Study of drosophila kinship from three regions in East Java, Indonesia

Drosophila has a massive number of members and a wide range of habitats. Different natural conditions make it possible to find diverse Drosophila populations, although several types of Drosophila are cosmopolitan. Drosophila diversity is characterized by morphological variations. The structural similarity of an individual can be used to determine various kinship relations between organisms. This research aimed to determine the relation between Drosophila from three regions in East Java, Indonesia, namely Malang, Mojokerto, and Pasuruan, based on similarities in morphological characteristics. Samples were taken from these three areas, and observations were carried out from January to May 2023 at the Genetics Laboratory, Biology Department, FMIPA UM. Identification of Drosophila is based on an identification key consisting of 52 traits. Drosophila was purified to the third generation to achieve a homogeneity of 87.5%. The kinship relation is sought by calculating the association coefficient and similarity index as a reference in preparing the dendrogram. The dendrogram illustrates that Drosophila from Malang had a closer relationship with Drosophila from the Pasuruan area than from the Mojokerto area. Drosophila kinship relations are influenced by internal and external factors, which include gene flow, mobility, distance, and natural conditions.


Introduction
Drosophila is considered the most important genus in the Drosophilidae family because of its diversity at the global level [1].The number of species in the genus Drosophila reaches more than 1.500, distributed widely in several areas [2].Genus Drosophila also has an expansive habitat in various altitudes and conditions [3].Species from the genus Drosophila can be found from the lowlands to the highlands [4].In addition, Drosophila's habitat is widespread in forests, mountains, swamps, deserts, tundra, and savanna [5].The different conditions of each area make it possible to find diverse Drosophila populations, although several types of Drosophila are cosmopolitan [6].
The differences in altitude and regional conditions can initiate internal conditions expressed in different morphological forms.Similarities and differences in morphological characteristics can be used to determine the relationship between species in Drosophila [7].Taxonomic-level kinship relationships can be viewed from two points of view: phyletic and phenetic [8].Phyletic is a type of kinship considered the phylogenetic relationship between one taxon and another.Phenotic is a type of kinship considered similarities in the characteristics possessed by each group [9].Thus, this research used a phenetic approach with morphological characteristics to determine kinship between species in the genus Drosophila.If many morphological characteristics are used for identification, phenetic relationships can describe phyletic relationships between species in the genus Drosophila [10].
Genus Drosophila has been widely used as the best model organism for research in various fields, such as molecular biology, genetics, and population biology [11].There are several advantages of using Drosophila as a model organism in research, such as short generation time, cosmopolitan distribution, clear morphology, small size, easy maintenance, high fecundity, small number of chromosomes, and polytene chromosomes [12].Additionally, Drosophila is neither a pest nor a vector [13].Drosophila

Research design
Ex post facto was chosen as a research design to investigate after the fact without the researcher's intervention.This research was an ex post facto study that aimed to trace the factors that cause kinship relations between species in the genus Drosophila.This research was carried out from January to May 2023 at the Genetics Laboratory, Biology Department, FMIPA UM.

Population and sample
The population used in this research was Drosophila from three different regions: Malang, Mojokerto, and Pasuruan Regency (Figure 1).In particular, Drosophila samples from Malang Regency were taken in the Tajinan District at three points: Ngawonggo, Pandanmulyo, and Randugading.Drosophila samples from Mojokerto Regency were taken in the Dlanggu District at three points: Dlanggu Market, Pekingan Hamlet, and Sumbersari Hamlet.Drosophila samples from Pasuruan Regency were taken in Purwosari District at Sekarmojo, Martopuro, and Purwosari Village.

Tools and materials
The tools used in this research were a stereo microscope, jam bottles as a place for Drosophila culture, clear plastic, and a laptop.The materials used in this research were Drosophila from three different regions.In addition, the culture medium was made from banana, cassava tape, and brown sugar (ingredient ratio 7: 2: 1).Then, the culture medium added yeast and a sponge to cover the jam bottle.

Procedures
This study observed 52 morphological characteristics of the head, thorax, abdomen, wings, and legs.Drosophila from each region was interbred until the third generation.Each interbred was repeated nine times.

