Valenzuela Lab

Dr. Nicole Valenzuela Department of Ecology, Evolution, and Organismal Biology

Iowa State University

251 Bessey Hall. Ames, IA 50011

(515) 294-1285



Sex Determination Why are there so many ways to establish the sexual fate of the developing embryo?

It is fascinating that sexually-reproducing organisms employ such diversity of mechanisms to produce males and females, ranging from systems under strict genetic control (GSD) [such as highly dimorphic or undifferentiated sex chromosomes (XY, ZW)], to genetic systems susceptible to some environmental influences [such as haplo-dyploidy, polygenic systems, socially-induced sex reversals], to systems under strict environmental control dependent on biotic or abiotic factors. Among vertebrates, an environmental system dependent on temperature (TSD) is commonly found in reptiles and fish.

To explain this diversity we investigate the mechanics, evolutionary dynamics and ecological context of sex determination through a series of complementary projects.

Research on sex determination has important implications for our understanding of multiple traits and phenomena related to sexual reproduction, such as Sex Allocation and Sex Ratio Evolution, Sexual Dimorphism and Sex-linked Traits.

Comparative Ecological and Functional Genomics How do the developmental networks that control sexual development function and evolve?

This work is funded in part by NSF IOS 0743284.

What renders TSD thermosensitive?

We use both candidate gene and global approaches address the crucial unanswered question of what molecular factor(s) renders TSD mechanisms thermosensitive. Potential candidates would be genes that express differentially by temperature prior to the onset of the thermosensitive period or TSP (genes organizing or activating the thermosensitive time window rather than those genes acting once the window has opened). By profiling gene expression early in development we have detected such differential expression in TSD turtles in two early-acting genes (Sf1 and Wt1) involved in the formation of the bipotential gonad (prior to the gonadal commitment to the ovarian or testicular differentiation pathways) (Valenzuela et al. 2006, Valenzuela 2008). Because Wt1 is a known regulator of Sf1, Wt1 is proposed as a more likely candidate TSD master switch. Our more recent data replicated these results and implicate Wt1 and Sf1 in the early activation of the thermosensitive period in a temperature-specific manner (Valenzuela et al. 2013).

The flavors of temperature-dependent sex determination

Our data on six critical sex differentiation genes (Aromatase, Sf1, Wt1, Dmrt1, Sox9, and Dax1) in painted turtles (Chrysemys picta: TSD) and softshell turtles (Apalone mutica: GSD) indicated that multiple molecular pathways that differ in the regulation of common genes have evolved to produce ecologically-equivalent TSD systems, dispelling the assumption that TSD refers to a single trait (in contrast to GSD which encompasses distinct mechanisms such as XX/XY, ZZ/ZW, among others (Valenzuela et al. 2006; Valenzuela and Shikano 2007, Valenzuela 2008b, 2010a). Further, we have shown that the fundamental difference that sets TSD and GSD apart is not driven by Aromatase as previously proposed (Valenzuela and Shikano 2007). Still unknown is how many distinct systems of sexual polyphenism, i.e. TSD, exist in nature, how do they work, and why did they come into being? More recent work reveals that substancial transcriptional evolution has accrued across all vertebrates and is not unique to turtles (Valenzuela et al. 2013)

Evolutionary transcriptomics of gonadal development

We are using a comparative transcriptomic approach for gene discovery to uncover the full composition and the regulation of the gene network underlying gonadal development at a genome-wide scale across TSD and GSD taxa and in an ecological context similar to that used for the candidate gene approach. This illuminates how this network responds to environmental perturbations at different time scales and what might be its evolutionary potential in the face of contemporary climate change.

Is gene expression totally thermoinsensitive in GSD taxa?

An additional evolutionary question is whether GSD species derived from TSD ancestors have lost all thermal sensitivity of the gonadal devevelopmental network. Data from A. mutica indicate that this is not always the case, as this GSD turtle has retained its ancestral sensitivity in the expression of Wt1, the first such case ever to be reported (Valenzuela 2008). This result is paramount as it reveals that GSD taxa can harbor thermal sensitivity even when it is non-functional for sexual differentiation (temperature does not bias sex ratios in A. mutica). Such finding is critical because theoretical models for the evolution of TSD rely on the premise that GSD taxa posses an ubiquitous thermal sensitivity that can be co-opted during the evolution of phenotypic plasticity (TSD), and our data provide the first empirical evidence for its existence at the level of gene expression.

