Morphometric analysis and bioclimatic distribution of Glebionis coronaria s.l. (Asteraceae) in the Mediterranean area

Abstract We present a revision of Glebionis coronaria in the Mediterranean area based on: a) micro-morphology of the disc floret cypselas observed with a high-resolution confocal microscopy; b) measurements of the disc cypsela with a stereoscopic microscope – duly scaled; c) its distribution in several bioclimatic belts; d) field observations; e) comparisons of herbarium samples. Because of this study, we propose the elevation of Glebionis coronaria var. discolor to the rank of species, as Glebionis discolor comb. & stat. nov., based on morphological and ecological characteristics such as the disposition of the intercostal glands, the size of the disc cypsela wings and its distribution according to the bioclimatic belts. Glebionis coronaria, with totally yellow ray florets and intercostal glands aligned, is exclusive to the thermo-Mediterranean bioclimatic belt, while Glebionis discolor, with white ray florets on a yellow base and intercostal glands arranged randomly, is found in the thermo- and meso-Mediterranean belt. Illustrations of micromorphological characteristics of the cypselas, an identification key, a taxonomic synopsis including information on nomenclatural types, synonyms, descriptions of the taxa, and, as supplementary information, a list of the specimens examined and bioclimatic classification of samples localities are also presented.


Morphometric analysis and bioclimatic distribution of Glebionis coronaria s.l. (Asteraceae) in the Mediterranean area Introduction
The genus Glebionis Cass. ex Spach is present in the Mediterranean area with two species: Glebionis coronaria (L.) Cass. ex Spach (= Chrysanthemum coronarium L.) and G. segetum (L.) Fourr. (= Chrysanthemum segetum L.). For the first species, d'Urville (1822) described the variety with yellow ray florets as Chrysanthemum coronarium var. concolor d'Urv., and the other with white ray florets with a yellow base as C. coronarium var. discolor d'Urv. The only character used by d'Urville to distinguish the two varieties was the colour of the ray florets. Cassini (1826) gave the first description of the genus Glebionis based on the species Chrysanthemum roxburghii Desf., and published the new combination Glebionis coronaria based on Chrysanthemum coronarium, wich was described later by Spach (1841). Subsequently, Pau described a new species under the name of Chrysanthemum merinoanum for the island of Ibiza with the following diagnosis: "Intermedio entre el coronarium y el segetum, pero más afine del primero, del cual difiere por las hojas simplemente pinado-cortadas; los aquenios son muy parecidos, pero carecen de alas tan pronunciadas, y sólo llevan una. ..... lígulas blanquecinas, en la base amarillas, apenas festonadas en la terminación;....." (Pau 1899). Recently, Rosselló and Sáez (2001) designated a lectotype of C. merinoanum Pau (MA 128240) from a specimen collected by Pau on the island of Ibiza, emphasizing that the type material is indistinguishable from other Balearic and Spanish accessions of C. coronarium L.
Turland (l.c.) also confirmed the differentiation of the two varieties and proposed a new combination under the name of Glebionis coronaria var. discolor (d'Urv.) Turland (Basionym: Chrysanthemum coronarium var. discolor d'Urv. in Mém. Soc. Linn. Paris 1: 368. 1822). Turland (l.c.) notes that the two entities appear to be widespread in the Mediterranean region and show no obvious correlation with geographic distribution.
From the karyological point of view the two varieties of G. coronaria are both diploid, with 2n = 18 (Pavone et al. 1981Strother and Watson 1997, Vogt and Aparicio 1999, Inceer and Hayirlioglu-Ayaz 2007, Paciolla et al. 2010, Lograda et al. 2013. Abd El-Twab et al. (2008) confirm this account and point out that the chromosome complement of G. coronaria consists of 18 median-centromeric chromosomes, while G. coronaria var. discolor consists of 16 median-and 2 sub-median-centromeric chromosomes.
The aims of this paper were: (a) to highlight and compare some important micromorphological characters of the two entities of Glebionis coronaria; (b) to relate their taxonomic differences with their bioclimatic characteristics; (c) to indicate new informative characters for identification of these two taxa; (d) to prepare a key, make a more complete description and provide notes on ecology and distribution of these two entities.

Sampling areas
To clarify the morphological and ecological characters of the two varieties, we carried out several samplings in different areas of the Mediterranean basin: Sicily, southern Italian Peninsula (Calabria), and Iberian Peninsula (southern Spain and Portugal) (Fig. 1).
The sampling was on bioclimatic criteria and according to the climate classification of Rivas-Martínez andRivas-Saenz (1996-2009). A statistical analysis was performed with T-Student to establish a possible relationship between the two entities and bioclimatic belts.

