1. Evolutionary Biology
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The rise and fall of the ancient northern pike master sex-determining gene

  1. Qiaowei Pan
  2. Romain Feron
  3. Elodie Jouanno
  4. Hugo Darras
  5. Amaury Herpin
  6. Ben Koop
  7. Eric Rondeau
  8. Frederick W Goetz
  9. Wesley A Larson
  10. Louis Bernatchez
  11. Mike Tringali
  12. Stephen S Curran
  13. Eric Saillant
  14. Gael PJ Denys
  15. Frank A von Hippel
  16. Songlin Chen
  17. J Andrés López
  18. Hugo Verreycken
  19. Konrad Ocalewicz
  20. Rene Guyomard
  21. Camille Eche
  22. Jerome Lluch
  23. Celine Roques
  24. Hongxia Hu
  25. Roger Tabor
  26. Patrick DeHaan
  27. Krista M Nichols
  28. Laurent Journot
  29. Hugues Parrinello
  30. Christophe Klopp
  31. Elena A Interesova
  32. Vladimir Trifonov
  33. Manfred Schartl
  34. John Postlethwait
  35. Yann Guiguen  Is a corresponding author
  1. INRAE, LPGP, France
  2. Department of Ecology and Evolution, University of Lausanne, Switzerland
  3. Swiss Institute of Bioinformatics, Switzerland
  4. Department of Biology, Centre for Biomedical Research, University of Victoria, Canada
  5. Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, United States
  6. Fisheries Aquatic Science and Technology Laboratory at Alaska Pacific University, United States
  7. Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Canada
  8. Fish and Wildlife Conservation Commission, Florida Marine Research Institute, United States
  9. School of Fisheries and Aquatic Sciences, Auburn University, United States
  10. Gulf Coast Research Laboratory, School of Ocean Science and Technology, The University of Southern Mississippi, United States
  11. Laboratoire de Biologie des organismes et écosystèmes aquatiques (BOREA), MNHN, CNRS, IRD, SU, UCN, Laboratoire de Biologie des organismes et écosystèmes aquatiques (BOREA), France
  12. Unité Mixte de Service Patrimoine Naturelle – Centre d’expertise et de données (UMS 2006 AFB, CNRS, MNHN), Muséum national d’Histoire naturelle, France
  13. Department of Biological Sciences, Northern Arizona University, United States
  14. Yellow Sea Fisheries Research Institute, CAFS, Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), China
  15. College of Fisheries and Ocean Sciences Fisheries, United States
  16. Research Institute for Nature and Forest (INBO), Belgium
  17. Department of Marine Biology and Ecology, Institute of Oceanography, University of Gdansk, Poland
  18. INRAE, GABI, France
  19. GeT‐PlaGe, INRAE, Genotoul, France
  20. Beijing Fisheries Research Institute & Beijing Key Laboratory of Fishery Biotechnology, China
  21. U.S. Fish and Wildlife Service, United States
  22. Conservation Biology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, United States
  23. Institut de Génomique Fonctionnelle, IGF, CNRS, INSERM, Univ. Montpellier, France
  24. INRAE, Sigenae, Genotoul Bioinfo, France
  25. Tomsk State University, Russian Federation
  26. Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk State University, Russian Federation
  27. University of Wuerzburg, Developmental Biochemistry, Biocenter, 97074 Würzburg, Germany; and The Xiphophorus Genetic Stock Center, Texas State University, United States
  28. Institute of Neuroscience, University of Oregon, United States
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Cite this article as: eLife 2021;10:e62858 doi: 10.7554/eLife.62858

Abstract

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

Introduction

Genetic sex determination (GSD) evolved independently and repeatedly in diverse taxa, including animals, plants, and fungi (Tree of Sex Consortium et al., 2014; Beukeboom and Perrin, 2014), but the stability of such systems varies drastically among groups. In mammals and birds, conserved male or female heterogametic sex determination (SD) systems have been maintained over a long evolutionary time with conserved master sex-determining (MSD) genes (Marshall Graves, 2008). In contrast, teleost fishes display both genetic and environmental sex determination (ESD), and a remarkable evolutionary lability driven by rapid turnovers of sex chromosomes and MSD genes (Kikuchi and Hamaguchi, 2013; Pan et al., 2017). These characteristics make teleosts an attractive group in which to study the evolution of SD systems.

In the past two decades, the identity of a variety of MSD genes has been revealed in teleosts thanks to advances in sequencing technologies. These findings generated new hypotheses on how the birth of MSD genes through allelic diversification or duplication with or without translocation may drive sex chromosome turnover in vertebrates (Kikuchi and Hamaguchi, 2013; Pan et al., 2017). These teleost MSD genes also provided empirical support for the ‘limited option’ idea that states that certain genes known to be implicated in sex differentiation pathways are more likely to be recruited as new MSD genes (Marshall Graves and Peichel, 2010). The majority of these recently discovered MSD genes, however, were phylogenetically scattered, making it challenging to infer evolutionary patterns and conserved themes of sex chromosomes and/or MSD gene turnovers. Although comparative studies have been accomplished in medakas, poeciliids, tilapiine cichlids, salmonids, and sticklebacks (Kikuchi and Hamaguchi, 2013), transitions of SD systems in relation to the fate of known MSD genes within closely related species have only been explored in medakas (Myosho et al., 2015) and salmonids (Guiguen et al., 2018).

Esociformes is a small order of teleost fishes (Figure 1) that diverged from their sister group Salmoniformes about 110 million years ago (Mya) and diversified from a common ancestor around 90 Mya (Campbell et al., 2013; Campbell and Lopéz, 2014). With two families (Esocidae, including Esox, Novumbra, and Dallia, and Umbridae, including Umbra) and 13 well-recognized species (Warren et al., 2020), Esociformes is an ecologically important group of freshwater species from the northern hemisphere (Campbell et al., 2013; Campbell and Lopéz, 2014). We demonstrated that a male-specific duplication of the anti-Müllerian hormone gene (amhby) is the MSD gene in Esociformes and that this gene is located in a small male-specific insertion on the Y chromosome of northern pike (Esox lucius) (Pan et al., 2019).

Species phylogeny with estimated divergence time (Campbell et al., 2013; Kumar et al., 2017) and technical approaches for investigating SD systems in Esociformes.

The two families within Esociformes, the Esocidae and the Umbridae, are highlighted with green and yellow background, respectively. Whole-genome sequencing (WGS), homology cloning (Cloning), restriction-site-associated DNA sequencing (RAD-Seq), and pooled-sequencing (Pool-Seq) approaches used in each species are shown by a black square on the right side of the figure. Fish silhouettes were obtained from phylopic.org.

Here, we took advantage of the small number of Esociformes species and their relatively long evolutionary history to explore the evolution of SD in relation to the fate of this amhby MSD gene. Generating novel draft genome assemblies and population genomic data in multiple species of Esociformes (Figure 1), we traced the evolutionary trajectory of amhby from its origin in a gene duplication event 65–90 Mya to its demise during species- or population-specific SD transitions. Our results highlight the diverse evolutionary fates experienced by an MSD gene. Among SD transitions, we uncovered a recent loss of the entire Y chromosome in some populations of northern pike. We hypothesize that drift, exacerbated by the bottleneck effect, may have facilitated the loss of the sex chromosome along with its MSD gene in these populations, a new mechanism for entire sex chromosome loss in vertebrates.

Results

The Esox lucius amhby gene originated from an ancient duplication event

Previously, we demonstrated that a male-specific duplication of the anti-Müllerian hormone gene (amhby) is the MSD gene in Esociformes and that this gene is located in a small, ~140 kb male-specific insertion on the Y chromosome of northern pike (Esox lucius) (Pan et al., 2019). To explore the evolution of the autosomal copy of amh (amha) and amhby in Esociformes, we collected phenotypically sexed samples of most species of this order (Figure 1 and Table 1). We searched for homologs of amh using homology cloning and whole-genome sequencing. We found two amh genes in all surveyed Esox and Novumbra species. In the more basally diverging species, Dallia pectoralis and Umbra pygmaea, we found only one amh gene in both species in tissue-specific transcriptome databases (Pasquier et al., 2016), while two amh transcripts were readily identified in E. lucius (Pan et al., 2019).

Table 1
Summary of the identification of amhby and its association with sex phenotypes in 11 Esociformes species, including six populations of E. lucius and three populations of Esox masquinongy.
SpeciesSampling locationGeographic regionMales with amhbyFemales with amhbyp-value of amhby association with sex
Esox luciusFranceWestern Europe161/161 (100%)0/60 (0%)<2.2E-16
Esox luciusSwedenNorthern Europe20/20 (100%)0/20 (0%)<2.2E-16
Esox luciusPolandCentral Europe20/20 (100%)0/20 (0%)<2.2E-16
Esox luciusXinjiang, ChinaEastern Asia5/5 (100%)0/5 (0%)0.0114
Esox luciusContinental USA and CanadaNorth America0/88 (0%)0/74 (0%)amhby absent
Esox luciusAlaska, USANorth America17/19 (89%)0/19 (0%)1.79E-07
Esox luciusSiberia, RussiaNorthern Asia9/9 (100%)0/8 (0%)0.0003
Esox aquitanicusFranceWestern Europe1/1 (100%)0/2 (0%)0.665
Esox cisalpinusItalyWestern Europe2/2 (100%)0/2 (0%)0.317
Esox reichertiiChinaEastern Asia11/11 (100%)0/10 (0%)3.40E-05
Esox masquinongyIowa, USANorth America18/27 (66.7%)4/18 (22%)2.01E-08
Esox masquinongyQuebec, CanadaNorth America9/20 (45%)4/20 (20%)0.18
Esox masquinongyWisconsin, USANorth America57/61 (93%)22/61 (36%)1.17E-10
Esox americanus (americanus)Mississippi, USANorth America6/6 (100%)0/4 (0%)0.0123
Esox nigerQuebec, CanadaNorth America5/5 (100%)0/8 (0%)0.0025
Novumbra hubbsiWashington, USANorth America21/23 (91%)0/22 (0%)5.28E-09
Dallia pectoralisAlaska, USANorth America0/30 (0%)0/30 (0%)amhby absent
Umbra pygmaeaBelgiumWestern Europe0/31 (0%)0/31 (0%)amhby absent

To clarify relationships among these amh homologs, we inferred phylogenetic trees from these sequences. These phylogenies provided a clear and consistent topology with two well-supported gene clusters among the amh sequences from Esocidae (Figure 2A, Appendix 1—figure 1). The first gene cluster contains the autosomal amha of E. lucius and an amh homolog from all other species of Esox, Dallia, and Novumbra. Therefore, we assigned these amh homologs as amha orthologs. The second gene cluster contains the Y-specific amhby gene of E. lucius, along with the other amh homolog from all species for which we identified two amh sequences. We assigned these amh homologs as amhby orthologs. The general topology of each cluster was in agreement with the Esociformes species taxonomy.

Phylogenetic analysis of amh homologs from the Esociformes revealed an ancient origin of amhby.

(A) Phylogenetic tree of amh coding sequences built with the maximum-likelihood method. Bootstrap values are provided on each node of the tree. The amha ortholog cluster is highlighted with blue background and the amhby ortholog cluster is highlighted with red background. (B) Time-calibrated species phylogeny of the Esociformes (Campbell et al., 2013). The three putative SD transitions are shown by black stars. The presence of pre-duplication amh, as well as amha and amhby along with the amhby sex linkage, is represented by colored dots at the end of each branch. In E. cisalpinus and E. aquitanicus, sex linkage is not significant (NS) due to low sample size. The earliest duplication timing of amh is denoted by a black arrow at the root of the divergence of Esocidae.

In U. pygmaea, the closest sister species to the Esocidae, we found a single amh homolog, which roots at the base of the amha/amhby clusters. This result indicates that the amh duplication happened after the divergence of Esocidae (including Esox, Dallia, and Novumbra) and Umbridae (including Umbra) lineages. In contrast to other Esocidae that have two amh orthologs, we found only one amh gene in D. pectoralis, which fell in the phylogeny within the amha ortholog cluster, suggesting that this species experienced a secondary loss of its amhby gene after the amh duplication at the root of the Esocidae lineage (Figure 2B and Appendix 1—figure 1). We confirmed the absence of an additional divergent amh gene in D. pectoralis by searching sex-specific Pool-Seq reads from 30 males and 30 females. In addition, only one amh homolog was found in an ongoing genome assembly project with long reads for a male D. pectoralis (Y. Guiguen, personal communication). Using information from an Esociformes time-calibrated phylogeny (Campbell et al., 2013), we estimated that the amhby duplicate arose between 65 and 90 Mya (Figure 2B).

Sex linkage of amhby in the Esocidae lineage

Given that amhby originated from an ancient duplication event and is the MSD gene in E. lucius, we investigated when amhby became the MSD gene and whether transitions of SD systems exist in this group. Because male sex linkage of a sequence is a strong indication that this sequence is located on a Y-chromosome-specific region, we first investigated the association of amhby with male phenotype. We found an association between male phenotype and the presence of amhby in most species of Esox and Novumbra (Table 1). This sex linkage was significant in European, Asian, and Alaskan populations of E. lucius, in two North American populations of E. masquinongy (Iowa and Wisconsin), and in all populations surveyed of E. reichertii, E. americanus, E. niger, and N. hubbsi. For two recently described species, E. cisalpinus and E. aquitanicus (Denys et al., 2014), we had insufficient samples with clear species and sex identification for a decisive result. We confirmed the presence of amhby in all males and its absence from all females in these two species, but the association was not significant due to low sample size (Table 1). Because amhby is associated with male phenotype in most species of Esox and Novumbra, it likely gained an MSD role shortly after its origin either at the root of Esocidae or before the split of Esox and Novumbra lineages (~56 Mya) (Campbell et al., 2013). Despite this global conservation of the linkage between amhby and male phenotype, however, we found some variations in amhby sex association across populations of E. lucius and E. masquinongy (Table 1). These population variations are further investigated below.

