Cytotaxonomy of North African species of Delphinium L . sect . Delphi nium ( Ranunculaceae )

BLANCHE, C , J . MOLERO & J. SIMON (1990). Cytotaxonomy of North African species of Delphinium L. sect. Delphinium (Ranunculaceae). Collect. Bot. (Barcelona) 18: 59-74. Both chromosme numbers and karyotype structure from 15 wild North-African popu­ lations of Delphinium L. sect. Delphinium were studied. Delphinium balansae Boiss. & Reuter, with a more symmetrical karyotype, appears as a possible perennial ancestor of the remaining annuals with highly assymetrical and shorter karyotype. A taxonomical reorgani­ zation of this section is proposed: two new series (ser. Macropeiala ser. nova and ser. Balansaeser. nova.) and two new nomenclatural combinations (D. nanum subsp. alboliliaceum and D. nanum subsp. elongatum) are proposed.


Introduction
The annual species of the genus Delphinium are grouped together in the section Delphi nium (= Sect.Delphinellum DC).Recent works of a general nature dealing with Delphinium C. BLANCHE, J. MOLERO & J. SIMON: Laboratori de Botánica.Departament de Productes Naturals, Biología Vegetal Sanitaria i Edafología.Faculta! de Farmacia.Universität de Barcelona.Avgda.Diagonal, s/n.08028 Barcelona.(EWAN, 1945;WANG, 1962;TAMURA, 1967;MALYUTIN, 1973 and1987) have devoted very little attention to the sect.Delphinium, probably on account of its small size in relation to the total number of species in the genus (approximately 5 %, cf.MALYUTIN, 1987) and because of its geographical distribution which is practically limited to the Mediterranean Region s.l.(Mediterranean Region, West of the Irano-Turanian Region and parts of the Saharo-Arabian region, sensu TAKHTAJAN, 1986).
On a different level, PAWLOWSKY (1963;1964), CHOWDHURI, DAVIS & HOSSAIN (1958) and DAVIS (1965) have contributed what in our view are fairly valid criteria for the taxonomic conception of the section Delphinium in their research on Europe and Turkey respectively.Of particular note is the treatment given relatively «small» species grouped together in series (cf.PAWLOWSKY, 1963 and1970, EWAN, I.e.), this last category being particularly useful for the Delphinium genus which, apart from a few species which are very isolated both taxonomically and geographically, presents a model of variation containing geographically and phylogenetically coherent and well defined natural groups, including a variable number of lesser taxa with a complex network of obvious degrees of relationship.For STEBBINS (1950:56): «In Delphinium (...) the pattern is that of closely related homogenous species more or less sharply isolated from each other, as well as of polytypic species, the two tipes occurring with about equal frequence)).
This new attempt to resolve this complex group follows upon our earlier works on taxa of the tribe DelphineaeWarming in the Iberian Peninsula (BLANCHE, 1985, BLANCHE & MOLERO 1984, 1985and 1986;BLANCHE, MOLERO & SIMON, 1987, etc.).In it we present cytogenetic data on the Nort African species of the section Delphinium about which very little is known (only one count by QUEZEL, 1957).The taxonomic solution by MAIRE (1964) differes substan tially from those proposed by Davis and Pawlowsky, followed by MUNZ (1967), and must still be considered provisional.Furthermore the study of the section Delphinium in this region (to which D. balansaeBo'iss.& Reuter, the only perennial species in the section, is endemic) may possible provide a clue to the evolution of the genus which cannot be sought elsewhere.

