A Further Investigation of the Morphology of Vessels in Marsilea

Pror Indian Acad. Sci., Vol. 87 B (Plant Sciences-4), No. I1, November 1978, pp. 335-346, 9 printed in India

A further investigation of the morphology of vessels in Marsilea

D S LOYAL and HARMOHINDER SINGH Botany Department, Panjab University, Chandigarh 160 014

MS received 3 March 1978

Abstract. Morphology of vessel members in 7 species of Marsilea is described. The roots in all species examined possess vessels with scalariform (original), scalariform-porous and simple (specialised state) perforation plates; these variables axe recorded in the different regions of the same root. Vesselsof distinct morphology are also recorded for the first time in the rhizome~root-petiolejuncture. Their origin and evolution in roots, and their absence in the internodal parts of the rhizome and petiole are discussed. Past comparison between Marsilea and Pteridium vessels is reviewed.

Keywords. Marsilea; morphology of vessels.

1. In~oducflon

Vessel members were reported for the first time by White (1961) in the roots of 3 species of Marsilea, M. quadrifolia, M. hirsuta and M. drummondi. He also recorded their absence in the rhizome and leaves of the same species. Mehra and Soni (1971) made a few sketchy observations on 4 species from the Indian subcon- tinent, namely, M. minuta, M. aff-sinensis,* M. quadrifolia and M. poonensis. More recently, Bhardwaja and Baijal (1977) have recorded vessel members purported to be occurring in the rhizome of M. drummondi and M. elata. Our present study encompassing 7 distinct species, M. minuta L. (diploid eytotype), M. minuta* (triploid eytotype), M. aegyptiaca Wall., M. diffusa A. Br., M. quadrifolia L. M. drummondi A. Br., M. vestita Hook. et. Grey. and M. rajasthanensis Gupta, has shown that the results published by the above mentioned authors from India do not faith- fully represent all the morphological diversity of the xylem elements involved. Hence, one of the objectives of the present study was to assess the validity of the previous conclusions based upon insufficient observations.

2. Materials and methods

Of the 7 species investigated, the material of M. minuta (diploid eytotype) and M. minuta (triploid eytotype) was coUected from a number of wild localities around

*The commonly occurring triploid cytotype of Maralea minuta from the Indo-gangetic plains (Mehra and Loyal 1959) was identified as M. affi-sinensis Hand-Mazz. (Cf. Mehra-Alston Panjab University Herbarium Sheet Nos. 84, 85). Gupta (1962) gave it a varietal rank, indica. However, pending typification of this cytotype, we denote it as M. minuta (triploid cytotype) in the present paper.

335 336 D S Loyal and Itarmohinder Singh

Chandigarh and District Ambala. The material of remaining 6 species, namely, M. quadrifolia, M. drummondi, M. aegyptiaca, M. diffusa, M. vestita and M. rajasthanensis was obtained through the courtesy of Dr T N Bhardwaja, Government College, Ajmer, Rajasthan. Plants were fixed in FAA and bits of rhizome, roots and petiole were macerated in Jeffrey's macerating fluid. After warming for about 5 rain over the spirit flame the material was left in the macerating fluid for nearly 2 hr after which the xylary elements were isolated and finally mounted in appropriate concentration of glycerol.

