Genome size for the species of Nerine Herb. - Springer Link

Pl. Syst. Evol. 257: 251–260 (2006) DOI 10.1007/s00606-005-0381-x

Genome size for the species of Nerine Herb. (Amaryllidaceae) and its evident correlation with growth cycle, leaf width and other morphological characters B. J. M. Zonneveld1 and G. D. Duncan2 1

Institute of Biology, Leiden University, Clusius Laboratory, Leiden, The Netherlands South African National Biodiversity Institute, Kirstenbosch Botanical Garden, Claremont, Cape Town, South Africa 2

Received August 8, 2005; accepted September 12, 2005 Published online: December 29, 2005 Springer-Verlag 2005

Abstract. Nuclear DNA content (2C) is used as a new criterion to investigate nearly all species of the genus Nerine Herb. The species have the same chromosome number (2n = 2x = 22), with the exception of three triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 18.0–35.3 pg. This implies that the largest genome contains roughly 2 · 1010 more base pairs than the smallest. The species, arranged according to increasing genome size, fell apart in three groups if growth cycle and leaf width were also considered. A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species, a broad-leaved winter growing group with four species has a DNA content from 25.3–26.2 pg and a broad-leafed summer growing group has a DNA content of 26.8–35.3 pg and contains six species. If the presence of filament appendages and hairiness of the pedicels were also considered, the thirteen evergreen species could be further divided into a group without filament appendages or hairy pedicels with a DNA content of 18.0–18.7 pg. A second group without filament appendages but with hairy pedicels had a DNA content of 19.7–22.3 pg. And a third group with both filament appendages and

hairy pedicels had a DNA content of 22.0–24.6 pg. The exception is N. marincowitzii that, despite a low DNA content and narrow leaves is summer growing. The broad-leafed group is further characterised by the absence of filament appendages and the absence of strongly hairy pedicels. The exception here is N. pusilla that, despite a high DNA content, has narrow leaves and minutely hairy pedicels. Nuclear DNA content as measured by flow cytometry is shown to be relevant to throw new light on the relationships between Nerine species. Key words: Nerine, Taxonomy, DNA content, genome size, flow cytometry.

Introduction Nerine Herb. (Amaryllidaceae) is a bulbous perennial closely related to Brunsvigia Heist. and Hessea Herb. They range from the Cape Peninsula to the northwest and northeast of South Africa, Botswana, Lesotho, Namibia and Swaziland. The first description was of N. sarniensis as Narcissus japonicus rutilo flore

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B. J. M. Zonneveld and G. D. Duncan: Genome size for the species of Nerine Herb.

in 1635 by J. Cornut. This was based on a plant of unknown origin which flowered in the garden of J. Morin in Paris in October 1634. In 1753 Linnaeus included this species under the name Amaryllis sarniensis, and in 1820 the genus Nerine was established by Herbert. During the ensuing period the number of species grew steadily, and in 1967 Traub published a scholarly review of the genus in which thirty species were recognised. This was followed by a fine review of Norris (1974) based on extensive field knowledge recognising 31 species. Based on an intimate knowledge of the species in culture Duncan (2002a, b, c) recognised 25 species. Nerine is gaining more and more horticultural interest for garden and pot use and large numbers are grown for the cut flower trade. The basic chromosome number is 11 (2n=22) but rarely plants with 2n=24 and triploids are encountered (Traub 1967). The main morphological characters used in Nerine taxonomy are, the presence of appendages to the filament bases, the presence of hairs on the scape, pedicels and ovary, and the shape and arrangement of the perianth segments. The latter is shown not to be very relevant. Holoploid (C-value) or monoploid (Cx-value) genome size (Greilhuber 1979, Greilhuber et al. 2005) have been receiving more attention in recent years (Bennett 1972, Ohri 1998). Flow cytometry was successfully used to investigate the genera Hosta (Zonneveld and Van Iren 2001), Helleborus (Zonneveld 2001), Galanthus (Zonneveld et al. 2003), Agapanthus (Zonneveld and Duncan 2003), Clivia (Zonneveld 2003) and Gasteria (Zonneveld and van Jaarsveld 2005). When species in a genus have the same chromosome number, as in Nerine, differences in nuclear DNA content have proven to be very effective in delimiting infrageneric divisions in a number of taxa (Ohri 1998). Moreover it is shown (Greilhuber 1998) that species vary less than often assumed and the so-called ‘fluid genome’ seems not very well founded. In this study the total amount of nuclear DNA as measured by flow cytometry was introduced as a novel criterion in Nerine. In 81 accessions the

