The karyotype of Adenia and the origin of the base number x = 12 in Passifloroideae (Passifloraceae)Abstract
Adenia is an Old World genus of Passifloroideae closely related to Passiflora. The two genera comprise the large majority of Passifloroideae species, although most studies are concentrated on Passiflora. Cytological analyses reveal that changes in chromosome numbers played an important role in the evolution of Passiflora, whereas in the remaining genera little is known, hindering the identification of the base number of the family. Here we analyzed the chromosome number and the 35S rDNA sites of three species of Adenia and reevaluated the base number (x) of the subfamily Passifloroideae and the family Passifloraceae, including chromosome data for Turneroideae and Malesherbioideae. The chromosome number of Adenia species seemed to be stable with 2n = 24 or 48 and one or two pairs of rDNA sites, very similar to Passiflora subgenus Astrophea, suggesting a common ancestral karyotype with x = 12. Differently, Turneroideae and Malesherbioideae present x = 7. A whole genomic duplication detected after the separation of Passifloroideae and Malesherbioideae suggests that the base number of Passifloraceae most probably was x = 7, which by dysploidy and polyploidy generated x = 12 for the subfamily Passifloroideae.
Key words
Cytotaxonomy; 35S rDNA sites; karyotype evolution; fluorescence in situ hybridization; Turneroideae; Malesherbioideae
INTRODUCTION
Adenia Forssk. is the second largest genus of the subfamily Passifloroideae (Passifloraceae) with approximately 100 species distributed in the Old World tropics and subtropics, the large majority of them in Africa (Feuillet & MacDougal 2007). The genus presents an uncommon diversity in growth form and ability to explore very different habitats (Hearn 2006). It is closely related to Passiflora, the largest and best studied genus of the family with over 560 species (Krosnick et al. 2013). Phylogenetic analyses revealed that both genera are monophyletic (Hearn 2006). Morphological (Feuillet & MacDougal 2007) and molecular analyses (Maas et al. 2019) placed Adenia in an intermediate position between the two tribes of Passifloroideae: Passifloreae and Paropsieae. According to APG III (2009), the former families Passifloraceae, Turneraceae and Malesherbiaceae should be included into the family Passifloraceae, as subfamilies Passifloroideae, Turneroideae and Malesherbioideae.
Cytological analyses of Passiflora revealed that chromosome number changes played an essential role in the early diversification of the genus, resulting in subgenera with different chromosome base numbers (x). The species of Passiflora are currently subdivided into four subgenera, according to Feuillet & MacDougal (2003), with a fifth subgenus (Tetrapathea) proposed latter by Krosnick et al. (2009). The two largest subgenera, Decaloba and Passiflora, have x = 6 and x = 9, respectively, whereas Astrophea, Deidamioides, and Tetrapathea possess x = 12 (Hansen et al. 2006), resulting in different interpretations of the base number of the genus (reviewed by Sader et al. 2019a). Differently, the chromosome number of Adenia species has been reported only for A. lobata (Jacq.) Engl. (2n = 24), A. mannii (Mast.) Engl. (2n = 24) and A. rumicifolia Engl. & Harms (2n = 48) (Mangenot & Mangenot 1962). In this sense, it would be important to have more chromosome counts of Adenia species to know if it experienced similar chromosome number radiation.
A key point to understand the chromosomal evolution of a taxon is the identification of its chromosome base number, which can be defined as the haploid number that most parsimoniously explains the cytological variability in a clade and shows a clear relationship with the base numbers of the closest related taxa (Guerra 2000). It may be inferred from a careful evaluation of the chromosome numbers reported for a clade, or it may be based on probabilistic models, some of them taking into consideration the possible ways of chromosomal evolution in that clade (Mayrose et al. 2010, Freyman & Höhna 2017). Since chromosome numbers are subjected to different rates of dysploidy and polyploidy and are under control of natural selection (Levin 2002), these probabilistic methods should be considered with caution. For Passiflora, the base number of each subgenus is clear since the haploid numbers do not vary, with a few exceptions, whereas the base number of the genus have been subject to a long debate. Strictly cytological analysis suggest x = 6 or x = 12 for the genus (reviewed by Melo et al. 2001), whereas probabilistic models suggest x = 6 (Hansen et al. 2006) or x = 12 (Mayrose et al. 2010, Sader et al. 2019a), depending on the algorithm used.
