Putative Independent Evolutionary Reversals from Genotypic To

Journal of Molecular Evolution (2018) 86:11–26 https://doi.org/10.1007/s00239-017-9820-x

ORIGINAL ARTICLE

Putative Independent Evolutionary Reversals from Genotypic to Temperature-Dependent Sex Determination are Associated with Accelerated Evolution of Sex-Determining Genes in Turtles

Robert Literman1 · Alexandria Burrett1 · Basanta Bista1 · Nicole Valenzuela1

Received: 12 May 2017 / Accepted: 18 November 2017 / Published online: 1 December 2017 © Springer Science+Business Media, LLC, part of Springer Nature 2017

Abstract The evolutionary lability of sex-determining mechanisms across the tree of life is well recognized, yet the extent of molecular changes that accompany these repeated transitions remain obscure. Most turtles retain the ancestral temperature-dependent sex determination (TSD) from which multiple transitions to genotypic sex determination (GSD) occurred independently, and two contrasting hypotheses posit the existence or absence of reversals back to TSD. Here we examined the molecular evolution of the coding regions of a set of gene regulators involved in gonadal development in turtles and several other vertebrates. We found slower molecular evolution in turtles and crocodilians compared to other vertebrates, but an acceleration in Trionychia turtles and at some phylogenetic branches demarcating major taxonomic diversification events. Of all gene classes examined, hormone signaling genes, and Srd5a1 in particular, evolve faster in many lineages and especially in turtles. Our data show that sex-linked genes do not follow a ubiquitous nor uniform pattern of molecular evolution. We then evaluated turtle nucleotide and protein evolution under two evolutionary hypotheses with or without GSD-to-TSD reversals, and found that when GSD-to-TSD reversals are considered, all transitional branches irrespective of direction, exhibit accelerated molecular evolution of nucleotide sequences, while GSD-to-TSD transitional branches also show acceleration in protein evolution. Significant changes in predicted secondary structure that may affect protein function were identified in three genes that exhibited hastened evolution in turtles compared to other vertebrates or in transitional versus non-transitional branches within turtles, rendering them candidates for a key role during SDM evolution in turtles.

Keywords Genotypic sex determination · Sex determination mechanisms · Substitution rate · Temperature-dependent sex determination · Turtles

Introduction differentiation of bipotential gonads into testes or ovaries, and (2) the environmental cues that individuals experience Among vertebrates, various sex-determining mechanisms throughout development, which may in some cases affect the (SDMs) direct the developmental fate of individuals towards deployment of those gene products. In many species, includ- the male or female condition (Beukeboom and Perrin 2014), ing all studied mammals, birds, snakes, and amphibians, the controlled primarily through the interaction of two major sex determination trigger is the individual’s genomic con- components: (1) the genes, whose products in a given devel- tent [genotypic sex determination (GSD)] (Valenzuela and opmental or genotypic background direct the morphological Lance 2004). Most commonly among vertebrates, these sex- specific genotypic differences are found in sex chromosomes that contain the sex-determining regions, which are present Electronic supplementary material The online version of this in one sex while absent in the other, or present in different article (https://doi.org/10.1007/s00239-017-9820-x) contains supplementary material, which is available to authorized users. doses between males and females. Sex chromosome systems often include male heterogamety (e.g., XX females, * Robert Literman XY males) and female heterogamety (e.g., ZZ males, ZW [email protected] females). In other species, including some fishes and squa- 1 Department of Ecology, Evolution and Organismal Biology, mates, most turtles, and all known crocodilians, no sex-lim- Iowa State University, Ames, IA 50011, USA ited genomic content exists and instead, sex is determined

Vol.:(0123456789)1 3 12 Journal of Molecular Evolution (2018) 86:11–26 by environmental cues experienced during development the molecular activity of a gene product including changes (environmental sex determination), the most common cue in enzymatic rates, co-factor binding identity, and efficiency, of which is temperature [temperature-dependent sex deter- as well as temperature’s effect on 3D protein conformation mination (TSD)] (Ashman et al. 2014; Valenzuela and (Bond et al. 1998; Kumar et al. 2000; Marnell et al. 2003). Lance 2004). Components of both the genotype and the SDM evolutionary transitions occurred in fishes, geckos environment may work within some taxa with intermediate and agamid lizards, and turtles (Sabath et al. 2016; Sarre mechanisms between the GSD and TSD extremes (Sarre et al. 2011; Valenzuela and Lance 2004). Molecularly, TSD- et al. 2004; Valenzuela et al. 2003), such as taxa with GSD to-GSD transitions that involved sex chromosome evolution susceptible to environmental influence (Lagomarsino and would be characterized by the appearance (or translocation) Conover 1993; Quinn et al. 2007; Shine et al. 2002); while in of a master sex-determining gene onto a proto-sex chromo- other taxa, populations may possess contrasting GSD SDMs some, whose overall effect on the sex determination network (Uno et al. 2008). would outweigh the ancestral effect of incubation tempera- Sex determination is fundamental to the life history of ture, as described in many taxa. Conversely, transitions from any species. Yet, the extent to which evolutionary changes GSD-to-TSD would involve either (1) molecular changes in the master sex-determining factor(s) trigger concerted that lead to temperature sensitivity on gene expression or on changes in other elements of the sex determination gene net- the activity of gene products at key points in this network work to maintain proper development of males and females or (2) the cooption of a novel thermosensitive element or remains an open question. Thus, understanding the molecu- pathway into the sex determination network. Fewer transi- lar evolution of core gene regulators of sexual development tions from a GSD ancestor towards TSD are documented across vertebrates could help uncover associations between in vertebrates (Pokorna and Kratochvíl 2009; Sabath et al. SDM transitions and changes in the underlying molecular 2016), perhaps because sex chromosomes are an ‘evolution- machinery that may be candidate key contributors to the ary trap’ (Pokorna and Kratochvíl 2009) given that during evolution of sex determination itself. the transition to TSD, YY, or WW individuals would be Most vertebrates (GSD or TSD) share a core suite of produced that may be suboptimal or lethal. Alternatively, genes which are integral for the development of the sexual GSD-to-TSD transitions are prevented perhaps because more phenotype (Beukeboom and Perrin 2014; Valenzuela and challenging changes may be required to de-differentiate sex Lance 2004). Individual genes can be classified as male-pro- chromosomes back into autosomes, a rare phenomenon that moting or female-promoting based on their role on the devel- can also have high fitness cost (Vicoso and Bachtrog2013 ). oping gonads and whether these roles are fairly conserved Finally, certain life history traits such as longevity may ren- among species (e.g., Aromatase [Cyp19a1], Dmrt1, Sox9 der TSD-to-GSD transitions more likely in some lineages genes) or not (e.g., Sf1 [Nr5a1]) (Beukeboom and Perrin as shorter life-spans accentuate the negative effects induced 2014; da Silva et al. 1996; Raymond et al. 2000; Valenzuela by TSD on population dynamics, such as sex ratio skews et al. 2013). Indeed, the relative functions and deployment (Sabath et al. 2016). of these genes have evolved among vertebrate lineages, with A definitive explanation for the evolutionary lability in different genes attaining the top-most position within the sex SDM remains elusive. Turtles represent a model clade to determination network in different systems (Beukeboom and study SDM evolution as they possess TSD and GSD. TSD Perrin 2014; Valenzuela and Lance 2004). Network changes has been reconstructed as the ancestral state for turtles, from include shifts in the timing of gene activation (Valenzuela which multiple independent transitions to GSD appeared to et al. 2013) or genes shifting positions in the regulatory have occurred, as well as reversals back to TSD (Sabath pathways between roles as master regulators or downstream et al. 2016; Valenzuela and Adams 2011; Valenzuela and actors (Cutting et al. 2013). Alternatively, temperature may Lance 2004). Interestingly, transitions in turtle SDM are become the key regulator of one or more genes at a crucial accompanied by drastic cytogenetic reshuffling, where line- developmental period [thermosensitive period (TSP)], tip- ages that experienced a SDM transition exhibit a ~ 20-fold ping the balance towards a male or female fate in TSD taxa. higher rate of chromosome number evolution (Valenzuela As SDMs evolve among lineages and genes and proteins and Adams 2011). This raises the question of whether an shuffle their task within the sex determination network, increased rate of molecular evolution at the gene level parallel molecular changes could be expected to accrue in accompanies SDM transitions in turtles, as does a greater two major genomic regions. First, molecular changes in the rate of chromosome evolution in this group (Valenzuela and cis- and trans- regulatory regions, which define the expres- Adams 2011). Addressing this and related questions is facili- sion landscape for a given gene, can lead to changes in the tated by the growing number of turtle genomic resources timing and conditions under which a gene may be expressed including a BAC library (Badenhorst et al. 2015; Janes et al. or silenced. Second, nucleotide changes in the functional 2008), candidate-gene expression analyses (Barske and domains of protein coding sequences themselves may alter Capel 2010; Maldonado et al. 2002; Valenzuela et al. 2013),

