Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(1): 57-64, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2232 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Praptosuwiryo, T.N., Gar- vita, R.V., Handini, E., Fijridiyanto, I.A., & Witono, J.R. (2024). Karyologi- cal studies of four species of Lady’s Slipper Orchids (Paphiopedilum) col- lected in the Bogor Botanical Garden, Indonesia. Caryologia 77(1): 57-64. doi: 10.36253/caryologia-2232 Received: July 6, 2023 Accepted: March 15, 2024 Published: July 8, 2024 Copyright: © 2024 Praptosuwiryo, T.N., Garvita, R.V., Handini, E., Fijridiyanto, I.A., & Witono, J.R. This is an open access, peer-reviewed article pub- lished by Firenze University Press (ht tps://www.fupress.com/caryolo- gia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Karyological studies of four species of Lady’s Slipper Orchids (Paphiopedilum) collected in the Bogor Botanical Garden, Indonesia Titien Ngatinem Praptosuwiryo1,*, R. Vitri Garvita2, Elizabeth Han- dini2, Izu Andry Fijridiyanto1, Joko Ridho Witono1 1 Research Center for Biosystematics and Evolution, National Research and Innovation Agency (BRIN), Jalan Raya Jakarta-Bogor Km. 46, Cibinong, West Java 16911, Indonesia 2 Research Center for Applied Botany, National Research and Innovation Agency (BRIN), Jalan Raya Jakarta-Bogor Km. 46, Cibinong, West Java 16911, Indonesia *Corresponding author. Email: tienpferns@gmail.com Abstract. Paphiopedilum is one of the most widely grown and hybridized of all orchid genera due to its distinctive flower morphology. This genus consists of 139 accepted species and is native to southern China to tropical Asia. Karyological studies on the genus Paphiopedilum have been reported by many cytologists in different countries. However, many Indonesian members of Paphiopedilum have remained comparatively limited in investigated cytologically. This study aimed to analyze karyological charac- ters of four species of Paphiopedilum collected in the Bogor Botanical Garden, Indo- nesia, namely P. armeniacum, P. hirsutissimum, P. primulinum, and P. superbiens. Kar- yological studies were conducted by root tips squash method. The results showed that four species of Paphiopedilum have a basic chromosome number of x = 15. They are diploid with 2n=30, instead of the common diploid chromosome number of Paphio- pedilum (2n = 26). Paphiopedilum hirsutissimum and P. primulinum possess 16 m + 14 sm chromosome formulae. Whereas Paphiopedilum armeniacum and P. superbiens revealed 16 m + 14 sm and 18 m + 12 sm chromosome formulae, respectively. A Rob- ertsonian change in chromosome number generated by the fission of chromosomes would best explain the origin of the new diploid chromosome number and karyotypes of these species. Keywords: Bogor Botanical Garden, chromosome, diploid, karyotype, Paphiopedilum. INTRODUCTION Paphiopedilum Pfitzer (Orchidaceae) is known as a lady’s slipper orchid and belongs to the subfamily Cypripedioideae Lindley (1840) with other four genera, namely Cypripedium, Mexipedium, Phragmipedium, and Selenipedi- um. The subfamily was separated from other subfamilies based on its special characteristic of having two separated fertile anthers (Cribb 1998). Paphio- pedilum has unique characters that distinguished from genera Cypripedium and Selenipedium, viz. conduplicate coriaceous leaves, as opposed to the pli- http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2232 https://doi.org/10.36253/caryologia-2232 https://www.fupress.com/caryologia https://www.fupress.com/caryologia mailto:tienpferns@gmail.com 58 Titien Ngatinem Praptosuwiryo et al. cate persistent leaves of the latter two genera. Paphiope- dilum differs from Phragmipedium and Mexipedium, as they have imbricate sepal vernation, different chromo- some base numbers, and a unilocular ovary (Albert and Chase 1992; Albert 1994). Paphiopedilum is the most popular orchid genus for ornamental plants, because of its distinctive flow- er morphology (Lee et al. 2011). Therefore, members of this genus are widely grown and hybridized of all orchids (Cribb 1998; Lan and Albert 