_??_1990 by Cytologia, Tokyo Cytologia 55: 1-14 , 1990

Relationship and Classification among incanum Complex

H. U. Anaso and J. O. Uzo

Department of Crop Science, University of Nigeria , Nsukka, Nigeria

Accepted November 28, 1988

Solanum incanum is a controversial because it was given different descriptions by different authors (Vide Nyas 1932, Dalziel 1936, Hutchinson and Dalziel 1963, Watt and Breyer-Brandijk 1962, van Epenhuijisen 1974, Omidiji 1975) and these descriptions vary widely. Vide Nyas (1932) regarded the thorny plant in Plate I (courtesy of Hutchinson and Dalziel 1963) as Solanum incanum. Plate 2 is another form of Plate 1, but without thorns; Vide Nyas (1932) reported that this first type of S. incanum (the thorny type) has wide range therapeutic applications. For example, he reported that extracts from the berries of the thorny S. incanum have the power of temporarily stimulating cardiac action and this opposite effect is different from that of poisonous properties. Dalziel (1936) and Hutchinson and Dalziel (1963) shared the view of Vide Nyas on the description for the same plant but Hutchinson and Dalziel (1963) reported that the thorny S. incanum was poisonous. Earlier work by Dalziel (1936) suggested that there was a rela tionship between the wild S. incanum and S. melongena, and that many edible forms are in cluded in S. incanum complex. Crossability studies and studies on chemotaxonomy by Nara simah Rao (1979) and Pearce (1975) respectively revealed that wild S. incanum and cultivated S. melongena are very closely related. The chromosome number of the wild species was discovered to be 2n=48 (Anaso 1982). Another school of thought made up of some West African workers e. g. van Epenhuijisen (1974) and Omidiji (1975) regarded the plant whose line drawing appears in Plate 3 as S. in canum. The life photograph of this specimen is shown in Plate 4. Earlier works by Opeke (1963), Oke (1965) and Oyenuga (1967) revealed that this plant species (Plates 3 or 4) is not poisonous at all. It had been in use as one of the most popular vegetable crops whose glabrous leaves and fruits are eaten raw in African salad. This latter plant species (Plates 3 or 4) which was shown to be a diploid (2n=24) (Omidiji 1975) is a dense herb about 0.8 metres in height and becomes woody with age. It has no spines and the fruits are oblate shaped. The petal of the flower is white in colour and the leaves are glabrous, ovate with slight lobulation. The description is in sharp contrast with the description given by earlier workers for the wild thorny S. incanum. The workers in West Africa recognised the plant form represented in Plates 1 and 2 as wild form of S. incanum, and these wild forms were shown to possess hairs and sometimes spines both on the petiole of the leaves, peduncle and stem. The flowers of these two wild forms are also violet coloured and the leaves lanceolate in shape, while the berries range in shape from round to short oval (Table 1). It is doubtful if earlier workers on these two morphologically different considered cytomorphology, proximate chemical analysis of fruits and crossability relationship before according them the same species name. It is therefore the aim of this investigation to study the cytomorphology, proximate chemi cal analysis of berries and crossability relationships between the two Solanum forms, wild and cultivated. Works by Dalziel (1936), Narasimah Rao (1979) and Pearce (1975) indicate that S. incanum may be a complex, extending from wild to cultivated taxa including intermediates. 2 H. U. Anaso and J. O. Uzo Cytologia 55

Plate 1. Wild thorny tetraploid S. incanum by courtesy of Hutchinson and Dalziel (1963). A, flowering shoot. B, longitudinal section of flower. C, anther. D, pistil. E, cross-section of ovary. F, section of fruit. Solanum incanum Linn. ().

Plate 2. Wild tetraploid S. incanum, without spines, hairs present on the leaves, flowers with violet coloured petals, leaves lanceolate to obovate without lobulation. Berries round and white green striped, golden yellow when ripe. 1990 Relationship and Classification among Solanum incanum Complex 3

Information gathered from our work may help in clearing some errors in S . incanum nomen clature and .

Plate 3. West African diploid cultivated S. incanum by courtesy of van Epenhuijisen (1974).