range of DNA involved is determined and it is shown that total amount of DNA is a very useful criterion to delimitate the species. Based on the amount of nuclear DNA found and all other relevant taxonomic criteria as published (Traub 1967, Norris 1974, Duncan 2002a) it is concluded that there are 23 species. Materials and methods Plant material. Leaf material was obtained from the Kirstenbosch Botanical Garden via one of the authors (G. Duncan). The species are maintained as a living collection in the Kirstenbosch Botanical Garden and vouchers are deposited in the Compton Herbarium (NBG) at Kirstenbosch, Cape Town, RSA. Flow cytometric measurement of nuclear DNA content. For the isolation of nuclei, about 0.5 cm2 of adult leaf was chopped together with a piece of Agave americana L. as internal standard. The nuclear DNA content (2C-value) of A. americana was measured as 15.9 picogram (pg) per nucleus with human leukocytes (=7 pg, Tiersch et al. 1989) as the standard. The chopping was done with a new razor blade in a Petri dish in 0.25 ml nucleiisolation buffer, with 0.01 % RNAse added, as described by Johnston et al. (1999). After adding 2 ml Propidium Iodide (PI) solution (50 mg PI/l in isolation buffer) the suspension with nuclei was filtered through a 30 lm nylon filter. The fluorescence of the nuclei was measured, 30 and 60 min. after addition of PI, 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. For each clone, two to twelve different runs (determinations) with at least 3000– 5000 nuclei were measured with two runs from a single nuclear isolation. Nerine turned out to be favourable material for flow cytometry and most histograms revealed a CV below 5 %.

Results and discussion DNA content of the species. In Table 1 the results are shown of the nuclear DNA measurements (2C-value) of 81 accessions,


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Table 1. Alphabetical list of Nerine species with accession number, locality or source, collector, nuclear DNA content (2C) with standard deviation, average for the species and number (n) of determinations Nerine species

2C DNA st.dev. average n in pg

acc. #

Locality

Collector

N. angustifolia Baker N. angustifolia Baker N. angustifolia Baker N. appendiculata Baker N. appendiculata Baker N. appendiculata Baker N. bowdenii W.Watson N. bowdenii W.Watson N. bowdenii W.Watson N. bowdenii W.Watson N. duparquetiana Baker N. filamentosa W.F. Barker N. filamentosa W.F. Barker N. filifolia Baker N. filifolia Baker N. filifolia Baker N. filifolia Baker N. filifolia Baker N. filifolia Baker N. frithii L.Bolus N. gaberonensis Bremek.&Oberm. N. gibsonii K.H.Douglas N. gracilis R.A.Dyer N. gracilis R.A.Dyer N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. humilis (Jacq.) Herb. N. krigei W.F.Barker N. krigei W.F.Barker N. krigei W.F.Barker N. krigei W.F.Barker N. krigei W.F.Barker

23.3 23.4 23.9 22.6 22.5 23.3 34.3 35.4 35.8 35.8 27.5 19.8 19.7 20.4 21.5 20.6 20.2 20.8 23.6 23.1 18.0

0.9 0.7 0.7 0.3 0.8 0.4 0.7 0.6 0.4 0.7 0.6 0.2 0.3 0.3 0.3 0.9 0.4 0.3 0.4 0.3 0.6

23.5

10 10 9 7 10 7 10 10 10 10 23 8 11 4 10 8 9 7 11 10 8

184/86 367/02 1052/82 877/75 135/86 44/79 1158/82 73/80 789/86 280/83 748/84 50/80 148/85 426/86 77/80 47/80 1049/82 75/80 449/66

Bosberg Harrismith Mt Anderson Harrismith Nkwezula Van Reenen Ex Hort. Weza Forest The Witches Sentinel Peak Ex Hort. Cathcart Cathcart Berlin E. Cape Athertone’s Kl. Stutterheim Ex Hort. Stanhoek Eastern Cape Malmans Oog Kakamas

McMaster s.n. Roux 3347 Schuur s.n. Schelpe s.n. Nichols 904 vd Zeyde s.n. Duncan Roux 628 NBI Staff Lavranos 20563 Kirstenbosch Roux 534 McMaster s.n. McMaster s.n. Roux 686 Roux 528 Ginsberg. Roux 527

