POLYPLOIDY IN POLYPODIACEAE— ITS EVOLUTIONARY AND ADAPTIVE SIGNIFICANCE

By G. Panigrahi and S. N . P a t n a i k Botanical Survey of India, Shillong

(Received for publication on December 26, 1962)

T h e family Polypodiaceae (Copeland, 1947), comprising of 65 genera in the world flora, are represented by 27 genera and more than 110 species in India. The family occur generally in the tropical and sub­ tropical evergreen forests of Eastern India, mostly as epiphytes on moss- covered tree trunks or on humus-covered rocks and boulders, though a few terrestrial species {viz., Dipteris wallichii) creep on the surfaces of sandy soil on rocky substratum. All the genera are characterized by creeping rhizome (short or extensive) and simple, lobed or pinnate fronds bearing exindusiate sori.

Although the family has been subjected to serious studies in orthodox taxonomy, very few genera were cytologically investigated until recently, owing to their inaccessibility to many fern cytologists on account of their occurrence in tropical and subtropical evergreen forests. However, Manton (1950, 1954) and Manton and Sledge (1954) reported the chromosome numbers of 40 species from Ceylon and Malaya including the Polypodium vulgare complex from temperate Europe and America. Nayar (1958), Bir (1960) and Abraham (unpublished) report chromosome numbers of only one species each from India. Realising the inadequacy of the cytological knowledge in this predomi­ nantly epiphytic family, Polypodiaceae and considering the great opportunities for such studies owing to our living in Shillong which is situated in the very heart of Assam’s “ Forest climate”, we have recently studied the cytology of 36 East Indian species (Panigrahi and Patnaik, 1961; Patnaik and Panigrahi, 1963a) collected from the montane ever­ green forests of the Khasi and Jaintia Hills and tropical evergreen and semi-evergreen forests of the lower and upper Assam and foothills of the North-East Frontier Agency. Very recently Malhotra (in Mehra, 1961a) and Pal (1961) have also reported the chromosome numbers in various Indian species. Thus, a total of 85 species belonging to 22 genera from the rain forests of South-East Asia have been cytologically investigated up to date.

A perusal of chromosome numbers of the 22 genera referred to above {cf. Table 1) brings out the following broad features of their cytological picture (c/. Panigrahi and Patnaik 1963 6):— (1) Basic chromosome numbers in tiie famil’v range between a : = 11 to X = 47, these two extreme numbers characterizing the species of Pleopehis (= Lepisorus) only. (2) The commonest basic chromosome numbers are x- == 36 charac­ terizing 14 genera and x ~ 37 characterizing 8 genera, whereas 5 genera, viz., Drynaria, Pleopehis (== Lepisorus), Polypodium, Pyrrosia and Goniophlehium each show both these numbers. (3) Only 4 genera possess .v = 35 whereas 2 genera share x — 33 only. (4) Pleopehis (= Lepisorus) is the only genus showing an astonish­ ing range of haploid and diploid chromosome numbers, viz., n — 22, 23, 26, 35, 36, 47, 74 and 2« = 39. (5) All but 14 species investigated are diploids showing regular bivalents at meiosis with the rare exception of Lepisorus pseudonudiis Ching with 2n = 39 and irregular meiosis. The phenomenon of euploidy in Polypodiaceae (sensu Copeland 1947) and its evolutionary and adaptive significance have alieady been discussed in a separate communication (cf., Panigrahi # Patnaik 1963 h). The discovery of tetraploidy in the most primitive terrestrial genus of Polypodiaceae, which are characterized by low percentage and low grades of euploidy in its epiphytic genera and species have led the authors to postulate an hypotehsis ‘Epiphytic habit in closed tropical and subtropical forests works as a ‘ bottle-neck” to the induction of euploidy whereas the terrestrial Iwibit is highly conducivc to it’.

