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hints at a prolonged courtship with males hanging around after fertilising the ‘hens’, perhaps to assist with rearing the young, perhaps feeding the hen during incubation; although the authors prefer that rather than being incubated bird-style the eggs were buried in the soft sediment, much like other reptiles do, as suggested by studies on predicted vapour conductance of pterosaur eggshells [16] and by assumptions about eggshell strength [17]. The phylogenetic analysis performed on Hamipterus allies it with such famous pterosaurs as the edentulous Pteranodon (the one with the big pointy head crest) from North America and Ornithocheirus, a toothy European form with a crest on the tip of its beak and made famous in the Giant of the Skies episode of the BBC TV blockbuster Walking With Dinosaurs. This group of pterosaurs, known as ornithocheiroids (although they are termed pteranodontians by Wang et al. [7]), were widespread during the Early Cretaceous and survived almost to the end of the period, which also marked the end of the dinosaurs. Most ornithocheiroids were very large with a wingspan in excess of 4 metres, and perhaps over 7 metres [18], but Hamipterus is comparatively small with a wingspan estimated to have been between 1.5 and 3.5 metres. An unusual aspect of Hamipterus as an ornithocheirid is the nature of its rostral crest. Most ornithocheirids possess a crest (located either on the upper and lower jaw tips, e.g. Coloborhynchus, on the posterior cranium, e.g. Ludodactylus, or both, e.g. Caulkicephalus), and in all cases the margin of the crest is entire, sharply defined and the bone surface smooth (although it may contain channels for depressed veins and arteries). This contrasts strongly with the irregular margin and fibrous bone of Hamipterus’ crest, and is of a type found in Dsungaripterids and some non-pterodactyloid pterosaurs, including Darwinopterus. Despite this anomaly, almost all other aspects of Hamipterus’ skeleton do indicate ornithocheiroid affinities. The authors have identified the remains of more than 40 individuals in the deposit so far, which is

unprecedented for a pterosaur site. Hundreds more probably remain to be found. Never before have so many remains attributed to a single taxon been found in such close association, and in the presence of eggs. This discovery represents a unique opportunity to investigate pterosaur growth, development, reproductive behaviour and ecology. Expect many more papers on this amazing deposit when the sedimentology and taphonomy have been studied in detail. References 1. Collini, C.A. (1784). Sur quelques zoolithes du cabinet d’Histoire Naturelle de S.A.S.E. Palatine et de Baviere, a Manheim. Acta Academiae Theodoro-Palatinae, Manheim, Pars Physica 5, 58–103. 2. Cuvier, G. (1809). Memoire sur le squelette fossile d’un reptile Volant des environs d’Aichstedt, que quelques naturalistes ont pris pour un oiseau, et don’t nous formons un genre de Sauriens, sous le nom de Ptero-Dactyle. Annales du Muse´um national d’Histoire Naturelle, Paris 13, 424–437. 3. Buckland, W. (1835). On the discovery of a new species of pterodactyle in the Lias at Lyme Regis. Trans. Geological Soc. Lond. 2, 217–222. 4. Owen, R. (1859). On remains of new and gigantic species of pterodactyle (Pter. Fittoni and Pter. Sedgwickii) from the Upper Greensand, near Cambridge. Report of the British Association for the Advancement of Science, for 1858, Leeds (London: John Murray), pp. 98–103. 5. Lu, J., Unwin, D.M., Jin, X., Liu, Y., and Ji, Q. (2010). Evidence for modular evolution in a long-tailed pterosaur with a pterodactyloid skull. Proc. R. Soc. B 277, 383–389. 6. Bramwell, C.D., and Whitfield, G.R. (1974). Biomechanics of Pteranodon. Philos. Trans. R. Soc. Lond. B 267, 503–581. 7. Wang, X., Kellner, A.W.A., Jiang, S., Wang, Q., Ma, Y., Paidoula, Y., Cheng, X., Rodrigues, T., Meng, X., Zhang, J., et al. (2014). Sexually dimorphic tridimensionally preserved

