vol. 194, no. 6 the american naturalist december 2019 Larger Genomes Linked to Slower Development and Loss of Late-Developing Traits Molly C. Womack,1,* Marissa J. Metz,1,2 and Kim L. Hoke1 1. Department of Biology, Colorado State University, Fort Collins, Colorado 80523; 2. Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80521 Submitted September 25, 2018; Accepted June 3, 2019; Electronically published October 18, 2019 Online enhancements: appendix. Dryad data: https://doi.org/10.5061/dryad.k02pq01. abstract: Genome size varies widely among organisms and is known 2002a), and slower development rate (Goin et al. 1968; to affect vertebrate development, morphology, and physiology. In am- Bachmann 1972; Bennett 1977; Oeldorf et al. 1978; Horner phibians, genome size is hypothesized to contribute to loss of late- and Macgregor 1983; Camper et al. 1993; White and McLaren forming structures, although this hypothesis has mainly been discussed 2000; Chipman et al. 2001; Roth and Walkowiak 2015; Liedtke in salamanders. Here we estimated genome size for 22 anuran species et al. 2018). Within amphibians, larger genome size and the and combined this novel data set with existing genome size data for associated larger cell sizes (Horner and Macgregor 1983; an additional 234 anuran species to determine whether larger genome Roth et al. 1994; Gregory 2001; Mueller et al. 2008; Roth size is associated with loss of a late-forming anuran sensory structure, the tympanic middle ear. We established that genome size is negatively and Walkowiak 2015) have been hypothesized to affect the correlated with development rate across 90 anuran species and found development of late-forming anatomical structures via two that genome size evolution is correlated with evolutionary loss of the potential mechanisms. Larger cells take longer to proliferate middle ear bone (columella) among 241 species (224 eared and 17 ear- (Shuter et al. 1983; Vinogradov 1999), which is thought to less). We further tested whether the development of the tympanic middle contribute to slower embryonic development (Bachmann 1972; ear could be constrained by large cell sizes and small body sizes during Oeldorf et al. 1978; Horner and Macgregor 1983; Chipman key stages of tympanic middle ear development (metamorphosis). To- et al. 2001), larval development (Goin et al. 1968; Camper gether, our evidence suggests that larger genomes, slower development rate, and smaller body sizes at metamorphosis may contribute to the loss et al. 1993), and limb regeneration (Sessions and Larson 1987) of the anuran tympanic middle ear. We conclude that increases in an- in amphibians with larger genomes. This slowing of devel- uran genome size, although less drastic than those in salamanders, may opment rate (neoteny) can lead to paedomorphosis, a form affect development of late-forming traits. of heterochrony (change in developmental timing) in which organisms become sexually mature before all traits have reached Keywords: trait loss, evo-devo, heterochrony, paedomorphosis, tym- panic middle ear loss, earless. their adult phenotype (Wake 1966; Gould 1977; Alberch et al. 1979; McNamara 1986). However, it also possible that larger cell sizes associated with large genomes constrain develop- Introduction ment at small body sizes due to limits on cell number, as pro- posed in salamanders (Wake 1991; Hanken and Wake 1993). Genome size evolution alters development, morphology, and fi However, the relationship between larger genome size and physiology across vertebrates. Speci c consequences of larger evolutionary lability of late-forming traits has limited empir- genomes include larger cell size (Ohno 1970; Szarski 1970, 1983; ical support. Olmo and Morescalchi 1975, 1978; Horner and Macgregor Most work focused on the association between genome size 1983; Walker et al. 1991; Gregory 2000, 2001, 2002a; Hardie and trait loss has occurred in salamanders, which have an ex- and Herbert 2003; Mueller et al. 2008; Roth and Walkowiak treme range of genome sizes (from 10.1 pg in Gyrinophilus 2015), slower metabolic rate (Vinogradov 1995, 1997; Gregory porphyriticus to 120.6 pg in Necturus lewisi; Gregory 2018) that are sometimes accompanied by toe and lung loss (Wake * Corresponding author. Present address: Department of Vertebrate Zoology, 2009). However, frogs and toads (anurans) provide an excel- National Museum of Natural History, Smithsonian Institution, Washington, lent opportunity to test whether less extreme genome size DC 20560; email: [email protected]. evolution could affect the development of late-forming struc- ORCIDs: Womack, https://orcid.org/0000-0002-3346-021X; Hoke, https:// orcid.org/0000-0001-5553-0138. tures. Genome size varies in anurans (from 0.95 pg in Limno- Am. Nat. 2019. Vol. 194, pp. 854–864. q 2019 by The University of Chicago. dynastes ornatus to 19 pg in Arenophryne rotunda;Gregory 0003-0147/2019/19406-58760$15.00. All rights reserved. 