Introduction

The family Orchidaceae comprises approximately 25,000 species (Dressler 2005) distributed in 850 genera (Atwood 1986; Pridgeon et al. 1999; Chase et al. 2003). It has a cosmopolitan distribution, with the exception of the regions always covered with snow and extreme deserts, but is most abundant and diverse in humid tropical and subtropical forests. As for the classification, the most recent systems for Orchidaceae are those proposed by Dressler (1993) and Pridgeon et al. (1999, 2001, 2003, 2005, 2009, 2014), the latter based also on molecular data, proposing the division of the family into five subfamilies: Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae. In the subfamily Orchidoideae, there are six tribes; one of them is the Cranichideae, with six subtribes: Cranichidinae, Galeottiellinae, Goodyerinae, Manniellinae, Pterostylidinae and Spiranthinae (Pridgeon et al. 2001, 2003).

Subtribe Cranichidinae has 16 genera and about 210 species, all of them endemic to the Neotropics (Cribb and Pridgeon 2003; Salazar et al. 2003), and is characterized by the non-resupinate flowers, comparatively short column and brittle pollinia (Dressler 1993). In turn, subtribe Spiranthinae comprises about 40 genera and 470 species almost exclusively restricted to the Neotropics, except for the cosmopolitan genus Spiranthes Rich. (Salazar 2003). Its members can be recognized by the tubular flowers, margins of the lip adhered to the sides of the column, forming a deep nectary, and the soft granulose pollinia (Dressler 1993).

There are few cytotaxonomic studies on Spiranthinae, and these are concentrated especially on the genus Spiranthes with x = 15, 22 and 2n = 30 to 74 (Vij and Vohra 1974; Sheviak 1982; Tanaka and Kamemoto 1984; Martínez 1985; Brandham 1999). For the Neotropical species, Martínez (1981, 1985), Felix and Guerra (2005), Daviña et al. (2009) and Grabiele et al. (2010, 2013) have made the most significant contributions. On Cranichidinae, chromosome counts are reported only for Ponthieva mandonii Rchb.f. (2n = 46; Martínez 1985) and six species of Prescottia R.Br. (one with n = 48 and the others with 2n = 48; Felix and Guerra 2005).

The aim of this study was to determine the chromosome numbers and other chromosomal features of eighteen species of Spiranthinae and two of Cranichidinae, as part of our ongoing systematic and evolutionary studies of these orchid groups (Guimarães 2014; Guimarães et al., unpublished data).

Materials and methods

Eighteen species of Spiranthinae from Brazil and Mexico, and two species of Cranichidinae from Ecuador were studied (Table 1). Samples were cultivated in the greenhouses of the “Núcleo de Pesquisa Orquidário do Estado/Instituto de Botânica” (NP-OE/IBt in São Paulo, Brazil) and of “Herbario AMO” (Mexico City, Mexico). Vouchers were deposited at the herbarium of the Institute of Botany (SP), Mexico’s National Herbarium (MEXU) and the herbarium of the Faculty of Superior Studies of the National Autonomous University of Mexico, campus Zaragoza (FEZA).

Table 1 List of species analysed of Spiranthinae and Cranichidinae with respective locality, vouchers and chromosome numbers (2n), previous counts and references
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Mitotic studies were performed in root tips pretreated with 8-hydroxyquinoline 0.2 mM (8-Hq) for 24 h at 4 °C (SP samples) and for 5 h at 18 °C (MEXU and FEZA samples), fixed in absolute ethanol:glacial acetic acid (3:1) for 3–24 h at room temperature (25 °C) and stored in a freezer at −20 °C. To prepare the slides, root tips were hydrolysed in 5 N HCl for 12–30 min at room temperature, frozen in liquid nitrogen to remove the coverslip, stained with 2 % Giemsa for the Brazilian specimens (Guerra 1983), hydrolysed in 1 N HCl for 12 min at 60 °C and stained according to the Feulgen technique for the remaining specimens (Mercado-Ruaro and Delgado-Salinas 1998). The meristems were soaked in a drop of 2 % aceto-orcein and then squashed.

