Mastozoología Neotropical / J. Neotrop. .; 9(2):159-170 ISSN 0327-9383 ©SAREM, 2002 Versión on-line ISSN 1666-0536

MORPHOLOGICAL DIVERSITY IN THE SPERMS OF CAVIOMORPH

Milton H. Gallardo1, F.C. Mondaca1, R.A. Ojeda2, N. Köhler1, and O. Garrido3

1 Instituto de Ecología y Evolución, Universidad Austral de Chile. 2 IADIZA-CONICET, GIB, Casilla de Correo 507, 5500 Mendoza, Argentina. 3 Instituto de Embriología, Universidad Austral de Chile, Casilla 567, Valdivia, Chile. .

ABSTRACT. The general sperm morphology of 20 species comprising five families of Caviomorph rodents was studied by light and electron microscopy. Large inter- and intrafamily differences in size and shape reflect the plastic nature of sperm morphology in the taxa analyzed. The sperm heads of the Abrocomidae and Myocastoridae are symmetrically rounded and tapered to the apical end. Paddle-like, asymmetric, and long-tailed sperms are reported in the monotypic family Ctenomyidae whereas symmetrically oval and inverted bulbous head morphologies are found in the Caviidae. Exceedingly large headed, truncated and paddle-like sperms differing from the common oval-shaped ones found in the , are char- acteristic of barrerae. The distinct, macrocephalic sperms of this octodontid are correlated with a quantum increase in DNA content as reflected by the species´ dupli- cated genome size. The implications of this finding in connection with sperm morphology and motility are discussed.

RESUMEN. Diversidad morfológica en los espermios de los roeodres caviomorfos. Mediante microscopía corriente y electrónica se estudió la morfología espermática de 20 especies que comprenden cinco familias de roedores caviomorfos. Grandes diferencias inter e intrafamiliares reflejan la naturaleza plástica de la morfología espermática en las especies analizadas. La cabeza del espermio de Abrocomidae y Myocastoridae es simétricamente redondeada, con un extremo apical más agudo. En Ctenomys se observaron espermios asimétricos, aplanados y de largos flagelos mientras que los espermios simétricos de los Caviidae son de cabeza ovalada o de forma de bulbo invertido. La enorme cabeza de los espermios de Tympanoctomys barrerae es plana y truncada en la base por lo que difiere de la forma ovalada que se observa en los otros octodóntidos. Los espermios macrocefálicos de este octodóntido están correlacionados con el aumento de su contenido de ADN nuclear que duplica al de sus congéneres. Se discuten las implicaciones de estos hallazgos con relación a la morfometría y la motilidad espermática.

Key words: caviomorph rodents, Octodontidae, sperm dimensions, sperm morphology, tet- raploidy, Tympanoctomys barrerae.

Palabras clave: roedores caviomorfos, Octodontidae, dimensiones espermáticas, morfología espermática, tetraploidía, Tympanoctomys barrerae.

Editor reponsable para el presente artículo: Carlos Borghi. 160 Mastozoología Neotropical / J. Neotrop. Mammal.; 9(2):159-170 M.H. Gallardo

