A NEW BASE NUMBER IN THE GENUS LYGODIUM

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1 A NEW BASE NUMBER IN THE GENUS LYGODIUM BY S. K. ROY AND I. MANTON The Botany Department, Leeds University {Received 28 August 1964) SUMMARY Chromosome counts have been carried out on thirteen specimens of Lygodium, mostly of known wild origin from various parts of the oriental tropics. Five specimens represent taxa not previously investigated from any source. The results confirm some previous records, notably the presence in the genus of two base numbers (n = 29 and n = 30) and their multiples, but they add a new base number (n = 28) and multiples. Some phyletic and other implications of these findings are discussed. INTRODUCTION The observations to be recorded below have been carried out on a small collection of living Lygodium specimens recently accumulated from expeditions, mainly in the oriental tropics, and brought into cultivation at Kew for the purpose of cytological study. They are of importance in showing that the chromosome numbers previously recorded in this genus, namely n = 29 and 30 and their multiples give an incomplete picture which, without the new information to be discussed below, could lead to considerable phyletic misconceptions. As is well known, the somewhat unusual prime number n = 29 is characteristic of the genus Pteris, and both 29 and 30 are also met with among a considerable range of genera of Cheilanthoid and Adiantoid affinity (cf. Manton and Sledge, 1954; Manton, 1958). The question of whether the possession of similar numbers by one genus of Schizaeaceae should or should not be regarded as a significant phyletic indicator is clearlyamatter of importance for the interpretation of fern taxonomy and phylogeny in general and this was the reason for the effort to accumulate data from the additional species which are now before us. The Schizaeaceae are an ancient group of ferns with four very dissimilar surviving genera: Schizaea, Lygodium, Anemia and Mohria. The first two are pantropic in distribution; Anemia is mainly tropical American with one species in Africa and one in south India; Mohria is confined to southern and eastern Africa and the Mascarene Islands. The antiquity of the family, as judged by sporangia and spores of extinct genera goes back to the Upper Carboniferous with Senftenbergia (Radforth, 1938, 1939). The surviving genera have a more recent fossil record. Anemia, Lygodium and Schizaea going no further back than the late Cretaceous and early Tertiary. Mohria appears to have no fossil record but fossils of Anemia and Lygodium include good leaf impressions and sporangia while those of Schizaea are limited to spores. For further information and literature see Holttum (1959) from which these statements are taken. All four genera have been investigated cytologically in a preliminary way, although the sampling is very incomplete. Table i summarizes the more important numerical facts for each genus, the details for each investigated species being given later (Tables 2-4). The numbers of accredited species listed in Table i are taken from Copeland (1947) except for Mohria which follows Alston and Schelpe (1952). 286

2 A new base number in the genus Lygodium 287 As will be seen, the genera are almost as diverse cytologically as they are in morphology. Anemia and Mohria probably share a common base number of n = 38 (or perhaps 19), but Lygodium and Schizaea are different. Beyond this it is not yet possible to go with certainty, since the basic monoploid for Schizaea cannot yet be worked out, all the numbers so far encountered being too high to be likely to represent it. The situation in Lygodium is as discussed above. Table i. Summary of previous cytological records for Schizaeaceae, further details in Table 4 Genus Schizaea Lygodium Anemia Mohria No. of accredited spp No. of investigated spp Lowest known chromosome number n = 77 n = 29 n = 30 n = 38 n = 76 MATERIAL AND METHODS The complete list of specimens examined is given below in Table 2 together with details of the country of origin and the collector