Proc. Indian Acad. Sci. ( Sci.), Vol. 97, No. 3, June 1987, pp. 235-246. Printed in India.

Anatomical contributŸ to the of some :

LILLYAMMA MATHEW and G L SHAH* Departmr of Botany, Alphonsa College, Palai 686 574, India *Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120, India Abstraer. The differentdimensional aspects of petiolar anatomy of 35 taxa of Verbenaceae has br traced with a view to reveal the structural diversity and to evaluate the cont¡ of petiolar anatomy to the taxonomy of Vr Seven parameters were quantitatively analysed for the patterns of variability. The different vascular configurations in va¡ taxa were found to be taxon specific and hence can be utilized asa taxonomic marker. The observation of intemal phloem in the petiole of Avicennia and Tectona is ah additional information relevant to the anatomy of Verbenaceae. The variation patterns in the l~tiolar anatomy of different taxa were found to have some adaptivr significance. Keywords. Pattems of variabifity; pr anatomy; structural diversity; taxonomy; Verbenaceae.

1. Introduction

Verbenaceae is predominantly a tropical family exhibiting a wide range of growth habit and inhabiting diverse habitats. In India there are indigenous and introduced, wild and cultivated . There is a considerable disagreement on the generic delimitation and relationships of different taxa in the family. Hence the present study has been taken up to supplement the exomorphic characters with anatomical studies in approaching the taxonomic problems of the family. Apart from the general study pertaining to the anatomy of Verbenaceae by Solereder (1908) and Metcalfe and Chalk (1950), limited studies on the petiolar anatomy of a few species of Cleroden- drum have been carried out by Inamdar (1968) and Inamdar and Patel (1969).

2. Materials and methods

The taxa studied, their source and abbreviations used in the dice diagrams etc are given in table 1. The voucher mate¡ are deposited in the herbarium of the Depart- ment of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat. Petiolar anatomy was studied from transections through the middle of the petiole. Small pieces of mature petiole were fixed in FAA, processed and embedded in paraffin (56-58~ following standard procedures (Berlyn and Miksche 1976). Sec- tions of 5-7 #m thick were cut on a rotary microtome, stained in toluidine blue O (O'Brien et al 1964) for general study and tannic acid-ferric chloride-resorcine blue (lacmoid) combination for testing internal phloem (Cheadle et al 1953). Sections were then dehydrated in Tertiary butyl alcohol (TBA)-xylene series, cleared in pure xylene and mounted in DPX. Quantitative characters such as (i) petiole length, (ii) cross sectional arca of the petiole, (iii) arca of the vascular bundle in TS, (iv) distance from vascular bundle to adaxial surface, (v) distance from vascular bundle to abaxial surface, (vi) number of pith bundles and (vii) number of vascular bundles were studied and statistically 235 236 Lillyamma Mathew and G L Shah

Table 1. Taxa investigated and their source of collection.

Code used in the dice Wild/ Names of the taxa Source diagram cultivated

Avicennia marina Vierh. M AA W A. officinalis L. K AO W Callicarpa tomentosa Murr. K CT W Citharexylum subserratum Sw. C CS C inerme Gaertn. G CI W/C C. multiflorum O. Ktze. G CM W C. indicum 0. Ktze. K CP C C. splendens G. Don G CE C C. thomsonae Balf. K CH C C. viscosum Vent. G CV W Duranta repens L. G DP C Gmelina arborea Roxb. G GA W G. philippensis Cham. G GP C Holmskioldia sanguŸ Retz. K HS C Lantana camara var. aculeata Mold. G LA W L. camara var. hybrida Mold. G LH C L. camara var. nivea Bailey G LN C volubilis Jacq. G PV W Phyla nodiflora (L.) Greene K PN W Premna latifolia Roxb. K PL W Stachytarpheta indica Vahl G SI W Tectona grandis L.f. G TG W/C T. hamiltoniana Wall. U TH C Verbena bipinnatifida Nutt. G VB C V. bonariensis L. GE VO C V. canadensis Britt. M VC C V. hispida Ruiz et Pav. GE VH C V. hybrida Voss M VY C V. incisa Hk. f. K VI C V. officinalis L. GE VP W/C V. stricta Vent. GE VS C V. urticifolia L. GE VU C Vitex negundo L. G VN W V. trifolia L. G VT W V. negundo var. intermedia L. M VX W

M, Maharashtra; K, Kerala; G, Gujarat; U, Uttar Pradesh; GE, Seeds obtained from Germany and glown in the botanical garden; C, cultivated; W, Wild. analysed for the patterns of variability using range, mean and standard error. Areas were calculated using area calculating device (Chavan et al 1979). All measurements were based on an average of 100 readings. The values were graphically represented in the form of dice diagrams. Photomicrographs were taken on Carl Zeiss photomicroscope using Ilford 35 mm black and white film.

