Karyological Study of Species of the Genus Paeonia (Paeoniaceae) from the Caucasus
Botanicheskij Journal 1987 72(11) 1504-1514
Translated from the Russian by Dr.Carsten Burkhardt, Cottbus, Germany
10 caucasian peony-species were analysed, of which 6 were diploid (2n=10): P.caucasica, P.mlokosewitschi, P.ruprechtiana , P.tenuifolia , P.lagodechiana , P.chamaeleon , and 4 were tetraploid (2n=20) : (P.steveniana, P.wittmanniana , P.tomentosa , P.macrophylla ). At the diploid species differences in the karyotypes were shown according to the absolute length and thickness of the chromosomes, but the centromer-index and the relative length of the chromosomes was constant. At the diploid species of hybrid origin P.chamaeleon and P.lagodechiana differences in the karyotypes were found, which can be seen as amphiplasty. Shown is the allopolyploid origin of the tetraploid species P.steveniana , P.tomentosa and P.macrophylla .
The genus Paeonia L. held the attention of investigation since long times. Their peculiarity of embryology, structure of the pollen and the number of chromosomes made it possible to include them in recent times not only to the monotypic family Paeoniaceae Rudolphi, but also to the monotypic section Paeoniales Nakai. But, although the embryological and anatomic markers of the genus are examined well, more detailed information about the karyology of the representatives of the genus Paeonia, especially the caucasians, is limited and restricted on the knowledge of the chromosome numbers. This is understandable, first, because the majority of investigators ad no sufficient material of the caucasian species, second, because they thought, that the karyotypes of the genus are uniform. This may be the reason for the decline of interest on the peonies as an object of karyosystematics, although significant and well distinguishable criteria within the karyotypes of the chromosomes exist.
The chromosome-number of the caucasian species of the genus Paeonia is 2n=10 at P.mlokosewitschi Lomak. and P.tenuifolia L., but also 2n=20 at P.tomentosa (Lomak.)N.Busch and P.wittmanniana Hartw.ex Lindl., as Langlet (1927), G.Hicks & G.Stebbins (1934), Stebbins (1938), H.Barber (1941), F.C.Stern (1944) noticed.
In the work of Stebbins (1938) about the relative length of chromosomes of P.tenuifolia , P.mlokosewitschi is referred. L.P.Zhgenti (1967) noticed 2n=10 and patterns of metaphasic discs at P.tenuifolia, P.mlokosewitschi and P.caucasica Schip. and 2n=20 at P.wittmanniana and P.steveniana Kem.-Nath..
Until recent times there is much confusion about the number of species in the Caucasus. Several authors name in the Caucasus:
5 species and 1 subspecies
4 species and 2 subspecies
5 species and 2 subspecies
Karyological examinations on peonies of the Caucasus may be helpful to answer the questions on the number of the species an their degree of relationships. Thats why we made this study on the caucasian representatives of the genus Paeonia. In the following study the author uses the classification of Kemularia-Nathadse (1961), because it is the most detailed.
To our regret it was not always possible to obtain material from wild habitats of the plants, so a part of this study has been done using material from botanical gardens. The author thanks the fellow-worker of the Botanical Institute of Tbilissi, AN GSSR B.D.Gavrilenko, the BG Tbilissi AN GSSR L.V.Asijeshvili and the BG Moscow, R.A.Karpisonovoj, for supplement of material.
Material and Methods
For the cytological preparates tops of young roots of mature plants were used. The plants were cultivated in containers in the ground at the Experimental Section of the institute. Fixations were made in May and October, all probes were fixed 1x. For processing was used 0,05% Colchicin-solution for 2h, and 0,02 M Oxychinolin for 1h, fixation in Acetyl-Alcohol (3:1). After cold hydrolysis (50% HCl, 20 min) and staining with Fjolgren sections were made. The best metaphasic discs were drawed with a magnification of 2000x using the drawing-apparate RA-6. Measuring of the chromosomes was made by hand of 8-12 drawings, equally. To obtain ideograms the classification of the chromosomes according to metacentrical, submetacentrical a.m. patterns the system of N.D.Agapov and V.G.Grifom (1981) was used. The examined species, their origination and chromosome numbers are shown on Table 1.
As visible in Table 1., under the examined caucasian species were diploid and tetraploid species, as well.
The karyotypes of P.caucasica , P.mlokosewitschi , P.ruprechtiana and P.tenuifolia consist of 3 pairs of metacentric, 1 pair of submetacentric 1 pair of subacrocentric chromosomes (Pict.1).
