome size Evolution 51:220-225[ISI]

    Warren A. M., J. M. Crampton, 1991 The Aedes aegypti genome complexity and organization Genet. Res 58:225-232[ISI][Medline]

    Wisotzkey R. G., I. Felger, J. A. Hunt, 1997 Biogeographic analysis of the Uhu and LOA elements in the Hawaiian Drosophila Chromosoma 106:465-477[ISI][Medline]

    Xia X., 1995 Body temperature, rate of biosynthesis, and evolution of genome size Mol. Biol. Evol 12:834-842[Abstract]

Accepted for publication March 5, 2002.