INTRODUCTION Vitullo and Cook, 1991; Freitas, 1995), the sister group of the Octodontidae (Gallardo and The mammalian spermatozoa is a highly dif- Kirsch, 2001). Moreover, the macrocephalic ferentiated cell displaying a complex morphol- sperm morphology correlated with a quantum ogy associated to its DNA content and chro- increase of genome size in the octodontid matin organization (Bedford and Hoskins, Tympanoctomys barrerae (Gallardo et al., 1990; Ferrari et al., 1998). These streamlined 1999) further emphasizes the plasticity of its cells consist of an elaborated motile apparatus sperm cells. Here, we present further data on and of a highly condensed, haploid nucleus cauda epididymal spermatozoa from a range (Breed, 1995). The spermatozoon's DNA is of caviomorph rodents. The morphological packed in a linear side-by-side array of chro- features of these sperms are compared to those matin that represents the most highly compacted previously described and the possible forces eukaryotic DNA (Ward and Coffey, 1991; involved in size variation are discussed. Ward, 1998). But the role of the sperm is rel- evant also in connection with the development METHODS of the future embryo. In fact, the sperm entry position determines the plane of initial cleav- Live adult males were live-trapped and sacrified in age of the egg, and thus defines the early antero- the field or in the laboratory, with ether anesthesia. posterior axis of mouse embryos (Piotrowska Testes and caudae epididymes were obtained from and Zernicka-Goetz, 2001). 55 of the following species: Abrocomidae: Abrocoma bennetti: Peñuelas Lake National Reserve, Although sperm dimensions and shape are Valparaíso province, Chile (1). Caviidae: Cavia constant within species, a wide variance in porcellus: captive bred specimen (1). Dolichotis morphology ranging from falciform to paddle- salinicola: El Balde Ranch, San Luis province, like sperm heads have been described among Argentina (1). Galea musteloides: Ñacuñán National higher taxa (Cummins and Woodall, 1985; Reserve, Mendoza, Argentina (3). Microcavia aus- Roldan et al., 1992). These differences have tralis: Ñacuñán National Reserve, Mendoza, Argen- been used to assess systematic relationships tina (4). Ctenomyidae: Ctenomys coyhaiquensis: both in eutherians (Roldan et al., 1992; Breed Fundo Los Flamencos, Coyhaique province, Chile and Aplin, 1994; Breed, 1995) and marsupials (2). Ctenomys eremicus: Ñacuñán National Reserve, Mendoza, Argentina (2). Ctenomys haigi: Cerro (Harding, 1987; Temple-Smith, 1987). Otto, Bariloche, Río Negro province, Argentina (6). Sperm morphology is also structurally diverse Ctenomys sociabilis: Confluencia, Río Negro prov- among rodents (Gage, 1998). The falciform ince, Argentina (1). Myocastoridae: Myocastor coy- sperm head found in most myomorphs is con- pus: Cruces river, Valdivia province, Chile (1). sidered to represent a plesiomorphic condition Octodontidae: Aconaemys fuscus: Radal-Siete Tazas according to the commonality criterium National Reserve, Curicó province, Chile (2). (Yanagimachi and Noda, 1970; Breed, 1984, Aconaemys porteri: Villarrica National Park, Cautín 1995; Breed and Yong, 1986; Breed and Aplin, province, Chile (2). Aconaemys sagei: Reigolil, 1994). Curarrehue, Cautín province, Chile (2). bridgesi: Unihue, Cauquenes province, Chile (2), Early studies on the sperms of South Ameri- Las Heras, Quirihue, Chillán province, Chile (1). can genera Lagidium, Myocastor, and Octodon degus: Cuesta Las Chilcas, Metropolitan Ctenomys, described a flattened, paddle-like Region, Chile (1); Peñuelas Lake National Reserve, and symmetrical sperm head supposed to rep- Valparaíso province, Chile (3), La Campana Na- resent the most common morphology for the tional Park, Valparaíso province, Chile (2), San Caviomorpha (Jones, 1974). The subsequent Felipe, Los Andes province, Chile (1). Octodon description of similar sperm heads in the lunatus: Peñuelas Lake National Reserve, Valparaíso octodontid Octodon degus further supported province, Chile (1). Octomys mimax: Ischigualasto this notion (Berríos et al., 1978; Roldán et al., National Park, La Rioja province, Argentina (2). Octodontomys gliroides: Chusmiza, Iquique prov- 1992). Nevertheless, large deviations from ince, Chile (2). Spalacopus cyanus: Los Remates, symmetry occur in the Ctenomyidae (Feito and Quirihue, Ñuble province, Chile (5). Tympanoctomys Gallardo, 1976, 1982; Vitullo et al., 1988; barrerae: EL Nihuil, Mendoza province, Argentina SPERM DIVERSITY IN CAVIOMORPHS 161