E u p l o id y a n d its E f j e c i s Now, let us examine the few examples of euploidy undoubtedly established within the family. Amongst the 85 species studied, only 14 taxa belonging to the following 12 genera turned out to be euj^loids, the respective positions of these 12 genera are shown below in the phylogenetic sequence (Copeland, 1947; Holttum, 1947):— Crypsinus Leptochilus (2n, 4n) Polypodium (2n, 4n) Arthromeris {In, 4 m, 6»i) Colysis {In, 4„) (2n, An) Pleopehis Behisia ( = Lepisorus) (2n, 4n) Loxogramme (2n, 4n) Pyrrosia (2k, 4n) Dipteris (2«, 4 « , 6«) Prosaptim (2«. 4 n ) iln , 4 » ) Xiphopteris ( 2 n , 4 « ) I

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IU5 CO II S C S I5J I o t

8 II

a c t

I I O f these, we have thoroughly studied the euploidy and its mor­ phological effects in Loxogramme, Pyrrosia and Dipteris, while a few others have been subjected to similar close analysis by Manton (1950) and Manton and Sledge (1954).

In the genus Loxogramme, L. lanceolata, L. scolopendrina and L. avenia are diploids, each with 3 5 clear bivalents at meiosis whereas L. involuta is a tetraploid with 70 bivalents at diakinesis. There is no indication of multivalent formation either in the diploid or tetraploid species. Although L. lanceolata is morphologically distinct from the remaining three, one has to look for differences (Table II) to distinguish L. involuta from L. avenia and L. scolopendrina, the shapes and sizes of simple fronds borne on tufted rhizomes being very variable in all the three species.

T able II

Rhizome scales Midrib on the upper side of the frond

L. scolopendrina Large, ovate-lanceolate Not raised, almost flat

L. avenia Linear-lanceolate with a Strongly raised and long narrow tip grooved towards base

L. involuta Ovate-lanceolate like that Raised like that of avenia of scolopendrina but but not grooved smaller

Thus, L. involuta appears intermediate in morphology betwera ijU. . aveniaavenuj and L. scolopendrina. But any suggestion of allopolyploid origin of L. involuta by the interspecific crossing between L. scolopendrina and L. avenia must await studies in hybridisation involving the three taxa. Again, Pyrrosia mannii and P. mollis are two very allied species practically indistinguishable from each other except for the colour differences in the scales and so both were graerally known as Niphobolus fissus Bak. or Cyclophorus porosus C. Chr. Ching (1935), however, recog­ nised two species within its limits, and assigned concolourous entire scales to P. mannii and discolourous fimbriate scales to P. mollis. This “ hair-splitting” in taxonomic approach finds, however, coixoboration from studies in their cytology. P. mannii is a diploid with » = 37 whereas P. mollis is a tetraploid with n ~ 74. Both behave as normal species at meiosis, form bivalwits without auy trace of multivalents and form good scores. The mode of origin of tetraploidy in P . mollis Iberefore, an open <^uestion, Manton’s (1950) researches on Polypodium vulgare complex showing 2n, 4n and 6n, which she subjected to a comprehensive hybridization programme, establish undoubtedly the well-known truth, that despite allopolyploid origin of the 6n taxon by the intercrossing of 2n and 4n taxa and subsequent doubling of chromosomes in the Fj hybrid, the morphological differences brought about by allopolyploidy with respect to its putative parents, are of minor nature and are expressed merely by an intermingling of characters already shared by the diploid and tetraploid parents respectively, although polyploidy confers greater plasticity on the organism to sudden changes in the environment.