3. Observations

3.1. Morphology of vessel members in roots

All the elements examined were exclusively those which comprised the metaxylem. However, owing to the mesarch condition of xylem it was difficult to ascertain the position of a given element in the early or late part of the metaxylem. In agreement with the earlier authors, vessel members are present in roots of all the species investigated. As depicted in figures 1-8, 21-25, 28, 31-34, the vessel members differ in: (a) length/diameter, (b) nature of perforation, (c) angle of inclination of the end wall i.e. long or short slants, transverse and (d) pitting pattern of the overlap and lateral wall area. Figures 3 and 4 illustrate elements with smallest diameter and those illustrated in figures 8, 33 and 34 are the largest in our sample; the remaining elements, as depicted in figures 1, 2, 5--7 may be regarded as intermediate in size. A comparison of figures 1-8, 9 with figures 20 and 21 reveals that the angle of inclination of the end walls of the elements not only differs from element to element in the same organ but also of the two ends of the same element. Generally, the transverse or truncate end walls are observed in the vessels of relatively large diameter. As regards the nature of perforation, the elements exhibit both the original scalariform perforation (figures 20, 21) as well as specialised state i.e. simple perforation (figures 1-3, 5, 8); the latter condition is however observable in overwhelmingly large number of elements in all the species investigated. The scalariform perforate plates may be present on long (figures 20, 21) or short slants and the number of intervening bars may vary, e.g. from 6 (figure 31) to as many as 20-26 (figures 20, 21). The elements with reduced width of intervening bars on long slants are sugges- tive of their being nearest to the ' presumptive vessels' recorded earlier in two terrestrial ferns, Woodsia ilvensis and Notholaena sinuata (White 1963b). Also, in certain scalariform perforate elements, for instance, as shown in figure 20, it was difficult to ascertain whether or not a few pores/pits situated on the lower as well as upper ends of the perforation plate are pores in the strict sense of the term, i.e. characterised by the absence of pit-closing membranes. In order to study the variation pattern of perforations in individual vessels throughout the length of a given root, we analysed the roots of 3 species, M. quadrifolia, M. drummondi and M. minuta (triploid cytotype). As schematically shown in figure 9, in the case of M. quadrifolia, the vessel members in the middle portion of the root are simple perforate with transverse end walls. Progressively toward the distal as well as the proximal part of the root, the elements are characterised by Morphology of vessels in Marsilea 337

Figures 1-9. Selected vessel members in the roots (• 875). 1, 2. M. minuta triploid cytotype. 3. M. aegyptiaca. 4. M. vestita. 5, 6. M. rajastltane~is. 7, M. quadrifolia. 8. M. drummondi. 9. Diagrammatic representation depicting the root-rhizome course of vessel members in M. quadrifolia. Note the variation in angle of inclination of the end wall and the perforation plates in different portions of the root. 338 Er~S Loyal and Harmohinder Singh

Figures 10-15. Selectedvessel members in the root-rhizome-petiole juncture (x 875). 10-12. M. minuta (triploid cytotype). 13. M. aegyptiaca. 14-15. M. rajasthanensis. Morphology of vessels in Marsilea 339

Figures 16-19. Selected vessel members in the root-rhizome-petiole juncture (x 875). 16. M. rayasthanensis. Note the presence of perforation (arrow) on one end of a branched vessel member, the other end is imperforate. 17. M. diffusa. 18. M. drummondi. 19. M. quadrifolia. Morphology of vessels in Marsilea 341

Figures 20-.38. (Seecaption~ in page 346) 342 D S Loyal and Harmohinder Singh soalariform or sealariform-porous perforations on long or short slants. As expected, the vessel members which are located in the root.shoot-petiole juncture, and are co- oriented with those of the root below, have perforation on one end only, the other end being invariably imperforate (figures 9, 16). In M. minuta (triploid cytotype) and M. drummondi, on the other hand, the elements tend to have simple perforations for most part of a given root. Pending further developmental study, it seems possible that the nature of perforation is determined by the hitherto unknown morpho- genetic factors which operate differentially at different stages of the root ontogeny. Further study is obviously necessary to elucidate this aspect of vessel differentiation in relation to their geography.

3.2. Vessel members in root-rhizome-petiole juncture

As pointed out earlier, White (1961) did not observe any vessel members in the rhizome and leaves of the species he investigated. During the course of our studies on water ferns in general (Loyal 1973) and while tracing the course of individual vessels in a given root, the last vessel members in the root were located in the nodal part of the shoot (figure 9). The vessel members in the above-mentioned region of the rhizome of M. minuta (triploid eytotype), M. aegyptiaea, M. diffusa, 3/I. rajasthanensis, M. quadrifolia and M. drummondi, are charaeterised by scalariform--porous perforations on bulbiform (figures 12, 19), curved, spatulate ends (figures 11-17, 19); simple-perforate elements on swollen ends are present in a tiny proportion only (figures 11-13). As depicted in figures 14, 15, 18, 19, simple perforations arise as a consequence of breakdown of bars. The perforation may be present on short (figures 11, 13) or on long slants (figure 27). It is clear from the foregoing that the simple perforate elements on truncate end walls such as those claimed to have been seen by Bhardwaja and Baijal (1977) are absent in our species which incidently include those worked out by the above authors. Stated succinctly, the perforate elements herein portrayed by us, serve as markers for the rhizome-root-petiole juncture and, therefore, a re-examination of Bharadwaja and Baijal's preparations will be necessary.