Material and methods
Fixations were performed directly in the field in the locations listed in Table I in the course of two expeditions through Morocco in 1985(cf.FERNANDEZ CASAS & al., 1987) and 1988 (J.Molero, A.M. Romo & A Susanna).Reference sheets are kept in herbariums BCF and BC.
Because of the difficulty in obtaining seeds (most of the populations were still in flower) and in getting them to germinate, the use of root meristems was not considered.The initial material in all cases consisted of flower buds fixed with Carnoy's reagent (absolute ethanolglacial acetic acid 3:1).After 24 hours, the fixative was renewed and kept in the laboratory at -18° C.
Samples were coloured with acetic carmine (boiled for 2 minutes) and then allowed to rest for 24-48 hours at room temperature.They were mounted using acetic acid 45%-glycerol (9:1) and subsequently prepared by means of the «squash» technique.All the specimens come from somatic metaphases of the staminal filaments.They were examined under a Nikon optic microscope where the camera lucida figures are drawn.The metaphasic plates with the most contracted chromosomes were selected for the study of size and morphology of chromoso mes, position of the centromere and presence and position of the satellites (10 data for each population).
For the observations regarding meiosis, pollen mother cells (P.M.C.) were used and the same procedure was followed.
The selected divisons were photographed with a Nikon Optiphot microscope.The negati-  1985), n = 8, 2n = 16 ves of the photographs and the orignal drawings are deposited at the Botany Laboratory of the Faculty of Pharmacy in Barcelona.
The terminology used to describe the karyotype is that of LEV AN & al. (1964) and from it the chromose formulae have been calculated.The degree of asymmetry of the karyotype is indicated in terms of the classes defined by STEBBINS (1971).In order to facilitate comparisons between different taxa and with data gathered by other authors, the centromeric index has also been calculated (100 x length of short arm / total length of chromosome) as well as the indexes of intrachrosomal asymmetry (Al) and of interchromosmal asymmetry (A2) propo sed by ROMERO (1986).

Results
A total of fifteen populations were studied and the corresponding chromosome numbers are shown in Table I.Apart from one previous count of D. balansae (QUEZEL, 1957), this is the first time, to our knowledge, that the remaining taxa have been counted All the populations studied present a diploid level with the same chromosome number (n = 8 and 2n = 16).Only a few meiosis (diakinesis or metaphase I) in D. balansae (Fig. 1) show short chromosomes in which the terminalization of the chiasmata had already finished and separation was beginning, while the rest of the complement was still at the equatorial plate.Some seemingly counts of n = 9 may appear, as already reported by N. Galland (com. pers.) with reference to the same taxon.
The karyotypes of the fifteen populations were studied.Their characteristics are given in Tables II (size) and III (asymmetry) while their respective idiograms appear in Figs. 2 and 3.The majority are fairly asymmetrical karyotypes belonging to STEBBINS' classes 3B and 4B (1971).The average length of the complement is relatively constant, being close to 3 um, between 3,87 +0.44 um for D. macropetalum and 3.02 ±0.37 pm for D. nanum subsp.elongatum (m ± SE, n = 10).However, the average length of the chromosomes ranges from 6,64 ± 0,16 um for pair I of D. macropetalum to 1,74 ± 0,06 pm for pair VIII of D. balansae (M±SE,n = 10).

