Genetic and demographic structure in a population of Ctenomys lami (Rodentia-Ctenomyidae)

Hereditas 140: 18/23 (2004) Genetic and demographic structure in a population of Ctenomys lami (Rodentia-Ctenomyidae) TARIK A. R. J. EL JUNDI and THALES R. O. DE FREITAS Genetic Department and Graduate Program in Genetics and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre / RS, Brazil El Jundi, T. A. R. J. and Freitas, T. R. O. 2004. Genetic and demographic structure in a population of Ctenomys lami (Rodentia-Ctenomyidae). */ Hereditas 140: 18/23. Lund, Sweden. ISSN 0018-0661. Received August 20, 2003. Accepted November 17, 2003 The variability at eight microsatellite loci was analyzed in 88 individuals of Ctenomys lami, a species of tuco-tucos. Initially considered as three subpopulations, the estimated F st values ranged from 0.0007 to 0.0589. Additionally, a Bayesian approach presented probabilities of 0.9933 for the existence of only one population. A F is value of 0.19 could indicate an incipient structure. Sexual dimorphism was observed. The reproductive period was short and a seasonal pattern was observed with sex ratio of 1:1 during all seasons. Presence of recent bottleneck is described suggesting further research in this population to assess more complete and detailed data. Thales Freitas, Genetic Department and Graduate Program in Genetics and Molecular Biology, Federal University of Rio Grande do Sul, C.P. 15053, Porto Alegre / RS, Brazil CEP: 91501-970. E- mail: thales.freitas@ufrgs.br The genus Ctenomys is reported to originate from Argentina around the beginning of Pleistocene, from where it started its expansion (REIG and KIBLISKY 1969; CONTRERAS et al. 1987; LACEY 2000). With a distribution restricted to the Neotropical region and comprising more than fifty nominated species (NOWAK 1991), it has been considered one of the genus with the most explosive radiation among mammals (COOK and LESSA 1998; LESSA and COOK 1998). Characteristics such as high chromosomic variability (2n /10 to 70), both inter and intra-specific (REIG and KIBLISKY 1969; KIBLISKY et al. 1977; FREITAS and LESSA 1984; MASSARINI et al. 1991; FREITAS 1997, 2001; SLAMOVITS et al. 2001), low vagility and high individuality have been described, suggesting the importance of chromosomic evolution associated to genetic drift in the speciation model of this genus (REIG and KIBLISKY 1969). In the state of Rio Grande do Sul, Brazil, four species of Ctenomys occur: C. flamarioni, C. minutus, C. torquatus and C. lami (FREITAS and LESSA 1984; FREITAS 1995, 1997). Out of these, only one of them (C. flamarioni) has karyotype with no variation in diploid number (FREITAS 1995). In the remaining three, intra-specific variations are observed (FREITAS 1997), as well as the occurrence of hybrid zones among different karyotypic forms (GAVA and FREITAS 2002) and among different species (GAVA and FREITAS 2003). Ctenomys lami is found endemically with a narrow distribution. Limited to a region of sandy soil known as Coxilha das Lombas, the first geological compartment in the Coastal Plain of Rio Grande do Sul, formed in the beginning of the Pleistocene, coasted and isolated by swamps and lakes (VILLWOCK 1983, 1989), this species is considered as the one presenting the highest variability in a smallest geographic distribution. Seven different diploid numbers, involving the fission/fusion of chromosomes 1 or/and 2 (2n/54, 55a, 55b, 56a, 56b, 57 and 58) are described in an area of 78/12 km (FREITAS 1995, 2001). The objective of this study is to enhance the knowledge of the species C. lami, allowing a better comprehension of the intricate patterns (environmental, demographic, social and genetic) involved in the large diversity presented by this species, and also the comparison with similar phenomena observed in other species of this genus. MATERIAL AND METHODS FREITAS (2001) described populations with individuals 2n/54 and 55 in the southwestern extreme of this species geographic distribution. In the period between March 1998 and October 1999, twenty-four field work outings were made in three subpopulations localized all in the same region (S 30821?35.6ƒ/WO 51800?32.9ƒ) with distances between subpopulations of 150, 300 and 450 m, A /B, B /C and A /C, respectively (Fig. 1). Using a method of capture, release and recapturing (Marinho and Freitas, unpubl.), ninety-three animals were collected and marked.