Data analysis technique
Data were analyzed by grouping Drosophila morphological characteristics.After the similarity was known, the calculation results were arranged in a matrix of the number of Operational Taxonomic Unit (OTU) character pairs.Next, the similarity index for each OTU pair was calculated using the following formula [14].
S = association coefficient of a pair of OTU's being compared Ns = Number of traits in common for a pair of OTU's being compared Nd = Number of different characteristics for a pair of OTU's being compared Groups were based on the highest level of similarity, then successively to lower levels of similarity.The results of calculating the association coefficient of OTU pairs were then analyzed using the similarity coefficient index with the following formula [14].
r AB = group with the greatest similarity r AC and r BC = groups other than group AB

Results and Discussion
Identifying were observed from the head, thorax, abdominal, leg, and wing.The results of the differences are shown in Table 1.Furthermore, we observed the morphological characteristics of Drosophila using a stereo microscope.There is a prominent carina √ √ √ 4.
The location of the interocellar bristles parallel to the ocellus √ √ √ 8.
The diameter of the compound eye is larger than the maxillary palp √ √ √ 12.
On the pedicel, there is a visible bristle √ √ √ Thorac 15.
Comparison of bristle thorax length aNP: Comparison of the number of aSC: pSC Comparison of length aSA: There is a microchaetae in the scutellum ---22.
The abdominal bands from the edges to the middle are interconnected √ √ √ 27.
The A5 segment is wider than the A6 segment √ √ √ Leg 29.
There is an apical seta in the tibia √ √ √ 30.
There Based on Table 1 and Figure 2, it is known that there were similarities and differences in the characteristics of Drosophila from each region.These characteristics were then compared in each region.Characteristics with the same code will be written with the number 1, while characteristics with different codes will be written with the number 0. The calculation results are shown in Table 2.The calculation results in Table 2 were used to compile a matrix of the number of OTU pair characters shown in Table 3.Based on the data in Table 3, the similarity index for each pair of OTUs can be calculated using the following formula.From this formula, the association coefficient results were: SAB (0.981), SAC (1), SBC (0.981).Association coefficients between regions are summarized in Table 4.The results of calculating the association coefficient of OTU pairs are then analyzed using the.similarity coefficient index.The results of similarity coefficient index as follows.Calculating a similarity index value close to 1.00 showed that the kinship relation was getting closer, while the similarity index value was away from 1.00, indicating that the kinship relationship was getting farther away [14].Drosophila pairs from Malang Regency and Pasuruan Regency have a similarity index value of 1.00.The index value showed that Drosophila from Malang Regency and Pasuruan Regency have a closer relation than Drosophila from Mojokerto Regency, which has a similarity index value of 0.981.
The close relations between two populations can be caused by internal factors like gene flow [15].In population genetics, gene flow is the transfer of alleles or genes from one population to another [16].Migration into or out of a population is responsible for changes in allele frequencies [17].Migration can also lead to adding new genetic variants to the established genes of a particular species or population [13].Gene flow tends to reduce genetic differences among populations [18].If extensive enough, gene flow can cause several populations to merge into a single population with a shared gene pool [19].
Several factors influence the rate of gene flow between different populations.One of the most significant factors is mobility because greater mobility tends to provide more tremendous migration potential [16].Drosophila gene flow mobility in Malang and Pasuruan Regency can occur without natural or artificial obstacles so that the populations from both regions merge into a single population with a shared gene pool.This is proven by the many similarities in the morphological characteristics of Drosophila from the two regions; the similarity index was 1.00.These results differ from Drosophila, which comes from Mojokerto Regency, whose similarity index was 0.981.
Schluter et al. [20] suggested that the factor that drives differences in morphological characteristics is the presence of natural obstacles.For example, the presence of natural obstacles in the form of vast oceans or high mountains will reduce the migration of Drosophila to other areas.This condition means that the types of Drosophila that exist in certain areas will very likely differ from those of Drosophila in other areas.The result follows the geographical conditions of Malang and Pasuruan Regency with Mojokerto Regency, bordered by natural obstacles in Arjuna Mountain (Figure 1), a barrier to Drosophila migration.
The distance between regions also influences the relationship between Drosophila in Malang and Pasuruan Regency.The distance between Malang and Pasuruan Regency is closer, which is 47 km, compared to the distance between Malang and Mojokerto Regency, which is 75 km, and between Mojokerto and Pasuruan Regency, which is 54 km.The distance between these regions causes geographic isolation, which is thought to significantly influence populations' genetic structure [21].However, let's look at the similarity index for Mojokerto Regency.It is 0.981, showing that the relationship between the Drosophila of Malang and Pasuruan Regency with Mojokerto Regency is still relatively close.This result can happen due to the migration of Drosophila from Malang or Pasuruan Regency through the transportation of agricultural products.Then, the Drosophila settled and adapted to Mojokerto Regency [17] Another external factor that influences Drosophila relationships is differences in height.Altitude is correlated with temperature [22].The average height above sea level in Tajinan District, Malang Regency, is 478 m; in Dlanggu District, Mojokerto Regency, is 120 m; and in Purwosari District, Pasuruan Regency, is 329 m.The height difference illustrates that the altitude of Malang Regency and Pasuruan Regency is not much different compared to Mojokerto Regency, so if it is related to temperature, the temperature difference in Malang Regency and Pasuruan Regency is not too much compared to Mojokerto Regency.This temperature difference can affect the Drosophila phenotype because high temperatures can denature enzymes, and conversely, low temperatures can inactivate enzymes responsible for regulating metabolism and phenotypic expression [23].Disruption of enzyme work means that the expressed genes may also differences [24].These differences in expressed genes result in differences in the phenotype of an organism [25].
According to Dyer et al. [26], environmental conditions can influence changes in genetic material.Events in nature allow the gene composition of each population to always change based on the probability that pairs of genes can unite [27].The phenotype of an individual is the result of genotypic factors and environmental influences [28].If the genotype changes, the environment changes, or both change, the phenotype will also change [29].It should be noted that phenotypic variations that occur due to environmental influences are not inherited, but what is inherited is the ability to form certain types or characteristics as a result of gene responses to changing environments [30].Likewise, variations in two populations cannot necessarily be called two different populations by having two different species if it is still possible to exchange genes between them [31].The environmental conditions of Malang and Pasuruan Regency are not much different, so there are similar morphological characteristics of Drosophila.This phenomenon is proven by a similarity index value of 1, which means the Drosophila relationship is very close.However, there are several limitations in this research.First, kinship studies still used morphological characteristics.Second, kinship relations were being viewed from the three regions.Further research can explore kinship relationships at the molecular level with more different regencies.