2011). Ecologi- cally, species of Paphiopedilum are important to nar- row endemics in various mainland and islands habitat and range from seaside cliffs to montane rainforests (Cribb 1998). Based on the morphological characteris- tics of inflorescence, leaf type, floral morphology, and molecular data on ITS sequences, Cribb (1997) divided this genus into three subgenera, namely Parvisepalum, Brachypetalum, and Paphiopedilum. After Cribb (1997), several new species and treatments have been described for this genus, therefore this genus consisted of approx- imately 98 species worldwide by the year 2000 (Koo- powitz 2000). The subgenus Paphiopedilum is classified into five sections, viz. Coryopedilum, Pardalopetalum, Cochlopetalum, Paphiopedillum, and Barbata (Lee et al. 2017; Tsai et al. 2020) based on morphological, cyto- logical and molecular data (Cox et al. 1997; Cribb 1998; Chochai et al. 2012). Paphiopedilum is distributed in four of 25 biodiver- sity hotspots (Myers et al. 2000), the Indo-Burma, Sun- daland, Wallacea, and Philippines, which are also the “major evolutionary hotspots” (de Bruyn et al. 2014). According to Tsai et al. (2020), phylogeny and historical biogeography of Paphiopedilum reveals the relevance of the differentiation of Paphiopedilum in Southeast Asia and geological history. The mountain forests of Indone- sia are home to great diversity of the endemic Paphio- pedilum species. It is predicted that at least 50% of the Lady’s Slipper Orchids of the world can be found natu- rally in the mountains forest of Indonesia, most of them are endemic to the mainland of Indonesia. For example, P. agusii Cavestro & N Bougourd, P. anitanum Cav- estro, P. braemii H.Mohr ., P. dodyanum Cavestro, P. glaucophyllum J.J. Smith, P. intaniae Cavestro, and P. javanicum (Reinw. ex Lindl.) Pfitzer are endemic to Java (Govaerts et al. 2018). Chromosome number and karyomorphological analysis is very significant for understanding the genome structure, its organization, and evolution within a genus of certain plant taxon at inter- and intra-specific levels (Ehrendorfer 1980). Analyses of chromosome numbers are very important, because they represent a fundamen- tal step in the study of any taxa of organisms. Chro- mosome counts provide indispensable information on genetic discontinuities within and among species, and they contribute to the understanding of phylogenetic relationships at all taxonomic levels (Windham and Yat- skievych 2003). Extensive chromosome account and karyotype analysis of the Lady’s Slipper Orchids have been pub- lished. Chromosome counts are published for almost all species of Paphiopedilum and the detailed karyo- types are also available for most species of the genus (Kamemoto et al. 1963; Karasawa 1979; Karasawa and Aoyama 1980; Karasawa 1982, 1986; Cox et al. 1998). However, chromosome account and karyotype analy- sis for Indonesian Paphiopedilum are scanty although most of species of this genus are distributed in Indone- sia. This study aimed to observe chromosome numbers and conduct the karyotipe analysis of some species of Indonesian Paphiopedilum. Accumulation of cytological data would be very important in providing reference to breeding programs. MATERIALS AND METHODS Plant materials Root tips of four species of Paphiopedilum in vari- ous subgenera were taken from the orchids in vitro cul- ture collection of the Plant Tissue Culture Laboratory and the orchid plants collection of the Bogor Botanical Garden (BBG). Paphiopedilum primulinum, P. super- biens were collected from the wild population, whereas P. armeniacum and P. hirsutissimum were introduced plants (Table 1). Somatic chromosome observations Procedures for somatic chromosome observation followed by Manton (1950) and modified by Praptosu- wiryo and Darnaedi (2008). The actively growing roots were used for chromosome preparation. Root tips pre- treated with 0.001 M 8-hydroxyquinolin at 4°C for 24-26 hours. The root tips then were fixed in 45% acetic acid for 10 minutes at room temperature after being rinsed with distilled water. Root tips were macerated with 45% acetic acid (CH3COOH): 1N