Plant material used in this study consists of 32 natural populations of genus Narcissus sampled in contrasting ecological conditions along the east-west biogeographic gradient of the northern Algeria (Table 1). Systematic determinations were made using the main Algerian floras (Munby 1847; Battandier and Trabut 1895, 1902; Maire 1959; Quézel and Santa 1962) as well as floras from the Iberian Peninsula (Aedo 2013), from Morocco (Fennane et al. 2014), and from Tunisia (Le Floc’h et al. 2010). Status of the species and synonyms have been checked on the two main specialized websites, World Check List of Selected Plant Families (Govaerts 2015) and African plant database (Dobignard and Chatelain (2010–2013)). The studied taxa are presented in Table 2 and Fig. 1: N. tazetta and N. elegans are represented by several populations. Two natural populations of N. pachybolbus were narrowly located in the north-west of Algeria on the Mounts of Tlemcen, while those belonging to N. papyraceus are naturalized relics of cultivated plants. N. serotinus sensu lato is represented by populations collected over all the sampling area, some of which belong to N. obsoletus. N. cantabricus is narrowly located in the NW of Algeria at Tlemcen and near the Algerian-Moroccan border. From each site, 3–10 plants per taxon, with bulb, leaves and flowers, were collected. Voucher specimens were deposited at the Official Herbarium of ENSA (Algiers, Algeria) under numbers: ENSA13367-68 (N. cantabricus), ENSA13369-73 (N. elegans), ENSA13374-75 (N. pachybolbus), ENSA13376-77 (N. papyraceus), ENSA13378-81 (N. serotinus), ENSA13386-93 (N. tazetta).

Figure 1. 

Habits and flowers of species of genus Narcissus from Algeria. N. tazetta: A, B Sidi Khélifa C, D Hammam Mélouane E Yakouren F–G Tizi Tghidet. N. pachybolbus: H–K El-Ourit. N. papyraceus: L–M Bologhine. N. elegans: N–Q. N. serotinus: R–S Ain Ftouh. N. obsoletus: T–U Sainte Salsa. N. cantabricus: V–X Mansourah. Photos by Rachid Amirouche.

Chromosomal analysis was based on metaphase plates of root-tip cells from cultivated bulbs. Young roots (6–10 mm long) were pre-treated with 1% colchicine for 5–6 hours at room temperature, then fixed in ethanol-acetic acid (3:1) for 48 hours and conserved at 4 °C in ethanol 70°.The protocol was adapted from the Feulgen method (Jahier et al. 1992). The root-tips were hydrolysed in 1N hydrochloric acid for 7–12 min at 60 °C before stained with Schiff’s reagent in darkness for 1–2 hours. The squash was made in a drop of 45% acetic acid or carmine acetic. Metaphase plates were examined with a Zeiss Axiostar-Plus Microscope. Cells with good spreading of chromosomes were photographed.

Karyomorphometric measurements and the homologous chromosome ordering were made using the KaryoType Software 2.0 (Altınordu et al. 2016). Chromosomes are described according to the nomenclature of Levan et al. (1964) based on the arm ratio (r = long arm / short arm) and the centromeric index (CI% = short arm / long arm + short arm × 100): metacentric (m), submetacentric (sm), subtelocentric (st) and telocentric (t). Ideograms were drawn from at least 5 well-spread metaphase plates of different individuals. Karyotype asymmetry indices were estimated following the proposal of Peruzzi and Eroğlu (2013). The intrachromosomal asymmetry index is represented by the mean centromeric asymmetry MCA = A × 100, where A is the average ratio of long arm-short arm/long arm + short arm (Watanabe et al. 1999). The interchromosomal asymmetry index is the coefficient of variation of chromosome length CV_CL = A2 × 100 (Paszko, 2006) where A2 is the standard deviation of chromosome length/mean chromosome length (Romero Zarco 1986). The coefficient of variation of the centromeric index CV_CI = SCI / X̄ CI × 100 is the ratio between the standard deviation SCI and the mean centromeric index X̄ CI (Paszko 2006).