For each species, chromosomes were counted in at least five cells, observed in a Carl Zeiss Jenaval (SP samples) and an Axioscop (MEXU, FEZA samples) optical microscopes and photographed using the Canon PowerShot S3 1S (SP samples) and AxioCam ERc5S (MEXU, FEZA samples) digital cameras.

The best mitotic metaphase spreads were selected from the SP samples for a general chromosome characterization. For five of them, we detailed the karyotypes and prepared the ideograms. Chromosomes were measured using Adobe Photoshop CS 5.1 software, determining the karyotype formula (KF), total chromosome length (TCL, calculated as the sum of all individual sizes of chromosomes), the average length of each chromosome (ALC) and the centromeric index (CI) for each chromosome pair (calculated as the ratio of the short arm and chromosome length). The chromosome types were classified according to the nomenclature of Levan et al. (1964).

Results

Within the subtribe Spiranthinae, Aulosepalum riodelayense (Burns-Bal.) Salazar presented 2n = 64; A. tenuiflorum (Greenm.) Garay, 2n = 60; Cyclopogon luteoalbus (A.Rich. & Galeotti) Schltr., 2n = 36; Dichromanthus aurantiacus (Lex.) Salazar & Soto Arenas, 2n = 40; Eltroplectris calcarata (Sw.) Garay & H.R.Sweet, 2n = 42; E. triloba (Lindl.) Pabst, 2n = 46; Pelexia funckiana (A.Rich. & Galeotti) Schltr., 2n = 46; Mesadenella cuspidata (Lindl.) Garay, 2n = 38 and 42 (variation possibility due to uncertainly or aneusomaty; see Discussion below); Sarcoglottis assurgens (Rchb.f.) Schltr., 2n = 46; Sarcoglottis cf. grandiflora (Lindl.) Klotzsch, 2n = 46; Sarcoglottis richardiana (Schltr.) Salazar & Soto Arenas, 2n = 50; Sarcoglottis rosulata (Lindl.) P.N.Don, 2n = 33; Sarcoglottis sceptrodes (Rchb.f.) Schltr., 2n = 46; Sarcoglottis schaffneri (Rchb.f.) Ames, 2n = 46; Sarcoglottis scintillans (E.W.Greenw.) Salazar & Soto Arenas, 2n = 46; Sauroglossum elatum Lindl., 2n = 46; Stenorrhynchos albidomaculatum Christenson, 2n = 46; and Stenorrhynchos cf. speciosum (Jacq.) Rich., 2n = 46 (Figs. 1–5, 11–26). All species studied, except A. riodelayense, A. tenuiflorum, C. luteoalbus, S. richardiana, S. scintillans, S. albidomaculatum and S. cf. speciosum, present a pair of acrocentric or submetacentric chromosomes, which are about twice the size of the remaining chromosomes, which are metacentric. Mesadenella cuspidata with 2n = 42 presents a pair of acrocentric chromosomes about four times as large as the remainder chromosomes (Table 2, Figs. 4, 8). The species A. riodelayense (Fig. 11) and A. tenuiflorum (Fig. 12) show a bimodal karyotype with six larger chromosomes and the others small.

Fig. 1–5
figure 1

Chromosomes of species of Spiranthinae studied. 1 Eltroplectris calcarata (2n = 42). 2 Eltroplectris triloba (2n = 46). 3–4 Mesadenella cuspidata (2n = 38 and 2n = 42, respectively). 5 Sauroglossum elatum (2n = 46). Arrows indicate largest chromosomes in bimodal karyotypes; asterisks in 3 and 4 indicate the satellites. Bar = 1 µm

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Table 2 Chromosome measurements of Mesadenella cuspidata
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Fig. 6–10
figure 2

Ideograms of some species of Spiranthinae studied. 6 Eltroplectris calcarata (2n = 42). 7 Eltroplectris triloba (2n = 46). 8–9 Mesadenella cuspidata (2n = 38 and 2n = 42, respectively, with one pair with satellites). 10 Sauroglossum elatum (2n = 46). Bar = 1 µm

