_??_1991 by Cytologia, Tokyo Cytologia 56: 31-41, 1991
Karyomorphology of Amaryllis Hybrids
Tasneem F. Khaleel, Susan Haven and Tim Gilg
Department of Biological Sciences, Eastern Montana College Billings, Montana 59101, U. S. A.
Accepted July 20, 1990
The genus Amaryllis (Amaryllidaceae) has attracted horticulturists and taxonomists alike because of the extensive hybridization of its species and the nomenclatural disputes (Traub 1983, Goldblatt 1984). In addition to the amazing diversity of hybrids , a large number of cultivated wild species are in circulation. The present day Amaryllis is the result of selections that have continued for over 200 years using whatever species were available. The quest for size and colour of flower led to indiscriminate use of hybrids and wild species in breeding, con sequently some species hybrids display low fertility and often will not set seed. According to Bell (1973) the genus Amaryllis illustrates a contemporary horticultural problem. Since hybrids are easier to grow than the species, the latter are frequently lost. Considering the importance of elemental species, an attempt should be made to conserve the valuable germ plasm for future hybridization. If parental species in the breeding programs are known, new varieties could be developed by systematic and organized hybridization programs. Where parental species are not well documented, pedigrees might possibly be traced or determined through cytological analysis. Cytological and embryological studies on the genus Amaryllis, are of immense value in assessing and evaluating the breeding potential of wild species and hybrids. They provide valuable information about the constitution of germ plasm, genetic compatibility of taxa involved in hybridization, reasons for low fertility and lack of or low seed set. A review of literature indicates that 25 of the 76 species have been studied (Flory and Coulthard 1981); although hundreds of hybrids are in circulation, only few have been studied (Guha 1979, Khaleel and Siemsen 1989). The basic chromosome number in the genus is x=11. While most species are diploid, the widely available hybrids are nearly uniformaly tetraploid. Other polyploids with 2n=33, 66, and 77 are also on record (Guha 1979). The present study involv ing nine hybrids Apple Blossom, Bold Leader, Cocktail, Desert Dawn, Dutch Belle, Intokazi, Miracle, Tangerine and Zanzibar is an attempt to furnish more comprehensive and exact information on the cytology and embryology of these hybrids. Since all of them show pollen sterility, degeneration of female gametophytes and low percentage of seed set, the data may be useful in evaluating the breeding potential of these hybrids and tracing their pedigrees. All hybrids more or less conform to the Division 4 (Reginae-type hybrids distinguished by open faced flowers with short tepal tubes, markedly imbricate or less markedly imbricate segments involving A. reginae, A. coreiensis and similar species), or Division 5 (Leopaldii type hybrids showing wide open flowers held horizontally indicative of A. leopaldii and A. paridina) of Traub's (1958) classification of cultivated Amaryllis (Table 1).
Materials and methods
The material used in the present study includes 192 bulbs of nine horticultural varieties of Amaryllis grown in the greenhouse of Eastern Montana College, Billings, Montana for the past five-seven years. Bulbs of Apple Blossom, Desert Dawn, Dutch Belle, Intokazi and Zanzibar were obtained from the Brown Bulb Ranch, Washington, and Bold Leader, Cocktail, 32 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56
Miracle and Tangerine from Park Seed Company, N. Carolina. Excised root tips from each bulb were pretreated with 0.25% colchicine for three and a half to four hours at room tem perature and fixed in Carnoy's fluid (3 ethanol: 1 glacial acetic acid V: V) for 24 hours, hy drolysed in IN HCl at 60•Ž for 8-9 minutes, stained with Schiff's reagent and squashed in 2% acetocarmine. Root tips of all varieties were treated similarly for karyological studies. Slides were made permanent by removing the coverslip by the dry ice method and processing through an n-butanol series, and mounting in euparal. Young anthers from each hybrid were fixed in modified Carnoy's fluid (6 ethanol: 1 glacial acetic acid: 3 chloroform, V:V:V) for 24 hours. They were squashed in 2% acetocarmine for meiotic studies. Permanent slides were made following the same steps as for the root tips. Camera lucida drawings of chromosomes were made from five mitotic metaphase plates of each variety. Chromosomes were karyotyped according to the increasing length of the short arm. Arm ratio was obtained by dividing the long arm by short arm and centromeric index was calculated by dividing short arm by the total length and x 100. The arm ratio and the centromeric index were both considered in determining the position of the centromere. Ave rage chromosome length was calculated by dividing the total chromatin length by the number of chromosomes in the karyotype. Photomicrographs were taken with Zeiss microscope. One thousand pollen grains from each hybrid were stained in 1% acetocarmine for 8-12 hours and scanned to determine fertility. Those appearing dense and darkly stained were considered fertile. Customary methods of dehydration, infiltration, microtoming and staining were followed for embryological studes of each hybrid. Since no significant differences were found among the hybrids in the present study or the ones studied previously (Khaleel and Siemsen 1989), results have not been described under the observations. However, pertinent reference has been made in the text. Voucher specimens of each variety have been placed in the Eastern Montana College herbarium.
