Karyological investigations and new chromosome number reports in Bellevalia Lapeyrouse, 1808 and Muscari Miller, 1758 (Asparagaceae) from Algeria

Abstract Karyological investigations were carried out on four species of Bellevalia Lapeyrouse, 1808 and Muscari Miller, 1758 (Asparagaceae) sampled in contrasting bioclimatic conditions of Algeria. The endemic Bellevalia mauritanica Pomel, 1874 was found to have a tetraploid cytotype 2n = 4x = 16 and an octoploid 2n = 8x = 32 which is a new report. The chromosome number 2n = 2x = 18 in Muscari comosum (Linnaeus, 1753) Miller, 1768 and Muscari maritimum Desfontaines, 1798 was in conformity with earlier reports. The latter species reveals a lesser bimodality of the karyotype. Within Muscari neglectum Gussone ex Tenore, 1842 pentaploid (2n = 5x = 45), hexaploid (2n = 6x = 54) and very rare octoploid cytotype (2n = 8x = 72) have been reported in Algeria. Principal component analysis performed on basis of karyotype parameters, showed a segregation of the different cytotypes. This study provides new karyological information, which is discussed in a taxonomic context.


Introduction
The Hyacinthaceae is one of the most important families of Asparagales, containing about 70 genera and 700-1000 species (Speta 1998, Pfosser and Speta 1999. Currently, they are included in the expanded Asparagaceae sensu APGIII (2009) as subfamily Scilloideae comprising four tribes Hyacintheae, Ornithogaleae, Urgineeae and Oziroëeae (Chase et al. 2009). Except Oziroëeae, they show a disjunct distribution pattern between the Mediterranean area, north-west Africa, western Asia and sub-Saharan Africa (Sanmartin et al. 2010. Hyacintheae is undoubtedly the most significant tribe, according to the number of species. Many of them present interesting patterns for examining karyological evolution and polyploidy in relation with their geographical distribution (Speta 1998, Stedje 2001, Hamouche et al. 2010, Goldblatt et al. 2012, Weiss-Schneeweiss and Schneeweiss 2013. Actually, many new chromosome records have lead to description of new species and/or change in taxonomic and nomenclatural status. That is the case of Bellevalia Lapeyrouse, 1808 and Muscari Miller, 1758.
Despite its biogeographical position in the south-western Mediterranean area, Algeria suffers from an obvious lack of cytotaxonomic data (Amirouche and Misset 2009). This is why it is necessary to start our research by karyological investigations. According to the ancient floras of Algeria (Desfontaines 1798-1799, Battandier and Trabut 1895-1902, Maire 1958, Quézel and Santa 1962, Bellevalia and Muscari comprise four and five species respectively. This paper is part of an ongoing program on Asparagales in Algeria and aims to complete chromosomal counts, karyotypes knowledge and geographical distribution of the polyploidy. It focuses on the endemic B. mauritanica Pomel, 1874, and M. comosum (Linnaeus, 1753) Miller, 1768, M. maritimum Desfontaines, 1798 Gussone ex Tenore, 1842.

Sampling and taxonomic determinations
Populations used in this study were sampled from March to May 2010-2012 in various ecogeographic areas of Northern Algeria (Table 1). In each site, 5-10 bulbs were collected and cultivated in the experimental garden of Houari Boumediene University of Sciences and Technology (Algiers). Taxonomic determinations were made based on several North-Africa and Mediterranean Floras: Desfontaines (1798-1799), Trabut (1895-1902), Maire (1958), Quézel and Santa (1962) and Davis and Stuart (1980). The specialized taxonomic and nomenclatural websites, the African Plant Database (Dobignard andChatelain 2010-2013) and the World Check List of Selected Plants (Govaerts 2015) were also consulted.