22.0 24.6 24.7 24.7 24.6 24.9 24.9 24.9 24.6 24.7 24.9 25.2 25.6 25.8 25.5 26.1 26.0 26.1 26.1 31.0 32.2 32.3 33.2 33.4

0.6 0.3 0.5 0.4 0.4 0.2 0.3 0.2 0.5 0.5 0.4 0.5 0.5 0.4 0.6 0.4 0.3 0.6 0.5 0.2 0.3 0.2 0.5 0.6

22.0 24.6

9 8 6 8 8 8 8 8 8 8 8 8 8 8 8 10 8 8 8 4 6 4 10 10

290/76 366/02

Lady Frere Devon,Gauteng Ex Hort. 461/59 Montagu 374/02 Vanwijksdorp 1045/82 Montagu 373/02 Ladismith 504/71 Meiringspoort 392/53 Du Toits kloof 521/72 Bredasdorp 1044/82 Robertson 1048/82 Langvlei 57/84 Ex Hort. 747/70 Worcester 380/04 Miaspoort 378/04 Du Toitskloof 719/91 Porterville 782/83 Scholtzberg 781/83 Geelhoutboskl. s.n. Pelindaba 221/99 Okavango? s.n. Rustenburg 567/65 Ex Hort. 878/75 Ex Hort.

McMaster s.n. Craib s.n. Duncan Leighton s.n. Harrower 523 Malan s.n. Harrower 490 Matthaei s.n. Compton s.n. Lewis s.n. Winter 138 Winter 142 Duncan Bayer s.n. van Eeden s.n. Duncan 488 Duncan 347 v.Jaarsveld7733 v.Jaarsveld714 Nieuwoudt s.n. Lotter s.n. Craib s.n. Bayliss 2591 Schelpe s.n.

22.8

35.3

27.5 19.7 20.7

23.6 23.1 18.0

25.3

32.4

674/86

Craib s.n. Duncan 249

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Table 1. (Continued) Nerine species

2C DNA st.dev. average n acc. # in pg

Locality

Collector

N. laticoma T.Durand&Schinz. N. laticoma T.Durand&Schinz. N. laticoma T.Durand&Schinz. N. laticoma ssp. huttoniae Schonland N. laticoma ssp. huttoniae Schonland N. laticoma ssp. huttoniae Schonland N. marincowitzii Snijman N. masoniorum L.Bolus N. masoniorum L.Bolus N. pancratioides Baker N. platypetala G.McNeil N. pudica Hook.f. N. pudica Hook.f. N. pudica Hook.f. N. pusilla Dinter N. rehmannii (Baker) L.Bolus N. rehmannii (Baker) L.Bolus N. ridleyi Phillips N. ridleyi Phillips N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. sarniensis (L.) Herb. N. undulata (L.) Herb. N. undulata (L.) Herb. N. undulata (L.) Herb. N. undulata (L.) Herb. N. undulata (L.) Herb. N. undulata (L.) Herb. (N. flexuosa)

26.6 26.9 27.0 27.3

0.5 0.3 0.5 0.3

10 6/96 10 206/87 7 49/03 8

Assen Kuruman Vryburg Kom.Drift Dam

Craib s.n. Lavranos 24350 Difoloko s.n. McMaster s.n.

26.6

0.4

10 259/86

Ex Hort.

26.6

0.1

14 114/86

Ex Hort.

Viviers

18.7 22.5 22.9 21.9 22.3 25.8 26.3 26.5 28.0 18.2 17.8 26.1 39.5 25.4 25.4 25.6 25.6 25.7 25.8 26.0 26.0 26.2 39.6 39.7 27.9 28.1 28.2 28.4 28.6 29.0

0.2 0.4 0.8 0.6 0.8 0.6 0.6 0.7 0.5 0.1 0.5 0.7 1.2 0.5 0.6 0.4 0.1 0.5 0.6 0.6 0.5 0.6 0.8 1.3 0.4 0.7 0.1 0.5 0.9 0.6

8 10 10 9 9 6 6 4 8 6 8 10 4 2 2 2 2 2 2 2 4 2 2 2 14 14 6 14 10 14

Leeu Gamka Mqanduli Umtata Mt. Currie Wakkerstroom Worcester Greyton Worcester Gobabis Namib. Belfast Mpum. Johannesburg Fonteintjiesberg Hex River Mnts Greyton Langeberg Betty’s Bay Ceres Genadendal Witzenberg Stellenbosch Table Mountain Table Mountain Guernsey, U.K. Ceres Stutterheim Bedford Engcobo Tiose River Grahamstown Somerset East

Vlok s.n. Mason s.n. Winter 483 Scott s.n. Craib s.n. Karoo Gardens Foster s.n. Perry s.n. Gildenhuys s.n. Wentzel s.n. Craib s.n. Winter 130 Helme 2900 Calitz s.n. Winter 271 Burrows s.n. Winter 437 Chaplin s.n. Barker 8877 Duncan 122 Kirstenb. White Duncan 322 Hort. le Pelley Hall s.n. McMaster s.n. McMaster s.n. Gilson s.n. Roux 533 Stayner s.n. McMaster s.n.

