Karyological study of the Caucasian Representatives of the genus PAEONIA (PAEONIACEAE) under application of the Giemsa - differential - chromsome staining
Botanitcheskij Journal 1989 74(3) 332-339
Translation from the Russian of Dr.Carsten Burkhardt, Cottbus
A differential staining and analysis of the distribution of the S - segments of metaphase chromosomes at 5 diploid (P.caucasica, P.mlokosewitschi, P.ruprechtiana, P.tenuifolia, and P.lagodechiana) and 4 tetraploid (P.wittmanniana, P.steveniana, P.macrophylla, P.tomentosa) species from the Caucasus was accomplished . A series of differences between the species was discovered. The autopolyploide origin of P.wittmanniana and the allopolyploide origin the other tetraploids species is demonstrated . There are described conjectures over the extent the phylogenetic kinship of these species. Karyological investigations of the Caucasian representatives of the genus Paeonia (Punina, 1987) have shown, that all explored species with a chromosome number of 2n=10 display very similar karyotypes, which differ only in the absolute length of the chromosomes, while the centromere-index and the relative length are equal at them. At the tetraploid species (2n=20) the karyotypes showed a larger variety.
There was expressed the conjecture, that the tetraploid P.wittmanniana are of autopolyploid, P.steveniana, P.macrophylla and P.tomentosa of allotetraploid origin. For the more exact judgment of this question a more detailed analysis of all species after the S - method with a differential chromsome staining was recommended.
Up to now became differential stainings of meiotic chromosomes at species of the genus Paeonia at P.tenuifolia accomplished with the S - method (Friebe, 1976), at P.tenuifolia and P.officinalis L. - at mitotic chromosomes with the help of Chromocin and silver - citrate (Schwarzacher-Robinson, 1986), the further at 6 species, which increase in Japan, with the help of the S - method (Nakamura, Nomoto, 1982). with the exception of from P.tenuifolia, was not accomplished a differential staining of the chromosomes by Caucasian representatives of this species up to now.
Material and methods
The differential staining was applied at the same species, at which the morphological chromosome analysis was accomplished in the earlier work (Punina, 1987), see Table 1 .
The plants were cultivated in containers in earth, in the experimental section of the botanical of institute 'AN USSR V.L.Komarová. Ends of young roots were treated successively with 0.05% Colchicin - solution for 2 hours and 0.02 M Oxychinolin for 1 hour. After that 16 hours was fixed with cold acetic acid in the refrigerator for at least, as by L.I.Abramova (1977) recommended. The meristematic part of the roots became with 45%-iger acetic acid for 30-40 min at room temperature macerated, after that were produced permanent preparates. The preparations were dried up to 48h at room temperature at the air (Nakamura, Nomoto, 1982). The dried preparates were treated with concentrated Ba(OH)2 - solution at 60°C for 10min, after that washed with 1n HCl, distilled water and incubated in 2xSSC first for 10min at room temperature, then for 60min at 60°C, again washed with distilled water, dried and with 1%-iger Giemsa-Solution in thinned phosphate - buffer at pH 6.8 stained. The staining lasted 30min to 24h, i.e. until chromosomes and interphasic nuclei assumed a bluish-lila shading, the Chromo - centers and blocks on the chromosomes however had no raspberry - or dark - violet color. After the staining the preparations were washed with distilled water, dried, conducted through Butyl - liquor and Xylol and subsequently balsamised. The best metaphasic chromosomes were drawn with a 2000x enlargement with the help of the drawing machine RA-6. If extent lay and the heterochromatic chromsome segments of the explored species was prepared using the analysis by several metaphasic plastines.
Results of the investigations
The distribution the heterochromatic segments on the chromosomes the diploid species shows picture 1.
At P.mlokosewitschi were the centromer - near segment of the I.chromosome pair large, at the II. - ++ V.pair medium-sized. Satellites were visible at all chromosome pairs with the exception of pair III. Intercalar segments did not appear. At the comparison of the plants from different places of origin (Kedi and Lagodechi) no differences were ascertained, therefore was presented only 1 drawing.
At P.ruprechtiana were all centromer - near segments medium-sized, satellites became at all chromosome pairs shown with the exception of pair III. At the II.nd. chromosome pair in 2 cases of 5 intercalar segments were found in the middle of the short arm.
At P.caucasica from Saguramo were the heterochromatic centromer - near segment on the I.-III. and V.chromosome pair large, on the IV.chromosome pair medium-sized, satellites were found on the II.,IV. and V.pair, on the I. chromosome the satellite was only at one of the homologous chromosomes present. In the middle of the short arm of the II.chromosome pair is found an intercalar segment.
At P.caucasica from Daba were the heterochromatic centromer - near segment on the I.-IV. chromosome pair medium-sized, on the V.chromosome pair large. Satellites were found on all chromosome pairs, besides pair III. Intercalar segments were not found.