Plate 4. Nsukka variety of diploid cultivated S. incanum (Photograph by Authors). 4 H. U. Anaso and J. O. Uzo Cytologia 55

Materials and methods

The materials used for this investigation were seeds of both Solanum types described by the two schools of thought; Vide Nyas (1932), Dalziel (1936), Watt and Breyer-Brandijk (1962), Hutchinson and Dalziel (1963) on one hand and van Epenhuijisen (1974), Omidiji (1975) on the other hand. The seed of Dalziel's (1936) type of S. incanum was obtained from the Solanum germplasm collection of Professor H. C. Choudhuri at the Ahmadu Bello University, Zaria, Nigeria. The seeds of van Epenhuijisen (1974) type of Solanum were obtained from Professor J. O. Uzo's Solanum germplasm collection at the University of Nigeria, Nsukka. Seedlings were raised in the sterilized nursery media and transplanted after three weeks into the field plots pretreated with poultry manure as a basal dressing. Experimental design was the Randomised complete block design with 5 replications.

Table 1. Comparison of morphological characters in diploid West Arican S. incanum and two wild tetraploid S. incanum, one with thorns and the other without

For cytological studies, young flower buds from both plant species were used. The buds

were harvested between 9.00 am and 12.00 noon each day and fixed in Carnoy's fluid (6:3:1) for 12 hrs. The next day the buds were hydrolysed in 0.1 N HC1, washed with 45 per cent acetic acid, squashed, and stained with 2 per cent aceto-orcein. The method of Choudhuri

(1980) was adopted. Good dividing stages were photographed. Morphological characters of the spiny S. incanum or Sodom apple and diploid S. incanum

were studied and presented in Table 1. For proximate chemical analyses, 10-healthy fresh green berries of each plant specimen

(including the two wild forms and the diploid cultivated form) were used. The berries were weighed fresh with a mettler balance and the weights were recorded. Then the berries were dried in Gallenkamp oven at a temperature of 60•Ž to a constant weight. The difference be tweenfresh and the dry weights gave the weight of moisture inthe berries, from which the per centage of moisture wass calculated. The dried berries were then pulverised into powder using an electric blender. Chemical analyses were carried out using 5 grams of the powdered berries for each estimation. 5 gram, 1990 Relationship and Classification among Solanum incanum Complex 5

of the powdered berries were also used for ashing by burning each measure in a fire clay furnace at 600•Ž for three days. The mineral elements were extracted from the ash with 5 per cent HCl and read from Unicam S. P. 1700 atomic absorption spectrophotometer. The method of Onyilagha and Lowe (1985) was adopted. The wave length needed for the various elements in the standard solution were shown in Table 2. The graphs obtained by plotting the concentrations of standard solutions (in parts per million) against their absorption spectra (calibration curve), were used in the determination of concentration of mineral elements in the sample. Nitrogen was estimated by the method of Onyilagha and Lowe (1985) and by Microkjeldahl as detained by Vogel (1961). Percentage of crude protein was estimated by multiplying the estimated value by 6.25 (Vogel 1961).

Crude fat determination was carried out through Soxhlet's extraction assembly using petro leum ether for 8 hrs. The fats condensed in the flask were cooled in the refrigerator and es timated by differences in weights adopting the method of Omidiji (1975).

Crude fibre was estimated from fat free residue left during crude fat determination using the method of Onyilagha and Lowe (1985). For crossability studies pure parental homozygous lines of wild tetraploid thorny S. incanum and diploid cultivated S. incanum were raised. Crosses were attempted in all possible combinations between these taxa. The developments of fruits, seed sets and the germination of hybrid seeds were examined to find out the nature of barriers at different levels. For suc cessful crosses a crossability index was used to measure the crossing affinity between each pair of parents. This index was derived from the commonly used seed set crossability index ( seed set in crosses/% seed sets in selfs•~100%) and the plants per pollination formula of Marks

(1965). For each cross the following data were recorded:

A=percentage of fruit set B=average number of seeds per berry C=percentage of germination of seeds (and time taken) D=percentage of survival of the germinated seedlings.

Crossability index= S Crossing efficiency of the cross •~100 elfing efficiency of the female parent 1

=Ac•~Bc•~Cc•~Dc •~100/1 As•~Bs•~CsxDs Pollen viability was assessed on the basis of stainability in acetocarmine and glycerine (l:1) of Omana Philip (1979). The deeply stained ones are regarded as viable, whereas the lightly stained, empty and denatured ones are regarded as non-viable.

