Facultative Parthenogenesis in a Critically Endangered Wild Vertebrate

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region) at sites of cell–cell contact: Correspondence 1900. It currently occurs primarily in Rac does not interact with LIMD1 even southwest Florida, U.S. [6], where though LIMD1 is recruited to adherens Facultative it may have stabilized and started junctions. to recover (http://www.iucnredlist. parthenogenesis org/details/18175/0 ). Between 2004 What is the link between Ajuba and 2013, 190 individuals ranging in proteins and the Hippo signaling in a critically stretched total length (STL) from 67.1 pathway? Through their interaction to 381 cm were sampled, tagged, with LATS/Wts kinases (via the LIM endangered wild and released in the Caloosahatchee region), Ajuba proteins and dJub vertebrate River, Peace River and Ten Thousand are negative regulators of the Hippo Islands regions. Sixteen microsatellite signaling pathway. LATS/Wts kinases Andrew T. Fields1, Kevin A. Feldheim2, loci were used to genotype these are components of the conserved Gregg R. Poulakis3, individuals (Supplemental Information). Hippo core kinase complex that directly and Demian D. Chapman1,* These loci had from 4 to 40 alleles phosphorylate the transcriptional (mean = 20) and conformed to Hardy- activator YAP/Yki, leading to its nuclear Facultative parthenogenesis — the Weinberg equilibrium expectations exclusion and degradation. Precisely ability of sexually reproducing species after correction for multiple tests where in the cell, how, and during to sometimes produce offspring (Supplemental Information). The what biological circumstances Ajuba asexually — is known from a wide program STORM [7] was used to proteins inhibit LATS/Wts activity range of ordinarily sexually reproducing calculate the internal relatedness is not fully appreciated, but during vertebrates in captivity, including some (IR) of each individual, a parameter Drosophila wing development dJub birds, reptiles and sharks [1–3]. Despite that expresses the relatedness of regulates cytoskeletal-tension-induced this, free-living parthenogens have the individual’s parents [8]. Outbred proliferation by inhibiting the Hippo never been observed in any of these individuals will have an IR close to pathway. taxa in the wild, although two free-living zero while individuals derived from snakes were recently discovered each close kin mating are expected to Where can I find out more? gestating a single parthenogen — exhibit IR values from ~0.25 to 0.50 Das Thakur, M., Feng, Y., Jagannathan, R., one copperhead (Agkistrodon (i.e., indicating that the individual’s Seppa, M.J., Skeath, J.B., and Longmore, G.D. (2010). Ajuba LIM proteins are negative regulators contortrix) and one cottonmouth parents were half or full-siblings, of the Hippo signaling pathway. Curr. Biol. 7, (Agkistrodon piscivorus) [1]. Vertebrate respectively). An IR value close to or 657–662. Hirota, T., Kunitoku, N., Sasayama, T., Marumoto, T., parthenogens are characterized as equal to 1 indicates near or complete Zhang, D., Nitta, M., Hatakeyama, K., and being of the homogametic sex (e.g., homozygosity across surveyed loci, Saya, H. (2003). Aurora-A and an interacting females in sharks, males in birds) and which is consistent with all known activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell 5, by having