Copyright 0 1991 by the Genetics Society of America Molecular Evidence for Multiple Origins of Hybridogenetic Fish Clones (Poeciliidae: Poeciliopsis) Joseph M. Quattro,* John C. Aviset and Robert C. Vrijenhoek* *Center for Theoretical and Applied Genetics, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231, and ‘Department $Genetics, University of Georgia, Athens, Georgia 30602 Manuscript received September 6, 1990 Accepted for publication October 1 1, 1990 ABSTRACT Hybrid matings between the sexual species Poeciliopsis monacha and Poeciliopsis lucida produced a series of diploid all-female lineages of P. monacha-lucida that inhabit the Rio Fuerte of northwestern Mexico. Restriction site analyses of mitochondrial DNA (mtDNA) clearly revealed that P. monacha was the maternal ancestor of these hybrids. The high level of mtDNA diversity in P. monacha was mirrored by similarly high levels in P. monacha-lucida; thus hybridizations giving rise to unisexual lineages have occurred many times. However, mtDNA variability among P. monacha-lucida lineages revealed a geographical component. Apparently the opportunity for the establishment of unisexual lineages varies amongtributaries of the Rio Fuerte. We hypothesize thata dynamic complex of sexual and clonal fishes appear to participate in a feedback process that maintains genetic diversity in both the sexual and asexual components. ENETIC studies have revealed that clonally re- versity among the hemiclonal monacha genomes of P. G producing, all-female “species” of vertebrates monacha-lucida fromthe Rio Fuerte (VRIJENHOEK, are the products of hybridization between congeneric ANGUSand SCHULTZ1977). For brevity, we refer to sexual species (SCHULTZ 1969;SITES et al. 1990; VRI- distinct hemiclones defined by electrophoresis as “E- JENHOEK et al. 1989).A new unisexual (all-female) types” and identify them by the Roman numerals lineage can be established if genetic differences be- following the biotype designation (e.g., ML/VIII). All tween the hybridizing entities are sufficient to disrupt the allozymic diversity distinguishing Rio Fuerte hem- recombinant processes during meiosis, without se- iclones also exists in P. monacha from this river. How- verely limiting viability, fecundity, and other charac- ever, endemic allozymes, found onlyin discrete P. teristics affecting fitness of the hybrids (MORITZ et al. monacha populations, often mark co-occurring hemi- 1989; VRIJENHOEK1989). With such a narrowwindow clones, suggesting that these ML lineages arose during of opportunity, it is not surprising thatthe origins and independent hybridization events in partially isolated establishment of new unisexual vertebrates have been tributaries. ecologically, geographically, and phylogenetically con- Subsequent tissue grafting studies of Rio Fuerte P. strained (DENSMOREet al. 1989; DENSMORE,WRIGHT monacha-lucida identified 18 histocompatibility clones and BROWN1989; MORITZ, WRIGHTand BROWN (H-types) hidden within the eight E-types (ANGUSand 1989; VRIJENHOEK1989; VYASet al. 1990). SCHULTZ 1979). What proportion of this additional An exception might be unisexual fishes of the genus variation was due to multiple hybridization events vs. Poeciliopsis which have arisen independently in several postformationalmutations within E-type lineages river systems of Sonora and Sinaloa, Mexico. Hybrid- could not be determined. However,evidence for post- ization between P. monacha and several sexual species formational divergence was found in expanded elec- in this genus has produced allodiploid and allotriploid trophoretic surveys that included more loci and uni- unisexual biotypes (SCHULTZ1977). In this study, we sexual fish from additional river systems (SPINELLA focus on the allodiploid biotype P. monacha-lucida and VRIJENHOEK1982; VRIJENHOEK1984). Further- (designated ML), which reproduces by hybridogene- more, genetic studies of several ML strains revealed sis, a hemiclonal mechanism that transmits only the that hemiclonal M genomes carried recessive lethals monacha (M) chromosome set to developing ova and subvitals that probably arose post-formationally (SCHULTZ1969). The lucida (L)chromosomes are (LESLIEand VRIJENHOEK1978, 1980). Apparently, excluded during a premeioticcell division, precluding some hybridogenetic strainshave existed long enough synapsis and crossing over (CIMINO1972). Sperm from to accumulate a substantial load of mutations, but the P. lucida males is required for fertilization and resto- data could not be used to infer relative evolutionary ration of diploidy. ages of hemiclonal lineages. Protein electrophoresis uncovered considerable di- Restriction site analysis of mitochondrial DNA has C;r~~ct~<a127: 391-9YH (February. 1991) 392 J. M. Quattro, J. C. Avise and R. C. Vrijenjhoek FIGURE 1.