3. Observafions

The transectional outline of the petiole is highly variable in different taxa (figures 1- 35). Epidermis is single layered, stomatiferous or estomatiferous with varying degree Contribution of petiolar anatomy to the taxonomy of Verbenaceae 237

,,:~~ ~~-

: s

lO

k:'::" xylem sc[erenchyma I ! ..... !..Y.Y .... Dhioern col~anchyrna I['2

. lOOq~rn

Figures 1-13. Diagrammatic representation of petiole in cross section. 1. Avicem~ia marina. 2. A. officinalis. 3, CaIlicarpa tomentosa. 4. Citharexylum subserratum. 5. Clerodendrum inerme, 6, C, multiflorum. 7. C. indicum. 8. C. splendens. 9. C. thom- sonae. 10. C. viscosum. 11. Duranta repens. 12. Gmelina arborea. 13. G. philippensis. 238 Lillyamma Mathew and G L Shah

14 16

24 25 26 ~J

'~~~s ~~

1000AJm

Figures 14-30. Diagrammatic representation of petiole incross section. 14. Holmskiotdia sanyuinea. 15, Lantana camara var. aculeata. 16. L. c. var. hybrida. 17. L. c. var. nivea. 18. Petrea t,olubilis. 19. Phyla nedifiora. 20. Premna latifolia. 21. Stachytarpheta indica. 22. V. bipinnatifida. 23. V. bonariensis. 24. V. canadensis. 25. V. hispida. 26. V. hybrida. 27. V. incisa. 28. V. o[~ficina/is. 29. V. urticifolia. 30. V. stricta. Contribution of petiolar anatomy to the taxonomy of Verbenaceae 239

~~:~,i,,+i + "

34

[.~.-7 ~a ~\ t:!N

~b3S !+ ~ .| +. xylem 0 ~~otm sr.|erlr~l~yn~ ;'~~.. "+ : ++/ :ti

9o'91 +,;':,~ "2?Ÿ ,.t,t"'+*.*+;; "~'~~*'+[+,..', ' +~,:-+.+..... ,++*'.+ ;..,:++'

Figures 31-35. Diagrammatic representation of petiole in cross section. 31. Tectona ,qrandis. 32. T. hamiltoniana. 33. Vitex negundo. 34. V. trifolia. 35. V. negundo var+ intermedia. of hairiness. In Avicennia trichomes are numerous on the abaxial surface and very few or absent on the adaxial surface (figures 36, 37). EpidermaI cells are mostly columnar, oval to oblong, rarely squarish of polygonal and usually with outer walls rounded, flat or rarely papillate as in Petrea (figure 39). 240 Lillyamma Mathew and G L Shah

...... : <

~a~91191237~" |

: ~

N

Figures 36-41. Photomicrographs of the structure of petio/e in cross section. 36. Avi- cennia marina (x 137). 37. A. oJficinalis (x 149). 38. V. incisa (x343). 39. Petrea votubilis ( x 514). 40. A. marina ( • 300). 41. C. inerme ( x 149). Contribution of petiolar anatomy to the taxonomy of Verbenaceae 241

