Mastozoología Neotropical; 6(2):129-133
ISSN 0327-9383
SAREM, 1999
REPRODUCTIVE BARRIERS BETWEEN THE 2N=42 AND 2N=36-38 CYTOTYPE OF Graomys (RODENTIA, MURIDAE) Gerardo R. Theiler1,3, Rubén H. Ponce1,4, Ricardo E. Fretes2, and Antonio Blanco1 1
Cátedra de Química Biológica, Fac. de Ciencias Médicas, C.C. 35, Suc. 16, Teléfono y Fax: 0351– 433-4024, Correo Electrónico:
[email protected]; 2 Cátedra de Histología y Embriología II, Fac. de Ciencias Médicas, 3 Cátedra de Introducción a la Química y la Física Biológicas, Fac. de Odontología; 4 Cátedra de Química Biológica, Fac. de Odontología; Universidad Nacional de Córdoba. ABSTRACT: Reproductive barriers between the 2n=42 cytotype (Graomys centralis) and the complex 2n=36-38 of G. griseoflavus have been previously observed. Those observations led to propose the existence of two separate species, being the 2n=36-38 cytotypes derived from 2n=42 by Robertsonian fusions. Sterile hybrids could be obtained in the laboratory colony by crossings between 36-38 chromosome females and 42 chromosome males. In contrast, sixty reciprocal crosses were always unproductive due to predominantly precopulatory barriers. There was no copula in 7 out of 8 controlled pairings. In the only mating resulting in fecundation, marked impairment of development and placental lesions making the embryos non viable were observed. The asymmetric precopulatory isolation agrees with the predictions of Kaneshiro’s hypothesis, stating that females of the new species mate either with conspecific as well as ancestral species’ males, while females of the ancestral species accept only conspecific males. RESUMEN: Barreras reproductivas entre el citotipo 2n=42 y el complejo 2n=36-38 de Graomys (Rodentia, Muridae). Se ha descripto la existencia de una serie de barreras reproductivas entre el citotipo 2n=42 y el complejo 2n=36-38 de Graomys . Estas observaciones llevaron a proponer que se trata de dos especies plenas, aunque morfológicamente indistinguibles. Los citotipos 2n=36-38 son derivados del 2n=42 por fusiones robertsonianas. En el laboratorio es posible obtener híbridos estériles por cruzamientos de hembras 2n=3638 con machos 2n=42. En cambio, 60 apareamientos recíprocos fueron siempre improductivos. Nuestras observaciones muestran que en este caso la barrera reproductiva es predominantemente precopulatoria, ya que en 7 de 8 apareamientos controlados no hubo cópula. Sólo en uno de estos cruzamientos se produjo fecundación, pero los embriones resultantes mostraban marcado retardo de su desarrollo y lesiones placentarias que los hacían inviables. El aislamiento precopulatorio asimétrico observado es compatible con las predicciones de la hipótesis de Kaneshiro: las hembras de la especie derivada aceptan tanto machos coespecíficos como heteroespecíficos, mientras las de la especie ancestral sólo aceptan machos coespecíficos. Key words: Graomys griseoflavus, G. centralis, cytotypes, reproductive barriers Palabras clave: Graomys griseoflavus, G. centralis, citotipos, barreras reproductivas
Recibido 30 octubre 1998. Primera aceptación enero 1999. Aceptación final junio 1999.