-The Rios Fuerte, Mayo, and Sinaloa of northern Mexico and associated tri- butaries. Sampling localities (arrows) are indi- cated by site abbreviations: CA, the Arroyo Aguajita de El Cajon;JA, the Arroyo deJaguari and its feeder streams (Arroyos de 10s Platanos and Nachapulon); SP, Rio San Pedro; RG, Ran- cho Guamichil; and GO, Arroyo Seco ca. Co- ronado. Refer to text for more detailed site descriptions. become a useful tool forstudying the origins and dogens from the Rio Fuerte. By integrating informa- evolutionary ages of unisexual vertebrates (MORITZ, tion from allozymes, tissue grafting, and mtDNA we DOWLINCand BROWN1987). Because it is maternally address the following questions: (1) is P. monacha the inherited, mtDNA contains in its sequence the evolu- maternal progenitor to all of P. monacha-lucida lin- tionary history of an all-female lineage. Additionally, eages; (2) are certain P. monacha populations more the molecule’s rapid rate of sequence evolution facil- prone to producingunisexual lineages; and (3) is there itates microevolutionary studies below the species evidence for mutationally divergent, potentially long- level. A preliminary analysis of mtDNA restriction lived unisexual lineages. Finally, we comment on the fragment polymorphism inPoeciliopsis (AVISEand recent decline of P. monacha populations in three river VRIJENHOEK1987), revealed three points relevant to systems encompassing its complete range, and hope- the present study. First, P. monacha and P. lucida have fully raise concern regardingconservation of the com- widely divergent mtDNAs, allowing unequivocal iden- mon ancestor to all unisexual lineages in the genus Poeciliopsis. tification of P. monacha as the maternal progenitor of two naturally occurring P. monacha-lucida strains MATERIALS ANDMETHODS (ML/VII and ML/VIII). Second, these natural hybri- dogens, plus five laboratory strains, clearly demon- Poeciliopsis monacha was originally described as having a strated that mtDNA is maternally inherited. Finally, relictual distribution in headwater tributaries of the Rios Fuerte, Mayo, and Sinaloa of northwestern Mexico (MILLER this study revealed no evidence that the naturalstrains 1960; VRIJENHOEK1979b). Prior to 1978, P. monacha were had diverged mutationally from one another or from found at a single locality in the Rio Sinaloa (Arroyo Seco, the coexisting P. monacha population. ca. Rancho Coronado, Sinaloa). It might be extinct in this In the presentstudy, we conducteda survey of river, since all subsequent attempts to capture this species were unsuccessful. No laboratory strains from this popula- mtDNA diversity in P. monacha, the common ancestor tion were available for study. Prior to 1989, P. monacha of all unisexual forms of Poeciliopsis, and compared were found at two sites in the Guamlichil tributary of Rio its variation to that found in P. monacha-lucida hybri- Mayo (ca. El Tabelo and ca. Rancho Guamlichil, Sonora). mtDNA Variation in Poeciliopsis 393 TABLE 1 according to the manufacturers' specifications with a battery of 17 restriction endonucleases having four (MsPI), five Sources of P. monacha and P. monacha-lucida and their (AuaI, AuaII, HincII), and six (BamHI, BclI, BglI, BglII, electrophoretic andmtDNA haplotypes BstEII, EcoRI, HindIII, NdeI, PstI, PuuII, SpeI, StuI, Xbal) base recognition sequences. DNA fragments were end-la- Strain beled with ["S]dNTP(s) and separated in 0.8-1.8% agarose Locality and biotype Wild Laboratory E-type" Mt-typeb gels. After electrophoresis, gels were dried under vacuum onto a filter paper backing and exposed to X-ray film at Rio Fuerte room temperature for 24-72 hr. A. San Pedro Each restriction digest profile for a specific endonuclease P. monacha 1 2 was given an arbitrary letter code. Composite scores, rep- P. monacha 1 8 resenting a letter code for each of 17 gel-fragment profiles, P. monacha 1 11 wereassigned to each individual. Sinceall gel-fragment P. monacha 1 14 patterns could be related by specific site gains or losses, a P. monacha-lucida 2 I 11 matrix of the minimum number of site changes necessary P. monacha-lucida 1 I1 14 to interrelate composite haplotypes was constructed. A min- P. monacha-lucida 1 IV 7 imum length network linking observed haplotypes was con- B. Guiricoba structed by inputting the mutation matrix into the minimum P. monacha 6 7 spanning tree algorithm of the NTSYS statistical package P. monacha-lucida 1 I 11 (ROHLF,KISHPAUCH and KIRK 1974). The resulting network P. monacha-lucida 1 I 12 was compared to the consensus of the most parsimonious P. monacha-lucida 1 I1 13 trees obtained with the CONSENSE and MIX computer 4 I1 14 P. monacha-lucida programs (FELSENSTEIN1990). We evaluated the fit of the P. monacha-lucida 1 Ill 9 resulting