Karyological and morphological investigations on a Hieracium putatively endemic to the National Park “Foreste Casentinesi, M. Falterona, Campigna” (northern Apennines, central Italy)

Abstract

A putatively endemic infraspecific taxon of the Hieracium villosum group from the northern Apennines was investigated from nomenclatural, morphological and karyological points of view. H. villosum Jacq. var. zangherii Vignolo-Lutati proved to be invalidly described, and for its morphological features was referred to H. dentatum Hoppe subsp. xanthostylophorum Furrer & Zahn, a taxon hitherto only known from Abruzzo (Central Apennines). Karyological investigations resulted in the first report for an Italian specimen belonging to the collective species H. dentatum. H. dentatum subsp. xanthostylophorum proved to be tetraploid, with a formula 2n = 4x = 36 = 2M + 14m + 18sm + 2st. Chromosomes were generally of large size and showed a low degree of polymorphism in their shapes. The karyological parameters of H. dentatum subsp. xanthostylophorum have been compared with some available karyological parameters from the literature, belonging to related entities of Hieracium subgen. Hieracium. The Italian Hieracium subgen. Hieracium populations proved to have the most asymmetric karyotypes with respect to other European populations. H. dentatum subsp. xanthostylophorum showed an intermediate karyotype asymmetry between Hieracium subgen. Hieracium populations of the Balkan Peninsula and those of southern Tuscany. From a broader point of view, this study confirms in the northern Apennines a trend pointed out in other central and southern European mountain areas: the core taxon (H. villosum) is surrounded by tetraploid apomictic lineages that show some morphological characters of other Hieracium species; in the case of H. dentatum subsp. xanthostylophorum (= H. villosum “var. zangherii”), characters of H. bifidum.

Keywords:

• Asteraceae
• Hieracium sect. Villosa
• H.villosum
• H. dentatum subsp. xanthostylophorum
• chromosome number
• morphology
• taxonomy

Introduction

While studying the flora and vegetation of the National Park “Foreste Casentinesi, M. Falterona, Campigna” (Viciani and Agostini 2009; Viciani et al. 2010), located in the Northern Apennines between Tuscany and Emilia Romagna (Figure 1), we collected several specimens of a Hieracium subgen. Hieracium (villosum/dentatum group) difficult to identify (Figure 2). It is well known that the genus Hieracium L. is one of the taxonomically most intricate groups of vascular plants, and that in Hieracium subgen. Hieracium group agamospermy is widespread (Chrtek et al. 2004). Moreover, according to Bedini et al. (2012a), Hieracium is one of the least studied genera of the Italian flora, on karyological grounds. This is further confirmed when one considers just the taxa endemic to Italy (Bedini et al. 2012b). The section to which our specimens could be referred is the sect. Villosa; H. villosum Jacq. belongs, together with its intermediate taxa, to one the most interesting groups of mountain hawkweeds in central and southern Europe (Severa and Chrtek 2002; Chrtek et al. 2004). In the studied territory, some specimens corresponded from a morphological point of view to an endemic taxon mentioned for this area as H. villosum Jacq. var. zangherii Vignolo-Lutati in Zangheri (1966). To check the actual distinctness of such an infraspecific taxon, karyological and morphological analyses have been carried out on fresh collected material and original herbarium specimens. The results are reported below.

Figure 1 Localization in Italy of the study area.

Figure 2 The plant in the wild (a whole plant with flowers; inset: a fruiting capitulum).

Materials and methods

Specimens

During the period 2008–2010, several specimens of plants morphologically corresponding to the original material and the description of H. villosum var. zangherii were collected in the locus classicus (northern slopes of Mt. Falco, Northern Apennines, altitude about 1600 m asl., 1 × 1 km UTM grid: 32T, 717 E, 4861 N). Mature seeds were used for the karyological analyses (seeds were present also in the original specimen but they proved not to be viable).