Discussion
1.-Our results as to the chromosome number confirm all previous counts of taxa belonging to sect.Delphinium (2n = 16).On the basis of present data, the section can thus be considered strictly diploid, in accordance with the basic number x = 8 proposed by TAMURA (1967) and AL-KELIDAR & RICHARDS (1981).This feature distinguishes the group of annual Delphinium from the sections with perennial taxa in which the levels 2 x and 4 x exist side by side (cf.JANKUN, 1973;WARNOCK, 1981or BLANCHE, 1985, among others).With respect to the karyotpe, very clear similarities are also observed (except in the case of D. balansae) among North African taxa belonging to the section, and also with Iberian and Balearic representatives (BLANCHE, 1985).
These resemblances could give support to MAIRE'S synthetic treatment (1964) in which all the annual species (except for D. balansae and D. cossonianum which are quite distinct) are grouped together in a single «large» species, D. peregrinum s.L, with a great number of dependent lesser taxa.On the other hand, these cytogenetic parallelism, added to the morpho logical similarities between the taxa studied and between Delphinium annuals in general, confirm that this group presents the same model of variation deduced by STEBBINS (1950) from his experiences with perennials, ie. a sort of phenotypic and genotypic «continuum».
2.-Another aspect of the problem which must be considered is whether the similarities,  in the process of crop expansion, seed mixing and the colonization of new arable land, provides far more frequent contact between populations with a close geographical relations hip.The possible entry of new genotypes into a particular population may explain the existence of intermediate forms, the fading of differences and finally a «mosaic» type model for the distribution of phenotypes.
4.-Despite the strong resemblance of the karyotypes studied, more or less noteworthy differences can be observed with a variety of meanings.When associated to variations in morphology and/or ecological preference, these make it possible to establish correlations of systematic value.
5.-D.balansae must be singled out first and foremost on account of the special characte ristic of its karyotype.The average length of the complement is among the greatest in the taxa under study (cf.Table II) and the presence of submetacentric chromosomes in pairs II and V, together with the relatively long short arms of the rest of the complement, set it clearly apart.These characteristics can be interpreted in the light of data relating to sect.Delphinastrum DC. which includes the perennial taxa of the Western Mediterranean (Table IV).In fact, D. balansae (the only perennial species of sect.Delphinium) occupies an intermediate position between annuals and perennials: the chromosomes are similar in size to those of the annuals, but the karyotype structure, is practically identical, in terms of asymmetry (centromeric index), to that of the perennials.This evidence is of great interest since, up to now, no possible link had been discovered between annual and perennial Delphinium species and this interme diate position makes D. balansae into a possible relictual ancestor of the annuals.
Analysis of Table IV reveals on the one hand an increase in asymmetry in the annual species (sect.Delphinium), illustrated by a decreasing centromeric index (average position of the centromeres closer to the end of the chromosomes, or, in other words, chromosome arms of more unequal size), and on the other a tendency for the size of annual karyotypes to decrease with respect to perennials.This reduction in length must be associated with a loss of genetic material, a hypothesis which is confimed by AL-KELIDAR & RICHARDS (1981) who evaluate the loss nuclear contents of DNA in annual Delphinium species at about 30 % less than in perennials, a figure approximately the same as the decrease in length reported here.
6.-In the various populations of D. cossonianum studied, there is little variation in the karyotype (cf.Tables II and HI and Fig. 3).The main characteristics are one submetacentric pair (pair I) and another between submetacentric and acrocentric (pair V and, accidentally, pair IV on account of the very small differences in their respective size in population CB. 046), together with the constant presence of satellites on the long arm of the chromosmes of pair IV (pair V in CB. 046 for the reason stated above).The centromeric index of pair IV (between 25.68 and 34.59, cf.Table III) is much closer to that of D. balansae (pair V, IC = 37.67) than to the remaining taxa (pair V, IC betwen 8.27 and 13.41).Taxonomically D. cossonianum is an extremely isolated species (it is the only annual with five carpels, the rest having three, and the seeds and structure of the inflorescence are clearly differentiated) and it is endemic to the N and NW of Morocco.
7.-In contrast, analysis of the results of D. macropetalum and D. nanum (subsp.elongatum and subsp.albolilaceum), two taxa with great phenotypic affinities which are included by MAIRE (1964) in various combinations in his concept of D. peregrinum s.l., reveals other characteristic traits.Thus while the karyotypes of D. balansae and D. cossonianum display great Constance and stability, these species display minor variations in karyotype structure which are apparently not correlated to other factors.Of significance for instance is the variability observed in the number and position of the satellites.In certain populations of all three taxa (CB.010, CB. 033, CB.045 and CB.009), no satellites were detected in any of the metaphases studied (Figs. 2 and 3).Other populations, however, have a pair of satellite-bearing chromosomes (pair II in CB.012, CB.013 and CB.018; pair VII in CB.005) or even two pairs (populations CB.016, pairs III and V; CB.003, pairs V and VII).Though satellites tend to some  II and III).extent to be present in particular chromosomes (II, III, V, and VII) and absent from the others, there is no obvious correlation with ecology or geographical distribution.Populations only 20-50 km apart and with practically identical phenotypes reveal differences in the number and position of the satllites.In a previous work (BLANCHE, MOLERO & SIMON, 1987) we suggested on the basis of data referring to the genus Consolida that this variability of satelli tes might have some geographical significance.That hypothesis must now be reconsidered in the light of the present data, as already pointed out to us by Prof. C. Favarger (com. pers.).This variability in the position of the satellites must therefore be interpreted as characteristic at population level only and indicative of a certain karyotype instability which may well be linked to adaptation processes.8.-Turning now to another topic, no significant differences are observed between the average lengths of the chromosomes of D. macropetalum and D. nanum (both subspecies).Their respective chromosome formulae always consist of Ism and only the proportion of st/t varies (and even with the values very close on either side to the numeric limit between them established by LEV AN & al., 1964).The asymmetry corresponds to 3B and 4B, using the classes defined by STEBBINS (1971).In an endeavour to obtain greater resolution, we have calculated indexes Al and A2 as established by ROMERO (1986) (see Fig. 4), together with data on perennial species (BLANCHE, 1985).D. cossonianum here appears to be highly individuali zed while D. balansae occupies a position of transition between annual and perennial taxa, as observed above, though it also falls into STEBBINS'S class 3B (I.e.).In contrast, the populations of D. macropetalum and D. nanum form interconnecting clusters of points, thus confirming the proximity of these two taxa.9.-In the light of these results and of the analysis of phenotypical and ecological characteristics, what might be the most valid taxonomic outline for the section?MAIRE (1964), probably the only author who has dealt with North African Delphinium and had direct knowledge of the annual species, proposed an integrating conception suggested by field experience which brought to light very subtle differences and numerous intermediate forms.
The outline we propone below endorses this view while taking into account both the criteria established by DAVIS (1965) and PAWLOWSKY (1963) and the geographic distribution of the section throughout the Mediterranean region.1985).