1 3 Journal of Molecular Evolution (2018) 86:11–26 13 transcriptomics (Czerwinski et al. 2016; Radhakrishnan Methods et al. 2017b; Zhang et al. 2017), methylation profiling (Mat- sumoto et al. 2016; Radhakrishnan et al. 2017a; Venegas Sample and Data Collection et al. 2016), as well as sequenced genomes of both TSD and GSD species (Shaffer et al.2013 ; Wang et al. 2013). Out of the ~ 60 known genes in the vertebrate gonadal devel- Here we examine the molecular evolution of a subset of opment network (Mizoguchi and Valenzuela 2016) coding 15 genes in the vertebrate sex determination network (tran- DNA sequences (CDS) from 15 genes (Table 1) were col- scription factors, hormone signaling genes, WNT signaling lected for 25 amniote species from various public databases genes, and temperature-sensing genes), using turtles as a for which CDS data were available (Fig. 1; Table 2), com- focal group to test whether lineages which have undergone plemented with sequences for a number of turtle species SDM transitions (TSD-to-GSD or GSD-to-TSD) are char- we obtained during parallel studies (Literman et al. 2017; acterized by higher rates of nucleotide or amino acid sub- Radhakrishnan et al. 2017b; Shaffer et al. 2013), includ- stitution in the target sex determination genes compared to ing both RNA-Seq and DNA-Seq data (Table 2). These turtle and other major vertebrate lineages where no SDM amniotes were chosen because of the availability of well- transition occurred. This comparative approach permits annotated sequences of the genes of interest which enabled us to investigate turtle evolutionary rates in a broader phy- comparison to turtles (our focal clade of interest). Samples logenetic context among major amniote clades to illumi- of Apalone spinifera, Chrysemys picta, Staurotypus tripor- nate the molecular underpinnings of SDM evolution. We catus, Glyptemys insculpta, and Carettochelys insculpta then examine changes in the predicted protein secondary derive from privately owned, pet trade or wild individuals structure within functional domains, which may alter pro- as described elsewhere (Literman et al. 2017; Montiel et al. tein activity for genes that exhibited exceptional evolution 2017; Radhakrishnan et al. 2017b). Emydura macquarii in turtles. The scarcity of comparable upstream sequences tissues were collected in the wild as part of other studies across the focal taxa precluded conducting the same analyses conducted at the University of Canberra (Australia) under on regulatory regions and thus, we focus on protein cod- appropriate permits. Podocnemis expansa samples were ing sequences whose evolution could profoundly affect the collected by FUDECI (Venezuela) from the wild as part of molecular architecture of gonadal development during SDM another study approved by local authorities, and imported transitions. under CITES permits. All procedures were approved by the IACUC of Iowa State University, University of Northern Iowa, and University of Canberra. Data were available for all genes in all taxa with the exception of the Rspo1 gene, which could not be found for the snake Ophiophagus hannah in any available datasets. In order to improve the quality of the sequence data and to

Table 1 Target genes with Gene symbol Gene name Gene class varied roles in vertebrate sex determination examined in this Ar Androgen receptor Hormone signaling study Cirbp Cold-inducible RNA-binding protein Temperature signaling Ctnnb11 Beta-catenin WNT signaling Cyp19a1 (Aromatase) Aromatase Hormone signaling Dmrt1 Doublesex and Mab-3-related transcription factor 1 Transcription factor Esr1 Estrogen receptor 1 Hormone signaling Esr2 Estrogen receptor 2 Hormone signaling Hsf2 Heat-shock factor 2 Temperature signaling Lhx9 LIM homeobox 9 Transcription factor Nr5a1 (Sf1) Steroidogenic factor 1 Transcription factor Rspo1 R-spondin 1 WNT signaling Sox9 SRY box 9 Transcription factor Srd5a1 Steroid 5-alpha reductase 1 Hormone signaling Wnt4 Wingless-type MMTV integration site family, member 4 WNT signaling Wt1 Wilms tumor 1 Transcription factor

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Fig. 1 Phylogenetic relationships of 25 vertebrate species analyzed in this study. Branch lengths are scaled to Time- Tree consensus divergence times. Branch thicknesses and darkness are scaled to average nucleotide substitution rates across all genes. SDMs are denoted in brackets. Specific branches discussed in the text are numbered (1 = Placental mammal root branch; 2 = Igua- nia lizard root branch; 3 = Rep- tile root branch; 4 = Trionychia root branch; 5 = Neoaves root branch). Temp. = temperature sex reversal or TSD reports. (Color figure online)

Table 2 Species used in this Species Taxonomic group NCBI genome ID and other data sources study along with data sources Gallus gallus Bird Gallus_gallus-4.0 Falco peregrinus Bird F_peregrinus_v1.0 + SRA Taeniopygia guttata Bird Taeniopygia_guttata-3.2.4 + SRA Alligator mississippiensis Crocodilian AllMis0.2 + SRA Alligator sinensis Crocodilian ASM45574v1 + SRA Crocodylus porosus Crocodilian CroPor_comp1 + SRA Gavialis gangeticus Crocodilian GavGan_comp1 Monodelphis domestica Mammal (Marsupial) MonDom5 + SRA Homo sapiens Mammal (Placental) GRCh38 Mus musculus Mammal (Placental) GRCm38.p2 Anolis carolinensis Squamate (Lizard) AnoCar2.0 + SRA + TSA + Genbank Gekko japonicus Squamate (Lizard) Gekko_japonicus_V1.1 Pogona vitticeps Squamate (Lizard) SRA Ophiophagus hannah Squamate (Snake) OphHan1.0 + SRA Python molurus Squamate (Snake) Python_molurus_bivittatus-5.0.2 Chelonia mydas Turtle (Americhelydia) CheMyd_1.0 + SRA Staurotypus triporcatus Turtle (Americhelydia) DNA-NGS Data Chrysemys picta Turtle (Emydidae) Chrysemys_picta_bellii-3.0.1 + RNA-Seq Data Glyptemys insculpta Turtle (Emydidae) DNA-NGS Data Trachemys scripta Turtle (Emydidae) SRA + Genbank mRNA Emydura macquarii Turtle (Pleurodira) DNA-NGS Data Podocnemis expansa Turtle (Pleurodira) RNA-Seq Data + SRA Apalone spinifera Turtle (Trionychia) RNA-Seq + DNA-NGS Data Carettochelys insculpta Turtle (Trionychia) DNA-NGS Data + Sanger Sequencing Pelodiscus sinensis Turtle (Trionychia) PelSin_1.0 + SRA