Observations
Morphology: Table 1 indicates some of the morphological features of the nine hybrids. The measurements given are means obtained from 8-10 mature flowering bulbs of approximate ly the same age for each variety. Two distinct behavior patterns were observed over a period of six years. Bold Leader, Desert Dawn, Dutch Belle, Intokazi, and Miracle flowered every year regardless of fruit set while Apple Blossom, Cocktail, Tangerine and Zanzibar flowered following a season of rest after fruiting. The classification used follows Traub's (1958) di visions of cultivated Amaryllis. Cytology: All nine hybrids are tetraploid with 2n=44 (Figs. 1-12). Tables 2 and 3 show the karyotype and genome analysis of each hybrid. The chromosomes can be clustered into three groups according to arm ratio, and centromeric index, which ranges from 1:3 to
1:6 (centromeric index: 1-25: acrocentric) in the fist group, 1:1.5 to 1:2.9 (centromeric index: 25.1-40: submetacentric) in the second, and 1:1 to 1:1.49 (centromeric index:40.1 50:metacentric) in the third. Chromosome length ranges from 4.3 to 17.7ƒÊm, with a bimodal distribution favoring long and short lengths (Figs. 1-12). Four length classes were recognized: small (4.3-6.6ƒÊm), medium (6.7-8.3ƒÊm), large (8.4-10ƒÊm), and very large (10 .1-17.7ƒÊm). All hybrids show various combinations of the three types and four sizes of chromosomes
(Talbes 2 and 3). Secondary constrictions are terminal or near terminal leading to very small, inconspicuous satellites.
Meiosis is normal and is characterized by the presence of bivalents , trivalents and quad rivalents. 1991 Karyomorphology of Amaryllis Hybrids 33
Appleblossom: Karyotype (Figs. 1, 4 and Table 2) consists of 22 pairs of chromosomes. Four pairs (2, 4-6) of chromosomes have subterminal constrictions (one small and three medium), 10 pairs (1,3 and 15-22) have submedian constrictions (2 small, 1 medium and 7 large) and eight pairs (7-14) show median or near median constrictions (all eight small). The
Figs. 1-3. Photomicrographs of somatic metaphase plates of Amaryllis hybrids. All figures •~1700. 1, Apple Blossom. 2, Bold Leader. 3, Cocktail. 34 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56
toatl chromatin length is 307.6ƒÊm with chromosome lengths from 4.33ƒÊm to 10ƒÊm. The average chromosome length is 6 .7ƒÊm. Four haploid genomes can be recognized (Table 3), each consisting of two acrocentric, five submetacentric and four metacentric chromosomes respectively. The karyotype formula for this hybrid, therefore is 4 (2A5SM4M)=44.
Figs. 4-12. Karyotypes of Amaryllis hybrids . All figures •~1750 . 4, Apple Blossom . 5, Bold Leader . 6, Desert Dawn. 7, Dutch Belle. 8, Intokazi . 9, Cocktail. 10 , Miracle. 11, Tangerine. 12, Zanzibar.