In order to link karyological data to morphological relationships between the studied species, multivariate analyses were carried out using the diagnostic descriptors of vegetative and reproductive parts, some from personal observations (Table 3). Principal Component Analysis (PCA) were performed using the program R Software 4.1.0 (2021).

≡ Narcissus tazetta subsp. tazetta

This species has many heterotypic synonyms. It is widespread in the north of Algeria and shows a high polymorphism with regard to the color of the perianth and corona (Fig. 1). The somatic chromosome number is generally 2n = 20 (Fig. 2A) and constant in all the karyologically examined populations (Table 4). Sometimes 1 to 2 supernumerary chromosomes have been observed such as in populations of Sidi Khélifa (Fig. 2B, C), and Oued Djenane (Fig. 2D). Three different cytotypes were observed (Table 3). The karyotypic formula 10sm + 10st was found in most of populations. Two other cytotypes concern populations located towards the east, Oued Djenane and Tabarka, with 2m + 8sm + 10st and 4m + 10sm (2sat) + 6st, respectively. The last two karyotypes are distinguished by a lower asymmetry indices MCA, 38.85 and 46.68 respectively, versus 50.27 for the remain populations. Satellites were observed in population of Tabarka only (Fig. 2E), which is characterized by a relative smaller total haploid length (THL = 114.75 μm).

≡ Narcissus tazetta subsp. pachybolbus (Durieu) Baker, 1888

≡ Narcissus papyraceus subsp. pachybolbus (Durieu) D.A. Webb, 1978

Narcissus pachybolbus is narrowly distributed in NW Algeria mainly in the region of Tlemcen. Two populations were sampled at Emir Abdelkader and El Ourit. Both are diploids with 2n = 2x = 22 and share the same karyotype formula 6m (2sat) + 6sm (2sat) + 8st + 2t (Table 3). This species has the highest total haploid length THL = 151.92 µm. The karyotype is distinguished by terminal satellites on the second and third largest submetacentric and subtelocentric pairs (Figs 2F, 3D).

≡ Narcissus tazetta subsp. papyraceus (Ker Gawler) Baker, 1888

This species has long been confused with the spontaneous N. pachybolbus due to strong similarities in the flower. N. papyraceus is an ancient cultivated species locally naturalized in Algeria. Two populations were found in the cemeteries of Algiers at Bologhine (ex. Saint Eugène) (Fig. 2G) and El Alia. Both populations show 2n = 2x = 22 chromosomes with the same karyotype formula 6m (2sat) + 12sm + 4st (Table 3). The karyotype of this species differs from that of N. pachybolbus by the presence of satellites on the 3^rd metacentric pair. For this taxon, the coefficients of variation of the length of the chromosomes (CV_CL = 37.62) as well as the centromeric index (CV_CI = 29.57) are lower. Despite their morphological similarity, the THL of N. papyraceus is closer to that of N. tazetta than that of N. pachybolbus (Table 3, Fig. 3E).

≡ Hermione elegans Haworth, 1831

N. elegans is encountered mainly in the Tell of the biogeographical sectors of Oranie, Algiers and the Kabylies. Seven representative populations were karyologically examined. The same diploid somatic chromosome number 2n = 20 are observed in all the samples with x = 10 (Table 4). However, two slightly different karyotypes were observed (Table 4, Fig. 2H, I). The most frequent concerns populations from the western region (Boutlélis, Santa Cruz, Tessala) and the center region (Chenoua, Sainte Salsa, Béni Messous) (Fig. 2H). The karyotype formula is 2m + 4sm + 14st. The second karyotype with formula 2m + 2sm + 14st + 2t (2sat) was observed only in the population of Ain Tagourait (Fig. 2J). It is distinguished by a coefficient of variation of centromeric index CV_CI (46.78 vs 32.92) and total haploid length THL (145.23 µm vs 125.32 µm).