26.9

26.8

18.7 22.7 21.9 22.3 26.2

28.0 18.0 26.2 25.9

28.4

comprising 23 species. These were classified as species based on their morphology and geography (Duncan 2002a). Growth cycle and leaf diameters turned out to be important characters as is shown by our results (Table 1,

4/96 828/20 733/96 1050/82 172/94 004/95 1046/82 108/83

1141/82 374/04 692/88 668/82 302/69 662/90 105/44 1047/82 643/84 193/79 212/89 1067/82 192/49 78/80 173/85 1069/60 76/80 1507/70 172/85

Table 2). It cannot be excluded that the nearly 1 pg difference between the lowest and highest DNA content for accessions found within a species like N. humilis is due to the presence or absence of B chromosomes that might con-


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Table 2. List of Nerine species arranged with increasing nuclear DNA content (2C), with standard deviation, numbers (according to Fig. 1 and Fig. 2), growth cycle (Duncan 2002a), leaf width, presence of filament appendages and hairy pedicels, section numbers according to Traub (1967) and group numbers according to Norris (1974) Number Nerine species

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18a 18 19 20 21 22 23

GROUP A N. gaberonensis N. rehmannii N. marincowitzii GROUP B N. filamentosa N. filifolia N. pancratioides N. platypetala GROUP C N. gibsonii N. masoniorum N. appendiculata N. frithii N. angustifolia N. gracilis GROUP D N. humilis N. sarniensis N. pudica N. ridleyi GROUP E N. l. ssp. huttoniae N. laticoma N. duparquetiana N. pusilla N. undulata N. krigei N. bowdenii

2C DNA st. grow cycle in pg dev. Duncan

leaf filament hairy section group width append. pedicels Traub Norris

18.0 18.0 18.7

0.6 0.3 0.2

Evergreen Evergreen Summer gr.

1–2 1 3–4

no no no

no no no

4 3 —

7 7 —

19.7 20.7 21.9 22.3

0.0 0.5 0.6 0.8

Evergreen Evergreen Evergreen Evergreen

1 1–2 2–3 2–4

no no no no

yes yes yes yes

3 3 4 —

8 8 9 9

22.0 22.8 22.8 23.1 23.5 24.6

0.6 0.3 0.4 0.3 0.3 0.0

Evergreen Evergreen Evergreen Evergreen Evergreen Evergreen

2 1 2 2 2–3 1–2

yes yes yes yes yes yes

yes yes yes yes yes yes

— 4 4 4 3 4

11 11 11 12 10 12

25.3 25.9 26.2 26.2

0.6 0.3 0.4 0.9

Winter Winter Winter Winter

6–18 15–37 7–11 25–37

no no no no

no no no no

3 2 3 3

5 2 6 4

26.8 26.9 27.5 28.0 28.4 32.4 35.3

0.4 0.2 0.5 0.5 0.4 1.0 0.7

Summer gr. Summer gr. Summer gr. Summer gr. *Summer gr. Summer gr. Summer gr.

14–20 12–18 12–18 1–2 7–14 8–30 13–23

no no no no no no no

no no **yes **yes no no no

1 1 1 3 3 1 3

3 3 3 7 5 3 5

gr. gr. gr. gr.

*summer and winter growing, ** only minutely/sparsely hairy

tribute to the total DNA value. The plants found occasionally with 24 chromosomes (Traub 1967) are probably due to splitting of a chromosome pair and as such don’t contribute to the total amount of nuclear DNA. However, chromosome counts in combination with nuclear DNA content are needed to corroborate these suggestions. In these mainly wild-collected plants, we found two plants of N. sarniensis and one of N. ridleyi with 50% more DNA that are presumed to be triploids.