DNA-NGS = inhouse generated next-generation DNA sequencing data (short read Illumina HiSeq data)

1 3 Journal of Molecular Evolution (2018) 86:11–26 15 ensure that similar isoforms were compared for each of these third-position substitutions are silent). Sites containing gaps genes, all coding sequences were manually extracted from were eliminated from the analysis. publicly available genomes (when present) rather than from In order to detect amino acid substitutions that could pre-existing gene annotations or other computer-based gene potentially alter protein function of a few genes identified as predictions. For species with additional publicly accessible undergoing exceptional molecular evolution in turtles in the sequence data, any existing data gaps in the target genes above analyses, the secondary structure of proteins was pre- were filled in via the NCBI Short Reach Archive (SRA) or dicted from the amino acid alignments using the EMBOSS through published mRNA sequences from the target taxa plugin as implemented in Geneious, and functional domains downloaded from Genbank. A dated pruned vertebrate phy- were annotated via the UniProt database. logeny of all species examined in this study was generated using the median time divergence estimates from the Time- Tree database, which are calculated from numerous pub- Data Analysis lished studies (Hedges et al. 2006) (Fig. 1). Parallel analyses were performed for the nucleotide and the Data Processing amino acid alignments. Due to unequal variances in rates among groups, non-parametric statistical tests (described For each gene, two datasets were generated: one dataset below) were performed to compare the differences among included sequence alignments from all 25 species (‘All Spe- groups. For both the ‘All Species’ and ‘Turtle’ datasets, cies’), and a second dataset included alignments from the ten pairwise Steel–Dwass tests were performed to test whether turtle taxa exclusively (‘Turtles’). In all cases, nucleotide substitution rate changes could be explained by phyloge- sequences were translationally aligned using MUSCLE with netic history, and to test whether gene classes differed in default parameters as implemented in Geneious v.9 (Kearse their substitution rates within taxonomic groups. These tests et al. 2012). In order to minimize the impact of taxa-specific were implemented using JMP, Version 12.0.1 (SAS Institute indels, all alignments were visually assessed to ensure that Inc., Cary, NC, 2015). Significance of the results from the homologous regions were aligned, and misalignments were Steel–Dwass test is reported at a Bonferroni-corrected alpha corrected manually. The lengths and sequence identity of the to account for multiple comparisons. final alignments for the ‘All Species’ and ‘Turtles’ datasets For each gene, in order to identify outlier phylogenetic after gaps were removed differ from each other (Table S1). branches experiencing faster substitution rates relative to all Alignments were generated for both nucleotide and amino other branches (‘Fast Branches’), a branch-specific Z score acid sequences. was calculated per gene as For each alignment, maximum likelihood was applied SSM(Rate of branch) − SSM(Rate mean for gene) to determine the most likely model of substitution using Z = . jModelTest v.2.1.4 (Darriba et al. 2012) for nucleotide align- SSM (Rate STDEV for gene) ments and ProtTest v.2.4 (Abascal et al. 2005) for amino acid alignments. Alignments were imported into MEGA Within each taxonomic group, a separate Z-score analysis v.7 (Kumar et al. 2016) and using the estimated model was carried out to identify outlier genes experiencing higher parameters, the number of substitutions per site along each substitution rates relative to all genes for that group (‘Fast branch was estimated using maximum likelihood against a Genes’), where a gene-specific Z score was calculated as well-supported species phylogeny (Fig. 1). Substitutions SSM(Rate mean for gene) − SSM (Rate mean for all genes) Z = . per site per million years (SSM) for each branch were then SSM (Rate STDEV for all genes) calculated by dividing the number of substitutions per site (generated from MEGA) by the divergence time estimates For the ‘Turtle’ dataset, Wilcoxon/Mann–Whitney U from TimeTree (Ho et al. 2005; Smith and Donoghue 2008) tests (2-way comparisons) and Steel–Dwass Tests (3-way to obtain a rate of molecular evolution that was used in all comparisons) were performed to investigate whether phylo- downstream statistical tests. This time-corrected approach genetic branches characterized by a sex determination transi- accounts for the impact of long branches when comparing tion (TSD-to-GSD or GSD-to-TSD) differed in substitution the extent of changes among lineages with differing time rate relative to non-transitional branches or to each other of divergence, because an older lineage could accumulate (TSD-to-GSD versus GSD-to-TSD). Each branch on the tur- more changes even if the rate of substitutions is identical tle phylogeny was scored as transitional or non-transitional to that of a younger lineage. To estimate neutral nucleotide based on two proposed hypotheses of the evolution of sex substitution rates for different taxonomic groups, the nucle- determination in turtles: (1) A hypothesis which reconstructs otide data were also analyzed using only the third codon five evolutionary transitions in turtles, all from the ancestral positions (note that this is an approximation since not all TSD condition to a derived GSD condition (Sabath et al.

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2016); and (2) a hypothesis that reconstructs five transitions were concordant with the findings of our first approach, from TSD-to-GSD (two of which overlap with the previ- below we detail only the CODEML results where differ- ous hypothesis) plus two reversals back to TSD from GSD ences were detected. in the lineages represented by C. insculpta and P. expansa (Valenzuela and Adams 2011) (Fig. 2). Considering both hypotheses, the focal taxa included in this study encom- pass representative species for four of the five TSD-to-GSD Results transitions predicted under each hypothesis. All comparisons, including Z-score analyses, Mann–Whitney U tests, Substitution Rates are Slowest in Turtles and Steel–Dwass tests were explored at α = 0.05 (threshold and Crocodilians and Fastest in Mammals Among Z > 1.644) to detect trends, but significance was assessed at Amniotes a Bonferroni-corrected α = 7.14E−3 (threshold Z > 2.45) for ‘All Species’ tests, and at α = 5.56E−3 (threshold Z > 2.54) For the ‘All Species’ dataset containing 25 amniotes, the for the ‘Turtles’ dataset. analysis across all 15 genes showed significant differences Although our main objective was to test whether transi- in nucleotide substitution rates at the third codon positions in SDM were accompanied by an acceleration in the tions among taxonomic groups indicative of differences evolution of the genes of interest, which requires the use in the neutral rates of evolution (Table 3, detailed sta- of an explicit time component as described above, we also tistical results in Table S2). Pairwise comparisons using analyzed the accumulation of substitutions in these DNA the Steel–Dwass test revealed a higher substitution rate sequences among taxonomic groups and SDM transition in mammals than in any other group (p < 0.002), while branches using CODEML as implemented in the program crocodilians and turtles exhibited a similar and lower rate ete3 (Huerta-Cepas et al. 2016), where divergence time than all other groups (Table 3). A similar observation was estimates are not incorporated explicitly. Using the same made when considering all codon positions, although in starting alignments, values for dN and dS, and dN/dS were this case turtle genes exhibited a significantly faster rate calculated for each branch of the tree and comparisons than crocodilians (p < 0.001) (Table 3, detailed statistical between groups were performed using Mann–Whitney U results in Table S3). At the amino acid level, mammals, tests for comparisons between two groups, or Steel–Dwass birds, and squamates formed a group with substitution tests for comparisons among more than two groups. Higher rates ~ 3× faster than turtles and crocodilians (p < 0.005) values of dN and dS are indicative of increases in non-syn- (Table 3, detailed statistical results in Table S4). Results onymous and synonymous substitution rates, respectively. from the CODEML analysis were concordant with the Complete methods for this analysis are described in the time-dependent analysis in terms of dS (silent) and dN Supplementary Material. Because results from CODEML (replacement) substitutions (Table S5).