= Narcissus serotinus var. emarginatus Chabert, 1889

Including N. obsoletus (Haworth) Steudel, 1841

≡ Hermione obsoleta Haworth, 1819

N. serotinus sensu lato is found in the same biogeographical areas than N. elegans, however with a much smaller occurrence. Sometimes, the two species grow in sympatry as in Ain Ftouh, Boutlélis, Ain Tagourait and Sainte Salsa. Five populations belonging to N. serotinus s.l. were examined and three chromosome numbers were observed, 2n = 20, 2n = 28 and 2n = 30 (Table 4). Most of the individuals of these populations from the central region, share the same chromosome number 2n = 30 corresponding to hexaploid level with base number x = 5. The karyotype formulas were slightly different particularly for THL and asymmetry indices A1 and MCA (Table 4, Fig. 2J, K, Fig. 3H, I). The cytotypes with 2n = 28 are unusual and concern individuals of two populations from the far west at Ain Ftouh and Boutlélis (Fig. 2L, Fig. 3J). The chromosome number 2n = 20 is observed for one population only of Aït Ali located toward east of the sampling area (Table 4, Fig. 2M, Fig. 3K). This tetraploid karyotype is moderately asymmetric and distinguished by a small total haploid length (THL = 66.01 µm).

= Narcissus bulbocodium subsp. monophyllus (Durieu) Maire, 1931

For this baetico-rifan species, two populations were sampled in NW Algeria, on clayey-marly slope in Mansourah forest near Tlemcen and on the edge of Lake Beni Bahdel towards the Algerian-Moroccan border. A diploid chromosome number was established 2n = 2x = 14 (Table 3, Fig. 2N, Fig. 3L). The karyotypic formula is 6m + 4sm + 4st with respectively intra and inter chromosomal asymmetry indices, A1 = 0.45 and A2 = 0.27 (Table 4). The total haploid length THL is 67.80 μm. One supernumerary chromosome was sometimes observed 2n = 14 + 1 (Fig. 2O).

All of the ten Algerian populations belonging to N. tazetta share the same chromosome number 2n = 20 with sometimes one or two B chromosomes. This somatic number was previously reported by Boukhenane et al. (2015) in the district of Constantine. This number is the most commonly observed in the Mediterranean region such as in Greece, Cyprus, Italy, and Southern France (Hong 1982; Garbari et al. 1988; Baldini 1990; Dominicis et al. 2002; Aquaro et al. 2007). Other chromosome numbers have been reported e.g., 2n = 14, 20, 22, 24, 28, 30 and 32 (Sharma and Sharma 1961; Brandham and Kirton 1987). The occurrence of one or two B chromosomes makes uncertain the base number (Baldini 1995; Dominicis et al. 2002; Zonneveld 2008). Indeed, most of the studies mention only the somatic chromosomal numbers (2n) without indication on the base number. Hong (1982) refer to x = 10 following the pioneering work of Fernandes (1951, 1966) who had already suggested three base numbers x = 7, x = 10 and x = 11 withing genus Narcissus. While, Brandham and Kirton (1987) have assumed a tetraploid (2n = 4x = 20) and hexaploid (2n = 6x = 30) levels for N. tazetta. On the basis of an exhaustive study on genome size measured by flow cytometry, Zonneveld (2008) has also assumed x = 5 as common base number for N. tazetta, N. elegans and N. serotinus. Most of the Algerian populations of N. tazetta show karyotypes expressing roughly similar formula. However, two populations collected in the eastern part near the Tunisian border (Oued Djenane, Tabarka), are distinguished by a less asymmetric karyotype. That of Tabarka, in Tunisia, was singularized by satellites on the 9^th submetacentric chromosome pairs contrary to those observed on the 6^th and 7^th subtelocentric chromosome pairs for some tazetta taxonomic units (Maugini1953; Hong 1982; Dominicis et al. 2002; Boukhenane et al. 2015).