Whole-genome analyses of the evolution of sex determination systems in Esociformes

Because amhby was not completely associated with male phenotype in E. masquinongy and N. hubbsi (Table 1) and the gene was not found in D. pectoralis and U. pygmaea, we also used population genomic approaches to search for whole-genome sex-specific signatures in these species. Because of the close phylogenetic distance (45 Mya) between E. lucius and E. masquinongy, we used the E. lucius genome assembly to remap reads from E. masquinongy. We performed Pool-Seq analysis of E. masquinongy (Iowa, USA) to compare the size and location of its sex locus with that of E. lucius (Pan et al., 2019). Comparison of the sex-specific heterozygosity across the entire genome revealed that a single region of less than 50 kb, containing the highest density of male-specific single-nucleotide polymorphisms (SNPs) in E. masquinongy, is located in a region homologous to the proximal end of LG24, where the sex locus of E. lucius is located. Besides LG24, no other linkage group showed enrichment for sex-biased chromosome differentiation (Appendix 1—figure 2), suggesting that the location and the small size of the sex locus are likely conserved between E. lucius and E. masquinongy.

No high-quality genome assembly was available from a closely related species to N. hubbsi. Therefore, we performed de novo RAD-Seq analysis on 21 males and 19 females for N. hubbsi. We found two markers showing a significant association with male sex and no female-biased marker (Figure 3A). This result supports the male heterogametic SD system (XX/XY) that was suggested by the amhby sex linkage and also indicates that the N. hubbsi sex locus is likely small (41.6 restriction enzyme cutting sites/Mb across the genome, on average, Supplementary file 2).

Characterization of sex determination systems in different Esociformes species through RAD-Seq analyses.

Each tile plot shows the distribution of non-polymorphic RAD-Seq markers shared between phenotypic males (horizontal axis) and phenotypic females (vertical axis). The intensity of color for a tile reflects the number of markers present in the respective number of males and females. Tiles that are significantly associated with phenotypic sex (chi-square test, p<0.05 after Bonferroni correction) are highlighted with a red border. (A) In N. hubbsi, two markers were present in 19 of 21 males and in 1 of 19 females, indicating a significant male association and thus an XX/XY SD system with a small sex locus. (B) In D. pectoralis, one marker showed a significant association with females, while no marker showed association with males, indicating a ZZ/ZW SD system. (C) In U. pygmaea, 140 markers showed a significant association with males, while no marker showed association with females, indicating a XX/XY SD system with a large non-recombining sex locus. (D) In the population of E. masquinongy from Quebec (Canada), no marker was associated with either sex. (E) In a population of E. masquinongy from Iowa (USA), five markers were significantly associated with male phenotype, indicating a XX/XY SD system.

Figure 3—source code 1

R Script to generate Figure 3.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig3-code1-v2.zip
Figure 3—source data 1

Distribution of RADsex markers of E. masquinongy from a Quebec population with a minimal marker depth of 10 reads.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig3-data1-v2.csv
Figure 3—source data 2

Distribution of RADsex markers of E. masquinongy from a Iowa population with a minimal marker depth of 10 reads.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig3-data2-v2.csv
Figure 3—source data 3

Distribution of RADsex markers of N. hubbsi a minimal marker depth of 10 reads.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig3-data3-v2.csv
Figure 3—source data 4

Distribution of RADsex markers of U. pygmaea a minimal marker depth of 10 reads.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig3-data4-v2.csv
Figure 3—source data 5

Distribution of RADsex markers of D. pectoralis a minimal marker depth of 10 reads.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig3-data5-v2.csv

In D. pectoralis and U. pygmaea, two species in which amhby is absent, we carried out RAD-Seq analyses comparing phenotypic males and females. In D. pectoralis, we found only three female-biased RAD markers, suggesting that this species also has a small sex locus region (30.8 restriction enzyme cutting sites/Mb on average, Supplementary file 2) under a female heterogametic SD system (ZZ/ZW) (Figure 3B). This ZZ/ZW SD system was further supported by an independent Pool-Seq analysis revealing 45 times more female-specific k-mers (N = 1,081,792) than male-specific k-mers (N = 23,816). This excess of female-specific k-mers indicates that females carry genomic regions that are absent from males and thus that females are the heterogametic sex. In contrast, 140 male-biased and no female-biased RAD markers were identified in U. pygmaea, supporting that this species has a large sex locus (26.2 restriction enzyme cutting sites/Mb on average, Supplementary file 2) under a male heterogametic SD system (XX/XY) (Figure 3C).

Some populations of Esox lucius lost their Y chromosome and ancestral master sex-determining gene

Although amhby is the MSD gene in European populations of northern pike (Pan et al., 2019), this gene was absent from the genome assembly (GCA_000721915.3) of a male specimen from a Canadian population (GCA_000721915.3). To explore this discrepancy, we surveyed the sex linkage of amhby in geographically isolated populations of E. lucius. We found significant male linkage in all investigated European and Asian populations and in one Alaskan population (Table 1). In contrast, amhby was absent from both males and females in all other North American populations.

To investigate whether the loss of amhby in most North American populations coincides with large genomic changes, we compared genome-wide sex divergence patterns between a European population carrying the amhby gene (Ille-et-Vilaine, France) and a North American population that lacks amhby (Quebec, Canada) using a Pool-Seq approach. We aligned Pool-Seq reads to an improved European E. lucius genome assembly (NCBI accession number SDAW00000000; assembly metrics presented in Supplementary file 3) in which all previously identified Y-specific contigs (Pan et al., 2019) were scaffolded into a single contiguous locus on the Y chromosome (LG24). In the European population, Pool-Seq analysis confirmed, with better resolution, previous results (Pan et al., 2019), showing the presence of a small Y chromosome region (~140 kb) characterized by many male-specific sequences at the proximal end of LG24 (Figure 4 A.1 and 4B.1). In comparison, virtually no reads from either male or female pools from the Quebec population mapped to this 140 kb Y-specific region (Figure 4 A.2 and 4B.2). Furthermore, we observed no differentiation between males and females along the remainder of the genome with either Pool-Seq or reference-free RAD-Seq (Appendix 1—figure 3). Together, these results suggest that the Quebec population and likely other mainland US populations, where the MSD gene amhby was not found, lack not only amhby, but also the surrounding Y-specific region identified in European populations. Moreover, the new sex locus of these North American populations, if it exists, is too small to be detected by the RAD-Seq and Pool-Seq approaches.

Loss of the ancestral sex locus and master sex determination gene in North American northern pike.

(A) The numbers of male-specific SNPs in 50 kb non-overlapping windows deduced from Pool-Seq data of a European population (A.1) and North American population (A.2) of E. lucius were mapped to the Y chromosome (LG24) of the sequence of E. lucius from a European male genome (SDAW00000000). The greatest number of male-specific SNPs is found in a single window located at the proximal end of LG24 (0.95 Mb–1.0 Mb) that contains 182 male-specific SNPs (highlighted by the gray box) in the European E. lucius population. The same region, however, shows no differentiation between males and females in the North American population. (B) Relative coverage of male and female Pool-Seq reads is indicated by blue and red lines, respectively, in contigs containing amhby in the European population (B.1) and in the North American population (B.2). Only zoomed view on the European population sex-specific region is presented to facilitate the visualization of the differences in coverage between two populations. We searched for Y-specific regions in 1 kb windows. A window is considered Y specific if it is covered by few mapped female reads (<3 reads per kb) and by male reads at a depth close to half of the genome average (relative depth between 0.4 and 0.6). Based on these depth filters, in the European population we identified 70 potential Y-specific 1 kb windows located between 0.76 Mb and 0.86 Mb and 70 Y-specific 1 kb windows located between 1.0 Mb and 1.24 Mb on LG24. This region is not covered by male or female reads in the North American population, indicating the loss of the entire Y-specific region, highlighted by the gray boxes. The region between 0.86 Mb and 1.0 Mb is not sex specific in both population and is likely an assembly artifact. 

Figure 4—source code 1

R script to generate Figure 4.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig4-code1-v2.zip
Figure 4—source data 1

Pool-Seq comparison of sex-specific SNPs in windows of 50 kb between males and female from a European population of E. lucius.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig4-data1-v2.csv
Figure 4—source data 2

Pool-Seq comparison of sex-specific coverage in windows of 1 kb between males and female from a European population of E. lucius.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig4-data2-v2.csv
Figure 4—source data 3

Pool-Seq comparison of sex-specific SNPs in windows of 50 kb between males and female from a North American population of E. lucius.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig4-data3-v2.csv
Figure 4—source data 4

Pool-Seq comparison of sex-specific coverage in windows of 1 kb between males and female from a North American population of E. lucius.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig4-data4-v2.csv
Figure 4—source data 5

E. lucius chromosome length file for the R script.

https://cdn.elifesciences.org/articles/62858/elife-62858-fig4-data5-v2.csv

Evolving sex determination systems: the case of the muskellunge, Esox masquinongy

As in E. lucius, we found variation in amhby sex linkage among different populations of E. masquinongy (Table 1). In addition, we found that two males and one female of E. masquinongy were heterozygous for amhby in the population from Quebec (Appendix 1—figure 4), a finding that conflicts with the expected hemizygous status of a Y-specific MSD gene. We thus used RAD-Seq to compare genome-wide patterns of sex differentiation in a population from Iowa (USA) where amhby was significantly associated with male phenotype and in a population from Quebec (Canada) where it was not. In the Iowa population, five RAD markers were significantly associated with male phenotype, while no marker was associated with female phenotype (Figure 3D). This result indicates a male heterogametic SD system (XX/XY) with a low differentiation between the X and Y chromosomes, as observed in northern pike (Pan et al., 2019) and N. hubbsi (this study). In contrast, we did not find a sex-specific marker in the Quebec population (Figure 3E). This result was further supported by the analysis of a publicly available dataset from another Quebec population (PRJNA512459, Appendix 1—figure 5), suggesting that the sex locus of E. masquinongy from these Quebec populations is too small to be detected with RAD-Seq (38.4 restriction enzyme cutting sites/Mb on average, Supplementary file 2) or that this population displays a multifactorial GSD or ESD system.

Evolution of the structure of the amhby gene in the Esocidae

The typical amh gene in teleost fishes comprises seven exons encoding a protein that contains 500–571 amino acids. The conserved C-terminal TGF-β domain is crucial for canonical Amh function (di Clemente et al., 2010). The predicted structures of most of the amha and amhby genes in Esociformes are consistent with this canonical structure, and most amha and amhby genes do not show signature of relaxation from purifying selection (Supplementary file 4 and Supplementary file 5). However, in both E. niger and N. hubbsi, the predicted Amhby protein is truncated in its N- or C-terminal part. In E. niger, this truncated amhby gene is flanked by repeated elements and encompasses only three of the seven conserved Amh exons, that is exons 5, 6, and 7 with a truncated TGF-β domain (Appendix 1—figure 6). In N. hubbsi, the truncated amhby gene contains the seven conserved Amh exons but with an N-terminal truncation of exon 1 encoding only eight amino acids with no homology to the conserved 50 amino acid sequence of the first Amh exon of other Esocidae (Appendix 1—figure 6). Together, these results show that in some species where amhby is sex linked, the protein sequence is strongly modified in the N- and C-terminal-conserved domains that are needed for proper protein secretion and correct conformation (di Clemente et al., 2010), leaving questions as to whether these proteins are functional.

Discussion

The northern pike MSD gene amhby substantially diverges from its autosomal copy amha, suggesting an ancient origin (Pan et al., 2019), unlike other cases of amh duplication in fishes, that appears to be comparatively young (Hattori et al., 2013; Li et al., 2015). Here, we show that the amhby duplicate emerged before the split of Esocidae and Umbridae between 65 and 90 Mya (Campbell et al., 2013) and was subsequently secondarily lost in the Dallia lineage. We also demonstrate that amhby presence is significantly associated with male phenotype in most species of Esox and in Novumbra. Although we cannot rule out the possibility that amhby was recruited independently as the MSD gene in Esox and Novumbra, our results suggest the more parsimonious hypothesis that amhby likely acquired an MSD function before the diversification of Esocidae at least 56 Mya.

Studies on SD systems of cichlid fishes and true frogs suggested that frequent turnovers can keep sex chromosomes undifferentiated (Gammerdinger et al., 2018; Jeffries et al., 2018). It was proposed that in these organisms, the turnover of sex chromosomes was facilitated by autosomes that host genes involved in sex differentiation pathways that could be readily recruited as new MSD genes, facilitating turnovers of sex chromosomes (Vicoso, 2019). Alternative evolutionary pathways could also involve conserved MSD genes that translocate onto different autosomes, as demonstrated for the salmonid sdY gene (Guiguen et al., 2018). Interestingly, this scenario does not seem to be the case with Esocidae. No highly differentiated sex chromosomes were observed in the surveyed population of E. masquinongy and N. hubbsi (this study), or in E. lucius, despite Esocidae having retained the same ancestral XX/XY system (likely controlled by amhby) for at least 56 million years. In addition, whole-genome analyses in E. masquinongy suggest that its male-specific locus on the Y chromosome is conserved in terms of size and location with that of E. lucius despite 45 million years of divergence. Such conservation is unusual in teleosts, where frequent turnover of SD systems is considered the norm (Kikuchi and Hamaguchi, 2013; Pan et al., 2017). In line with our present results, substantially undifferentiated sex chromosomes have also been maintained over relatively long evolutionary periods in some Takifugu fish species (Kamiya et al., 2012), supporting the idea that theoretical models of sex chromosome evolution cannot be generalized across taxa and that additional, as-yet unknown evolutionary forces can prevent sex chromosome decay.

Even though this MSD gene has been maintained for a long evolutionary period, our results revealed that it has been subjected to several independent SD transitions, potentially driven by different mechanisms in the Esocidae lineage. In D. pectoralis, we observed a transition from the ancestral XX/XY male heterogametic system shared by Esocidae and U. pygmaea to a ZZ/ZW female heterogametic system. This transition also coincided with the loss of amhby. This shift from male to female heterogamety could have been driven by the rise of a new dominant female determinant, leading to the obsolescence and the subsequent loss of the ancestral male MSD gene, similar to the transition of SD documented among the Oryzias and also in the housefly (Kozielska et al., 2008, Myosho et al., 2012).