where this is known. The naming of all specimens has been verified either by Professor R. E. Holttum or by Dr. F. M. Jarrett of Kew, to whom we are greatly indebted for this and other assistance. Herbarium specimens of all the worked plants are deposited in the Kew herbarium and almost all the species listed are still alive in the Kew fern houses at the time of writing. Species L. fiexuosum (L.) Sw. L. fiexuosum (L.) Sw. L. microphyllum (Cav.) R.Br. (= -L. scandens auct non L.) L. microphyllum (Cav.) R.Br. {= L. scandens auct non L.) L. microphyllum (Cav.) R.Br. {= L. scandens auct non L.) L. microphyllum (Cav.) R.Br. (= -L. scandens auct non L.) L. volubile Sw. L. cirdnnatum (Burm Sw L. longifolium (Willd.) Sw. L. longifolium (Willd.) Sw. L. dimorphum Copel. L. dimorphum Copel. L. salicifolium Presl L. japonicum (Thunb.) Sw. Table 2. Sources of the specimens of Lygodium used in this paper Locality Sapong estate, Tenom Minipe Singapore jungle Sapong estate, Tenom Kew ) Menkukor stream, Sapong estate Singapore jungle Singapore jungle Nr. Lae Nr. Lae Perak Canton Distr. Country N. Borneo Ceylon Ceylon New Guinea Collector Giles & WooUiams T. G. Walker L Manton Jury R. E. Holttum N. Borneo Giles & WooUiams 'W. Indies' N. Borneo Giles & WooUiams R. E. Holttum R. E. Holttum New Guinea R. E. Holttum New Guinea R. E. Holttum R. E. Holttum S. China R. E. Holttum Date No. Kew No PB50 417/ Ts9 22/ P / /61 i / PB / PB /63 i i /60 268/64 545/63 578/63 399/60 The cytological methods employed are two. For sporangia the aceto-carmine squash method is exactly as described in Manton (1950). For root tip squashes a modification

3 288 S. K. ROY AND I. MANTON of the Chambers's snail cellulase method (Chambers, 1955) has worked excellently. The details are as follows: The enzyme is obtained from Helix pomatia (two-dozen) starved for 24 hours and then immobilized with chloroform. The digestive sac is dissected out and the juice squeezed into a glass bottle. The accumulated juice is dried by exposure to P2O5 under vacuum overnight, the dry extract being subsequently stored in a sealed bottle in the deep freeze. When required for use 0.01 g of the dried extract is made up in i ml distilled water. Before fixation, detached root-tips are immersed for 4-5 hours in a saturated solution of either ^-dichlorbenzene or 8-hydroxyquinoline kept cool in running water. They are then rinsed in three to four changes of distilled water, blotted to remove excess water and fixed overnight in 113 acetic alcohol. After this they can be stored in a deep freeze indefinitely. In order to separate the cells, the fixed roots are first rinsed in three to four changes of water to remove the fixative and are then left in a freshly prepared enzyme solution for 2 hours at room temperature. They are then again rinsed and returned to the acetic alcohol fixative for -J-i hour. After this treatment, preparations are made by teasing out the cells of the meristem with needles into a drop of aceto-carmine on a slide. Unwanted solid parts of the root are removed, a coverslip is put on and the preparation gently heated over a spirit lamp, although it should not be allowed to boil. Whilst still hot the cells are squashed by considerable manual pressure applied over a filter paper pad. Subsequent procedure for making the preparation permanent is standard. OBSERVATIONS Samples of the more important types of specimen examined are assembled in Plates 7 and 8; Plate 7 illustrating species agreeing with previous records in having twenty-nine or thirty chromosomes or their multiples while Plate 8 illustrates species with numbers based on n = 28. The complete list of chromosome counts for Lygodium is given in Table 3, omitting two very uncertain records, by de Litardiere, (1920) and Clarke (1936) respectively (cf. Chiarugi, i960) for which accuracy was not claimed and which cannot now be interpreted. Among the remaining counts discrepancies occur in three places. L. japonicum and L. circifinaticm have each given two different numbers, obviously in a polyploid relation. Though one of these counts is in each