At P.tenuifolia were the heterochromatic centromer - near segment on the I.-IV. chromosome pair medium-sized, on the V.chromosome pair small. Satellites were found on the III.,IV. and V. chromosome pair. It is worth mentioning, that the satellites could not be observed on the III.chromosome pair at a staining with Fjolgren (Punina, 1987), but only with the help of the differential staining.
At P.lagodechiana, which is a introgressive natural hybrid between P.caucasica and P.mlokosewitschi , centromer - near heterochromatic segments of medium size were located on the I. - IV.chromosome pairs, at the V.pair is visible a heteromorphy - at the one chromosome of the pair was the segment medium-sized, at the other chromosome large. The I., II. and IV.pairs showed a heteromorphism also at the satellite, at the I. and II.pair was found satellites only on one chromosome of the pair, the IV.pair owns a chromosome an especially large satellite, how already before could be shown (Punina, 1987).
At the tetraploids species put itself the distribution the heterochromatic segments differentiated there ():At P.wittmanniana () all chromosome paire own centromer - closely heterochromatic segments medium size. The I. - ++ II. and VII.-X.pairse have satellites. Besides, single a heterochromaticr was found satellite on one of the homologous the IV.pair. On the short arm of a chromosome the III.pair is found a intercalars segment.
At P.steveniana() the chromosome paire own I - ++ IV,VI,VII and IX centromer - closely heterochromatic segments medium size, which X.pair small, at which V. and VIII.pair are heteromorph, only a homologous chromosome the V. Pair owns a centromer - near segment medium size, and only a Homologous the VIII.pair a small. heterochromatic satellites became on the short arm the II. and Vii. - X. chromosome paires observes, and episodically on a chromosome the III. Pair. intercalar segments were found episodically on different segments the II.-IV.pair.
At P.macrophylla from Adsharien () the II own. and IX.pair large centromer - closely heterochromatic segments, I., II., IV., VI.pair medium-sized, V., VII and X.pair small. The VIII.pair owns no centromer - near heterochromatine. Satellites are found on the short poor the Vii. - X. chromosome paires, on the short arm of a chromosome the I. and III.pair and on the long arm of a chromosome the II.pair. On the short poor the IV. and V. Chromosomes appeared the satellites episodically. intercalar segments appeared singularly on the long arm of a chromosome the VII.pair .
At P.macrophylla from Svanetien () the III. own, VI. and IX.pair and a chromosome the IV.pair large centromer - closely heterochromatic segments. Segments medium size were ascertained at the I.und II.pair, small segments at the VII. and X.pair and on a chromosome the V.pair. At the VIII.pair the centromer are missing - near heterochromatic segments, likewise at a chromosome the V.pair. Satellites are found on the short poor the Vii. - X. chromosome paires and on a chromosome the VII.pair, likewise on the long arm of the chromosomes the II.pair. Episodically satellites on the short poor were found the III.-VI.chromosome paire. intercalar segments are found always on the long arm of the chromosome the VII.pair, which carries also the satellite. Singularly intercalar segments on the short arm of a chromosome stepped the III. and VI.pair on.
At P.tomentosa() are large centromer - closely segments on the X., medium-sized on the III., indeed only on the short chromosomes - arm, and to find small on the I.,II.,IV.,VII and IX.chromosome pair, besides on one of the homologous the V. and VIII.pair. Centromer - ++ near heterochromatine was missing at the VI.chromosome pair and one of the chromosomes the V.und VIII.pair. Satellites could ascertained become on the short poor the I.,III.,VII., IX. and X.chromosome paires and episodically on the short arm of a chromosome the II. and VIII.pair. intercalar segments medium size are found on the long arm of the chromosomes the VII.pair.
The existing investigation shows essential similarities in the character of the order the heterochromatic chromsome segments at the diploid species the species - P.mlokosewitschi, P.caucasica and P.ruprechtiana. Solely P.mlokosewitschi presents itself unquestionably as sovereign species, which can be distinguished likewise as in the entirety the outer morphologicaln features of the other diploid species well. It could be shown, that the karyotypes of this species own a large similarity with the karyotypes of P.caucasica and P.ruprechtiana (Punina 1987). Besides could the lightness of his crossing both on natural way, as well as artificially with P.caucasica are shown (Kakheladse, 1965; Kemularia-Nathadse, 1980), likewise a similarity in the anatomical construction the Spermoderms of this species with it by P.caucasica (Melikjan, Astrazatran, 1971). universe that attests them closely kinship of P.mlokosewitschi and P.caucasica, and so it appears us inexpediently, to arrange these species in different sections, as from Kemularia-Nathadse (1961) happen, or subsectionen, like at M.S.Uspenskaja (1987). We hold back however before it, to undertake a final settlement of the place of P.mlokosewitschi in the system of the species to this time, as long as further knowledge do not exist over these species.
