SEED MORPHOLOGY AND CLASSIFICATION OF IMPATIENS ( BALSAMINACEAE )

The Balsaminaceae, a family consisting mostly of subsucculent annual or perennial herbs, is usually regarded as comprising two genera, the monotypic Hydrocera Blume ex Wight & Arn. and the prolific Impatiens L. with an estimated number of about 850 species (Grey-Wilson, 1980). The genus Impatiens is concentrated in mountainous regions of South-East Asia, South China, India and Africa. Like most genera of comparable or larger size Impatiens suffers from a rather confused taxonomy and the absence of recent comprehensive revisions. Studies on seed coat morphology have been carried out by several researchers. Wunderlich (1967), Corner (1976) and Kumar & Singh (1990) pointed out the significance of seed coat morphology in solving problems of classification and in phylogenetic considerations. The seed coat ornamentation in the genus Impatiens has been given attention since Hooker & Thomson (1859), but without comprehensive surveys. In the following account the seeds of 65 species and 10 sections of Impatiens in the sense of Warburg & Reiche (1895) are described and the contribution that seed coat may make to the taxonomy of the family is considered.


INTRODUCTION
The Balsaminaceae, a family consisting mostly of subsucculent annual or perennial herbs, is usually regarded as comprising two genera, the monotypic Hydrocera Blume ex Wight & Arn. and the prolific Impatiens L. with an estimated number of about 850 species (Grey-Wilson, 1980).The genus Impatiens is concentrated in mountainous regions of South-East Asia, South China, India and Africa.Like most genera of comparable or larger size Impatiens suffers from a rather confused taxonomy and the absence of recent comprehensive revisions.
Studies on seed coat morphology have been carried out by several researchers.Wunderlich (1967), Corner (1976) and Kumar & Singh (1990) pointed out the significance of seed coat morphology in solving problems of classification and in phylogenetic considerations.The seed coat ornamentation in the genus Impatiens has been given attention since Hooker & Thomson (1859), but without comprehensive surveys.In the following account the seeds of 65 species and 10 sections of Impatiens in the sense of Warburg & Reiche (1895) are described and the contribution that seed coat may make to the taxonomy of the family is considered.

MATERIALS AND METHODS
Seed samples of 65 species of Impatiens representing two subgenera, 10 sections (2 sections of subg.Acaulimpatiens and 8 sections of subg.Impatiens) taken from the field or herbarium specimens in Kanazawa University (KANA), Japan and the Herbarium Bogoriense, Bogor Indonesia (BO) (See Table 1).The samples were air dried, mounted, coated with gold and observed with SEM (AKASHI ALPHA) 30A at 15 kV and magnification 30-300.