elevated homozygosity cases of facultative parthenogenesis in 585–598. compared to their mother [1–3], vertebrates [1–3]. Kadrmas, J., and Beckerle, M.C. (2004). The LIM domain: from the cytoskeleton to the nucleus. which may reduce their viability [4]. The mean IR of sampled individuals Nat. Rev. Mol. Cell Biol. 5, 920–931. Although it is unknown if either of the was 0.033 (s.d. = 0.199, with >85% Kanungo, J., Pratt, S., Marie, H., and Longmore, G.D. parthenogenetic snakes would have of individuals having an IR <0.10; (2000). Ajuba, a cytosolic LIM protein, shuttles into the nucleus and affects embryonal cell been carried to term or survived in Figure 1), indicating that mating pairs proliferation and fate decisions. Mol. Biol. Cell 10, the wild, facultative parthenogenesis were typically unrelated. Seven outliers 3299–3313. Langer, E.M., Feng, Y., Zhaoyuan, H., Rauscher, F.J., might have adaptive significance (3.7% of the sample) with IR values 3rd, Kroll, K.L., and Longmore G.D. (2008). Ajuba [1]. If this is true, it is reasonable to ranging from 0.84 to 1.0 were found, LIM proteins are snail/slug corepressors required hypothesize that parthenogenesis with two individuals being homozygous for neural crest development in Xenopus. Dev. Cell 3, 424–436. would be found most often at low at all loci and five being homozygous Nola, S., Daigaku, R., Smolarczyk, K., Carstens, M., population density, when females at all loci but one or two (Figure 1). Martin-Martin, B., Longmore, G., Bailly, M., and Braga, V.M.M. (2011). Ajuba is required for risk reproductive failure because All of these individuals were female, Rac activation and maintenance of E-cadherin finding mates is difficult5 [ ]. Here, we even though the overall sample sex adhesion. J. Cell Biol. 5, 855–871. document the first examples of viable ratio excluding these individuals was Rauskolb, C., Sun, S., Sun, G., Pan, Y., and Irvine, K.D. (2014). Cytoskeletal tension inhibits parthenogens living in a normally close to unity (1 male to 1.12 females). Hippo signaling through an Ajuba-Warts complex. sexually reproducing wild vertebrate, Five of the outlier individuals were Cell 158, 143–156. Sharp, T.V., Al-Attar, A., Foxler, D., Ding, L., de, A., the smalltooth sawfish Pristis( captured in the Peace River in 2011 Vallim, T., Zhang, Y., Nijmeh, H., Webb, T., pectinata). We also provide a simple (n = 4) and 2012 (n = 1). Their size at Nicholson, A., et al. (2008). The chromosome approach to screen any microsatellite capture indicated they were all born in 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer DNA database for parthenogens, which 2011 [9] and their genotypes indicate development. Proc. Natl. Acad. Sci. USA 50, will enable hypothesis-driven research they were full siblings (Supplemental 19932–19937. on the significance of vertebrate Information). We consider these five parthenogenesis in the wild. individuals to be members of a single ICCE Institute, Department of Medicine and The smalltooth sawfish is a brood of parthenogenetic offspring, Cell Biology and Physiology, Washington University School of Medicine, St. Louis, large, critically endangered ray which has previously been observed MO 63110, USA. that is estimated to have declined in captive white-spotted bamboo E-mail: [email protected] to 1–5% of its population size in sharks, Chiloscyllium plagiosum (brood

