Genetic Variation at Microsatellite Loci in Odorrana hosii (Boulenger, 1891)

The true frog species, Odorrana hosii, found in West Sumatera Indonesia, has high morphological differentiation and also estimated has high genetic variation. A total of 35 O. hosii at a seven location have analyzed using DNA microsatellite markers. Genetic variation of five microsatellite loci provided the highest value of expected heterozygosity (He) for the population in Padang (He = 0.618), while the lowest was the population in Merapi (He = 0.427). There are genetic differences in moderate levels among populations of O. hosii in West Sumatra (FST = 0.108) with inbreeding intrapopulation value (FIS = -0.559), and high value of gene flow among the populations (Nm = 2.061). This study becomes the first molecular data for establishing effective population management conservation.


INTRODUCTION
The distribution of Odorrana hosii is wide, from Thailand, Malay Peninsula, Sumatra, Java, to Borneo. This species can live in an altitude interval from 300 masl to 1430 masl. The widespread and the altitude interval for survival provide a possibility of morphological and genetic variation to O. hosii (van Kampen, 1923;Inger et al., 2017).
According to a previous study, O. hosii in West Sumatra from different altitudes interval shows a high morphological differentiation and variation to the relative length of chromosomes (Agesi, 2011). Several environmental factors affected differentiation in phenotypes, such as temperature and altitude, genetic factor, or interaction of both factors (Conover & Schultz, 1995;Vences et al., 2002). Therefore, further research is necessary to examine the DNA level among O. hosii in West Sumatra to describe the species morphological variation that also happens genetically. This research aimed to analyze the genetic variation level of the O. hosii population in West Sumatra based on microsatellite markers. DNA Microsatellite markers are used because of these markers have a high polymorphism level. Hence, it is very appropriate to determine the genetic variation level within and between populations (Allendorf et al., 2012). Besides, the analysis of DNA variation using DNA microsatellite can also determine the genetic differentiation of a frog population-based on geographical location with a range of 10 kilometers (Morgan et al., 2008).

MATERIALS AND METHODS
The samples were collected at seven sites in West Sumatra (Alahan Panjang, Padang, Pasaman, Palupuah, Payakumbuh, Gunung Merapi, and Sijunjung). Those locations have been chosen based on altitude and distance shown in Figure 1 and Table 1.
DNA isolation. Analysis of genetic variation used DNA microsatellite markers (Funk et al., 2005;Arruda et al., 2017). DNA isolated from the liver tissue of five individuals from each population using QIAGEN kit protocol DNAeasy® Blood and Tissue Kit. PCR reaction was performed using PROMEGA PCR Core kit by mixing 2 μl isolate DNA, 12 μl GoTaq Green Master Mix, 1 μl forward primer, 1 μl reverse primer, and sterile water until the total volume is 25 μl. All loci have an optimal temperature. Information of nine microsatellite loci showed in Table 2. Amplification of DNA was programmed with an initial denaturation 95ºC for 2 min, 33 cycles of denaturation: 95ºC for 30 s, annealing: at optimal temperature 30 s, extension: 72ºC for 30 s, and final extension 72ºC for 5 min. The product was detected by electrophoresis in  Data analysis. Quantification of the results analyzed using the GENEPOP version 4.0 to determine the genetic variation within and between populations. The parameters used to determine the genetic variation within a population were allele frequencies, the percentage of polymorphic loci (Pp), the average number of alleles per locus (Na), and expected heterozygosity (He). Genetic variation between populations can be seen from the Fstatistic, genetic distance and similarity values dendrogram based on Nei's genetic distance matrix (1978) and the value of gene flow (Nm) (Raymond & Rousset, 1995).

RESULT AND DISCUSSION
Amplification results of nine loci showed that only five loci produce the amplicon (Rnh-3, . The optimal temperature has been searched previously for the nine loci with different annealing temperatures. The annealing temperature for the five loci were modified from Gong et al. (2013) shown in Table 2.