Bold Leader: Karyotype (Figs. 2, 5 and Table 2) consists of 22 pairs of chromosomes -5) of chromosomes have subterminal . Fourpairs(2 constrictions (2 medium, 1 large and 1 very large) , 9 pairs (1, 6-10, 20-22) have submedian constrictions (1 small , 2 medium, 3 large and 3 very large) and nine pairs (11-19) have median or near median constrictions (8 small and Table 1. Classification, morphology, and behavior of Amaryllis hybrids
Table 2. Karyotype analysis of Amaryllis hybrids
All hybrids 2n=44; All Measurements are in micrometers. S=Small (4.3ƒÊ-6.6ƒÊ) A=Acrocentric M=Medium (6.7ƒÊ-8.3ƒÊ)
SM=Submetacentric L=Large (8.4ƒÊ-10ƒÊ) M=Metacentric VL=Very large (10.1ƒÊ-18ƒÊ) 36 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56
Table 3. Genome analysis of
* Since genomes I and II are the same , composition is only given once.
1 medium). The total chromatin length is 350.7ƒÊm with lengths from 5.3ƒÊm to 13 .7ƒÊm. The average chromosome length is 7.9ƒÊm. Four haploid genomes are recognized (Table 3) . Two of these consist of two acrocentric, five submetacentric and four metacentric chromo
somes; the other two are composed of two acrocentric, four submetacentric and five metacen tric chromosomes respectively. The karyotype formula is 2(2A5SM4M)+2(2A4SM5M)= 44. Desert Dawn: Karyotype (Fig. 6 and Table 2) is composed of 2n=44 in 22 pairs . Ten pairs (1-4, 10, 13, 15, 19, 21 and 22) show subterminal constrictions (2 large and eight very large), 10 pairs (5, 6, 8, 9, 11, 12, 16-18, and 20) show submedian constrictions (1 small , 4 medium, 1 large and 4 very large) and 2 pairs (7 and 14) show median or near median con
strictions (1 small and I medium). The total chromatin length is 477 .8ƒÊm with chromosome lengths from 6.2ƒÊm to 16.8ƒÊm. The average chromosome length is 10 .8ƒÊm. Four haploid genomes are recognized (Table 3), each consisting of five acrocentric, five submetacentric and one median chromosome. The karyotype formula is 4 (5A5SMIM)=44 . Dutch Belle: Karyotype (Fig. 7 and Table 2) is composed of 2n=44 in 22 pairs . Ten pairs (1-6, 11, 12, 15 and 16) show subterminal constrictions (1 large and 9 very large) , 5 pairs (10 , 17 and 20-22) show submedian constrictions (all 5 very large) and 7 pairs (7-9 , 13, 14, 1991 Karyomorphology of Amaryllis Hybrids 37
Amaryllis hybrids; x=11 in all genomes
18 and 19) show median or near median constrictions (2 small, 3 medium and 2 large). The total chromatin length is 492ƒÊm with chromosome lengths from 5.3ƒÊm to 16.8ƒÊm. The average chromosome length is 11.8ƒÊm. Four haploid genomes are identified (Table 3).
Two of these consist of five acrocentric, three submetacentric and three metacentric chro mosomes each; while five acrocentric, two submetacentric and four metacentric chromosomes are recognizable in each of the other two genomes. The karyotype formula for this hybrid is 2(5A3SM3M)+2(5A2SM4M)=44. Intokazi: Karyotype (Fig. 8 and Table 2) is composed of 2n=44 in 22 pairs. Six pairs
(1-3, 5, 6 and 11) show subterminal constrictions (4 large and 2 very large), 11 pairs (4, 7-10, 12, 17, 18 and 20-22) show submedian constrictions (3 medium, 2 large and 6 very large) and 5 pairs (13-16, and 19) show median or near median constrictions (4 medium and 1 large).
The total chromatin length is 420.4ƒÊm with chromosome lengths from 6.6ƒÊm to 15.1ƒÊm. The average chromosome length is 9.5ƒÊm. Table 3 shows the composition of each haploid genome. Two of these are composed of three acrocentric, six submetacentric and two met acentric chromosomes; each of the remaining two consist of three acrocentric, five submeta centric and three metacentric chromosomes. The karyotype formula is 2 (3A6SM2M)+2
(3A5SM3M)=44. 38 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56
Cocktail: Karyotype (Figs. 3, 9 and Table 2) consists of 2n=44 chromosomes of which 21 are paired and two unmatching. Thirteen chromosomes (pairs 1-6, and 22a) have sub
terminal constrictions (4 medium, 2 large and 7 very large), 16 (8, 10, 13, 14, 17, 18, 20 and 21) show submedian constrictions (4 large and 12 very large) and 15 chromosomes (pairs 7, 9, 11, 12, 15, 16, 19 and 22b) possess median or near median constrictions (2 small and 13 me dium). The total chromatin length is 421.8ƒÊm. The smallest chromosome in the karyotype is 6.2ƒÊm and the largest is 13.7ƒÊm in length. The average chromosome length is 9.5ƒÊm.
