MORPHOLOGICAL AND CHROMOSOMAL CHARACTERIZATION OF ORCHID Peristylus goodyeroides Lindl. FROM CURUG SETAWING, KULONPROGO

Peristylus goodyeroides is a terestrial orchid that scattered around Southeast Asia. Morphological characters of P. goodyeroides can vary, depending on the ecological factors and habitat. Cytological characters in the form of chromosome configurations can be used as a taxonomic tool for the process of identifying and understanding variations in taxa. The purpose of this study was to determine the morphological characters and chromosome configuration of the P. goodyeroides from Curug Setawing, Kulonprogo. The method used was morphological characterization and plant chromosome preparation by squash method with the main steps of fixation, maceration, staining and observation. Data were analyzed with the help of Image raster 3, Corel Draw X7, and Microsoft Excel 2013. P. goodyeroides from Curug Setawing has the morphological characters of root tubers, cylindrical stems, ovate leaf shape, convolutive leaf arrangement and creamy white flowers. The orchid has a number of chromosomes 2n = 10 with a karyotype formula of 2n = 2x = 8m + 2t. Metacentric chromosomes are found on chromosomes 1-8 and telocentric chromosomes 9-10. The absolute arm length of the chromosomes has a range of 2.03-3.44 μm, the relative arm length of the chromosomes is 2.21-3.32 μm, the length of the p arm is 1.13-1.58 μm and the q arm is 1.23-2.12 μm.


INTRODUCTION
Orchids are flowering plants with high diversity and economic value (Zhang et al., 2018;Pamarthi et al., 2019). Orchidaceae consists of 763 genera and 28,000 species (Christenhusz & Byng, 2016). Curug Setawing is a tourist park area located in the Menoreh Mountains, Kulonprogo, Special Region of Yogyakarta. This area has latosol and grumusol types of soil (Nagara & Buchori, 2013), dry soil condition and humid air. There are 15 species of orchids found with three types of habitus, namely terrestrial, amoebophyte and epiphyte. One of the most attractive orchids found in this area is Peristylus goodyeroides with a total of 20 individuals and a frequency of 0.4 (Kurniawan et al., 2018). In addition to Kulonprogo, this species can also be found on the South Slope of Mount Merapi, Sleman Regency (Setiaji et al., 2018). P. goodyeroides is an orchid commonly found in teak forests and grasslands with an altitude of about 1,750 meters above sea level. The characters of this orchid are white flowers with a diameter of 7-8 mm. The leaves are 5-8 cm wide and up to 12 cm long (Comber, 1990;Nurfadilah, 2017). The phenotypic characters of P. goodyeroides appeared due to the interaction between genotype and environmental factors (Gienapp et al., 2017;Bocianowski et al., 2019).
Chromosomes are structures containing genetic material which have roles in the process of inheritance, growth and development of organisms (Shetty, 2018). The most common number of basic chromosomes (n) found in Orchidaceae are n = 19, 20, and 21 with 2n = 38, 2n = 40, and 2n = 42 (Hartati et al., 2017).
Each species has significantly different shape and number of chromosomes, so has its karyotype. (Bolcun-Filas & Schimenti, 2012). Kariotype plays important role in providing information about taxonomic relationships, genetic abnormalities and the origin of a species (Young et al., 2012). Karyotype abnormalities can be related to abnormalities in the anatomical, morphological and physiological characteristics of an individual (Samatadze et al., 2020). Chromosome identification is carried out during metaphase or prometaphase based on cytometric images and morphometric data. This way is classified as conventional method (Ata et al., 2017).
Chromosome analysis has several basic functions. One of them are as an indication of the evolutionary process of a species based on the structure and number of chromosomes (Lusinska et al., 2019), species identification tools (Lukhtanov & Shapoval, 2017) and could determine the genetic diversity of species indicated by variations in the population (Bzdega et al., 2016).
P. goodyeroides is a natural orchid with botanical value. It is because the prospects of this species has not been deeply explored. In addition to Yogyakarta, this species can also be found in Purwodadi (East Java) (Nurfadilah, 2017), Maluku and Sulawesi (Nurfadilah, 2020) searching, there is no research has been found on cytogenetic data or the number of chromosomes in the orchid P. goodyeroides from Kulonprogo, so that this cytogenetic research could complete and enrich the taxonomic evidence on this orchid using cytotaxonomic data.

