The involvement of Narcissus hispanicus Gouan in the origin of Narcissus bujei and of cultivated trumpet daffodils ( Amaryllidaceae ) by

Zonneveld, B.J.M. 2010. The involvement of Narcissus hispanicus Gouan in the origin of Narcissus bujei and of cultivated trumpet daffodils (Amaryllidaceae). Anales Jard. Bot. Madrid 67(1): 29-39. To investigate the origin of cultivated trumpet daffodils the genome size (2C-value) of more than 100 old and new cultivars were measured. A large number of concolored yellow tetraploid cultivars with large coronas seem to be derived from a doubling of chromosomes of a hybrid of the two species N. hispanicus Gouan and N. pseudonarcissus L. This can also still be recognized by the presence of a black spot at the top of the anthers in about 15 % of the tetraploid cultivars. Assuming N. pseudonarcissus as one of the parents of the allotetraploid cultivars, species of trumpet daffodils of section Pseudonarcissi investigated earlier were compared. Doubling of the nuclear DNA content of 23.8 pg of N. pseudonarcissus falls short of the values found for the trumpet daffodils. Species of trumpet daffodils of section Pseudonarcissi with a complementing higher amount of nuclear DNA like N. hispanicus with 25.8 pg, N. poeticus L. with 26 pg and the hexaploid N. pseudonarcissus ssp. bicolor (L.) Baker with 67.7 pg (Zonneveld,2008) and other species were evaluated. N. hispanicus with 25.8 pg clearly differs from N. pseudonarcissus with 23.8 pg and is accepted here also as a species. The nuclear DNA content of N. bujei (Fern. Casas) Fern. Casas with 30 pg fits with the (ancient) hybrid origin for N. bujei between N. longispathus Pugsley (36 pg) and N. hispanicus Gouan (25.8 pg). The tetraploids with white tepals and yellow corona’s can be obtained by crossing a diploid N. pseudonarcissus with the hexaploid and bicolored N. pseudonarcissus ssp. bicolor (L.) Baker.


Introduction
Daffodil culture started with the import of large numbers of bulbs collected from the field in the 16 th century.Especially the large concolored yellow N. hispanicus (Gouan, 1773) was imported and consequently likely became exterminated in southern France and most of the Pyrenees and later also most of Spain.From these in culture at first triploids originated at the end of the 19 th century (Wylie, 1952) with N. 'Golden Spur' found in the Estate Bakkershage in Wassenaar, The Netherlands (De Mol, 1923).At roughly the same time the triploids N. 'Emperor' and 'Empress' origi-nated with Backhouse UK (Wylie, 1952).At the start of the 20th century the first tetraploids became available (N. 'King Alfred' 1899, N. 'van Waverens Giant', 1900) and since that time the tetraploid trumpet daffodils have taken over the market.
Previous work in Hosta Tratt.(Zonneveld & Van Iren, 2001), Galanthus L. (Zonneveld & al., 2003a), Agapanthus L'Hér.(Zonneveld & Duncan, 2003b), Nerine Herb (Zonneveld & Duncan, 2006), Narcissus L. (Zonneveld, 2008) and Tulipa L. (Zonneveld, 2009) has shown that nuclear DNA content of species and cultivars can contribute to the taxonomy of species and the origin of hybrids.The N. pseudonarcissus L. complex has given rise in nature to many forms now accommodated in eight subspecies (Zonneveld, 2008) The taxonomy of all species of Narcissus (Amaryllidaceae) has been investigated recently by flow cytometry (Zonneveld, 2008).The somatic nuclear DNA contents (2C) were shown to range from 14 to 38 picogram for the diploids.The total number of Narcissus species was determined as 36, nine more than in Flora Europaea and they were divided up in two subgenera and eleven sections.Section Pseudonarcissi DC was much more heterogeneous in nuclear DNA content than expected.Sixty five accessions of N. pseudonarcissus possessed, with 23.8 pg, similar amounts of DNA.However several species from this section were clearly distinctive in nuclear DNA content.It runs from the diploid N. primigenius Fern.Casas with 21.7 pg to the also diploid N. nevadensis Pugsley with 38.2 pg.Also N. cyclamineus DC, N. abscissus (Haw.)Schult.f. and N. moleroi Fern.Casas are with about 26 pg clearly different from N. pseudonarcissus.For the first time, in eleven accessions, hexaploidy was found in N. pseudonarcissus ssp.bicolor.A new section Nevadensis Zonn.with 30-39 pg of nuclear DNA was split off from the section Pseudonarcissi with now 21-27 pg.
The aim of the present study was the desire to know whether nuclear DNA value was of use in determining the origin of trumpet daffodils and of the controversial Narcissus bujei (Fern.Casas) Fern.Casas.This is of value for the taxonomy of Narcissus and also for improving commercial varieties.Moreover, important pharmacological substances like galanthamines are found in daffodils (Codina, 2002) making it worthwhile to be able to discriminate between the species and show the origin of the main groups of cultivars.