We considered the original specimen (Figure 3) [M. Falco (M. Falterona) dirupi volti a settentrione, alt. 1600 m., 8 agosto 1934, leg. P. Zangheri, det. F. Vignolo-Lutati in 1953, Herb. Zangherii n. 13779, VER] and all the other original allied Hieracium specimens (H. villosum, H. dentatum Hoppe, H. bifidum Hornem.) coming from the same area and belonging to Herbarium Zangherii, hosted in the herbarium of the Verona Natural History Museum (VER). We checked the main macro-morphological characters with regard to recent research on Italian Peninsula Hieracium (Gottschlich 2005, 2009a, 2009b, 2011).

Figure 3 The original specimen on which Hieracium villosum var. zangherii was invalidly described.

Karyological analyses

We took into consideration several recent cytogenetic analyses concerning other taxa of genus Hieracium and related genera (Coşkuncelebi and Hayirlioğlu-Ayaz 2006; Ilnicki et al. 2010; Ozcan et al. 2011) and other Asteraceae (Kiran et al. 2012; Meng et al. 2012).

Karyological analyses were obtained from somatic mitoses taken directly from the first phases of the developing embryo root tips after seed germination. After a pre-treatment of about 3 hours in 8-hydroxichinoline saturated solution at room temperature, the material was fixed in Carnoy’s fluid, second fixative solution (ethanol and acetic acid 3:1) (Johansen 1940) for several hours, also used as a preservative solution in the test tubes stocked at 5°C temperature. Afterwards, the material was washed in distilled water and then hydrolyzed in HCl 1N at 60°C for 5 minutes. Finally, after HCl removing and after briefly washing the tips in distilled water, they were stained with 40% water solution of lacto-propionic-orceine (Dyer 1979) for 18–48 hours at room temperature.

Karyological investigations were carried out on mitotic metaphase plates of meristematic cells, with the technique of fresh squashes of root tips (Dyer 1979): a very small piece of stained meristematic tissue was placed on a slide in a drop of 45% acetic acid, a clean coverslip was lowered over the drop and then gently pressured with the tip of a mounted needle until all the cells were dispersed in a large squash. Chromosome counts were made both during direct observations with the microscope, and on enlarged images of the original micrographs. Images were recorded with a microscope ZEISS Axiophot and digital photocamera Canon G5 connected to a personal computer. Measurements and values were processed in order to obtain chromosome ordering and homologue recognition, idiogram, karyotype formula and chromosome nomenclature proposed by Levan et al. (1964). Several other caryological parameters were calculated: [TF%] index by Huziwara (1962), centromeric index [As.k%] (Arano and Saito 1980), asymmetry [A] by Watanabe et al. (1999), the intra-[A1] and inter-chromosomal [A2] asymmetry indices proposed by Romero Zarco (1986), and also more recently proposed indices as the CV_CL, CV_CI (Paszko 2006), or M_CA (Peruzzi and Eroğlu 2013) (Table 1). Some of these parameters have been recently criticized (for an updated review see Peruzzi and Eroğlu 2013). The aim of the performed comparative analysis was to have more karyotype information for comparing among populations or taxa supposed to be more or less closely related. Morphological or biochemical characters depend on the expression of some genes, but chromosomal characters do not depend on gene expressions: karyotype is nothing less than the “shape” of the genome itself (Guerra 2012), adding a priori information, so that karyological analyses are very important in biosystematic research.

Results

Nomenclature and taxonomy

According to art. 39 ICBN (McNeill et al. 2012) H. villosum var. zangherii is invalidly described (only with an Italian and not a Latin description). Despite this, the checks both on fresh material (Figure 4) and on the original specimen demonstrated that the plants must be attributed not to an infraspecific taxon of H. villosum but to H. dentatum, a taxon considered as an intermediate between H. villosum and H. bifidum (Pignatti 1982; Severa and Chrtek 2002). Also H. villosum, H. pilosum Froel. and H. bifidum are present in the same restricted area where H. villosum var. zangherii (= H. dentatum) lives. The interpretation of H. villosum var. zangherii as belonging to H. dentatum is due to the fact that the original specimen and the recently collected material have the following morphological main characters: basal leaves +/– petioled; stem leaves only rounded at the base and not more or less amplexicaul as in H. villosum; involucrum less hairy than in H. villosum. Because of the broadly spathulate basal leaves the plant can be assigned to H. dentatum subsp. xanthostylophorum Furrer & Zahn, a taxon hitherto only known from Abruzzo (Gottschlich 2009a, there erroneously mentioned as “xanthostylomorphum”).