Results

Cytological studies-Meiosis Because of the close similarity between the thorny and non thorny tetraploid S. incanum, the cytological studies were limited to a comparison between the wild thorny tetraploid and the cultivated diploid forms. The chromosome numbers in the diploid West African S. incanum and thorny tetraploid S. incanum or Sodom apple were 2n=24 and 2n=48 respectively. The nuclei of meiotic cells of the two plants reacted differently to aceto-orcein stain, with that of the thorny S. incanum or Sodom apple appearing to stain deeper. Darlington (1965) suggested that the area most deeply affected by staining is the centromere region. 6 H. U. Anaso and J. O. Uzo Cytologia 55

Plate 5 (Figs. 1-12). Comparative cytological studies of the diploid cultivated West Aftrican S. incanum (van Epenhuijisen 1974) and wild tetraploid S. incanum (Hutchinson and Dalziel 1963). 1, lepto-zygotene stage in the diploid edible West Aftrican S. incanum with early pairing taking place. 2, lepto-zygotene stage in the thorny tetraploid S. incanum or Sodom apple with early pairing and deeper staining. Note, the preferential pairing involving fewer number of chromo somes. 3, pachytene stage with some loops and impaired segments in cultivated West African S. incanum. 4, pachytene stage with full pairing and some loops in thorny tetraploid S. incanum or Sodom apple. 5, diakinesis in West African diploid S. incanunt with two quadrivalents (arrowed) and 8 bivalents. 6, diakinesis in thorny S. incanum or Sodom apple with presumably 4 quadri- 1990 Relationship and Classification among Solanum i ncanum Complex 7

Pairing started early in both species although differences became obvio us later. For purposes of clarity, the stages were compared side by side . In Figs. 1 and 2 of Plate 5, the b earded appearance of the chromosomes and their indistinct length made it impossible f or the exact length to be traced at this stage . Figs. 3 and 4 show pachytene stages with some im paired segments (Fig. 3) i.e. in diploid West African S. incanum and near full pairing in Fig. 4 ( or thorny S. incanum). There were pachytene loops (point of similarity) in b oth Figs. 3 and 4. Darlington (1965) reported the presence of loops and impaired segments i n Fritillaria and explained the occurrence of such a phenomenon to be either as a result of diff erences in h omology of chromosomes as a result of mechanical interference with pairing or due to pre sence of too many homologous chromosomes . At diakinesis (Figs. 5, 6) there were cases of multivalents (arrowed) and bivalents . At this stage, the number of chiasmata decreased presumably because the chiasma moved from interstitial to terminal position . It is also presumed that the chiasma frequency was reduced as a result of multiple association of some chromosomes . The number of multivalents was higher in Sodom apple or thorny S . incanum (Fig. 6) than in cultivated diploid S. incanum (Fig. 5). At first metaphase, all the multivalents disjoined and were oriented at the equatorial plate ready to move to the poles . Fig. 7 is a polar view of first metaphase stage in the diploid cul tivated S. incanum still showing 2 trivalents and 9 bivalents . Fig. 8 is also a metaphase stage (polar view) in thorny tetraploid S. incanum with 1 quadrivalent (q) , 1 trivalent (arrowed), 1 univalent (u) and 20 bivalents. One eliminated bivalent (el) was also shown . The distribution and movement of chromosomes to the poles was not smooth probably because of presence of some multivalents in the two specimens . This led to the formation of bridges and fragments due to the difficulty shown by some chromosomes at point of separa tion. Fig. 9 shows a first anaphase stage (in diploid cultivated S. incanum) with two broken bridges (arrowed) while Fig. 10 is a first anaphase stage in the thorny S. incanum or Sodom apple. As expected, all the abnormalities shown during the first division were reflected in the second division. The second anaphase division was not very smooth becaused of presence of laggards (arrowed) in diploid West African S. incanum (Fig. 11). In Fig. 12 is shown a normal tetrad in the tetraploid wild S. incanum alongside with a diad (D, arrowed) separated by a chromatid bridge. All the abnormalities detected during pollen formation led to abnormal development of pollen, deformity and abortion of pollen grains in both the wild thorny S. incanum and the diploid West African S. incanum. The percentages of morphologically deformed pollen in diploid West African S. incanum and wild thorny tetraploid S. incanum were 13.5 per cent and 18.5 per cent, respectively. The percentages of aberrations during second divisions in diploid S. incanum and wild tetraploid S. incanum were 14.3 per cent and 17.5 per cent, respectively. Pollen malformations in both the diploid cultivated S. incanum and the wild tetraploid thorny S. incanum were brought about by ordinary cytological aberrations.