Each haploid genome consists of 11 chromosomes (Table 3). Three of these are similar in having three acrocentric, four submetacentric and four metacentric chromosomes; while the
fourth consists of four acrocentric, four submetacentric and three metacentric chromosomes respectively. The karyotype formula is 3(3A4SM4M)+(4A4SM3M)=44. Miracle: Karyotype (Fig. 10 and Table 2) consists of 2n=44 chromosomes of which 21 are paired and two unmatching. Eleven chromosomes (pairs 1-4, 10 and 22a) have sub
terminal constrictions (2 small, 2 large and 7 very large), 11 pairs (5-9, 13, 15, 16 and 19-21) show submedian constrictions (4 medium, 4 large and 14 very large) and 11 chromosomes
(pairs 11, 12, 14, 17, 18 and 22b) possess median or near median constrictions (5 medium and 6 large). The total chromatin length is 461.5ƒÊm. The smallest chromosome in the karyotype is 6.2ƒÊm and the largest is 16ƒÊm in length. The average chromosome length is 10.4ƒÊm.
Four haploid genomes are recognized (Table 3). Two of these are similar in consisting of three acrocentric, six submetacentric and two metacentric chromosomes. The third genome is composed of three acrocentric, five submetacentric and three metacentric while the fourth
consists of two acrocentric, five submetacentric and four metacentric chromosomes respective ly. The karyotype formula is 2 (3A6SM2M)+(3A5SM3M)+(2A5SM4M)=44.
Tangerine: Karyotype (Fig. 11 and Table 2) consists of 2n=44 chromosomes of which 21 are paired and two unmatching. Sixteen chromosomes (pairs 1-4, 6, 8, 10 and 19) have
subterminal constrictions (2 large and 14 very large), 15 chromosomes (pairs 5, 11, 12, 15, 16, 18, 21 and 22a) show submedian constrictions (2 medium, 2 large and 11 very large) and 13 chromosomes (pairs 7, 9, 13, 14, 17, 20 and 22b) possess median or near median constrictions
(8 medium and 5 large). The total chromatin length is 502.5ƒÊm. The smallest chromosome in the karyotype is 6.6ƒÊm and the largest is 17.7ƒÊm in length. The average chromosome length is 11.4ƒÊm. Four haploid genomes are recognized (Table 3). Three of these consist
of four acrocentric, four submetacentric and three metacentric chromosomes each. The fourth shows four acrocentric, three submetacentric and four metacentric ones. The karyo type formula is 3(4A4SM3M)+(4A3SM4M)=44. Zanzibar: Karyotype (Fig. 12 and Table 2) consists of 2n=44 chromosomes of which
21 are paired and two unmatching. Fifteen chromosomes (pairs 1-5 , 9, 19 and 22a) have
subterminal constrictions (5 large and 10 very large), 17 chromosomes (pairs 6 , 10, 14, 16-18, 20, 21 and 22b) show submedian constrictions (4 medium, and 13 very large) and 12 chro mosomes (pairs 7, 8, 11-13 and 15) possess median or near median constrictions (4 small and 8 medium). The total chromatin length is 449.1ƒÊm. The smallest chromosome in the karyo
type is 5.1ƒÊm and the largest is 15.7ƒÊm in length. The average chromosome length is 10 .2 ƒÊ m. Table 3 shows the genome analysis of this hybrid to consist of three similar haploid
genomes (each with four acrocentric, four submetacentric and three metacentric) and the fourth consisting of three acrocentric, five submetacentric and three metacentric chromosomes respectively. The karyotype formula for this hybrid is 3 (4A4SM3M)+(3A5SM3M)=44 . Embryology: All nine hybrids are protandrous . Microsporogenesis and the devel opment of anther wall occur completely inside the bulb long before the emergence of the in florescence. The structure and development of the anther wall , male gametophyte, mega sporogenesis and the development of the female gametophyte are similar to the previously 1991 Karyomorphology of Amaryllis Hybrids 39 studied hybrids (Khaleel and Siemsen 1989). Polygonum as well as Allium type of female gametophyte development was observed with the former being the most frequent type in all hybrids. All hybrids show 80-95% degeneration of female gametophytes at the two or four nucleate stage. Apple Blossom and Dutch Belle showed the maximum degeneration; Cocktail and Zanzibar were next, followed by Desert Dawn, Tangerine, Intokazi, Bold Leader and Miracle. This is reflected in the percentage of fruit set in these hybrids (Table 1).
