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A somatic mosaic of the gynogenetic Amazon molly

A somatic mosaic of the gynogenetic Amazon molly
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  Journal of Fish Biology  (2002)  60,  1417–1422doi:10.1006/jfbi.2002.1939, available online at on A somatic mosaic of the gynogenetic Amazon molly D. K. L  *‡, M. S  †    M. S  * *Lehrstuhl fu¨r Physiologische Chemie I, Theodor-Boveri-Institut and   † Institut fu¨rHumangenetik, Biozentrum, Am Hubland, D-97074 Wu¨rzburg, Germany ( Received 27 September 2001, Accepted 26 March 2002 ) Breeding experiments with the gynogenetic Amazon molly,  Poecilia formosa , proved thatmosaicism, detected by flow cytometry with the fluorescent dye DAPI was restricted to thesoma, and that gynogenetic reproduction was maintained by the o ff  spring. Potential pathwaysfor how mosaicism might have arisen in  P. formosa  are discussed.  2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved. Key words: flow cytometry; diploid; triploid; unisexual vertebrate;  Poecilia formosa . INTRODUCTION Unisexual vertebrates are generally thought to arise through interspecific hybrid-ization (Dawley, 1989). The combination of two genomes from genetically divergent species is believed to be responsible for the key features of unisexualvertebrates, namely that they are almost exclusively female, that genetic recom-bination is inhibited, and that they often include polyploids (Dawley, 1989).TheAmazon molly  Poecilia formosa  (Girard) is an all-female fish of hybrid srcinthat lives in freshwater habitats ranging from northern Mexico to southernTexas. The parental species that were involved in this hybridization event atleast 100 000 years ago were female  Poecilia mexicana  Steindachner (mm) (Avise et al. , 1991; Schartl  et al. , 1995 a ) and male  Poecilia latipinna  (Lesueur) (ll)(Schartl  et al. , 1995 a ).  Poecilia formosa  reproduce by sperm-dependent parthe-nogenesis (gynogenesis). Sperm of males of the parental species is used only totrigger the onset of embryogenesis. Normally, males do not contribute geneticmaterial to the F 1  but rarely paternal introgression of subgenomic DNAamounts (Schartl  et al. , 1995 b ) occurs, or even actual syngamy of the diploid ovaand the haploid sperm can result in triploid individuals (Prehn & Rasch, 1969;Schlupp  et al. , 1998). These triploids carry one haploid genome of the sympatricspecies (m n  or l n ) in addition to their hybrid genome (ml). In the Rı´o Purificacio´n(Tamaulipas, Mexico) mixed, i.e. diploid–triploid breeding complexes of  P. formosa  are found.In order to understand the evolutionary significance of triploid individualsamong the mostly diploid  P. formosa , natural populations were analysed fromthe Rı´o Purificacio´n for ploidy level by flow cytometry (Lamatsch  et al. , 2000).A diploid-triploid mosaic was found in  P. formosa  indicating that triploidy mightnot be a stable situation. ‡Author to whom correspondence should be addressed. Tel.: +49 931 888 4152; fax: +49 931 888 4150;email: lamatsch@biozentrum.uni-wuerzburg.de1417 0022–1112/02/061417+06 $35.00/0   2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.  To test whether gynogenetic reproduction was maintained the o ff  spring weremated to black molly males. Black mollies ( P. latepinna ) carry a dominant genefor jet black whole body and fin pigmentation (Schartl  et al  ., 1995 a , 1997;Schlupp  et al  ., 1998). Potential sexual reproduction would lead to interspecificheterozygotes showing black blotches. MATERIAL AND METHODS FISHAll fish tested in this study were wild type individuals from two collection sites,Barretal (24  02   N; 98  22   W) and Nuevo Padilla (24  02   N; 98  54   W), of the Rı´oPurificacio´n, Tamaulipas, Mexico. Until analysis, they were kept in tanks under a12L : 12D cycle with temperature of   c . 