Morphological studies

valents and 18 bivalents. 7, first metaphase (polar view) in diploid West African S. incanum with

2 trivalents and 9 bivalents. 8, first metaphase (polar view) in tetraploid thorny S. incanum with I

quadrivalent, 1 trivalent, 20 bivalents and 1 univalent. 9, first anaphase stage in West African diploid S. incanum with broken bridges. 10, first anaphase division in thorny S. incanum or Sodom apple with clear separation of chromosomes. 11, late stage of second anaphase with laggards in diploid West Aftrican S. incanum. 12, normal tetrad and a diad with a bridge in thorny S. incanum

or Sodom apple. •~400. 8 H. U. Anaso and J. O. Uzo Cytologia 55

Table I compares the morphological characteristics shown by diploid cultivated S. incanum and the two wild forms of tetraploid S. incanum. It is apparent from the table that characters like spines, hairs on the leaves, round shape of fruits, golden yellow colour of ripe berries and violet colour of petals which are intensively represented in the two wild tetraploid forms of S. incanum were entirely lacking in the diploid cultivated S. incanum. This means a wide range variation. It is also apparent that the non thorny wild tetraploid S. incanum is a direct derivative from the thorny wild form, because of similarity in genetic attributes shown by both plants.

Proximate chemical composition of berries of the diploid cultivated S. incanum and the tetraploid thorny S. incanum A comparative proximate chemical composistion of Sodom apple, or thorny S. incanum, non thorny tetraploid S. incanum and the West African diploid S. incanum is shown in Table 4. A mean of ten determinations shows wide differences in moisture content, crude protein, crude fats, carbohydrates and some mineral elements (e. g. Na, K, Fe, Mg, Ca and P) between the diploid cultivated S. incanum and tetraploid thorny S. incanum and close values between the two wild tetraploid S. incanum. It is apparent from this table that moisture contents, carbohydrates and some nutrients (e. g Na, K, Mg, Ca and P) were higher in the diploid cul tivated S. incanum than in the two wild tetraploid varieties . For example, the percentage of moisture in diploid S. incanum was 85.56 per cent as against 71.59 ane 73.49 per cent reported in thorny and non thorny wild tetraploid S. incanum, repsectively. The amounts of crude proteins, fats and solid materials were higher in the berries of the two wild tetraploid forms than in the diploid cultivated S. incanum. For example crude proteins , fats and solid materials were higher in the two wild tetraploid S. incanum than in the diploid cultivated S. incanum. These figures were given as follows: Protein=5 .30 per cent, fats=7.03 per cent and solid drymatter=28.41 per cent compared to 5.25 per cent of protein , 7.00 per cent fats and 28.40 per cent solid material obtained in thorny and non thorny tetraploid wild S. incanum, respectively. The berries of diploid cultivated S. incanum had 2.35 per cent of crude protein, 0.80 per cent of crude fats and 14.44 per cent of solid matter. These figures show wide range variation and suggest that the non thorny S. incanum is a direct derivative of the thorny wild tetraploid S. incanum and none of them is closely related to the diploid cultivated S. incanum. Crossability

Table 2. Wave lengths used for various elements in the standard solution from pure salt samples

relationships between the diploid West African S. incanum, thorny wild S . incanum and the non thorny S. incanum are shown in Table 3B. Table 3A is a diagrammatic representation of the results of the crosses attempted between species within the S. incanum complex. In Solanum incanum complex, barriers to crossability appeared to have developed to vari ous degrees at different levels. Successful cross with normal seeds which grew well resulted i n diploid S. incanum and wild thorny tetraploid S. incanum crosses, only when the diploid incanum was used as the pistillate parent. When the wild tetraploid S. incanum was used 1990 Relationship and Classification among Solanum incanum Complex 9 pistillate parent, the percentage of successful cross was very low (about 4 per cent) and this value was as a result of bud-pollination. When the seeds germinated , the percentage of germi nation was also about 4.35per cent , because most of the seeds obtained in the F1 of this cross were shrunken and non viable. Table 3B shows the results of crossability relationships of species within S. incanum complex. The crossability indices for the wild tetraploid thorny S. incanum and diploid cultivated S. incanum calculated from Table 3B were 0.002 and 90, respec tively. This shows a very wide variation.