Beside the chromosome numbers, extensive genomic and cytomolecular studies have been done for Passiflora species (Melo & Guerra 2003, Munhoz et al. 2018, Pamponét et al. 2019, Dias et al. 2020, Xia et al. 2021), whereas nothing similar is known for Adenia. Most cytomolecular studies include the chromosome mapping of 5S and 35S rDNA sites by fluorescence in situ hybridization (FISH), bringing further details about the chromosome variability of the group (e.g., Melo & Guerra 2003, Silva et al. 2018, Sader et al. 2019b). The analysis of 20 species of Passiflora revealed that the number of 5S rDNA sites was generally proportional to the ploidy level of the species, while the number of 35S rDNA sites varied from 2 to 10 among diploid species (Melo & Guerra 2003).
In the present study, we analyzed the chromosome number and the distribution of the 35S rDNA sites in three Adenia species, aiming to evaluate the karyotype variability of the genus. Further, we reappraised the basic number of Adenia, Passiflora, Passifloroideae and Passifloraceae based on the most recent phylogenetic arrangements and genomic data.
MATERIALS AND METHODS
The three species analyzed, Adenia fruticosa Burtt Davy, A. spinosa Burtt Davy, and A. glauca Schinz, were grown in pots in the greenhouse of the Botanical Garden of the University of Vienna, Austria. Actively growing shoot meristems and young root tips were cut in small pieces and immediately pretreated with 8-hydroxyquinoline (0.002 M) for 5 h at 6 °C. After pretreatment they were washed in distilled water for 5 min, fixed in Carnoy solution [ethanol-acetic acid (3:1, v/v)] for 24 h at room temperature, and stored in the freezer at –20 °C.
For cytological preparations, the meristems were digested in 2% cellulase-20% pectinase at 37 oC for 90 min. The meristems were squashed in 45% acetic acid and the coverslips were removed in liquid nitrogen. The slides were air-dried and stained with 2 μg/ml DAPI–glycerol (1:1) to allow selection of the best preparations. The best slides were fixed again in Carnoy, for 30 min, dehydrated in 100% ethanol and stored at –20 oC until required for in situ hybridization.
For in situ hybridization, the same protocol described by Melo & Guerra (2003) for Passiflora species was used. Probes SK18S and SK25S containing, respectively, 18S and 25S rDNA of Arabidopsis thaliana L. (Unfried et al. 1989, Unfried & Gruendler 1990) were used to localize the 35S rDNA sites. They were labelled with biotin-11-dUTP and detected with TRITC (tetramethyl rhodamine isothiocyanate). Chromosomes were counterstained with DAPI and the slides mounted in Vectashield (Vector). Cells were photographed with a DMLB Leica epifluorescence photomicroscope using Kodak Ultra color film ASA 400. The images were later digitalized and edited in Adobe Photoshop CS3 version 10.0.
RESULTS AND DISCUSSION
The karyotype of Adenia
Adenia spinosa and A. fruticosa presented the same chromosome number, 2n = 24, with symmetrical karyotypes and small chromosomes, which were slightly smaller in the former, whereas A. glauca exhibited 2n = 48, with some chromosomes nearly twice as larger as the smaller ones (Figure 1a-d). These data reinforce the assumption that the basic chromosome number of the genus is x = 12. At prophase, most chromosomes exhibited less condensed terminal regions (Figure 1e), as observed in most Passiflora species (Melo et al. 2001). Noteworthy, the tetraploid A. glauca had a more asymmetrical karyotype than its sister species, the diploid A. spinosa (Hearn 2006), suggesting that A. glauca most probably is an allopolyploid derived from A. spinosa and another species with larger chromosomes. Likewise, A. rumicifolia (2n = 48) is the sister species of A. lobata (2n = 24) (Mangenot & Mangenot 1962, Hearn 2006), but in this case there is no information about their karyotype symmetry. A parental relationship between diploid and tetraploid sister species by allopolyploidy with increasing karyotype asymmetry has been demonstrated in several other genera (see, e.g., Moraes & Guerra 2010, Ibiapino et al. 2019).