Hereditas 140 (2004) Genetic and demographic structure in Ctenomys 19 Fig. 1. Map of Itapua Park with the indicated the collected sites. The weight and female reproductive conditions were analyzed. Tissue and blood samples were collected and transferred to the laboratory for further analysis. DNA extration, PCR amplification and karyotypes DNA was isolated from tissue samples using the method described by MEDRANO et al. (1990) with modifications. Eight sets of primers for microsatellites developed by LACEY et al. (1999) from the co-generic species C. haigi, which showed to be polymorphic for this species (El Jundi and Freitas, unpubl.) were used / Hai 2, Hai 3, Hai 4, Hai 5, Hai 6, Hai 9, Hai 10, and Hai 12. Amplification reactions were made in volumes of 20 ml (0.27 mm dntp; 0.27 mm of each primer; 1 / Taq buffer; 1.5 mm MgCl2 and 0.65 U Taq DNA polymerase), in the conditions that follow: 1 initial cycle of denaturation at 948C by 1 min, 30 cycles with annealing temperatures varying between 58 to 618C for the different primer sets, ending with a final extension of 5 min at 728C. PCR products were run in 8% denaturing polyacrylamide gel and visualized with silver-nitrate. Cytogenetics analysis Fifteen drops of total blood collected from the animals were seeded in culture medium containing RPMI and incubated at 378C for 70 h, 50 ml of colchicine (0.025%) was then added and incubated for 2 h at 378C. The material was treated with a hypotonic solution (0.075 M KCl) for 15 min and fixed with methanol:acetic acid (3:1). Slides were prepared and conventional staining was used. Diploid numbers were determined after the analysis of 10 metaphases per individual. Data analysis Demographic structure. */ The ratio between total number of males and females, as well as in the different yearly seasons were analyzed using the binomial distribution (SOKAL and ROHLF 1969). Weight means between adult males and females were compared using Student s t-test. The reproductive condition of the captured females was compared between the different seasons. Age structure was defined according WILKS (1963), and is divided in

20 T. A. R. J. El Jundi et al. Hereditas 140 (2004) three categories: young, less than mean between the heaviest individual found in the mother s tunnel system and the lightest one found in its own; subadult (male), less than mean between the heaviest and lightest individual in its own tunnel system; sub-adult (female), less than lightest female sexually mature and: adult, more than sub-adult s superior limit. Genetic structure. */ Relations among sub-populations were examined using the program Structure (PRITCHARD et al. 2000), where a Bayesian subdivision test was used in order to access the number of existing sub-populations based only on the genotypes without information of their origin (100 000 burn-in periods and 100 000 reps.). Hardy-Weinberg disequilibrium was tested for each locus, by the exact values of p, and linkage disequilibrium among loci were tested by Fisher s method, both using the Markov chain method (100 dememorization, 100 batches, 100 iterations) (GUO and THOMPSON 1992; ROUSSET and RAYMOND 1995), with posterior Bonferroni correction (RICE 1989). Relations among the sub-populations and population structure were tested using F st and F is (WEIR and COCKERHAM 1984). All analyses were implemented in Genepop 3.1 (RAYMOND and ROUSSET 1995). RESULTS Cytogenetics analysis Karyotypes obtained from 17 individuals showed diploid number 2n /54. Demographic structure Reproductive condition of females. */ The presence of perfurated females starts in June, extending until October (Fig. 2). During the winter these numbers increase with the appearance of pregnant females. The presence of not perfurated females is maintained throughout all seasons, decreasing slightly during the winter. Births are observed during the spring, going on until the beginning of summer, with an accentuated decrease. Fig. 2. Reproductive condition of Ctenomys lami females found in the three subpopulation. N. perf. /not perfurated; perf. /perfurated; cicat. /females who has copulated; preg. /pregnant; Lact. /lactating. The difficulty in defining appropriate criteria for an objective distinction of age groups through external characteristics can be responsible for the absence of young males in the sample. The weight limit among young and sub-adult individuals was established according to a female found in its own tunnel system, where the significant difference observed between the weight of males and females must be considered. Subadult and adult males are found all year round, with an increase in the number of sub-adults during the summer. Young and sub-adult females were observed in the spring, with the presence of sub-adults being prolonged until summer (Fig. 3). Genetic structure. */ Of the eight microsatellite loci analyzed, two (Hai 9 and 10) were monomorphic. In the remaining, a small number of alleles varying from one to three were observed (Fig. 4). Using the Bayesian analysis method to check the number of existing populations, probabilities of Sexual structure. */ No significant deviation of the proportion 1:1 was observed when considering the total of individuals, nor when considering only adults or by season. Age structure and sexual maturity. */ The limits established for age identification were 112.5 g for young individuals; 222.5 g for male sub-adults and 150 g for female sub-adults. The adult average weight was 264.99/25.1 g (n /22) for males and 1949/21.4 g (n /38) for females (p B/0.001). Fig. 3. Age Structure found in three subpopulations of Ctenomys lami. Recaptures not considered.