At P.caucasica , P.mlokosewitschi and P.ruprechtiana is a satellite on the short arm of pair I, II, IV and V.
At P.tenuifolia satellites are only on the IV. and V.pair. Table 2 demonstrates the absolute length (L a) and the centromer-index (I c) of the diploid species.
The chromosome pairs I and II are in all 4 diploid species significantly distinguishable in the absolute length, at P.caucasica and P.ruprechtiana in the centromer-index too.
The chromosome pairs II and III are distinguishable in these parameters at all species, except at P.tenuifolia, where the differences are only in the absolute length significant. Between the pairs II, IV and V the differences are in all these parameters at all species significant. (For the examination of the significance we used here and later the t-criteria after Student).
Inter-species comparisons were made by help of the absolute length and thickness of the chromosomes (Pict.1). The longest and thickest chromosomes were found at P.mlokosewitschi and P. ruprechtiana, from these clearly distinguishable were the chromosomes of P.caucasica (they are shorter and more slender), the shortest and most slender were found at P.tenuifolia .
Comparing the homologue pairs only by their relative length and the centromer-index, practically no differences are visible. A comparison of the karyotypes of P.caucasica from Saguramo and Daba, and of P.mlokosewitschi from Lagodekhi and Kedi demonstrates the absence of significant differences between the different populations (pict.2).
The diploid species P.lagodechiana u. P.chamaeleon are hybrids between P.caucasica and P.mlokosewitschi. P.lagodechiana is a natural hybrid originating from Lagodekhi-valley, P.chamaeleon is a hybrid, which accrued spontaneously in the BG of Tbilissi, what is also confirmed experimentally (Kakheladse, 1965; Kemularia-Nathadse,1980).Through these species we've got descendants with intermediate patterns. At present time it's still controversy, which of these hybrid taxons deserve the rank of a species, but for convenience we use these names in the following to demarcate these natural and artificial hybrids. By analysing the chromosomes of these species we found a heteromorphism between the homologue pairs. That's why in Table 3 and on the ideograms the full chromosome garniture of these diploid species is shown (pict.3).
A heteromorphism is notable in the length of the 1./2., 7./8. and 9./10. chromosomes in both species, at the same time at P.chamaeleon a difference between the non-homologue chromosomes 2/3 and 4/5 is absent.
Beside this, has at P.chamaeleon of the II. chromosome pair only the 4th chromosome a satellite, while at P.lagodechiana only from the 1st chromosome (I.pair) a satellite is absent, but the 7th chromosome (IV.pair) has a very large satellite (pict.3,a).
The karyotypes of the tetraploid species P.wittmanniana , P.steveniana , P.macrophylla u. P.tomentosa consist of 6 pairs of metacentric, 2 pairs of submetacentric and 2 pairs of subacrocentric chromosomes (pict.4, Table 4).
At P.steveniana are differences in the absolute length between the I./II., V./VI., VII./VIII. and IX./X. chromosome pair. Pair IV and V differ significantly in their centromer-index, between the pairs II and III, but also III and IV significant differences are not visible. All chromosome pairs except V and VI have satellites on the short arm.
At P.wittmanniana all chromosome pairs differ in the absolute length, except pairs V/VI and IX/X, which are not distinguishable in the centromer-index too. Satellites are on all chromosomes except pairs V and VI.
At P.tomentosa all pairs differ significantly in the absolute length, according to the centromer-index also the chromosome-pairs IV/V, V/VI, VII/VIII and IX/X. Satellites on the short arms are on pairs I, VII, IX and X.
At P.macrophylla from Adsharia all pairs differ in the absolute length significantly, pairs II/III, IV/V, V/VI, VII/VIII also in the centromer-index. Satellites were found on the short arms of pair I, IV, VII, IX and X .
At P.macrophylla from Svanetia all pairs differ in the absolute length with exception of pair IV and V, but the centromer-index of both pairs is significantly different. In the centromer-index also differences are between the pairs III/IV, V/VI, VII/VIII. The II.chromosome-pair has a satellite on the long arm, while the pairs VII-X have such on the short arm.
In contrast to our examinations at the diploids the differences at the tetraploid caucasian species are of another nature. At the diploid species the summarised length of the haploid garniture varied considerably (55.5, 59.5, 62.1, 70.2, 70.3, 71.1 mm), at the tetraploids much less (111.9, 114.9, 127.1, 127.9, 128.0 mm).