The ground tissue is differentiated into hypodermal collenchyma which forms continuous or discontinuous strands and parenchyma. In Avicennia large intercel- lular spaces occur towards the adaxial side (figure 37). In Phyla nodiflora, Verbena bipinnatifida, V. bonariensis, V. incisa and V. stricta the dorsal margins of the petiole are dilated (figures 19, 22, 23, 27, 30) consisting of radially elongated parenchyma cells, containing denser contents and chloroplasts (figure 38). The patterns of vascular configurations observed in the middle region of the petiole are: (i) Crescent shaped in Clerodendrum inerme, Gmelina philippensis, Holmskioldia san9uinea, Phyla, Stachytarpheta and Verbena (figures 5, 13, 14, 19, 21- 30, 41). (ii) Crescentic with invaginated ends in Avicennia marina (figure 1). (iii) Crescentic with medullary bundles in Duranta repens, Lantana Camara vars. aculeata, hybrida and nivea and Vitex (figures 11, 15-17, 33-35). (iv) Two arcs with 4-5 dorsal bundles in Citharexylum subserratum (figure 4). (v) Dissected crescent of collateral bundles in Avicennia officinalis and Clerodendrum (except C. inerme and C. splendens) (figures 2, 6, 7, 9, 10, 43). (vi) Dissected crescent with invaginated ends in Callicarpa tomentosa and Premna latifolia (figures 3, 20). (vii) Dissected cylinder of collateral bundles without medullary bundles in Clerodendrum splendens and Gmelina arborea (figures 8, 12). (v¡ Dissected cylinder of collateral bundtes with medullary bundtes in both species of Tectona (figures 31, 32). (ix) Medullated cylinder in Petrea volubilis (figures 18, 45). The number of vascular bundles ranges from 1-many (figures 41, 43, 45). The maximum number 12-21 is observed in Tecwna 9randis (figure 31). Each bundle consists of an adaxial xylem and abaxial phloem with cambium in between (figure 44). Vessels may be solitary, in radial multiples or in clusters with the cross sectional outline circular or angular. The radial multiple arrangement is the prominent type in all the species studied. In Avicennia and Tectona additional groups of phloem occur internally towards the pith region (figures 40, 42). The perivascular sclerenchyma occurs asa continuous cylinder of 2-3 layers in Petrea oras discontinuous strands in A. officinalis, Citharexylum, Gmelina, Clerodendrum indicum, C. splendens, C. viscosum, Tectona and Vitex. One or two or seldom 4-5 lateral bundles are observed below each dorsal lateral rib in all taxa except Premna (figure 17). The lateral bundles are collateral and open in all the taxa (figure 47) except Callicarpa and Tectona where they are amphicribral or hardocentric (figure 46). Those taxa which have perivascular sclerenchyma are provided with a sclerenchymatous bundle cap outside the lateral bundles. In both species of Tectona the sclerenchymatous bundle sheath surrounds the whole bundle (figure 46). Medullary bundles are observed in Duranta, Lantana, Tectona and Vitex. The number varies in different taxa (2-12) with the maximum number in T. grandis (figure 31).

3.1 Patterns of cariability in the petiolar anatomy

The length of the petiole is within a range of 0-2-13 cm, with the rnaximum in Clero- dendrum viscosum and minimum in Phyla (figure 48). The cross sectional area of the petiole in different taxa varies significantly (figure 49). It is highest in T. 9randis (19971200 ,aro2) and lowest in Phyla (384000 #m2). Though the range is very high 242 Lillyamma Mathew and G L Shah

Figures 42--47. 42. Tectona hami(toniana ( • 126). 43. C. viscosum ( • 143). 44. Duranta repens ( • 343). 45. Petrea volubilis ( • 126). 46. 7('. grandis ( • 329). 47. P. volubili~ ( x 514). Contribution of petiolar anatomy to the taxonomy of Verbenaceae 243

PL inr |

AA A Cl C.S C~ C~ CP CE CM CV 0P GA GP ~45 CA LH kN P'~ PN pL S~ "{G ;H VB V0 vC VIl VY V~ VF VS VU V~l ~T ~},

1300000 CSA (petio~e) ir,, umŸ 20000000 t , | 1000000 t*, l

700000 t [ t + 5000000

+ t 4- J I00Q000 CP wS PW ~S "/r V~ v~ '~~ Yf: v~ AA ~0 C~ C$ ~4 ~;W Cs C~ C~ OP GA C~p ~A t.H k~ P/ pt. St "~G ~H V0 u vk ~/T WX

V. B.A. (petiohl| inl~m2 ~?$0001 t~l t | t t t

1500000 13S000

t tf ~ I ft t ~OSOOO , T ?00000

§ i. l J ttt* t + t lOOO00 ..... DL , ,It,AAOCI:'CH0P 8PNS LN PllYEIVD VC "#14 Vy u I/~ b'tJVNCT CS. CI CMCECV GA, LAI.H pv p L $1 TG IN V~u yJi

Figures 48-50, For caption, see p, 245. 244 Lillyamma Mathew and G L Shah

| 22

VB Number (petiole) T § ti #4

O B O t Q a O @ Q t O O O @ i l, AA AO CT CS CI CM CP ~)P GP H5 LA uH LN PV PN PL 51 VEI VO ve VH VV VI ~.'F ~$ ~~ ~h ~T r CE Cd CV G=. TG TH

DVB TO ADS (pehole) in iJm

& 9BO

360 320 tt 7 280

2Z.C SBO

20C t tttt ~ I ttt ~ 15C t t t t r

12C 3 II C$ CM CP CH ~ PN Vil VO ~ ~ Vu VI ~1~ VS VU VN VX &A ~ CI CI CI~ CV OP GP HS LA LH LN PV PL SI TG IH VI 191 D V B TO A BS Ipetiolel in pm (~3"~ t loco II l flt~ ~ 800 280 G00 t

160 AA CI CP C~4 CV OPGA HS LH PN PL VlB V0 VC VH VY Vi VF VU VN VT VX AO CT CS CM CE GP LA LN PV St TG TH VS

P.VB. Number (pettole)