Plant material
A micro-and macro-morphological study was made of sampled plants from pure non mixed populations. All the specimens collected in the field are conserved in the herbaria of Jaén (JAEN) and Reggio Calabria (REGGIO). We have also consulted the following herbaria which have specimens proceeding from eastern Mediterranean regions, the source location of the species originally described by Linnaeus: REGGIO, JAEN, FI, MS, CAT, SEV, VAL, COFC, MA. All 194 examined specimens are listed alphabetically by country in Appendix 1.
Seeds of G. coronaria var. coronaria obtained from pure populations in southern Portugal and Sicily and seeds of G. coronaria var. discolor obtained from pure populations in Jaén (Spain) were cultivated for three years. Both specimens were cultivated in the thermo-Mediterranean town of Andújar (Spain) and in the meso-Mediterranean town of Jaén, where they were grown separately and together to determine their vigour and the permanence of the characters.
High-resolution confocal microscopy was used to study the micro-morphology of the disc floret cypselas. A total of 880 cypselas (322 of the entity with yellow ray florets and 558 of the entity of white ray florets) were measured by taking images with a stereoscopic microscope -duly scaled-of both entities from different populations of plants cultivated in Portugal, Spain and Italy. The measurements were based on several observations ranging from 296 for the variety with yellow ray florets, to 425 for the variety with white ray florets; a statistical treatment was then applied using the XLSTAT programme.
Using these samples, measurements were taken of the length and width of the disc cypselas (excluding ventral and dorsal wings) and the width of the ventral wings (Table 1). We added a measure of the glands dispersion in each cavity formed between the ribs of the disc cypselas. To measure the degree of glands dispersion, a linearity coefficient (Lc) is proposed. A two-pixel wide straight line was drawn on the image between the two most separated glands in length within the group. The glands in contact with the straight line (A) were counted, and these glands were related to all the glands occupying the cavity (T). For cypselas whose morphology was not straight, but whose glands were aligned, two or more lines were used to count the aligned glands, applying a correction factor depending on the number of lines used (C). The formula and its correction are as follows: where (C-1)/A is the correction factor. If only one line is used, it is = 0.

Lc
Linearity coefficient A Aligned glands T Number of glands in the valley C Number of straight lines used

Results
To verify the observations made in the field, both varieties (from pure populations in different regions) were cultivated from seeds in the two bioclimatic belts for three years. In the thermo-Mediterranean belt, the seeds of both entities sprouted and produced plants that maintained their characters unchanged from year to year. In the meso-Mediterranean belt both seed entities sprouted initially; however only the white floret variety completed its life cycle and maintained its characters. According to Heywood (1976), sessile non-mucilaginous glands are present between the ribs of cypselas in both varieties. However, after careful observation (Tab. 1), we noticed that in the variety with yellow ray florets these glands were neatly arranged between the ribs (Fig. 2a), while they were disordered in the variety with white ray florets (Fig. 2b).
Other characters that differentiate the two entities are the width and shape of the abaxial wing of the disc floret cypselas. In the yellow floret variety, this wing is wider Both the arrangement of the glands in the intercostal spaces and the wing width are good characters -among others-for differentiating the two entities, as can be seen from the statistical study (Figs 3,4). The linearity coefficient was used to measure objectively the arrangement of the glands in the intercostal spaces.
In the boxplot (Fig. 3), the Linearity coefficient of the glands present in the intercostal valleys of the inner cypselas can be observed. In both species, they do not overlap, so it is an important differentiator character: it is therefore that both taxa present morphological differences in the arrangement of the glands.
As for the boxplot analysis of the wing width measurements of the cypsela (Fig. 4), it is observed as this character is also different in both taxa, by not overlapping measures significantly and having a bounded variance.
However, the ratio cypsela-wing width (Fig. 5), the measures of width (Fig. 6) and length (Fig. 7) of the disc cypselas, are not adequate parameters to differentiate both taxa, since the overlap of the measurements is evident. Although the cypsela length is statistically different between both taxa, as can be seen in Table 1.
An average confidence interval of 95% was used in the statistical treatment. A parametric distribution analysis was applied and gave a P-value with a significance of less than 0.05 in the Student's T test and the Z test. The margin of error is < 1.62 % in the case of the length of the disc cypsela, and < 0.01% for the arrangement of glands (linearity) and the ratio cypsela-wing width of the disc cypsela (Table 1).
In the analysis of the width of the disc cypsela for the two species, the P value is > 0.05, The character of width and length of disc cypsela therefore does not have much strength in differentiating the species (Figs 6, 7).      The thermo-Mediterranean belt is differentiated into the lower (with 400 <Itc <450) and upper thermo-Mediterranean belt (350 <Itc <400). We have collected both G. coronaria entities in pure and/or mixed populations in these belts.
Specifically, G. coronaria var. coronaria was sampled in 84% of the 32 stations in the thermo-Mediterranean belt, while G. coronaria var. discolor was sampled in 34% ( Table 2).
The two entities are more or less equally distributed in 50 stations in the upper thermo-Mediterranean belt: G. coronaria var. coronaria was sampled in 50% and G. coronaria var. discolor was sampled in 56% (Table 2).
Only G. coronaria var. discolor was sampled in the single station in the lower mesotemperate belt (Table 2).
On this basis, the application of the X 2 test (=0,00247) highlighted the high significance of the preferential distribution of G. coronaria var. coronaria samples in the warmer belts (infra-and lower thermomediterranean), while G. coronaria var. discolor was observed to have a significantly greater presence in cooler belts (meso-and upper thermomediterranean) than G. coronaria var. coronaria.