(7). Specimens were deposited in the Collection of The terminology for symmetry, indicating the , Institute of Ecology and Evolution, displacement of the tail insertion from the central Universidad Austral de Chile, and in the Mammal axis of the caudal end of the head, follows Feito Collection, Iadiza-Cricyt, Argentina. and Barros (1982). Ventral referred to the tail ap- Semen was extruded from the lacerated caudal pearing at the left side of the flat surface of the end of the epididymes and placed in Hanks bal- head facing the observer. When the tail appeared to anced salt solution. Smears were air-dried, fixed in the right, that surface was referred to as dorsal (Feito Carnoy (methanol-acetic 3:1), and stained either with and Barros, 1982). Images of sperms were digitally DAPI or with 10% Giemsa solution in pH 7.0 captured and contrast enhanced with the Adobe Sörensen buffer (Feito and Gallardo, 1982). Sper- Photoshop 7.0 software. matozoa were examined under a light microscope and measured using an ocular micrometer at a mag- RESULTS nification of 1,250X. Linear dimensions of tail length, head length, and greatest width of the head Only slight morphological variation of the sper- were recorded in 150 gametes chosen at random matozoa from any species was generally found, from different smears. The ultrastructure of the sperms of T. barrerae was studied by transmission but there was considerable interspecific varia- electron microscopy (TEM). The epididymes were tion. Among members of the Octodontidae, fixed in 2.5% glutaraldehyde and 2% paraformalde- sperms were examined for representatives of hyde in 0.1% cacodylate buffer for 4 hrs at room all traditional genera, excluding the recently temperature (Lin and Rodger, 1999). The tissues described genera Salinoctomys and were post-fixed in 1% osmium tetroxide in 0.175M Pipanacoctomys (Mares et al., 2000). Consid- cacodylate buffer at pH 6.8, dehydrated in acetone ering the interspecific variation across fami- and embedded in epoxy resin as described by Feito lies, the description of the medium-sized, sym- and Barros (1982). The sperms for scanning elec- metrical sperm of the octodontid Octodon tron microscopy (SEM) were prepared by using the same fixative procedures as for TEM. After dehy- degus will provide a descriptive standard. The dration and critical point drying, the sperms were sperm head of this species was 6.0 mm long gold-coated and examined in a Hitachi H 700 scan- and 4.6 mm wide, and the tail was 40.9 mm. ning electron microscope. (Fig. 1E). The head was ovate in outline, flat- The spermatozoa smears were Feulgen-stained tened dorso-ventrally, basally broad and taper- according to Itikawa and Ogura (1954) and the ing apically as previously described (Berríos emissions recorded with a Zeiss MPM 400 scan- et al., 1978). Slight differences in sperm head ning microdensitometer. The smears were processed measurements and shape were observed in the and analyzed simultaneously with sperm cells of other Octodon species (Fig. 1D-F). The sperm Mus musculus used as an internal standard. Two slides of each specimen tested were analyzed, and head of O. bridgesi was more elyptical than the fluorescent emission was read at 542 nm. The rounded (Fig. 1D) whereas in O. lunatus it statistical analysis and graphic ellaboration of data was paddle-shaped and basally broader (Fig. were both carried out with the computer program 1F; Table 1). The acrosome was about 60% PHOTAN, provided by the manufacturers. DNA of the head length in the three species (Fig. content was calculated by dividing X by the emis- 1D-F). sion value of the tested, multiplied by the The head and flagellar dimensions of the 3.3 pg DNA for the sperm cells of the Mus muscu- three fossorial Aconaemys species were smaller lus standard (Pogany et al., 1981). Descriptive sta- than in O. degus. The sperm heads were sym- tistics and linear regressions between DNA content and sperm dimensions were obtained with Excel. metrical, moderately spatulated or ovoid, but Additional estimates of gametic DNA content to narrower and shorter than those of O. degus the one published elsewhere (Gallardo et al, 1999) (Fig. 1 A-C; Table 1). The head tapered to- were assesed by microdensitometry in 150 Carnoy- wards its tip in A. fuscus and A. sagei but it fixed spermatozoa of each of several species of was longer and narrower in A. porteri. The octodontids. Small differences attributed to slide acrosome covered more than 50% of the head handling and staining procedure may explain the length in these species (Fig. 1A-C). difference between that information and the one Among the desert specialists, the moderately provided here. spatulated, symmetrical sperm heads of 162 Mastozoología Neotropical / J. Neotrop. Mammal.; 9(2):159-170 M.H. Gallardo

Fig.1. Sperm morphology in the Octodontidae. A) Aconaemys fuscus, B) Aconaemys porteri, C) Aconaemys sagei, D) Octodon bridgesi, E) Octodon degus, F) Octodon lunatus, G) Octomys mimax, H) Octodontomys gliroides, I) Spalacopus cyanus, J) Tympanoctomys barrerae.