Plant material
Plant material of the species was mainly obtained from the collections of J. Blanchard (UK) and D.
Origin of Narcissus bujei and trumpet daffodils Donnison-Morgan (UK).However, most cultivars were obtained from The Netherlands (KAVB Lisse, Hortus Bulborum Limmen, C.P. Breed, C. van der Veek, H. Meeuwissen, W. Lemmers, and S. de Groot) and a few from the UK (J.M. Grimshaw, M. Salmon and I. Young), from France (L. de Jager), from Germany (G.Knoche) and from the USA (N.Wilson and H. Koopowitz).Care was taken to ensure correct identification of all material, relying also on the expert opinions of J. Blanchard, D. Donnison-Morgan and S. de Groot.In most cases fresh leaves were used from the wild or available in the different collections.

Determination of nuclear DNA content
For the isolation of nuclei, about 0.5 cm of a fresh full-grown leaf was chopped together with a piece of Agave americana L. 'Aureomarginata' as internal standard.The nuclear DNA content (2C-value) of A. americana was measured as 15.9 picogram (pg) per nucleus with human leukocytes (2C = 7 pg, Tiersch & al., 1989) as the standard.The chopping was done with a new razor blade in a Petri dish in 0.25 ml nuclei-isolation buffer, with 0.01 % RNAse added (Zonneveld & Van Iren, 2001b).After adding 1.8 ml propidium iodide solution (50 mg/l in isolation buffer) the suspension with nuclei was filtered through a 30 µm nylon filter.The fluorescence of the nuclei was measured, 30 and 60 min.after addition of propidium iodide, using a PARTEC CA-II flow cytometer.The more DNA is present in a nucleus, the higher is the intensity of the fluorescence.The 2C DNA content of the sample was calculated as the sample peak mean, divided by the Agave peak mean, and multiplied with the amount of DNA of the Agave standard.From chopping of a leaf piece of 0.5 cm 2 about 50 000 nuclei could be isolated.For each clone, two to six different runs (determinations) with at least 3000-5000 nuclei were measured with two runs from a single nuclear isolation.