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Fig. 11–16
figure 3

Chromosomes of species of Spiranthinae studied. 11 Aulosepalum riodelayense (2n = 64). 12 Aulosepalum tenuiflorum (2n = 60). 13 Cyclopogon luteoalbus (2n = 36). 14 Dichromanthus aurantiacus (2n = 40). 15 Pelexia funckiana (2n = 46). 16 Sarcoglottis assurgens (2n = 46). Arrows in 11–12, 14–16 indicate largest chromosomes in bimodal karyotypes; asterisks in 16 indicate the satellites. Bar = 5 µm (11–15); 10 µm (16)

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The chromosome numbers for E. calcarata (Table 3, Figs. 1, 6), A. riodelayense (Fig. 11), A. tenuiflorum (Fig. 12), C. luteoalbus (Fig. 13), D. aurantiacus (Fig. 14), P. funckiana (Fig. 15), S. assurgens (Fig. 16), S. richardiana (Fig. 18), S. rosulata (Fig. 19), S. schaffneri (Fig. 20), S. sceptrodes (Fig. 21), S. scintillans (Fig. 22), S. albidomaculatum (Fig. 23) and S. cf. speciosum (Fig. 24) represent the first mitotic records for those species.

Table 3 Chromosome measures of Eltroplectris calcarata, E. triloba and Sauroglossum elatum
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Fig. 17–22
figure 4

Chromosomes of species of Spiranthinae studied. 17 Sarcoglottis cf. grandiflora (2n = 46). 18 Sarcoglottis richardiana (2n = 50). 19 Sarcoglottis rosulata (2n = 33). 20 Sarcoglottis schaffneri (2n = 46). 21 Sarcoglottis sceptrodes (2n = 46). 22 Sarcoglottis scintillans (2n = 46). Arrows in 17, 19–21 indicate largest chromosomes in bimodal karyotypes; asterisks in 19 indicate the satellites. Bar = 5 µm (18–20); 10 µm (17, 21–22)

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Fig. 23–26
figure 5

Chromosomes of species of Spiranthinae and Cranichidinae studied. 23 Stenorrhynchos albidomaculatum (2n = 46). 24 Stenorrhynchos cf. speciosum (2n = 46). 25 Ponthieva andicola (2n = 26). 26 Ponthieva pilosissima (2n = ±42). Bar = 5 µm (23, 25–26); 10 µm (24)

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A secondary constriction (satellite) was observed on the largest chromosome pair of M. cuspidata (Table 2, Figs. 3, 4, 8, 9), S. assurgens (Fig. 16) and S. rosulata (Fig. 19).

The chromosome number (CN), total chromosome length (TCL) and karyotype formula (KF) of E. calcarata, E. triloba, M. cuspidata and S. elatum are given in Table 4.

Table 4 Karyological features of the species analysed
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Regarding the studied representatives of the subtribe Cranichidinae, Ponthieva andicola Rchb.f. has 2n = 26 (Fig. 25) and Ponthieva pilosissima (Senghas) Dodson, 2n = ±42 (Fig. 26). Both are first chromosome counts for these species. Ponthieva pilosissima presents 10 large chromosomes, 20 median chromosomes and ca. 12 small chromosomes in a trimodal karyotype. This difficulty of counting is due to the distinct degree of contraction of the different types of chromosomes, so that the small chromosomes may be earlier or later condensed and some of them have a secondary constriction. Thus, we were unable to determine the precise chromosome number of this species.

Discussion

Some of the counts obtained here differ from those found in literature. Martínez (1985) and Daviña et al. (2009) reported a 2n = 46 cytotype for a specimen of Mesadenella cuspidata collected in Argentina, while in this study we found a chromosomal number variation of 2n = 38/42 for a single specimen collected in Brazil. This difference in chromosomal number could be due to the difficulty of counting, to the possibility that these specimens could actually represent different species of Mesadenella, or merely to intra-polymorphism in this feature. Another possible explanation is that Martínez (1985) and Daviña et al. (2009) interpreted the secondary constrictions (satellites) of some chromosomes as distinct chromosomes, as was the case with the legume Oxyrhynchus (Palomino and Mercado 1983). Analysis of additional specimens of this species, preferably from different geographic origins, would help to clarify the issue.