The other SD transitions that we characterized, those in E. lucius and E. masquinongy, are probably more recent because these transitions are found within populations of the same species. In E. masquinongy, we observed population-specific variation in SD systems, supporting the conservation of an XX/XY system with amhby as the MSD gene in two US populations belonging to the northern lineage, but not in a Quebec population belonging to the Great Lakes lineage (Turnquist et al., 2017). This result suggests that although amhby is still present in the genome of the Great Lakes lineage, it is no longer the MSD gene and that a new population-specific SD mechanism emerged after the split of the lineages. Previously, female heterogamety was inferred for the Great Lakes lineage based on the presence of males in the offspring of gynogenetic females (Dabrowski et al., 2000). With RAD-Seq, we did not find evidence for female-specific genomic regions in the Great Lakes lineage of E. masquinongy. Higher resolution approaches are needed to identify differentiation between male and female genomes to reveal the new SD locus and its interaction with amhby in the Great Lakes lineage.

In E. lucius, we found that amhby is present and completely sex linked in European, Asian, and Alaskan regions, but is not present elsewhere in North America (Figure 5). This finding suggests that amhby functions as an MSD gene in Eurasian and Alaskan populations, but that populations from elsewhere in North America lost amhby together with the entire sex locus. The current geographic distribution of E. lucius results from a post-glacial expansion from three glacial refugia ~0.18 to 0.26 Mya. The North American populations lacking amhby belong to a monophyletic lineage with a circumpolar distribution originating from the same refugia as the other population we surveyed that carry amhby (Skog et al., 2014). Fossil records from Alaska support the idea of an ‘out of Alaska’ North American expansion of E. lucius within the last 100,000 years (Wooller et al., 2015), suggesting that the Y-specific sequences containing amhby were lost during this dispersal period (Figure 5, Appendix 1—figure 7).

Figure 5 with 1 supplement see all
Schematic representation of a hypothesized route of post-glacial E. lucius expansion from a Eurasian refugium ~0.18 to 0.26 Mya.

The presence (blue silhouettes)/absence (red silhouettes) of amhby in different populations, showing that this MSD gene was lost in some North American populations during an out-of-Alaska dispersal around 0.1 Mya. The hypothesized refugium in the Ponto Caspian region is highlighted in beige. An alternative route of post-glacial dispersal is shown in Figure 5—figure supplement 1.

The loss of the entire sex locus in such a short evolutionary time is unlikely to have resulted from the pseudogenization of the amhby gene, as is the case for the ancestral MSD gene of the Luzon medaka (Myosho et al., 2012). Rather, it is probably the result of colonization by a small pool of females and sex-reversed XX males. Such a founder-effect hypothesis is supported by the fact that North American populations of E. lucius display a much lower genetic diversity compared to other populations from the same lineage (Skog et al., 2014). This hypothesis is also supported by the fact that environmental influence on GSD systems is a well-documented phenomenon in fishes (Baroiller et al., 2009; Chen et al., 2014; Sato et al., 2005) and that XX sex reversal induced by environmental factors has been observed occasionally in captive E. lucius (Pan et al., 2019). Our whole-genome analyses failed to identify any sex-associated signals in these North American populations, meaning that if such a new sex locus exists, it would lack detectable signatures of sequence differentiation, similar to the one-SNP sex locus of some Takifugu species (Kamiya et al., 2012). Whether ESD, which is common among poikilothermic animals including teleosts (Navara, 2018), could be the SD system in these North American populations or whether these populations already have a new GSD system in place is still unresolved. Notably, this Y chromosome loss in natural populations of E. lucius mirrors the loss of the W chromosome in lab strains of zebrafish after just a few decades of selective breeding (Wilson et al., 2014).

A few evolutionary models have been formulated to capture the dynamics of sex chromosome turnover (Vicoso, 2019). The replacement of the ancestral SD locus by a new one could be driven by drift alone, by positive selection (Bull and Charnov, 1977), by sexual antagonistic selection (van Doorn and Kirkpatrick, 2007), or by the accumulation of deleterious mutations at the sex locus (Blaser et al., 2013). In all of these models, the ancestral sex chromosomes would revert to autosomes only when the new SD locus is fixed in the population. However, in North American populations of E. lucius, the ancestral sex chromosome was likely lost due to drift in bottlenecked populations. Interestingly, this process would not require the simultaneous emergence of a new GSD system, given the flexibility of SD mechanisms in teleosts, where environmental cues generate phenotypic sexes in the absence of a GSD system. ESD, which is easily invaded by a new genetic system (Muralidhar and Veller, 2018; van Doorn, 2014), may serve as a transitional state between sex chromosome turnovers.

We found two cases of altered amhby copies. In N. hubbsi and E. niger, amhby is strongly sex linked, which suggests that it is located in a Y-specific region of the genome. In both species, however, the amhby gene lacks parts of the conserved C- and/or N-terminal Amh regions, which are needed for proper protein secretion and interaction between Amh and its receptor (di Clemente et al., 2010). Although we cannot exclude that these are non-functional amhby copies, this hypothesis seems unlikely because the tight sex linkage of amhby in these species would require the independent emergence of a new MSD gene in close proximity to amhby. It is more likely that these copies still function as MSD genes, but with altered protein sequences. A similar example of a truncated amh duplicate acting as an MSD gene is found in the teleost cobalt silverside Hypoatherina tsurugae (Bej et al., 2017). Additional cases of truncated duplicated genes functioning as MSD genes have been described in Salmonids, yellow perch (Perca flavescens), and Atlantic herring (Clupea harengus), suggesting that the preservation of ancestral domains is not necessary for a duplicated protein to assume a novel role (Feron et al., 2020b; Rafati et al., 2020; Yano et al., 2012). These cases are all consistent with domain gains and losses contributing to new protein functions (Moore and Bornberg-Bauer, 2012).

Collectively, our results depict the evolutionary trajectories of a conserved MSD gene in an entire order of vertebrates, highlighting both the potential stability of MSD genes as well as non-differentiated sex chromosomes in some lineages, and the dynamics of species- or population-specific SD evolution in teleost fishes. Our results highlight the importance of careful consideration of the population demographic history of SD systems, and of the potential buffering role of ESD during transitions between genetic SD systems in sex chromosome evolution.

Materials and methods

Sample collection

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Information on the Esociformes species, collectors, sexing method, and experiments is provided in Supplementary file 6.

Genomic DNA extraction

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Fin clips were preserved in 75–100% ethanol at 4°C until genomic DNA (gDNA) extraction. For genotyping, samples were lysed with 5% Chelex and 25 mg of Proteinase K at 55°C for 2 hr, followed by incubation at 99°C for 10 min. For Illumina sequencing, gDNA was obtained using NucleoSpin Kits for Tissue (Macherey-Nagel, Duren, Germany) following the producer’s protocol. DNA concentration was quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA) and a Qubit3 fluorometer (Invitrogen, Carlsbad, CA). For Pool-Seq, DNA from different samples was normalized to the same quantity before pooling for male and female libraries separately. High-molecular-weight gDNA for long-read sequencing was extracted as described by Pan et al., 2019.

Genome and population genomics sequencing

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Draft genomes of northern pike, muskellunge (E. masquinongy), chain pickerel (E. niger), Olympic mudminnow (N. hubbsi), and Alaska blackfish (D. pectoralis) were sequenced using a whole-genome shotgun strategy with 2 × 250 bp Illumina reads. Libraries were built using the Truseq nano kit (Illumina, FC-121–4001) following instructions from the manufacturer. gDNA (200 ng) was briefly sonicated using a Bioruptor (Diagenode). The gDNA was end-repaired and size-selected on beads to retain fragments of ~550 bp, and these fragments were a-tailed and ligated to Illumina’s adapter. The ligated gDNA was then subjected to eight PCR cycles. Libraries were checked on a fragment analyzer (AATI) and quantified by qPCR using a library quantification kit from KAPA. Libraries were sequenced on a HIseq2500 using the paired end 2 × 250 bp v2 rapid mode according to the manufacturer’s instructions. Image analysis was performed by HiSeq Control Software, and base calling was achieved using Illumina RTA software. The output of each run is presented in Supplementary file 7. To improve the European male genome of E. lucius, we generated an extra coverage of Oxford Nanopore long reads using a higher fragment size (50 kb) library made from gDNA extracted from a different male from the same European population as the sample used for the previous genome assembly. Library construction and genome sequencing were carried out as in Pan et al., 2019 and 12.7 Gbp of new data were generated from one PromethION flowcell.

Pool-Seq was performed on E. masquinongy, D. pectoralis, and the North American populations of E. lucius. Pooled libraries were constructed using a Truseq nano kit (Illumina, FC-121–4001) following the manufacturer’s instructions. Male and female DNA Pool-Seq libraries were prepared for each species using the Illumina TruSeq Nano DNA HT Library Prep Kit (Illumina, San Diego, CA) with the same protocol as for the draft genome sequencing. The libraries were then sequenced on a NovaSeq S4 lane (Illumina, San Diego, CA) using the paired-end 2 × 150 bp mode with Illumina NovaSeq Reagent Kits following the manufacturer’s instructions. The output of each run for each sex is presented in Supplementary file 7.

RAD-Seq was performed on E. masquinongy, N. hubbsi, D. pectoralis, U. pygmaea, and the North American populations of E. lucius. RAD libraries were constructed from gDNA extracted from fin clips for each species using a single Sbf1 restriction enzyme as in Amores et al., 2011. Each library was sequenced on one lane of an Illumina HiSeq 2500. The summary of the output of each dataset is presented in Supplementary file 8.

Analysis of population genomic data for sex-specific signals

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Raw RAD-seq reads were demultiplexed with the process_radtags.pl script from stacks version 1.44 (Catchen et al., 2013) with all parameters set to default. Demultiplexed reads for each species were analyzed with the RADSex computational workflow (Feron et al., 2020a) using the radsex software version 1.1.2 (10.5281/zenodo.3775206). After generating a table of markers depth with process, the distribution of markers between sexes was computed with distrib and markers significantly associated with phenotypic sex were extracted with signif using a minimum depth of 10 (−d 10) for both commands and with all other settings at the default. RAD-Seq figures were generated with sgtr (10.5281/zenodo.3773063).

For each Pool-Seq dataset, reads were aligned to the reference genome using BWA mem (version 0.7.17) (Li and Durbin, 2009). Alignment results were sorted by genomic coordinates using samtools sort (version 1.10) (Li et al., 2009), and PCR duplicates were removed using samtools rmdup. A file containing nucleotide counts for each genomic position was generated with the pileup command from PSASS (version 3.0.1b, 10.5281/zenodo.3702337). This file was used as input to compute FST between males and females, number of male- and female-specific SNPs, and male and female depth in a sliding window along the entire genome using the analyze command from PSASS with parameters: window-size 50,000, output-resolution 1000, freq-het 0.5, range-het 0.15, freq-hom 1, range-hom 0.05, min-depth 1, and group-snps. The analysis was performed with a snakemake workflow available at https://github.com/SexGenomicsToolkit/PSASS-workflow. Pool-Seq figures were generated with sgtr version 1.1.1 (10.5281/zenodo.3773063).

For k-mer analysis, 31-mers were identified and counted in the reads of the male and female pools using the ‘count’ command from Jellyfish (version 2.2.10) (Marçais and Kingsford, 2011) with the option ‘-C’ activated to count only canonical k-mers and retaining only k-mers with an occurrence >5 and <50,000,000. Tables of k-mer counts produced by Jellyfish were merged with the ‘merge’ command from Kpool (https://github.com/INRA-LPGP/kpool), and the resulting merged table was filtered using the ‘filter’ command to only retain k-mers present >25 times in one sex and <5 times in the other sex; such k-mers were considered sex biased.

Sequencing of amha and amhby genes

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To survey the presence of amha (the canonical copy of amh) and amhby in Esociformes, we collected samples of 11 species (Table 1): all species in the genus Esox (E. americanus americanus, E. americanus vermiculatus, E. aquitanicus, E. cisalpinus, E. masquinongy, E. reichertii, and E. niger), N. hubbsi (the only species in its genus), D. pectoralis (the only well-recognized species in its genus), and one species from the genus Umbra (U. pygmaea). To search for amh homologs in the genomes of these 11 species, we either sequenced PCR amplicons with custom primers (Supplementary file 9) and/or sequenced and assembled the genome (Supplementary file 10) and searched in the assembly and in the raw reads for the presence of one or two amh genes.

For species closely related to E. lucius (E. aquitanicus, E. cisalpinus, and E. reichertii), amh homologs were amplified with primers designed on amha or amhby of E. lucius. Complete sequences of amh homologs were obtained from overlapping amplicons covering the entire genomic regions of both amha and amhby with primers anchored from upstream of the start codon (SeqAMH2Fw1 and SeqAMH1Fw1) and downstream of the stop codon (SeqAMH2Rev3 and SeqAMH1Rev4). All PCR amplicons were then Sanger sequenced from both directions and assembled to make consensus gene sequences.

For three other Esocidae species (E. niger, E. masquinongy, N. hubbsi), only partial amplifications of amh sequences were obtained with primers designed on amha or amhby of E. lucius. To acquire the complete amh sequences, we generated draft genome assemblies from amhby-carrying individuals.

For the two species from the more divergent genera (D. pectoralis and U. pygmaea), we were unable to amplify amhby with primers designed on regions that appear conserved on Esox species. Therefore, we also generated a genome assembly from a phenotypic male of each species.

For E. niger, E. masquinongy, N. hubbsi, and D. pectoralis, we used amha and amhby genomic sequences from E. lucius as queries and searched for amh homologs with blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, version 2.10.0+) (Altschul et al., 1997) using the parameters ‘Max target sequences’=50 and ‘Max matches in a query range’=1.

For U. pygmaea, the most divergent species from E. lucius in this study, blasting with E. lucius amha and amhby sequences did not yield any result. We used the blastn strategy with the coding sequence of amh as well as tblastn of the protein sequence from Salmon salar, which returned only one contig.

No genome was available for the other two more distantly related species of E. lucius, E. americanus americanus, and E. americanus vermiculatus; therefore, we designed primers on regions conserved in multi-sequence alignments of amha and amhby in the other Esox species (Supplementary file 9).

To investigate whether truncations of Amhby in E. niger and N. hubbsi could be assembly artifacts, we searched for potential missing homologous sequences in raw genome reads of both species using tblastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, version 2.10.0+) (Altschul et al., 1997) using the exon 1 protein sequence from E. lucius as a query. Homologous sequences were determined with a maximum e-value threshold of 1E-10.