Hereditas 140 (2004) Genetic and demographic structure in Ctenomys 21 Fig. 4. Frequency and proportion of alleles in eight microsatellite locus of Ctenomys lami. 0.9933; 0.0067 and 8.38/10 3 were obtained for one, two or three populations, respectively. The differentiation analysis among sub-populations pair by pair indicates F st values of 0.0007; 0.0463 and 0.0589 (populations A/B, B/C and A/C, respectively). Therefore, analyses of the data considered individuals as belonging to only one population. Linkage disequilibrium analyses suggest linkage between the Hai-5/Hai-6, Hai-3/Hai-12 and Hai-3/Hai-4 loci. However, after Bonferroni correction was applied, significant disequilibria were not found. Hardy- Weinberg equilibrium analysis demonstrated significant deviation in only one locus (Hai 4), after Bonferroni correction, indicating a heterozygote deficit (pb/0.05). F is was observed to be positive in five loci and negative in one locus (Table 1). DISCUSSION The results obtained from F statistics and Structure suggest the existence of one population instead of the Table 1. Total number of alleles, heterozygosity and F is observed in a Ctenomys lami population. Ho /observed heterozygosity; He /expected heterozygosity. Locus Number of alleles Ho He F is Hai 2 2 0.348 0.391 0.109 Hai 3 3 0.545 0.654 0.167 Hai 4 3 0.215* 0.414 0.482 Hai 5 2 0.341 0.469 0.274 Hai 6 2 0.375 0.474 0.210 Hai 9 1 / / / Hai 10 1 / / / Hai 12 3 0.636 0.633 /0.005 Total 17 0.41 # 0.506 # 0.19 # *pb/0.05; # not considered monomorphic loc. three sub-populations initially admitted. On the other hand, data should be considered as evidence of a single population: a) three pairs of unrelated individuals, genotypically identical, were found within and among populations A and B; b) one of the alleles in the Hai 4 locus is found only in sub-populations A and B; c) F st was low among sub-populations A and B, increasing to a value higher than 0.05 (A /C); d) during field work no animal was observed or recaptured in a different sub-population from the ones it had been collected for the first time. These factors could indicate an incipient structuring in this population as described for C. rionegrensis (WLASIUK et al. 2003) and C. flamarioni (Fernandez and Freitas, unpubl.). Analysis of the allelic frequencies distribution indicates the existence of a recent bottleneck in this population (LUIKART et al. 1998). However, the small number of alleles observed and the low vagility of this species associated with processes of local extinction and recolonization of populations (El Jundi and Freitas, unpubl.) could indicate the possibility of a founder effect being responsible for this low variability, as observed by LACEY (2001) in C. sociabilis. A lot has been published on the demography and social structure effects on gene dynamics in populations (DOBSON 1998). The behavior of Ctenomys species is described as varying from marked individualism to others where tunnels are shared by adult individuals, as is the case of C. sociabilis. (LACEY et al. 1997; LACEY 1998, 2000). Data such as the absence of adult individuals sharing the same tunnel system and presence of sexual dimorphism (FREITAS 2001), probably associated with the competition for resources and/ or reproduction, suggest that C. lami is a solitary species, such as C. flamarioni (Fernandez and Freitas, unpubl.), C. talarum (MALIZIA and BUSCH 1991) and C. haigi (LACEYet al. 1998). The results demonstrate a sex ratio of 1:1, perhaps being caused by densitydependent factors, as described for C. talarum (MAL- IZIA AND BUSCH 1991) AND C. FLAMARIONI (FERNAN- DEZ AND FREITAS, UNPUBL.) AND/OR BY THE LOW DISPERSION OF YOUNG MALES. HARDY-WEINBERG EQUILIBRIUM ANALYSES DID NOT DEMONSTRATE A SIGNIFICANT DECREASE ON THE NUMBER OF HETERO- ZYGOTE OBSERVED, WHICH WOULD BE EXPECTED IN CASES OF INBREEDING, POLYGYNY AND/OR STRUC- TURING (DOBSON 1998; ZENUTO ET AL. 1999). Females analyzed presented a seasonal reproduction pattern, as is the case in many of subterranean rodents (BENNETT et al. 2000), possibly connected to environmental factors such as the availability of resources. The short duration of this reproductive period contrasts with the one described by WEIR