Octomys mimax and Octodontomys gliroides stituted more than half of the head length. were similar to the ones of O. degus (Fig. 1G- The sperm dimensions of T. barrerae were H). The tail, shorter in O. mimax than in O. significantly larger than those of the remaining degus, attached to the truncated posterior end octodontids (Table 1, Fig. 1J) or of any other of the head (Table 1, Fig. 1H). A large species so far reported (Cummins and acrosomic region, covering approximately 60% Woodall, 1985). The sperm head had a dis- of the head length was also observed in these tinctive spatulated shape with a broad and flat species. Although previous data reported lateral face (Gallardo et al., 1999). Unlike the slightly wider than larger sperm head dimen- symmetrical sperms of the remaining sions for O. gliroides (Cummins and Woodall, Octodontidae, the flagellum of T. barrerae was 1985), the general morphology of its sperma- attached submedially to the truncated end of tozoa coincides with our description. the head (Fig. 2A). Although the flagellum was The sperm dimensions of the subterranean longer relative to other cofamily members, it Spalacopus cyanus were smaller than in O. was only two thirds the flagellum length of degus but similar to the sperms of A. fuscus Cavia porcellus (Table 1). The hood-like and O. lunatus (Table 1). The symmetrical head acrosome is more than 50% of the anterior was elyptical and had rounded, apical and part of head (Fig. 2A, 2B). Using pooled val- posterior borders (Fig. 1I). The acrosome con- ues of the octodontid genera, and assuming an SPERM DIVERSITY IN CAVIOMORPHS 163

Table 1 Mean values and standard deviations for head length, width, and total length of the tail in 20 species of caviomorph rodents. Measurements in mm. DNA content (pg DNA) is also provided for different taxa (Gallardo et al., in press).

TAXON Mean head Mean head Mean tail Gametic length width length DNA Content

ABROCOMIDAE Abrocoma bennetti (1) 6.4 ± 0.3 4.4 ± 0.1 35.0 ± 1.2

CAVIIDAE Cavia porcellus (1) 7.5 ± 0.3 6.6 ± 0.1 92.3 ± 0.9 Dolichotis salinicola (1) 5.5 ± 0.3 3.3 ± 0.2 47.3 ± 0.8 Galea musteloides (3) 5.3 ± 0.2 4.9 ± 0.4 47.2 ± 1.0 Microcavia australis (4) 5.3 ± 0.2 4.8 ± 0.4 44.5 ± 0.7

CTENOMYIDAE Ctenomys coyhaiquensis (2) 7.6 ± 0.4 4.3 ± 0.3 66.3 ± 2.2 Ctenomys eremicus (2) 9.7 ± 1.4 5.8 ± 0.5 70.6 ± 1.8 Ctenomys haigi (6) 8.1 ± 0.6 5.5 ± 0.2 61.7 ± 1.6 Ctenomys sociabilis (1) 6.7 ± 0.3 5.4 ± 0.2 61.8 ± 1.2

OCTODONTIDAE Aconaemys fuscus (2) 5.6 ± 0.3 3.3 ± 0.2 35.2 ± 2.3 Aconaemys porteri (2) 5.5 ± 0.2 3.1 ± 0.1 33.2 ± 0.8 2.1 ± 0.3 Aconaemys sagei (2) 5.5 ± 0.4 3.3 ± 0.2 40.9 ± 1.4 Octodon bridgesi (3) 7.0 ± 0.7 4.4 ± 0.4 39.1 ± 1.7 Octodon degus (7) 6.0 ± 0.7 4.6 ± 0.4 40.9 ± 1.6 2.7 ± 0.1 Octodon lunatus (1) 7.7 ± 0.6 5.3 ± 0.4 35.8 ± 2.4 Octomys mimax (2) 5.4 ± 0.8 3.9 ± 0.7 33.4 ± 0.8 4.1 ± 0.3 Octodontomys gliroides (2) 6.5 ± 0.3 4.7 ± 0.5 39.7 ± 1.0 Spalacopus cyanus (5) 5.2 ± 0.6 3.4 ± 0.5 36.0 ± 1.6 3.3 ± 0.5 Tympanoctomys barrerae (7) 14.2 ± 0.3 13.7 ± 0.3 69.5 ± 1.0 8.7 ± 0.5