Discussion Chromosome studies carried out previously on species and cultivars of Amaryllis (Narain and Khoshoo 1968, Vij et al. 1978, Guha 1979) have revealed the basic chromosome number to be x=11 for the genus. Narain and Khoshoo (1968) identified a basikaryotype to be com posed of 2 metacentric (median), 5 submetacentric (submedian) and 4 acrocentric (subterminal) chromosomes. The nine hybrids studied in the present investigation are all tetraploid with 2n=44 and conform to the basic chromosome number for the genus. The basikaryotype is not traceable in any of the genomes in all hybrids. Tangerine, conforms to the number of acrocentric chromosomes (4) in the basikaryotype in all four of its genomes, Zanzibar in three of its genomes, Cocktail in one and all others in none of their genomes. Apple Blossom and Desert Dawn, shows five submetacentric chromosomes (as in the basikaryotype) in all four genomes, Bold Leader, Intokazi and Miracle in two, Zanzibar in one and the others in none of their genomes. Only Intokazi and Miracle show two metacentric chromosomes (as in the Basikaryotype) while others have between 1-5 metacentric ones in their haploid genomes. Sim ilar deviations from the basikaryotype due to chromosome repatternings have been recorded in other Amaryllis cultivars (Vij et al. 1978, Khaleel and Siemsen 1989). Narain and Khoshoo (1977) considered such heterozygous karyotypes to result from amphiplasty and other structural changes. The modified basikaryotypes in the present study are perhaps due to such structural alterations and the hybrid origin involving different genomes. Naranjo and Andrada (1975) suggested a basic karyotype of four metacentric (median), four submetacentric (submedian) and three acrocentric (subterminal) chromosomes in the genus Hippeastrum. (=Amaryllis). This basic karyotype was found in eight of the nine species studied by these authors while H. vittatum showed a derived karyotype involving small changes. Only Cocktail in the present study shows this karyotype in three of its genomes and the same number of submetacentric chromosomes in the fourth genome. Apple Blossom shows similarities only in the number of metacentric chromosomes in all four genomes; Bold Leader in metacentric in two genomes and submetacentric in the other two genomes; Dutch Belle in the number of metacentric in two genomes; Miracle in the number of metacentric in one genome; Tangerine in the number of submetacentric in three genomes and only metacentric in one genome; Zanzibar in submeta centric in three genomes. The most common combination found in the present study is 2 acrocentric, 5 submetacentric and 4 metacentric found in Apple Blossom, Bold Leader and Miracle or 4A4SM3M as in Cocktail, Tangerine and Zanzibar; the next most frequent com bination is 3A5SM3M as seen in two genomes of Intokazi, and one each of Miracle and Zanzibar. 3A6SM2M is found in two genomes each of Intokazi and Miracle; all four genomes of Desert Dawn show a 5A5SMIM combination, three of Cocktail show 3A4SM4M, two of Bold Leader show 2A4SM5M; two of Dutch Belle show 5A3SM3M and the other two show 5A2SM4M; 4A3SM4M is seen in only one genome of Tangerine and is therefore the rarest combination of all. Based on genome similarities, it can be concluded that Miracle, Intokazi and Zanzibar; Tangerine and Cocktail are more closedly related; Apple Blossom and Bold Leader show similarities in possessing one genome in common and the same number of ac rocentric chromosomes in the other two; Desert Dawn and Dutch Belle have only the number 40 Tasneem F. Khaleel, Susan Haven and Tim Gilg Cytologia 56
of acrocentric chromosomes in common in all genomes. These similarities are also reflected in the flower colour, number of flowers per scape and the fruiting pattern of some of these hybrids. Cocktail, Miracle, Tangerine and Zanzibar all have red flowers, at least three per scape; except Miracle they all flower-fruit and rest. Intokazi is the only hybrid with white flowers in this group; however, it resembles Miracle in its fruiting behaviour and Cocktail that has white tepal centers. Appleblossom and Bold Leader, show similarities in their genomes. These are seen not only in their possessing two diploid genomes of similar composition, but also in having relatively smaller chromosomes, (average chromosome length is 6.9ƒÊm in