How are TSD males and females produced in nature, and how will TSD respond to climate change?

Through an NSF-funded project we are elucidating the effect of fluctuating temperature on gene expression underlying gonadogenesis in turtles, and testing the ecological relevance of our observations made under constant temperature. This approach is essential because most molecular studies of TSD have been carried out at constant temperatures, yet sex ratios produced under constant conditions often differ from sex ratios obtained in the field where temperature fluctuates daily (e.g. Valenzuela et al. 1997, Valenzuela 2001a). Thus, the relationship between fluctuating temperatures in natural nests and offspring sex ratio remains obscure.

The Ecological Context of Evolution

Because we are interested in the comparative evolutionary genomics in an ecologically-relevant context, some of the research in my lab investigates questions in population and ecological genetics, life history evolution and conservation biology following previous research (Lance et al. 1992, Valenzuela et al. 1997, Valenzuela 2000, Valenzuela 2001a,b,c, Valenzuela & Janzen 2001, Morjan & Valenzuela 2001, Valenzuela et al. 2003, 2004, Pearse et al. 2006). This component, which addresses basic questions in evolutionary ecology, provides a critical view of the ecological framework in which sex determining mechanisms evolve and their evolutionary potential in the face of climate change.

Population and ecological genetics of Neotropical turtles

We conducted a metapopulation genetic study of an endangered freshwater turtle (Podocnemis unifilis) inhabiting the Amazon and Orinoco river basins (Escalona et al. 2009). This study parallels a previous collaboration done on a sister taxon (P. expansa) (Pearse et al. 2006). This project was funded in part by the NSF DBI 0511958, and the Scott Neotropical Fund from the Cleveland Zoo.

Life History Evolution, Herpetology, and Conservation

Another important component of our research is the characterization of reptilian life histories, theoretical and empirical TSD thermal ecology, mating systems, and reproductive behavior. In particular, we study South American river turtles of the genus Podocnemis, the North American painted turtle (Chrysemys picta), softshell turtle (Apalone spinifera), and snapping turtle (Chelydra serpentina).

A recent study in Podocnemis unifilis test multple hypotheses to explain female nesting behavior and finds support for a social facilitation model rather than an adaptive nest site selection model (Escalona et al. 2009).

Reptiles are a very good taxon for study because TSD and GSD co-occur in this group. We use turtles as a model system to study the evolution of sex determining mechanisms, and compare both TSD and GSD species from the tropics and the temperate zone.

Many TSD reptiles are endangered. An important contribution to conservation is providing managers with essential biological information to design, evaluate, and enhance effective management programs. Part of our research is devoted to addressing basic questions that have conservation implications, such as the assessment of population structure, migratory and mating behavior with the use of molecular markers. The understanding of how TSD works in nature and under semi-natural or laboratory conditions is also important for conservation practices.

Phylogenomics What are the genomic causes and consequences of the evolutionary transitions in sex determination?

Sex Chromosome Evolution

TSD appears to be the ancestral state in turtles, and GSD has evolved multiple times in different lineages (Valenzuela and Adams 2011, Sabath et al. 2016), yet little is known about the sex chromosome systems that evolved during the repeated transitions from TSD to GSD. Through a collaborative NSF-funded project (MCB 0815354 - S.V. Edwards CoPI, Harvard University), we tackle the evolution of sex chromosomes and of sex-linked genes, identifying additional sex chromosome systems in turtles and investigating the molecular evolution of the genes they contain.

During an early collaboration, we used comparative genome hybridization and discovered the existence of a cryptic XY sex chromosome system in a GSD turtle from Australia (Chelodina longicollis), which involved a pair of micro-chromosomes (Ezaz et al. 2006). This was the first such report for turtles. A follow up study in a closely related species (Emydura macquarii) revealed a similar cryptic XY system of macro-chromosomes (Martinez et al. 2008). More recent data on Apalone spinifera uncovered a ZW system in this species, and revealed an intriguing conservation in the sex chromosomes of Trionichid turtles compared to the morphological divergence seen in Chelid turtles (Badenhorst et al. 2013). Our work also permitted the identification of the youngest turtle sex chromosome system in Glyptemys insculpta (XX-XY) (Montiel et al. 2016).

