STUDIES IN THE VII. Floral Anatomy of Hakeu Schrad.

By C. Venkata R ao Department o f Botany, Andhra University, Waltair

(Received for publication on February 24, 1966)

I ntroduction

Hakea Schrad. belongs to the tribe Grevilleae of the subfamily Grevillc- oideae of the Proteaceae. It is a large genus with 110-124 species all of which are endemic in Australia. About 80 spccies arc found in W. Australia, 40 in E. Australia and 7 in Tasmania. Four of the Tasmanian species are common with E. Australia and one is given by Bentham (1870) as a variety of the mainland species (H. adcularls var. lissosperma). Some species are introduced into the hilly legions of other countries. H. saligna is grown in the Nilgiris. A few spccies introduced into S. Africa are said to have run wild.

Hakea resembles closely Greviltea both in morphological features as well as in the chromosome number of « = 10. I placed both genera in the sub-tribe Grevillinac (Venkata Rao, 1957 h).

P revious W ork

The cytology of a few species was studied by Ramsay (1963). The floral anatomy of H. cyclocarpa is described by Haber (1959). There is no other floral anatomical or morphologicsil work in the genus.

M aterials and M ethods

This paper deals with the floral anatomy of 17 spccies of Hakea ranging from different geographic regions. In all cases material fixed in formalin-acetic-alcohol was used. The material was collected by me during my stay in Tasmania and Australia. Customary methods of microtechnique were followe and the preparations were stained in Delafield’s haematoxylin.

F low er .

As in other , the flowers in Hakea arise in paijs in the axik of common bracts. They are pedicellate, zygomorphic, 4-merous and hermaphrodite. The thalamus may be horizontal or 10 slightly or markedly oblique (Text-Figs. 1, 6, 8). In some species th^ pjrianth is gibbous at base (Text-Fig. 2). In H. laurina and H. victoria^ the flowar-bud remiins straight during development (Text-Fig. 1) while in other species like H. acicularis its tip becomes strongly reflexed early in development (Text-Fig. 6). The four stamens are anti- tepalous and epiphyllous (Text-Figs. 3, 7). The anthers are sessile and non-apiculate. The pistil is monocarpellary and stipitate or sessile; it bears two laterally attached hemianatropous ovules (Text-Fig. 15). There is a hpyogynous, zygomorphic nectary formed by the fusion of 3 alternitepalous lobes, the fourth one of the anterior side having been suppressed (Text-Figs. 4, 8). Only in H. purpurea a small vestigial anterior lobe is noticcd. The nectary is absent in H. murophylla. The stigma is either conical, simple and terminal as in H. laurina (Text-Fig. 5) or lateral, papillate and subtended by a pollen-collecting apparatus as in H. acicularis (Text-Figs. 8, 10).

T ext-F ig s. I-IO . Floral structure in Hakea. Figs. 1-5. H . laurina. Fig. 1. Flowcr-bud, x 2. Fig. 2. Open flower, x 1. Fig. 3. Part of tepal with stamen, X 4. Fig. 4. Base of pistil with nectary, x 4. Fig. 5. Top of style and stignia, X 4. Figs. 6-10. H. acicularis. Fig. 6. Flower-bud, X 1. Fig. 7. Part of tepal with stamen, x 4. Fig. 8. Pistil with nectary, x 6. Fig. 9. T.s. nectary, X 100. Fig. 10. L.s. top of flower-bud, x 25.