Procedures 1. Morphological Characterization
Morphological characterization was carried out by recording the morphological characters of orchids including habitus, root type, stem shape, leaf shape, leaf tip shape, leaf size, leaf color and arrangement, type of flower, flower color, sepal and petal shapes and special characters on labellum. After the morphological characters obtained, identification of orchids is carried out by matching the Jurnal Pendidikan Matematika dan IPA Vol. 12, No. 2 (2021) h. 110-122

Chromosome Preparation
The preparation method used is the squash method (Aristya et al., 2015; Aristya et al., 2019) with modifications. The roots of orchid are washed with water before preparation. The root tips of the young P. goodyeroides orchids were cut ± 5 mm from the tip at 09.00 am. The root pieces were then immersed in a flakon bottle filled with distilled water for 24 hours, then placed in a refrigerator at 5° C. The root pieces were then washed with distilled water three times.
Fixation was conducted by adding 45% glacial acetic acid solution to the root sample. Then the samples were incubated at 4° C for ± 30 minutes in the refrigerator. The root pieces were then washed with distilled water three times. After that, maceration is carried out. The root sample was put into a flakon bottle then added with 1 N HCl and incubated in a block heater with a temperature of 62° C for ± 30 minutes. Staining was carried out with the help of a fulgen solution which was added to the root sample and then the sample was incubated at room temperature for 1 hour.
After the coloring stage, then the root pieces are squashed (squashing). The cut of purple root tip is placed on a glass preparation and glycerin is added, then covered with a cover glass. The root tip is then squashed (squashing) with the base of pen slowly until distributed. The preparations were then observed using a light microscope with a magnification of 10x40. The variables observed included: number, size, chromosome shape and karyotype pattern (Hartati et al. 2014).

Visualization of chromosome shape and karyotyping
Visualization of chromosome shapes is assisted by Image raster 3 and Corel Draw X7 softwares. First, the chromosome shape is reconstructed with the help of the "shape tool" and "freehand tool" menus in Corel Draw X7. Second, labeling chromosomes with a temporary number is carried out using the "text tool" menu. Next, the chromosome arm length measurements (p and q arms) were carried out with the help of Image raster 3. The measurement results obtained were then used to calculate the absolute arm length, centromere index, chromosome arm length ratio, identification of chromosome shape, actual chromosome numbering, and manufacturing idiogram using Microsoft Excel 2013.