At the analysis of the S - segments of the chromosomes of P.ruprechtiana showed itself, that them from P.caucasica and P.ruprechtiana of each other no more differ, as which from P.caucasica from different places of origin. morphologic is P.ruprechtiana with P.caucasica very similar, what allowed, to dispute their independence. On the other hand are, how already formerly was shown (Punina, 1987), the chromosomes of P.caucasica of different origins in the absolute length immediately long, while they are larger at P.ruprechtiana, than at P.caucasica. Therefore hold back we once more before a final judgment over the systematic Einordnung of P.ruprechtiana to this point in time, as long as further statements about the DNA at the Caucasian species of the species do not be available Paeonia. P.tenuifolia differs in the Verteilungsmuster the S - ++ segments well from the other diploid species. So became never satellites on the I. and II.chromosome pair found, while they were observed on the III.pair, at what these here only with the help of the differential staining could be demonstrated. The V.pair owns only very little centromer - near heterochromatine, besides does not appear the segment as zone, like at the other species, but as small pairige points. From it follows, that the order shown by us of the S - segments agrees on the chromosomes of P.tenuifolia in wide trains with the order of the kernel - organization - regions, which was noticed by Schwarzacher - ++ Robinson at staining with silver nitrate.
At the tetraploids species fashioned itself the Kartierung of the order of the S - segments more differentiated than at the diploid.
At P.wittmanniana we found altogether no essential differences in the distribution the S - ++ segments between I./II, III./IV., V./VI., VII./VIII. and IX./X. chromosome pair. The similarity in the absolute length and the centromere-indexwas shown already formerly (Punina, 1987).Bei of this species can one not only from chromosome pairen, but rather from groups of four speak. We consider P.wittmanniana as auto-tetraploide species and the differential chromsome stainingen confirm this view.
An other picture of the order the heterochromatic segments shows itself at P.steveniana and P.macrophylla. At these species differ the chromosomes well in the distribution it centromer - near segment. Distinguished in the absolute length and in the centromere-indexbetween I./II, III./IV., V./VI., VII./VIII. and IX./X.chromosome pair were ascertained already formerly (Punina, 1987), what the conjecture admitted, that these species Allo-Tetraploide are, i.e. hybrid origin. The Verteilungsmuster at the differential staining confirm that likewise. Like on Abb.2 (b-g) visibly, is contained in each group of four of chromosomes, which are similar themselves in the Centromer - index, a pair with large or medium-sized centromer - near segments and a pair with small or altogether missing segments.
It remains to notice, that number and order of the satellites did not agree at these species sometimes with that, which at the morphologicaln chromosome analysis was observed after staining with Fjolgren (Punina, 1987). At this staining was not a distinction by well visible and bent satellite possible, and if at individual chromosome pairen satellites were noticed, although they appeared only singularly, these pairs were marked carrying as satelliten -. In the existing investigation chromosomes were marked carrying only as satelliten -, if their Vorhandensein could be confirmed at least 3 times. At the staining with Giemsa showed itself, that further chromosome paire own heteromorphe satellites, how for example the VII.pair of P.macrophylla from Svanetien. A comparison of the chromosomes of P.macrophylla from different Naturstandorten yielded, that the differences were very slight between these populations, if one pulls the remarkable geographical distance between both in consideration.
At P.steveniana the Kartierung yielded the heterochromatic segments a similar picture, as at P.macrophylla, which doubts leaves emerge, to consider this species as independently, as it was proposed by Kemularia-Nathadse (1961). Uspenskaja (1987), which 17 species of the species Paeonia after 24 morphologicaln features analyzed, ascertained, that P.macrophylla and P.steveniana differed only in a feature. The Areale of these species lie closely together. Therefore we observe it for possibly, that P.steveniana and P.macrophylla no independent species, but only variants and the same species present.
If one considers the distribution of heterochromatine at P.tomentosa, notices one before everything, that it is quantitatively little. From 20 chromosomes no centromer own 4 altogether - near heterochromatine and 8 have only small centromer - closely blocks. On the long arm the VII.pair is found a intercalars segment, which appeared at the other species never. The IX. and X. chromosome paire look in comparison with their homologous at P.steveniana and P.macrophylla like exchanged places: the pair with the long poor owns a small, punktshaped centromer - near block, which with the shorter a large, so like at P.steveniana and P.macrophylla, only conversely. In this regard P.tomentosa differs clearly by the other tetraploids species, apart from P.wittmanniana, them, as further above executed, as Autotetraploid is to be examined. Because of these fundamental differences in the descent we examine it as unauthorized, that these species are arranged in a series, as from Kemularia - ++ Nathadse (1961) and Uspenskaja (1987) happen, but propose, P.steveniana and P.wittmanniana in different series einzugliedern.