Chromosome preparations
Mitotic preparations were performed on young root-tips obtained from potted plants. The chromosome observations were performed using the standard Feulgen technique for staining tissues (Jahier et al. 1992), with little modifications. Root-tips were pre-

Karyotype and idiogram constructions
Measurements for karyotype and idiogram constructions were based on at least five well-spread metaphase plates of different individuals. The arrangement of homologous pairs was made using MICROMEASURE Software version 3.3 (Reeves 2001). Chromosomes are described according to the nomenclature of Levan et al. (1964) based on the chromosomal 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). Karyotype asymmetry indices were estimated following the proposal of Peruzzi and Eroğlu (2013). The intrachromosomal asymmetry index is represented by the mean centromeric asymmetry M CA = A × 100, where A is the average ratio of long arm-short arm/long arm + short arm, according to Watanabe et al. (1999). The interchromosomal asymmetry index is the coefficient of variation of chromosome length CV CL = A 2 × 100 (Paszko 2006) where A 2 is the standard deviation of chromosome length/mean chromosome length (Romero Zarco 1986). The coefficient of variation of the centromeric index CV CI = S CI /X CI × 100 is the ratio between the standard deviation S CI and the mean centromeric index X CI (Paszko 2006).

Multivariate analysis
In order to compare the karyotypes of the studied species, a Principal Component Analysis (PCA) was performed using STATISTICA Software version 6. Analysis was based on six fundamental karyological parameters as proposed by Peruzzi and Altinordu (2014): chromosome number (2n), chromosome base number (x), total haploid chromosome length (THL), mean centromeric asymmetry M CA , coefficient of variation of chromosome length CV CL and coefficient of variation of the centromeric index CV CI .

Results
Chromosome numbers, ploidy level and karyotype characteristics of the four studied species of Bellevalia and Muscari occurring in Algeria are summarized in Tables 2-3. Representative metaphases and the idiograms are shown in Figs 1-2.

Bellevalia mauritanica Pomel, 1874
Mitotic observations showed tetraploid and octaploid cytotypes with base number x = 4. The tetraploid cytotypes 2n = 4x = 16 (Fig. 1A-B; 2A-B) was found in two populations from two contrasted biogeographical sectors. Plants from biogeographical sector of Constantine (Tiddis) grow on clayey-marly soil. Their chromosomes show a total haploid length THL = 93.05 μm with mean length per chromosome (CLR) ranging from 7.00 to 17.1 μm ( Table 3). The karyotype consists of 4m + 4st + 8sm. Specimens  (Table 3) with a karyotype formula 4msat + 4st + 8sm. This karyotype is distinguished by two terminal satellites on the first largest metacentric pair (Fig. 2B). Except the occurrence of the satellites, the structure of the two karyotypes is similar regarding the centromeric asymmetry values and the coefficient of variation (Table 3). The octoploid cytotype 2n = 8x = 32 (Fig. 1C, 2C) was found in two populations from Ouled Fayet and AinTorki of the biogeographical sector of Algiers. This cytotype is characterized by a larger THL 171.40 μm and CLR values more extensive (6.05-18.05 μm). The karyotype formula is quite similar to that of the tetraploids. One submetacentric supernumerary chromosome was occasionally observed in octoploid individuals (Fig. 1C). The centromeric asymmetry indices (M CA ) of tetraploid and octaploid cytotype are rather different while the coefficients of variation (CV CL ) are much closer.

Muscari comosum (Linnaeus, 1753) Miller, 1768
This species is widespread in the north of Algeria. Examined populations were diploids with 2n = 18 chromosomes and a base number x = 9 (Fig. 1E). The mean length per chromosome is comprised between 1.94 μm to 10.49 μm and total length THL = 33.51 μm ( Table 3). The karyotype is distinguished by two large pairs of chromosomes and seven other pairs much smaller. The first pair is telocentric; the second pair constituted by one metacentric and one submetacentric chromosome is polymorph due to structural heterozygosity (Figs 1E, 2D). All the remaining small chromosomes are metacentric. The karyotype formula is 2t + (1m + 1sm) + 14m. The values of the centromeric asymmetry (M CA ) and the coefficient of variation (CV CL ) are 73.8 and 19.97 respectively.