A n e u p l o id B ase N u m b e r s a n d T h e ir E volutionary Significance A perusal of Table 1 and analysis of the cytological pictures provide overwhelming evidences in favour of the great role played by aneuploid base numbers in the evolution of the order Poypodiales (Pichi-Sermolli, 1959), both at generic and family levels. Pichi-Sermolli {he. cit.) and Mehra (1961 b) both recognize 4 families, viz., Dipterid- aceae, Cheiropleuriaceae, Polypodiaceae and Grammitidaceae. Although they do not indicate the genera included in the respective families they consider these families as more or less natural. The discovery of the base numbers x = 11 (viz., n — 33, 66) in Dipteris and x = 11 to jr == 47 with a large array of haploid and diploid chromosome numbers {viz., n — 22, 23, 26, 35, 36, 47, 74 and 2n — 39) in the genus Pleopeltis (— Lepisorus), undoubtedly provides the answer to the successive steps in progressive evolution within the epiphytic family, Polypodiaceae through the mechanism of aneuploid base num­ bers. Thus, the hypothetical phylogenetic relationships of Dipteris with Matoniaceae and of Grammitidaceae with Gelicheniaceae are corroborated by the discovery of very low base numbers, viz., x = 9 {i.e., n = 36), 11 and 13 in Polypodiaceae and x — \'i both in Mato­ niaceae and Gleicheniaceae. With this cytological picture outlining the main lines of evolution of the order Polypodiales (through Dipteridaceae to Pleopeltis-Wkt: ancestors) from the Gleichenioid ancestors {see Pichi-Sermolli, 1959) or from Gleicheniaceous stock {see Mehra, 1961 b), further evolution within the order'Polypodiales follows a stereotyped pattern. Although the basic chromosome numbers in Polypodiales vary between = 11 and x — Al the commonest base numbw, viz., x = 36 characterizes 8 genera, i.e., Microsorium, Phymatodes, Colysis, Leptoohilus, Aglao- morpha and Photinopteris all included within Microsorieae and Lemma- phyllum and Neooheiropteris included in Pleopeltideae (Copeland, 1947). These genera share the common base number, viz., x = 36 with the genus Pleopeltis ( = Lepisorus, 2n = 72 found in one species, viz., longifolius). The similar cytological features and possession of a large array of low base numbers in Pleopeltis ( = Lepisorus) considered together with evidences from comparative morphology postulating divergent lines of evolution, as represented by these eight genera amongst Others, from P lw peltis ( = Lepisorus) {qf. Panigrahi and Patnaik, 1961 b) undoubtedly establish Pleopeltis (= Lepisorus) as an evolutionary plexus (Text-Fig. 1) holding the key to the macro-evolution at generic and family levels within the order Polypodiales Pichi-Sermolli,/oc.aV.). The next common base number within the family is ;c = 37 ^:harac- terizing 5 genera of which Arthromeris and Platycerium represent tvvo end points in evolution within the family Polypodiaceae whereas Ctenopteris, Prosaptia and Xiphopteris are segregated as members of the family Grammitidaceae (Jc/JJM Pichi-Sermolli, 1959). The next base number ;c = 35 characterizes two genera Belvisia and Loxogramme, both of which are supposed derivatives from Pleopeltis (which has also x — 35 in three taxa) and represent again two other evolutionarily stagnant ends. But the 5 genera characterized by the possession of more than one basic chromosome numbers, viz.. Polypodium (x — 36, 37), (Gonio- hlebium (x = 36, 37), Crypsinus (x — 33, 35, 36), Drynaria (x = 36, 37) and Pyrrosia (x ~ 36, 37) seem to possess greater plasticity and appear to be in a state of evolutionary activity. While commenting on the discovery of discordant base numbers in Thelypteris (viz., x = 31, 35, 36) and in Crypsinus (viz., x = 33, 36) Manton (1953) considered “ this type of aberration” as uncommon in fern genera and suspected that generic boundaries had been wrongly drawn in these genera. In the great majority of fern genera, she observed uniformity in base num­ bers rather than diversity and this she considered as characteristic as euploidy in ferns “ in spite of the small amount of wobbling which is apparently liable to occur round most of the main base numbers”.

M o d e OF O r ig in o f A n e u p l o id Base N u m b e r s Whatever may be mode of origin of the base number ;c = 36 in Pleopeltis (— Lepisorus), evolution of all the genera cytologically investi­ gated with the exception of Dipteris, Ctenopteris, Prosaptia and Xipho­ pteris, from the Lepisorus-TpXewii may be visualized by the decrease or increase of one pair of chromosomes from the base number x = 36 in Lepisorus. Stebbins (1950) outlines the main steps for the reduction of base number ;c = 8 to jc — 7 and finally to ;c = 5 in a natural popula­ tion of Ixeris and cites the example of the reduction of x = 7 to = 6 in an artificially synthesized fertile biotype of Goditia whit-neyi. This reduction in base number is postulated to arise from the translocation of active distal portions of a chromosome having geneticaUy inert seg­ ments next to the centromere, to a non-homologous chromosome and subsequent loss of the centromere with th». inert segment. Thus, mere rearrangement of chromosome segments in structural heterozygotes is sufficient to bring about the reduction of the base number. If this process repeated several times in Lepisorus-\\k6 ancestors, involving different pairs of chromosomes in the remote past, origin of Pleopeltis (Lepisorus) sp. with n = 35 and again, that of genera Hke Belvisia with jc = 35, Loxogramme with = 35 and Crypsinus with X ==i 33 and 35, is not difficult to visualize. But progressive increasi in basic numbers is mainly due to duplica- tion of one whole pair of cHromosomes (viz., tetrasomics) but swcn biotypes are bound to be unstable and are unlikely to be precursors of new genera. Stebbins (1950), therefore, postulates progressive increase in base numbers in nature as arising from the duplication of a centro­ mere plus a system of translocations. The discovery of n = 36 in Drynaria propinqua from Shillong in contrast to the prevailing number n = 37 in D. quercifolia from Garo Hills and the nature of pairing at meiosis in the latter species (Table III) may be cited here as of cytological and evolutionary interest.