Data analysis
The determination of chromosomes number is known by calculating the number of chromosomes visualized at prometaphase.
Karyotypes and idiograms are made using Corel Draw X7 software and Microsoft Excel 2013. According to Levan et al. (1964) the value of the Centromere Index (CI) can be calculated by the following formula: The ratio value of long arm to short arm of chromosomes (CAR) can be calculated by the following Jurnal Pendidikan Matematika dan IPA Vol. goodyeroides is ± 40-50 cm high, a perennial herbaceous plant and terrestrial type ( Figure 2A). P. goodyeroides has adventitious roots with rhizomes. It has sympodial stem growth type with a cylindrical stem shape. It has yellowish green leaves, ovate with ± 18 cm long and ± 7 cm wide, convolutive arrangement of young leaves, flat leaf edges with pointed leaf tips. The leaves are terminal totaling 3-7 leaves. Alternate leaves arrangement. Parallel leaf venation.
Clustering (raceme) inflorescence ( Figure 2B Figure 2C) with respective characters are ovate-shaped dorsal sepalum, triangular lateral sepala with flat edges and pointed tips, lateral petala has a narrower base with a rounded tip and the edges are flat while on the lateral part it is shaped like a wing. There are parallel veins on the petals and sepals of the P. goodyeroides orchid. The labellum has three lobes that are tapered at the ends and narrower at the sides.
Based on morphological characters identified from samples collected, it is known that the species is P. goodyeroides according to the similarity of morphological characters in Comber (1990). Other species from the genus Peristylus such as P. gracilis have different morphological characteristics from P. goodyeroides. The difference lies in the loose leaf arrangement at the top of the stem and the smaller flower size with longer spurs than the sepals. P. constrictus has more oval sepal morphological character with a conduplicate-shaped dorsal sepalum and lateral sepala having irregular edges, the petala spreads upward but does not form a cap with the dorsal sepalum, the spura has a groove in the middle and has a short column structure. P. coeloceras has stem with a tubular sheath and opposite leaf arrangement, the leaves are elliptical and the petals are rhombic and oblique (Raskoti et al. 2017).
Based on morphological observation P. goodyeroides collected from Kulonprogro have the same characteristics as the literature. Although morphologically similar but could not be ascertained accurately that the sample is P. goodyeroides. Ecological factors can influence the genotype of an individual as a form of adaptation to the environment. In natural populations, we often encounter the phenomenon of phenotypic plasticity as in behavior or morphology.
This phenomenon causes the process in analyzing the interaction between genotype and phenotype becomes difficult (Gienapp et al., 2017). Besides the cryptic species also play a role in genetic variation accounted for although phenotypically alike. Therefore, it is necessary to have complementary data to support the hypothesis such as cytogenetic data.
Based on observation of P. goodyeroides chromosomes, it can be seen that the number of diploid chromosomes observed is 10 chromosomes (2n = 10) (Figure 3). In Orchidaceae, there are variations in the number of chromosomes in each member of species. The number of diploid chromosomes 2n = 10 can be found in members of the subtribe Oncidiinae (Daviña et al., 2009). Members of the subfamily Spiranthoideae and Orchidoideae are known to have a varying number of chromosomes, ranging from n = 10 to n = 50. In earlier studies, genus Peristylus has varied number of basic chromosomes between 2n = 21, 23, 44, and 18 (Felix & Guerra, 2005). In other studies, it is stated that the variation in the number of chromosomes in this genus is 2n = 28, 36, 38, 42, 44, 46 and 88 (Luo, 2004).
Based on data obtained from The Chromosome Counts Database (CCDB) (Rice et al., 2015) it is known that there are variations in the number of chromosomes in P. goodyeroides, namely n = 47, 46, 42, 28, 23, 21 (CCBD, 2021  The number of chromosomes can provide information about the genetic diversity of orchids in Indonesia. Such information can be used to determine the phylogenetic relationship between species of orchids in Indonesia (Bzdega et al., 2016). Based on literature studies, the number of chromosomes in P. goodyeroides species in Kulonprogo have not been reported before. Comparing with P. constrictus, the number of chromosomes in P. constrictus 2n = 38 (Hartati et al., 2017).
It can be used as a comparison to the number of chromosomes P. goodyeroides because both species are still classified in the same genus although each species can basically have a different number of chromosomes (Suliartini et al., 2004). In this study, the chromosome number of P. goodyeroides orchids was 2n = 10. The difference in the number of chromosomes between species can be caused by differences in geographical conditions, habitat, the domestication process, and so on. This difference can lead to a process of speciation and evolution. Products from a speciation will rise to new species of the same genus, but with a different chromosome configuration (Djordjevic et al., 2016;Erzurumlu et al., 2018). Not all variations in the number of chromosomes in a species necessarily lead to the speciation process. Many orchid species vary in the number of chromosomes, even within the same species. This variation may also arise due to the sample preparation is not exactly at the active division, instability measurement and so on. In essence, each species has a fixed number and structure of chromosomes. This consistency can be utilized for taxonomic purposes (Wang & Zheng, 2017).  The process of arranging a karyotype and an idiogram is based on the appearance of chromosomes in the mitotic prometaphase phase. The arrangement of the karyotype is based on the shape, size and location of the centromere (Singh, 2003). Based on the results of the karyotype of P. goodyeroides ( Figure  4), the karyotype formula for this orchid species is 2n = 2x = 10 = 8m + 2t with an absolute chromosome length range of around (2.03-3.44) μm. Based on the results of the karyotype visualized in the form of an idiogram, it can be seen that the chromosome pairs number 1-4 (chromosome number 1-8) are metacentric type and chromosome pair number 5 (chromosomes number 9 and 10) are telocentric type (Figures 4 and 5).  (Singh, 2003). P. goodyeroides orchids have relative chromosome length ranging from 2.21-3.32 μm with short arm length (p) ranging from 1.13-1.58 μm and long arm chromosome (q) between 1.23-2.12 μm ( Figure 5).

CONCLUSION
Based on the result of study, it can be concluded that the morphological characters observed in P. goodyeroides orchids are perennial herbaceous habitus, terrestrial orchids, root tubers, sympodial cylindrical stems, ovate leaf shape, convolutive leaf arrangement and creamy white flowers, ovate and triangular sepals with flat edges and pointed tips, wingshaped petals with flat edges and rounded tips and also three lobes of labellum with pointed tips used as a characteristic for the orchid. The number of P. goodyeroides chromosomes observed is 2n = 10 with the karyotype formula 2n = 2x = 10 = 8m + 2t which consists of five pairs of chromosomes with a metacentric (chromosome number 1-8) and telocentric (chromosome number 9-10). Molecular data is needed to strengthen the results obtained such as RAPD, ISSR, SSR, RFLP, sequencing, FISH (Flourescence In-Situ Hybridization), and other methods.