3.3. Pitting patterns of overlap and lateral wall area

In all the three organs, viz., rhizome, root and petiole we have observed 6 types of pitting patterns: (a) scalariform, Co) trans-edge opposite, uniseriate (figures 31, 32), (c) trans-edge opposite (figures 33, 34), (d) trans-edge alternate, (e) irregular or mixed (figures 35, 36) and (f) obscalariform (figure 38). Of these, in agreement with White (1963b), opposite pitting was predominant on the lateral walls of all three organs. A quantitative analysis of pitting patterns in M. minuta (triploid eytotype) (table 1) reveals that the highest percentage (89.67) of opposite pitting was present in the root and the lowest (60"35) in the case of petiole. Obscalariform pits observed in the rhizome of M. vestita are structurally comparable with those recorded earlier in Angiopteris eveeta (Marattiacaeae), Bleehnum and Dennstaedtia (Bierhorst 1960). Completely circular pits (figure 37) show indistinct borders and in none of the species examined, did they conform to large, circular bordered pits seen in Ophioglossales (Bierhorst 1960). Also, exceptionally large-sized, circular pits such as shown by Bhardwaja and Baijal (1977 figure ID) in 31. drummondi were not observed by us. Morphology of vessels in Marsilea 343

Table 1. Percentage frequency of perforate, imperforate elements, nature of perforation and pitting patterns in three organs of Marsilea rainuta (triploid cytotype)

Elements Nature of perforation Pitting patterns Organs Alternate Perforate Imper- Scalari- Simple Helical and Oppo- forate form opposite site

Root 83 17 15 83 2.00 8.33 89.67 Rhizome root juncture 21 79 8 3 4.52 6"66 88.82 Petiole 0 100 -- -- 8.62 31.03 60.35

We studied the ontogenetic sequence of three pitting patterns only, namely, trans- edge opposite uniseriate, (figures 31, 32), (b)trans-edge opposite (figures 33, 34) and (d) irregular or mixed (figures 35, 36). Following Bierhorst and Zamora's 0965) terminology, the primary order framework of the secondary wall is helical in all the sequences but they differ in respect of the deposition of the second order framework as is illustrated in figures 30 A, B, C. (a) Sequence for trans-edge opposite uniseriaie (figure 30 A): The second order framework appears in the form of sheets between the gyres of the helix which are restricted to the cell edges. By further addition of the wall material, the pit boun- daries become well-defined. The last stage of this sequence is shown in the roots of M. minuta and M. drummondi (figures 31, 32). (b) Sequence for trans-edge opposite (figure 30 ]3): This sequence is similar to the preceding one except that the sheets of second order framework are deposited on the cell faces as well as the edges. As a consequence, opposite pits of variable size are formed as shown on the lateral wall area in two perforate elements (figures 32, 33). (c) Sequence for mixed or irregular pitting Herein, the oblique strands of the second order wall material appears on the cell faces as well as cell edges. Later, deposition of vertically-oriented strands may appear along the edges as well as cell faces. In consequence, the final pitting pattern is in part alternate and in part opposite (figures 35, 36). Besides the above ontogenetic pathway, the irregularity in pitting pattern may arise due to deposition of vertically or obliquely-oriented second order framework along with the fusion of contiguous bars of the helical system on faces as well as cell edges.

4. Discussion and conclusions

4.1. Extent of variation--an over view

In the present study, vessel members have been observed in roots of all the species investigated. In accord with White's data (White 1961, 1963b), the perforations may vary from scalariform, scalariform-porous to simple ones; all the three conditions are connected through intermediate grades of inclined (long or short slants) to transverse end walls in the same vessel. Such characteristics of the vessel members as; 344 D S Loyal and Harmohinder Singh