The differences in the thickness were also lower (1.3 - 1.8 mm at the diploids, 1.3 - 1.6 mm at the tetraploids). Number and arrangement of satellites is at the tetraploids of greater diversity. Satellites were found on the following pairs:
P.wittmanniana on 8 pairs (I.-IV.,VII.-X.)
P.steveniana on 8 pairs (I.-IV.,VII.-X.)
P.tomentosa on 4 pairs (I.,VII.,IX.,X.),
P.macrophylla (Adsharia) on 5 (III.,IV.,VII.,IX.,X.pair), and
P.macrophylla (Svanetia) also on 5, but on other chromosomes,(namely II.,VII.-X.), and one single satellite is not, like in the other species on the short arm, on the II.pair it is on the long one.
In the relative length differs the I.chromosome pair of P.tomentosa and P.macrophylla from the I.pair of P.wittmanniana and P.steveniana , the VI.pair of P.tomentosa from its homologues at the other species. The VIII.pair of P.steveniana differs from its homologues at P.wittmanniana and P.tomentosa . The centromer-index of the IV.pair of P.macrophylla from Svanetia is different from all homologues in the other species. The same way differ the V.-IX.chromosome pairs too.
Between the karyotypes of P.macrophylla from several populations are differences notable in the location of satellites and the centromer-index of several chromosome pairs.
All authors, who examined the karyotypes of species of the genus Paeonia before, (Stebbins 1938, Sopova,1971, Tzanoudakis, 1983), were only interested in their comparisons to the relative length (L rel), what the reason for their fallacy is, to see them all being similar. Comparing the karyotypes of the diploid species P.mlokosewitschi P.ruprechtiana , P.caucasica and P.tenuifolia in accordance to the relative length, they are in fact uniform. But the individual chromosomes of the species differ substantially in their absolute length and thickness. In connection with this we tried to count the summarised volume of the chromosomes.
The measured thickness of a chromosome (d) is equal with the double thickness of the chromatide. The Chromatid has a cylindrical form. Its volume can be calculated with the formula (d/2)²p x La hapl/4.
In the same sense the volume of a chromosome is (d/2)² p x La/2. The summarised volume of the diploid garniture is given with d² p x La hapl/4 (La hapl means the absolute length of the haploid garniture).
For the diploid species, the results are P.mlokosewitschi 178.5, P.ruprechtiana 161.3, P.caucasica 110.2, P.tenuifolia 73.6 cubic mm³, respectively.
This means, that the volume of the karyotypes of P.mlokosewitschi is the double of that of P.tenuifolia . It is less probable, that these differences are caused only by a different level of spiralisation of the chromosomes, what is seen as one pattern of the evolution of species, accompanied by simultaneous transformation and fixation. Most probably a difference in the DNA-content of these species is reasonable for this, so in a following investigation the DNA-content will be compared with the absolute sizes of the chromosomes.
The diploid species P.chamaeleon and P.lagodechiana are hybrids between P.caucasica and P.mlokosewitschi, what could be verified experimentally (Kakheladse, 1965; Kemularia-Nathadse, 1980). We could not identify the chromosomes of the parent species in the karyotypes of these plants. 3 chromosomes were indistinguishable.
We were confronted with an analogue problem yet before, when we studied natural hybrids of the genus Pulsatilla (Ranunculaceae), namely hybrids between Pulsatilla patens x P.vernalis, where it was impossible to discriminate 3 chromosome pairs of 8, whereas we could distinguish all 8 chromosome pairs of the parent species (Punina, Grif, 1984).
Surprising were the results of the measured sizes of the thickness of the chromosomes of Paeonia chamaeleon and P.lagodechiana .As noticed before, the chromosomes of P.mlokosewitschi were 1.8, those of P.caucasica 1.5 mm thick. Logical would be, that the current sizes in the hybrid species would be between them or close to the size of one parent. But they were in P.chamaeleon 1.4 and in P.lagodechiana 1.3 mm respectively (statistical divergence only 0.03 mm).
M.S.Navashin (1985) examined chromosome-alterations at artificial hybrids of the genus Crepis. He was able to show the disappearance of satellites the prolongation and abridgement of all chromosomes of one species in the cytoplasm of the hybrid, but also the disappearance of differences in the thickness of the chromosomes at the hybrids. For these phenomena he created the terminus "amphiplastia", demarcating a differentiate (only a single chromosome is alterated, for instance by disappearance of a satellite) and a neutral (alteration of the length and thickness of all chromosomes of one species) amphiplastia.