HCl (1:3) at 60°C for 4 min- utes, and then stained in 1% aceto-orcein. The meris- tematic cells were squashed in a drop of 1% acetic acid orcein under a coverslip of 22 x 22 mm on a microscope slide. Chromosome observation was performed under the microscope using a 100x magnification objective with the addition of immersion oil. An Olympus micro- 59Karyological studies of four species of Lady’s Slipper Orchids (Paphiopedilum) collected in the Bogor Botanical Garden scope U-TV0 with the objective 100x connected to a dig- ital camera (5XC-3 5H12344) with a computer monitor was used to capture the images of well-spread chromo- some complements. Data analysis The long-arm and the short-arm length of each chromosome were recorded using 5 µm as the unit. Each chromosome picture was cut out and arranged in descending order of length. Karyotype analysis was based on mitotic metaphase cells from each species. Chromosome shape at metaphase was classified based on arm ratio (AR) (Levan et al. 1964) (Table 2). Chro- mosome characteristics were measured using Ideokar 1.2 software (Mirzaghaderia and Marzangib 2015). RESULTS In the present investigation, the numbers of somat- ic chromosomes of four species of Paphiopedilum were counted and presented in Table 3 and Figures 1. The four species of Paphiopedilum disclosed the same chromosome number of 2n=30. Somatic chromo- somes at metaphase and the idiogram of four species of Paphiopedilum are shown in Figure 1. The chromosome shapes of P. armeniacum, P. hirsutissimum, P. primuli- num, and P. superbiens are 16 m + 14 sm, 24 m + 6 sm, 24 m + 6 sm, and 18 m + 12 sm, respectively (Table 3.). DISCUSSION Paphiopedilum possesses unusually large chromo- somes for orchids (Kamemoto et al. 1963). The basic cytology of Paphiopedilum is reasonably well stud- ied and chromosome numbers have been published for many species (Karasawa 1979, 1986; Karasawa and Aoy- ama 1980, 1988; Karasawa and Tanaka 1980, 1981; Kara- sawa and Saito 1982; Cox et al. 1998). However, almost all species reported were outsite of Indonesia. Four species of Paphiopedilum cultivated in the Bogor Botanical Garden have chromosome number of 2n = 30 (Figure 1). Chromosome numbers of the four species were reported for the first time. Karyological studies of Paphiopedilum have shown considerable chro- mosomal variation, which ranges from 2n = 26 to 2n = Table 1. Living material for karyological studies of the genus Paphiopedilum collected in the Bogor Botanical Garden, Indonesia. No. Species Subgenus/Section Distribution Status 1. P. armeniacum S.C.Chen & F.Y.Liu Parvisepalum/ Parvisepalum China introduced 2. P. hirsutissimum (Lindl. ex Hook.) Stein Paphiopedilum /Paphiopedilum China, India, Lao PDR, Myanmar, Thailand, Vietnam introduced 3. P. primulinum M.W.Wood & P.Taylor Paphiopedilum/Cochlopetalum Sumatra wild, collected from Aceh 4. P. superbiens (Rchb.f.) Stein Paphiopedilum /Barbata Sumatra wild, collected from North Sumatra Table 2. Chromosome shape classification based on long and short arms ratio. Arm Ratio (AR) Sentromer position Chromosome shape 1.0 ≤ AR < 1.7 median metasentric (m) 1.7 ≤ AR < 3.0 submedian submetasentric (sm) 3.0 ≤ AR < 7.0 subterminal subtelosentric (st) 7.0 ≤ AR < ∞ near terminal acrosentric (a) ∞ terminal telosentric (t) Table 3. Karyotypic characters of four species of Paphiopedilum collected in the Bogor Botanical Garden. No. Species Chromosome Chromosome number (2n) Long arm (µm) Short arm (µm) Total arm Length (µm) Arm ratio (µm) Chromosome shape 1. P. armeniacum S.C.Chen & F.Y.Liu 30 3.23–5.24 1.12–4.60 4.32–9.80 1.13–1.25 16 m + 14 sm 2. P. hirsutissimum (Lindl. ex Hook.) Stein 30 2.36–5.73 1.21–4.33 3.58–10.05 1.14–2.03 24 m + 6 sm 3. P. primulinum M.W.Wood & P.Taylor 30 1.58–7.07 1.21–5.31 3.00–12.38 1.11–1.76 24 m + 6 sm 4. P. superbiens (Rchb.f.) Stein 30 2.36–5.73 1.21–4.33 3.58–10.05 1.27–1.95 18 m + 12 sm 60 Titien Ngatinem Praptosuwiryo et al. Figure 1. Somatic and ideogram metaphase chromosomes of Paphiopedilum, Ideogram of each species is on the right side of the image: (A) P. armeniacum (2n = 30), (B) P. hirsutissimum (2n = 30), (C) P. primulinum (2n = 30), (D) P. superbiens (2n = 30). Scale bars: 5 µm. 61Karyological studies of four species of Lady’s Slipper Orchids (Paphiopedilum) collected in the Bogor Botanical Garden 42 (Karasawa 1979). However, species of Paphiopedil- lum have the multiple chromosomes of the basic chro- mosome number x = 13 as reported by Lee et al. (2017). Species in the sections Barbata and Cochlopetalum of subgenus Paphiopedilum have a variety of chromosome numbers ranging from 2n = 28 to 42 and 2n = 30 to 37, respectively (Cox et al. 1998). Paphiopedilum armeniacum S.C.Chen & F.Y.Liu Paphiopedilum armeniacum belongs to subgenus Parvisepalum, section Parvisepalum (Lan and Albert 2011). This species is currently listed as endangered in The IUCN Red List of Threatened Species, version 2014.3 and grows in the restricted area to a river valley in Yun- nan, China on limestone substrates in rocky and brushy habitat (Rankou and Averyanov 2015). The chromosome account of P. armeniacum culti- vated in the Bogor Botanical Garden with 2n = 30 is a new record for chromosome number for this species. Lee et al. (2018) reported a cytotype of P. armeniacum with 2n = 26 from Taiwan. This species is usually reported to have chromosome 2n = 26 (Lan and Albert 2011). The new karyotype of P. armeniacum, with 2n = 30, with the centromeric formula of chromosomes 16 m + 14 sm reported here (Figure 1A), expands the chromo- some number range for subgenus Parvisepalum, which was previously considered to be conserved at 2n = 26 (Chochai et al. 2012). The members of subgenus Parvise- palum usually show 2n = 26m (Lee et al. 2011). Paphiopedilum hirsutissimum (Lindl. ex Hook.) Stein. Paphiopedilum hirsutissimum is a member of sub- genus Paphiopedilum, section Paphiopedilum (Lan and Albert 2011). This species is distributed in northern and western Guangxi, southern and western Guizhou, south- ern and eastern Guizhou, northern and eastern India, Laos, Thailand, and northern Vietnam (Liu et al. 2009; Li et al. 2015; Chen et al. 2018). This species usually grows on the crevices on shaded cliffs or rocky and well- drained places in forests or thickets in limestone areas at the elevation of 700–1500 m asl. Chromosome number of P. hirsutissimum cultivat- ed in the Bogor Botanical Garden reported here is 2n = 30. Formerly cytological records showed that most spe- cies of the subgenus Paphiopedilum, section Paphiopedi- lum, have diploid chromosome number 2n = 26 (P. fair- rieanum, P. henryanum, P. hirsutissimum, P. tigrinum), except P. druyi ( 2n = 30) (Lan and Albert 2011). The chromosome formula of P. hirsutissimum reported here is 2n = 24 m + 6 sm (Figure 1B). Chromo- some formula in the subgenus Paphiopedilum are vary, for example, 2n = 26 m of P. rothschildianum (section Coryopedilum), 2n = 20m + 12t of P. callosum (section Barbata), 2n = 14m + 22t of P. glaucophyllum (section Cochlopetalum) (Lee et al. 2011). Paphiopedilum primulinum M.W.Wood & P.Taylor. Paphiopedilum primulinum is endemic to Sumatra (southern Aceh). It is a member of section Cochlopetalum and closely related to P. liemianum based the molecular data, such as nuclear ribosomal ITS, plastid trnL intron, trnL-F spacer, and atpB-rbcL spacer (Tsai et al. 2020). The chromosome account of P. prmulinum with 2n = 30 is reported here for the first time. Formerly cyto- logical study P. primulinum of Sumatra has been report- ed to have 2n = 32 (Lan and Albert 2011). The chromo- some formula for P. primulinum reported here is 2n = 24 m + 6 sm (Figure 1C). Paphiopedilum superbiens (Rchb.f.) Stein. Paphiopedilum superbiens belongs to subgenus Sig- matopetalum, section Barbata (Karasawa and Saito 1982). According to Braem and Chiron (2003), this spe- cies is included in the Ciliolare complex of subsection Loripetalum. Based on the phylogenetic relationships resulting from analysis of the combined molecular data matrix, such as nuclear ribosomal ITS, plastid trnL intron, trnL-F spacer, and atpB-rbcL spacer, P. superbiens is closely related to P. curtisii and come into section Bar- bata (Tsai et al. 2020). The chromosome account of