12

A A ~,L~ C1 CS Ct CM CP CE CH CV DP GA GP HS LA LH LN PV PN PL SI TG Vil TN YO ~'C Vil VV VI VF '~S VU VN VI VX

Figures 51-54. For caption, see p. 245. Contribution of petiolar anatomy to the taxonomy of Verbenaceae 245 within the group it is very small within a taxon. A similar trend is noticed in the case of the area occupied by vascular tissue in cross section (figure 50). There is a single vascular bundle in the petioles of most of the taxa b,ut more than one bundle is recorded in woody forms except Vitex which has only one bundle (figure 51). The distance from vascular bundle to the adaxial and abaxial surfaces also varies in different taxa (figures 52, 53)+ The pith bundles are observed only in a few woody members (figure 54). The range of variation is 2-12 with the lowest in Vitex and highest in T. grandis.

4. Discussion

The observations on the petiolar anatomy of different species of Clerodendrum do not agree with those recorded earlier by Inamdar (1968) and Inamdar and Patel (1969). The median vascular strand is a dissected crescent of collateral bundles in the different species of Clerodendrum except in C. inerme and C. splendens. In C. inerme ir is crescentic (figure 41), while in C. splendens a dissected cylinder. Phloem is seen as a continuous arc in C. inerme a feature also recorded by Metcalfe and Chalk (1950). There are 9 patterns of petiole vascularization. In most cases it is taxon specific and these variations can be utilized in differentiating different taxa. Solereder (1908) reported a circular median strand for the Avicennia which is not in accordance with the present observation. Similar types of vascula¡ in different species of Lantana, Verbena, Vitex, etc suggest the close relationship among them, and the different types in the species of Avicennia, Clerodendrum and Gmelina show their heterogeneity. Petrea is distinct among all other taxa in having a medullated cylinder. Internal phloem has been observed in the petioles of Avicennia and Tectona during the present study and it appears that there is no previous reference about the occur-

Figures 48-54. Dice diagrams showing the patterns of variability. 48. Lcngth of the petiole (standard error below 0-1% is not represented). 49. Cross sectional area of the petiole (standard error below 2% is not represented). 50. Area occupied by vascular tissue in cross section (standard error below 2% is not represented). 51. Number of vascular bundles in cross section (standard error below 0-1% is not represented). 52. Distante from vascular bundle to the adaxial surface in petiole TS (standard Error below 2% is not represented). 53. Distance from vascular bundle to the abaxial surface in petiole TS (standard error below 2% is not represented). 54. Number of pith bundles in petiole TS (standard error below 0"1% is not represented). Key to the dice diagrams .A

I IC B A--Range B--Mean C--Standard error.

IPH, internal phloem; XY, xy[em; P. phloem. 246 Lillyamma Mathew and G L Shah rence of internal phloem in the petiole of these taxa. The presence of perivascular sclerenchyma in a few taxa can also be used asa taxonomic marker. The distance from vascular bundle to the adaxial and abaxial surface depends on the depth of the curve of the bundle and in the present study, herbaceous taxa are found to have shallow crescentic forms. The number of vascular bundles is found to be positively related with the size of the plant. The medutlary bundles are observed in woody and large shrubs such as Tectona, Vitex, Duranta and Lantana. The variation patterns in the petiolar anatomy of different taxa is found to have some adaptive significance.

Acknowledgements

We gratefully acknowledge the assistance of Dr C R Babu, University of Dethi for the statistical analysis. One of the authors (LM) is also thankful to the University Grants Commission, New Delhi for the award of a fellowship under the faculty improvement programme.

References

Br G P and Miksche J P 1976 Botanical microtechnique and cytochemistry (Amsterdam: The Iowa State Univ. Press) Chavan R R, Kothari I L and Patel J D 1979 A simple area calculating device (ACD) for biological systems; Curr. Sci. 48 792-793 Cheadle V I, Gifford E M Jr and Esau K 1953 A staining combination for phloem and contiguous tissues; Stain Technol. 28 49-53 Inamdar J A 1968 Anatomical studies in Clerodendrum splendens G. Don; Proc. lndian Acad. Sci. 1367 8-17 Inamdar J A and Patel R C 1969 Vascular cambium in the leaf of three species of Cterodendrum; Beitr. Biol. Pflanz. 45 403-407 Metcaife C R and Chalk L 1950 Anatomy ofdicotyledons II (London: Oxford Univ. Press) O'Brien T P, Feder N and McCully M E 1964 Staining for plant cell by toluidin blue O; Protoplasma 59 367-378 Solereder H 1908 Systematic anatomy of the Dicotyledons (eds) L A Boodle and F E Fritsch (Oxford: Clarendon Press)