Fig. 2 Phylogenetic relationships of ten turtle species analyzed in this study. Branch lengths, thicknesses, and darkness as in Fig. 1. Arrows indicate branches where a transition in sex determination mechanism was reconstructed to have occurred under two evolutionary hypotheses: (1) Open arrowheads above branches indicate transitions under Sabath et al. (2016) hypothesis where all transitions occur from TSD-to-GSD. (2) Closed arrowheads below branches indicate transitions under Valenzuela and Adams (2011) hypothesis, where C. insculpta and P. expansa lineages underwent reversals from GSD-to-TSD, denoted by asterisks

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Table 3 Overall substitution rates per million years of nucleotide and amino acid sequences across target genes in each taxonomic group, and relative rate (%) compared to turtles Average number of substitutions per site per Myr across all genes in “All Species” Third codon position All nucleotides Amino acids Substitutions per Myr % From turtles Substitutions per Myr % From turtles Substitutions per Myr % From turtles

Mammals 2.60E−03 [A] 387.5 1.02E−03 [A] 328.4 5.66E−04 [A] 241.9 Squamates 2.28E−03 [B] 326.7 8.79E−04 [B] 269.3 5.40E−04 [A] 227.3 Birds 1.73E−03 [B] 223.5 6.64E−04 [B] 179.3 3.76E−04 [A] 127.2 Turtles 5.34E−04 [C] – 2.38E−04 [C] – 1.65E−04 [B] – Crocodilians 4.37E−04 [C] − 18.0 1.67E−04 [D] − 29.6 9.03E−05 [B] − 45.4

For each comparison (third codon, all nucleotides, and amino acids), statistically indistinguishable rates share bracketed letters. Myr = million years

Among Turtles, Substitution Rates are Slowest than in Emydidae (Table 4, detailed statistical results in in the Emydidae Species Table S8). Emydidae also exhibited a lower dN and dS (Table S9). No other comparisons were significant. Turtle branches were also analyzed separately using the ‘Turtles’ dataset (N = 10), which are more complete align- Root Branches Tend to Experience Higher ments than the “All Species” dataset (turtle sequences were Substitution Rates in More Genes Than Other longer because they contained fewer gap positions that Branches needed to be removed), and because mammals, squamates, and birds exhibited significantly faster overall substitution For each gene alignment, certain branches on the phylo- rates relative to turtles which could obscure biologically genetic tree (Fig. 1) display significantly faster nucleotide important shifts among turtles. Clade-level comparisons substitution rates (“fast branches”) relative to the overall revealed that turtle sub-groups differed in third-position sub- average rate for that gene (Table S10). This was true for stitution rates, which were slower in Emydidae (Glyptemys, several root branches across many genes (9 out of 15 genes Chrysemys, plus Trachemys) than in other turtles, while no at the root of the Iguania lizards, 6 out of 10 genes at the root rate differences were detected between Trionychia (Apalone, of reptiles, and 7 out of 10 genes for the placental mammal Pelodiscus, plus Carettochelys), Americhelydia (Staurotypus root), whereas in turtles this was true for a smaller subset of plus Chelonia), or Pleurodiran turtles (Emydura plus Podoc- genes. For the ‘All Species’ dataset, 8 of 48 branches in the nemis) (Table 4, detailed statistical results in Table S6). phylogenetic tree exhibited a significantly faster nucleotide Overall nucleotide substitution rate mimicked the third- substitution rate relative to other branches for at least one position substitution rate differences qualitatively, which gene (Table S10). Despite the lower nucleotide substitution was slower overall in Emydidae than in Trionychia and rate observed in turtles as a whole, one turtle branch dis- Pleurodira (Table 4, detailed statistical results in Table S7). played a significantly faster nucleotide substitution rate for At the amino acid level, the rates in Trionychia were faster one gene each (Sox9 in the Trionychia root branch), while

Table 4 Overall substitution rates per million years of nucleotide and amino acid sequences for all genes examined in each focal turtle clade, and relative rate (%) compared to Emydidae Average number of substitutions per site per Myr for all genes in “Turtles” Third codon position All nucleotides Amino acids Substitutions per Myr % from Substitutions per Myr % from Substitutions per Myr % from Emydidae Emydidae Emydidae

Trionychia 9.37E−04 [A] 244.5 4.09E−04 [A] 219.5 3.14E−04 [A] 231.6 Pleurodira 5.34E−04 [A] 96.3 2.53E−04 [A] 97.7 1.70E−04 [AB] 79.5 Americhelydia 5.12E−04 [A] 88.2 2.38E−04 [AB] 85.9 2.39E−04 [AB] 152.4 Emydidae 2.72E−04 [B] – 1.28E−04 [B] – 9.47E−05 [B] –

For each comparison (third codon, all nucleotides, and amino acids), statistically indistinguishable rates share bracketed letters. Myr = million years