case based on botanic garden material of unknown wild origin it is to be expected that populations of different ploidy will be discovered in some parts of the natural range of both taxa when more wild samples have been examined. The conflict of information for L. flexuosum on the other hand suggests the presence of some cytological or taxonomic error since it seems unlikely that one species will genuinely contain polyploids on two different monoploid numbers. The fact that our own specimens, from Ceylon and North Borneo, are unequivocally polyploid on the new number n = 28 and not on either of the numbers previously known in the genus is indicated in Nos. 6 and 9, Plate 8. Since photographic evidence was not included in the publication of the two Indian records claiming n = 60 for this species it is possible that re-investigation of Indian material might show that the earlier counts may in fact have been slightly too high. The other new observations listed in Table 3 and illustrated in Plate 8 need little further comment since in themselves they are all unambiguous. L. flexuosum and L. longifolium with n = 56 we interpret as tetraploids on 28 and L. salicifolium and

4 A new base number in the genus Lygodium 289 L. dimorphum as diploids. The new gametic number n = 28 has been seen directly in L. salicifolium although the root in this case (No. 7) makes a more important comparison. The other diploid (No. 8) has only recently been collected and is still too young to bear sporangia. Table 3. Summary of cytological information for species of Lygodium Species L. japonicum (Thunb.) Sw. L. japonicum (Thunb.) Sw. L. circinnatum (Burm.) Sw. L. circinnatum (Burm.) Sw. L. circinnatum (Burm.) Sw. L. volubile Sw. L. microphyllum (Cav.) R.Br. L. microphyllum (Cav.) R.Br. L. microphyllum (Cav.) R.Br. L. microphyllum (Cav.) R.Br. L. microphyllum (Cav.) R.Br. L. palmatum (Bemh.) Sw. L. flexuosum (L.) Sw. L. flexuosum (L.) Sw. L. fle.xuosum (L.) Sw. L. flexuosum (L.) Sw. L. longifolium (WiUd.) Sw. Cat. L. longifolium (Willd.) Sw. Cat. L. salicifolium Presl L. dimorphum Copel. L. dimorphum Copel. L. articulatum A. Rich. Country South China Ceylon N. Borneo Ceylon S. India N. Borneo New Guinea Michigan, U.S.A. Darjeeling S. India N. Borneo Ceylon New Guinea New Guinea New Zealand ') 60 ) c.-jo Author Abraham, Ninan and Mathew (1962) Wagner (1963) Mehra(i96i) Abraham, Ninan and Mathew (1962) Brownlie (1961) DISCUSSION The discovery of a new base number within part of the genus Lvgodium is important in two rather different respects. On the one hand it clarifies the phyletic interpretation of the presence of the problematical number n = 29, and on the other hand it provides, for the first time, evidence of a cytological condition within the family which might be primitive. Taking the phyletic problem first, a high prime number such as n =29 can in theory be synthesized in many different ways, but if wholly imaginary ancestral conditions are to be ruled out the alternatives are fewer. Where only one other number is known in the genus, as formerly in Lygodium with n = 29 and 30, the alternatives may be so restricted as to be virtually useless. Thus 29 can be thought of arithmetically as 30 i or conversely 30 can be derived in imagination from but without positive guidance from established facts as to which, if either, direction of imagined change might reasonably be regarded as probable, neither of these alternatives by itself is an acceptable solution. Scrutiny of other numbers present in the family (Table 4) is not helpful and, therefore, on previous evidence the ancestry of these numbers could not have been worked out. This notwithstanding, unless the second alternative mentioned above, i.e = 30, could be excluded on positive grounds, the possibility that n = 29 might be primitive and denote some measure of common ancestry with Pteris or other genus outside the family would remain open. This however can now be done. With the knowledge that the number 29 is not one of two but the middle member of three, serial, gametic numbers (i.e. n = 28, 29, 30) the H NP.