2002b) and is positively associated with length of embryonic DOI: 10.1086/705897 (Bachmann 1972; Oeldorf et al. 1978; Horner and Macgregor Larger Genomes Linked to Middle Ear Loss 855 1983; Chipman et al. 2001) and larval (Goin et al. 1968; middle ear development at sparse ontogenetic time points Camper et al. 1993) development in the limited taxa studied among nine anuran species and attributed middle ear varia- to date. Furthermore, Roth et al. (1994) and Roth and Wal- tion to truncated development (progenesis). Smirnov (1991) kowiak (2015) found that anurans (and salamanders) with compared tympanic middle ear development in 10 anuran larger genome size had lower complexity of late-forming brain species and argued that three different paedomorphic mech- structures. Thus, genome size variation in anurans may alter anisms could explain middle ear variation in three families morphology, although the prevalence of such morphological (Microhylidae, progenesis; Bombina, neoteny; Bufonidae, changes and the underlying developmental mechanisms re- postdisplacement) based on lineage-specific differences in main unclear. developmental timing of cranial structures. However, none One late-forming structure that may be affected by genome of these studies explicitly tested for differences in develop- size evolution in anurans is the tympanic middle ear. The tym- ment rate among species to determine whether slower devel- panic middle ear facilitates detection of airborne sounds and opment rate (neoteny) could explain middle ear loss. One is a key tetrapod adaptation for hearing on land (reviewed reason development rate has not been rigorously compared in Manley 2010). Surprisingly, within anurans, the tym- between species that vary in middle ear presence/absence is panic middle ear has been lost at least 38 times (Pereyra et al. a lack of development rate data for earless species. However, 2016) despite reduction of high-frequency (above 1 kHz) genome size, which is more easily measured and correlates hearing sensitivity (Lombard and Straughan 1974; Womack positively with length of embryonic (Horner and Macgregor et al. 2017) and lack of clear environmental selection pres- 1983) and larval (Goin et al. 1968) development in the few sures to explain tympanic middle ear loss (Jaslow et al. 1988). anurans species studied, may provide indirect evidence that Furthermore, some frogs that are earless (completely lack slower development promotes middle ear loss. all tympanic middle ear structures) or have reduced tympanic A non-mutually-exclusive mechanism by which genome middle ears, such as Bombina bombina and B. orientalis, also size could affect lability of late-forming structures is cell size have large genomes (Roth and Walkowiak 2015). Therefore, to body size ratios that constrain the space available for struc- we hypothesize that the loss of peripheral auditory structures tures to form. Genome sizes are associated with larger cell in anurans may result from larger genome sizes, which either sizes in anurans (Olmo and Morescalchi 1978; Horner and result in paedomorphic loss of the tympanic middle ear or Macgregor 1983; Gregory 2001; Mueller et al. 2008; Roth result in larger cell sizes that impose space constraints on and Walkowiak 2015) as well as all other vertebrates (Ohno small-bodied anurans. 1970; Szarski 1970, 1983; Olmo and Morescalchi 1975, 1978; The tympanic middle ear forms quite late, making it vul- Walker et al. 1991; Gregory 2000, 2001, 2002a; Hardie and nerable to genome size expansion and associated shifts in de- Herbert 2003). Wake (1991) and Hanken and Wake (1993) velopment rate, such as paedomorphosis. Development of the proposed that large cell sizes and small body sizes prevent middle ear structures begins just prior to metamorphosis and formation of late-developing peripheral digits in salaman- proceeds proximally to distally (Hetherington 1987; Voro- ders. Furthermore, the large cells and small bodies of the sal- byeva and Smirnov 1987; Smirnov 1991; Womack et al. amander Thorius appear to generate a developmental trade- 2018b). Middle ear structures are lost in a distal to proximal off between the eyes and the brain, resulting in deformation fashion; hence, loss may reflect incomplete development and posterior placement of the brain (Hanken 1983; Wake (Vorobyeva and Smirnov 1987; Smirnov 1991; Pereyra et al. 1991). The anuran middle ear emerges just before metamor- 2016). Furthermore, the middle ear can take up to a year to phosis (Sedra and Michael 1959; Hetherington 1987; Voro- complete development (Sedra