It seems obvious to us, in the case of hybrids between species of the genus Paeonia also to see an amphiplastia, which is shown by alterations in the chromosome length of the parent species or a levelling of differences between the chromosome pairs.
For the understanding of the evolution of the genus Paeonia the tetraploid representatives are of largest interest. (Other ploidy is not existent). The origination of the tetraploids of this genus has been an object of discussion. Several authors mean, that the tetraploids are of auto-polyploid origin (Barber, 1941; Stern, 1944).
Their argument for this thesis is the quantity of the diploid and tetraploid species in the european and asian extension of the genus. Consequently practically every of the 8 diploid species has 2-3 morphologically closely related tetraploid species or variant. Singly P.tenuifolia has up to date no tetraploid analogues. Another picture is found in the Far East. Only 1 diploid species, P.japonica, is morphologically related with a tetraploid species, P.obovata. The other 8 diploid species have no tetraploid analogue. The authors interpret this with the extreme circumstances during the ice age in Europe. After the retreat of the glaciers the tetraploids had a better ability for migration to north and south to settle in wider areas. This hypothesis could interpret, why the area of the tetraploids is usually much larger than the area of the adequate diploids. For instance, P.clusii occurs only on Crete, whilst her related P.officinalis in Albania Triest, Northern Italy, Northern France, Central Spain.
P.mlokosewitschi too, occurs also only on a single spot in the Caucasus, but P.wittmanniana and other tetraploids are found in the entire Caucasus. We don't want to go deeper into the western european peonies, but we have to summarise, that the knowledge of Barber (1941) and Stern (1944) according to the caucasian peonies with yellow or white flowers was altogether not very precise.
First, there are at least 3 sufficiently separate distant areas of P.mlokosewitschi (Lagodekhi reserve, near Kedi in Georgia and upper course of the river Avarskij Kois in Dagestan). More areas are named in Kemularia-Nathadse (1961). Second, it seems misleading to us to enclose all tetraploid species with yellow or white flowers into one species, P.wittmanniana , separating only some subspecies.
In our examination of the tetraploid caucasian species of the genus Paeonia many differences in the karyotypes of these species were observed, and in combination with the particularities of the morphology and the geographic distribution we feel right to state, that P.wittmanniana , P.steveniana , P.macrophylla and P.tomentosa are independent species, as it was recommended in the classification of Kemularia-Nathadse 1961).
In the karyotypes of P.macrophylla , P.steveniana and P.tomentosa are differences in the absolute length (and in many cases in the centromer-index and the location of satellites, too) between the homologues within the limits of the karyotypes (I./II., V./VI., VII./VIII., IX./X.chromosome pair). That's why it's possible to state, that all these karyotypes have resulted by the duplication of an ancestral karyotypes and the visible differences are the result of the forther evolutionary process.
It is probable, that species are of allo-polyploid origin, if the differences in the absolute length of their chromosomal homologues are explicable with differing lengths in the garniture of the parent-species.
Probably one of the parent-species may be seen in P.mlokosewitschi or in one of its direct ancestors, and the other parent may be possibly P.caucasica .If we try to construct a model of such a hypothetical amphi-diploid (pict.5) the resulting karyotype is very similar to that of P.steveniana .From this primary karyotype the species P.macrophylla and P.tomentosa may have developed by forther chromosome-remodellations.
The karyotype of P.wittmanniana can be seen on two ways. On the one hand, the difference between the homologue pairs is not as gross as at the other tetraploids, which allows us to think that this species is of auto-polyploid origin, and the small differences between the I./II. and VII./VIII. pair may be a result of subsequent chromosome-remodellations. On the other hand, it's possible, that during the hybridisation of 2 species with similar chromosome length a reduplication followed subsequently, that means amphi-diploidy.
Our opinion is, that forther examinations using a differential staining of the chromosomes and an analysis of the meiosis may be helpful to decide finally about the nature of this species.
Analysis of other species of the genus Paeonia (Sopova, 1971; Tzanoudakis, 1983) showed the allo- and auto-tetraploid origin of the tetraploid species native to Macedonia and Greece. In this sense we confirm with the theory of Stebbins (1948), that the tetraploid peonies of the Mediterranean region (we include the caucasian species in this sense) can be seen as a polyploid complex, in which auto-polyploidy possibly exists, but the majority of the species is of an allo-polyploid origin resulted from crosses of present diploids or their ancestors, or from auto-polyploid forms.