Muscari maritimum Desfontaines, 1798
M. maritimum is less common. The studied population lives on the sand dunes in the steppe high plains of the Saharan border (Guelt es Stel). It is also diploid with 2n = 18  (Fig. 1F). The mean length of chromosomes is between 2.37 μm and 9.38 μm with a THL = 47.64 μm (Table 3). The karyotype is characterized by 6st-sat + 6sm-sat + 6m (Fig. 2) showing two satellites: terminal on the first subtelocentric pair, and intercalary on the fifth submetacentric pair. Compared to M. comosum, M. maritimum have a less asymmetrical karyotype reflected in a low value of its centromeric asymmetry index (M CA ).

Muscari neglectum Gussone ex Tenore, 1842
In this species, three cytotypes were observed: pentaploid 2n = 5x = 45, hexaploid 2n = 6x = 54 and octaploid 2n = 8x = 72 (Figs 1G-I). All cytotypes were encountered in the eastern biogeographical sector of Constantine (Ain El Bey, Tiddis and Djemila). The western population of Mansourah (Tlemcen) is pentaploid (Tables 1-2). Compared to the previous species, chromosomes are markedly small with mean lengths between 1.80 μm and 5.39 μm and no significant difference among the three karyotypes (Table 3). This species is characterized by a rather symmetrical karyotype comprising only metacentric chromosomes (Figs 1-3, Table 3) with total length depending on the ploidy level. The centromeric asymmetry indices (M CA ) and the coefficients of variation are also similar.

Karyotype relationship
In order to estimate the karyological relationship among the studied taxa, a principal component analysis (PCA) was carried out on the 8 populations, each representing different species and/or cytotypes (Fig. 3). The pattern of correlation loadings of the variables (Fig. 3A) highlights the major role of PC1 and PC2. Cumulative variance explained by these two first components approaches to 85% of the total information. The formation of PC1 was due to intrachromosomal asymmetry parameter M CA, the coefficient of variation of the centromeric index CV CI (negative values) and to chromosome numbers 2n and x (positive values) which have a discriminant power > 0.79 (data not shown). PC2 is well described by the variables THL and CV CL (inversely correlated) and, in least degree, once again, by the chromosomal numbers 2n and x (Fig. 3A). As expected, the projection of the taxa on the first two axes confirms the divergence between the cytotypes representing the genus Muscari from those of genus Bellevalia (Fig. 3B).
Cytotypes of Bellevalia mauritanica constitute a clearly distinct group, in which the two tetraploid cytotypes (from Stidia and Tiddis) shows close relationship. The octoploid cytotype (2n = 8x = 32) can be discreetly distinguished probably because of a higher value of the total haploid length (THL).
The karyotypes of the studied species of Muscari constitute two other clusters significantly different from each other (
Usually, in the genus Bellevalia, the karyotypes show satellites on either the first, the second or the third pair of chromosomes (Bothmer and Wendelbo 1981, Bareka et al. 2008Bareka et al. , 2012. Our tetraploid B. mauritanica from Stidia shows a similar chromosome arrangement and bears one pair of satellites on the first metacentric pair.