5 290 S. K. ROY AND I. MANTON picture changes substantially. The most probable interpretation now is that none of these numbers is in itself primitive. Species with n = 28 and n = 30 are Hkely to be polyploid derivatives from precursors with n = 14 and n = 15 respectively from which, by an act of hybridization i'ollowed by chromosome doubling, a new number n = 29 could have been synthesized. There are of course other alternatives (such as = 29) but all virtually rule out any reasonable likelihood that the number 29 could have been the starting point for the genus as we find it. Its importance as a character shared with Pteris or other genus outside the Schizaeaceae is thus minimal. While, therefore, there are now strong positive reasons for believing that the gametic number 29 cannot be primitive in the genus Lygodium but must be a relatively late innovation, it is important to subject to similar scrutiny the other numbers now present with it since further conclusions might thereby be forthcoming. If, as seems likely, some ances- Table 4. Summary of cytological information for other genera of Schizaeaceae {Compiled from Chiariigi, i960 and Eabbri, 1963) Species Country 2n n Author Anemia rotundifolia Schrad. Berlin B.G. A. rotundifolia Schrad. Kew - A. rotundifolia Schrad. S. India A. phyllitidis (L.) Sw. Berlin B.G. A, phyllitidis (L.) Sw. Ceylon, alien - A. obliqua Schrad. Berlin B.G. A. adiantifolia (L.) Sw. Jamaica - A. adiantifolia (L.) Sw. Jamaica A, adiantifolia Jamaica c. 152 A, adiantifolia Jamaica pairs 38 univ. Friebel (1933) Abraham, Ninan and Mathew (1962) Friebel (1933) Friebel (1933) Walker (1962) Walker (1962) Walker (1962) Walker (1962) Mohria caffrorum (L.) Desv. S. Africa 152 Schizaea dichotoma (L.) Smith New Zealand - 5. digitata (L.) Sw. S. India S. fistulosa l^ahiw. New Zealand S. fistulosa var. aiistralis Hook f. New Zealand S. pusilla Pursh New Jersey, U.S.A. is. robusta Baker Hawaii S. asperula Wakefield New Zealand 76 ' ^ c. 270 c Lovis and Roy (1964) Brownlie (1961) Abraham, Ninan and Mathew (1962) Brownlie (personal communication) Brownlie (personal communication) Wagner (1963) W^agner (1963) Lovis (1958) tral species of Lygodium possessed lower chromosome numbers than those so far detected in the genus, further search among the twenty odd species not yet examined might yield direct evidence of them. It is by no means impossible that Lygodium species possessing the postulated n = i4orn = 15 may still exist and it is also possible that other numbers relevant to the general inquiry might be found. The imperfect count offered by Brownlie (1961) for L. articulatum (n = c. 70) would already be evidence of this kind if the details could be established with finality, for here is exactly the type of information to expect if, as is possible, the number 28 should be polyploid not on 14 but on 7. The scientific interest of a successful demonstration of the truth of the latter possibility would be considerable. The high chromosome numbers possessed by the overwhelming majority of extant ferns is one of their most distinctive qualities yet there are reasons for thinking that the Pteridophyta, as such, must have started with low chromosome numbers comparable to those still retained by all the other major groups of land

6 A new base number in the genus Lygodium 291 plants. This matter was first discussed in Manton (1950) and everything that has happened since has tended to confirm this conclusion although direct evidence for low numbers is scarce. Whereas in flowering plants some of the commonest monoploid numbers are 7, 8, 9 and 11, in ferns (see especially Manton and Sledge, 1954) high prime numbers, notably 29, 37 and 41 are abundant. In practically no case can an origin for these numbers be deduced with certainty although the analysis of the situation in Lygodium discussed above, in spite of its incompleteness, is one of the best documented examples of a relatively successful attempt to do this. Actual low numbers have so far only been found in Salvinia, where n = 9 was reported by Loyal (1958) and in the Hymenophyllaceae where n = 11 was found in a New Zealand species by Brownlie (1958) and n = 13 in a British species by Manton (1950). Traces of similar numbers may perhaps be recognized in several genera now showing multiples of 13, e.g. Paesia and IMatotiia with n = 26 (Brownlie, 1958 and Manton, 1954 respectively), Pteridium with n = 52 (Manton, 1950). Arithmetic alone is however an unreliable