F l o r a l A n a to m y The anatomical features of the flower of H akea present slight variation about a mean ground plan. The pedicel receives 4-8 vas­ cular bundles (Text-Figs. 12, 26, 52) which branch and increase in number in the region of thalamus (Text-Fig. 57). If the thalamus is horizontal, the origin and emergence of the traces for the floral organs is symmetrical on all sides (Text-Figs. 11, 13, 19, 32, 40) while if it is oblique, the traces in the anterior and posterior sectors arise at slightly or markedly different levels depending on the oblicity of the thalamus (Text-Figs. 23, 25, 29. 36, 42, 45. 47, 49, 51, 53, 54. 56). In all spccies studied the tepals are diagonal in position and 3-traced; the tepal marginals are connate. Eight traces are first given off from the floral slele (Text-Figs. 13. 37, 53, 58). Of ihese, the four along the antero-posterior and lateral radii repiesent the conjoint marginals. These divide radially within the cortex of the thalamus and demarcate the laterals of adjacent tepals (Tcxt-Figs. 30. 43. 59. 71. 72). They traverse the lepal without any branching.

There is some difference in the morphology of the foui diagonal traces. In some species like H. aciciilaris var. lissosperma. H. adnata. H.erinacea, H. trifurcata, H. rostrata, and H. purpurea, the stamina! traces and tepal midribs are not adnate. So the four diagonal traces represent only the tepal midribs. The slaminal traces in these species arise as pairs of bundles close to the lepal midribs and from the same gaps, as is noticed in most members of the family. The bundles of each pair fuse together to form a concentric strand, either close to the origin or higher up in the tepal (Text-Figs. 14-16,20. 21,24, 33. 38.41. 43, 44. 46. 48). The staminal bundle thus formed traverses parallel to the tepal midrib and emerges into the stamen when this separates. So, as in most other members of the family, the tepal and stamen in these species show con­ genital concrescence. Though the antheis are apparently sessile, microtome sections reveal short filaments in H. ackuhris var. Usso- sperma (Text-Fig. 17).

In some other species like H. aciciilaris, H. Jaciyloidt's. H. undu- lata, H. salicina, H. victoriae and H. laurina, the tepal midrib and stamen traces are adnate. The 4 diagonal traces, therefore, represent the conjoint tepal midrib-stamen traces (Text-Figs. 18, 27. 28. 30, 50. 58, SI). These traverse the tepals as such and divide tangentially below the level of the stamens so as to demarcate the tepal midrib and staminal bundles (Text-Fig. 31). In some species like H. acicularis. the staminal bundle enters the connective of the anther when it becomes free from the tepal. In others like H. victoriae and H. laurina the anther is fused throughout its length with (he tepal midrib as in (Venkata Rao, 1965). So the staminal bundle traverses in the tissue of the tepal to the inside of the midrib bundle (Text-Figs. 31, 62). After the emergence of the traces for the lepals and stamens, the stelar bundles bend inwards and function as the carpel supply, leaving off some strands which feed the nectary. The nectary is the organ which shows the greatest variation. There is much difference in Ihe number and behaviour of the strands feeding it, and also in its size and shape in different species. In species in which the nectary is horse-shoe-shaped (least reduced), it rcjeives vascular strands from the posterior as well as lateral sectors of the stele; these branch further and form a brush of strands (Text-Figs. 20. 24, 33, 38. 43, 44, 46, 48). When the nectary gets reduced in the lateral sectors it becomes cushion-like or even spherical and receives vascular strands only from posterior sector of floral stele (Tcxt-Figs. 30. 41, 49, 50. 56, 59-61). In If. acicularis vai. lisso.