MYOCASTORIDAE Myocastor coypus (1) 4.4 ± 0.2 3.3 ± 0.1 31.1 ± 1.5

* The number of animals analyzed is given in parenthesis.

isometric growth of the head/flagellum ratio, sperms of T. barrerae (Gallardo et al., 1999). tail length in T. barrerae should range between Head dimensions were 14.2 mm in length, 13.7 82 mm and 121mm relative to head length and mm in width, and 0.4 mm in height (Fig. 2). head width, respectively. This means that the The length and width of the mid-piece (7 x 1.7 flagellum has grown only 55% to 81% of the mm, respectively) was within the range of other value predicted by the enlargement of its head mammalian sperms (Bahamonde, 1999). dimensions. Slightly larger head dimensions The dimensions of the sperm head of were obtained by electron microscopy of the Abrocoma bennetti were similar to those of O. 164 Mastozoología Neotropical / J. Neotrop. Mammal.; 9(2):159-170 M.H. Gallardo

Fig. 2. Electron microscopy of the sperms of Tympanoctomys barrerae. A) Ventral view of a scanning electron micro- graphy. Note the asymmetric position of the tail insertion. B) Sagital view of a transmission electron micrograph. Note the flat appearance of the head including the hood-like acrosome that covers its apical area. degus but the flagellum was shorter (Table 1). apex (Fig. 3C). Its acrosome covered approxi- The head was also symmetrical and bilaterally mately 70% of the head length and the nuclear flattened. It had an elypsoid, less elongated caudal extension was comparatively shorter shape compared to the octodontids and the head than in the C. haigi and C. sociabilis. The sperm did not taper apically (Fig. 3A). This general head of C. haigi, although rounded at the apex, morphology coincides with a previous descrip- was rather rectangular. Its acrosome covered tion (Herrera et al., 1976; Berríos et al., 1978). approximately 70% of the head length (Fig. The sperm head of the monotypic 3D). The largest sperm dimensions in the Myocastoridae Myocastor coypus was rounded Ctenomys species reported here (including a and tapered to the apical end (Fig. 3B). Al- 10.7 mm long nuclear caudal extension) were though no micrograph was originally provided, found in C. eremicus (Table 1). The sperm the general characteristics and dimensions here head of this species was spatulated but more recorded (Table 1) coincide with the ones re- rectangular than in the other species. Its ported by Cummins and Woodall (1985). The acrosome covered three fourths of the head acrosome cap covered more than 50% of the length (Fig. 3E). head. The tail attached to a rounded, posterior Variation in sperm size and morphology was end was observed also in the octodontid gen- extensive among the Caviidae. Larger sperms era Aconaemys and Spalacopus. compared to those of O. degus were found The sperm morphology in the Ctenomys (Table 1; Fig. 4A-D). Differing in size and species (Fig. 3) conformed to the asymmetri- shape, the sperms of the guinea pig, Cavia cal pattern already described for several spe- porcellus were the longest among the cies (Feito and Gallardo, 1982). The paddle- caviomorphs reported here. The sperm head shaped sperm heads had a nuclear caudal ex- had a broad, disc-shaped and symmetrical form tension displaced toward the opposite side of with a truncated posterior end. The acrosome the flagellum (Feito and Barros, 1982). These covered 50% of the head length (Fig. 4A). large-headed, long-tailed sperms exceeded the Although no micrograph of the sperm morphol- dimensions recorded for O. degus (Table 1). ogy was provided by Cummings and Woodall The sperm head of C. sociabilis was more (1985), our estimates are highly coincidental rectangular than elyptical and tapered to the with theirs. A very distinctive, symmetrical SPERM DIVERSITY IN CAVIOMORPHS 165

