Variability and genetic structure in fragments of Eugenia involucrata De Candolle established through microsatellite markers

ABSCTRACT: Eugenia involucrata DC. is a forest species with high environmental and economic potential. The objective of this study was to quantify the genetic variability and analyzed the genetic structure of three natural fragments located in the central region of the Rio Grande do Sul state, Brazil. We used four microsatellite loci developed for the congener species Eugenia uniflora and using GenAlEx 6.5 software, parameters of genetic variability and its partition among and within fragments were estimated for each locus. We observed high levels of genetic variability (3.67 alleles per locus; HO = 0.815; HE = 0.625; FIS = −0.294), most of which (93%) were distributed within the fragments, suggesting that these individuals came from a single original population. Gene flow between fragments was high (2.35 to 4.56 migrants per generation), resulting in low genetic differentiation indexes (FST values ranging from 0.052 to 0.096). The fragments showed high genetic variability, distributed within the remnants themselves, and low genetic differentiation. Our results have repercussions for planning locally adapted germplasm collections for forest restoration programs, thereby avoiding the implantation of populations with an exogamous depression.

However, the perpetuation of this important forest resource is threatened because of the growing fragmentation that the biome suffered, primarily during the last 60 years, by anthropic activities, such as logging, construction of cities and highways, agricultural practices, and pollution. This conversion of a continuous area into small fragments leads to the spatial isolation of individuals and under these circumstances the performance of genetic processes, such as inbreeding and drift, can affect the viability of natural plant populations, leading to their extinction (CARVALHO, 2009;MAtESANZ et al., 2017).
Studies related to the quantification of genetic variability and analyses of the genetic structure of their natural populations using DNA markers can provide subsidies for the conservation of forest resources, providing the appropriate selection of management strategies (CARVALHO, 2009;CONSON et al., 2013). In this study, microsatellite primers developed for the congener species Eugenia uniflora were used to quantify the variability and analyze the genetic structure in three natural fragments of E. involucrata located in the central region of the state of Rio Grande do Sul, Brazil.
Leaf samples were collected from 24 adult individuals from the three fragments, located in the municipalities of Quevedos (29°21′09″S; 54°04′18″W), Santa Maria (29°41′03″S; 53°4825″W), and Silveira Martins (29°38′33″S; 53°35′08″W). According to reports by residents in nearby places (data not shown), individual trees from Quevedos were approximately 100 years old, whereas those from Santa Maria and Silveira Martins, were approximately 60 years old. Genomic DNA was obtained using the CtAB protocol (Doyle and Doyle, 1990). Working solutions at 50 ng μL -1 were amplified via polymerase chain reaction (PCR), and 11 microsatellite loci developed by SARZI et al. (2019)  the amplification reactions were performed in a BIO-RAD C1000 Touch ® thermocycler (BioRad Co., Hercules, CA, USA) in a final volume of 12.5 μL, which were prepared using Eppendorf epMotion ® 5070 (Eppendorf AG, Hamburg, Germany), containing 50 ng of DNA, 0.25 μM of buffer, 0.5 μM of MgCl 2 , 1 U of taq DNA polymerase, 0.05 μM of each dNtP, 0.125 μM of forward primer, 0.125 μM of reverse primer, and 0.125 μM of the M-13 primer (50-TGTAAAACGACGGCCAGT-30) labeled with AlexaFluor 680 fluorescence (Invitrogen ® ). PCR reactions consisted of an initial denaturation step at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C (Pit 26,34,48,and 57) or 52 °C (the other primers) for 30 s, an extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. Subsequently, the fragments were separated by electrophoresis on a 6% (w/v) polyacrylamide gel using a Li-Cor 4300S ® automatic DNA sequencer (LiCor Inc., Lincoln, NB, USA) and were analyzed using SAGA-GT Software ® (LiCor Inc.). Population genetic parameters were estimated using GenAlEx 6.5 software (PEAKALL & SMOUSE, 2012). For each locus, the total number of alleles (A), observed heterozygosity (H O ), expected heterozygosity (H E ), population fixation index (F IS ), genetic differentiation index (F ST ), and Hardy-Weinberg equilibrium (HWE) were tested. to quantify the distribution of genetic variability between and within fragments, an analysis of molecular variance (AMOVA) was performed. Gene flow was also estimated for each pair of fragments, considered to be the number of migrants by generation (Nm), based on the equation: where Nm is the number of migrants per generation and F ST is the index of genetic differentiation (WRIGHt, 1949). Of the tested loci, only four (Pit 26, Pit 57, Pit 64 and Pit 66) were amplified in all studied individuals, indicating a low transferability rate between the two species. Polymorphism was observed, with Pit 57 producing the highest number of alleles (5) in Quevedos, whereas Pit 26, Pit 57, and Pit 66 in Santa Maria, and Pit 64 and Pit 66 in Silveira Martins exhibited the lowest number (three alleles). Similarly, 3.5 alleles per locus were recorded in E. brasiliensis, whereas higher numbers were detected in other similar species: eight in E. uniflora, 7.9 in E. piriformis, and 8.3 in E. francavilleana (FERREIRA-RAMOS et al., 2014). Quevedos, which in general presented the highest levels of genetic diversity (A = 4.250; H O = 0.850, H E = 0.690; Table 1), also indicated the presence of exclusive alleles (table 1). Values close to diversity indices were observed in E. uniflora in the Pampa biome, also with microsatellites (SARZI et al., 2019).
The largest proportion of genetic variability was distributed within (93%) the studied remnants, suggesting that the individuals studied came from a single original population. this result also indicated that crosses in this population would be predominant, which is contrary to the classification of the species as autogamous (CARVALHO, 2009). thus, the fragments studied in the present work were classified as mixed with a predominance of crossings (DEStRO & MONtALVÁN, 1999). Corroborating this result, Golle (2010) recorded greater variability within five populations of E. involucrata.
The analyzed loci revealed average observed heterozygosity (H O ) that did not differ significantly from the expected heterozygosity (H E ), with values of 0.815 and 0.625 (Table 1), respectively, indicating that they were in HWE. As a result, the fixation index did not differ from zero (F IS = −0.294; table 1). this behavior, observed in three of the four loci being studied, was the opposite of that expected in autogamous population and even more so in populations in the process of fragmentation, suggesting that the time in which fragmentation occurred has not been long enough to promote genetic differentiation between fragments. Pit 64 was an exception, in that its heterozygosity estimates indicated a deficiency (H O = 0.475 versus H E = 0.564; table 1), demonstrating that this locus was not in HWE, which suggested the existence of some evolutionary factor.
Gene flow estimates were high (2.35 for Quevedos and Santa Maria, 3.85 for Quevedos and Silveira Martins, and 4.56 for Silveira Martins and Santa Maria), and subsequently, resulted in low F ST values, ranging from 0.052 (Silveira Martins and Santa Maria) to 0.096 (Quevedos and Santa Maria). Among populations of the same species in the South, Southeast, and Northeast regions of Brazil, moderate genetic differentiation was obtained (F ST = 0.211) (SALGUEIRO et al., 2004). Our research revealed high genetic variability in the studied fragments, a greater proportion of which was distributed within the fragments as a result of crosses between individuals. there was also high gene flow, and consequently, low genetic differentiation among the remnants, which guarantees the maintenance of high levels of genetic variability, which is important to the survival of populations facing changes in their environment. The observed results also have consequences in the planning of germplasm collection for local adaptation for forest restoration programs, thereby avoiding the implantation of populations with an exogamous depression.

ACKNOWLEDGSMENTS
This study was financed in part by the Conselho Nacional de Desenvolvimento Científico e tecnológico -Brasil (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES -Finance Code 001).

DECLARATION OF CONFLICT OF INTERESTS
the authors declare no conflict of interest. the founding sponsors had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

AUTHOR CONTRIBUTIONS
the authors contributed equally to the manuscript.