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pterosaurs and their eggs from China. Curr. Biol. 24, 1323–1330. Unwin, D.M. (2001). An overview of the pterosaur assemblage from the Cambridge Greensand (Cretaceous) of eastern England. Mitteilungen des Museum fu¨r Naturkunde, Berlin, Geowissenschaften. Reihe 4, 189–221. Benton, M.J. (1999). Scleromochlus taylori and the origin of dinosaurs and pterosaurs. Philos. Trans. R. Soc. Lond. 354, 1423–1446. Wang, X.L., and Zhou, Z.H. (2004). Pterosaur embryo from the Early Cretaceous. Nature 429, 621. Ji, Q., Ji, S.A., Cheng, Y.N., You, H.L., Lu¨, J.C., Liu, Y.Q., and Yuan, C.X. (2004). Pterosaur egg with a leathery shell. Nature 432, 572. Chiappe, L.M., Codorniu´, L., Grellet-Tinner, G., and Rivarola, D. (2004). Palaeobiology: Argentinean unhatched pterosaur fossil. Nature 432, 571. Lu¨, J., Unwin, D.M., Deeming, D.C., Jin, X., Liu, Y., and Ji, Q. (2010). An egg-adult association, gender, and reproduction in Pterosaurs. Science 331, 321–324. Tomkins, J.L., LeBas, N.R., Witton, M.P., Martill, D.M., and Humphries, S. (2010). Positive allometry and the prehistory of sexual selection. Am. Nat. 176, 141–148. Bennett, S.C. (1992). Sexual dimorphism of Pteranodon and other pterosaurs, with comments on cranial crests. J. Vertebrate Paleontol. 12, 422–434. Grellet-Tinner, G., Wroe, S., Thompson, M.B., and Ji, Q. (2007). A note on pterosaur nesting behavior. Hist. Biol. 19, 273–277. Unwin, D.M., and Deming, D.C. (2008). Pterosaur eggshell structure and its implications for pterosaur reproductive biology. Zitteliana 28, 199–207. Martill, D.M., and Unwin, D.M. (2012). The world’s largest toothed pterosaur, NHMUK R481, an incomplete rostrum of Coloborhynchus capito (Seeley 1870) from the Cambridge Greensand of England. Cretaceous Res. 34, 1–9.

School of Earth and Environmental Sciences, University of Portsmouth, Portsmouth, PO1 3QL, UK. E-mail: [email protected]

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Sex Determination: Ciliates’ Self-Censorship Differentiation involves the expression of certain latent cellular characteristics and the repression of others. A new study has revealed how Paramecium uses short RNAs to delete information from the somatic genome of one of its two sexes. Gareth Bloomfield Sex involves the most fundamental kind of differentiation in biology. Most interbreeding populations are divided into separate classes of organism that are mutually sexually

compatible: sexes or mating types. Crucially, gametes must be different enough for their correct partners to be distinguished. Although in many organisms sperm and egg cells are different in size and shape, unicellular species typically

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Figure 1. Simplified depiction of ciliate conjugation and cytoplasmic inheritance. After meiosis (not shown), conjugating cells each possess two identical haploid micronuclei. One from each cell is then passed into its partner, and then the micronuclei in each cell fuse, making both progeny cells’ diploid micronuclei identical to each other. The macronuclei then degrade, and are replaced by new versions with sequences deriving from the new micronucleus. For some traits (here shown as blue and red), inheritance follows the genotype of the previous macronucleus, irrespective of the new micronuclear genotype.

have gametes that differ in only a small number of proteins. All of the major lineages of eukaryotes have sexual species, and across them sex is determined in startlingly different ways [1–4]. One group, the Ciliophora (or ciliates), is of special interest because it provides examples of species with more than the usual two sexes, and also unconventional modes of inheritance [5,6]. A recent study published in Nature by Singh et al. [7] has revealed how different species of one ciliate genus ensure cytoplasmic inheritance of mating type [7]. Ciliates, though unicellular, are highly advanced organisms. Most strikingly, ciliates maintain germline and somatic genetic material in separate organelles, the micronucleus and macronucleus, respectively. Micronuclei are maintained in the diploid state, and are transcriptionally silent, while macronuclei are highly polyploid and active. The macronucleus is destroyed when sex takes place, and is afterwards made anew, incorporating reorganised and amplified segments deriving from the micronuclear chromosomes. Ciliates are also unusual in that meiosis is not accompanied by a

change in cell number (Figure 1). When two parental cells conjugate, their micronuclei undergo meiosis, with only one of the four haploid products surviving in each cell (Paramecium cells actually start conjugation with two micronuclei, and seven of their eight meiotic products are destroyed). The remaining nucleus divides by mitosis, and one of the resulting pair is transferred from each of the conjugating cells to the other. The two nuclei in each cell then fuse, forming a new diploid nucleus containing one copy from each parent of every chromosome. So two parents give rise to two progeny, and these have genetically identical micronuclei. Macronuclei are then formed, incorporating rearranged fragments of a portion of the germline genome. The progeny cells grow and then multiply through binary fission, micronuclei going through mitosis and macronuclei dividing amitotically. Some species can undergo meiosis and renew their macronucleus without conjugation. This is called autogamy, and follows the same sequence as conjugation, but in a single cell; the duplicate recombinant haploid nuclei formed

