Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(4): 11-16, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-811 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: A. Turco, A. Albano, P. Meda- gli, S. D’Emerico (2020) Contribution to the study of wild Orchidaceae, genus Platanthera L.C.M. Richard. Karyotype and C-banding analysis of two species from Italy. Caryologia 73(4): 11-16. doi: 10.13128/caryologia-811 Received: January 08, 2020 Accepted: November 18, 2020 Published: May 19, 2021 Copyright: © 2020 A. Turco, A. Albano, P. Medagli, S. D’Emerico. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, 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. ORCID AT: 0000-0001-9071-344X AA: 0000-0002-0874-320X Contribution to the study of wild Orchidaceae, genus Platanthera L.C.M. Richard. Karyotype and C-banding analysis of two species from Italy Alessio Turco1,*, Antonella Albano1, Pietro Medagli1, Saverio D’Emerico2 1 Dept. of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy 2 “Aldo Moro” University of Bari, Bari, Italy *Corresponding author. E-mail: alessio.turco@unisalento.it Abstract. This study examined the chromosome numbers and karyotypes of two taxa of the genus Platanthera (Orchidaceae) from Italy. Cytological analyses showed 2n = 2x = 42 in P. chlorantha and P. algeriensis. Karyotype analysis revealed similarity between the species. The karyotypes are as follows: P. chlorantha consists of 34 metacentric + 8 submetacentric pairs and P. algeriensis consists of 36 metacentric + 6 submetacen- tric pairs. Both species possess a rather symmetrical karyotype. P. chlorantha has a very similar C-banding pattern to P. algeriensis. DAPI bright blocks were observed in P. chl- orantha. These analyses also show the close relationship between the studied species. Keywords: Chromosome number, C-Banding, heterochromatin content, karyotypes, Orchidaceae, Platanthera algeriensis, P. chlorantha. INTRODUCTION The genus Platanthera Rich., also known as “butterfly orchids”, belongs to the subtribe Orchidinae (subfamily Orchidoideae) and consists of 100 to 200 species (Wood 2001; Delforge 2016; Efimov 2016 and references there- in). The geographical distribution of Platanthera species covers most of the temperate areas of Europe, North Africa, Asia, New Guinea and North and Central America (Hulténand Fries 1986; Wood 2001; Efimov 2016), with 12 species widespread in Europe, six of which are found in Italy (Delforge 2016). This genus is divided into 5 sections (Efimov, 2016), three of which are found in Europe: P. hyperborea, the most ancient clade, which has a Far- Eastern and North-American distribution, with only P. hyperborea (L.) Lindl. present in Iceland; the P. oligantha clade, with a circumpolar distribution, and finally the Eurasian section Platanthera (Delforge, 2016). Platanthera species are terrestrial, photosynthetic and – in very few cases – epiphytic or lithophytic. They are found in a variety of habitats, including meadows, temperate and boreal forests, bogs, fens, marshes and prairies. European Pla- 12 Alessio Turco, Antonella Albano, Pietro Medagli, Saverio D’Emerico tanthera species are geophytes, characterised by a broad anther, 2 elongated root-tuberoids, a dorsal sepal and petals combining to form a helmet, a stigma without processes and an enlarged receptive surface with a nec- tariferous spur. In cytological analyses performed on taxa of the genus Platanthera, the chromosome number was found to be 2n = 2x = 42 (Cauwet-Marc and Balayer 1986; Yokota 1987; Yang and Zhu 1988; Dalgaard 1989; D’Emerico 2001 and references therein). To date howev- er, only four taxa exhibit polyploidy: Platanthera hyper- borea (Dalgaard 1989) and P. huronensis (Nutt.) Lindl. (Sheviak and Bracht 1998), both with 2n = 4x = 84 chro- mosomes; P. obtusata (Banks ex Pursh) Lindl., which may be triploid in some populations with 2n = 63 (Tan- aka and Kamemoto 1984); and the Nordic–Siberian P. oligantha Turcz. (= P. obtusata subsp. oligantha), which according to Webb (1980) is hexaploid (2n = 126). As already mentioned above, six of these species are found in Italy: Platanthera algeriensis Batt. & Trab. 1892, P. bifolia subsp. bifolia (L.) Rich. 1817, P. bifolia subsp. osca Lorenz, Romolini, Romano & Soca 2015, P. bifolia subsp. subalpine Brügger, P. chlorantha (Custer) Rchb. and P. kuenkelei subsp. kuenkelei var. sardoa Lorenz, Akhalk, Baumann, Cortis, Cogoni & Scrugli 2012. In this study, karyotype morphology and the dis- tribution of heterochromatin in Italian specimens of P. chlorantha and P. algeriensis were studied for the first time. The aim of this study was to verify chromosome numbers and to compare the heterochromatin pattern of the above-mentioned species in order to verify similari- ties between them. MATERIALS AND METHODS The material studied in this investigation was gath- ered from natural populations of Platanthera chlorantha and P. algeriensis in