Results and discussion
In Table 2 nuclear genome size is presented for more than 60 trumpet daffodils.These were compared with the genome sizes of all species (Zonneveld, 2008, Table 1).It turns out that doubling of the nuclear DNA content of 23.8 pg of N. pseudonarcissus falls short of the values found for the trumpet daffodils (Table 3).Morphological similar looking species of trumpet daffodils of section Pseudonarcissi with a com plementing higher amount of nuclear DNA like N. hispanicus with 25.8 pg, N. poeticus with 26 pg and the hexaploid N. pseudonarcissus ssp.bicolor with 67.7 pg (Zonneveld, 2008) were selected and data on Table 2. Narcissus cultivars with their amount of nuclear DNA, average, standard deviation, chromosome number, origin and remarks.Chromosome counts are from Mol, 1923;Nagao, 1933;Fernandes & Fernandes, 1946;Fernandes & Neves, 1971;Sañudo, 1985, andBrandham &Kirton, 1985 these and other related species are here presented (Table 1).Species of other sections can be excluded as these have mainly short trumpets and/or result (with exception of N. poeticus) in sterile offspring.For comparison the results for 61 other cultivars are also given.
The establishment of N. hispanicus as a species In 2008 I found a Narcissus in fair numbers in the Parc Natural Los Quejigales in the Sierra de las Nieves amidst Abies pinsapo Boiss.forests that turned out to have a 2C-value of 25.8 pg.Other accessions from the same area have similar 2C-values (Table 1).An exception was a plant found 10 km west of Los Quejigales that with 39.3 pg clearly is a triploid.Meadows (1972) found N. hispanicus in a remote just opened spot in the Pyrenees, Val Cardos, Pleta de Tornapigol, but this was not available for study.This amount of on average 25.8 pg of nuclear DNA is clearly different from the 23.8 pg for N. pseudonarcissus.The plants from Los Quejigales conform with N. hispanicus as described by Gouan (1773).It is characterized by big bright yellow colored flowers with spiraled tepals, an ascending posture and a minute black spot on the anthers.In 1788 Curtis described the same species as N. major.Baker (1888) included it as a subspecies of N. pseudonarcissus.The term 'Major' or 'Maximus' was later used for (m)any large yellow daffodils.N. 'Hispanicus Maximus' in culture turns out to be a triploid (Table 1).Narcissus hispanicus 'Pinetorum' PB436 with 25.8 pg was found by P. Bird in Sierra de las Nieves and via M. Salmon grown by J. de Groot from seed.It is presumably the same plant as described by Smythies (1973) from the Sierra de las Nieves in southern Spain as growing amidst Abies pinsapo.According to Smythies (1973) Willkomm collected this species in 1861 in the Sierra de Yunquera (now Sierra de las Nieves) at 1350 m and considered them to be N. major.Webb (1980) considered N. hispanicus as a form of N. pseudonarcissus ssp.major.However Pugsley (1933), Fernandes (1968) and Fernandes Casas (1986) regarded N. hispanicus as a good species.Moreover, Graham & Barrett (2004) could separate N. hispanicus from N. pseudonarcissus in their phylogeny of Narcissus based on DNA sequences of two chloroplast genes.The arguments presented above supports species rank of N. hispanicus.Its origin seems to be the Pyrenees, Southern France (rare nowadays in both cases) and the Sierra de las Nieves in southern Spain.rra de Gredos, and from Embalse de Abraham, Sierra Higuera, described by Fernández Casas as allied to N. obvallaris (Henon) Pugsley are just plain N. pseudo narcissus with 23.8 pg (Zonneveld, 2008).Narcissus obvallaris Salisb.and N. gayi Salisb., both of doubtful origin, have an amount of nuclear DNA similar to N. pseudonarcissus.However they have a black spot on the anthers (Blanchard 1990;1998).So it could be suggested that occasionally N. pseudonarcissus has a black spot.More likely, they show the influence of N. hispanicus, although their amount of DNA does not reflect that anymore.
The N. bujei with 30.2 pg is described from the Serrania de Ronda, southern Spain.It is clearly separate in nuclear DNA content but hardly morphologically from both N. pseudonarcissus and N. longispathus or N. nevadensis.However, the latter two are characterized by having usually more than one flower to a stem.Narcissus bujei was first described as a variety of N. longispathus (N.longispathus var.bujei Fern.Casas: "amicissimo Eugenio Domínguez Vilches, familiariter nobis 'Buje', ex animo dicata", the orthography therefore is with an "j", not a "g"), later as a variety of N. hispanicus and then as a species all by the same author (Fernández Casas, 1986, "bugei"), who in 2000 changed his mind again and made it a subspecies of N. hispanicus.Both N. bujei and N. hispanicus are described as having bright yellow flowers, with spirally twisted tepals and leaves and a black spot on the anthers.These data fit with the (ancient) hybrid origin for N. bujei (30.2 pg) between N. longispathus (36 pg) and N. hispanicus (25.8 pg).

The origin of cultivated trumpet daffodils
In the most recent taxonomical treatment of Narcissus (Zonneveld, 2008) seven species are attributed to the section Pseudonarcissi DC. with a 2C-value of about 23.8 pg for all subspecies of N. pseudonarcissus.The calculations in Table 3 show that plain tetraploids from N. pseusonarcissus (23.8 pg) will have a 2C-value of 47.6 pg.The nuclear DNA content of the large trumpet daffodils have higher values (Table 2).This suggests that a species of trumpet daffodil with a higher amount of nuclear DNA is also involved in the formation of the trumpet daffodils that are grown nowadays.Three morphological similar species of trumpet daffodils of section Pseudonarcissi have a 2C-value of about 26 pg: N. abscissus Schult.f., N. moleroi Fern.Casas and N. hispanicus Gouan.N. abscissus has a cut-off corona, a character rarely encountered in today's daffodils, whereas the rare N. moleroi has pale colored flowers.So both seem hardly involved in today's breeding.Although N. cyclamineus DC. and N. poeticus L. from section Narcissus (Zonneveld, 2008) have a similar genome size of about 26 pg, they differ too much in flower morphology to con-  (Wylie, 1952).The varieties N. 'Emperor' and N. 'Empress' were also triploid.In 1890 tetraploids arose from them like N. 'King Alfred', that is supposed to be a hybrid of N. 'Empress' with N. hispanicus (Williams, 1929), and in The Netherlands the triploid N. 'Golden Spur' was followed by the tetraploid N. 'van Waverens Giant'.However, the plant in culture as N. 'Golden Spur' turned out to be diploid (Table 2).In these cases the involvement of N. hispanicus was already suggested at the time when they appeared on the market.
The cultivars mainly from Hortus Bulborum, Limmen, The Netherlands with 48.1 pg and with yellow tepals and trumpets most conveniently can be explained as being allotetraploids of N. pseudonarcissus and N. hispanicus.Autotetraploid N. pseudonarcissus would have a 2C-value of 47.6 pg and autotetraploid N. hispanicus would have a 2C-value of 51.6 pg.Counts of Melissa Reading and Boyce Tankersley in the USA and myself in the Hortus Bulborum of Limmen (NL) have shown that up to 15 % of the narcissi with large trumpets (division 1 and 2) have black spots at the tip of the anthers.These black spots, the bright yellow colors and upright posture shows the influence of N. hispanicus in today's daffodil breeding.