Text-Fios. 11-35. Floral anatomy of Hakea sp. Figs. 11-17. H. acicularis var. lissosperma. Fig. II. L.s. base of bud. Fig. 12. T.s. pedicel. Figs. 13-17. T.s. bud. All figs., X 25. Fig. 18. T.s. bud of H. acicularLi, > 25. Figs. 19 and 20. L.S. and T.s. buds of H. erlnacea, 25. Figs. 21 and 22. H. epiglottis. Pig. 21. T.s. bud, X 30; Fig. 22. T.s. slipe and nectary, x 30. Figs. 23 and 24. L.s. and T.s. bud of H. adnata, k 20. Figs. 25-27. H. ducrvloides. Fig. 25. L.s. base of bud. Fig. 26. T.s. pedicel. Figs. 27 and 28. T.s. base of bud. All figs., X 30. Figs. 29-31. H.vicioriae. Fig. 29. L.s. base of bud. x 20. Fig. 30. T.s. base of bud, x 25. Fig. 31. T.s. top of bud. x 20. Figs. 32 and 33. L.s. and T.s. bud of H. trifurcata, x 20. Figs. 34 and 35. T.s. base of stipe and nectary of H, pugioniformis, x 25. Explanation in text. core (Text-Figs. 9, 18). In H. epiglottis the nectary is cup-likc in the upper part (Text-Fig. 22) while in H. pugioniformis. H. rostivia and H.platysperma the approximation of the margins of the nectury with the stipe results in the enclosure of a cup-like cavity inside which probably nectar is stored (Text-Figs. 35, 39, 44). In some spccies like H. pugioniformis and H. rostnita the ncctary is adnate to the base of the stipe (Text-Figs. 34. 38) while in others like H. platysperma it is slightly adnate to the base of the perianth (Text-Figs. 44. 46). The nectary is made up of thick-walled, glandular and tannin-bearing cells (Text-Figs. 9, 64). These become demaicated much below the level at which it separates out externally. In H. iieurapliylh the flowers arc glandless probably due to reduction. All the bundles of floral stele after the emergence of tepal and stamen traces function as carpel supply (Text-Figs. 53-55). The number of carpellary traces varies from 5 to many (cf. Text- Figs. 27, 30). These may branch further inside the ovary wall. Some of the larger bundles extend towards the top of the style (Text-Figs. 16, 31). In H. laurina the dorsal carpellary bundle splits into two and each strand becomes associated with sclerenchyma towards the top of the ovary (Text-Fig. 62). Probably this facilitates the dehiscence of the fruit into two separate valves. The core of the style is filled with elongated glanduUir cells of transmitting tissue. The stigma consists of glandular cells which adjoin the transmitting tissue. Just below the stigma a space appears duo to the separation of the glandular cells (Text-Figs. 10, 63). The perianth parts show pcllately attached unicelluhr hairs.

A n a t o m y o f A b n o r m a l F lo w e r s

Abnormalities in floral structure are very rare in Proteaceae. Brown (1867) states that he did not notice a single variation from the tetramerous plan of structure in the flowers of 400 spccies he examined. This shows that Proteaceae is a very ancient family with well-fixed characters. 1 described pentamerous flowers in (Venkata Rao, 1964). Flowers with 5-8 perianth lobes with as many ante- tepalous stamens and two carpels noticed in sp. (Venkata Rao, 1963) were supposed to be due to the fusion of flowers of a pair. Longman and White (1918) and Gillies and White (1919) de.scnbed abnormal flowers of celsissima (Tclopecae) with Iwo Tevt-Figs. 36-55. Floral anatomy of Hakea sp. Figs. 36-39. H. rostrata- Fig. 36. L.s. base o f bud, x 25. Figs. 37 and 38. T.s. bud. x 20. Fig. 39- T.s. stipe and gland, x 25. Figs. 40 and 41. L.s. and T.s. buds of H. amplexi- cauHs, X 20. Figs. 42-44. H. plaiysperma; Fig. 42. L.s. base of bud, x 20: Figs. 43 and 44. T.s. bud, x 10. Figs. 45 and 46. L.s. and T.s. buds o> H. cyclocarpa, x 10. Figs, 47 and 48. H. purpurea. Fig. 47. L.s. bud, x 20- Fig. 48. T.s. bud, X 12. Figs. 49 and 50. L.s. and T.s. buds of H. saUcina, X 25. F i» . 51-55. H. neurophylla. Fig. 51. L. S. base of bud. x 30. Fi«. 52. T.S. pedicel, x 50. Figs. 52-55. T.s. bud. Figs. 52-54, x 50. Fig. 55. X 40 Explanation in text. ’

styliform outgrowths which they interpreted as accessory lobes of the nectary or extra carpels. These abnormalities were also noticed by me and interpreted differently (Venkata Rao, 1957 h).