The number of species. The amount of DNA per somatic nucleus (2C) and earlier morphological and geographical arguments (Duncan 2002a) determined our number of species at 23. Maintaining all the subspecies as recognised or introduced by Traub (1967) seems not very fruitful. Groups of species. The recognised species were arranged according to their nuclear DNA content (Table 2). This nicely correlated with several morphological parameters and with the

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three growth cycles as observed in temperate climates. This might indicate, as already suggested by Grime (1998), an important role of genome size in a functional classification of plants. This could consequently predict the response of vegetation to regional and global changes in climate. It does not correlate well with the four sections of Traub (1967), based mainly on filament characters and scape length as his section 3 is found in all five groups as proposed here. The twelve groups of Norris (1974) seem to fit much better, provided several are taken together, but his group 5 and 7 is found in two of our groups. As shown in Table 2, plants with narrow leaves (1– 4 mm) have 2C-values of 18.0–24.6 pg and were thus separated from the broad leafed plants (6–37 mm) that all have higher 2Cvalues ranging from 25.3–35.3 pg. Based on this, the species can be divided into three main groups as already done by Duncan (2002a). A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species. A broad-leafed, winter growing group (Group D) with four species has a DNA content from 25.3–26.2 pg and the six species of the broad leafed, summer growing group (Group E) have a DNA content of 26.8– 35.3 pg. The broad-leafed group is further characterised by the absence of filament appendages. Five of the seven species have glabrous pedicels, and in the two species with hairy pedicels the hairs are very minute or sparsely present. The thirteen evergreen species can be divided further into three groups by taking other morphological characters into account too. Three species without filament appendages and with glabrous pedicels have a DNA content of 18.0–18.7 pg (Group A). A group of four species without filament appendages but with hairy pedicels have a DNA content of 19.7–22.3 pg (Group B) and six species with both filament appendages and hairy pedicels have a DNA content of 22.0– 24.6 pg (Group C). Two of the 23 species don’t fit this nice pattern. The recently described N. marincowitzii (Snijman, 1995), despite a low DNA content and narrow leaf, is summer

growing. Compared to the other twelve evergreen, narrow-leafed plants it is one of the most south-westerly growing species, actually growing close to the area of the winter growing plants (Fig. 1, Fig. 2). N. marincowitzii could therefore be a hybrid between a winter growing species and an evergreen plant, but the genome size seems to exclude this, at least as a recent event. In the broad-leafed, high DNA group the very rare N. pusilla has, despite a high DNA content, narrow leaves. These narrow leaves could be a secondary adaptation to its habitat in Namibia. It also has pedicels that are only minutely hairy. This could be a similar adaptation as found in N. duparquetiana that also has minutely hairy pedicels and also occurs in Namibia. The high pollen fertility of 97 % seems to exclude that the measured N. pusilla is a triploid. N. duparquetiana was described by Baker (1896) and discussed in more detail by Dyer (1951) and Norris (1974), but considered by Obermeyer (1993) to be synonymous with the glabrous N. laticoma. It is restored here to species status based on its fewer-flowered umbels of larger flowers with much narrower perianth segments that are prominently arching and strongly recurved in the upper part, its hairy pedicels, and geographical isolation in central and northern Namibia and western and eastern Botswana. The cytological structure of N. duparquetiana and N. laticoma was investigated by Gouws (1949), and his findings also support the maintenance of both species. Nerine huttoniae occurs in the southeastern parts of the Eastern Cape. It was described by Scho¨nland in 1903 and further discussed by Dyer (1952) and Norris (1974). Traub (1967) treated it as a subspecies of N. laticoma but Obermeyer (1993) restored it to species level. The only real morphological difference between this species and N. laticoma is that N. huttoniae has fleshy scales at the base of its filaments, and geographically it is well separated from N. laticoma. We concur with Traub’s treatment and it is here restored to subspecific status. A special case is a collection of N. filifolia (449/66) from the Eastern Cape. It


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Fig. 1. The main localities of the evergreen species of Nerine altered after Norris (1974). Group A : 1: N. gaberonensis. 2: N. rehmannii. 3: N. marincowitzii. Group B : 4: N. filamentosa. 5: N. filifolia. 6: N. pancratioides. 7: N. platypetala. Group C : 8: N. gibsonii. 9: N. masoniorum. 10: N. appendiculata. 11: N. frithii. 12: N. angustifolia. 13: N. gracilis