Figure 4 A recent specimen of Hieracium dentatum subsp. xanthostylophorum (= “H. villosum var. zangherii”).

Karyological analyses

Hieracium dentatum subsp. xanthostylophorum (= “H. villosum var. zangherii”) – 2n = 36 (Figure 5a, 5b). This is the first report for an Italian specimen belonging to the collective species H. dentatum (Bedini et al. 2010).

Figure 5 (a) Photomicrograph of mitotic metaphase plate of Hieracium dentatum subsp. xanthostylophorum (= “H. villosum var. zangherii”); (b) karyogram.

The count corresponding to the tetraploid number 2n = 36 (x = 9) was made on 12 mitotic metaphase plates, but the karyotype is related to the selected micrograph for Figure 5a. The formula is 2n = 4x = 36 = 2M + 14m + 18sm + 2st (Figure 5b). The total chromosome length varied from 6.4 μm to 3 μm. The karyotype therefore was a little asymmetric, because of a large number of subtelocentric and telocentric chromosomes. Chromosomes were generally of large size and showed a low degree of polymorphism in their shapes. All karyological parameters of H. dentatum subsp. xanthostylophorum (labeled as DX) are shown in Table 1. They have been compared with some available karyological parameters from the literature, belonging to taxa of the large agamic complex H. villosum/H. morisianum (sect. Villosa) (Pignatti 1982) or other related entities of Hieracium subgen. Hieracium. The Table 1 dataset allowed us to make the scatter plots of Figure 6. The first scatter plot (Figure 6a), between A1 and A2 indices (Romero Zarco 1986), points out that the most symmetric karyotypes are those of taxa living in northern Europe, the Carpathian Region and Turkey (on the left side of the plot). On the other hand, the Italian Hieracium subgen. Hieracium populations appear to be the most asymmetric ones (on the right side of the plot). The second scatter plot (Figure 6b), between CV_CL and M_CA indices (Paszko 2006, Peruzzi & Eroğlu 2013), which, according to Peruzzi & Eroğlu (2013) are the most appropriate parameters to estimate respectively interchromosomal and intrachromosomal asymmetries, points out that H. dentatum subsp. xanthostylophorum lies to the right end of the plot, quite far from Polish H. villosum population (VP), that should be the most similar, from a morphological and taxonomical point of view. This could be due to the different evolutionary processes that the two populations, inhabiting distant lands, have undergone over time.

Table 1. Karyological parameters of some taxa belonging to Hieracium subgen. Hieracium.