Abnormal flowers are noticed in H. laurina, a W. Australian species cultivated in Hobart, with acccssory outgrowths which bear superficial resemblance to the outgrowths noticed in Buckinghamia (Text-Figs. 65-68 and 77, 78). In some flowers, the development of the outgrowth resulted in splitting of the nectary into two lobes (Text- Fig. 66), while in others the nectary was pushed to one side (Text-Figs. 65, 67, 75, 76). Sometimes the outgrowth was slender and solid and did not show any loculus or ovules, though it showed a stigma and the vasculature characteristic of the style (Text-Figs. 66, 69, 70). In other flowers, the outgrowths were true carpels and bore normal ovules. When the flower showed two carpels they were always juxtaposed (Text-Figs. 65, 73, 74). After the emergence of traces for the peiianth and stamens, the remaining stelar bundles arrange themselves into two inversely oriented groups of bundles for the two carpels (Text-Figs. 71, 72). The nectary in such cases is dev,.*lopcd on the ventral margin of the main carpel and is vascularised by strands derived from ventral carpellary bundles (Text-Figs. 69, 70, 74). The ovules in the accessory carpels were also collaterally attached (Text-Figs. 70, 74), though in one case they were superposed (Text-Fig. 73). The extra carpcis showed normally developed stigmas (Text-Fig. 68).

D isc u ssio n

Roral anatomical studies in the Proteaceae have shown that moniana, the Tasmanian monotypic endemic, belonging to the tribe Persoonieae, is the only member of the family in which the stamens are completely free from the tepals. In all other members they are adnate and show congenital concrescence, i.e., fusion of the organs but not their traces. In Oriies, however, a characteristic type of adnation is seen in which the stamen and tepal traces are fused throughout their length. The common trace does not divide but enters the stamen bodily so that the lamina of the tepal is devoid of a midrib (Venkata Rao, 1963). Though this represents the most extreme type of adnation, the flowers in Orites sp, show primitive features in the regular perianth, its antero-posterior position, straight style, simple terminal stigma and the non-vascular, 4-lobed nectary. So the adnation of the traces in this genus looks like a premature specialisation. Species of Hakea show a difl'erent type of adnation of the tepal and stamen traces. In some species like H. acicularis, the anthers are free from the tepal as in Orites-, the staminal bundle formed by the tangential division of the common trace enters the con- qective of the anther. In others like H. victoriae and H. laurina th o u ^ Te:cr-Fias.l56-76 Text-Figs. 56-76. Floral anatomy of H. laiirina. Figs. 56-64. Anatomy of normal flowers. Fig. 56. L.s. base of bud, ■: 30. Figs. 57-62 T.s bud Fig. 61, X 25; rest, x 30. Fig. 63. T.s. stigma, \ 60. Fig. 64. T.s. part of nectary, x 610. Fim. 65-76. Abnormal flowers and their anatomy. Figs. 65-67. Abnormal pistils, x 5. Fig. 68. L.s. styles of extra pistils. 18, Figs. 69-76. T.s. abnormal flowers, ■ 20. Explanation in text. a stiminal bundle is demarcrited by the tangential division of the common trace, il remains included inside the tepal tissue sincc the anther does not separate out from the tepal. This seems to represent a very advanced type of adnation noticed in the family. None of the 35 species of studied floral anatomically show this feature. It is interesting to notice that the most advanced type of adnation within the genus is found in the two W. Australian species of Hakea. This fact supports the conclusion of Smith-White (1948) drawn in'the Epacridaceae, that E. Australian taxa are lelativeiy more primitive and the W, Australian ones seem to be their derived relatives.