Chromosomes of Adenia fruticosa (a-b, 2n = 24), A. spinosa (c, 2n = 24), and A. glauca (d-e, 2n = 48). Note the similarity in chromosome size and morphology (a,b), the occurrence of four 35S rDNA sites (red) in b, d and e, and only two sites in c. Prophase chromosomes of A. glauca (e) show the chromosome condensation pattern. Bar in and corresponds to 5 µm.
The in situ hybridization experiment detected only two sites of 35S rDNA in A. spinosa (2x) and four sites in A. fruticosa (2x) and A. glauca (4x) (Figure 1), indicating instability in the number of rDNA sites between diploid species. Similarly, among diploid species of Passiflora the number of 35S rDNA sites varied from two to six with 2n = 12 or 18 (Melo & Guerra 2003, Viana & Souza 2012, Silva et al. 2018, Dias et al. 2020). However, in Passiflora subgenus Astrophea, the most basal lineage of Passiflora, the two species investigated had also 2n = 24 and four 35S rDNA sites (Melo & Guerra 2003), as A. glauca, emphasizing the similarity between the karyotype of these two taxa. Reduction of 35S rDNA sites to a single pair was observed in some species of Passiflora (Melo & Guerra 2003) as well as in most angiosperms (Roa & Guerra 2012).
The base number of Passifloraceae
The finding of three other species of Adenia with n = 12, 24, in the present work, reinforces the assumption that its ancestral base number is x = 12. However, the six species of Adenia cytologically investigated belong to Clade V, the largest and most diversified among the five clades of the genus, with approximately 25 species and all of them endemic to Madagascar, one of the two centers of diversity of the genus (Hearn 2006). Therefore, additional chromosome counts are necessary to confirm the apparent chromosome stability of Adenia.
The elevated base number x = 12 has probably been originated by the Whole Genomic Duplication (WGD) that occurred after the separation of Passifloroideae from the monospecific Malesherbioideae (One Thousand Plant Transcriptomes Initiative 2019). Figure 2 shows the phylogenetic relationships within Passifloraceae (modified from Maas et al. 2019), highlighting only the genera with known chromosome numbers. Violaceae, the sister group of Passifloraceae, has a huge variation in chromosome numbers and an uncertain basic number (Raven 1975). The two species of Malesherbia cytologically known displayed n = 7 and n =14 (Ricardi 1967). For the Turneroideae, the base number x = 7 occurs in Piriqueta, Adenoa, and in most series of Turnera (Shore et al. 2006, Gonzalez et al. 2012).
Cladogram of Passifloraceae (based on Maas et al. 2019) highlighting the genera with known base numbers. Adenoa was added to the cladogram as sister to Turnera and Piriqueta, according to Arbo et al. (2015). The base number of each clade is indicated within the circles. Triangles indicate three or more genera. The base number 11 refers to Crassostemma only.
The base number x = 7 in Malesherbioideae and Turneroideae suggests that the WGD has occurred after the separation of Turneroideae and Passifloroideae with x = 12 (Figure 2). In this case, there are two alternative scenarios: the sister group of Turneroideae, with n = 7, experienced a descending dysploidy to n = 6 followed by a WGD generating n = 12, or, the sister group had a WGD, resulting in n = 14, which by descending dysploidy generated n = 12. Further chromosome counts for other genera of Turneroideae and Passifloroideae are necessary to elucidate this point.
Besides Adenia and Passiflora, the only other chromosome count for Passifloroideae is n = 11 for the monospecific genus Crossostemma (Gadella 1970), suggesting that n = 12, or near 12, was on the origin of several Passifloroideae genera (Figure 2). Within Passiflora, the number n = 12 seems to have been conserved in the subgenera Astrophea, Deidamioides and Tetrapathea, whereas the subgenera Passiflora and Decaloba evolved by descending dysploidies to n = 9 and n = 6, as indicated by recent genomic analyses of P. edulis Sims (n = 9) (Xia et al. 2021) and P. organensis Gardner (n = 6) (Costa et al. 2021). Intermediate numbers between the extremes of this dysploid series have been reported for a few species of Passiflora with n = 11, 10, and 7 (Melo et al. 2001), supporting the assumption that descending dysploidy played a central role on chromosome number variation and in the origin of the subgenera.
ACKNOWLEDGMENTS
This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (grant numbers 308903/2011-0, 311924/2016-6 to M. Guerra), and Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Brazil (grant number SEG 22.16.04.007.00.03.002 to N.F.Melo).
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