guide and it would not be safe for example to infer an ancestry based on x = 11 for the Osmundaceae which at present display only n = 22 in all investigated species of the three extant genera (Manton, 1950) since 22 could be reached in many other ways which are unlikely now to be traceable. Strong presumptive evidence for the former presence of n = 7 in the Hymenophyllaceae was supplied by finding n = 21 and n = 28 (Manton and Sledge, 1954) though n = 7 itself has not yet been found (Vessey and Barlow, 1963). Further search may nevertheless still reveal it since in no large fern group with a cosmopolitan distribution is the C)i:ological sampling anything like adequate to remove a reasonable possibility of further significant discoveries. While, therefore, it is important that the inquiry should be extended as opportunity offers in all groups of this kind, the discovery that Lygodium may in fact be favourable from this point of view is perhaps the most significant conclusion to which the present inquiry has led. It is greatly to be hoped that further collecting will contribute more specimens for cytological study from other areas of the globe than those so far sampled. REFERENCES ABRAHAM, A., NINAN, C. A. & MATHEW, P. M. (1962). Studies on the cytology' and phylogeny of the Pteridophytes. VII. Observations on one hundred species of South Indian ferns. J. Indian bot. Soc, 41, 339. ALSTON, A. H. G. & SCHELPE, E. A. C. L. E. (1952). An annotated checklist of the Pteridoph>'ta of Southern Africa, yi S. Afr. Bot., 18, 153. BROWNLIE, G. (1958). Chromosome numbers in New Zealand ferns. Trans. R. Soc. N.Z., 82, 665. BROWNLIE, G. (1961). Additional chromosome numbers New Zealand ferns. Trans. R. Soc. N.Z., Bot., I, I. CHAMBERS, T. C. (1955). Use of snail stomach cytase in plant cytology. Nature, Lond., 175, 215. CHIARUGI, A. (i960). Tavole cromosomiche delle Pteridophyta. Caryologia, 13, 27. CLARKE, H. M. (1936). The morphology' and anatomy oi Lygodium japonicum. Am. J. Bot., 23, 405. CoPELAND, E. B. (1947). Genera Filiciim. Chronica Botanica Co., Waltham, Mass. FABBRI, F. (1963). Primo supplemento alle Tavole cromosomiche delle Pteridophyta di Albert Chiarugi. Caryologia, 16, 237. FRIEBEL, H. (1933). Untersuchungen zur Cytologie der Fame. Beitr. Biol. Pfl., 21, 167. DE LiTARDiERE, R. (1920). Recherches sur l'el^ment chromosomique dans la caryocin^se somatique dans les Filicinees. Cellule, 31, 255. HoLTTUM, R. E. (1959). Flora Malesiana. Series II, vol. i, part i, p. 37. Lovis, J. D. (1958). A chromosome count in Schizaea. Nature, Lond., 181, Lovis, J. D. & ROY, S. K. (1964). A chromosome count in Mohria. Nature, Lond., 201, LOYAL, D. S. (1958). Cytology of two species of Salviniaceae. Curr. Sci., 27, 357. MANTON, I. (1950). Problems of Cytology and Evolution in the Pteridophyta. Cambridge University Press. MANTON' I. (1954). Cytology of meiosis in Matonia. Nature, Lond., 173, 453. MANTON, I. (1958). Chromosomes and fern phylogeny with special reference to 'Pteridaceae'. J. Linn. Soc. (Bot.), 56, 73.

7 292 S. K. ROY AND I. MANTON MANTON, I. & SLEDGE, W. A. (1954). Observations on the cytology and taxonomy of the pteridophyte flora of Ceylon. Phil. Trans. R. Soc, B238, 127. MEHRA, P. N. (1961). Chromosome numbers in Himalayan ferns. Res. Bull. Panjab Univ., N.s., 12, 139 RADFORTH, N. W. (1938). XIV. An analysis and comparison of the structural features of Dactylotheca plumosa Artis sp. and Senftenbergia ophiodermatica Goppert sp. Trans. R. Soc Edinb., 59, 385. RADFORTH, N. W. (1939). XXVII. Further contributions to our knowledge of the fossil Schizaeaceae; genus Senftenbergia. Trans. R. Soc Edinb., 59, 745. VESSEY, J. & BARLOW, B. A. (1963). Chromosome numbers and phylogeny in the Hymenophyllaceae. Proc Linn. Soc N.S.W., 88, 301. WAGNER, W. H. JR. (1963). A biosystematic survey of U.S. ferns. Preliminary abstract. Am. FernJ., 53, i. WALKER, T. G. (1962). The Anemia adiantifolia complex in Jamaica. Neiv PhytoL, 61, 291. EXPLANATION OF PLATES 7 AND 8 Lygodium: permanent acetocarmine squashes from root tips or spore mother cells x PLATE 7 No. I. L. scandens,, 2n = 60. No. 2. L. japonicum, S. China, 2n = 58. No. 3. L. circinnatum, N. Borneo, 2n =: 58. No. 4. L. volubile, Kew, n = 29. PLATE 8 No. 5. L. longifolium,, n = 56. No. 6. L. fiexuosum, Ceylon, n = 56. No. 7. L. salicifolium,, 2n ^ 56. No. 8. L. dimorphum. New Guinea, 2n = 56. No. 9. L. flexuosum, N. Borneo, 2n = 112.

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