1 3 18 Journal of Molecular Evolution (2018) 86:11–26 the trend for Lhx9 in C. insculpta was not significant at the the ‘All Species’ dataset, amino acid sequences of hormone Bonferroni-adjusted alpha (Table S10). signaling genes tend to evolve faster than certain other gene Somewhat similar results were obtained at the protein classes in squamates, crocodilians, and turtles (Table S15). level, where 12 of 48 branches had at least a single gene For the ‘Turtles’ dataset, the amino acid sequences of hor- with a faster than average amino acid substitution rate mone signaling genes evolve faster than the WNT signaling (Table S11). This list was topped by the root of Iguania liz- genes in the Emydidae and Trionychia turtles (Table S17). ards (5 of 15 genes), placental mammals (5 of 15 genes), and When broken down to the level of individual genes, few the reptile root branch (4 of 15 genes). In turtles, additional genes stand out as experiencing faster than average substi- fast branches were detected at the amino acid level beyond tution rates, and this varied by dataset and sequence type. those detected at the nucleotide level. Indeed, four turtle For instance, no such gene was detected using the ‘All Spe- branches exhibited faster amino acid substitution rates for at cies’ nucleotide dataset (Table S18), and for the ‘Turtles’ least one gene: C. insculpta (Lhx9), and the Americhelydia dataset Srd5a1 shows a trend for faster evolution in the root branch (Wt1) (Table S11), while the tendency detected Americhelydia (p < 0.02, Table S19). At the amino acid in the Trionychia root branch (Esr1, Hsf2, Sox9) and the level, DMRT1 tends to evolve faster in birds (p < 0.01) and turtle root branch (Cirbp) is not significant after Bonfer- SRD5A1 in mammals (p < 0.05) (Table S18), whereas for roni correction. Using the turtle only dataset also revealed the ‘Turtles’ dataset, SRD5A1 evolved faster in the Amer- faster than average nucleotide substitution rates in 2 of 18 ichelydia and shows the same tendency in Trionychia branches relative to other turtle branches for at least one (p < 0.025, Table S19). However, other than SRD5A1 in the gene (Table S12). Indeed, 10 of 15 genes evolved at a faster Americhelydia, all other trends were not significant after than average rate in the Trionychia root branch, while this Bonferroni correction. was true only for a single gene in any other branch. At the protein level, 6 of 18 branches exhibited a faster amino acid GSD‑to‑TSD Reversal Hypothesis Helps Explain substitution rate for at least one gene (Table S13). The Tri- Molecular Evolution in Turtles onychia root branch showed this pattern for 5 of 15 genes. We examined sequence data for five turtle species with Genes on Z (But Not X) Sex Chromosomes Evolve TSD (P. expansa, C. insculpta, Chelonia mydas, C. picta, Faster at the Amino Acid Level and Trachemys scripta) and five turtles with GSD (E. macquarii, A. spinifera, Pelodiscus sinensis, S. triporcatus, G. Three target genes in this study, Ar, Dmrt1, and Ctnnb1, are insculpta) under two contrasting evolutionary hypotheses sex-linked (located in the X or Z chromosomes) in certain that posit contrasting evolutionary scenarios of turtle SDM focal taxa, and their protein sequences evolve faster when transitions (Fig. 2). they are Z-linked than on taxa where they are autosomal or First, no nucleotide or amino acid substitution rate dif- X-linked. For instance, Dmrt1 is Z-linked in birds (Nanda ferences were detected between transitional and non-tran- et al. 1999), and three of the five avian branches (Taeniopy- sitional branches (Prob > ChiSq > 0.4) under the hypothesis gia guttata, Falco peregrinus, and the Neoaves root branch) that turtles underwent exclusively transitions from TSD- have a significantly faster amino acid substitution rate than to-GSD (Sabath et al. 2016) (Fig. 3), whereas significantly all other branches (although interestingly, this faster rate is higher dN and dS rates were detected in transitional branches absent in chicken [Gallus gallus] or the bird root branch) relative to non-transitional branches using CODEML (~ 40 (Table S11). Second, Ctnnb1 is Z-linked in snakes (Vicoso and ~ 63% higher, respectively, Table S20). et al. 2013), and O. hannah snakes show a significantly Second, significantly higher nucleotide and amino acid faster amino acid substitution rate relative to all other spe- substitution rates were detected in transitional branches cies, including the other snake examined, Python molurus. In relative to non-transitional branches (Prob > ChiSq < 0.001) contrast, Ar is X-linked in mammals (Ross et al. 2005), yet under the hypothesis that C. insculpta and P. expansa rep- no mammalian branch exhibits faster amino acid substitution resent lineages where a GSD-to-TSD reversal occurred rates (Table S11). (Valenzuela and Adams 2011) (Fig. 3). Moreover, the GSD-to-TSD reversal branches exhibit significantly faster Hormone Signaling Genes Evolved Faster in Most nucleotide substitution rates relative to both TSD-to-GSD Vertebrates and Srd5a1 in Mammals and Some transitional branches (p = 0.0043) and non-transitional Turtles branches (p < 0.001) across all genes (Fig. 3). TSD-to-GSD nucleotide sequences tended to evolve faster than non- Most gene classes display similar rates of substitution at the transitional branches (p = 0.0057). Amino acid sequences nucleotide and amino acid levels across our target taxa with evolved faster in GSD-to-TSD reversal branches relative to some noticeable exceptions (Tables S14–S17). Namely, for non-transitional branches (p < 0.001), and tended to do so

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Fig. 3 Tempo of molecular evolution among lineages that experience and P. expansa lineages underwent reversals back to TSD from a sex determination stasis or turnovers under two competing evolution- GSD ancestor. Under this second hypothesis, proteins evolve faster ary hypotheses. Acceleration in nucleotide and amino acid substitu- in GSD-to-TSD branches than in both non-transitional and TSD- tion rates across all genes are not correlated with turtle SDM tran- to-GSD branches, while proteins on TSD-to-GSD branches do not sitions under Sabath et al. (2016) hypothesis, where all transitions evolve significantly faster than non-transitional branches. (*p < 0.05; occur from TSD-to-GSD, but are correlated at the nucleotide level **p < 0.005; N.S = not significant). (Color figure online) under Valenzuela and Adams (2011) hypothesis, where C. insculpta relative to TSD-to-GSD transitional branches albeit not sig- other species or, (2) tended to evolve faster in transitional nificantly (p = 0.0406). Notably, no significant differences versus non-transitional turtle branches, and subjected them in the amino acid substitution rate were detected across to secondary protein structure predictions in order to identify genes between non-transitional branches and TSD-to-GSD amino acid substitutions in transitional turtle branches that branches (p = 0.0862, Fig. 3), suggesting that the trend in may induce functional changes. Structural changes within the transition-dependent substitution rate at the protein UniProt functional domains were detected in the HSF2 pro- level is driven by the GSD-to-TSD reversal branches. This tein, which evolved faster in the Trionychia turtles relative pattern was also observed using CODEML (Table S21). to all other turtle clades, and displayed a similar tendency Namely, GSD-to-TSD reversal branches showed higher dN in turtle transitional branches relative to non-transitional and dS rates relative to non-transitional branches (> 500 and branches. Likewise, we detected structural changes within > 300%, respectively) as well as to TSD-to-GSD branches functional domains of the RSPO1 protein, which tended to (> 400 and > 250%, respectively), whereas TSD-to-GSD evolve faster in transitional branches relative to non-tran- transitional branches and non-transitional branches displayed sitional branches, and in the LHX9 protein, which evolved similar rates. Broken down by individual genes, nucleotide faster in C. insculpta relative to all other species (Fig. 4). sequences of Dmrt1, Hsf2, Sox9, and Wnt4 exhibited a non- Specifically, a series of amino acid substitutions that significant tendency to evolve faster in transitional branches change the secondary protein structure were identified in relative to non-transitional branches (p < 0.05), and the same turtle transitional branches in both the positive and negative was true for the amino acid sequences of HSF2, RSPO1, transactivation domains of the HSF2 protein, which form its and SOX9 (p < 0.05). Further transition type-specific com- C-terminal domain and contribute to transcriptional activa- parisons were precluded because there are only two GSD-to- tion of target genes (Yoshima et al. 1998). In the positive TSD branches and four TSD-to-GSD branches. transactivation domain 1, which binds co-regulators during cellular stress (Fig. 4a), P. expansa has a single substitu- Potentially Functional Evolutionary Changes tion predicted to cause the loss of two helices present in Accrued in Turtle Proteins all other species, while in a separate site, C. insculpta has a single substitution that is predicted to join two shorter We searched for target proteins in our dataset that either (1) helices found in all other species into a single, longer helix. evolve faster in a transitional turtle branch compared to all In the associated negative regulator domain, which keeps

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Fig. 4 Amino acid substitutions in functional domains of sex deter- under Valenzuela and Adams (2011). a, b Transactivation and nega- mination genes in turtle lineages experiencing shifts in sex determi- tive regulatory domains of HSF2. c LIM domains of LHX9. d FU and nation. Boxes highlight focal regions with substitutions. SDMs for TSP1 domains of RSPO1. Zigzags denote trimmed sequence between each species noted in brackets. TSD* = GSD-to-TSD reversal branch domains. aa Amino acids trimmed