Discussions Omidiji (1981) observed that among other things, the relationships of species are usually assessed on the basis of one or more of several criteria. These criteria include morphological characteristics, eco-geographical distribution, karyotypic distinction, meiotic chromosomal association, chemotaxonomic principles and crossability. In many studies with Solanum species, the evaluations of morphological characters and crossability have both been widely used in the establishment of relationships between Solanum species (Bhaduri 1951, Seithe 1971,

Table 3A. Representation of results of various crosses

•¡ =Normal seed set on selfing •œ =Normal seeds which grew well

_??_ =Cross unsuccessful _??_ =Fruit sets but seeds shrunken

Schelling and Heiser 1971). The present investigation considered more parameters viz. mor phological characters, meiotic chromosomal association, proximate chemical analysis of ber ries and crossability relationships. Results from cytological studies show differential reactions of the chromosomes of the meiotic cells of the three plants under consideration to aceto-orcein stain, and suggests that the centromere region of the two wild tetraploid species contain higher concentration of nucleic acid. The behaviour of chromosomes in both wild tetraploid S. incanum shows close similarities with the diploid cultivated S. incanum in such cytological aspects like formation of lower per centage of multivalents and in pollen deformity being caused by cytological aberration instead of by gene mutation. The fact still remains that pairing in the two wild tetraploid species was autosyndetic while in the diploid cultivated S. incanum pairing was allosyndetic. This pre ferential pairing of chromosomes in the wild tetraploid S. incanum might possibly be the factor that limited multiple association of chromosomes often witnessed in some tetraploid plants and some interspecific hybrids. That is in other words, the wild tetraploid S. incanum behaved like diploids. Sodom apple and its direct non thorny derivative are amphidiploids. Results from the study of morphological characters revealed that the only difference be tween the thorny and non thorny wild tetraploid S. incanum was in presence of spines in the Table 3B. Crossability relationships of the different Solanum incanum 1990 Relationship and Classification among Solanum incanum Complex 11 thorny forms and absence of spines in non thorny type . All other attributes including genetic, cytological, biochemical constituents and crossability show that the non thorny tetraploid S. incanum is direct derivative of the thorny S. incanum . The absence of thorns in the non thorny S. incanum may be due to a one point mutation leading to the loss of spines . It was also seen that each species breeds true to type without offtypes . In other words, tetraploid wild thorny S. incanum only produces spiny tetraploid varieties with non-edible fruits while the diploid cultivated S. incanum only produces edible cultivars with oblate shaped fruits . This fact was further supported by induction of polyploidy in diploid cultivated West Aftrican S . incanum which yielded a tetraploid form, var. marvelum (Anaso and Uzo 1987). The berries of this new derivative of diploid S. incanum is edible, very large, oblate shaped with grooves at the

Table 4. Comparative proximate chemical analysis based on determinations of moisture, dry matter, ash, crude protein, crude fats, crude fibres, carbohydrates and some micro nutrients (or trace elements), e. g. Na, K, Fe, Mg, Ca and P

sides and navels or deep scars at the blossom end. This is a very sharp contrast with what obtains in the wild tetraploid forms of incanum. Proximate chemical analyses showed lower percentage of moisture (71.59 per cent and 73.49 per cent) in thorny and non thorny wild tetraploid S. incanum respectively and 85.56 percentage of moisture in diploid cultivated S. incanum. Also the percentage of crude proteins and crude fats were 5.30 and 5.25 per cent for proteins and 7.03 and 7.00 per cent for fats in thorny and non thorny wild tetraploid S. incanum, respectively. In the diploid cultivated S. incanum the percentage of crude proteins and lipids were 2.35 per cent and 0.80 per cent, re spectively. These wide variations in moisture, protein and lipids could account for the poi sonous properties attributed to the two wild tetraploid taxa. 12 H. U. Anaso and J. O. Uzo Cytologia 55