T able 111 Meiotic analysis in D. quercifolia from Garo Hills of Assam

Total No. of No. of No. of No. of No. of spore cells cells cells cells mother showing showing showing showing cells 37" 35" — !■'' 36“ - 2' 35" - 4' studied

56 20 5 22

The variation in pairing as observed above and the formation of one tetravalent or 4 univalents in 14 of the 56 cells analyzed may suggest the duplication of one pair of chromosomes in the haploid complement of n = 36 as in D. propinqua, the duplicated set showing, however, either incomplete homology or structural translocations in varying degrees in different cells. The meiotic behaviour in Pleopeltis pseudonuda* (PI. I, with In — 39 is highly significant. While a few cells in Figs. 3-6) p. pseudonuda (Ching) Paniar. cf. Patn. Comb. nov. show 11 bivalents and 17 univalents or 17 bivalents and 5 univalents, majority of the cells at diakinesis form varied numbers of clumps of odd shapes or only a massive chain while the young pollen-grains show micronuclei. The very good staining of these nuclear bodies with aceto- carmine undoubtedly indicate their DNA nature. Such peculiar cytological features uncommon in ferns and angiosperms may find parallel only in the nucleus of the members of Cyanophyceae. While one is tempted to interpret P. pseudonuda as an autotriploid based on = 13, the discovery of the unorthodox numbers, viz., n — 22, 23 and 47 (PI. I, Figs. 1 and 2) within Lepisorus itself (= Pleopeltis linearis of authors) and occasional formation of strictly bivalents and univalents without any multivalents in P. pseudonuda warns us against hasty con­ clusions regarding the nature of polyploidy in this species. Whether

* Pleopeltis pseudonuda (Ching) Panigr. & Patn. Basionyni— pseudo- nudus CWn^. Bull. Comb, nov. Fan. Mem. Inst. Biol. 4; 83, m } , O o

< i 3 -i s HI POLYfLOW r IN POLYPODIACEAE 3l$ p. pseudonuda could be assigned an allopolyploid origin from the inter­ crossing of the diploid species (« = 22) with a hypothetical diploid species having « = 17, may only be conjectured at present. The meiotic behaviour of Drynaria quercifoUa is, therefore, a pointer to the important role the structural heterozygous translocations must have played in the evolution of higher base numbers within Polypodiales. Accordingly, it is not difficult to postulate the origin of x = 37 from a: = 36 in different species of the same genus, viz.. Polypodium, Gonio- phlebium, Drynaria and Pyrrosia, of x = 37 in Arthromeris from species of Crypsinus-Vike ancestors with x = 36 and of x = 37-38 in members of the Grammitidaceae from the vast array of basic chromosome num­ bers, including x = 37, characterising Pleopeltis (= Lepisorus). It may, therefore, be seen that while the very low percentage of euploidy in this family has been responsible in originating rather a fewer new species only, aneuploid base numbers have provided the “ evolutionary pool” and presumably have acted as originators of species in the first instance but as potential genera in the long run (Manton, 1950).