HSF2 inactive during non-stress conditions, E. macquarii, substitutions are predicted to yield a unique secondary P. expansa, and C. insculpta share a unique predicted sec- structure among turtles with a highly truncated alpha ondary structure characterized by the addition of two short helix and the expansion of the beta sheet predicted in P. alpha helices and notably, these result from three different expansa. Further upstream substitutions in C. insculpta and independent mutations in each lineage. In the positive cause the shifting of a beta sheet relative to all other tur- transactivation domain 2 (Fig. 4b), S. triporcatus accrued tles. Additionally, the 5′ end of the upstream LIM domain several amino acid substitutions predicted to drastically in C. insculpta also contains a split helix, where in all change its secondary structure from that of other turtles by other turtles the helix is unbroken. inducing multiple alpha helices, while G. insculpta has a Finally, the thrombospondin-1 domain of the RSPO1 single substitution which also induces a novel helix among protein in two XX/XY turtle species (E. macquarii and S. turtles in this domain. In the corresponding negative regula- triporcatus) shares a secondary structure unique among tor domain, P. expansa, A. spinifera, and P. sinensis expe- turtles, lacking an alpha helix in the C-terminal end of the rienced a reduction or loss of alpha helices that exist in all domain. Notably, this pattern evolved through two independ- other turtle species. ent amino acid substitutions at separate sites in this domain The 3′ end of the downstream LIM domain of LHX9 in each lineage (Fig. 4d). S. triporcatus has also accumulated in turtles generally contains a 10-amino acid long alpha substitutions in the second of two upstream Furin-like repeat helix which is disrupted in P. expansa and C. insculpta, the domains which cause both the truncation of an alpha helix, representatives of the two putative GSD-to-TSD reversals which is longer in all other species, as well as the insertion in turtles (Fig. 4c). In P. expansa, an inserted short beta of a helix at the 3′ end of the domain, which is unique to S. strand splits the helix, while in C. insculpta additional triporcatus.

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Discussion a combination of their slower metabolic rates and longer generation times relative to endothermic mammals, birds, Numerous independent transitions between GSD and TSD and to most squamates (Green et al. 2014; Shaffer et al. have occurred throughout vertebrate evolution (Beuke- 2013). Whether population sizes of turtles and crocodil- boom and Perrin 2014; Valenzuela and Lance 2004), yet, ians might also affect their molecular evolution alone or in the gene network that regulates sex determination has combination with other life history traits (Bromham 2002) remained remarkably similar in its composition, whereas remains unknown. the roles of this shared set of genes have shuffled as SDMs Despite the relatively low rates of molecular evolution evolve (Cutting et al. 2013; Valenzuela et al. 2013). Here in turtles, significant differences in evolutionary rates were we advance our understanding of other changes that have detected among turtle lineages. For instance, both the nucle- accompanied SDM transitions, as we detected substantial otide and amino acid substitution rates of the target genes molecular evolution among major vertebrate clades in the in Emydidae (Fig. 2; Table 2) were slower than in other coding sequence of a subset of 15 genes involved in sex turtle lineages (85–244% depending on the dataset used, determination, for which adequate CDS data were avail- Table 4). Furthermore, the branch leading to the Trionychia able across the focal taxa (25 selected amniotes). Among turtles (softshell plus pig-nose turtles) (Fig. 2; Table 2) stood those, we highlight in particular the changes identified in out by its faster nucleotide and amino acid substitution rate turtle lineages which have experienced SDM transitions, relative to the turtle average for most genes (Tables S10 including predicted structural changes of potential func- and S11), revealing that a major acceleration in molecular tional importance. Results using dN and dS analysis were evolution accompanied the divergence of this morphologi- mostly concordant with our time-explicit approach and cally intriguing lineage. However, whether this hastened thus we discuss CODEML results only for cases were dif- molecular evolution is functionally linked to SDM transi- ferences were observed. Analysis of the upstream regula- tions and restricted to genes in the sex-determining network, tory regions associated with gene activation and silencing or whether it is a more generalized phenomenon associated of these genes was precluded due to lack of data in the with other ecological, genomic, or morphological innova- low-coverage genomes available for non-model taxa. We tions in this clade remains to be tested. The higher neutral note also that data scarcity prevented a broader taxonomic rate of evolution in Trionychia compared to other turtles sampling such that our conclusions should be interpreted supports the notion that this lineage follows a unique evo- as working hypotheses to guide future research facilitated lutionary trajectory. Notably, members of this turtle family by growing genomic information of non-model organisms. underwent a cytogenetic revolution, as they contain the spe- Additionally, future genome-wide analyses are warranted cies with the highest numbers of chromosomes among tur- to test whether the patterns detected here with a limited tles (A. spinifera/P. sinensis: 2n = 66; C. insculpta: 2n = 68) set of genes related to sexual development might be more (Badenhorst et al. 2013; Bickham and Legler 1983; Sato general and present in other regulatory networks. and Ota 2001). It is tempting to speculate that perhaps the same drivers might be responsible for the acceleration of molecular evolution and chromosomal fissions observed in Genes in the Sex Determination Network Evolve this group (Montiel et al. 2016). Slowly in Turtles and Crocodilians But Show an Acceleration in Trionychia Turtles Faster Branches Are Mostly Root Branches

Among the five major vertebrate clades examined, the tar- Major clade root branches, such as the branches leading to get genes evolve at a significantly lower rate in the turtles the placental mammals, Iguania lizards, and the root of all and crocodilians, a pattern observed at both the nucleotide reptiles had the most genes with a faster than average sub- and amino acid levels and accentuated in crocodilians rela- stitution rate for both nucleotides and amino acids (Tables tive to turtles when considering all nucleotides (Table 3). S8 and S9), suggesting that the sex determination network These observations agree with recent genome-wide reports experienced major molecular changes during significant for turtles, Anolis lizards, and crocodilians (Fujita et al. taxonomic diversification events. Although appealing, fur- 2011; Green et al. 2014; Shaffer et al.2013 ), but coun- ther research is warranted to test the biological relevance ter the expectation that the evolutionary lability of SDMs of this hypothesis under a more extensive taxonomic sam- in turtles, when compared to mammals and birds, might pling than is currently possible, as genome-level data for be associated with higher molecular evolution overall. additional species become available. Our results on the evo- The lower genome-wide substitution rates of crocodil- lutionary rate of sex-determining genes among model and ians and turtles have been hypothesized to derive from non-model vertebrates complement our knowledge of the evolution of sex chromosome content and gene synteny that

1 3 22 Journal of Molecular Evolution (2018) 86:11–26 is well documented among diverse vertebrate clades (Ezaz proposed, such as GC content and expression levels that may et al. 2017; Graves 2017; Montiel et al. 2016; Rovatsos explain the difference in the rate of molecular evolution in et al. 2016) by illuminating the evolution of protein coding mammalian sex-linked genes (Nguyen et al. 2015). sequences that accompanies shifts in SDM. Fast Genes: the Hormone Signaling Gene Srd5a1 Protein Sequences Evolve Faster on Sex Evolves Faster in More Lineages Than all Other Chromosomes, But Not Always Genes

