683 The nucleotypic effects of cellular DNA content in cartilaginous and ray-finned fishes David C. Hardie and Paul D.N. Hebert Abstract: Cytological and organismal characteristics associated with cellular DNA content underpin most adaptionist interpretations of genome size variation. Since fishes are the only group of vertebrate for which relationships between genome size and key cellular parameters are uncertain, the cytological correlates of genome size were examined in this group. The cell and nuclear areas of erythrocytes showed a highly significant positive correlation with each other and with genome size across 22 cartilaginous and 201 ray-finned fishes. Regressions remained significant at all taxonomic levels, as well as among different fish lineages. However, the results revealed that cartilaginous fishes possess higher cytogenomic ratios than ray-finned fishes, as do cold-water fishes relative to their warm-water counterparts. Increases in genome size owing to ploidy shifts were found to influence cell and nucleus size in an immediate and causative man- ner, an effect that persists in ancient polyploid lineages. These correlations with cytological parameters known to have important influences on organismal phenotypes support an adaptive interpretation for genome size variation in fishes. Key words: evolution, genome size, DNA content, cell size, erythrocyte size, fishes, nucleotypic effect. Résumé : Des caractéristiques cytologiques et de l’organisme entier, lesquelles sont associées avec le contenu en ADN, sous-tendent la plupart des interprétations adaptivistes de la variation quant à la taille des génomes. Puisque les pois- sons constituent le seul groupe de vertébrés chez lequel les relations entre la taille du génome et certains paramètres cellulaires clés sont incertains, les corrélations entre les caractéristiques cytologiques et la taille du génome ont été exa- minées chez ce groupe d’espèces. Il existait une corrélation positive très significative entre les volumes de la cellule et du noyau chez les érythrocytes ainsi qu’entre ceux-ci et la taille du génome chez 22 espèces de poissons cartilagineux et chez 201 espèces de poissons à nageoires rayonnées. Les régressions sont demeurées significatives à tous les niveaux taxonomiques ainsi que chez tous grands groupes de poissons. Les résultats ont cependant révélé que les pois- sons cartilagineux possédaient des ratios cytogénomiques plus élevés que les poissons à nageoires rayonnées, tout comme le font les poissons d’eau froide par rapport aux poissons d’eau chaude. Des accroissements de la taille des génomes dus à des changements de ploïdie influençaient directement et immédiatement la taille de la cellule et du noyau, un effet qui persiste chez les anciennes lignées évolutives polyploïdes. Ces corrélations avec des paramètres cy- tologiques connus pour exercer des influences importantes sur le phénotype de l’organisme viennent appuyer une inter- prétation adaptative de la variation de la taille du génome chez les poissons. Motsclés:évolution, taille du génome, contenu en ADN, taille de la cellule, taille des érythrocytes, poissons, effets nucléotypiques. [Traduit par la Rédaction] Hardie and Hebert 706 Introduction variable, they are also strongly associated with cell size (Cavalier-Smith 1978 and references therein). Since this Early observations that cellular DNA amounts varied in a early work, the cytological correlates of genome size have non-random fashion both within and among groups of or- been confirmed in many groups across a 200 000 fold range ganisms (Mirsky and Ris 1951; Vendrely 1955) were soon of genome size (Gregory 2001b and references therein), followed by the discovery that cell and nuclear sizes varied such that the relationships between C value and both nucleus in concert with DNA content in protists (Shuter et al. 1983), and cell size rank among the most fundamental rules of plants (Martin 1966), and animals (Olmo and Morescalchi eukaryote cell biology (Cavalier-Smith 1993). 1975). Although prokaryotic genome sizes are much less Other than shifts in ploidy level, differences in amounts of non-coding DNA account for most genome size diversity Received 3 December 2002. Accepted 15 April 2003. (Cavalier-Smith 1985b), and five main explanations have Published on the NRC Research Press Web site at been advanced to explain this fact. The earliest of these hy- http://genome.nrc.ca on 13 June 2003. potheses proposed that non-coding DNA consists of extinct Corresponding Editor: P.B. Moens. genes (Ohno 1972), or “junk DNA”, which accumulates in the genome until constrained by selection. This term was D.C. Hardie1,2 and P.D.N. Hebert. Department of Zoology, more recently extended to include any genetic elements that University of