of extinction. Screening existing and emerging microsatellite databases from wild populations in the manner we

120 describe will enable hypothesis-driven research on the adaptive significance 100 of this alternative mode of vertebrate reproduction. 80

60 SUPPLEMENTAL INFORMATION

40 Supplemental Information including experi- Frequency (n) mental procedures, results and acknowledge- 20 ments can be found with this article online at 0 http://dx.doi.org/10.1016/j.cub.2015.04.018. 4 7 3 6 9 ≤ 0 0.30 0.60 0.90 0.03 0.06 0.09 0.12 0.15 0.18 0.21 0.24 0.27 0.33 0.36 0.39 0.42 0.45 0.48 0.51 0.54 0.57 0.63 0.66 0.69 0.72 0.75 0.78 0.81 0.84 0.87 0.93 0.96 0.99 REFERENCES Internal relatedness (IR) 1. Booth W., Smith, C.F., Eskridge, P.H., Hoss, S.K., Mendelson, J.R., and Schuett, G.W. Figure 1. Internal relatedness of smalltooth sawfish. (2012). Facultative parthenogenesis discovered Individual internal relatedness (IR) of smalltooth sawfish n( = 190) sampled in the Caloosahatchee in wild vertebrates. Biol. Lett. 8, 983–985. River, Peace River, and Ten Thousand Islands region (2004–2013). The box highlights putative 2. Feldheim, K.A., Chapman, D.D., Sweet, D., parthenogens (n = 7, all female) from the Peace and Caloosahatchee rivers. Fitzpatrick, S., Prodöhl, P.A., Shivji, M.S., and Snowden, B. (2010). Shark virgin birth produces multiple, viable offspring. J. Hered. 101, 374–377. size = 4 hatched eggs; [2]). Two fully because they were immature, but there 3. Olsen, M.W. (1975). Avian Parthenogenesis. homozygous individuals were captured, is evidence that parthenogens are (Beltsville: Agricultural Research Service). one in the Caloosahatchee River in capable of sexual reproduction in both 4. Booth, W., and Schuett, G.W. (2011). Molecular genetic evidence for alternative reproductive 2005 at 156.6 cm STL (estimated birth birds and reptiles [3,10]. strategies in North American pitvipers year 2004), and one in the Peace River The idea that sexually reproducing (Serpentes: Viperidae): long-term sperm storage and facultative parthenogenesis. Biol. J. Linn. in 2011 at 87.8 cm STL (estimated species sometimes reproduce asexually Soc. 104, 934–942. birth year 2011). This additional 2011 in the wild is novel, but we provide 5. Lampert, K.P. (2008). Facultative individual was unrelated to the other evidence that it is occurring in a critically parthenogenesis in vertebrates: reproductive error or chance? Sexual Dev. 2, 290–301. five putative parthenogens collected endangered population of smalltooth 6. Waters, J.D., Coelho, R., Fernandez- from the Peace River that year. The sawfish. Establishing that an individual Carvalho, J., Timmers, A.A., Wiley, T., Seitz, J.C., McDavitt, M.T., Burgess, G.H., probability of an individual being is a parthenogen has typically involved and Poulakis, G.R. (2014). Use of encounter homozygous at 14 to all 16 of these obtaining DNA fragment-based profiles data to model spatio-temporal distribution loci regardless of allele identity and of mother–offspring pairs that reveal an patterns of endangered smalltooth sawfish, Pristis pectinata, in the western Atlantic. Aquat. assuming sexual reproduction was absence of unique paternal alleles in Conserv. Mar. Freshw. Ecosyst. 24, 760–776. calculated by multiplying the observed the offspring’s genotype [1]; however, 7. Fraiser, T.R. (2008). STORM: software for testing hypotheses of relatedness and mating patterns. homozygosity of each locus. This this approach is logistically unrealistic Mol. Ecol. Res. 8, 1263–1266. probability ranged from 2.29 x 10-12 (14 when sampling many taxa in the wild, 8. Amos, W., Worthington, W.J., Fullard, K., Burg, loci) to 4.18 x 10‑15 (all loci). especially those without maternal care T.M., Croxall, J.P., Block, D., and Coulson, T. (2001). The influence of parental relatedness Five of the putative parthenogens (e.g., rays, including the smalltooth on reproductive success. Proc. R. Soc. B. 268, were heterozygous at one or two sawfish). As an alternative, we have 2021–2027. 9. Scharer, R.M., Patterson III, W.F., Carlson, J.K., loci (involving Ppe-8 and Ppe-114), shown that it is also possible to infer a and Poulakis, G.R. (2012). Age and growth which demonstrates that they are parthenogenetic origin for a free-living of endangered smalltooth sawfish Pristis( diploid. This indicates that they were individual when it exhibits a level of pectinata) verified with LA-ICP-MS analysis of vertebrae. PLoS One 7, e47850. most likely produced by automictic homozygosity that is abnormally high 10. Booth, W., Schuett, G.W., Ridgway, A., parthenogenesis with the heterozygous for sexual reproduction. We suggest Buxton, D.W., Castoe, T.A., Bastone, G., Bennett, C., and McMahan, W. (2014). New loci being close to the telomere and that any microsatellite database can insights on facultative parthenogenesis in undergoing recombination prior to be rapidly screened using the program pythons. Biol. J. Linn. Soc. 112, 461–468. fusion of the ovum and the sister polar STORM [7] to identify potential body [1–3]. Automictic parthenogenesis parthenogens based on IR values. 1School of Marine and Atmospheric Science, reduces genome-wide heterozygosity, The ability to reproduce via facultative Stony Brook University, Stony Brook, raising questions about the viability and parthenogenesis is taxonomically NY 11794, USA. 2Pritzker Laboratory for reproductive competence of individuals widespread in normally sexually Molecular Systematics and Evolution, Field produced in this manner [4]. The sawfish reproducing vertebrates (except those Museum of Natural History, 1400 South Lake 3 parthenogens were normal size for up with genomic imprinting [5]) and may be Shore Drive, Chicago, IL 60605, USA. Florida Fish and Wildlife Conservation Commission, to one-year-old individuals [9], thus more common than suspected in nature. Fish and Wildlife Research Institute, Charlotte demonstrating that individuals produced We hypothesize that it may be found Harbor Field Laboratory, 585 Prineville Street, in this way can survive in the wild. Their most often in low-density populations Port Charlotte, FL 33954, USA. reproductive competence is not known such as those that are on the verge *E-mail: [email protected]