after meiosis simply fuse together, resulting in a homozygous new micronucleus. Further information on ciliate biology can be found in recent reviews [8,9]. Given the complexities of nuclear organisation, one might expect ciliate genetics to be similarly complex. In fact it gets even worse. Despite the fact that it is destroyed after meiosis, it is the parental macronucleus that governs the inheritance of certain traits [10]. In some species, mating type is inherited in this way [5]. In Paramecium tetraurelia, there are two mating types, E (for even) and O (for odd). Conjugation occurs between cells of different mating type, and so every progeny cell is a heterozygous diploid for mating type. But each cell only expresses one mating type: macronuclei are differentiated for this trait, and this differentiation is normally inherited by the succeeding macronucleus, despite its origins from a new heterozygous micronucleus. How can this be? First, it is important to remember that ciliate cells do not fuse during sex: nuclei are transferred between cells, but their cytoplasm is not. While the macronuclear genome degrades, there are still organelles, proteins and RNA within the cytoplasm remaining from the parent cell. It is this RNA that governs the reorganisation of chromosomes during production of a new macronucleus [11]. In the meiotic micronucleus, small RNAs (scnRNAs) are produced that correspond to germline DNA sequences; these are thought to be compared to noncoding RNAs produced from the old macronucleus in a form of natural ‘subtractive hybridisation’, allowing DNA elements not present in the old macronucleus to be recognised and then deleted (Figure 2) [12]. Singh et al. [7] provide the molecular details that confirm decades of classical genetics findings on mating type inheritance in P. tetraurelia. They began by comparing the transcriptomes of E and O cells under conditions when they are most mating-competent, and identified a candidate transmembrane protein whose mRNA is strongly enriched in E cells. A GFP-tagged version of the protein localises to the surface of the anterior half of the cell, and depletion of the native gene by RNAi switched E cells to the O phenotype. Conversely,

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microinjection of the gene into O cell macronuclei switched them to the E phenotype. Further, a nonsense mutation was found within the coding sequence of the gene in a classically defined mtA mutant. Together these results prove conclusively that the gene is mtA, the determinant of the E mating type. How then is O mating type specified? Sequencing of the mtA gene in the micronucleus and in E and O macronuclei revealed that a segment of the germline sequence containing the start codon, transcriptional start site, and a critical promoter element is excised in O macronuclei, but retained in those of E cells. Flanking this region are short sequences matching the consensus for elements that promote the directed removal of germline-only regions during macronuclear genesis. So mtA expression is actively prevented in O cells by a deletion of part of the gene. Introduction of a plasmid containing the short deleted sequence into O cells prevented its excision in newly forming macronuclei, while introduction of double-stranded RNA corresponding to the sequence into E cells undergoing autogamy enabled the production of O type offspring. Further, knockdown of components involved in excision or in the scnRNA system interfered with the removal of the promoter sequence from O macronuclei. Thus, this Paramecium species has co-opted the system normally used to streamline its somatic genome to stably determine mating type. A sibling species, P. octaurelia, was found to use the same excision site in mtA to produce its O cells. However, another, more distantly related species, P. septaurelia, which also inherits mating type through its macronuclei, showed no differences in mtA processing between E and O cells. Singh and colleagues solved this puzzle by making use of further classical genetic findings from P. tetraurelia. Mutations in two genes other than mtA had been defined that are necessary for expression of the E phenotype [13]. Resequencing of the genomes of mutant strains identified mtB and mtC as putative transcription factors; strikingly, mtA is the only gene downregulated in mtB mutant cells. And macronuclei of O cells of P. septaurelia contain either of two different deletions in their mtB sequences, again between sites matching the consensus for sequences

Transcription in old macronucleus

Hybridization of scnRNAs with transcripts

Selected scnRNAs

Deletion of genomic sequences in new macronucleus

Micronucleus

Macronucleus

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Figure 2. Schematic of targeted removal of macronuclear DNA sequences in Paramecium. In parental macronuclei, non-coding RNA (white wavy line) is transcribed from existing chromosomes. Numerous short scnRNAs (purple lines) are meanwhile produced and transported from the micronucleus to the old macronucleus, where some can hybridise with the non-coding RNA. scnRNAs that do not hybridise subsequently target sequences in the new macronucleus for deletion.