Validating amh gene number in D. pectoralis and U. pygmaea

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To check whether the two copies of amh were not collapsed into a single sequence during assembly, we computed the total number of apparent heterozygous sites in the amh region in population genomic data with the expectation that the presence of two divergent gene copies should result in high apparent heterozygosity when remapped on the single copy assembled in the genome. We compared these heterozygosity data in species where we only identified one amh gene, that is D. pectoralis and U. pygmaea, to results observed when mapping sex-specific pooled libraries of E. lucius (n = 30 males and 30 females, respectively) to a female reference genome containing only amha (GCA_004634155.1). Because there are two amh genes in male genomes of E. lucius and one amh gene in female genomes, this result from mapping E. lucius sex-specific Pool-Seq reads serves as a reference for expected number of variant sites. Reads from the male and female pools of E. lucius and from male pool of D. pectoralis were aligned separately to the reference genome (GCA_004634155.1) and our draft genome, respectively, using BWA mem version 0.7.17 (Li and Durbin, 2009) with default parameters. Each resulting BAM file was sorted and PCR duplicates were removed using Picard tools version 2.18.2 (http://broadinstitute.github.io/picard) with default parameters. Variants were then called for each BAM file using bcftools mpileup version 1.9 (Li et al., 2009) with default parameters on genomic regions containing amh, which locates between 12,906,561 and 12,909,640 bp on LG08 of E. lucius (GCA_004634155.1: CM015581.1), between 23,447 and 25,910 bp on the flattened_line_2941 contig of the draft genome of D. pectoralis, and between 760,763 and 757,962 bp on contig 633485 of our draft genome of U. pygmaea.

Gene phylogenetic analysis

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Phylogenetic reconstructions were performed on all amh homolog sequences obtained from Esociformes with the S. salar amh used as an outgroup. Full-length CDS were predicted with the FGENESH+ (Solovyev et al., 2006) suite based on the genomic sequence and Amh protein sequence from E. lucius. Sequence alignments were performed with MAFFT (version 7.450) (Katoh and Standley, 2013). Both maximum-likelihood and Bayesian methods were used for tree construction with IQ-TREE (version 1.6.7) (Minh et al., 2020) and Phylobayes (version 4.1) (Lartillot et al., 2007), respectively.

Additional analyses were carried with or without the truncated amh/Amh sequences of E. niger and N. hubbsi using the amh/Amh homolog of S. salar as an outgroup. CDS and proteins were predicted with the FGENESH+ suite (Solovyev et al., 2006), based on genomic sequences. These putative CDS and protein sequences were then aligned using MAFFT (version 7.450) (Katoh and Standley, 2013). Residue-wise confidence scores were computed with GUIDANCE 2 (Sela et al., 2015), and only well-aligned residues with confidence scores above 0.99 were retained. The resulting alignment file was used for model selection and tree inference with IQ-TREE (version 1.6.7) (Minh et al., 2020) with 1000 bootstraps and the 1000 SH-like approximate likelihood ratio test for robustness. To verify that the topology of the single amh homolog of D. pectoralis was not an artifact of long-branch attraction, we also constructed phylogenetic trees with only the first and second codons of the coding sequences (Lemey et al., 2009) as well as with complete protein sequences. For additional confirmation of the tree topology for the amh homologs, the same alignments were run in a Bayesian framework implemented in Phylobayes (version 4.1), (Lartillot et al., 2007; Lartillot and Philippe, 2006, Lartillot and Philippe, 2004) using the CAT-GTR model with default parameters, and two chains were run in parallel for approximately 1000 cycles until the average standard deviation of split frequencies remained ≤0.01.

Detection of signatures of selection on amha and amhby genes

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To compare selection pressure between amha and amhby genes, dN/dS ratios were computed between each ortholog sequence and the amh sequence of U. pygmaea, which diverged from the other Esociformes prior to the amh duplication. Sequences from E. niger and N. hubbsi were excluded because their amhby orthologs were substantially shorter and could introduce bias in the analysis. The ratio of nonsynonymous to synonymous substitution (dN/dS = ω) among amha and amhby sequences was estimated using PALM (version 4.7) (Yang, 2007) based on aligned full-length CDS, and the phylogenetic tree obtained with the CDS was used in the analysis. First, a relative rate test on amino acid substitution (Hughes, 1994) was performed between amha and amhby pairs with the amh of U. pygmaea used as an outgroup sequence. For each species with amh duplication, ω was calculated between the amha ortholog and amh of U. pygmaea and was compared to the ω calculated between the amhby ortholog and amh of U. pygmaea. A Wilcoxon test was used to compare the resulting ω values for the amh paralogs of these species. Then, several branch and site models (M0, M1a, M2a, M7, M8, free-ratio, and branch-site) implemented in the CODEML package were fitted to the data. The goodness of fit of these models was compared using the likelihood ratio test implemented in PALM.

Genome assembly

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Raw Illumina sequencing reads for D. pectoralis, E. masquinongy, E. niger, and N. hubbsi were assembled using the DISCOVAR de novo software with the following assembly parameters: MAX_MEM_GB = 256, MEMORY_CHECK = False, and NUM_THREADS = 16. Assembly metrics were calculated with the assemblathon_stats.pl script (Earl et al., 2011), and assembly completeness was assessed with BUSCO (version 3.0.2) (Simão et al., 2015) using the Actinopterygii gene set (Supplementary file 10).

To facilitate the comparison of sex loci in different populations of E. lucius, we improved the continuity of the previously published assembly of an E. lucius European male (GenBank assembly accession: GCA_007844535.1). This updated assembly was performed with Flye (version 2.6) (Kolmogorov et al., 2019) using standard parameters and genome-size set to 1.1 g to match theoretical expectations (Hardie and Hebert, 2004). Two rounds of polishing were performed with Racon (version 1.4.10) (Vaser et al., 2017) using default settings and the Nanopore reads aligned to the assembly with minimap2 (version 2.17) (Li, 2018) with the ‘map-ont’ preset. Then, three additional rounds of polishing were performed with Pilon (version 1.23) (Walker et al., 2014) using the ‘fix all’ setting and the Illumina reads aligned to the assembly with BWA mem (version 0.7.17) (Li and Durbin, 2009). Metrics for the resulting assembly were calculated with the assemblathon_stats.pl script (Earl et al., 2011). The completeness of the assembly was assessed with BUSCO (version 3.0.2) (Simão et al., 2015) using the Actinopterygii gene set (4584 genes) and the default gene model for Augustus. The resulting assembly was scaffolded with RaGOO (version 1.1) (Alonge et al., 2019) using the published female genome (GenBank accession: GCA_011004845.1) anchored to chromosomes as a reference.

Appendix 1

Appendix 1—figure 1
Additional phylogenetic reconstruction of amha and amhby orthologs from the Esociformes with amh sequence from Salmo salar as an outgroup.

(A) Phylogenetic tree built by Maximum likelihood method implemented in IQ-TREE putative protein sequences of all identified amh homologs. (B) Phylogenetic tree built by Maximum likelihood method with only the 1st and 2nd codon of putative coding sequence of all identified amh homologs. (C) Phylogenetic tree built by Maximum likelihood with putative protein sequences of amh homologs without one highly truncated sequence from E. niger. (D) Phylogenetic trees built by Maximum likelihood with putative coding sequences of amh homologs without one highly truncated sequence from E. niger. (E) Phylogenetic tree built by Bayesian method implemented in PhyloBayes with putative protein sequences of all identified amh homologs. (F) Phylogenetic tree built by Bayesian method with putative coding sequences of all identified amh homologs. Bootstrap values are given on each nod of the tree and all trees are rooted with amh sequence from S. salar.

Appendix 1—figure 2
Analysis of sex differentiation in the genome of a male E. masquinongy with Pool-Seq data.

(A) The relative to genome average coverage depth of male and female Pool-Seq reads on the region containing amhby. The male data are represented in blue and female in red. The 3 kb region containing amhby (unplaced_scaffold_RaGOO_chr0: 24,967,172–24,968,938) is covered by virtually no female reads and by male reads at a depth about half of the genome average depth. (B) Number of male-specific SNPs in 50 kb non-overlapping windows in each linkage group in the male genome of E. masquinongy. The window containing the highest number of male-specific SNPs on LG24 is highlighted. (C) Number of male and female-specific SNPs from Pool-Seq in 50 kb non-overlapping windows is plotted along LG. we found the highest number of male-specific SNPs (MSS) in a single window located around 100–150 kb on LG24 that contained 212 MSS and zero female-specific SNPs, while genome average was 1.57 MSS per 50 kb window (3.17 MSS per window when excluding 50 kb windows without MSS).This result indicates that the sex locus in E. masquinongy is homologous to the proximal end of LG24 in E. lucius. Besides LG24, no other linkage group contained a window enriched with MSS. Overall, this Pool-Seq analysis confirms the XX/XY SD system with a low differentiation between the X and Y chromosomes identified with the sex linkage and RAD-Seq analyses, as observed in E. lucius (Pan et al., 2019) and N. hubbsi (see above), and supports the hypothesis that the sex locus of E. masquinongy is similar to that of E. lucius.

Appendix 1—figure 3
No differentiation between male and female genomes revealed by RAD-Seq and Pool-Seq in some North American populations of E. lucius.

(A-B) Distribution of RADSex markers in males and females of two populations of E. lucius from Canada. The distribution of markers in males and females was computed with RADSex with a minimum depth of 5 to consider a marker as present in an individual for both datasets. In each tile plot, the number of males and number of females are represented on the horizontal and vertical axes respectively, and intensity of color of a tile indicates the number of markers present in the corresponding number of males and females. In both populations, we did not find any sex-linked markers among a total of 8,440,899 and 4,526,552 markers identified in each population, indicating that if there is a new sex locus in these populations its detection escapes the resolution of RAD-Seq (31.8 RAD markers per Mb on average) due to a very low differentiation between the new sex chromosome pair. (C–E) Analysis of male/female differentiation across Canadian E. lucius genome with Pool-Seq reads from 30 males and 30 females mapped to the reference genome (Accession number: GCA_004634155.1). Between sex FST (C), female-specific SNPs (D) and male-specific SNPs (E) are computed for 50 kb non-overlapping windows across the 25 linkage groups (LGs) and unplaced scaffolds. We searched for 50 kb non-overlapping windows enriched with either male or female-specific SNP. Overall, the level of differentiation between males and females was low, and the highest Fst observed across the entire genome is 0.07 located between 0.397 Mb and 0.398 Mb on linkage group 11. Very low number of sex-specific SNPs were found for both male and female pools in this population, especially when comparing to the same analysis performed with data from an European population. In the European population, we found a genome average of 3.5 male-specific and 2.7 female-specific SNPs per 50 kb window, with peak windows containing 625 male-specific SNPs near the LG24 sex locus, and 217 female-specific SNPs at the proximal end of LG5. In comparison, the Canadian pools had roughly similar values of average genome sex-specific SNPs (3.3 male-specific and 2.9 female-specific SNPs per window), but with peak windows of sex-specific SNPs of 49 and 45 for males and females, respectively. Given this low amount of differentiation between the sexes, and that no particular chromosome was enriched with windows containing a high number of sex-specific SNPs, no region stood out to be the candidate region for the sex locus. Besides that, no 1 kb window with sex-specific read depth was found. Overall, no clear signal of a sex locus was observed across the entire genome supporting the hypothesis that the mainland USA and Canadian populations lack a well-differentiated sex determining region.

Appendix 1—figure 4
Sanger sequencing results for E. masquinongy one female and one male carrying two different alleles of amhby and one male carrying only one amhby allele.

The base with a bi-allelic SNP is highlighted with the red dashed-line box.

Appendix 1—figure 5
RAD-Seq analysis in a second Esox masquinongy population from Quebec (Canada) supports the lack of sex-specific marker in this population.

Each tile plot shows the distribution of non-polymorphic RAD-Seq markers between phenotypic males (horizontal axis) and phenotypic females (vertical axis). The intensity of color for a tile corresponds to the number of markers present in the respective number of males and females. No tiles for which the association with phenotypic sex is significant (chi-square test, p<0.05 after Bonferroni correction) were detected in this analysis. RAD-Seq reads data and samples information are found under NCBI bioproject PRJNA512459.

Appendix 1—figure 6
ClustalW alignment of Esocidae Amhby putative protein sequences.

The signal peptide was predicted with SignalP (Nielsen, 2017). No signal peptide was detected in the Amhby sequences of both E. niger and N. hubbsi. N-terminal region (indicated by a black bar) and the Transforming growth factor beta like domain (indicated by a red bar) were predicted with the Motif Scan tool at ExPASy (Gasteiger et al., 2003) with the Pfam motif database (Finn et al., 2014) optimized for local alignments (pfam-fs, Pfam 32.0, September 2018). The seven exons of amhby are shown by the alternating blue and yellow colors on the sequence ruler. The seven conserved cysteines of the TGF-beta domain are indicated by red arrowheads. The region containing the putative Amh cleavage site (Cleav) is boxed in red. E. luc (Esox lucius), E. aqu (E. aquitanicus), E. cis (E. cisalpinus), E. rei (E. reichertii), E. mas (E. masquinongy), E. aam (E. americanus americanus), E. ave (E. americanus vermiculatus), E. nig (E. niger) and N. hub (Novumbra hubbsi).

Appendix 1—figure 7
Hypothesized scenarios of sex determination system transition in North American populations of E. lucius.

In the ancestral population, Linkage group 24 (LG24) was the sex chromosome (Y). Males were the heterogametic sex carrying one Y chromosome with a small (~300 kb) sex-locus region (SLR) containing amhby and one X chromosome without the SLR and amhby, while females carried two X chromosomes. During post-glaciation re-colonization of North America, the Y chromosome with amhby gene was not carried by individuals that made up the small populations that dispersed out of Alaska. These populations could still produce phenotypic males in the absence of a master sex determining gene via environment-induced sex reversal, a well-documented phenomenon among teleosts. In this transitional state, both males and females carry two X sex chromosomes (LG24). The current sex determination mechanism in these North American populations is still unknown: it could rely entirely on environmental sex determination (ESD), random sex determination, or new male (XY) or female (ZW) heterogametic genetic sex determination system with highly undifferentiated sex chromosomes. SLR: sex-locus region. ESD: environmental sex determination. NA: North America. LGN: un-specified Linkage group, that is ancestral autosomes (A) that are not LG24.