Rnh-12
Rnh The number of alleles at each locus ranged from five to seven, with as much as 27 alleles and an average of 5.4 alleles per locus. All alleles microsatellite showed a high polymorphism with the percentage of polymorphic loci (Pp) ranged from 75 (Rnh-13) to 100% (Rnh-9). Analysis of genetic variation showed that population of Padang have a high genetic variation with 17 allele per locus, 0.618 expected heterozygosity (He), Shannon Index of 1.00, and 100% of polymorphic loci (Pp), and Merapi have a lower (Na = 11; He = 0.427; I = 0.616; Pp = 80%) (Table 3).  (Arens et al., 2006;Alberto et al., 2013). It has known that Padang was likely to become the habitat of O. hosii. According to Iskandar (1998), O. hosii was likely to live on a clear and strong water stream and a river streamline in the primary forest. A large population will reduce Vol 8(1), June 2020 Biogenesis 65 the chances of inbreeding and increase random mating so that heterozygosity will also be increased (Cervantes et al., 2011;Frankham, 2015;Hedrick & Garcia-Dorado, 2016).
The lowest He value was found in the Merapi population. It can expect these values a small number of population, which will increase the occurrence of inbreeding (Frankham et al., 2002). Inbreeding associated with the loss of genetic variation, drift and less heterozygous levels related to the loss of some alleles and low levels of polymorphism. As a result, it would increase inbreeding depression (Arens et al., 2006;Taylor et al., 2010;Jamieson, 2011).
Inbreeding depression is mating between individuals from the same parental offspring, which produce offspring with lower live quality level (Arens et al., 2006). Besides, Allendorf et al. (2012) also stated that a low level of heterozygosity could reduce viability because most of the population recessive alleles were lethal. Lethal recessive alleles, predominantly covered by dominant alleles in a heterozygote locus, have a big opportunity to express in a few populations. The extinction will probably led faster in a small number of frog populations (Rog et al., 2013;Soto-Azat et al., 2013). The categorization of a population can be looked at from its frequency of occurrence. The frequency of occurrence of O. hosii from Merapi is relatively lower than another population.   (Table 4) can determine the level of genetic differentiation between the O. hosii population in West Sumatra. According to Wright (1938), there are four categories of FST values that ranged from 0 to 0.05; 0.05-0.15; 0.15-0.25; and >0.25. Each showed a level of genetic differentiation, which indicated low, moderate, high, and very high genetic differentiation. Based on the statements above, we can conclude that O. hosii between populations in West Sumatra has genetic differentiation value that shows moderate genetic differentiation (F ST = 0.108). From the F ST value, we can determine that 10.82% of total genetic variation is among populations, and 89.18% is within the population.
The FIT value that is less than 0 indicated that inbreeding in a population is low and random mating still occurs, so the probability for closely related species cross is low (Charlesworth & Willis, 2009). It is also supported by the value Nm among populations (2.061; Nm ≤0.5). Based on that value, we know the migration of individuals among the population of O. hosii in West Sumatra is high (Slatkin, 1981). According to the obtained value, it can be concluded that O. hosii among populations in West Sumatra does not have genetic differentiation significantly based on microsatellite DNA. The pattern of O. hosii grouping among populations is presented in the dendrogram (Figure 2). Based on the UPGMA cluster analysis, O. hosii grouping in West Sumatra is divided into two major groups and provides a unique grouping pattern. O. hosii populations form groups following Bukit Barisan highlands (Padang and Malampah populations create a group on the western side of Bukit Barisan highlands; Alahan Panjang, Sijunjung, Harau, Merapi, and Palupuah populations create a group on the eastern side of Bukit Barisan), as well as the existence of rivers that facilitate the migration of individuals among populations. O. hosii grouping based on UPGMA cluster analysis is supported by genetic distance value among populations (Table 5).  Table 5 presents that several populations are showing the great value of genetic distance differences among the population. It is caused by factors such as geographical isolation in the form of physical barriers, environmental factor differences, and reproduction. One of those factors will affect genetic structure formation (Arioli et al., 2010;Fouquet et al., 2012;Twomey et al., 2016)). It can be concluded that geographical isolation factor, which is Bukit Barisan highlands locating from northern to the southern side of Sumatra that serves as a physical barrier separating among populations, has affected genetic structure formation of O. hosii.

CONCLUSION
The highest genetic variation of O. hosii is from the population in Padang (Na = 17, He = 0.618, I = 1.000), while the lowest genetic variation is from the population in Merapi (Na = 11, He = 0.427, I = 0.616). The existence of Bukit Barisan Mountain potentially becomes a barrier as well as the classification of O. hosii in West Sumatra. The analysis of the population's genetic structure has shown a moderate genetic variation (FST = 0.108).