Apulia and Sardinia (Table 1). Mitotic chromosomes were prepared from immature ovaries, pre-treated with 0.3% colchicine at room tem- perature for 2h. For Feulgen staining they were fixed for 5 min in 5:1:1:1 (v/v) absolute ethanol, chloroform, gla- cial acetic acid and formalin, hydrolysed at 20 °C in 5.5 N HCl for 20 min (Battaglia 1957) and stained in freshly prepared Feulgen solution. For C-banding, ovaries were fixed in 3:1 (v/v) etha- nol–glacial acetic acid and stored in a deep-freeze for up to several months. Subsequently, they were squashed in 45% acetic acid; coverslips were removed by the dry ice method and the preparations were air-dried over- night. The slides were then immersed in 0.2N HCl at 60 °C for 3 min, thoroughly rinsed in distilled water and then treated with 4% Ba(OH)2 at 20°C for 4 min. After thorough rinsing they were incubated in 2xSSC at 60°C for 1h, and then stained in 3-4% Giemsa (BDH) at pH 7 (D’Emerico et al. 1996). For DAPI (4–6-diamidino- 2-phenylindole) staining, ovaries were treated as for C-banding and stained using a buffered DAPI solution (0.6 mg/mL) for 5 min, followed by rinsing and mount- ing in glycerol buffer (1:1 v/v). Chromosome pairs were identified and arranged on the basis of their length and any other evident kar- yomorphological feature. Heterochromatin content was assessed using MicroMeasure 3.3, a freeware program from Colorado State University (Reeves 2001). Karyo- ty pe symmetry indices – Mca (Mean Centromeric Asymmetry) and CVcl (Coefficient of Variation of Chro- mosome Length) – were used for the evaluation of kary- otype asymmetry (Peruzzi et al. 2009). The nomenclature used for describing karyotype composition followed Levan et al. (1964). A list of the examined specimens and their sampling locations is giv- en in Table 1. RESULTS AND DISCUSSION Analysis of the somatic metaphases showed that the diploid chromosome number is 2n = 2x = 42 in both Platanthera chlorantha and P. algeriensis. P. chlorantha, known as the “Greater Butterf ly Orchid” (Lima-de-Faria 2020) was found to be diploid with 2n = 2x = 42 chromosomes (Fig. 1a), in agreement with previous reports (Scrugli 1980; Averyanov et al. 1985; Cauwet-Marc and Balayer 1986), with chromo- Table 1. Taxon, sites, chromosome number, formula and percent heterochromatin in set of the chromosomes of species Platanthera chlor- anta and P. algeriensis. m, metacentric; sm, submetacentric. Taxon Site Chromosome number (2n) Formula % Het in set P. chlorantha Martina Franca (TA) Balvano (PZ) 42 34m+8sm 25.50 P. algeriensis Aritzo (NU) 42 34m+2m(sm)+6sm 22.43 13Contribution to the study of wild Orchidaceae, genus Platanthera somes that range in size from 4.04 to 1.8 µm at meta- phase, and the arm length ratio was from 1.03 to 3.00. Six well spread-out metaphases were paired on the basis of chromosome size and centromere position and used for chromosome measurements. The karyotype con- sisted of 34 metacentric and 8 submetacentric chromo- some pairs (Fig. 2a). The complement showed two chro- mosome pairs with secondary constrictions on the long arm (pair 2) and the short arm (pair 8). This species possesses a fairly symmetrical karyo- type (Mca = 18.50±1.09 and CVcl = 21.53±0.29), with metacentric chromosomes being the most frequent. It is interesting to note that this species has similarities in terms of dimensions and structure (such as the visibil- ity of centromeres) with karyotypes of the Anacamptis group (2n = 2x = 36) (D’Emerico et al. 1996). Similari- ties with Chamorchis alpina (L.) Rich. (2n = 2x = 42) (D’Emerico and Grünanger 2001) and Dactylorhiza romana (Sebast.) Soò (2n = 2x = 40) (D’Emerico et al. 2002) can also be observed. The C-banding analysis shows that constitutive het- erochromatin was located in the centromeric regions of numerous chromosomes (Fig. 3a). One pair of chromo- somes had the subtelomeric C-bands only on the short arm. Interphase nuclei exhibited a number of chro- mocentres equal to that of the constant bands (Fig. 3b). The centromeric regions of numerous chromosomes had bright fluorescence after staining with DAPI (Fig. 3c). In Platanthera algeriensis, somatic cells showed 2n = 2x = 42 chromosomes (Fig. 1b). This species is simi- lar to P. chlorantha apart from its greener flowers and its very different preferred habitat. In Europe this species is restricted to a few sites in Corsica, Sardinia, mainland Italy and Spain, but it also occurs in Algeria. In this spe- cies, similarities to the karyotype structure and C-band- ing of P. chlorantha were observed. Chromosome lengths were found to be between 3.90 and 2.18 µm. The karyo- type consisted of 34 metacentric, 2 metacentric/submeta- centric and 6 submetacentric chromosome pairs (Fig. 2b). In addition, this species possesses a symmetrical karyotype (Mca = 21.21±0.83 and CVcl = 18.19±0.60). Constitutive heterochromatin was also detected in the centromere regions of numerous chromosomes (Fig. 3d). The present analysis of chromosome evolution showed that the species P. chlorantha and P. algeriensis are very close. Indeed, specimens of the two species in the present study exhibited practically the same karyo- type and C-banding pattern. However, P. algeriensis seems to differ from P. chlorantha in that it has lower heterochromatin content (Fig. 3-d vs. Fig. 3-a). The small Figure 1. Mitotic metaphase with Feulgen staining of Platanthera chlorantha (a) and P. algeriensis (b); 2n = 2x = 42. Bar = 5 µm. Figure 2. Diploid karyotypes of Platanthera chlorantha (a) and P. algeriensis (b). Bar = 5 µm. 14 Alessio Turco, Antonella Albano, Pietro Medagli, Saverio D’Emerico differences in the C-banding patterns found between the two species seem to indicate limited rearrangement of constitutive heterochromatin during their evolution. In a study based on plastid DNA sequence varia- tion, Pavarese et al. (2011) found that Platanthera alge- riensis was characterized by haplotypes A and B, both of which are shared with P. chlorantha. In another study, Italian Platanthera chlorantha were found to form a sin- gle group with P. algeriensis from Tunisia and Sardinia (Bateman et al. 2012). CONCLUDING REMARKS The chromosome number 2n = 2x = 42 has been reported in 13 of the 17 genera for which data are avail- able, although the subtribe Orchidinae includes about 50 genera (D’Emerico 2001; Felix and Guerra 2005). In this subtribe, the Internal Transcribed Spacer (ITS) phy- logenies (Bateman et al. 2001; Bateman et al. 2003; Jin et al. 2017) show that the group Pseudorchis-Amerorchis- Galearis-Platanthera s.l. includes genera with the chro- mosome number 2n = 2x = 42 (Löve and Simon 1968; Löve 1981; Cauwet-Marc and Balayer 1986). Also it is suggested that Dactylorhiza s.l. and Gymnadenia s.l., which have 2n = 2x = 40, probably derive from 42 chro- mosomes (Pridgeon et al. 1997; Bateman et al. 2009). As pointed out in the discussion, it is interesting to note the remarkable karyomorphological similarity in the species Platanthera chlorantha, P. algeriensis (this work), Chamorchis alpina (D’Emerico and Grunanger 2001), Galearis diantha (Schltr.) P. F. Hunt (Luo 2004), which have 2n = 2x = 42 chromosomes and Dactylorhiza romana (D’Emerico et al. 2002), which has 2n = 2x = 40 chromosomes. Last but not least, in spite of the extensive cytoge- netic literature that has built up over the years, little is known about the karyotype structure in other species of the genus Platanthera and related genera. Furthermore, to our knowledge, there are few studies of constitutive heterochromatin content, despite the fact that C-band- ing patterns provide extra information useful in assign- ing genomes. We have a limited quantity of data on the Platan- thera genus but it is possible to make some considera- tions on heterochromatin content. Indeed, Platanthera chlorantha and P. algeriensis show centromeric and subtelomeric heterochromatin, with a higher percent- age in the former. On this basis, an evolutionary com- parison is possible with the genera Dactylorhiza and Gymnadenia. Previous cy tological studies using the traditional Giemsa C-banding technique have shown significant heterochromatin content in some species of these two genera (D’Emerico et al. 2002; D’Emerico and Grunanger 2001; Baumann et al. 2012). For example, in the genus Gymnadenia, G. rhellicani (Teppner and Klein) Teppner and Klein, G. conopsea and G. odorat- issima (L.) Rich. have been found to possess numerous chromosomes with centromeric and telomeric hetero- chromatin. Similar banding patterns were previously observed in three species of the genus Dactylorhiza, including two diploids (D. romana, D. saccifera (Brogn.) Soò) and one polyploid (D. urvilleana subs. phoenissa B. Baumann and H. Baumann). Moreover, the distribution of C-banding patterns in Dactylorhiza karyotypes is of great cytological interest, although its nature can only be conjectured for the time being. Specifically, D. roma- na specimens have shown the chromosome numbers 2n = 40+1B and 2n = 40+2B, with one or two hetero- chromatic supernumerary chromosomes, which seems to suggest a possible evolutionary trend from 2n = 42 to 2n = 40 (D’Emerico et al. 2002). Figure 3. Giemsa C-banded mitotic metaphase of Platanthera chlo- rantha (a) and P. algeriensis (d); in P. chlorantha, interphase nuclei exhibit numerous chromocentres (b); P. chlorantha DAPI stained mitotic metaphase (c). Bar = 5 µm. 15Contribution to the study of wild Orchidaceae, genus Platanthera For a better understanding of phylogenetic relation- ships within the genus Platanthera, cytogenetic analy- sis should be extended to other species. Moreover, new chromosome methods such as fluorescent in situ hybrid- ization (FISH) and genomic in situ hybridization (GISH) will help to solve these problems using chromosomal analysis techniques. 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