References

    1. Beukeboom LW
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    In: A Fostier, editors. Sex Control in Aquaculture. John Wiley & Sons. pp. 249–280.
    https://doi.org/10.1002/9781119127291.ch11
    1. Hardie DC
    2. Hebert PDN
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    Canadian Journal of Fisheries and Aquatic Sciences 61:1636–1646.
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    1. Navara KJ
    (2018) Mechanisms of Environmental Sex Determination in Fish, Amphibians, and Reptiles
    In: K. J Navara, editors. Choosing Sexes: Mechanisms and Adaptive Patterns of Sex Allocation in Vertebrates, Fascinating Life Sciences. Springer International Publishing. pp. 213–240.
    https://doi.org/10.1007/978-3-319-71271-0_10
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    Predicting Secretory Proteins with SignalP. InProtein Function Prediction
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    Evolution of Sex Determining Genes in Fish Encyclopedia of Reproduction
    Laboratoire de Physiologie et Génomique des Poissons.
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Decision letter

  1. Molly Przeworski
    Reviewing Editor; Columbia University, United States
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Developmental Biology, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This paper investigates the evolution of a sex determination system in closely related species of Esociformes, focusing on the fate of a known master sex determining locus (SDL). By carefully tracing the evolution of this gene in an entire order of fishes, the work provides a more integrative picture of SDL evolution than previously available for any group. The authors suggest that genetic drift and in particular bottlenecks can facilitate loss of master SDLs in natural populations.

Decision letter after peer review:

Thank you for submitting your article "The rise and fall of the ancient northern pike master sex determining gene" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The reviewers have opted to remain anonymous.

As you will see from the comments, the reviewers find the results of interest and would be happy to see them published. However, there was also unanimity that the writing requires considerable work: several points are imprecise or very difficult to parse and the structure is needlessly repetitive. We include individual comments below to guide you in what we expect will be fairly extensive revisions to the text.

Reviewer #2:

The manuscript presents the analysis of the evolution of the sex chromosomes across an entire teleost order. Specifically, the authors trace the evolution of a master sex determining gene (amhby), allowing to test recent hypothesis on how the birth of master sex determining (MSD) genes drive sex chromosome turnover in vertebrates. Data so far on evolution of sex-determination is based on distantly related clades, making it challenging to infer evolutionary pathways because data is obscured by large evolutionary times. Here the authors study closely related teleost species exhibiting sex determining (SD) system transitions, which allow to gain important insights on causes and mechanisms underlying SD turnover. Clearly, this is a topic that is a adapted to the broad readership of eLife.

The authors use draft genome assemblies and population genomic datasets in several species which allows them to investigate the evolution of amhby as a master SD gene across 65-90 MY. They propose that drift, exacerbated by bottleneck effects, can facilitate loss of MSD genes in wild populations. This is an interesting idea, that would provide a mechanism for the observed loss of sex chromosomes in some vertebrates. Overall the manuscript is well written and provides a wealth of interesting data and ideas. I believe the work in principle deserves publication in eLife. I have very few major comments, except that I find the Discussion a bit unstructured and lengthy and would be worth separating in sections. I would also like to see clear arguments supporting the XY -ZW system transition in Dallia pectoralis, I worry that just a few female markers from the RAD-seq are insufficient.

Reviewer #3:

Previous studies have found turnovers of sex-determining systems in fish, including Oryzias, tilapiine cichlids, salmonids, and sticklebacks, and this study uses another fish taxon, the order Esociformes, an old teleost group in which the amhby gene was known from previous studies by Pan's group to be the sex-determining gene. The study shows that, in 2 species, this gene does not behave as expected in all populations. The manuscript is well organized, but repeats things unnecessarily, and the only new information in the long Discussion is the statement about the amhby gene having "undergone an accelerated evolution", but I did not find the evidence for this described.

The manuscript suffers from creating the impression that these fish have Y chromosomes (which readers will understand to mean non-recombining chromosomes, or at least chromosomes with substantial non-recombining regions). This is not the case, and the Abstract and the text should not claim "Y-chromosome loss" or very recent loss of "the entire Y-chromosome". This suggests that there has been a Y-chromosome that became completely degenerated and was lost, creating an X0 sex-determining system. This is not the case, as the title is clear that this is not about loss of an entire Y chromosome. Instead, a single male-determining gene seems to characterize this fish group, and it has sometimes been lost, presumably in species that nevertheless have genetic sex-determination (though the text fails to make this clear). Overall, the study adds another example of changes in sex-determining genes in fish, so the results are less exciting than the text suggests,.

Confusion between Y chromosomes and sex linkage occurs in several parts of the text. For example, the start of subsection “Sex-linkage of amhby in the Esocidae lineage” should probably read simply "We tested whether the presence of amhby was strictly associated with the male phenotype, which indeed proved to be the case in populations of many of the species investigated in the genus Esox and Novumbra". As the associations were not significant in some cases, the text should not say "in all species". This association shows that amhby is probably the sex-determining gene in these species, but not that there is a non-recombining region that might be termed a Y-linked region or a Y chromosome.

Another related confusion is seen where the text mentions accumulation of deleterious mutations in the non-recombining sex-determining region. In the fish under study, however, there appear to be no non-recombining regions, so the reference to this is merely confusing to the reader.

The discussion of situations that favour changes in sex-determining genes could be replaced by citing a couple of reviews, as this study did not test any of the ideas, and is purely descriptive. In addition, this section speculates that genetic drift during a strong population bottleneck following postglacial dispersal might have facilitated the loss of the MSD gene (the text should not say "loss of the Y chromosome along with its MSD gene", as the chromosome is presumably not lost at all, and presumably the real meaning is that such an event might, for some reason have led to a turnover event). While a bottleneck may indeed be the reason for a change in the sex determining gene, and may have involved XX males due to sex reversal (as sometimes seen in captive E. lucius) after a colonization event that resulted in an all-female population, a single observation cannot be considered evidence in support of the hypothesis. Moreover, this suggestion is not a case of genetic drift, as stated, because it clearly involves selection favouring the ability of some XX genotypes to function as males.

Reviewer #4:

This paper presents interesting results from a family of fishes about the gain and loss of a master sex determining gene, one of the first studies of this kind. The conclusions are generally well justified and I would support publication after major revisions. The main problem is the writing, which has problems so distracting that at times it is hard to follow the point being made.

1) There are a large number of grammatical and stylistic errors. Two examples occur in the very first sentence of the Abstract:

• "Sex determination is an evolutionary highly dynamic process…." No, sex determination is a developmental process. The phrase "evolutionary highly dynamic process" is both factually wrong in many taxa (such as mammals) and grammatically incorrect.

• "… this dynamic evolution requires…" By definition, all evolution is dynamic.

It is very evident that different authors have written different sections. The Introduction and start of the Results are well-written, for example, but the other sections (e.g. the Discussion) are much weaker. The paper needs to be rewritten in a consistent style by a senior author with a command of the English language.

2) The organization of the paper is awkward and makes the story hard to follow. The two sections with big phylogenetic pictures ("Sex-linkage of amhby in the Esocidae lineage" and "Whole genome analyses of the evolution of sex determination") are interupted by sections that delve into two individual species (Esox lucius and Esox masquinongy). I don't know whether simply reordering the sections will fix the problem or whether some other organizational scheme would be best.

3) The Discussion is confusing. The Discussion paragraphs three and four are particularly rambling and disorganized. It's odd that ESD is not mentioned here.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "The rise and fall of the ancient northern pike master sex determining gene" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mark Kirkpatrick (Reviewer #4).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This paper investigates the evolution of a sex determination system in closely related species of Esociformes, focusing on the rise and fall of a known master sex determining gene. By carefully tracing the evolution of this gene in an entire order of fish, it provides a bigger evolutionary picture than previously available for any group. The authors suggest that genetic drift and in particular bottlenecks can facilitate loss of master sex determination genes in natural populations.

Two of the reviewers appreciated the careful attention paid to their previous suggestions and concerns, and had no more comments, other than a minor tweak: "in subsection “Some populations of Esox lucius lost their Y chromosome and ancestral

master sex determining gene”, many Canadian readers will not be amused to see Quebec described as American." A third reviewer had more extensive suggestions about how the manuscript might be reframed; we leave it to your discretion to what extent you'd like to incorporate these in a revised version.

Reviewer #3:

Although the sentence should be revised to make the English correct, the conclusions described in the Abstract are potentially interesting: that (i) the sex-determining gene in the northern pike (Esox lucius) originated from a duplication at least 65 to 90 million years ago, and remained sex-linked for at least 56 million years in multiple related species without the chromosomes becoming differentiated into heteromorphic sex chromosome-like haplotypes, and that (ii) several independent species- or population-specific sex determination transitions have also occurred, including a recent loss of all genes in the Y-linked region of the E. lucius sex chromosome. However, the text does not have a clear conceptual focus on result (i), which is probably the most interesting result. I believe that long-term maintenance of small sex-linked regions has not previously been documented, although claims are made that cases exist, and such claims are plausible, in principle. Yet the manuscript appears not to mention previous suggestions, or explain that the case in pike may be the first adequately documented one. The lack of a conceptual focus should be corrected by a further revision. However, viewing this as the most interesting conclusion focuses attention on the evidence supporting it, I am not convinced that the conclusion is indeed supported. An alternative possibility is that the duplication arose long ago, and has independently, and more recently, become a sex-determining gene, and this is not excluded, as far as I can see. I highlight in my comments below some internal evidence that seems to support this alternative. The authors should be encouraged to think more carefully about this conclusion and whether the alternative can be excluded.

The Introduction, and much of the focus, is concentrated on result (ii) that turnovers appear to have occurred. However, turnovers are already well documented in many different taxa, and reporting new ones is less novel than conclusion (i). The Introduction has a lengthy section about the "limited option" hypothesis, which is almost untestable, as it has never seemed likely that the number of genes that could be involved in turnover events could be unlimited, but is much more likely that only a limited set of genes could have such functions. What is especially interesting about the evolution of sex-determination in fish such as the Esociformes is the possibility that, although many fish have young sex-linked regions that evolved by turnovers, some might have ancient ones. The Esociformes are stated to (i) have genetic sex- determination and (ii) to have diverged from the Salmoniformes (which are presumably the closest related group, though this is not stated explicitly) about 110 million years ago, making them of interest in relation to this question.

On this view, the main question is whether the evidence shows convincingly that the same gene as in E. lucius has remained the sex-determining in multiple related species, and still forms a small Y-linked region, as in E. lucius, where it is called amhby, and is a ~ 140 kb male-specific insertion of an autosomal gene, amha. If so, it is of interest to ask whether the chromosome region has become differentiated into a larger heteromorphic sex chromosome-like region, with a pair of haplotypes.

The relationships and taxonomy of the species are not well explained. The Introduction should explain that the two families (Esocidae and Umbridae) and 13 well-recognized species include the Esocidae species in the Esox and Novumbra genera studied (when these genus names are mentioned, we have not been told that they come from the Esocidae, and readers should not have to guess whether they are from the two separate families mentioned earlier, or the same one; it would be helpful to refer explicitly to the figure that shows the species phylogeny – I think it is Figure 1, but the figures are not labelled with their numbers). The important point readers need to understand at this place in the text is that all 7 Esox species and one Novumbra species surveyed (N. hubbsi) had two amh genes, whereas only one was found in two species, Dalliapectoralis and Umbra pygmaea, representing more basally diverging, outgroup species. This result suggests that Esox and Novumbra species could both have an amhby gene, as well as an amha one. Then we need to have the evidence explained that Dallia pectoralis and Umbra pygmaea have only the amha one (information is confusingly revealed in this and the next paragraph). It is misleading to write "all Novumbra species" unless more than one was surveyed, which Table 1 suggests was not the case. I also did not understand why the sentence says "However, two amh transcripts were readily identified in E. lucius (Pan et al., 2019), as well as in whole genome sequencing reads and assemblies (Supplementary file 1)". This sentence seems to confirm what was already stated above about E. Lucius, and perhaps it should be moved to an earlier place, saying "Moreover" rather than "However".

However, the next paragraph says that phylogenetic trees of the amh sequences showed "two well-supported clusters in all Esox, Dallia and Novumbra species", suggesting Dallia has both copies. Some conclusions about these species are based on limited information in transcriptome databases, and some on other information, and the evidence should be more clearly and coherently explained.

At this point, if I have guessed the correct meaning, I think that all Esox species, plus one species each of the genera Novumbra (but not Dallia and Umbra), have sequences that cluster with the E. lucius amha one, and are likely to be orthologs of this gene, and also sequences that cluster with the E. luciusamhby one, suggesting that the amh gene duplication preceded the split between the genera Esox and Novumbra, but perhaps occurred after the split from U. pygmaea (and perhaps Dallia). The text says that the "duplication happened after the divergence of Esocidae and Umbridae lineages", which suggests that Novumbra (with both copies) is not in the Umbridae. Please can the relationships be made clear before the results from them are described.

Clearly, the important questions are now whether the amhby copies are sex-determining genes in all species that carry the duplicate copy, and whether they acquired this function in the common ancestor of Esox and Novumbra, and retained this ever since. To test this, the study tested whether amhby was found in all males and absent from all females, in species additional to the E. lucius sample previously studied.

The tests indeed detected associations between the male phenotype and the presence of amhby in most species of Esox (Table 1), but the associations were absent or weak in some Esox samples, including some North American E. lucius populations, and incomplete in the Novumbra species sample.

Overall, it is clear that the duplication is ancient, and pre-dates the split between Esox and Novumbra, but there does not seem to be strong evidence for the conclusion that it has maintained a male-determining function since the duplication occurred. The alternative cannot be excluded that the duplicate copy has acquired a sex-determining function independently in Esox and Novumbra, as well as losing amhb several times. In fact, this study seems a nice example of how much evidence is needed before one can be sure that a sex-determining gene has been maintained for a long time.

The claim that the same region has remained as an undifferentiated sex-determining or Y-linked region for a long evolutionary time comes from the Pool-Seq analysis of E. masquinongy, one of the species with an incomplete association between the presence of a copy of the amh duplicate, to compare its size and location (as a putative sex locus) with that of E. lucius. Male-specific variants (described rather vaguely as "sex-specific heterozygosity") were detected in just a single genome region of less than 50 kb, and the region is homologous to the proximal end of the E. lucius LG24, where its sex-determining locus is located. This indeed suggests that both E. lucius and E. masquinongy have a physically small male-determining locus at the same location. However, it appears to have a weakened or partially lost function in E. masquinongy.