Due to their morphological similarities, Maire (1959) had considered N. pachybolbus and N. papyraceus as subspecies of N. tazetta. Although N. papyraceus has never been reported in the ancient flora of Algeria (Munby 1847; Battandier and Trabut 1895, 1902). N. pachybolbus first described in NW of Algeria by Durieu (1846), is currently considered as an Ibero-Mauritanian species quoted in Morocco (Fennane et al. 2014) and Spain (Aedo 2010). For the Algerian populations of N. pachybolbus we have counted a diploid number of 2n = 2x = 22 consistent with previous studies (Maugini 1953; Brandham and Kirton 1987). However, in Flora Iberica, Aedo (2013) mentions 2n = 36. These two different chromosome numbers in two distinct territories suggest the need for a revision of this taxon. In our knowledge, the karyotypic formula is here provided for the first time: 6m (2sat) + 6sm (2sat) + 8st + 2t. A few karyological studies were devoted to this species. Brandham and Kirton (1987) have described just talk about a karyotype significantly different consisting of “…8 large acrocentric and 14 smaller acrocentric or submetacentric chromosomes”. Our samples of N. papyraceus exhibit also 2n = 22 chromosomes confirming previous reports (D’Amato 2004; Aedo 2013; Samaropoulou et al. 2013; Marques et al. 2017). The structure of the karyotype of N. papyraceus has been widely discussed by Brandham and Kirton (1987) and D’Amato (2004). Satellites have been observed on the 6^th and 7^th chromosomes pairs in contrast to Algerian samples which exhibit satellites on the 3^rd pair only. Although the karyotypic structures of these two species were considered as similar by Brandham and Kirton (1987), the Algerian samples of N. pachybolbus and N. papyraceus differ notably in the asymmetry indices. Contrary to the karyological diversity observed between N. pachybolbus and N. papyraceus, trees resulting from molecular phylogenies reconstruction show a polytomy indicating a very close relationship between these two species (Santos-Gally et al. 2012; Marques et al. 2017).

Morphologically N. tazetta, N. pachybolbus and N. papyraceus constitute three distinct clusters (Fig. 4). In respect to PC2, N. pachybolbus and N. papyraceus (2n = 22) are clearly in opposition to N. tazetta (2n = 20). The main morphological characters involved in this differentiation, relate to the color of the corona, the size and color of the outer layers of the bulb as well as the number of flowers per scape. Although sharing the same chromosome number 2n = 22, N. pachybolbus differs from N. papyraceus by higher values in the size of the bulb, the number of flowers per scape and emerging stamens from the corona (Fig. 1, Table 2). N. papyraceus is in intermediate position between N. pachybolbus and N. tazetta. The latter shows a high morphological variability expressed by small to medium bulb with rather brown outer tunics, a perianth white to yellow and a corona lemon to orange. These results agree with molecular phylogenies (Santos-Gally et al. 2012). The specific statute of N. pachybolbus and N. papyraceus agree with recent typification and taxonomic updating on daffodils (Aedo 2010; Koopowitz et al. 2017).

Narcissus elegans and N. serotinus s.l. have been described in all ancient floras of Algeria (Desfontaines 1798; Munby 1847; Battandier and Trabut 1895, 1902; Maire 1959; Quézel and Santa 1962) and several intermediate forms and putative hybrids have been reported. In Zonneveld (2008) and Marques et al. (2017), these two taxa were placed in section Serotini and section Tazettae, respectively. Some authors have grouped them together in the section Tazetteae (Santos-Gally et al. 2012). Regarding the Algerian material, these two species show close morphological relationships (Fig. 5). N. serotinus sensu lato within the meaning of Maire (1959) and Quézel and Santa (1962), is distinguished from N. elegans by its hysteranthous and smaller habit, and by “stable” characters such as single, or rarely 2, flowers per scape, larger and obtuse outer tepals. The other diagnostic descriptors, in particular the color and the shape of the corona, are variable and therefore difficult to use in practice. The inconstancy of these characters was noted by Maire (1959) and Quézel and Santa (1962) who had described around Algiers, intermediate forms attributed to × N. obsoletus (= Hermione obsoleta), as a putative hybrid N. elegans × serotinus. These two species are also distinguished by their karyological characteristics. The natural hybrid × N. obsoletus was underlined by DNA content of specimens from Spain and Morocco (Donnison-Morgan et al. 2005).