Text-Fios. 77-80. Abnormal flowers and their anatomy of Buckinghamia cehissima. Figs. 77, 78. Pistils with 1 or 2 styliform outgrowths, ■ 3. Figs, 79 and 80. T.s. abnormal flowers, x 25.

The vegetative as well as floral features of Hakea also show that it is a very advanced genus. Several sj^ ies are dwarf undershiubs; in view of the absence of herbs in the family, this seems to be the climax habital form. The leaves arc generally small and sclerophyllous; several species show the ecologically specialised ‘acicular’ form of leaf. The inflorescence in many species is small and few-flowered; in some species reduction seems to have led to the loss of even the paired-flower arrangement so characteristic of the Grevilleoideae (Briggs and Johnson, 1963). The flowers are zygomorphic with dia­ gonal perianth, a well-vascularised 3-lobed nectary, a curved style and lateral stigma subtended by pollen-collecting apparatus. The pollen grains are of a very advanced type and fairly uniform in all species. Specialised endosperm haustoria. fruits and seeds arc also noticed which point to Hakea being the most advanced genus of the family.

There is difference in the anatomical features of the flowers of ff. acicularis (Sydney) and the Tasmanian spccies which is given as a variety of the above. In H.acicularis the tepal and stamen traces are a nate while in H. acicularis var. lissosperma they are separate. The ncctary strands in H. acicularis fuse at the base to form a solid core while in the Tasmanian species they remain separate. The two also differ in size of the leaf, pollen morphology and fruit characters. So the Tasmanian taxon seems to deserve the rank of a separate species.

There has been much discussion regirding the morphology of the nectary in the Proteaceae. Kausik (1941), Haber (1959) and Eames (1961) think that it is homologous to the corolla; t consider that it is only a nectar secreting outgrowth of the thalamus. In addi­ tion to the great diversity of form it shows in the family, the variation in the number, origin and behaviour of the vascular strands supplying it, the origin of these bundles to the inside of the whorl of tepal-stamen traces, the diversity of its external form within the genus and its adnation either to the stipe or to the base of perianth or even to the carpel (in abnormal flowers of H. laurina) show that the nectary cannot be homologous to the corolla.

In view of the rarity of abnormalities in floral structure in the Proteaceae, it would be worthwhile examining these with a view to drawing any phylogenetic conclusions. The two styliform outgrowths noticed in flowers of Buckinghamia were interpreted by Longman and White (1918) as accessory lobes of the nectary, the production of which resulted in splitting of the original one into three lobes. This abnormality seems to be heritable since Gillies and White (1919) found it in a grown from the seeds of the above. Though these authors took micro­ tome sections of the flowers they could not make out the histological details or the vasculature due to bad fixation of the material. From the external appearance Gillies and White concluded that the out­ growths were abortive carpels. However, they did not observe either loculi, or ovules or even the stigmas characteristic of the genus Bucking­ hamia. 1 also noticed these abnormalities in B. celsissima. In the material collected from Brisbane, two outgrowths were seen like those described by the above authors, while in the material obtained from Sydney, only one such outgrowth was found occupying a position similar to that of one of the outgrowths in Brisbane material. Micro­ tome sections show that the original nectary in these abnormal flowers is vascularised by strands derived from stelar bundles of the posterior sector as in normal flowers. Out of these, one strand becomes pro­ minent and feeds each styliform outgrowth. So they cannot be accessory carpels but only specialised regions of nectary (as Longman and White thought) which seem to have assumed a meristematic activity. It is interesting to notice that they always arose in the alterni- tepalous position. I consider that this change has potentialities towards e aboration of floral structure. If these outgrowths were to become flat and expanded, they would form typical petals both in position and vasculature. In such a case the glands would look like another whorl since in these abnormal flowers they stand alternate to th? styliform outgrowths (Text-Figs. 79, 80). D qcs this point tp ^ probable way in which, the dichlamydeous flower may originate from a monochlamydeous one? The accessory organs noticed in the abnormal flowers of H. laurina are true carpels and not homologous to the outgrowths noticed in the flowers of Buckinghamia. Two carpels were occasionally found in abnormal flowers of GreviUea vestita also. The presence of extra carpels may indicate that the present monocarpellary condition is derived by reduction from a multicarpellary one. The flower pairs in some genera like , , , etc., show carpels which have their ventral sutures oriented in opposite directions. The antero-posterior position of carpels in the abnormal flowers of Hakea would necessitate the assumption that the ancestral flowers had four carpels of which the antero-posterior and proximal carpels of each flower got suppressed in order to give rise to the carpel position noticed in the flower pairs of the above-named geneia.