Conclusions
Genus Drosophila found in various habitats has diverse morphological characteristics.Morphological characteristics can be used as a reference to determine the kinship of Drosophila.The results concluded that Drosophila from the Malang area has a closer relationship with Drosophila from the Pasuruan area than from the Mojokerto area.The Drosophila similarity index value shows this for the Malang and Pasuruan areas is 1, while the Drosophila similarity index value for the Malang and Pasuruan areas with the Mojokerto area is 0.981.The main factor that influences the difference in similarity index is gene flow.Apart from that, several external factors, such as landscape, distance, and natural conditions, also affect the continuity of gene flow.Although this research can reveal the kinship between three regions, several limitations are derived from kinship studies still used morphological characteristics and viewed from the three areas.Further research can explore kinship relationships at the molecular level with different characteristics and involve more regencies to determine kinship between species in the genus Drosophila.

Figure 1 .
Figure 1.The Three regions of the sample are Malang, Mojokerto, and Pasuruan.Note: The yellow color shows the three regions, and the orange color shows Arjuna Mountain.(Source:www.googleearth.com)

8 A
dendrogram graph is created based on the similarity coefficient index value, as shown in Figure3.

Figure 3 .
Figure 3. Drosophila dendrogram in three different areas.

Table 3 .
Matrix of number of Operational Taxonomic Unit (OTU) pairs *: Number of morphological characteristics that are not the same.Without *: The exact number of morphological characteristics.