Figure 5. 

Principal components analysis focused on populations of Narcissus elegans and N. serotinus sensu lato A scatter plot on the first two PC of individuals of each taxon B loading of the morphological variables on the circle of correlations (see Table 3 for abbreviations). Morphologically N. elegans is well separated from N. serotinus sensu lato, by its synanthous habit (Syn), the number of flowers per scape (Nf), a full section of the scape (SSfill). With respect to PC2, individuals of N. serotinus s.l. are distributed in two opposed groups by the color of the corona. In the negative side individuals with yellow corona (Corcol6) correspond to N. serotinus type. Others individuals with orange corona (Corcol5) belong to N. obsoletus type. N. serotinus s.l.: black circle - St Salsa, white circle - Ahfir, white triangle - Ain Ftouh, gray circle - Boutlélis. N. elegans: gray star - Boutlélis, white square - Ain Tagourait, black triangle - Santa Cruz, black square - Chenoua, black star - St Salsa.

In our study, N. elegans has a constant somatic chromosome number 2n = 20 reported also in the literature but often without mention of the base number (D’Amato 2004; Díaz Lifante et al. 2009; Aedo 2013; Troia et al. 2013). The reconstructed ideograms of N. elegans show groupings preferentially in pairs of homologous suggesting a diploid level with x = 10. This is inconsistent with Donnison-Morgan et al. (2005), Zonneveld (2008) and Marques et al. (2012) who have assumed that N. elegans is tetraploid with x = 5. The karyotypic structure of N. elegans compared to that of N. tazetta from Algeria, shows similarities in agreement with the first assumptions of Fernandes (1966). The values of THL and the asymmetry indices of these two species vary within the same interval, except for CV_CI and CV_CL which are different. These differences would be due to chromosome structural changes as suggested by D’Amato (2004).