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INTRODUCTION The South American murid rodent Graomys griseoflavus (Waterhouse, 1937) is widely distributed throughout Argentina (Rosi, 1983). Populations from different regions show a striking chromosomal polymorphism (Pearson and Patton, 1976; Rosi, 1983; Zambelli et al., 1994; Theiler and Blanco, 1996a; Theiler, 1997). Whole arm translocations (Robertsonian fusions, RF) are the dominant type of rearrangement observed in Graomys griseoflavus (Ortels et al., 1989). Cytogenetic studies on different populations have indicated that two fusions between chromosome pairs 15 and 17 (RF 15-17) and 16-18 (RF 16-18) from animals with 2n=42 originated the 2n=38 cytotype (Zambelli et al., 1994). A third fusion between pairs 1 and 6 (RF 1-6) produced the cytotype 2n=36. Individuals with 2n=37 are heterozygotes for the 1-6 Robertsonian fusion. In addition, individuals with four Robertsonian fusions (RF 2-5 plus those mentioned previously) were found. Homozygotes for RF 2-5 have 34 chromosomes and heterozygotes are 2n=35. All these different cytotypes are morphologically undistinguishable. The existence of reproductive barriers between the 2n=42 cytotype and the complex of 2n=36, 37 and 38 have been reported by Theiler and Blanco (1996a). It was also demonstrated that females are able to discriminate olfactory stimuli from males and show marked preference for conspecific males (Theiler and Blanco, 1996b). This finding supports the existence of an effective premating reproductive barrier that prevents heterospecific crosses and explains the absence of hybrids in nature. The demonstration of the presence of these reproductive barriers between cytotypes 2n=3638 and 2n=42 led us to propose that they are two sibling species, and to assign the name Graomys centralis to the 2n=42 species and to maintain the name Graomys griseoflavus for the 2n=36-38 complex (Theiler and Blanco, 1997); a similar nomenclature was proposed by Tiranti (1998). Cytogenetic studies on indi-
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viduals from the type locality of G. centralis (Cruz del Eje, Córdoba province) would confirm the pertinence of this specific name for the 2n=42 species. Although some heterospecific crosses were possible in the laboratory colony, matings between Graomys centralis females and G. griseoflavus males were always unproductive. New data presented here indicate that the reproductive barrier is not only pre-, but also post-zygotic. The asymmetry in the premating barrier agrees with Kaneshiro’s hypothesis proposing that the phylogenetic direction can be inferred from the direction of asymmetric reproductive barriers.
MATERIAL AND METHODS Individuals studied of the Graomys centralis species were F1 and F2 descendants from animals trapped in Santiago Temple (Córdoba province) and those of G. griseoflavus were descendants from Salicas (La Rioja province) specimens. They were maintained in a temperature-controlled room (25 ± 2ºC), with a 12 h light : 12 h dark photoperiod. Food and water were given ad libitum. Eight females of Graomys centralis (2n=42) were mated with G. griseoflavus males (2n=36-38) within polycarbonate mouse cages. Additional crosses were carried out with 10 conspecific pairs: five 2n=42 females were paired individually with 2n=42 males, and five 2n=36-38 females were paired with 2n=3638 males. To determine whether successful mating had occurred, a daily observation of vaginal smears was performed under the microscope to reveal the presence of spermatozoa. The couples were maintained in the cage until copula was confirmed or during 25 days in negative cases. Twelve days after coitus, females were sacrificed by ether inhalation. The uteri of all pregnant females were removed and dissected, and the embryos counted. Fixation of embryos and uteri was performed with Bouin’s solution, then dehydrated, embedded in paraffin wax, sectioned transversally at 5 mm and stained with haematoxylin and eosin. Four normal embryos were dissected to be measured. The material was observed under microscope at 100x. Degree of development of the nervous system, mesodermic and endodermic structures, and placental tissue was examined and compared with homologous normal specimens.
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RESULTS Observations under light microscope revealed the presence of spermatozoa in the vaginal tract of all intraspecific crosses performed with sexually receptive females. Evidence of coitus was observed in only one out of the eight interspecific crosses. Four out of the seven females that not mated with heterospecific males kept their estrual cycle during the 25-day period of cohabitation with the male and three showed pseudopregnancy, as indicated by a continuous diestrus. When the male was removed from the cage they recovered the estrual cycle. All ten females mated with conspecific males, and the only Graomys centralis female mated with a 36-chromosome male, had embryos twelve days after corroboration of sperm in vaginal smears. Size of gravid uterus of females crossed with conspecific males were visibly larger than uterus of the 2n=42 female crossed with a 2n=36 male (Fig. 1).