However, the results do not tell us that they the male-determining function is ancestral, rather than independently evolved, in Esox and Novumbra. Indeed, subsection “Evolution of the structure of the amhby gene in the Esocidae” reports information that may suggest independent evolution in E. niger (in which amhby is strongly associated with maleness) and N. hubbsi. In both, the predicted Amhby protein is truncated in regions known to be important for this gene's function, but in E. niger the truncation is in its C-terminal part, whereas it affects the N-terminal part of the N. hubbsi sequence, and exon 1 encodes only eight amino acids, with no homology to the amino acid-sequence in other Esocidae. It is not explained whether the E. lucius amhby copy is complete, or how it differs from amha. On the interpretation that these changes evolved independently, the similarity of other parts of the sequence (which provides the signal in the phylogeny) is potentially rather misleading.

The manuscript also reports evidence that sex-determining systems evolved independently in the 2 species that do not have the amh duplicate, D. pectoralis and U. pygmaea.

It then turns to plausible suggestions about why Esox species might lose their male-determining factor. This section is rather unorganised, and should be greatly shortened to make clear what hypotheses were tested, and what the results were. Loss of genetic sex-determination is plausible in colonizing or low density situations, and the findings should be related to this concept.

https://doi.org/10.7554/eLife.62858.sa1

Author response

Reviewer #2:

The manuscript presents the analysis of the evolution of the sex chromosomes across an entire teleost order. Specifically, the authors trace the evolution of a master sex determining gene (amhby), allowing to test recent hypothesis on how the birth of master sex determining (MSD) genes drive sex chromosome turnover in vertebrates. Data so far on evolution of sex-determination is based on distantly related clades, making it challenging to infer evolutionary pathways because data is obscured by large evolutionary times. Here the authors study closely related teleost species exhibiting sex determining (SD) system transitions, which allow to gain important insights on causes and mechanisms underlying SD turnover. Clearly, this is a topic that is a adapted to the broad readership of eLife.

The authors use draft genome assemblies and population genomic datasets in several species which allows them to investigate the evolution of amhby as a master SD gene across 65-90 MY. They propose that drift, exacerbated by bottleneck effects, can facilitate loss of MSD genes in wild populations. This is an interesting idea, that would provide a mechanism for the observed loss of sex chromosomes in some vertebrates. Overall the manuscript is well written and provides a wealth of interesting data and ideas. I believe the work in principle deserves publication in eLife. I have very few major comments, except that I find the Discussion a bit unstructured and lengthy and would be worth separating in sections. I would also like to see clear arguments supporting the XY -ZW system transition in Dallia pectoralis, I worry that just a few female markers from the RAD-seq are insufficient.

The manuscript has been deeply revised to incorporate supplementary notes that are not allowed by the eLife format and also indeed to take into consideration reviewers' comments. The Discussion has been shortened and organized.

Concerning the Dallia pectoralis sex determination system, we now provide results from an independent Pool-Seq analysis supporting the existence of female-specific sequences in the Dallia genome. Results are in agreement with the ZZ/ZW SD system revealed by the RAD-seq analysis. We answered this comment in more detail below.

Reviewer #3:

Previous studies have found turnovers of sex-determining systems in fish, including Oryzias, tilapiine cichlids, salmonids, and sticklebacks, and this study uses another fish taxon, the order Esociformes, an old teleost group in which the amhby gene was known from previous studies by Pan's group to be the sex-determining gene. The study shows that, in 2 species, this gene does not behave as expected in all populations. The manuscript is well organized, but repeats things unnecessarily, and the only new information in the long Discussion is the statement about the amhby gene having "undergone an accelerated evolution", but I did not find the evidence for this described.

As mentioned above, the manuscript has been thoroughly revised to take into consideration all reviewers' comments and to incorporate our previous supplementary notes that are not allowed by the eLife format. The Results section has been changed to incorporate some information that was previously in the supplementary notes and the Discussion has been greatly shortened.

Regarding the statement “undergone an accelerated evolution”, we agree that it is not a result we have support for, and we then thus changed our sentence to “ The northern pike MSD gene amhby substantially diverges from its autosomal paralog amha, suggesting an ancient origin (Pan et al., 2019), unlike other cases of amh duplication in fishes which appear to be comparatively young (Hattori et al., 2013; Li et al., 2015)”. We attribute the high level of sequence divergence between amhby and its autosomal paralog amha to ancient origin rather than to accelerated evolution.

The manuscript suffers from creating the impression that these fish have Y chromosomes (which readers will understand to mean non-recombining chromosomes, or at least chromosomes with substantial non-recombining regions). This is not the case, and the Abstract and the text should not claim "Y-chromosome loss" or very recent loss of "the entire Y-chromosome". This suggests that there has been a Y-chromosome that became completely degenerated and was lost, creating an X0 sex-determining system. This is not the case, as the title is clear that this is not about loss of an entire Y chromosome. Instead, a single male-determining gene seems to characterize this fish group, and it has sometimes been lost, presumably in species that nevertheless have genetic sex-determination (though the text fails to make this clear). Overall, the study adds another example of changes in sex-determining genes in fish, so the results are less exciting than the text suggests,.

The reviewer’s comment revolves around the definition of a sex chromosome. A sex chromosome is generally defined as a chromosome that contains a major sex determining genetic factor that controls whether an individual becomes a male or female. It does not mean that one of the sex chromosome pairs has to be nearly devoid of genes that do not directly control sex, even though this is true of the mammalian Y chromosome. In our previous study, besides the identification and functional demonstration of amhby as the MSD gene in northern pike, we also identified a 300kb male specific region on a specific chromosome and demonstrated an absence of recombination in male around this region with linkage mapping. According to the generally accepted definition of a sex chromosome, this then would be a Y chromosome even though it shares a substantial number of genes with the X chromosome. Although this species doesn’t not have cytologically heteromorphic sex chromosomes, it thus has a Y-chromosome with a small, yet clearly Y-specific region. In this current study, when we say the loss of the Y-chromosome, we are not implying that it was replaced by a X-O system, but rather that all the 300kb of Y-specific sequences are absent in the North American populations of northern pike. This loss is not just limited to a single male-determining gene but to the entire, albeit small, Y-chromosome-specific sequence (inserted sequence on Y chromosome, as well as differentiated regions from the X chromosome). We have added Appendix 1—figure 7 to clarify this point. There is little divergence time (~8000 years) between the population with amhby (Alaska populations) and without amhby (Canada and mainland USA). Considering this, we suggest that this loss of all Y-specific sequences could have been caused by having all founders in a bottle-necked population being genetic females (XX individuals), some of which were sex-reversed phenotypic males, during postglacial dispersal. Hence, no ancestral Y-chromosomes remained in these populations that re-colonized North America out of Alaska. We agree that it is probably another case of change in sex-determining gene in fish, however, we think that the potential mechanism underlying this change has not been described before as it puts forward a non-adaptive hypothesis for such a transition and highlights the importance of considering population demography in understanding the drivers for transitions in sex determination systems.

We realize, however, that we probably didn’t describe clearly enough our previous finding on the Y chromosome of E. lucius (Pan et al., 2019); thus, we added additional information in the Introduction : “Previously, we demonstrated that a male-specific duplication of the anti-Müllerian hormone gene (amhby) is the MSD gene in Esociformes, and that this gene is located in a small, ~ 140 kb male-specific insertion on the Y chromosome of northern pike (Esox lucius) (Pan et al., 2019).”

Confusion between Y chromosomes and sex linkage occurs in several parts of the text. For example, the start of subsection “Sex-linkage of amhby in the Esocidae lineage” should probably read simply "We tested whether the presence of amhby was strictly associated with the male phenotype, which indeed proved to be the case in populations of many of the species investigated in the genus Esox and Novumbra". As the associations were not significant in some cases, the text should not say "in all species". This association shows that amhby is probably the sex-determining gene in these species, but not that there is a non-recombining region that might be termed a Y-linked region or a Y chromosome.

We agree we cannot say that a “significant association” is found in all species given the small sample size for the two species where we only had access to a limited number of museum samples. We have followed the reviewer’s suggestion and updated the text to “A significant association between male phenotype and the presence of amhby was found in most species investigated in the genus Esox and Novumbra”.

But we are confused over the reviewer’s view on the relationship between master sex determining genes and sex chromosomes. From both classic literature (Bull, 1983; Charlesworth et al., 2005) to more recent reviews on sex chromosome formation and evolution (Vicoso, 2019; Wright et al., 2016), new sex chromosomes arise after the acquisition of a new master sex-determining gene (if brought by a duplication / insertion event) or a new master sex determining allele (if brought by an allelic diversification process). From there, this new sex locus on a new sex chromosome can either become heteromorphic by extension of the non-recombining region around the original sex locus, or it might remain in its current state with as little difference from the X chromosome as one SNP on the sex-determining gene as found in Takifugu (Ieda et al., 2018; Kamiya et al., 2012). But whatever their differentiation level, both at the molecular and karyotype levels, a chromosome containing a sex locus is (always) considered as a sex chromosome. So, unless we missed something important, or we misunderstood the reviewer’s comment, we decided to stick with the idea that the presence of a master sex determination gene in one sex indirectly informs about sex determination systems and sex-chromosomes.

Another related confusion is seen where the text mentions accumulation of deleterious mutations in the non-recombining sex-determining region. In the fish under study, however, there appear to be no non-recombining regions, so the reference to this is merely confusing to the reader.

We agree that we only have evidence for that in the European populations of Northern pike (Pan et al., 2019), from our linkage map that is showing that recombination is suppressed around the 300kb of the Y-specific region on the proximal end of LG24. We agree that we do not know precisely the size of the sex locus in many of the species investigated, except for a very rough estimation based on number of RAD-Seq markers. However, these sequence differences between male and female genomes result from reduced recombination around the sex determining locus. If X and Y chromosomes are free to recombine in these fish, fixed differences between the male and female genomes would involve solely the few nucleotides specifically within the MSD gene itself. Nevertheless, to help readers, we updated our text to use either “regions of X-Y differentiation” or “Y-chromosome-specific regions” instead of non-recombining regions.

The discussion of situations that favour changes in sex-determining genes could be replaced by citing a couple of reviews, as this study did not test any of the ideas, and is purely descriptive.

We have followed the reviewer’s suggestion and shortened significantly discussions of our hypothesis of how the transitions could have happened.

In addition, this section speculates that genetic drift during a strong population bottleneck following postglacial dispersal might have facilitated the loss of the MSD gene (the text should not say "loss of the Y chromosome along with its MSD gene", as the chromosome is presumably not lost at all, and presumably the real meaning is that such an event might, for some reason have led to a turnover event). While a bottleneck may indeed be the reason for a change in the sex determining gene, and may have involved XX males due to sex reversal (as sometimes seen in captive E. lucius) after a colonization event that resulted in an all-female population, a single observation cannot be considered evidence in support of the hypothesis. Moreover, this suggestion is not a case of genetic drift, as stated, because it clearly involves selection favouring the ability of some XX genotypes to function as males.

Again, we have shown that this loss is not restricted to just to the MSD gene, but to the entire sex locus with at least 140kb of Y-chromosome specific sequences. Indeed, the former X chromosome (=LG24) is still present in the population and we never suggested that the X chromosome is in a hemizygous state in these populations. We changed large parts of our Discussion and we hope that it is more clearly stated now that it is not a transition from an XY to XO system but a rapid disappearance of the ancestral Y chromosome in some of the Northern American populations we survey. To clarify this point, we have added a diagram to better illustrate our hypothesis that can be found in Appendix 1—figure 7.

Regarding whether there is selection favoring the ability of some XX genotypes to function as males, we think this would be a process independent from the loss of the Y chromosome. We didn’t exclude the involvement of selection in generating a potential new mechanism for sex determination, but we are suggesting that the loss of the Y-chromosome was not due to selection (for instance, the “hot-potato” model of sex-chromosome turnover (Blaser et al., 2013) but to a sudden drift occurring in a bottlenecked population with environmental or stochastic sex reversal.

Reviewer #4:

This paper presents interesting results from a family of fishes about the gain and loss of a master sex determining gene, one of the first studies of this kind. The conclusions are generally well justified and I would support publication after major revisions. The main problem is the writing, which has problems so distracting that at times it is hard to follow the point being made.

As mentioned above, the manuscript has been thoroughly revised to take into consideration all reviewers' comments and to incorporate our previous supplementary notes that are not allowed by the eLife format. The Results section has been changed to incorporate some information that were previously in the supplementary notes and the Discussion has been greatly shortened.

1) There are a large number of grammatical and stylistic errors. Two examples occur in the very first sentence of the Abstract:

• "Sex determination is an evolutionary highly dynamic process…." No, sex determination is a developmental process. The phrase "evolutionary highly dynamic process" is both factually wrong in many taxa (such as mammals) and grammatically incorrect.

• "… this dynamic evolution requires…" By definition, all evolution is dynamic.

It is very evident that different authors have written different sections. The Introduction and start of the Results are well-written, for example, but the other sections (e.g. the Discussion) are much weaker. The paper needs to be rewritten in a consistent style by a senior author with a command of the English language.

Thanks for pointing out these mistakes in the Abstract. We have now changed the first sentence to “The evolution of sex determination mechanisms varies across taxa, and an understanding of this variation requires comparative studies among closely related species.”

The whole manuscript has been edited for style and grammar by native English-speaking senior co-authors.

2) The organization of the paper is awkward and makes the story hard to follow. The two sections with big phylogenetic pictures ("Sex-linkage of amhby in the Esocidae lineage" and "Whole genome analyses of the evolution of sex determination") are interupted by sections that delve into two individual species (Esox lucius and Esox masquinongy). I don't know whether simply reordering the sections will fix the problem or whether some other organizational scheme would be best.

We changed the ordering of these sections according to the reviewer’s suggestion. The placing of “Whole genome analyses of the evolution of sex determination" right after "Sex-linkage of amhby in the Esocidae lineage" was our original ordering of the sections and we agree it is probably more fluid to give the big picture before venturing into the population level of SD variation it the two species.