Chromosome number and polyploidy in genus Muscari
Karyological results on M. comosum and M. maritimum agree with previous findings on the subgenus Leopoldia in which species are mostly diploids (Ruiz Rejón et al. 1985, Nersesian 2001, Jafari and Maassoumi 2011, Jafari 2012a, 2012b.
All the examined specimens of M. comosum have 2n = 2x = 18 with a markedly asymmetric karyotype consisting of 2 pairs of large chromosomes and 7 pairs of small and metacentric chromosomes. Slight variations were observed in the first pair of chromosomes, sometimes viewed as telocentric (Ruiz Rejón et al. 1981, Kostovic-Vranjes 1999 or as subtelocentric (Cuñado et al. 2000, Jafari et al. 2008. Similarly, the second pair is polymorphic with submetacentric and subtelocentric chromosomes (Ruiz Rejón et al. 1985, 1990, Cuñado et al. 2000, Kostovic-Vranjes 1999. Concerning M. maritimum, the chromosome number 2n = 18 was previously quoted by Garbari and Di Martino (1972) for specimens with unspecified origin. It is also quoted by Troìa et al. (2014) for one Tunisian population at the Cap Bon. However, in our knowledge, the karyotype structure of M. maritimum is reported here for the first time. It would be related to that of M. gussonei (Parlatore) Todaro, 1872, an endemic species to Sicily Di Martino 1972, Davis andStuart 1980). The karyotype of this species consists of 10 large and 8 small chromosomes (Ruiz Rejón et al. 1985). However, karyotype of Algerian specimens collected in the Saharan border is distinguishable in having satellites located on the 1 st and the 5 th large chromosome pair.
Muscari neglectum belongs to the subgenus Botryanthus which contrasts considerably with the precedent by the occurrence of ploidy series of 2x, 3x, 4x, 5x and 6x levels (Davis and Stuart 1980, Karlén 1984, Ruiz Rejón et al. 1985, Garbari 2003, Suárez-Santiago et al. 2007. Previous chromosomal counts for M. neglectum indicate several numbers: 2n = 18, 36, 44, 54, 55, 63 and 72 (Karlén 1984). The three ploidy levels (5x, 6x, 8x) observed in Algeria confirm the extent of polyploidy in this complex. However, no diploids or tetraploids were detected in our country. In contrast, the presence of octaploid plants is significant because the 8x level was extremely rare and only few individuals having 2n = 72 were previously quoted in a population from the northern Greece (Karlén 1984). So far, only tetraploid, pentaploid and hexaploid populations of this taxon have been observed in the western Mediterranean area, precisely in the Iberian Peninsula (Ruiz Rejón et al. 1985, Suárez-Santiago et al. 2007. This is what justifies the statement generally accepted that the diploids occur only in Greece and Turkey (Karlén 1984, Garbari 2003.

Taxonomical remarks
Morphologically, both 4x from Tiddis and 8x from Ouled Fayet and Ain Torki, are similar and belong to the endemic B. mauritanica precisely to var. eu-mauritanica Maire & Weiller, 1958. This variety is known with a geographic distribution from Central and NE Algeria throughout Tunisia and Cyrenaica. A second variety, B. mauritanica var. tunetana Battandier, 1911 is restricted to Tunisia. Concerning, the 4x population from Stidia (NW Algeria), the karyotype is distinguished by large chromosomes and satellites on the first chromosomal pair. This population of Bellevalia cf. mauritanica grows on sandy soil and differ from the type in some variable features as small scape, perigone campanulate-oblong, tepals white to sky-blue and style white. In regard to these characters and its restricted location in the NW Algeria, specimens from Stidia may be attributed to B. dubia var. variabilis (Freyn) Maire, 1941 as quoted previously (Maire 1958, Quézel andSanta 1962). However, the recent phylogenetic studies by Borzatti Von Loewenstern et al. (2013), demonstrated that B. dubia is diploid and narrow endemic to Sicily. Therefore, the taxonomic status of 4x samples from Stidia, considered here as B. cf. mauritanica, needs to be re-evaluated.
In conclusion, our results contribute to a better knowledge of Hyacinthaceae in Algeria. Beside the earlier chromosomal counts, new chromosomes numbers were ascertained from Algerian populations. That is the cases of the new reports of octoploid cytotypes in Bellevalia mauritanica and Muscari neglectum. All karyological data are illustrative and reflect the east-west pattern of polyploidy at the Mediterranean scale. Further studies are needed to reconsider the taxonomic status and the evolutionary relationships of diploid and polyploid taxa in North Africa.