10 mm

Fig 3. Light microscopy of the sperms of A) Abrocoma bennetti, B) Myocastor coypus, C) Ctenomys sociabilis D) Ctenomys haigi, and E) Ctenomys eremicus.

sperm head with a broad apical end resem- variations. To explore their relationships, the bling an inverted bulb was found in Galea head dimensions and flagellum length was re- musteloides (Fig. 4B). The acrosome formed a gressed against the somatic DNA content of broad cap that mimics the bulbous shape of different rodents listed in Table 1. Significant the head. Slightly larger head dimensions (but relationships were found between DNA con- no picture) had been reported previously for tent and head length (F = 98.7, P < 0.001, N this species (Cummins and Woodall, 1985). = 15), or head width (F = 78, P < 0.001, N = The sperm heads of Microcavia australis (Fig. 15), but not with tail length (F = 3.66, P = 4C) were also bulbous and, as the ones of 0.07, N = 15). Nevertheless, if T. barrerae is Dolichotis salinicola, bullet-shaped in ventral excluded from the comparison between sperm view. The flagellum length was similar in both dimensions and DNA content, non significant species, but larger than in O. degus (Table 1, results are obtained (P > 0.68). Fig. 4D). The acrosoma was poorly defined and covered more than 50% of the head length. DISCUSSION As expected, genome size estimates from sperm cells of several octodontids were ap- Contrasting differences in size and morphol- proximately half the values obtained from so- ogy within and across the families characterize matic tissues (Gallardo et al., in press). The the spermatozoa of the caviomorph rodents gametic DNA content fluctuated from 2.1 pg studied here. The sperms of C. porcellus dras- in Aconaemys to 8.7 pg in T. barrerae (Table tically differed in head and tail dimensions from 1). The difference between this estimate for T. those of the remaining caviids (Table 1). The barrerae and its previous recorded value (9.2 macrocephalic and distinctive paddle-like sperm pg DNA, Gallardo et al., 1999) is attributed to head of T. barrerae is the largest reported for the staining procedure and inter individual rodents and it is as large as the record-sized, 166 Mastozoología Neotropical / J. Neotrop. Mammal.; 9(2):159-170 M.H. Gallardo

10 mm

Fig. 4. Sperm morhology within the Caviidae. A) Cavia porcellus, B) Galea musteloides, C) Microcavia australis, D) Dolichotis salinicola. mega-acrosomal sperms of the insectivore tion of the eutherian spermatozoa (Gomendio Suncus murinus (Cummings and Woodall, and Roldán, 1991, 1993; Roldán et al., 1992). 1985). These morphological divergences to- The rationale behind these models is that more gether with the different asymmetric shapes time and energy is required to manufacture described in Ctenomys (Vitullo et al., 1988; sperms of greater dimensions. Nevertheless, the Vitullo and Cook, 1991) emphasize the plas- production of giant heteromorphic sperms in ticity of the sperm morphology in the Drosophila challenge the adaptive explanation Caviomorpha. and favors one based on phylogenetic constraints Several adaptive models, including female (Pitnick and Markow, 1994; Pitnick, 1996). selection, sperm competition and coevolution Critical assumptions of reproductive strategy of the reproductive biology in both sexes have evolution rely on the direct or inverse relation- been advanced for the morphological evolu- ships between gamete size and life-history traits SPERM DIVERSITY IN CAVIOMORPHS 167