3) The Discussion is confusing. The Discussion paragraphs three and four are particularly rambling and disorganized. It's odd that ESD is not mentioned here.

We agree that our original Discussion was too long and not clearly structured. We have since substantially shortened the Discussion, especially for the paragraphs pointed out by the reviewer. We actually mentioned ESD, but it was probably buried by our “rambling”. The discussion on ESD can now be found in Discussion paragraph six.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

This paper investigates the evolution of a sex determination system in closely related species of Esociformes, focusing on the rise and fall of a known master sex determining gene. By carefully tracing the evolution of this gene in an entire order of fish, it provides a bigger evolutionary picture than previously available for any group. The authors suggest that genetic drift and in particular bottlenecks can facilitate loss of master sex determination genes in natural populations.

Two of the reviewers appreciated the careful attention paid to their previous suggestions and concerns, and had no more comments, other than a minor tweak: "in subsection “Some populations of Esox lucius lost their Y chromosome and ancestral

master sex determining gene”, many Canadian readers will not be amused to see Quebec described as American."

Thanks for this positive feedback on our revised manuscript. We have changed the text to read: “an American population that lacks amhby (Quebec, Canada)” to “ a North American population that lacks amhby (Quebec, Canada)” to make sure that Quebec is not to be confused as a part of America (USA), but the continent of North America.

A third reviewer had more extensive suggestions about how the manuscript might be reframed; we leave it to your discretion to what extent you'd like to incorporate these in a revised version.

We carefully read reviewer #3 comments and all his(her) “extensive suggestions”, and we tried to provide answers and to take into consideration his(her) suggestions to the largest possible extent without totally rewriting the manuscript

Reviewer #3:

Although the sentence should be revised to make the English correct,

We agree that some Abstract sentences were not easy to understand. This was due to last minutes changes to fit with the tight word count required for the Abstract during submission. We slightly rewrote the Abstract to clarify these sentences. This new Abstract now reads as:

“The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex-linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y-chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.”

the conclusions described in the Abstract are potentially interesting: that (i) the sex-determining gene in the northern pike (Esox lucius) originated from a duplication at least 65 to 90 million years ago, and remained sex-linked for at least 56 million years in multiple related species without the chromosomes becoming differentiated into heteromorphic sex chromosome-like haplotypes, and that (ii) several independent species- or population-specific sex determination transitions have also occurred, including a recent loss of all genes in the Y-linked region of the E. lucius sex chromosome. However, the text does not have a clear conceptual focus on result (i), which is probably the most interesting result.

We understand that some colleagues, like reviewer #3, will find point (i) more interesting than point (ii) and may be frustrated by the fact we did not develop further the discussion on this point. However, we do not think that point (i) should deserve more attention than point (ii), and our current manuscript currently provides a balanced discussion on these two points. As this prioritization is purely subjective we would prefer not changing our manuscript with regards to this comment.

I believe that long-term maintenance of small sex-linked regions has not previously been documented, although claims are made that cases exist, and such claims are plausible, in principle. Yet the manuscript appears not to mention previous suggestions, or explain that the case in pike may be the first adequately documented one.

To our knowledge there are a few known cases of long-term maintenance of small sex-linked regions in fish that we actually discussed in our manuscript like for instance in some Takifugu species in which a very small sex locus (restricted to a single causal SNP in the amhr2 gene, probably the smallest sex locus described in any species) has been conserved for more than 10 million years. See our Discussion “In line with our present results, substantially undifferentiated sex chromosomes have also been maintained over relatively long evolutionary periods in some Takifugu fish species (Kamiya et al., 2012) “. We then do not think that we can claim that the pike case is the first documented one.

The lack of a conceptual focus should be corrected by a further revision. However, viewing this as the most interesting conclusion focuses attention on the evidence supporting it, I am not convinced that the conclusion is indeed supported.

With respect to “long-term maintenance of small sex-linked regions” what we found is multiple species with a conserved MSD gene (amhby) and a small (or undifferentiated) sex locus. But we didn’t claim that it is a conserved ancestral locus as we only provided evidence that homologous regions containing amhby have been used as the sex locus in two species that diverged 45 million year ago i.e., E. lucius and E. masquinongy. As we don’t have a chromosome level genome assembly for E. masquinongy, we chose not to make inferences on the age of the sex chromosome or this shared sex locus. In other species we only found a conservation of the MSD gene, but this does not necessarily means that such a conserved ancestral MSD gene is located in a conserved sex locus and /or sex chromosome. For instance, in salmonids, the MSD gene sdY has been conserved over 50 millions year, but the linkage group hosting this MSD gene is different in many species.

An alternative possibility is that the duplication arose long ago, and has independently, and more recently, become a sex-determining gene, and this is not excluded, as far as I can see. I highlight in my comments below some internal evidence that seems to support this alternative. The authors should be encouraged to think more carefully about this conclusion and whether the alternative can be excluded.

We agree that in theory, amhby could have been recruited independently as a MSD gene long after the divergence of Esox and Novumbra. However, the hypothesis that amhby is the ancestral MSD is more parsimonious than amhby gaining function, independently and much more later, in the Esox and Novumbra lineage. We now discuss this point in the main text, which reads “Although we cannot rule out the possibility that amhby was recruited independently as the MSD gene in Esox and in Novumbra, our results suggest the more parsimonious hypothesis that amhby likely acquired an MSD function before the diversification of Esocidae at least 56 Mya.“

The Introduction, and much of the focus, is concentrated on result (ii) that turnovers appear to have occurred. However, turnovers are already well documented in many different taxa, and reporting new ones is less novel than conclusion (i).

As mentioned above, whether point (i) or point (ii) is more “novel” is purely subjective and we would prefer not changing our manuscript with regards to this point (i) prioritization. We also disagree with reviewer #3 idea that “turnovers are already well documented in many different taxa” and in our Introduction we wrote that “Although comparative studies have been accomplished in medakas, poeciliids, tilapiine cichlids, salmonids, and sticklebacks (Kikuchi and Hamaguchi, 2013), transitions of SD systems in relation to the fate of known MSD genes within closely related species have only been explored in medakas (Myosho et al., 2015) and salmonids (Guiguen et al., 2018).”. This clearly states that “turnovers” (SD transitions) have not been clearly documented in many teleost groups, and that outside, medakas and salmonids, most of these transitions were documented without any knowledge on the identity and the fate of the MSD gene.

The Introduction has a lengthy section about the "limited option" hypothesis, which is almost untestable, as it has never seemed likely that the number of genes that could be involved in turnover events could be unlimited, but is much more likely that only a limited set of genes could have such functions.

We agree with reviewer #3 that the “limited option” hypothesis is not a testable hypothesis but more a conceptual idea. We then changed the wording to “limited option” idea. However, as far as MSD gene turnovers are concerned, the list of potential candidates maybe bigger than reviewer #3 expectation (if his(her) expectation is that all genes with a key function in the sex differentiation network could be selected as a new MSD gene), as unexpected genes like the sdY MSD gene of salmonids can also evolve to become new MSD genes. Finding these unusual MSD genes may be just more difficult, leading to a hidden complexity that could actually challenge this “limited option” idea.

Concerning our lengthy section on the "limited option" hypothesis it is just a single sentence “These teleost MSD genes also provided empirical support for the “limited option” hypothesis that states that certain genes known to be implicated in sex differentiation pathways are more likely to be recruited as new MSD genes”, that is used to introduce the following idea that “The majority of these recently discovered MSD genes, however, were phylogenetically scattered, making it challenging to infer evolutionary patterns and conserved themes of sex chromosomes and / or MSD gene turnovers”. We then do not think that this is a particularly lengthy section and we would like to keep our text as it was.

What is especially interesting about the evolution of sex-determination in fish such as the Esociformes is the possibility that, although many fish have young sex-linked regions that evolved by turnovers, some might have ancient ones. The Esociformes are stated to (i) have genetic sex- determination and (ii) to have diverged from the Salmoniformes (which are presumably the closest related group, though this is not stated explicitly) about 110 million years ago, making them of interest in relation to this question.

We indeed agree with reviewer #3 that the evolution of sex determination is Esociformes is an interesting question. But we cannot introduce our manuscript by saying that Esociformes are known to have a genetic sex determination mechanism as this was not known before our present manuscript (with the exception of northern pike). We also think that the fact that salmoniformes are the closest order to Esociformes is not the main point that drove our study (may be just interesting in light of our results as they do not share the same MSD gene). Based on that we feel that we do not need to change this introduction part.

We however slightly changed one sentence of our Introduction to clearly mention that salmoniformes are the closest order to Esociformes in line with reviewer #3 suggestion. This sentence now reads as “Esociformes is a small order of teleost fishes (Figure 1) that diverged from their sister group Salmoniformes about 110 million years ago (Mya) and diversified from a common ancestor around 90 Mya (Campbell et al., 2013; Campbell and Lopéz, 2014) “.

On this view, the main question is whether the evidence shows convincingly that the same gene as in E. lucius has remained the sex-determining in multiple related species, and still forms a small Y-linked region, as in E. lucius, where it is called amhby, and is a ~ 140 kb male-specific insertion of an autosomal gene, amha. If so, it is of interest to ask whether the chromosome region has become differentiated into a larger heteromorphic sex chromosome-like region, with a pair of haplotypes.

We agree that the question on how an initial small sex locus on a sex chromosome can differentiate into a larger sex locus is indeed a very interesting question. However, the E. lucius sex locus (that we investigated more extensively in our previous paper (Pan et al. 2019) with both a linkage map to delineate the region of suppressed recombination and population data to explore X/Y differentiation) is probably not large enough to consider its sex chromosomes as heteromorphic at least if we follow a currently well accepted definition like the one given by Wright et al., 2016 (10.1038/ncomms12087) in which homomorphic sex chromosomes were defined as “sex-chromosomes that exhibit few differences from each other in size and gene content, and are difficult or impossible to distinguish from karyotype data alone”.

The relationships and taxonomy of the species are not well explained. The Introduction should explain that the two families (Esocidae and Umbridae) and 13 well-recognized species include the Esocidae species in the Esox and Novumbra genera studied (when these genus names are mentioned, we have not been told that they come from the Esocidae, and readers should not have to guess whether they are from the two separate families mentioned earlier, or the same one; it would be helpful to refer explicitly to the figure that shows the species phylogeny – I think it is Figure 1, but the figures are not labelled with their numbers).

We anticipated this taxonomy and species phylogeny difficulties for non (fish and pike) specialist readers and because of that we have dedicated the first main figure of the manuscript for this purpose. But to make it even more clear we now mention that both the Esox and Novumbra genera belong to the Esocidae. “With two families Esocidae, including Esox, Novumbra and Dallia and Umbridae, including Umbra, and 13 well-recognized species (Warren et al., 2020)”.

We however, do not understand why figures were not labelled with their corresponding numbers in the final pdf provided for reviewing as we clearly filled that information in the eLife submission website. We apologize for this inconvenience but it is clearly not our fault.

The important point readers need to understand at this place in the text is that all 7 Esox species and one Novumbra species surveyed (N. hubbsi) had two amh genes, whereas only one was found in two species, Dallia pectoralis and Umbra pygmaea, representing more basally diverging, outgroup species.

We agree and this is exactly what is stated in our manuscript i.e., “We found two amh genes in all surveyed Esox and Novumbra species. In the more basally diverging species, Dallia pectoralis and Umbra pygmaea, we found only one amh gene …”.

This result suggests that Esox and Novumbra species could both have an amhby gene, as well as an amha one. Then we need to have the evidence explained that Dallia pectoralis and Umbra pygmaea have only the amha one (information is confusingly revealed in this and the next paragraph).

We understand that this may be difficult to follow. To solve that specific point we propose to switch the following sentence : “We confirmed the absence of an additional divergent amh gene in D. pectoralis by searching sex-specific Pool-seq reads from 30 males and 30 females. In addition, only one amh homolog was found in an ongoing genome assembly project with long-reads for a male D. pectoralis (personal communication Y. Guiguen). “ just after the sentence stating that “In the more basally diverging species, Dallia pectoralis and Umbra pygmaea, we found only one amh gene in tissue-specific transcriptome databases (Pasquier et al., 2016).”

It is misleading to write "all Novumbra species" unless more than one was surveyed, which Table 1 suggests was not the case.

We agree that this would be misleading, but we have searched for this “all Novumbra species” mention in our current manuscript and we did not find it. The closest sentence is "… two well-supported clusters in all Esox, Dallia and Novumbra species" and even if our sentence is correct (as we do not refer to Novumbra species alone) we now changed it to "with two well-supported gene clusters among the amh sequences from Esocidae." in line with our answer to another comment below.

I also did not understand why the sentence says "However, two amh transcripts were readily identified in E. lucius (Pan et al., 2019), as well as in whole genome sequencing reads and assemblies (Supplementary file 1)". This sentence seems to confirm what was already stated above about E. Lucius, and perhaps it should be moved to an earlier place, saying "Moreover" rather than "However".

We agree that this sentence, that was changed between our initial submission and this first revision, is not understandable like it is written. We have now changed it to “Two amh genes were found in all surveyed Esox and Novumbra species. In more basally diverging species i.e., Dallia pectoralis and Umbra pygmaea, only one amh gene was found in both species in tissue-specific transcriptome databases (Pasquier et al., 2016), while two amh transcripts were readily identified in E. lucius (Pan et al., 2019)”

However, the next paragraph says that phylogenetic trees of the amh sequences showed "two well-supported clusters in all Esox, Dallia and Novumbra species", suggesting Dallia has both copies.

The reviewer is right about the interpretation of the phylogeny tree that the duplication of amh happened before the divergence of Esocidae and that Dallia had two copies. But from our extensive search, we only found one copy (in transcriptomes and in genomic reads of multiple individuals), and this result led to the idea that Dallia has lost its amhby. But we agree that this sentence might be confusing and we have hence changed it to “ These phylogenies provided a clear and consistent topology with two well-supported gene clusters among the amh sequences from Esocidae. “.

Some conclusions about these species are based on limited information in transcriptome databases, and some on other information, and the evidence should be more clearly and coherently explained.

As the reviewer suggested above we have moved these additional evidences just after the transcriptome evidence. We hope it will be easier to follow this way.