At first glance, sex-linkage effects were observed at the Hormone signaling genes were the only gene class exhibit- amino acid level for a number of species. For example, the ing a consistent tendency for faster evolution at the nucleo- DMRT1 protein evolved faster in birds where this gene is tide or amino acid levels (Tables S14–S17). On a gene by Z-linked (Nanda et al. 1999), in particular in the branches gene level, Srd5a1 exhibited a trend towards a faster nucleo- of the zebra finch T.( guttata), falcon (F. peregrinus), and tide substitution rate in the Americhelydia turtles (Fig. 2; their root branch (Neoaves) (Table S10). Interestingly how- Table 2, Table S18) as well as a faster amino acid substitu- ever, the root branch for all birds and the branch leading to tion rate (a trend also observed in mammals and Trionychia chicken (G. gallus) have substitution rates more comparable turtles, Tables S17 and S18) relative to all other genes for to their autosomal homologs in the other taxa examined, sug- those groups. Srd5a1 is also the only gene that exhibits gesting that DMRT1 evolution has been significantly accel- greater dN/dS ratio in all taxa compared to all other genes erated in the Neoaves relative to the more basal birds, and (results not shown) indicating that it is under relaxed purify- that some driver specific to Neoaves, other than the evolution ing or positive selection. Srd5a1 encodes a 5-alpha reductase of avian sex determination, might be causal. Second, beta- protein which converts weaker androgens to more potent catenin (CTNNB1) is a highly conserved signaling protein, androgen compounds (Chang et al. 2011), a critical step in exhibiting the lowest average amino acid substitution rate sexual differentiation towards a male fate (Urbatzka et al. of all target genes such that no substitutions were detected 2007). Deficiencies in human 5-alpha reductase activity are in 33 of 48 branches. Moreover, Ctnnb1 is the only gene linked to partial or complete sex reversal of genotypic males that exhibited a lower dN/dS ratio in all taxa compared to (XY) at birth (exhibiting intersex or even female phenotype all other genes (results not shown) indicating that it is under due to low levels of potent androgens) that is reverted par- strong purifying selection. However, Ctnnb1 is Z-linked in tially at puberty when appropriate hormones are synthesized snakes (Vicoso et al. 2013) and its protein sequence evolved (Wilson et al. 1993). We hypothesize that the increased rate faster in the king cobra (O. hannah) (Table S10) than in the of molecular evolution of Srd5a1 observed here may reflect Indian python P. molurus, which displayed only the substitu- more profound changes in the hormone signaling pathways tions accumulated during the diversification of the Serpentes or how androgens are utilized during sexual development as a group. These results in birds and snakes suggest that among taxa, but this hypothesis requires further testing. the differential molecular evolution is not uniform for sex- linked genes, but that important lineage-specific effects are Contrasting Hypotheses of the Evolution of Turtle also at play. Furthermore, the other sex-linked protein in the Sex Determination Affect the Interpretation analysis, the mammalian X-linked AR (Ross et al. 2005), did of Transition‑Dependent Shifts in Substitution Rates not show evidence of ‘Fast X’-type evolution (Table S10). Combined, all these findings refute the absolute generality, Our ‘Turtle’ dataset permitted us to examine the influence even among closely related taxa, of the ‘Fast X’ and ‘Fast of SDM transitions on molecular evolution, but the same Z’ hypotheses, which state that genes residing in sex-lim- analyses were precluded for the ‘All Species’ dataset. The ited (non-psuedoautosomal) portions of sex chromosomes evolutionary history of SDM transitions among the turtles should evolve faster than their autosomal counterparts due remains uncertain, and current contrasting evolutionary to their smaller effective population size relative to auto- hypotheses (Fig. 2) were evaluated here (Sabath et al. 2016; somes (Bachtrog et al. 2011; Charlesworth et al. 1987; Mank Valenzuela and Adams 2011). First, some research recon- et al. 2007). Instead, our data suggest that gene-specific, structs all transitions in turtle sex determination as occur- lineage-specific, or other chromosome-specific effects may ring from a TSD ancestor to a derived GSD state (Sabath override the evolutionary dynamics driven by sex-linkage et al. 2016). Under this scenario, no significant change alone. Similarly, a recent study detected chromosomal and in substitution rate was seen between the transitional and lineage effects on molecular evolution when contrasting non-transitional branches at either the nucleotide or amino sex-linked sequences to the same sequences linked to auto- acid levels. However, CODEML detected higher dN and somes in turtles and other amniotes (Radhakrishnan et al. dS rates in transitional branches (Table S20), underscoring 2017b). Alternative drivers of molecular evolution have been that the accumulation of substitutions alone can provide a

1 3 Journal of Molecular Evolution (2018) 86:11–26 23 misleading estimate of the tempo of evolution when diver- genes in transitional turtle branches renders them important gence time is not taken into account. The time-dependent candidates for further study, as they span the key processes rates observed counter the notion that accelerated molecu- of temperature-sensing, hormonal regulation, and gonadal lar evolution in the genes examined here is associated with differentiation that underlie sexual development. overall turnovers in sex determination. Whether genes in this network, other than those analyzed here, might exhibit such Secondary Protein Structure Changes in Transitional an association remains to be tested. Alternatively, miniscule Turtle Branches in Functionally Important Domains genetic changes such as point mutations, rather than more dramatic shifts in substitution rate, may alter sex determina- Our results revealed structural changes that may play a role tion profoundly and affect SDM evolution. For instance, the in SDM evolution in three proteins which showed an accel- sex determination network of Takifugu fish was hijacked by erated rate of amino acid evolution in transitional turtle a single nucleotide polymorphism in the kinase domain of branches relative to all species, or in transitional relative the gene Amhr2 (Kamiya et al. 2012). to non-transitional branches within turtles. These findings Second, another study reconstructed seven total transi- should help guide future studies to determine the effect that tions in turtles, five TSD-to-GSD transitions (three of which these substitutions may have on protein function, if any. overlap with the previous hypothesis) plus two GSD-to-TSD First, the HSF2 protein is involved in stress response, ‘reversals’ in the lineages represented by C. insculpta and P. temperature-signal transduction, and spermatogenesis expansa (Valenzuela and Adams 2011). Further, these tran- (Garolla et al. 2013; Yoshima et al. 1998). The HSF2 sition braches display ~ 20× faster rate of evolution of chro- C-terminal domain contains two pairs of transactivation mosome number (Valenzuela and Adams 2011), suggesting domains that modulate transcription during cell stress that although GSD-to-TSD transitions might be rare, turtle through co-regulatory ligand binding, plus two pairs of genomes are generally more evolutionarily labile in tran- negative regulation domains that maintain HSF2 inactive sitional branches. Interestingly, under this hypothesis, we when cells are not stressed (Yoshima et al. 1998). These found that transitional branches, irrespective of their direc- four sub-domains exhibit predicted secondary structure tion (TSD-to-GSD or GSD-to-TSD), had a faster nucleo- changes with potentially functional consequences specific tide and amino acid substitution rate than non-transitional to transitional turtle branches. For instance, the two rep- branches (Fig. 3), but this did not translate into accelerated resentatives of the putative GSD-to-TSD transition line- protein sequence evolution in TSD-to-GSD branches. How- ages (P. expansa and C. insculpta), each have separate ever, genes in the GSD-to-TSD ‘reversal’ branches evolved single amino acid substitutions in different sites of the significantly faster at the nucleotide level (~ 20% faster) HSF2 first transactivation domain that lead to structural relative to the TSD-to-GSD transitional branches, a pattern changes unique among turtles (Fig. 4a). Specifically, P. supported by the dN and dS results (Table S21). This find- expansa’s single substitution deletes two short alpha heli- ing is concordant with the idea that regaining TSD during ces present in all other turtles, while C. insculpta’s cre- GSD-to-TSD transitions requires a more dramatic rewiring ates one longer alpha helix where all other taxa have two of the sex determination network, perhaps reflecting the dif- helices separated by a beta sheet. On the other hand, E. ficulty of escaping the evolutionary trap of sex chromosomes macquarii (representing a TSD-to-GSD transition line- (Pokorna and Kratochvíl 2009), and helping explain why age), P. expansa, and C. insculpta share a similar second- these transitions are considerably rarer than TSD-to-GSD ary structure in the negative regulator domain of the first transitions, at least in turtles. transactivator domain compared to all other turtles, but When broken down by gene, the nucleotide sequences caused by different substitutions in each lineage, suggest- of Dmrt1, Hsf2, Sox9, and Wnt4, and the protein sequences ing that they result from somewhat convergent evolution of HSF2, RSPO1, and SOX9 tended to evolve faster in rather than from shared ancestry. Additionally, the HFS2 transitional branches. HSF2 is a member of the heat-shock second transactivation domain of S. triporcatus (repre- family of proteins, which is able to transduce temperature senting a TSD-to-GSD transition) accumulated multiple signals into transcriptional responses (Sarge et al. 1991). substitutions, which create several novel alpha helices not RSPO1 is an integral signaling molecule in the WNT sign- present in any other turtle (Fig. 4b), and G. insculpta (rep- aling pathway regulating expression of Ctnnb1 and Wnt4, resenting a TSD-to-GSD transition) exhibits yet another which helps direct proper ovarian development (Parma novel alpha helix in this domain. Finally, A. spinifera and et al. 2006). SOX9 is a male-promoting transcription factor P. sinensis, (representing a TSD-to-GSD transition) along in most vertebrates, and in many taxa it is critical for both with P. expansa, show a drastic reduction or loss of alpha the initiation and maintenance of the male developmental helices in the negative regulator domain for HFS2’s sec- pathway (da Silva et al. 1996). Because of their identity ond transactivator present in all other turtle species. We and function, the accelerated amino acid evolution of these hypothesize that because the regulatory role of HSF2 is