26   C and fed twice a day  ad libitum  with  Artemia naupliae and commercially available fish food.FLOW CYTOMETRYMeasurements of the ploidy level were performed from small biopsies of the dorsal finas described by Lamatsch  et al  . (2000). Briefly, the fin clips were chopped in small piecesin 2·1% citric acid/0·5% Tween and blown through a 0·6  0·25 mm syringe. Theresulting cell suspension was gently shaken for 20 min at RT. After centrifugation thecells were suspended in 1 ml 0·5% pepsin/0·1    HCl and incubated for 15 min at RT withgentle agitation. By adding an equal volume of DAPI solution (5·9% tri-sodiumcitrate  2H 2 O/0·0002% DAPI) the DNA was stained for at least 3 h. Before analysis thesamples were filtered (50   m, Partec) to avoid blocking the flow cytometer (CAII, Partec).Chicken  Gallus gallus  erythrocytes were used as internal standard (2·5 pg nucleus  1 ). Atleast 10 000 cells were measured per sample.The x-axis refers to the fluorescence intensity expressed as channels. The DNA contentof the sample was calculated by the relative position of the sample peak to the standardpeak multiplied by the DNA content of the standard: (channel P.formosa ) (channel chicken )  1 2·5 pg nucleus  1 . RESULTS Among 367  P. formosa  from the Rı´o Purificacio´n, Tamaulipas, Mexico,routinely analysed by flow cytometry for triploidy, one specimen exhibitingsomatic diploid–triploid mosaicism (Fig. 1) was found that was phenotypically indistinguishable from diploid and triploid  P. formosa . The measurement wasrepeated twice after the dorsal fin regenerated within 4 weeks. In the firstmeasurement [Fig. 1(a)] 16·6% triploid cells were detected. In the followingmeasurements the proportion of triploid cells decreased to 10·9% [Fig. 1(b)] and 6·8% [Fig. 1(c)]. Theoretically, the germ line of mosaics could contain diploid cells, triploidcells, or both, resulting in diploid or triploid o ff  spring, respectively, or both. Toinvestigate whether the germ line of the  P. formosa  female exhibited mosaicism,the fish was mated to a black molly male. All o ff  spring specimens turned out tobe diploid (unpubl. data). Therefore, the mosaicism appeared to be restricted tothe soma.All o ff  spring ( n =23) were mated to black molly males to see whether asexualreproduction was maintained. Lacking any black body colouration, all the F 2 proved to be produced gynogenetically.1418   .   .       .  DISCUSSION As far as is known, this is the first evidence for a mosaic in  P. formosa .Mosaicism has so far only been reported from some other vertebrate complexessuch as the side-necked turtle  Platemys platycephala  (Bickham  et al. , 1985, 1993), the ginbuna crucian carp  Carassius auratus langsdorfii   (L.) (Murayama  et al. ,1986), the hybrid freshwater minnow  Phoxinus eos-neogaeus  (Cope) (Dawley& Goddard, 1988; Goddard & Schultz, 1993), the rock lizard  Lacertaunisexualis  (Kupriyanova, 1989), and the green frog  Rana esculenta  (Berger &Ogielska, 1994) but is still a rare phenomenon (Kraus, 1991). In the gynogenetic P. formosa  triploids arose by paternal introgression and make up a stableproportion of the asexual population (Rasch  et al. , 1965, 1970; Schultz & Kallman, 1968; Rasch & Balsano, 1989; Lamatsch  et al. , 2000). Among themany animals investigated since its detection in 1932 (Hubbs & Hubbs, 1932) only this single mosaic has been detected. It might be possible that thisphenomenon escaped detection so far due to a lower sensitivity of the previouslyavailable methodology, especially if the degree of the mosaicism was very low.Dawley & Goddard (1988) discuss two possible pathways by which mosaicismmight arise, namely ‘ genome loss ’ and ‘ delayed fertilisation ’. The former canbe seen in meiosis of hybridogenetic unisexuals in which the paternal genome isdiscarded during oogenesis, e.g.  Poeciliopsis  (Schultz, 1969; Cimino, 1972) and Rana esculenta  (Graf & Mu¨ller, 1979), or during mitosis in diaspidid insects(Brown & Bennett, 1957). The latter has been proposed for  C. auratuslangsdorfii  . In this case the sperm nucleus fails to fuse with the female pronucleusand does not participate in the first mitotic division. It persists, however, as acondensed mass in one of the resulting two blastomeres (Kobayashi, 1971). If the sperm nucleus at a later stage joined the maternal chromosomes and 0 1002200(a)    N  u  m   b  e  r  o   f  c  e   l   l  s 5501100165025 50 75 0 1002400DAPI-fluorescence(b)6001200180025 50 75 0 1501800(c)450900135050 100bcabcabca F  . 