Subgenus
Section Species Locality Voucher

RESULTS AND DISCUSSIONS
The Balsaminaceae have small-to medium-sized seeds, that are exarillate, exalbuminous and in most cases pachychalazal, the integument is not or scarcely multiplicative.The seed coat is formed by the expanded chalaza; the testa (outer or exotesta) is unspecialized or the cells have a papilla or a hair; the mesophyll is unspecialized, more or less crushed; the tegmen when present becomes crushed.The vascular bundle ends at the chalaza.The endosperm is cellular but absorbed.The embryo is straight (Corner, 1976).Parenchyma, tannin, crystal, mucilage, cork and sclerenchyma or collenchyma cells all contribute to the differentiation and build-up of the seed coat.The seed coat of Balsaminaceae usually contains crystals.However, the function of the crystals in the seed coat is unknown.They may serve as storage organs of waste products, defence against animal predators or, especially in the case of silicate crystals, assist in mechanical protection (Boesewinkel & Bouman, 1984).
In the last decade the SEM technique has opened an entire new field of micromorphological research on seed coats.Brisson & Peterson (1976) reviewed the use of SEM in the study of seed coats.They found that several features can be distinguished on the surface of the seed by SEM.Our study focuses on seed morphology of the genus Impatiens.The descriptions of the seed coats follow Grey-Wilson's (1980) and Barthlott's (1990) terminology.Scanning Electron Micrographs (SEM) of the representative seed coat types of Impatiens species examined are presented in Plate 1-4.This indicates that the subgenus may be divided at least into two sections, supporting  Bhaskar & Razi, 1972).Shimizu et al. (1990) reported two anther types in this subgenus: tetrasporangiate (I.acaulis and I. scapiflora) and trisporangiate (I.modesta Wight and I. stocksii).
The species I. acaulis and I. scapiflora have a seedling morphology of the 'third type' (Shimizu, 1982).Together seed coat morphology, seedlings, anther type and chromosome numbers support the classification given by Warburg & Reiche (1895).
The most interesting problems are to be found in subg.Impatiens in which the species show considerable diversity in their seed coat pattern.Eight sections were examined in this study.They are as follows: 1. Section Enantiophyllon Warb.This section is variable in seed coat patterns, which can be divided into two groups as shown in Plate 1c-f (I.chinensis L., I. gardneriana Wight, I. patula Craib, I. henslowiana Arn.).Group I is characterized by black globose seeds with a glabrous and shining surface as found in I. chinensis (Plate 1c), I. kleinii Wight & Arn., I. masoni Hook.f. and I. proctrata Hook.f.All species of this group share opposite or whorled leaves, but differ in seedling morphology.The seedling morphology of I. chinensis belongs to 'subgroup F', while I. masoni is assigned to 'subgroup H' (Shimizu, 1982).The chromosome number of I. chinensis and I. kleinii is 2n = 16 (Shimizu, unpubl.;Zinov'Eva-Stahevitch, 1981).Group II: Seeds densely covered with long hairs on the surface, whorled leaves and seedling morphology 'subgroup E' (Shimizu, 1982).The chromosome number of I. gardneriana (Plate 1d) and I. platypetala L. is 2n = 16 (Bhaskar & Razi, 1974;Zinov'Eva-Stahevitch, 1981).Bhaskar & Razi (1978) divided sect.Enantiophyllon into two subsections on the basis of phyllotaxis, subsect.Oppositifoliae Bhaskar & Razi, including all of the members of Group I and subsect.Verticillatae Bhaskar & Razi which refers to Group II.As a result, his subdivision is well concordant with the present grouping, although it is not always supported by seedling type and chromosome number.

Section Macrocentron Warb.
This section is characterized by alternate leaves and only one or two flowers with long spurred lips per inflorescence.The seed coat morphology suggests division of the species examined into three groups.Group I has a testa with tufts of helical 'hairs' (Plate 1e, I. patula) and is further characterized by pink flowers, turgid and tomentose capsules and seedling type 'subgroup C' (Shimizu, 1982).Their distribution is strictly restricted to South-East Asia.This is a natural group to be treated as an independent taxon.Impatiens henslowiana of Group II (Plate 1f) is characterized by an irregular testa with isodiametric cells with reticulate thickened outer walls (compare Plate 2b, I. siamensis T. Shimizu).The seedling belongs to 'subgroup E' (Shimizu, 1982).Group III, I. siamensis (Plate 2b), is characterized by a four-carpellate ovary and connate winged petals like Group II and III of section Microcentron Warb.The chromosome numbers of I. violiflora Hook.f. are 2n = 10 and 12 (Larsen, 1981) and that of I. santisukii T. Shimizu is 2n = 10 (Shimizu, unpubl.).3. Section Microcentron Warb.This section can be divided into six groups of species.Group I, I. balsamina L. is characterized by a verrucose seed coat (Plate 2c) and inflated tomentose capsules.These capsules are very characteristic and not seen in any of the other groups.The seedling type of this species belongs to 'subgroup C' (Shimizu, 1982).Its chromosome number is variable, 2n = 12, 14, 18, 20, 24 and 14 + 2 (Khoshoo, 1955variable, 2n = 12, 14, 18, 20, 24 and 14 + 2 (Khoshoo, , 1956variable, 2n = 12, 14, 18, 20, 24 and 14 + 2 (Khoshoo, , 1957;;Chatterjee & Sharma, 1970).Group II is characterized by testa cells with reticulate thickened outer walls.This group includes I. arriensii (Zoll.)T. Shimizu, I. bunnackii T. Shimizu, I. charanii T. Shimizu, I. harmandii Hook.f.(Plate 2d), I. kanburiensis T. Shimizu, I. macrosepala Hook.f., I. obesa Hook.f. and I. saraburiensis T. Shimizu.Group III, including I. hongsonensis T. Shimizu, I. kerriae Craib, I. parishii Hook.f. and I. psittacina Hook.f.(Plate 2e), is characterized by a finely granulate seed coat.These species are very unique in having a glabrous, four-carpellate ovary.The species mentioned above, together with some others, should be treated as a third subgenus of Impatiens.This group is restricted to South Asia, from South China to South Indonesia.Group IV, represented by I. boni Hook.f., I. cardiophylla Hook.f. and I. namkatensis T. Shimizu (Plate 2f), is characterized by a 'warted' epidermis.These 'warts' appear to be glands derived from a single epidermal cell and are distributed over the entire surface of the testa.These species have four carpellate ovaries and connate winged petals.Like Group III, Group IV should be transferred to a new subgenus.Impatiens siamensis of section Macrocentron probably belongs to the same new subgenus.Group V is heterogeneous in floral characters: I. salaengensis T. Shimizu has a four-carpellate ovary, while I. tripetala Roxb.has a five-carpellate ovary.In this case, seed characters are considered as homoplastic.Group VI, I. muscicola Craib, should be regarded as a member of section Macrocentron on the basis of flower morphology and seedling type.