S um m ary The floral anatomy of 17 species of Hakea is described. The tepals are always diagonal and show conjoint marginals. In several species the tepal and stamen show congenital concrescence as in the majority of members of the family; in a few their traces are also adnate. In H. victoriae and H. laurina (W. Australia) not only are the traces united but the anthers are fused throughout their length with the tepals. This represents the most complete adnation not only in Hakea but in the whole family. The predominantly under-shrubby habit, the morphological features of leaf, inflorescence, flower, fruit and seed, the pollen grain characters and the presence of endosperm haustoiia of a specialised type show that Hakea is the most advanced genus of the family. The anatomy of some abnormal flowers of H- laurina with acces­ sory cirpels is d-;scribed and a comparison is made with some abnormal flowers of Buckinghamia. The probability that the present mono- cirpellary pistil of the proteaceous flower being the result of reduction from multicarpellary condition is suggested and the potentialities of the proteaceous flower towards further elaboration are considered.

ACK.NOW1.EDGEMENTS

I am thankful to the Staff of the Botany Departments of the Sydney University and W. Australian University for help in collection of miterial of Hakea sp. I am thankful to Mr. McKee of Sydney University for some material of Buckinghamia.

R e fer en c es

Bentham, G. 1870. Flora Auslraliensis 5: London. Brioos, B. G. and Johnson, L. A. S. 1963, Evoluiion in th e Proteaceae. Aust, /. Bot. « : 21-61. B r o w n , R. 1867. The M(seellaneous Bottmieal Works 2; London. Eames, a. J. t%l. Morphology o f the Angiosperms. McGraw-Hill, N.Y.

G i l l i e s , C. D. ano W hite, C. T. 1919. On the occurrence of abortive styles in Buckinghamla celshsima F.v.M. Proc. R. Soc. Q. 31; 42-45. Haber, J. M. 1939. The comparative anatomy and morphology of flowers and infknvscences of the Proteaceae. I, Some Australian ta*a. Phytomorphology 9 ; 325-58. Kausik, s. B. 1941. Studies in the Proteaceae. VI. Vascular anatomy of the flower of Crevillea robusta Cunn. Proc. not. Inst. Sci. India 7 : 257-66.

Longman, H. A. AND W h ite , C. T. 19)8. Mutation in a Proteaceous tree. Proc. S . Soe. Q. 30: 162-65. Ramsay, H. P. 1963. Chromosome numbers in the Proteaceae. Aust. J. Bot. U : 1-20.

Smith-White, S. 1948. A survey of chromosome numbers in the Epacridaceae. Proc. Linn. Soc. N.S.W ., 73; 37-56. Venkata Rao, C. 1957 o. Cytotaxonomy of the Proteaceae. [bid. 82; 257-71. 1957 6. Cytotaxonomic Studies in the Proteaceae. 2; Ph.D. Thesis. University of Tasmania, Hobart. 1963. Studies in the Proteaceae. IIL Tribe Oriteae. Proc. nat. Inst. Scl. India 29 8 : 489-510. 1964. Studies in the Proteaceae. IV. Tribes Banksieae, ^ and Embothrieae. Ibid., 27B: 126-5f. 1965. Studies in the Proteaceae. VL Tribe Franklandieae. J. Indian bot. Soc. 44; 479-94.