The Algerian populations belonging to N. serotinus sensu lato, display three somatic chromosome numbers 2n = 20, 2n = 28 and 2n = 30. The karyotype formula and the ideograms let suppose a base number x = 5 and consequently tetraploid and hexaploid levels. The tetraploids (2n = 20) were encountered in Sicily (Garbari et al. 1973) and in Greece (Phitos and Kamari 1974), the hexaploids (2n = 30) were quoted in Italy (D’Amato 2004; Troia et al. 2013). Diploid forms 2n = 2x = 10 were mentioned in Iberian Peninsula and Morocco by Fernandes (1968, 1975), Brandham and Kirton (1987) and Aedo (2013). This diploid cytotype (2n = 10) is considered very rare and would represent the N. serotinus type narrowly distributed in this region (Zonneveld 2008). In the literature, the most accepted and widespread ploidy level for N. serotinus remains the tetraploid 2n = 20. The hexaploid would raise controversy over its systematic statute. Analysis of genome size by flow cytometry led Zonneveld (2008) to attribute the hexaploid cytotype to N. miniatus which would be also confused with N. serotinus. Subsequent studies (Díaz Lifante et al. 2009; Marques et al. 2010, 2012, 2017) support that N. miniatus is an allohexaploid from N. serotinus (2n = 10) × N. elegans (2n = 20). This hexaploid form, firstly located in Spain, have a geographic range through the northern Mediterranean edge from Italy toward Lebanon, Palestine until Syria (Zonneveld 2008). On the contrary, the hexaploid specimens found by Troia et al. (2013) in Mazara del vallo (Sicily, Italy) have been attributed to N. obsoletus, which would have a larger geographic distribution area, especially in North Africa. Díaz Lifante et al. (2009) confirmed that the hexaploid cytotype of Spain and Greece belong to N. obsoletus. In our study, the karyologically examined populations are all mixed and would include individuals belonging to N. serotinus and N. obsoletus. The PCA focused on specimens of N. serotinus sensu lato and N. elegans (Fig. 5) show that the cytotypes with 30 and 28 chromosomes are all distributed along PC2. This distribution is determined by the color of the corona. All individuals located in positive pole of PC2, have orange corona and would correspond to N. obsoletus. At the opposite, individuals with yellow corona correspond to N. serotinus. This differentiation is consistent with the observations of Díaz Lifante and Andrès Camacho (2007) and Koopowitz et al. 2017. In Algeria, N. obsoletus was often misidentified and sometimes confused with N. serotinus. In our opinion, the two species N. serotinus (4x, 6x) and N. obsoletus (6x) are well present in Algeria in mixed populations. The hexaploid cytotypes are located mainly in the center region near Algiers (Ain Tagourait, Sainte Salsa). The unusual cytotypes 2n = 28 were encountered in the northwest near Oran (Boutlélis) and Tlemcen (Ain Ftouh), could be due to aneuploidy event (Figs 4, 5). The tetraploid cytotypes (2n = 20) belongs to N. serotinus are rare in Algeria and its encountered rather in pure populations, localized mostly in the eastern region. In the IUCN Red List of Threatened Species, N. serotinus was considered uncertain in our country (Juan Vicedo et al. 2018).

The presence of N. cantabricus in Algeria, was subject to controversy with N. bulbocodium. N. cantabricus was not mentioned previously in the floras of North Africa. Maire (1959) had described this species under N. bulbocodium subsp. monophyllus var. typicus with Corbularia monophylla as synonym. C. monophyla was initially reported in Algeria by Battandier and Trabut (1895) and then considered as synonym of N. monophyllus before being accepted by Quézel and Santa (1962) under N. cantabricus. N. cantabricus is distinguished from N. bulbocodium by a “white or slightly yellowish flower” (Battandier and Trabut 1895). These two species are mentioned in Flora Iberica (Aedo 2013) and Flore Pratique du Maroc (Fennane et al. 2014). Phylogenetic analyzes carried out successively by Fonseca et al. (2016) and Marques et al. (2017) confirmed their separation. The Algerian populations of N. cantabricus is diploid (2n = 14) with sometimes one B chromosome. The karyotype established here for the first time for this species, is rather symmetrical comprising mostly meta and submetacentric chromosomes. In the literature, diploid cytotypes were reported on the Cantabrian Mounts in the north, and in the center of Spain, while tetraploids are quite rare and found in Morocco on the Anti-Atlas (Zonneveld, 2008). Therefore, the Algerian diploids would be the southernmost within the geographic range of this species. Although the haploid amount of DNA is similar in the two species, it seems that N. cantabricus derived from N. bulbocodium following structural changes (Zonneveld, 2008). N. bulbocodium is distinguished by a high polyploid series from 2x to 8x with 2n = 72 as the highest chromosome number (Fernandes 1963; Fernandes and Franca 1974; Brandham and Kirton 1987; Marques et al. 2017). N. bulbocodium is an Ibero-Mauritanian whose polyploids propagate from North to South towards Morocco and from West to East through the Maghreb as already hypothesized by Fernandes (1951). This geographical distribution of the polyploidy is similar for the two species, and therefore the Algerian diploids of N. cantabricus constitute original and interesting material. The supernumerary chromosomes in the Algerian peripheral diploids, would express an adaptive response to aridity.