Discussion
D'Urville (1822) describes two varieties of Chrysanthemum coronarium -discolor and concolor-taking into consideration only the external female ray floret colour.
Specimens with totally yellow ray florets are now treated as Glebionis coronaria (L.) Cass. ex Spach. Also, Turland (2004) considers these two entities as distinct taxa treated at the rank of variety, and proposes a new combination in Glebionis coronaria for var. discolor (Glebionis coronaria var. discolor (d'Urv.) Turland, comb. nov. -Basionym: Chrysanthemum coronarium var. discolor d'Urv. in Mém. Soc. Linn. Paris 1: 368. 1822). This author also maintains that the two varieties may appear in independent or mixed populations, with no difference in distribution. We cannot agree with this author, as our sampling carried out in Sicily, southern Italy, Spain and Portugal, and our observations of specimens from Great Britain (Gibraltar), France, Croatia, Greece, Turkey, Cyprus, Malta, Israel, Egypt, Morocco and Libya reveal that the G. coronaria var. coronaria is distributed exclusively throughout the whole of the thermo-Mediterranean belt with thermo-climatic values of It/Itc = 350-450; while G. coronaria var. discolor is found throughout the thermo-and meso-Mediterranean belt with values of It/Itc =220-350 (Tab. 2), but it is more represented in percentage terms in stations in the meso-Mediterranean belt. An entity at the specific level of Chrysanthemum with bicolour ray florets was previously described by Pau (1899) as C. merinoanum. In our opinion, this species is different from Chrysanthemum coronarium var. discolor d'Urville. According to the analysis of the herbarium sample (MA 128240) and from the description given by Pau (1899): "Intermedio entre el coronarium y segetum…. lígulas blanquecinas...; aquenios calvos, los externos trigonos con una sola ala…", C. merinoanum Pau is a probable hybrid of C. coronarium var. discolor and C. segetum. In fact, C. coronarium var. discolor lacks the characters of C. segetum and has external cypselas with two wings and two dorsal ribs. C. merinoanum, however, has only one wing on the cypsela and leaves that are clearly like those of C. segetum.
Moreover, our studies on the morphology of disc cypselas using high-resolution confocal microscopy, morphometric analysis and statistical techniques have revealed sufficient differences to justify raising the variety to a higher rank. Since two subspecies cannot coexist in the same geographic area and even less in the same habitat (criterion of allopatry), we consider them to be two distinct species.
For all these reasons, we propose a lectotypification and a change in rank for Chrysanthemum coronarium var. discolor d'Urville. The two species are listed below, with their differential characteristics highlighted.

Conclusions
The two entities traditionally included in Glebionis coronaria (L.) Cass. ex Spach based on external female ray floret colour have differences in their morphological and ecological features that enable them to be attributed to two different species.
In the study of the material collected in the Mediterranean area, we can confirm that the two varieties given by d'Urville (1822) present major differences in their micro-and macro-morphological characters and their distribution. Moreover, the aforementioned characters of the cypselas are very important for the determination of herbarium specimens, as the colours of the ray florets do not persist when the plants are dried.
Since Glebionis coronaria is conserved in the form of plants with yellow ray florets, corresponding to Chrysanthemum coronarium var. concolor d'Urv. and necessarily to G. coronaria var. coronaria, we establish a change of rank for the var. discolor d'Urv. Both entities present clear differences in the colour of their ray florets, the shape and size of their disc cypselas and in the disposition of their glands. For this reason, based strictly on the ICN (McNeill et al. 2012), we maintain the species Glebionis coronaria and propose G. discolor comb. & stat. nov.

Taxonomic treatment
Identification key