Our more recent research avenues involved the study of dosage compensation in turtles and the epigenetics of sex determination.

Co-evolution of chromosome number and sex determination

We detected an startling association in turtles, where chromosome number evolves 20 times faster in phylogenetic branches of the turtle tree of life where there is an evolutionary transition in sex determining mechanism (from TSD to GSD or viceversa), and this transitions coincide with peaks or lows in global temperature over more than 200 years of turtle evolution, linking climate change as a potential ecological force fueling this process (Valenzuela and Adams 2011).

Thanks to another NSF grant (MCB 1244355), we combine molecular cytogenetics and transcriptomics approaches to better understand the functional nature of this association and the ultimate forces responsible for shaping this pattern. We are using multidirectional chromosome painting to reconstruct the ancestral turtle karyotype and to identify chromosomal breakpoints that accrued during genome evolution across turtle lineages, and the newly sequenced turtle genomes and transcriptomic resources we have developed to identify the link of genome rearrangements and sex determination.

Evolution of Dosage Compensation using turtles as a model system When sex chromosomes (e.g. XX/XY or ZZ/ZW) evolve in diploid species, genes are lost from the Y or W (or lose their function) as they degenerate over time due to their lower recombination. The inequality of gene copy number causes an imbalance in transcription which may produce sub-optimal or lethal phenotypes, unless a mechanism existed to re-establish a balance. Dosage compensation (DC) is a system to equalize the activity of X- or Z-linked genes to counterbalance the deleterious effects of differential gene copy number. The diversity of dosage compensation remains obscure as well as its causes and consequences. A new NSF grant (IOS 1555999), will help fund a study on the evolution ofturtle dosage compensation. Turtles are ideal to study dosage compensation because XX/XY and ZZ/ZW sex chromosomes have evolved independently multiple times, and there are increasing turtle genomic resources.


Phenotypic Plasticity How does the phenotype respond to environmental inputs?

Sex determination

Most of evolutionary biology relies on the assumption that genetic variation (i.e. differences in genomic composition) underlies the diversity of the phenotypes that are exposed to natural selection and allows its evolution. But a great proportion of the phenotypic variation we observe in nature derives from environmental sensitivity of the genome which influences its expression during development (regulatory differences). Discrete traits such as alternative morphs (e.g. threshold traits) may result from environmental modulation of the expression of major genes, rather than from the added expression of quantitative genes.

TSD represents a form of phenotypic plasticity (a thermal sexual polyphenism) where identical genomes can permanently differentiate into either sex depending on the environmental conditions. Our work on sex determination helps us understand the basis of phenotypic plasticity and the recurrent evolution of developmental canalization (GSD) in a group of closely related taxa.

The role of growth plasticity on sexual dimorphism

We found that sex-specific plasticity, the differential response of the genome of males and females to different environments, mediates the degree of sexual dimorphism in the snapping turtle (Chelydra serpentina), a species with male-larger sexual size dimorphism (Ceballos and Valenzuela 2011).

We are currently studying if the same is true in species with female-larger dimorphism and whether sex-specific plasticity plays a role in shaping macroevolutionary patterns such as Rensch's rule in turtles (Ceballos et al. 2012). For this, we investigated the plasticity of body growth in Podocnemis expansa, a species we have studied intensely from a sex determination and ecological genetics perspective (Valenzuela 2000, Valenzuela 2001a,b,c). his project was funded in part by the National Science Foundation DEB 0808047 and the Turtle Conservation Fund.

Development of Genomic Resources Painted turtle genome sequenced

Our lab participated in the collaborative project to sequence the painted turtle genome, as part of the Steering Committee for the project, developing transcriptomes of multiple turtle species, carrying out the classic and molecular cytogenetic analyses for its annotation and physical mapping, and conducting analyses to understand the evolution of functional traits such as sex determination.

Classic and molecular cytogenetic resources

We have developed cell lines for chromosome prepartions of a multitude of turtles as well as chromosome-specific paints which we are using for our phylogenomic research.

Developmental transcriptomes


We have developed embryonic series of transcriptomes for multiple TSD and GSD turtle species that we are employing for our functional and evolutionary investigations.

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