is morphologically indistinghuisable from the other N. filifolia collections investigated, but has nearly 3 pg more DNA. It could be an aneuploid, however it has a good pod set. Two species mentioned by Duncan (2002a) were not available for study. N. transvaalensis is a doubtful species, related to N. frithii, of which the exact location is unknown. It has not been found again since its original description in 1928. N. hesseoides grows in an area overlapping that of the closely related N. frithii. It differs from N. frithii by the differently shaped stamen appendages, and its much smaller flowers are said to have actinomorphic perianth segments (Bolus 1938). Its flowers are the smallest in the genus and are of all nerines most like those of Hessea. It could be a subspecies or variety of N. frithii. Evolutionary considerations. Kalender et al. (2000) suggested that in dry, growth-limited situations species adapt over evolutionary time with an increase in DNA content and a decrease

in permanently wet situations. This was confirmed for Hosta (Zonneveld and van Iren 2001), and seems also to be true for Agapanthus africanus that grows in dry and hot locations (Zonneveld and Duncan 2003), but not for Galanthus (Zonneveld et al. 2003). Nerine also seems to fit this course as from a supposed area in eastern South Africa where mainly evergreen plants occur, an increase in DNA is found both extending to the north with dry winters, and to the south-west with dry summers. DNA content does not give direct clues about evolution. However its correlation here with the grow cycles is striking, suggesting that plants with similar nuclear DNA contents are related. This was found earlier for e.g. the genera Hosta (Zonneveld and van Iren 2001), and Helleborus (Zonneveld 2001). Assuming an increase in DNA content only, the following can be envisioned (Fig. 1, Fig. 2). From a supposed proto-Nerine, Group A arose with the lowest amount of DNA. This amount of

258


Fig. 2. The main localities of the winter and summer growing species of Nerine altered after Norris (1974). Group D (winter growing, heavily shaded): 14: N. humilis. 15: N. sarniensis. 16: N. pudica. 17: N. ridleyi. Group E (summer growing): 18: N. laticoma. 18a: N. laticoma ssp. huttoniae . 19: N. duparquetiana. 20: N. pusilla. 21: N. undulata. 22: N. krigei. 23: N. bowdenii

DNA gradually increased, resulting in Group B, acquiring hairy pedicels at the same time. A further increase in DNA in Group C also was accompanied by the formation of appendages to the filaments. Then two possibilities present themselves for the broad-leafed groups. Going further south for the winter growing group and further north for the summer growing group, broad-leafed, high DNA groups arose from Group C by also losing the appendages and the hairy pedicels. Or, alternatively, they arose separately from Group A, adapting to summer or winter growing conditions. However, a very strong increase in DNA of 7–17 pg must then be envisioned. It must also be remarked that two taxa of the summer growing group (N. krigei and N. laticoma ssp. huttoniae) occur in the same area of eastern South-Africa as the evergreen group, but occupy different, drier niches. Parallels can be seen in the spread of Agapanthus (Zonneveld and Duncan 2003)

and Gasteria (Zonneveld and van Jaarsveld 2005) in which a spread to the north and south-west also resulted in an increase in DNA. It would be interesting to compare the spread of these three genera in relation to their genome size in more detail, but that is beyond the scope of this article. Another anomaly is seen in certain forms of N. undulata that lose their leaves for a short period in midwinter under temperate conditions, but new growth starts shortly afterwards. This is contrary to the other summer growing species that sprout again later in spring. A form of the normally deciduous Agapanthus inapertus from Fraser Falls in the Eastern Cape behaves similarly. It seems to be a summer growing plant that is in the process of acquiring the evergreen habit (again). The ‘anomalous’ pedicels of N. pusilla and N. duparquetiana are only minutely/sparsely hairy, also suggesting a secondary adaptation.


The above conclusions are based on the available data collected. The DNA content confirms several relationships that were already suggested by Norris (1974) and Duncan (2002a) based on morphological and geographical arguments. The results give a firm footing to what were earlier educated guesses. Each species has a certain amount of DNA, which is of systematic value. Flow cytometry is used, combined with morphological and geographical data, to delimitate the species, to show relationships between members of the genus Nerine and to show the presence in three of the investigated plants of triploidy. The authors would like to thank Eloy Boon who kept the flow cytometry apparatus going, P. Hock for drawing Fig. 1 and Fig. 2. and the Kirstenbosch Botanical Garden for their generous supply of plant material.

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Address of the authors: Ben J. M. Zonneveld (e-mail: [email protected]), Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarse Weg 64, 2333 AL Leiden, The Netherlands. Graham D. Duncan, South African National Biodiversity Institute, Kirstenbosch Botanical Garden, Private bag X7, Claremont 7735, Cape Town, South Africa.