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Hybrid embryos were macroscopically undetectable, whereas the body length of normal embryos was 5 mm. Twelve day old control embryos showed structures derived from ectoderm, mesoderm and endoderm. Histological sections from the same age hybrid embryos revealed ectodermic structures and formation of a neural tubule where the lumen and encephalic vesicle were visible. In the layer next to the lumen neuroepithelial cells were proliferating and forming neuroblasts (Fig. 2). There was no formation of the mantle and marginal layers. This picture is similar to that observed in normal embryos at the second or third day of development. A very complex structure could be seen in control embryos, showing organs derived from the telencephalic, diencephalic, mesencephalic, metencephalic and mielencephalic vesicles, and from mesoderm and endoderm. Mesodermic and endodermic structures were absent in hybrid embryos (Fig. 2).
Fig. 1. Gravid uteri, dissected at 12 days of pregnancy. A) 2n=42 x 2n=42; B) 2n=38 x 2n=37; C) 2n=42 x 2n=36.
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Fig. 2.
A) Transversal section of gravid uterus product of a mating 2n=42 x 2n=36 (x 5.6). E: embryo; Nt: Neural tube; P: placenta showing degenerative lesions. B) Neural tube (x 8.5), showing encephalic vesicle (V).
The placental tissue from hybrids showed hemorrhagic areas and necrosis (Fig. 2). These degenerative lesions were serious enough as to be incompatible with prosecution of embryonic development.
DISCUSSION In a previous work Theiler and Blanco (1996a) reported that sterile hybrids were obtained from matings between Graomys griseoflavus (2n=36-38) females and G. centralis (2n=42) males, while none of sixty reciprocal crosses produced descendants. Absence of mating with heterospecific males in seven out of eight of the 2n=42 females, confirm the existence of premating reproductive barriers as previously proposed (Theiler and Blanco, 1996b). The presence of sperm in vaginal smears or the formation of embryos was not investigated previously; however, it is possible that there were some matings and fecundations, but resorption of defective embryos could have halted the gestation process. The present observations indicate retarded development of embryos in the only case resulting in pregnancy. Further experiments will be required to define the causes of the inviability of hybrid embryos from 2n=42 female and 2n=36 male crosses. Anyway, our observations indicate that
the reproductive isolation between 2n=42 females and 2n=36-38 males is predominantly precopulatory. An asymmetric premating reproductive isolation has been observed in several species of Drosophila (Kaneshiro, 1976, 1980; Gidding and Templeton, 1983) and rodents such as Spalax ehrenbergi (Heth and Nevo, 1981) and Geomys (Bradley et al., 1991). In order to explain his observations in Drosophila, Kaneshiro (1976, 1980) proposed that during the origin of a new species some elements of the premating behavior could change by genic drift and relaxation of the sexual selection along explosive colonization. It is also argued that females from the ancestral species are able to discriminate between the conspecific male courtship and that from males of the new species, whereas females from the new species can accept the courtship of conspecific and heterospecific males. Our results are compatible with the predictions of the Kaneshiro hypothesis. However, the model of Kaneshiro (1976, 1980) is applicable only to species whose isolation mechanisms are originated from a founder-flush event. Previous allozymic data (Theiler and Gardenal, 1994; Theiler, 1997; Theiler et al., 1999), in populations of both species of Graomys indicated similar, and in some cases
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lower, values of interspecific genetic divergence than pairs of populations of the same species. These results indicate that the cladogenetic process observed in Graomys has occurred without the genetic drift characteristic of founder events. The Kaneshiro’s hypothesis offers criteria to infer which is the ancestor from the direction of the one-way reproductive isolation between a pair of species. This idea is supported by many authors (Mc Phail, 1969; Kaneshiro, 1976, 1980; Heth and Nevo, 1981; Gidding and Templeton, 1983; Bradley et al., 1991) and questioned by others (Ehrman and Wasserman, 1987; Moodie, 1982; Markow, 1981). If the asymmetric reproductive isolation really reflects the direction of evolution, alternative theoretical models would be required to explain our observations on the Graomys species without evidence of a founder-flush origin.
ACKNOWLEDGEMENTS We thank Dr. M. Gallardo for valuable suggestions and Mr. V. Tomasi for his technical assistance. We also acknowledge the anonymous reviewers. This work has been partially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Fondo para la Investigación Científica y Tecnológica (FONCYT), the Consejo de Investigaciones Científicas y Tecnológicas de Córdoba (CONICOR), and the Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba (SeCyT). A. B. is a Career Investigator of CONICET.
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