Taxon	Geographical location	Label inscatter plots	TF%	A	A1	A2	Ask%	CVCI	CVCL	MCA	Average total length (μm)	Total length (μm)	Ploidy	2n	Karyotype formula(Levan et al. 1964)
H. dentatum subsp . xanthostylophorum	Italy, northern Apennines between Tuscany and Emilia	DX	37.31	0.25	0.38	0.16	62.7	18.41	16.46	25.4	4.35	156.75	4x	36	2M + 14m + 18sm + 2st
H. racemosum [1]	Italy, southern Tuscany	RA	36.4		0.41	0.16					6.21	108.3	2x	18	8m + 10sm
H. sabaudum [1]	Italy, southern Tuscany	SA	37.1		0.4	0.18					4.80	131.2	3x	27	9m + 18sm
H. umbellatum [1]	Italy, southern Tuscany	UA	35.5		0.44	0.17					5.6	150.6	3x	27	9m + 18sm
H. murorum [1]	Italy, southern Tuscany	MA	38		0.37	0.25					4.6	123.5	3x	27	3M + 9m + 15sm
H. grovesianum [1]	Italy, southern Tuscany	GA	37.8		0.38	0.22					5.05	131.3	3x	27	12m + 15sm
H. sabaudum [2] ^∗	Slovakia, western Carpathians	SC	40.33	0.19	0.30	0.16	59.60	23.05	15.81	19.3	3.76	101.50	3x	27	6M + 12m + 6sm + 3st
H. umbellatum [3] ^∗	Southern Poland	UP	39.86	0.20	0.32	0.17	60.09	13.12	16.73	20.3	3.05	55.00	2x	18	10m + 8sm
H. racemosum subsp . barbatum [3] ^∗	Southern Poland	BP	40.78	0.18	0.31	0.16	58.88	11.41	16.63	18.4	2.52	68.10	3x	27	21m + 6sm
H. villosum [3] ^∗	Southern Poland	VP	38.93	0.22	0.34	0.23	61.26	16.26	22.80	22.1	3.14	112.95	4x	36	4M + 16m + 16sm
H. sabaudum [3] ^∗	Southern Poland	SP	38.9	0.22	0.34	0.20	60.99	18.86	20.11	22.1	3.13	84.60	3x	27	16m + 11sm
H. umbellatum [4] ^∗	Turkey, Trabzon	UT	41.65	0.17	0.28	0.15	58.47	4.55	14.73	16.7	3.03	54.62	2x	18	12 m + 6 m sat
[1] Selvi and Fiorini (1996), [2] Chrtek et al. (2004), [3] Szeląg and Vladimirov (2005), [4] Coşkuncelebi and Hayirlioğlu-Ayaz (2006).^∗Measurements and values were processed directly from the pictures of the cited papers.Index references – TF%: Huziwara (1962); As.k%: Arano and Saito (1980); A: Watanabe et al. (1999); A1 and A2: Romero Zarco (1986); CVCL and CVCI: (Paszko 2006); MCA: Peruzzi and Eroğlu (2013).

Figure 6 Scatter plots comparing the karyological parameters of some taxa belonging to Hieracium subgen. Hieracium, obtained (a) between A1 and A2 indices, and (b) between CV_CL and M_CA indices. For the meaning of the labels and index references, see Table 1.

Discussion and conclusion

Hieracium s. str. comprises an immense agamic complex with a base-number of x = 9. Triploids and tetraploids form the bulk of taxa investigated so far. Diploids (2n = 18) are rather rare and supposed to be confined to restricted geographical areas (Chrtek et al. 2004), reported mostly from SW Europe (e.g. in southern Tuscany, see Selvi and Fiorini 1996). In central and southern European mountains, H. villosum proved to be both an apomictic triploid (2n = 27) and an apomictic tetraploid (2n = 36) (Schuhwerk 1996; Severa and Chrtek 2002; Chrtek et al. 2004; Szeląg and Vladimirov 2005; Kruk 2011). In the same territories, H. dentatum was found to be an apomictic tetraploid (2n = 36) with, surprisingly, the same genotype as detected in H. villosum (Severa and Chrtek 2002). In the Balkan Peninsula (Niketić et al. 2006), in Austria and France (Tyler and Jönsson 2009), taxa considered of the H. dentatum group were found to be triploid or tetraploid. Our analysis seems to point out similar trends in the northern Apennine study area. The core taxon (H. villosum) is surrounded by tetraploid apomictic lineages that show some morphological characters of other Hieracium species, in the case of H. dentatum subsp. xanthostylophorum (= H. villosum “var. zangherii”), characters of H. bifidum.

This biosystematic interpretation well agrees with karyological analysis made on H. dentatum subsp. xanthostylophorum, which in the northern Apennines confirms the presence of tetraploid populations having an intermediate karyotype asymmetry between the populations of the Balkan Peninsula and those of southern Tuscany.

Even if this supposed locally endemic Hieracium variety cannot be confirmed, at least from a nomenclatural and morphological point of view, the presence in this small area of a high number of Hieracium taxa corroborate the highly relevant role in plant diversity and conservation (Viciani et al. 2010) of this territory, that for its geographical position can be considered a “trait d’union” between two very different phytogeographical typologies, a northern European one and a southern Mediterranean one.

Acknowledgements

We would like to thank Lorenzo Peruzzi and two other anonymous reviewers for their comments and suggestions that helped to improve this work.