Section Megalocentron Warb.
This section is characterized by alternate leaves, racemes with 2-5 flowers with long spurred lips.This section can be divided into two groups.Group I is characterized by a testa with papillae (Plate 3a, I. garrettii Craib).Impatiens garrettii has violet and red striped flowers with curved spurs.Impatiens tapanuliensis Grey-Wilson has yellow flowers with stripes and straight spurs.No features common to these species were found.Group II is represented by rheophytic plants with flabellate wing petals.

Section Brachycentron Warb.
This section is divided into two groups.Group I is characterized by a reticulate seed coat as seen in I. hypophylla Makino, I. textori Miq.(Plate 3b) and I. noli-tangere L. (Plate 3c).The seedlings of I. noli-tangere and I. textori belong to 'subgroup C' (Shimizu, 1982).Group II, I. alboflava Miq. and I. larsenii T. Shimizu, is characterized by a seed coat with two kinds of granules: large and small (Plate 3d, e).These two species are peculiar in having non-pendulous flowers and asymmetric wing petals (Utami & Shimizu, 1998).Warburg & Reiche (1895) classified I. alboflava as a member of sect.Brachycentron together with I. noli-tangere and I. textori.However, the seed coat features indicate that I. alboflava and I. larsenii may better be placed in sect.Microcentron.

Section Longicornes Warb.
This section is characterized by alternate leaves, many-flowered umbels and flowers with a long spur.Impatiens cordata Wight of India and I. walleriana Hook.f.ex Oliv. of Africa belong to this section.These two species differ greatly in their seed coat structure: I. cordata (Plate 3f) is peculiar in having ciliate seeds, while I. walleriana (Plate 4a) has a pattern similar to that of sect.Macrocentron, Group I.However, the chromosome number of I. walleriana is 2n = 16 (Arisumi, 1980), while Group I of sect.Macrocentron has 2n = 10 or 12. Impatiens goughii Wight (Plate 4b) and I. benthami Steenis (Plate 4c) of India are characterized by opposite leaves and inflorescences with few flowers with a short spur.These two species have a seed coat similar to that of several species of sections Enantiophyllon (Group II), Megalocentron (Group III) and Choniochillon Warb.

Section Choniochillon Warb.
This section is characterized by opposite leaves and inflorescences with many flowers with a long spur.The two species of this section I. omissa Hook.f.(Plate 4d) and I. viscosa Bedd. of India, have the same type of seed coat as section Enantiophyllon (Group II), Megalocentron (Group III) and Salpingochillon.The chromosome numbers of these two species are 2n = 28 for I. omissa and 2n = 16 for I. viscosa (Rao, 1972).

CONCLUSION
The genus Impatiens has a most diverse and elaborately sculptured seed coat.The seed coat patterns can be used for distinguishing groups or aggregates.However, seed coat morphology alone does not provide universally applicable key characters for identification, but it can be as helpful as many other characters used in taxonomy.These As a result of the morphological and seed coat morphological examinations carried out, it is concluded that a number of good species groups should be recognized in Impatiens as more natural.Several species in sect.Microcentron which are characterized by four carpellate ovaries, connate winged petals and seed coat pilose with long hairs or granulate and finely granular form a new subgenus in Impatiens that will be published in a separate paper.

Table 1
From Plate 1a & b (I.acaulis Arn., I. stocksii Hook.f.& Thomson) it can be seen that subg.Acaulimpatiens has two types of seed coat.The seeds of I. acaulis (Plate 1a) are covered with broad and ring patterned hairs of 40-50 μm diam., while those of I. stocksii (Plate 1b) have long and slender hairs of 30-37 μm diam.at both ends only.