Daffodils with white tepals and with yellow trumpets
These have a 2C-value of 49.6 pg (Table 2) and have likely been derived from crosses between hexaploid N. pseudonarcissus ssp.bicolor from the Pyrenees and Southern France and/or ssp.leonensis (Pugsley) Ferd.
Casas & Laínz from Northern Spain and a diploid species, giving a tetraploid in the first generation.Both wild hexaploid daffodils have white tepals and large yellow trumpets.

Daffodils with pink or orange to red corona's
All tetraploid cultivars around with orange to pink corona's are derived from N. poeticus and especially from the tetraploid form N. poeticus 'Actaea'.Tetraploids with white tepals and pink trumpets with large corona's have on average a 2C-value of 49.4 pg.These are clearly derived from N. poeticus with a 2C-value of 26 pg or its tetraploid cultivar 'Actaea' with 52 pg and N. pseudonarcissus.Fernandes (1968a;1968b) remarks on the similarity of the karyotypes of N. poeticus of section Narcissus and section Pseudonarcissi.As the hybrid between these two seems to be the only fertile intersectional hybrid one may wonder whether or not these two sections should be united.This is also indicated by the results of Graham & Barrett (2006).The red or pink color in today's daffodil corona's that is exclusively derived from N. poeticus and the black spot on the anthers are both unique characters.However in the first case there was and still is a very strong selection for the red color.It is very unlikely anyone has given any attention to the minute black spot on the anthers.The black spots are absent from the investigated cultivars that show the red color of N. poeticus or the white tepals and yellow corona's derived from N. pseudonarcissus ssp.bicolor.
In Table 3 2C-values of different combinations of species are calculated.If these are compared with the actual data found (Tables 1, 3) and also taking the flower colors into account, it is clear that N. hispanicus played a role in the tetraploid yellow cultivars, N. poeticus in the cultivars with red or pink corona's and N. pseudonarcissus ssp.bicolor in the tetraploids with white tepals and yellow corona's.This is the first time that the difference in nuclear DNA content is shown between N. hispanicus and N. pseudonarcissus and is used to explain the origin of N. bujei and the yellow tetraploid cultivars.Also the DNA hexaploidy of N. pseudonarcissus ssp.bicolor and its use in obtaining tetraploid bicolored trumpet daffodils was not recorded earlier.These data allowed giving a firm footing to the supposed involvement of these species in the origin of the trumpet daffodils.
Plants from Zezere Portugal described by Fernandes as N. hispanicus, N. confusus Pugsley from Sie-

Table 1 .
Narcissus species with their amount of nuclear DNA, standard deviation, chromosome number and origin.Chromosome counts are from literature or derived from the genome size.

Table 3 .
Calculated genome sizes of Narcissus species, hybrids and cultivars compared with the actual measurements.siderthem as being involved with the origin of the yellow trumpet daffodils.This leaves us with N. hispanicus.N. hispanicus is characterized by big bright yellow colored flowers with spiraled tepals, an ascending posture and minute black spots on the anthers.Apart from the spiraled tepals these characters can be found back in today's cultivars.A plain tetraploid N. hispanicus (25.8 pg) will have a 2C-value of 51.6 pg, higher than any of the cultivated trumpet daffodils.This suggests that both N. hispanicus and N. pseudonarcissus are involved.The diploid plant from Sierra de las Nieves has a 2C-value of about 26 pg.The following can be envisioned: this mountain range is one of the few remaining places were N. hispanicus survived.N. hispanicus has been imported repeatedly in the old days.This might be due to the fact that it is not very well adapted to culture in N. Europe and it might resent the summer wetness and winter frosts.Despite that and also despite its spiraled tepals, N. hispanicus has several characters worthwhile to introduce.First it has big bright yellow colored flowers, whereas those of N. pseudonarcissus are usually whitish to pale yellow.Further, contrary to N. pseudonarcissus that usually has drooping flowers, the flowers have an ascending posture.The main character contributed by N.