At this point, if I have guessed the correct meaning, I think that all Esox species, plus one species each of the genera Novumbra (but not Dallia and Umbra), have sequences that cluster with the E. lucius amha one, and are likely to be orthologs of this gene, and also sequences that cluster with the E. lucius amhby one, suggesting that the amh gene duplication preceded the split between the genera Esox and Novumbra, but perhaps occurred after the split from U. pygmaea (and perhaps Dallia). The text says that the "duplication happened after the divergence of Esocidae and Umbridae lineages", which suggests that Novumbra (with both copies) is not in the Umbridae.

With all respect we have to say that the reviewer has wrongly guessed the meaning. Novumbra is not an Umbridae and our sentence is totally right. We think that this comment is mainly due to the fact that reviewer #3 had problems following the track of the Esociformes phylogeny despite our efforts to be clear on that (Figure 1 and its corresponding legend). As we understand that it may be difficult (Novumbra sounds like Umbra and might suggest that it is also a member of Umbridae) and that this difficulty may be also shared by many other readers we modified this sentence to "duplication happened after the divergence of Esocidae (including Esox, Dallia and Novumbra) and Umbridae (including Umbra) lineages”

Please can the relationships be made clear before the results from them are described.

We have always been concerned about this potential difficulty and this is why we provided a main figure in our first submitted version, to better explain these phylogenetic relationships. No comments have been made by any reviewers, including reviewer #3, on this point during the first revision step. We believe phylogenetic relationships are clearly presented in the text. The main figure of the manuscript (Figure 1) should remove any doubts if any.

Clearly, the important questions are now whether the amhby copies are sex-determining genes in all species that carry the duplicate copy, and whether they acquired this function in the common ancestor of Esox and Novumbra, and retained this ever since. To test this, the study tested whether amhby was found in all males and absent from all females, in species additional to the E. lucius sample previously studied.

The tests indeed detected associations between the male phenotype and the presence of amhby in most species of Esox (Table 1), but the associations were absent or weak in some Esox samples, including some North American E. lucius populations, and incomplete in the Novumbra species sample.

We agree and this is exactly what we did and wrote. We are very aware of the weak association due to low sample size for two of the Esox species for which our samples were obtained from museum collection. The reason for this low sample size was explained in the main text: “For two recently described species, E. cisalpinus and E. aquitanicus (Denys et al., 2014), we had insufficient samples with clear species and sex identification for a decisive result”. As the reviewer mentioned, in Esox lucius we actually showed here that amhby is lost in all North American species investigated outside of Alaska. So there is no association between amhby and sex phenotype. The entire section “Some populations of Esox lucius lost their Y chromosome and ancestral master sex determining gene” in the Results section is dedicated to this case study on these populations.

Overall, it is clear that the duplication is ancient, and pre-dates the split between Esox and Novumbra, but there does not seem to be strong evidence for the conclusion that it has maintained a male-determining function since the duplication occurred. The alternative cannot be excluded that the duplicate copy has acquired a sex-determining function independently in Esox and Novumbra, as well as losing amhb several times. In fact, this study seems a nice example of how much evidence is needed before one can be sure that a sex-determining gene has been maintained for a long time.

This is correct that we cannot completely exclude that amhby has acquired its sex determining function independently in Esox and Novumbra, but this alternative hypothesis is not the more parcimonious one. However as we agree that it could be still a possibility we introduced a short sentence in the Discussion stating that this is an alternative hypothesis clearly mentioning that this hypothesis is less parsimonious than the first one “Although we cannot rule out the possibility that amhby was recruited independently as the MSD gene in Esox and in Novumbra, our results suggest the more parsimonious hypothesis that amhby likely acquired an MSD function before the diversification of Esocidae at least 56 Mya.”.

The claim that the same region has remained as an undifferentiated sex-determining or Y-linked region for a long evolutionary time comes from the Pool-Seq analysis of E. masquinongy, one of the species with an incomplete association between the presence of a copy of the amh duplicate, to compare its size and location (as a putative sex locus) with that of E. lucius. Male-specific variants (described rather vaguely as "sex-specific heterozygosity") were detected in just a single genome region of less than 50 kb, and the region is homologous to the proximal end of the E. lucius LG24, where its sex-determining locus is located. This indeed suggests that both E. lucius and E. masquinongy have a physically small male-determining locus at the same location. However, it appears to have a weakened or partially lost function in E. masquinongy.

Yes, we agree. The amhby gene seems to have lost its association with male phenotype in some populations of E. masquinongy, like in some populations of E. lucius. Both species show population level variation in their SD systems. However, for comparison with the E. lucius sex locus, we selected a E. masquinongy population where amhby is associated with male phenotype. This association was actually also confirmed by the specific mapping of males-only reads on this amhby genomic region. Furthermore, although we are comparing one population from E. masquinongy and one population from E. lucius, the divergence time is comparable to what has been estimated between the two species and supports our result that homologous regions are constituting the SD locus in the two species with 50 million year of divergence.

However, the results do not tell us that they the male-determining function is ancestral, rather than independently evolved, in Esox and Novumbra. Indeed, subsection “Evolution of the structure of the amhby gene in the Esocidae” reports information that may suggest independent evolution in E. niger (in which amhby is strongly associated with maleness) and N. hubbsi. In both, the predicted Amhby protein is truncated in regions known to be important for this gene's function, but in E. niger the truncation is in its C-terminal part, whereas it affects the N-terminal part of the N. hubbsi sequence, and exon 1 encodes only eight amino acids, with no homology to the amino acid-sequence in other Esocidae.

As we explained above, we didn’t reject this alternative hypothesis based on our data, but considered the “ancestral MSD gene” as the more parsimonious scenario. As mentioned above we now provide a short sentence in the Discussion stating that this is an alternative hypothesis clearly mentioning that this hypothesis is less parsimonious than the first one.

For the other comments on truncated Amhby in E. niger and N. hubbsi we agree with reviewer #3. All what the reviewer mentioned is actually written in our manuscript and we do not see any question in this comment.

It is not explained whether the E. lucius amhby copy is complete, or how it differs from amha.

The characterization of amhby, its comparison with amha, and the functional demonstration of its role in the initiation of male sex determination in E. lucius is based on our already published results (Pan et al., 2019).

On the interpretation that these changes evolved independently, the similarity of other parts of the sequence (which provides the signal in the phylogeny) is potentially rather misleading.

This similarity is undoubtedly the result of common origin (as seen in the phylogeny as the topology of the tree is also in agreement with the taxonomy tree) and thus confirmatory rather than misleading.

The manuscript also reports evidence that sex-determining systems evolved independently in the 2 species that do not have the amh duplicate, D. pectoralis and U. pygmaea.

It then turns to plausible suggestions about why Esox species might lose their male-determining factor. This section is rather unorganised, and should be greatly shortened to make clear what hypotheses were tested, and what the results were.

In our revision, we have already substantially restructured the Discussion following the suggestions from all three reviewers. As only reviewer #3 asked for additional re organizations (that will somehow also disorganize the current version and disrespect the agreement of the two other reviewers with this version) we would prefer keeping our discussion as it is.

Loss of genetic sex-determination is plausible in colonizing or low density situations, and the findings should be related to this concept.

This concept of a strong bottleneck effect in small founding populations during post-glaciation re-colonization is the main hypothesis we presented for the loss of amhby in some North American populations of E. lucius (see our Discussion section). A similar hypothesis could be eventually made for the Dallia transition but we feel that we don't have enough data to support this hypothesis in this species. The case of Umbra should not be considered as a transition, more like an ancestral state, followed by the emergence of a new SD system and eventually some transitions (in Esocidae). We agree that “the loss of genetic sex-determination is plausible in colonizing or low density situations” but because of that lack of data support in Dallia and also because our Discussion was already considered too lengthy, we do not want to add additional text on our Discussion on this point.

https://doi.org/10.7554/eLife.62858.sa2

Article and author information

Author details

  1. Qiaowei Pan

    1. INRAE, LPGP, Rennes, France
    2. Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8501-0405
  2. Romain Feron

    1. INRAE, LPGP, Rennes, France
    2. Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland
    3. Swiss Institute of Bioinformatics, Lausanne, Switzerland
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
  3. Elodie Jouanno

    INRAE, LPGP, Rennes, France
    Contribution
    Software, Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
  4. Hugo Darras

    Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland
    Contribution
    Investigation, Visualization, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Amaury Herpin

    INRAE, LPGP, Rennes, France
    Contribution
    Supervision, Investigation, Visualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0630-4027
  6. Ben Koop

    Department of Biology, Centre for Biomedical Research, University of Victoria, Victoria, Canada
    Contribution
    Resources, Supervision, Investigation
    Competing interests
    No competing interests declared
  7. Eric Rondeau

    Department of Biology, Centre for Biomedical Research, University of Victoria, Victoria, Canada
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  8. Frederick W Goetz

    Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, Seattle, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  9. Wesley A Larson

    Fisheries Aquatic Science and Technology Laboratory at Alaska Pacific University, Anchorage, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  10. Louis Bernatchez

    Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, Canada
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  11. Mike Tringali

    Fish and Wildlife Conservation Commission, Florida Marine Research Institute, St. Petersburg, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  12. Stephen S Curran

    School of Fisheries and Aquatic Sciences, Auburn University, Auburn, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  13. Eric Saillant

    Gulf Coast Research Laboratory, School of Ocean Science and Technology, The University of Southern Mississippi, Ocean Springs, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  14. Gael PJ Denys

    1. Laboratoire de Biologie des organismes et écosystèmes aquatiques (BOREA), MNHN, CNRS, IRD, SU, UCN, Laboratoire de Biologie des organismes et écosystèmes aquatiques (BOREA), Paris, France
    2. Unité Mixte de Service Patrimoine Naturelle – Centre d’expertise et de données (UMS 2006 AFB, CNRS, MNHN), Muséum national d’Histoire naturelle, Paris, France
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  15. Frank A von Hippel

    Department of Biological Sciences, Northern Arizona University, Flagstaff, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9247-0231
  16. Songlin Chen

    Yellow Sea Fisheries Research Institute, CAFS, Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, China
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  17. J Andrés López

    College of Fisheries and Ocean Sciences Fisheries, Fairbanks, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  18. Hugo Verreycken

    Research Institute for Nature and Forest (INBO), Brussels, Belgium
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2060-7005
  19. Konrad Ocalewicz

    Department of Marine Biology and Ecology, Institute of Oceanography, University of Gdansk, Gdansk, Poland
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  20. Rene Guyomard

    INRAE, GABI, Jouy-en-Josas, France
    Contribution
    Resources, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  21. Camille Eche

    GeT‐PlaGe, INRAE, Genotoul, Castanet-Tolosan, France
    Contribution
    Resources, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  22. Jerome Lluch

    GeT‐PlaGe, INRAE, Genotoul, Castanet-Tolosan, France
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  23. Celine Roques

    GeT‐PlaGe, INRAE, Genotoul, Castanet-Tolosan, France
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  24. Hongxia Hu

    Beijing Fisheries Research Institute & Beijing Key Laboratory of Fishery Biotechnology, Beijing, China
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  25. Roger Tabor

    U.S. Fish and Wildlife Service, Lacey, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  26. Patrick DeHaan

    U.S. Fish and Wildlife Service, Lacey, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  27. Krista M Nichols

    Conservation Biology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Seattle, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  28. Laurent Journot

    Institut de Génomique Fonctionnelle, IGF, CNRS, INSERM, Univ. Montpellier, Montpellier, France
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  29. Hugues Parrinello

    Institut de Génomique Fonctionnelle, IGF, CNRS, INSERM, Univ. Montpellier, Montpellier, France
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  30. Christophe Klopp

    INRAE, Sigenae, Genotoul Bioinfo, Toulouse, France
    Contribution
    Resources, Software, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7126-5477
  31. Elena A Interesova

    Tomsk State University, Tomsk, Russian Federation
    Contribution
    Resources, Software, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1148-6283
  32. Vladimir Trifonov

    Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk State University, Novosibirsk, Russian Federation
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  33. Manfred Schartl

    University of Wuerzburg, Developmental Biochemistry, Biocenter, 97074 Würzburg, Germany; and The Xiphophorus Genetic Stock Center, Texas State University, San Marcos, United States
    Contribution
    Conceptualization, Resources, Funding acquisition, Investigation, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  34. John Postlethwait

    Institute of Neuroscience, University of Oregon, Eugene, United States
    Contribution
    Conceptualization, Funding acquisition, Investigation, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  35. Yann Guiguen

    INRAE, LPGP, Rennes, France
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Project administration, Writing - review and editing
    For correspondence
    yann.guiguen@inrae.fr
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5464-6219

Funding

Agence Nationale de la Recherche (ANR-13-ISV7-0005)

  • Yann Guiguen

Deutsche Forschungsgemeinschaft

  • Manfred Schartl

Agence Nationale de la Recherche (ANR-10-INBS-09)

  • Celine Roques
  • Laurent Journot

Korea National Institute of Health (R01GM085318)

  • John Postlethwait

ANR (SCHA 408/10–1)

  • Yann Guiguen
  • Manfred Schartl

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to the fish facility of INRAE LPGP for support in experimental installation and fish maintenance and to the GenoToul bioinformatics platform Toulouse Midi-Pyrenees (Bioinfo GenoToul) for providing help, computing, and storage resources. This work was supported by the Agence Nationale de la Recherche, the Deutsche Forschungsgemeinschaft (ANR/DFG, PhyloSex project, 2014–2016, SCHA 408/10–1, to YG and MS), and the U.S. National Institutes of Health (R01GM085318 to JHP). The MGX and Get-Plage core sequencing facilities were supported by France Genomique National infrastructure, funded as part of the ‘Investissement d’avenir’ program managed by the Agence Nationale pour la Recherche (contract ANR-10-INBS-09). We thank the MNHN curators for providing access to the collections of E. aquitanicus and E. cisalpinus and for the ONEMA. We thank GB Delmastro for help with specimen sampling. We also thank Mackenzie Garvey and Penny Swanson for assistance with dissection of N. hubbsi.

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Developmental Biology, Germany

Reviewing Editor

  1. Molly Przeworski, Columbia University, United States

Publication history

  1. Received: September 6, 2020
  2. Accepted: January 27, 2021
  3. Accepted Manuscript published: January 28, 2021 (version 1)
  4. Version of Record published: February 8, 2021 (version 2)

Copyright

© 2021, Pan et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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