Crossability The easy crosscompatibility (Tables 3A and B) between the West Aftrican diploid S. incanum and its autotetraploid derivative var. marvelum, with production of profuse flowering and fertile triploid hybrid plants, confirms the fact that these two specimens have similar genetic attributes and close affinity. Failure of some of the crosses occurred by complete failure of fruit set, production of shrunken seeds which may be as a result of genetic incompatibility pre sumably resulting from interspecific lethal genes and by seedling mortality. This collapse of the embryo in the early stages of development called "somato plastic sterility" had earlier been reported by Brink and Cooper (1947) in crosses of Nicotiana rustica using pollen grains of other species of Nicotiana, Petunia and Lycopersicon. Also the wide variation in the values of crossability index of wild tetraploid S. incanum (0.002) and diploid cultivated S. incanum (90) indicates that this partial incompatibility barriers permitted crosses of many combina tions only in one direction. If indeed Sodom apple (wild thorny tetraploid S. incanum) or its direct non thorny deriva tive are ancestors of the diploid cultivated S. incanum, it follows that the tetraploid wild types gave rise to the diploid cultivated forms by retrogressive evolution. But Stebbins (1969, 1971) pointed out that the trend in angiosperm evolution is for the lower genome to give rise to the higher genome and not vice versa. With this view in mind one is forced to say that the di ploid cultivated S. incanum is not so closely related to the two wild tetraploid S. incanum to be accorded the same species name. Based on the findings of this work, the conclusion is reached that the two wild tetraploid forms are not sufficiently related to the diploid cultivated forms (all of which belong to the same complex) and therefore they merit separate species ranking. The name S. spinosum is suggested for the thorny S. incanum while the non thorny tetraploid form is recognised as a botanical variety of the thorny form. The name S. incanum should be reserved for the cultivat ed diploid S. incanum.

Summary

Preliminary investigations were carried out on cytomorphology, proximate chemical analy sis and crossability relationships of three members of the genus Solanum both classified as S. incanum, with a view to establishing the genetic basis of their taxonomic relationships. Results from these investigations revealed variations in morphological characters, behaviour of chromo somes, proximate chemical composition and crossability relationships. With basic chromosome numbers of n=24 in both thorny and non thorny S. incanum (Plates 1 and 2) and preferential pairing of chromosomes, it is quite clear that these two wild types are not the same as the diploid West African S. incanum whose genomic number was n= 12 and in which pairing was allosyndetic. Chemical composition of the two wild forms of S. incanum showed 71.59 per cent for the thorny and 73.58 per cent moisture for the non thorny S. incanum as against 85.56 per cent mois ture content in the diploid cultivated S. incanum. Also protein and lipid contents of the wild tetraploid species were 5.30, 5.25 (for protein) and 7.03 and 7.00 per cent respectively as against 0.83 per cent reported in the diploid S. incanum. There was high cross-compatibility between the wild tetraploid thorny and wild tetraploid non thorny S. incanum but strong pistil pollen incompatibility between the two wild forms and the diploid cultivated S. incanum. The similarity in some behaviour of chromosomes between the wild tetraploid forms and the cultivated diploid species was probably due to the fact tha_??_ wild tetraploid forms of Solanum incanum were amphidiploids. It is suggested that the non thorny wild tetraploid S. incanum (Fig. 2) is a direct derivati_??_ 1990 Relationship and Classification among Solanum incanum Complex 13 of the thorny wild tetraploid S. incanum (Fig. 1), since both are similar morphologically, in behaviour of their chromosomes and chemical composition of their berries. It is also suggested that the non thorny tetraploid S. incanum is a botanical variety of the thorny tetraploid type and that the absence of thorns in the former was as a result of point mutation. If either the thorny wild tetraploid S. incanum or its non thorny derivative was truly the ancestor of the di ploid S. incanum, then retrogressive evolution had taken place. But the trends in angiosperm evolution is for the lower genomic form to give rise to the higher genomic form. It is therefore concluded that the wild tetraploid S. incanum is not related to the cultivated diploid S. incanum. The name spinosum is suggested for the wild thorny S. incanum while in canum is reserved for the cultivated form of Solanum incanum. The non thorny wild derivative is a botanical variety of the thorny wild tetraploid form.

Acknowledgements

Anaso thanks Professor J. O. Uzo for providing the facilities used in this research and proof reading the manuscripts.

References

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