(a) angle of inclination of the end wall and (b) the nature of perforation could be uniformly correlated with the location of a given element in the root, e.g.M, quadrifolia. Two other species, M. drummondi and M. minuta (triploid cytotype) however, showed simple perforate elements throughout the length of a given vessel. As regards the mechanism of simple perforation, our data show indisputably that they arise due to breakdown of bars. Vessel members in the rhizome-root-petiole juncture may have scalariform, sealariform-porous perforations on deformed end walls which in disposition, connect the last vessel members in the root. In agreement with White (1961) we did not find vessel members elsewhere in the rhizome of all the species investigated. In a previous report of their occurrence in the rhizome, Bhardwaja and Baijal (1977) did not speci- fically mention the geographical location i.e. nodes or internodes, of the vessel members they observed. From a functional standpoint, the presence of vessel members in the rhizome-root- petiole juncture indicates that water column can move without obstruction from the root into the shoot and thence upto the base of the petiole. It is apparent from our results that the variation in length and diameter of vessel members in Marsilea is caused by the growth of rhizome-root complex; in the rhizome, for instance, the internodal length and plastoehron intervals are quite considerably altered by habitat conditions, submerged, amphibious and finally dry, parched. That the length of internode and habitat do affect the fern tracheid length has earlier been demonstrated by White (1963a). In our opinion, this generalisation of White is especially applicable to Marsilea in view of its very wide ecological tolerance in terms of water. The vessel member length, therefore, does not appear to be a reli- able criterion on which to base taxonomic or phylogenetie relationships among the various species of this genus. This may well be possible if the plants grown under perfectly identical conditions are compared for tracheary length parameter rather than sampling the data from plants of wild origin.

4.2. Evolution of vessels in Marsileales

An attempt at this point to trace the evolution of vessels in Marsileales is beset with three obstacles. First, of the three genera, Marsilea, Regenellidium and Pilularia, vessel members have so far been reported in tbe roots of a little less than dozen species out of approximately 60 extant species of Marsilea and in the roots of monotypie Regnellidium (Tewari 1975); compara,ble information in the genus Pilularia is lacking. Secondly, to the writers' knowledge, vessels have not been seen among the known fossil members of this alliance. In the absence of fossil records, it would be unwise, in our opinion, to decide upon the question as to which characteristics of the vessel members are primitive and which are derived in strict Darwinian sense. Thirdly, the relationships of Marsileales is highly conjectural and as recently pointed out by Hall (1974), ' the Marsileales appear just as suddenly and as mysteriously as do the contemporaneously arising angiosperms in the lower Cretaceous '. Therefore, any comparison drawn of similarities and differences with Pteridium, provides no worth- while clue to the resolution of this problem and like other groups of vascular plants, the occurrence of vessels in the filiealean ferns is undoubtedly an event of homoplasy. Yet our existing information suggests two possibilities. Analogous with the situa- tion in monocotvledoneae, especially the family Liliaceae (Cheadle 1944; Cheadle Morphology of vessels in Marsilea 345 and Kosakai 1971), it seems that in Marsilea the vessel members evolved in the root, and as far as can be judged from the present data, their evolution did not proceed beyond the rhizome-root-petiole juncture. This is supported by the fact that both the scalariform (basic type) and simple perforate elements (derived condition) occur in the roots of all the species examined. A still more significant evidence pertinent in the present context is that the so-called 'presumptive vessels' (trachaery elements with scalariform pitting on long slants and with the intervening bar width highly reduced), comparable to scalariform perforate elements in our material, have so far been reported properly in the roots of two unrelated, terrestrial ferns, Woodsia ilvensis, and Notholaena sinuata (White 1963b). Alternatively, their absence in the internodal part of the rhizome as well as petiole may be interpreted as an evolutionary loss in these organs as a sequel to neotenic reduction (Allsopp 1965) (if one follows this line of reasoning ?) under xeric conditions; hence, it is not improbable that the extant members have evolved from their harbingers which had vessels in all three organs of the sporophytic generation. It is obvious from the foregoing that the vessel members must be studied in a more critical and comprehensive way before they can be used as indicators of interrelationships among the species of this genus as well as their phylogenetic origin among the Marsileales.

4.3 Past comparison between Marsilea and Pteridium vessels

While comparing the morphology of the vessel members in Marsilea and Pteridium, Mehra and Soni (1971) have stated that '(b) the perforation plate of the vessel members in Marsilea may not possess bars (simple perforation) while in Pteridium bars are present (Cfi figures 11 and 2A); (c) the vessel members of Marsilea have transverse perforation plates as compared to those of Pteridium which are oblique'. Our present data as well as White's warrant refutation of Mehra and Soni's conclusions. In the first place, their using the term ' bar' to distinguish between scalariform and simple perforation is confusing because in all the contemporary publications (White 1963 a, b; Bierhorst and Zamora 1965; Cheadle and Kosakai 1971), the term ' bar' has often been used either to specify any horizontal/oblique portion of the wall separating the two pits or the wall area intervening the individual pore in a scalariform perforation plate. Secondly, a glance at our illustrations shows unequivocally that Mehra and Soni's premise is factually inco .rrect, because in all the species of Marsilea investigated which include those worked out by Mehra and Soni, the root vessel members exhibit end walls varying all the way from oblique (long or short slants) to transverse with scalariform and simple perforation. It is true that most of the elements in the roots of Marsilea possess simple perforation but like Pteridium- scalariform perforate elements were also recorded both in the roots as well as rhizome- root-petiole-juncture.