Conclusions
1.-Cytogenetic analysis reveals a growing tendency towards karyotype asymmetry in annual species of Delphinium.In the light of the suggestions made by STEBBINS (1971:92) this phenomenon must be considered a «secondary» evolutionary characteristic, since it is superimposed over the already high «primary»asymmetry of the perennial Delphinium species due to the specialization of the floral structure.STEBBINS (I.e.) explains the mechanism governing this increase in karyotype asymmetry as the result of pericentric inversions and unequal translocations of fragments of chromoso me arms which occur without any change in the number of centromeres or of independent chromosomes.This process is not detected in long-living perennial species occupying relati vely stable habitats.On the other hand, it is usually associated with the colonization of new, open habitats and the transition to annual cycles ocurring at the same time as continued allogamy which is often rendered necessary by floral structure.This model indeed matches our observations perfectly and is an alternative to the fairly well known strategies of auto gamy, polyploidy or apomixis.
2.-In addition to this overall mechanism there is the role played by geographical, ecological and/or phenological isolation which have facilitated (in the case of D. balansae and D. cossonianum) or hindered (in the case of D. macropetalum or D. nanum) the processes of differentiation and indeed of speciation in populations with few genetic barriers to separate them (LEGRO, 1961, indicates the existence of over 4000 cultivars of Delphinium for horticul tural use which were obtained from 211 hybridations).
3.-Finally, the biogeographical analysis of the whole of section Delphinium reveals the existence of two major focusses of speciation in the Mediterranean Region: a) a first centre of diversity in the Eastern Mediterranean (Turkey-Greece) which pre sents a high degree of endemism, characteristic of explosions of annuals caused by the arrival of Irano-Turanian elements in the Mediterranean Region (cf.DAVIS, 1971).b) a secondary focus of diversity in the Western Mediterranean, with its centre in the Atlas mountains, where D. balansae has been conserved as a relictual element, the only surviving perennial ancestor of this section.There are also a large number of endemisms.
This model is analogous to that reported by EHRENDORFER (1988) for the Achillea-group (Anthemideae, Compositae), which includes both centres of diversity, but in which the ancestral groups are also conserved in the region of the North West Africa (the monotypic perennial genus Heliocauta and the greater diversity of the circum-Mediterranean genus Anacylus).

Fig. 1 .
Fig. 1. -Asynchronous meiosis of D. balansae (CB.220, n = 8) in CMP.In metaphase I the presentee of a ring-shaped bivalent corresponding to the long pair (I) is observed.In other bivalents with shorter chromosomes (arrows), the terminalizacion of chiasmas has taken place more rapidly and separation strarts earlier.

TABLE I .
-Chromosome numbers of Delphinium L. sect.Delphinium.

TABLE II .
-Karyotype size of Delphinium L. sect.Delphinium.North African Populations.