1 3 24 Journal of Molecular Evolution (2018) 86:11–26 self-modulated via the negative regulator domains, which an important pathway in ovarian development, providing varies depending on the degree of cell stress (e.g., high another target for future functional testing. temperature), the structural modifications described here could impact how HSF2 is deployed during bouts of high temperature (changes in transactivation domains), and Conclusions perhaps also under more moderate temperatures (changes in negative regulator domains). This hypothesis requires Evolutionary transitions in sex-determining mechanism future functional testing. require at least a partial reorganization of the regulatory Second, LHX9 is a transcription factor which is essen- network involving, among other possibilities, the molecular tial for the formation and general development of the bipo- evolution of protein coding regions of its gene members. tential gonad and typically shows equal levels of activity Our work revealed a significant acceleration of substitution in both developing male and female embryos among ver- rates during putative GSD-to-TSD reversals than during tebrates (Barske and Capel 2010; Birk et al. 2000; Mazaud TSD-to-GSD transitions or SDM stasis. Evolutionary state et al. 2002; Oréal et al. 2002; Ottolenghi et al. 2001). It reconstruction has a major influence on our understand- activates the NR5A1 protein (also known as SF1) (Shima ing of SDM evolutionary history, as no differences on the et al. 2012) whose transcription is evolutionarily labile molecular evolution of this regulatory network were detected across vertebrates (Valenzuela et al. 2013). In order to when GSD-to-TSD reversals are not considered by one of bind co-regulator proteins, LIM-containing transcription the evolutionary hypotheses evaluated. These discrepancies factors possess tandem protein-binding LIM domains sep- highlight the importance of reliable methods for ancestral arated by a linker sequence (Rétaux et al. 1999). Here we state reconstruction, and the need for additional genomic and C. insculpta found that ’s LHX9 is characterized by two SDM data across the tree of life to enable robust analyses. predicted structural changes unique among vertebrates. Our data predicted modifications in the functional domains C. insculpta Namely, ’s downstream LIM domain has a of proteins in the sex determination network which may smaller conserved C-terminal alpha helix and beta strands play a role in SDM evolution in turtles, providing targets shifted within the center of the domain, and the N-terminal for future research. Among the genes that experienced sig- helix in the upstream LIM domain is also disrupted. This nificant molecular evolution, hormonal signaling genes raises the question as to whether such predicted modifica- (Srd5a1 in particular) and HSF2 emerged as being dramati- tions to the protein:protein ligand-binding domain might cally altered during SDM shifts in turtles. No single substi- operate as part of a shifting regulatory network where old tution or structural change was present in all of the studied interactions are lost, and new molecular relationships may GSD turtles, and some transitional branches appear to evolve be forged. much faster than others, a testament to the independent Finally, RSPO1 is a critical co-regulator of the WNT/ nature of turtle SDM evolution and the many trajectories CTNNB1 pathway, primarily involved in ovarian develop- taken by nature to produce males and females. ment through Ctnnb1 activation and stabilization (Parma et al. 2006). RSPO1 is also a SOX9 antagonist, working Acknowledgements We greatly appreciate the assistance in speci- to both promote ovarian development while silencing male men collection for G. insculpta (Jeff Tamplin), E. macquarii (Arthur P. expansa C. insculpta developmental pathways (Chassot et al. 2008). RSPO1, as Georges), (Omar Hernandez), and (Gabriel Rivera). This work was funded in part by NSF Grants DEB 1310793 other –spondin protein family members, contains a throm- to NV and RL, and MCB 1244355 and IOS 1555999 to NV. bospondin-1 repeat domain which appears unnecessary to modulate Ctnnb1 (Parma et al. 2006), and its precise role is still unclear. Two independent single amino acid substitu- References tions in the TSP1 domain in E. macquarii and S. triporcatus are predicted to cause the convergent loss of an alpha Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit helix found in all other turtles (Fig. 4d). While the role models of protein evolution. Bioinformatics 21:2104 of the TSP1 domain remains elusive, the furin-like repeat Ashman T, Bachtrog D, Blackmon H, Goldberg E, Hahn M, Kirkpat- domains appear integral to R-spondins’ ability to modulate rick M, Kitano J, Mank J, Mayrose I, Ming R, Otto S, Peichel C, the Ctnnb1/Wnt pathway (Kim et al. 2008, 2006). Intrigu- Pennell M, Perrin N, Ross L, Valenzuela N, Vamosi J (2014) Tree of Sex: a database of sexual systems. Sci Data 1:140015 ingly, in S. triporcatus a single amino acid substitution in the Bachtrog D, Kirkpatrick M, Mank JE, McDaniel SF, Pires JC, Rice second of two tandem Furin-like repeat domains truncates W, Valenzuela N (2011) Are all sex chromosomes created equal? an alpha helix and adds a beta strand, a predicted structure Trends Genet 27:350 unique among all vertebrates examined. We hypothesize Badenhorst D, Stanyon R, Engstrom T, Valenzuela N (2013) A ZZ/ZW microchromosome system in the spiny softshell turtle, Apalone that these modifications may impact RSPO1-mediated reg- spinifera, reveals an intriguing sex chromosome conservation in ulation of the Ctnnb1/Wnt pathway in this XX/XY turtle, Trionychidae. Chromosome Res 21:137

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