Guelph, Guelph, ON N1G 2W1, Canada. increase in the genome by chance and lack a coding or regu- 1Corresponding author (e-mail: [email protected]). latory function (Pagel and Johnstone 1992). Second, the 2Present address: Department of Biology, Dalhousie “selfish DNA” theory argues that self-replicating DNA seg- University, Halifax, NS B3H 4J1, Canada. ments like transposable elements persist and increase within Genome 46: 683–706 (2003) doi: 10.1139/G03-040 © 2003 NRC Canada 684 Genome Vol. 46, 2003 the genome solely for their own benefit, and account for negatively to cell metabolic rate (Smith 1925; Goniakowska much non-coding DNA (Doolittle and Sapienza 1980; Orgel 1970) and both mitotic (Van’t Hof and Sparrow 1963) and and Crick 1980). More recently, the “mutational equilib- meiotic (Bennett 1971) division rates. rium” model proposed that genome size varies in a lineage until the loss of DNA through frequent small deletions is equal to the rate of DNA increase owing to long insertions DNA content and cell size: organismal phenotypes (Petrov 2002). Under the “nucleoskeletal” theory, DNA con- Given the ubiquitous and apparently causative relationship tent is secondarily selected owing to selection on cell and between genome size and a diversity of cytological parame- nuclear size (Cavalier-Smith 1978). Lastly, Bennett (1971) ters, the adoption of an adaptive interpretation for observed proposed the “nucleotypic” theory that DNA content affects patterns of genome size variation requires only that these cellular parameters in a causative manner, and is therefore cellular characteristics extend in some way to the organismal subject to secondary selection via selection on cytological level, so that they (and by turn, DNA content) are subject to and organismal phenotypes. Since each of these theories selection. That cell volume is subject to selection is hardly identifies factors that might contribute to genome size diver- debatable owing to its many physiological and developmen- sity, a pluralistic approach may provide the best explanation tal implications (Gregory 2001a). Most obviously, cell size of genome size evolution. Chipman et al. (2001) provide a affects body size in organisms with a fixed or constrained clear statement of this prospect — “One of the problems in number of cells, resulting in strong selection on cell size via many attempts at explaining the evolution of genome size is its effect on body size (Gregory et al. 2000). In fact, some the search for a single evolutionary model that holds for all organisms are known to exploit the causative effects of ge- taxa. We believe the situation is more complicated. Changes nome size on cytological parameters, undergoing in genome size are probably the result of a complex interac- endopolyploidy “on demand” to increase cell size in certain tion of heritable factors…random factors…and adaptive fac- tissues such as defensive or secretory structures (Perdix- tors…”. Nonetheless, the view that DNA content exerts Gillot 1979; Beaton and Hebert 1997). The negative associa- causative “nucleotypic” effects on cellular and related tion between cell size and division rates clearly subjects the organismal characteristics explains commensurate changes former to strong selection, particularly during development in cell size after both increases and decreases in genome and reproduction, when mitotic and meiotic rates are para- size. As such, the nucleotypic theory best addresses ob- mount. This relationship is best established in the amphibi- served relationships between nucleus, cell, and genome sizes ans, where a large volume of literature outlines (Gregory 2001a). However, this theory requires that cell size developmental correlates of cell (and genome) size and genome size be associated in a causative manner and (Chipman et al. 2001 and references therein), including neg- that cell size itself be of adaptive significance. ative associations with developmental rate (Camper et al. 1993) and complexity (Roth et al. 1997). Negative develop- DNA content and cell size: cellular phenotypes mental and growth rate correlates of cell size have also been A positive relationship between cell and genome size has identified in both protozoan and eukaryotic unicells (Shuter been identified in every group of organisms where it has et al. 1983), in plants (Van’t Hof and Sparrow 1963), and in been examined (Cavalier-Smith 1985a)exceptinfishes. invertebrates (Bier and Müller 1969), as well as in some ver- Boveri’s classic experiments first demonstrated this in ur- tebrate groups (Cavalier-Smith 1985b). Cavalier-Smith first chins, where manipulations of chromosome number gener- implicated r-selection (organisms adapted for ephemeral en- ated changes in cell size (Mirsky and Ris 1951). Importantly,