normally excised during macronucleus generation. Expression of the intact mtB switched O cells to the E phenotype, and mutations were found within the mtB sequence in a P. septaurelia strain compromised in both expression and inheritance of the E mating type. This work is the culmination of decades of painstaking genetic analysis of mating behaviour in Paramecium [14], demonstrating how this surprising and unconventional system operates at the molecular level to differentiate cells through targeted deletion of germline genetic material. The convergent outcomes in different species suggest that many further examples of this phenomenon will be found. Sex determination in the distantly related ciliate Tetrahymena also involves deletion of germline elements, but apparently stochastically, and giving rise to one of multiple mating types [15]. Several puzzles still remain in Paramecium: while mtA is almost certainly the cell surface recognition protein in E cells, the equivalent factor on the O cell surface is not known, and until it is found an understanding of how mtA exerts its dominant effects, and of how O becomes the default in its absence is still wanting. Perhaps these results will also shed light on enigmatic observations of switching between mating types in Paramecium species [16,17]; and perhaps, conversely, investigation of even stranger examples can help elucidate the core conserved mechanisms.

3. Heitman, J., Sun, S., and James, T.Y. (2013). Evolution of fungal sexual reproduction. Mycologia 105, 1–27. 4. Zhang, J., Boualem, A., Bendahmane, A., and Ming, R. (2014). Genomics of sex determination. Curr. Opin. Plant Biol. 18, 110–116. 5. Sonneborn, T.M. (1977). Genetics of cellular differentiation: stable nuclear differentiation in eucaryotic unicells. Annu. Rev. Genet. 11, 349–367. 6. Phadke, S.S., and Zufall, R.A. (2009). Rapid diversification of mating systems in ciliates. Biol. J. Linn. Soc. 98, 187–197. 7. Singh, D.P., Saudemont, B., Guglielmi, G., Arnaiz, O., Gouˆt, J.-F., Prajer, M., Potekhin, A., Przybo`s, E., Aubusson-Fleury, A., Bhullar, S., et al. (2014). Genome-defence small RNAs exapted for epigenetic mating-type inheritance. Nature 509, 447–452. 8. Beisson, J., Be´termier, M., Bre´, M.-H., Cohen, J., Duharcourt, S., Duret, L., Kung, C., Malinsky, S., Meyer, E., Preer, J.R., et al. (2010). Paramecium tetraurelia: the renaissance of an early unicellular model. Cold Spring Harb. Protoc. 2010, pdb.emo140. 9. Orias, E., Cervantes, M.D., and Hamilton, E.P. (2011). Tetrahymena thermophila, a unicellular eukaryote with separate germline and somatic genomes. Res. Microbiol. 162, 578–586. 10. Sonneborn, T.M. (1947). Recent advances in the genetics of Paramecium and Euplotes. Adv. Genet. 1, 263–358. 11. Nowacki, M., and Landweber, L.F. (2009). Epigenetic inheritance in ciliates. Curr. Opin. Microbiol. 12, 638–643. 12. Lepe`re, G., Be´termier, M., Meyer, E., and Duharcourt, S. (2008). Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia. Genes Dev. 22, 1501–1512. 13. Byrne, B.C. (1973). Mutational analysis of mating type inheritance in Syngen 4 of Paramecium Aurelia. Genetics 74, 63–80. 14. Preer, J.R., Jr. (1997). Whatever happened to Paramecium genetics? Genetics 145, 217–225. 15. Cervantes, M.D., Hamilton, E.P., Xiong, J., Lawson, M.J., Yuan, D., Hadjithomas, M., Miao, W., and Orias, E. (2013). Selecting one of several mating types through gene segment joining and deletion in Tetrahymena thermophila. PLoS Biol. 11, e1001518. 16. Barnett, A. (1966). A circadian rhythm of mating type reversals in Paramecium multimicronucleatum, syngen 2, and its genetic control. J. Cell. Physiol. 67, 239–270. 17. Taub, S.R. (1966). Unidirectional mating type changes in individual cells from selfing cultures of Paramecium aurelia. J. Exp. Zool. 163, 141–149.

References

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK. E-mail: [email protected]

1. Gempe, T., and Beye, M. (2011). Function and evolution of sex determination mechanisms, genes and pathways in insects. BioEssays 33, 52–60. 2. Kikuchi, K., and Hamaguchi, S. (2013). Novel sex-determining genes in fish and sex chromosome evolution. Dev. Dyn. 242, 339–353.

http://dx.doi.org/10.1016/j.cub.2014.05.041

Sex determination: ciliates' self-censorship.

Differentiation involves the expression of certain latent cellular characteristics and the repression of others. A new study has revealed how Parameci...
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