P. superbiens with 2n = 30 is reported for the first time. A formerly cytological study of the members of the section Barbata revealed 2n = 36 for P. curtisii and P. dayanum, 2n = 38 for P. acmo- dontum and P. sangii, 2n = 40 for P. sukhakulii and P. venustum (Lan and Albert 2011). The chromosome for- mula for P. superbiens reported here is 2n = 18 m + 12 sm (Figure 1D). Paphiopedilum (Orchidaceae: Cypripedioideae) is characterized by considerable chromosome number vari- ation (2n = 26–42). Chromosome numbers vary from 2n = 28–42 and 2n = 30–37 in the sections Barbata and Cochlopetalum of subgenus Paphiopedilum, respective- ly (Lee et al. 2018). According to Cox et al. (1998), the most common diploid chromosome number in Paphio- pedilum is 26 metacentric chromosomes, as displayed in subgenus Parvisepalum, Brachypetalum and Paphiope- dilum, mainly sections Pardalopetalum, Coryopedilum, and Paphiopedilum. However, the chromosome number 2n = 30 was also reported. This diploid chromosome number occured in P. druryi and P. spicerianum (Cox et al. 1998). The chromosome account of Paphiopedilum obtained from this current study supported the existence of 2n = 30 for the genus. Prior to this study, 2n = 30 was 62 Titien Ngatinem Praptosuwiryo et al. reported for the hybrid species of P. rothschildianum × P. moquetteanum (Lee et al. 2011). The changes in chromosome number and karyo- type symmetry were considered as a consequence of Robertsonian centric fission (Jones 1998). A Robert- sonian relationship between the different karyotypes whereby changes in chromosome number are generated by the fission or fusion of chromosomes at or near the centromere to generate either telocentric or metacen- tric chromosomes, respectively (Robertson 1916). Dun- can and MacLeod (1949, 1950) proposed Robertsonian change to explain the maintenance of arm number in the two genera of Orchidaceae, namely Paphiopedilum and Phragmipedium. Cox et al. (1997) postulated cen- tric fission of metacentric chromosomes into telocentrics as the predominant mechanism of karyotype evolution in Phragmipedium and Paphiopedilum and that karyo- type orthoselection is operating in some species groups. Paphiopedilum species display many chromosomal rear- rangements like duplications, translocations, and inver- sions. Therefore the genus is a good model system for the study of complex chromosomal evolution in plants (Lan and Albert 2011). The phylogeny of Paphiopedilum has been report- ed by Chochai et al. (2012) and Tsai et al. (2020) using molecular data (nuclear and plastid DNA) which each species studied here are separated in different clades both subgenus and section levels. However, their chro- mosome shape does not correlate with clade separation, mainly in subgenus Paphiopedilum. P. hirsutissimum (sect. Paphiopedilum) and P. primulinum (sect. Cochlo- petalum) have similar chromosome shape of 24 m + 6 sm, but based on ITS nrDNA and plastid DNA sequence data, they were separated in different clade. It tends that clade separation among subgenus and section within subgenus Paphiopedilum are strongly correlated with morphological and molecular data. However, extensive chromosome account and karyotype analysis of Paphio- pedilum are necessary to construct their relationships and evolution in the future. CONCLUSION Chromosome account and the karyotypes of four species of Paphiopedilum cultivated in the Bogor Botani- cal Garden, Indonesia were investigated. New diploid chromosome number was reported. The chromosome number was uniformly 2n = 30. Chromosome account of Paphiopedilum used in the study supports the find- ing that this genus has variations in the number of chro- mosomes. P. hirsutissimum and P. primulinum exhibited remarkably similar karyotypes, with centromeric formu- lae 24 m + 6 sm. Whereas Paphiopedilum armeniacum and P. superbiens possessed 16 m + 14 sm and 18 m + 12 sm chromosome formulae, respectively. These findings supported the statement that Paphiopedilum is a good model system for the study of complex chromosomal evolution in plants. 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