(Snook, 1997). For example, no relationship of response in erythocyte’s size in T. barrerae exists between sperm head dimension and ei- remains paradoxical, and certainly conflicts ther genome size, chromosome number, the with the direct effects predicted by the bulk duration of oestrus, or the volume of the mito- DNA hypothesis. Apparently this hypothesis is chondrial sheath in mammals (Gage, 1998). valid only within the gradual range of genome Nevertheless, a positive correlation has been size fluctuations in diploid mammals. By the found between diploid number and the sperm’s same token, the departures from isometric tail length in mammals as well as between the growth observed in T. barrerae’s sperm heads mid-piece and cell dimensions in other organ- are more likely to reflect the complex nature isms (Stocker and Hafen, 2000, and references of ploidy-dependent interactions than the di- therein). Optimal sperm motility and head/tail rect effects of DNA content itself. ratios are also central to the adaptive models. The mechanistic view to the puzzling C-value Motility is generated by the mitochondrion- paradox focusses on the genetic mechanisms dense mid-piece (Bedford and Hoskins, 1990), causally connected to genome-size differences and its number is considered species-constant (Flemming et al., 2000; Petrov, 2001). As it in mammals (Cummins et al., 1998). The di- has been shown in nematodes, a regulatory mensions of the mid-piece of the T. barrerae’s signalling pathway causally involved in body sperm (7 x 1.7 mm) are within the range re- size (since cell number remains constant) and ported for other caviomorph rodents, but sig- dependent on ploidy level rather than on DNA nificantly smaller than those of Cavia porcellus content itself was identified in nematodes (11.1 x 1 mm; Cummins and Woodall, 1985). (Flemming et al., 2000). Cell size in Droso- Thus, the limited size of Tympanoctomys’mid- phila is dependent on an insulin receptor sig- piece as energy-producer begs the question of nalling as the increasing repression of the G1 efficient motility assumed by the adaptive ciclins is causally associated to cell size and models (Pitnick, 1996; Snook, 1997). More- ploidy level in yeast (Galitski et al., 1999). over, the sperm head of T. barrerae has grown These instances favor a pleitropic explanation beyond the predicted isometric value of the in which the genetic control mechanisms de- flagellum length and thus imposes an additional termine cell size by altering the cell cycle burden to mobility. duration (Flemming et al., 2000). Since the From varied positive correlations, the so- processes directing the epigenetic homeostasis called bulk DNA hypothesis predict a direct is shared by a wide range of organisms causality between cell size and DNA content (Flemming et al., 2000; Stocker and Hafer, even in enucleated cells of diploid mammals 2000), the suggestion is made that the (Gregory and Ebert, 1999; Gregory, 2000). nucleotypic effects observed in T. barrerae These nucleotypic effects link genome size with result from similar regulatory biases to com- sperm dimensions as exemplified in the abnor- pensate the gene dosage as demonstrated in mally-produced, diploid sperms of rabbits isogeneic strains of yeast differing in ploidy (Beatty and Fechheimer, 1972), Bos taurus level (Galitski et al., 1999). (Ferrari et al., 1998) and in Tribolium beetles Contrary to previous suggestions of cladistic (Alvarez-Fuster et al., 1991). Nevertheless, a differences between both basic sperm mor- high-variance in nucleotypic effects are char- phologies in Ctenomys (Feito and Gallardo, acteristic of T. barrerae’s different cell lin- 1982), DNA sequencing of the cytochrome b eages. For example, the large increase in the gene does not support its reciprocal monophyly diameter of lymphocytes (50% to 70%), and (D’Elía et al., 1999). Although an independent the moderate increase in liver cells (17% to origin is implied by the data, such an interpre- 22%) contrast with the lack of increase in the tation is questionable considering the sperm’s erythrocyte’s diameter of the species (Gallardo central role in reproduction and its coevolu- et al., 1999; Gallardo et al., in press). In this tion with the female’s reproductive organs. respect, if DNA content by itself determines These results may reflect the lack of resolution cell diameter, the large variance and the lack of a single gene and not the evolutionary con- 168 Mastozoología Neotropical / J. Neotrop. Mammal.; 9(2):159-170 M.H. Gallardo