22 T. A. R. J. El Jundi et al. Hereditas 140 (2004) (1974) for caviomorphs, as well as the results obtained for other species (C. talarum, MALIZIA and BUSCH 1991 and C. opimus, PEARSON 1959), approximating itself in duration to C. peruanus (PEARSON 1959). The pregnancy period described for Ctenomyidae as being from 90 to 120 days (WEIR 1974; BENNETT et al. 2000) and the observation that we did not find females simultaneously lactating and pregnant, indicates that C. lami, differently from C. talarum (MALIZIA and BUSCH 1991), have only one reproductive period per year. Females were considered sexually mature with weight above 150 g, considering intermediary when compared to C. australis and C. talarum (MALIZIA et al. 1991) with weights above 250 and 95 g, respectively. Data also indicate individuals capable to mate in the year following of their birth. The overall data suggests a population with peculiar patterns, which seems to be relevant for a better understanding of phenomena associated with this species chromosomic variability. Genetic and demographic patterns indicate a species with little movement and low genetic flow. Dispersion does not seem to be a characteristic that stands out in C. lami, which contributes to the fact that populations remain isolated and allows genetic drift to act in more efficient manner. Acknowledgements / This work would not have been possible without the contribution of Lucas Klasmann and Camila Castilho for the field work. This research was supported by CNPq, FAPERGS, and FINEP. REFERENCES Bennett, N. C., Fulkes, C. G. and Molteno, A. J. 2000. Reproduction in subterranean rodents. / In: Lacey, E. A., Patton, J. L. and Cameron, G. N (eds), Life underground: the biology of subterranean rodents. The Univ. of Chicago Press, p. 145/177. Contreras, L. C., Torres-Miura, J. C. and Yañez, J. L. 1987. Biogeography of Octodontid rodents: an eco-evolutionary hypotesis. / Fieldiana Zool. 39: 401 /411. Cook, J. A. and Lessa, E. P. 1998. Are rates of diversification in subterranean South American tuco-tucos (genus Ctenomys, Rodentia: Octodontidea) unusually high? / Evolution 52: 1521/1527. Dobson, F. S. 1998. Social structure and gene dynamics in mammals. / J. Mammal. 79: 667 /670. Freitas, T. R. O. 1995. Geographic distribution and conservation of four species of the genus Ctenomys in southern Brazil. / Studies Neotrop. Fauna Environ. 30: 53 /59. Freitas, T. R. O. 1997. Chromosome polymorphism in Ctenomys minutus (Rodentia-Octodontidae). / Rev. Bras. de Genética 20: 1/7. Freitas, T. R. O. 2001. Tuco-tucos (Rodentia: Octodontidae) in southern Brazil: Ctenomys lami Spec. Nov. Separated from C. minutus Nehring, 1887. / Studies Neotrop. Fauna Environ. 36: 1 /8. Freitas, T. R. O. and Lessa, E. P. 1984. Cytogenetics and morphology of Ctenomys torquatus (Rodentia-Octodontidae). / J. Mammal. 65: 637 /642. Gava, A. and Freitas, T. R. O. 2002. Characterization of a hybrid zone between chromosomally divergent populations of Ctenomys minutus (Rodentia Ctenomyidae). / J. Mammal. 83: 843 /851. Gava, A. and Freitas, T. R. O. 2003. Inter and intra-specific hybridization in tuco-tucos (Ctenomys) from Brazilian coastal plains (Rodentia: Ctenomyidae). / Genetica 119: 11 /17. Guo, S. W. and Thompson, E. A. 1992. Performing the exact tests for Hardy-Weinberg proportion of multiple alleles. / Biometrics 48: 361 /372. Kiblisky, P., Brum-Zorrilla, N., Perez, G. et al. 1977. Variabilidad cromosómica entre diversas poblaciones uruguayas del roedor cavador del género Ctenomys (Rodentia-Octodontidae). / Mendeliana 2: 85/93. Lacey, E. A. 2000. Spacial and social systems of subterranean rodents. / In: Lacey, E. A., Patton, J. L. and Cameron, G. N (eds), Life underground: the biology of subterranean rodents. The Univ. of Chicago Press, p. 257 /296. Lacey, E. A. 2001. Microsatellite variation in solitary and social tuco-tucos: molecular properties and population dynamics. / Heredity 86: 628 /637. Lacey, E. A., Braude, S. H. and Wieczorek, J. R. 1997. Burrow sharing by colonial tuco-tucos (Ctenomys sociabilis). / J. Mammal. 