The variation of the chromosome number in M. cuspidata in the present study may be due to the difficulty of counting or to the occurrence of aneusomaty. Davide et al. (2007) defined polysomaty and aneusomaty as the intraindividual variations in chromosome number, related to the occurrence of polyploidy and aneuploidy, respectively. In the case of aneusomaty, it would represent the first observation of this condition in Spiranthinae, although it has been documented in orchids of the rupicolous epidendroid genus Hoffmannseggella of Laeliinae (Yamagishi-Costa and Forni-Martins 2009, 2013). Few cases of aneusomaty in plants are described in literature, but this phenomenon is common in families such as Orobanchaceae (Greilhuber and Weber 1975), Boraginaceae (Bigazzi and Selvi 2003) and Fabaceae (Rodrigues et al. 2009).

Martínez (1985) also noted that the species of Spiranthinae with 2n = 46 often show a bimodal karyotype, with one pair of large chromosomes and the remainder ones much smaller. The present study confirms this bimodal karyotype (with some exceptions), as has been found in other species of the subtribe by Felix and Guerra (2005). This karyotype bimodality supports the inclusion of the genera Eltroplectris and Mesadenella in the Stenorrhynchos clade sensu Salazar et al. (2003). On the other hand, Sarcoglottis assurgens, S. cf. grandiflora, S. sceptrodes, S. schaffneri and S. scintillans share the same chromosome number (2n = 46) of other species of Sarcoglottis and Pelexia previously analysed (Martínez 1985; Dematteis and Daviña 1999; Felix and Guerra 2005; Daviña et al. 2009), which reinforces the proposal that Sarcoglottis really belongs in the Pelexia clade sensu Salazar et al. (2003). The meaning of the discordant chromosome numbers of Sarcoglottis richardiana (2n = 50) and S. rosulata (2n = 33) remains unclear and must be carefully studied by analysis of additional specimens of both species.

According to Martínez (1985), the basic number x = 23 for the Spiranthinae, as well as the bimodal chromosome condition, seems to be the distinguishing characteristic of the subtribe, which Felix and Guerra (2005) suggest as a significant feature to separate it from subtribe Goodyerinae, which belongs to a different lineage within the tribe Cranichideae (Salazar et al. 2003). The origin for this high basic number, compared to Goodyerinae (with x = 10, 14?, Brandham 1999; Felix and Guerra 2005), is not clear but could be due to polyploidy, although the single pair of large chromosomes does not support this hypothesis, unless the polyploids have chromosomes derived from different species, i.e. the origin would be fully allopolyploid as supposed by Martínez (1985).

The evolutionary significance of a single pair (or three pairs in Aulosepalum ssp.) of larger chromosomes in a bimodal karyotype also remains obscure, especially in cases where this condition is present in species with different chromosome numbers, e.g. Eltroplectris sp. with 2n = 26 (Martínez 1985; Daviña et al. 2009) and Sacoila sp. with 2n = 46 (Cocucci 1956; Martínez 1985; Felix and Guerra 2005; Daviña et al. 2009; Grabiele et al. 2010). Furthermore, this kind of karyotype appears to be quite rare in other groups of Orchidaceae. Felix and Guerra (2000) analysed 44 species of cymbidioid orchids and found bimodality in only two of them. Apparently, a bimodal karyotype is a specialized genomic architecture that could be selected for its functionality (Stebbins 1971; Kenton et al. 1990).

Ponthieva pilosissima, with 2n = ±42, is a typical case of trimodal karyotype. This condition was observed only in Hyacinthaceae (Forrest and Jong 2004), Ruscaceae (Yamashita and Tamura 2004; Meng et al. 2005) and Orchidaceae (this study). All these families belong to the order Asparagales, and this type of karyotype could be a homoplasy (parallel/convergent character) among those groups.

This study confirms some previous observations (Martínez 1985; Daviña et al. 2009) on chromosome numbers in Spiranthinae, but also contributes new chromosome counts, expanding the cytogenetic knowledge of the subtribe, and also of the family Orchidaceae. Further analyses are needed to clarify the different chromosome numbers found for M. cuspidata and to understand the trimodal karyotype reported for P. pilosissima.