1. Flow cytometric measurement of somatic cells of the diploid–triploid mosaic  P. formosa  showingdecreasing proportions of triploid cells in subsequent measurements of the regenerated fin. (a)Peak a refers to diploid (83·4%, CV 2·56%) and peak c refers to triploid (16·6%, CV 2·50%) fish fincells, respectively. Peak b shows the DNA content of chicken erythrocytes (2·5 pg nucleus  1 )which serve as internal standard (CV 2·04%). (b) The first regenerate shows 10·9% triploid (CV1·72%) and 89·9% diploid (CV 2·70%) cells. (c) The second regenerate shows 6·8% triploid (CV2·05%) and 93·2% diploid (CV 2·50%) cells. The peaks on the far left are due to the scatter lightof the mercury lamp using higher channels. The chicken DNA peak was arbitrarily set to Channel50 [(a) and (b)] or 100 (c).         1419  participated in the next mitotic division, a mosaic would result containing‘ fertilised ’ triploid and ‘ unfertilised ’ diploid cells.In  P. formosa  the actual syngamy of a diploid egg nucleus with a haploidsperm nucleus leads to a triploid zygote. Mosaics could result from thesubsequent loss of whole genomes from single dividing cells in the triploidembryo (‘ genome loss ’). This could happen early or late in development.Occurring early in development, due to the lower DNA content thesere-diploidized cells should probably replicate their DNA faster than the triploidcells, and would therefore be able to divide more often than triploid cells. Thiscould explain the large fraction of diploid somatic cells, which is also underlinedby the fact that the proportion of diploid cells in the regenerated dorsal finsincreased in comparison to the previous measurement.Alternatively, mosaics could occur if a sperm nucleus, entering a diploid ovum,initially remains quiescent but later undergoes amphimixis with an early cleavagecell (‘ delayed fertilisation ’). Such a delayed-fertilization mechanism wouldresult in unfertilized diploid cells mixed with fertilized triploid cells. Also thismechanism would result in a greater proportion of diploid cells in comparison totriploid cells, as seen in the mosaic  P. formosa . Up to now, insu ffi cient data existto determine whether ploidy di ff  erences are established early or late in develop-ment and by which mechanism. Nilsson & Cloud (1993) have postulated that inorgans, in which cells are rapidly replicating, triploid cells are prone to looseextra chromosomes and assume diploidy.Triploids (D. K. Lamatsch & M. Schartl, unpubl. data) as well as the reportedmosaic  P. formosa  do not change their asexual reproductive mode due to ploidychanges which is in good agreement with the  P. eos-neogaeus  system. Here, thetriploids and the diploid–triploid mosaics mostly produce o ff  spring that carry theclonal genome of   P. eos-neogaeus  (Goddard & Schultz, 1993). They also produce some o ff  spring, however, that did not carry the clonal genome as couldbe shown by tissue graft analyses (Goddard & Schultz, 1993). Unisexual hybridsare a potential source of mosaics, because they employ atypical reproductivemechanisms that often give rise to polyploids (Bogart, 1980; Schultz, 1980). In particular in sperm-dependent parthenogenesis, a failure of the mechanismnormally clearing the egg from the sperm nucleus directly leads to (partial)fertilization.The fact that mosaicism is a very rare event in  P. formosa  may be explained bythe uncompromised viability and fertility of triploids (mlm n ) and mosaics(ml/mlm n ), so that rediploidization is not under selective pressure. Alternatively,triploids may loose the third set in all cells, and therefore reinstall their diploidstatus without being noticed. If the lost set was not the new one (m n ), theindividual gained fresh genetic material, resulting in a m n l-genotype. Byexchanging a whole chromosome set through ‘ genome gain and loss ’, this couldhelp to compensate for Muller’s ratchet (Muller, 1964). Asexual organisms cannot purge deleterious mutations and are therefore expected to experience aso-called mutational ‘ melt-down ’ leading to a gradual decrease of fitness in thecourse of many generations.This process is known as Muller’s ratchet (Muller,1964; Lynch  et al  ., 1993). Progressing further in this direction,  P. formosa  couldeven return to the ‘ pure ’  P. mexicana  (mm n ) genotype by excluding the‘ unequal ’ genome (l) (minority set). To date, however, there are no data1420   .   .       .  indicating that a whole set of chromosomes was excluded. Therefore, informa-tion about the srcin of the third set and which chromosomes are passed on tothe following generations is needed. We thank G. Schneider, H. Schwind and P. Weber for raising the fish, and M. Sto¨ckfor critically reading the manuscript. This work was supported by grants from theDeutsche Forschungsgemeinschaft to M.S. (SFB 567) and a fellowship to D.K.L.(Graduiertenkolleg, Regulation des Zellwachstums). References Avise, J. C., Trexler, J. C., Travis, J. & Nelson, W. S. (1991).  Poecilia mexicana  is therecent female parent of the unisexual fish  P. formosa. Evolution  45,  1530–1533.Berger, L. & Ogielska, M. (1994). Spontaneous haploid-triploid mosaicism in theprogeny of a  Rana kl. esulenta  female and  Rana lessonae  males.  Amphibia-Reptilia 15,  143–152.Bickham, J. W., Tucker, P. K. & Legler, J. M. (1985). Diploid-triploid mosaicism: anunusual phenomenon in side-necked turtles ( Platemys platycephala ).  Science  227, 1591–1593.Bickham, J. W., Hanks, B. G., Hale, D. W. & Martin, J. E. (1993). Ploidy diversity andthe production of balanced gametes in male twist-necked turtles ( Platemys platycephala ).  Copeia  1993,  723–727.Bogart, J. P. (1980). Evolutionary significance of polyploidy in amphibians and reptiles.In  Polyploidy: Biological Relevance  (Lewis, W. H., ed.), pp. 341–378. New York:Plenum Press.Brown, S. W. & Bennett, F. D. (1957). On sex determination in the diaspine scale insect Pseudaulacaspis pentagona  (Targ.) (Coccoidea).  Genetics  46,  510–523.Cimino, M. C. (1972). Egg-production, polyploidization and evolution in a diploidall-female fish of the genus  Poeciliopsis. Evolution  26,  294–306.Dawley, R. M. (1989). An introduction to unisexual vertebrates. In  Evolution and Ecology of Unisexual Vertebrates  (Dawley, R. M. & Bogart, J. P., eds), pp. 1–18.Albany, NY: New York State Museum.Dawley, R. M. & Goddard, K. A. (1988). Diploid-triploid mosaics among unisexualhybrids of the minnows  Phoxinus eos  and  Phoxinus neogaeus. International Journal of Organic Evolution  42,  649–659.Goddard, K. A. & Schultz, R. J. (1993). Aclonal reproduction by polyploid members of the clonal hybrid species  Phoxinus eos-neogaeus  (Cyprinidae).  Copeia  1993, 650–660.Graf, J.-D. & Mu¨ller, W. P. (1979). Experimental gynogenesis provides evidence of hybriddogenetic reproduction in the  Rana esculenta  complex.  Experientia  35, 1574–1576.Hubbs, C. L. & Hubbs, L. C. (1932). Apparent parthenogenesis in nature, in a form of fish of hybrid srcin.  Science  76,  628–630.Kobayashi, H. (1971). A cytological study on gynogenesis of the triploid ginbuna( Carassius auratus langsdorfii  ).  Zoological Magazine  80,  316–322.Kraus, F. (1991). Intra-individual ploidy consistency among unisexual  Ambystoma.Copeia , 38–43.Kupriyanova, L. A. (1989). Cytogenetic evidence for genome interaction in hybridlacertid lizards. In  Evolution and Ecology of Unisexual Vertebrates  (Dawley, R. M.& Bogart, J. P., eds), pp. 236–240. Albany, NY: New York State Museum.Lamatsch, D. K., Steinlein, C., Schmid, M. & Schartl, M. (2000). Noninvasivedetermination of genome size and ploidy level in fishes by flow cytometry:detection of triploid  Poecilia formosa. Cytometry  39,  91–95.Lynch, M., Burger, R., Butcher, D. & Gabriel, W. (1993). The mutational meldown inasexual populations.  Journal of Heredity  84,  339–344.         1421
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