Apple Blossom and 7.9ƒÊm in Bold Leader) lowest number of acrocentric chromosomes (8 in each) closer numbers of submetacentric (20 and 18) and metacentric (16 and 18) chromo somes and the total chromatin length (307ƒÊm and 350.7ƒÊm). Despite these common cy tological features they do not show any morphological similarities. Desert Dawn and Dutch Belle, however, share a common fruiting behaviour thereby reflecting the partial homologies of the constituent genomes. Of the nine hybrids, Appleblossom shows not only the largest number of small chromosomes (22) but also the lowest total chromatin length (307.6ƒÊm) while the karyotype in Dutch Belle consists of the largest number of very large chromosomes (28); the highest total chromatin length (502.5ƒÊm) however, is seen in Tangerine. There have been conflicting reports on the type of embryo sac development in Amaryl lidaceae. Shadowski (1925) reported an Allium type of embryo sac in Pancratium maritimum, while Brown (1951) described an Adoxa type in Zephyranthes texana. Dutt (1964, 1970) based on studies of Crinum species and a reinvestigation of Pancratium maritimum considered the previous reports to be erroneous and Polygonum type to be the norm of development in Amaryllidaceae. Khaleel and Siemsen (1989) reported some interesting features in the embryo sac development of four Amaryllis hybrids which not only clarified the earlier confusion but also shed light on the low fertility of the hybrids. According to Khaleel and Siemsen (1989) the megasporocyte undergoes meiosis I to form dyads. The chalazal dyad member is more precocious in development and meiosis II in this dyad member is not always accompanied by cytokinesis, therefore occasionally leading to a bisporic Allium type of development. Both linear and T-shaped tetrads were observed in their study. All four hybrids showed 30-40 pollen sterility and degeneration of embryo sacs at various stages of development which in their opinion accounted for the low percentage or lack of seed set. The present study confirms the previous observations of Khaleel and Siemsen (1989). All nine hybrids showed 20-40% pollen sterility. Polygonum as well as Allium type of female gametophyte development was observed with the former being the most frequent type in all hybrids. All hybrids showed 80-95% degeneration of female gametophytes at the two or four-nucleate stage. Apple Blossom and Dutch Belle showed the maximum degeneration and highest percentage of pollen sterility; Cocktail and Zanzibar were next, followed by Desert Dawn, Tangerine , Intokazi, Bold Leader and Miracle. This is correlated to the percentage and behaviour of fruit set in these hybrids.
Summary
Karyotype analysis, genome analysis and development of gametophytes is described for nine hybrids of garden Amaryllis. The basic chromosome number is x=11 and all nine hybrids are tetraploid with a bimodal distribution favoring long and short lengths . The basikaryotype proposed by Narain and Khoshoo (1968) which consists of two median, five submedian and four subterminal chromosomes, is not traceable in any of the nine hybrids . 2A5SM4M and 4A4SM3M are the most common basikaryotypes in six out of nine hybrids . All hybrids show altered karyotypes. Chromosomes range from 4 .3-17.7ƒÊm in length. The total chromatin 1991 Karyomorphology of Amaryllis Hybrids 41
ranges from 307.6ƒÊm-502.5ƒÊm. Bivalents, trivalents and quadrvalents are formed during meiosis. About 20-40% of the pollen grains are sterile. Development of the megagameto phyte is monosporic in 90% of the ovules and bisporic in the remaining 10%. All nine hybrids show 80-90% degeneration of embryo sac at various stages of development . The fruiting behaviour and low percentage of seed set are associated with pollen sterility, and degeneration of embryo sacs.
Acknowledgements
This work was supported by an operating grant from Eastern Montana College . We thank Arlene Dreezen for growing hybrids in the greenhouse and Dave Simonson for as sistance.
Literature cited
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