78: 556/562. Lacey, E. A., Braude, S. H. and Wieczorek, J. R. 1998. Solitary burrow use by adult Patagonian tuco-tucos (Ctenomys haigi). / J. Mammal. 79: 986 /991. Lacey, E. A., Maldonado, J. E., Clabaugh, J. P. et al. 1999. Interspecific variation in microsatellites isolated from tuco-tucos (Rodentia-Ctenomyidae). / Mol. Ecol. 8: 1754 /1756. Lessa, E. P. and Cook, J. A. 1998. The molecular phylogenetics of tuco-tucos (genus Ctenomys, Rodentia:Octodontidae) suggests an early burst of speciation. / Mol. Phylogenet. Evol. 9: 88 /99. Luikart, G., Allendorf, F. W., Cornuet, J. M. et al. 1998. Distortion of allele frequency distributions provides a test for recent population bottlenecks. / J. Heredity 89: 238 / 247. Malizia, A. I. and Busch, C. 1991. Reproductive parameters and growth in the fossorial rodent Ctenomys talarum (Rodentia-Octodontidae). / Mammalia 55: 293 /305. Malizia, A. I., Vassallo, A. I. and Busch, C. 1991. Population and habitat haracteristics of two sympatric species of Ctenomys (Rodentia-Octodontidae). / Acta Theriol. 36: 87 /94. Massarini, A., Barros, M. A., Ortells, M. O. et al. 1991. Evolutionary biology of fossorial Ctenomyine rodents (Caviomorph:Octodontidae). I. Chromosomal polymorphism and small karyotypic differentiation in Central Argentinian populations of tuco-tucos. / Genetica 83: 131 /144. Medrano, J. F., Aesen, E. and Sharrow, L. 1990. DNA extraction from nucleaded red blood cells. / Biotechniques 8: 43. Nowak, R. M. 1991. Walker s mammals of the world. / Johns Hopkins Univ. Press.

Hereditas 140 (2004) Genetic and demographic structure in Ctenomys 23 Pearson, O. P. 1959. Biology of subterranean rodents, Ctenomys, in Perú. / Mem. Mus. Hist. Nat Javier Prado 9: 1/56. Pritchard, J. K., Stephens, M. and Donelly, P. 2000. Inference of population structure using multilocus genotype data. / Genetics 155: 945 /959. Raymond, M. and Rousset, F. 1995. GENEPOP (Version 1.2). Population genetics software for exact test ecumenism. / J. Heredity 86: 248 /249. Reig, O. A. and Kiblisky, P. 1969. Chromosome multiformity in the genus Ctenomys (Rodentia: Octodontidae). / Chromosoma 28: 211 /244. Rice, W. R. 1989. Analyzing tables of statistical tests. / Evolution 43: 223/225. Rousset, F. and Raymond, M. 1995. Testing heterozygote excess and deficiency. / Genetics 140: 1413 /1419. Slamovits, C. H., Cook, J. A., Lessa, E. P. et al. 2001. Recurrent amplifications and deletions of satellite DNA accompanied chromosomal diversification in South American tuco-tucos (Genus Ctenomys, Rodentia: Octodontidae): a phylogenetic approach. / Mol. Biol. Evol. 18: 1708 /1719. Sokal, R. R. and Rohlf, J. 1969. Biometry. / W. H. Freeman and Company. Villwock, J. A. 1983. Geological aspects of the coastal province of RS. A synthesis. / Pesquisas 16: 5/49. Villwock, J. A. 1989. Summary of the geology of the coastal province of Rio Grande do Sul. / Proc. Int. Symp. on utilization of coastal ecosystems: planning, pollution and productivity 2: 471 /484. Weir, B. J. 1974. Reproductive characteristics of hystricomorph rodents. / Symp. Zool. Soc. Lond. 34: 265/301. Weir, B. S. and Cockerham, C. C. 1984. Estimating F- statistics for the analysis of population structure. / Evolution 38: 1358 /1370. Wilks, B. J. 1963. Some aspects of ecology and population dynamics of the pocket gopher (Geomys bursarius) in southern Texas. / Texas J. Sci. 15: 241/283. Wlasiuk, G., Garza, J. C. and Lessa, E. P. 2003. Genetic and geographic differentiation in the Río Negro tuco-tuco (Ctenomys rionegrensis): inferring the roles of migration and drift from multiple genetic markers. / Evolution 57: 913 /926. Zenuto, R. R., Lacey, E. A. and Busch, C. 1999. DNA fingerprinting reveals polygyny in the subterranean rodent Ctenomys talarum. / Mol. Ecol. 9: 1529 /1532.