Acknowledgements

We wish to thank Dr T N Bhardwaja, Government College, Ajmer, Rajasthan for supplying us the material used in the present study and to Miss Harviner K Chopra and Miss Raman Ratra for help in illustrations.

Prec. B.---5 346 D S Loyal and Itarmohinder Singh

References

Allsopp A 1965 Heteroblastic development in Cormophytes, Encyclopedia of plant physiology (eds) W Ruhland et al (Heidelberg : Springer-Verlag) 15 1172-1222 Bhardwaja T N and Baijal J 1977 Vessels in rhizome of Marsilea; Phytomorpholngy 27 206-208 Bierhorst D W 1960 Observations on tracheary elements; Phytomorphology 10 249-305 Bierhorst D W and Zamora P M 1965 Primary xylem elements and element associations of angiosperms; Am. J. Bet. 52 657-710 Cheadle V I 1944 Specialisation of vessels within the xylem of each organ in the monocotyledoneae; Am. J. Bet. 31 81-92 Cheadle V I and Kosakai Hatsume 1971 Vessels in Liliaceae; Phytomorphology 21 320-333 Gupta K M 1962 Marsilea. Botanical Monograph (New Delhi: CSIR) pp 1-113 Hall J W 1974 Cretaceous Salviniaceae; Ann. Missouri Bet. Gard. 61 354-367 Loyal D S 1973 Chromosome size and structure in some heterosporous ferns with a bearing on evolutionary oroblems Advancing frontiers in cytogenetics ed. P Kachroo (New Delhi) pp. 293-298 Mehra P N and Loyal D S 1959 Cytological studies in Marsilea with particular reference to M. minuta L.; Res. Bull. Panjab Univ. 10 357-374 Mehra P N and Soni Sarvjit L 1971 Morphology of tracheary elements in Marsilea and Pteridium; Phytornorphology 21 68-71 Tewari R B 1975 Structure of vessels and tracheids of Regnellidium diphyllum Lindm. (Marsileaceae); Ann. Bet. N.S. 39 229-231 White R A 1961 Vessels in roots of Marsilea; Science 133 1073-1074 White R A 1963a Tracheary elements of the ferns I. Factors which influence tracheid length; cor- relation of length with evolutionary divergence; Am. J. Bet. 50 447-455 White R A 1963b Tracheary elements of the ferns II. Morphology of tracheary elements, conclusions; Am. J. Bet. 50 514-522

Caption for figures 20-38

Figures 20-38. (All approximately x 600). 20. Vessel member in the root of M. minuta (triploid cytotype). 21. Vessel member in the root of M. minuta (diploid). 22. Explanatory diagram to 15. 23-24. Multiple-perforate element arising from breakdown of bars in the root of M. quadrifolia. 25. simple perforate element from the middle portion of the root of M. quadrifolia. 26. Explanatory diagram to 19. 27. Multiple perforate vessel member in the rhizome-root-petiole juncture. Note the breakdown of bars. 28. Scalariform, multiple perforate end wall from the proximal portion of the root of M. quadrifolia. 29. Origin of simple perforation in the subdistal part of the element in the root-rhizome-petiole junctures of M. diffusa. 30. A, B, C. Schematic diagram showing three ontogenetic sequences of: trans-edge opposite, uniseriate pitting (30 A), trans-edgr opposite (30 B), and mixed or irregular (30 C). 31, 32. Scalariform perforate element with broad pits on faces (31, M. minuta) and trans-edge opposite, uniseriate. 32. M. drummondi). 33, 34. Simple perforate element with trans-edge opposite pits in the roots of: M. minuta. (33) and M. rajasthanensis (34). 35, 36. Imperforate elements showing irregular pitting pattern in the petiole of M, vestita. 37. Part of imperforate element showing nearly circular, alternate pits in the petiole of M. minuta (triploid cytotype). 38. Imperforate element from the rhizome of m. vestita showing obscalariform pitting pattern.