vergence suggested by the authors. Since pe- nome duplications to trigger cytological novel- culiar molecular results have been shown to ties in mammals. occur in other caviomorphs (Cao et al., 1997), further studies including other molecular mark- ACKNOWLEDGMENTS ers will be needed to sustain D’Elía et al.’s (1999) proposition and to propose a genetic The facilities of I. Greenbaum (Texas A&M University) explanation, so far lacking. and the comments of J. Kirsch are greatly appreciated. The senior author thanks the Fulbright Commission for The extensive variation in sperm morphol- the scholarship granted to study the octodontids. This ogy reported for the monophyletic subset of research was funded by Fondecyt, Grant 1010727 to MHG, caviomorphs, the Octodontoidea, occurs con- and CONICET PIP 4684 to RAO. comitantly with a wide variation in allozymic variation (Köhler et al., 2000), chromosome LITERATURE CITED number and genome size variation (Gallardo and Kirsch, 2001; Gallardo et al., 2002). For ALVAREZ-FUSTER, A.; C. JUAN, and D.E. example, the explosive radiation of the PETTITPIERRE. 1991. Genome size in Tribolium flour beetles: inter and intraspecific variation. Ctenomyidae (the succesive outgroup to the Genetical Research, 58:1-5. Octodontidae), and its departures from sperm BAHAMONDE, R. 1999. Estudio de la espermiogénesis symmetry have taken place in 25 mybp as in- y morfología espermática de Tympanoctomys barrerae dicated by DNA-DNA hybridization data (Rodentia, Octodontidae) a nivel de microscopía óptica y electrónica. Unpublished thesis, Universidad (Gallardo and Kirsch, 2001). Although the Austral de Chile, 44 pp. phylogenetic composition of the Cavioidea is BEATTY, R.A. and S. FECHHEIMER. 1972. Diploid complex, the monophyly of the Caviidae is well spermatozoa in rabbit semen and their experimental established by sequence analyses (Rowe and separation from haploid spermatozoa. Biology of Reproduction, 7:267-270. Honeycutt, 2002) BEDFORD, J.M. and D.D. HOSKINS. 1990. The Male DNA annealing data suggest that the diver- Reproductive System. The mammalian spermatozoon: sification within the octodontids started nine morphology, biochemistry and physiology. Pp. 379- myrbp and that a 6.5 myrbp gap separates the 568. In: Marshall’s Physiology of Reproduction. 2. (Lamming, G. E., ed.). Churchill Livingstone, London. desert-adapted T. barrerae from its sister-spe- BERRÍOS, M.; J.E. FLECHON, and C. BARROS. 1978. cies, Octomys mimax. Allozymic analyses also Ultrastructure of Octodon degus spermatozoon with depict the close association between the desert special reference to the acrosome. The American specialists Octomys mimax and T. barrerae Journal of Anatomy, 151:39-54. BREED, W.G. 1984. Sperm head structure in the (Köhler et al., 2000). Hydromyinae (Rodentia: Muridae): a further The recent addition of two new halophytic evolutionary development of the subacrosomal space desert genera and species to the Octodontidae in mammals. Gamete Research, 10:31-44. (Pipanacoctomys aureus and Salinoctomys BREED, W.G. 1995. Spermatozoa of murid rodents from Africa: morphological diversity and evolutionary loschalchalerosorum; Mares et al., 2000) have trends. Journal of Zoology, 237:625-651. increased drastically the diversification of the BREED, W.G. and H.S. YONG. 1986. Sperm morphology desert-adapted octodontids. These two species of murid rodents from Malasia and its possible are phenetically successive sister-taxa to T. phylogenetic significance. American Museum Novitates, 1856:1-9. barrerae and could have diverged genetically BREED, W.G. and K.P. APLIN. 1994. Sperm morphology from a common ancestor even more recently of murid rodents from New Guinea and the Solomon than did O. mimax. If the preliminary phenetic islands: Phylogenetic implications. Australian Journal analyses of Mares et al. (2000) reflects genetic of Zoology, 43:17-30. CAO, Y.; N. OKADA, and M. HASEGAWA. 1997. relationships, either one or both new species Phylogenetic position of guinea pigs revisited. might be tetraploid. Chromosomal data and Molecular Biology and Evolution, 14:461-467. detailed molecular analyses of these new taxa CUMMINS, J.M. and P.D. WOODALL. 1985. On would be necessary to assess the complex mammalian sperm dimensions. Journal of Reproduction and Fertility, 75:153-175. karyotypic evolution of this family. Neverthe- CUMMINS, J.M.; T. WAKAYAMA, and R. less, the sole existence of a tetraploid rodent YANAGIMACHI. 1998. Fate of microinjected with giant sperms emphasizes the role of ge- spermatid mitochondria in the mouse oocyte and embryo. Zygote, 6:213-222. SPERM DIVERSITY IN CAVIOMORPHS 169

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