A n e u p l o id B ase N u m b e r s a n d T h e ir A d a p t iv e S ignificance The association of antheridia and archegonia on the same pro- thallus germinating on the moss-covered tree trunks or within its cre­ vices high above the forest floor and the consequent obligate inbreeding in ferns must be considered as assets in aiding the taxon to bring together gametes with identical chromosome numbers (either reduced or increased from normal haploid complement) and then to sort out the biotype with the best selective advantages. The creeping rhizome (either short or long), invariably characterizing almost all the genera and species of Polypodiaceae and other methods of vegetative propagation, confer very great advantages in tiding over periods of instability that inevitably follows the origin of aneuploid base numbers in a biotype. Thus, these aneuploid methods of reduction or increase in the base numbers have played the major role in the evolution and adaptation of the epiphytic family Polypodiaceae to the peculiar “ mid-air” habitats which they generally occupy under closed canopy of evergreen forests. But the problem whether the haploid numbers, viz., n — 35, 36 37 and 47 seen against the discovery o f x = 11, 13, etc., mthin Pleopeltis (which also possesses n — 35, 36, 37 and 47) may be looked upon as tips of a number of divergent polyploid series (whose bases must be searched for in the low numbers already discovered or in wild popula­ tions of the family awaiting cytologica! studies) needs further extensive studies in the family.

S u m m a r y 1. Analysis of the cytologica] data of 85 species of the predomi­ nantly epiphytic family Polypodiaceae studied up-to-date shows that only 10 species are tetraploids and 4 are hcxaploids, whereas the remain­ ing species are diploids characterized by an array of aneuploid numbers varying between n = 22 and « = 47. 2. The epiphytic habil inside the closed evergreen forest canopy is presumed to serve as “bottleneck” to euploidy but as “ stimulant” to the origin of aneuploid base numbers. The terrestrial habit on the other hand may play the reverse role in identical situations.

3. Evidences both from comparative morphology and cytology suggest the genus Pleopeltis ( = Lepisorus) as a highly potential “evolu­ tionary plexus”. Origin of a large number of genera of the Polypodi- aceae is visualised from Pleopeltis (— Lepisorus)-\iks ancestors through the mechanism of aneuploid base numbers. The latter provide the “ evolutionary pool” and appear to have played the major role in the evolution and adaptation of the epiphytic family to the peculiar “ mid­ air” habitats in the tropical and subtropical evergreen forests.

4. The finding of so low base numbers as x = 11 and x ~ 13 in Pleopeltis {— Lepisorus) may point to the haploid numbers, viz., n = 33, 35, 36, 37, 38 and 47, etc., as tips of a divergent polyploid series.

A c KNOWLEDGEMENTS

Grateful thanks are due to the authorities of the Council of S'-ienli- fic and Industrial Research, New Delhi, for the award of a Junior Research Fellowship and other financial assistance to the junior author for carrying out cytotaxonomic studies on East Indian Polypodiaceac and to Director, Botanical Survey of India, Calcutta, for his keen interest in the progress of the research scheme. The junior author expresses his thanks to the Education Department, Government of Orissa, for granting him leave during the period of this investigation.

R eferences

Bir, s. s. I960. Cytology of some “Acrostichoid’’ Ferns. Nucleus 3: 121-24.

C h in g , R. C. 1935. On the genus Pyrrosia Mirbal from th e mainland of Asia including Japan and Formosa. Bull. Chin. hot. Soc. 1: 36-72. Coi'ELAND, E. B. 1947. Genera Filiriim. Waltham, Mass., U.S.A.

H o l tt u m , R. F. 1954. A Revised Flora o f Malaya. Vol. II. The Ferns of Malaya. Government Printing Office, Singapore. M a n to n , I. 1950. Problems of Cytology and Evolution in the Pteridophyta. Uni­ versity Press, Cambridge, England. 1953. The cytological evolution of the Fern Flora of Ceylon. Evolution Symp. Soc. exp. Biol. 174-85. ------. 1954. Cytological notes on one hundred species of Malayan Ferns. Appendix in Holttum’s Flora of Malaya 2: 623-28. - AND S lk d g e , W. a. 1954. Observations on the cytology and taxonomy of the Pteridophyte Flora of Ceylon. Phil. Trans. 239; 127-85. . * 1 - V ,, ‘ ^ % ^ V

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\ G- Panisrahi and S. N. Patn«ik Facing page 320 wran r- M e h r A , p . N. 1961 a. Chromosome Numbers in Himalayan Ferns. Research Bull. Punjab Univ. N.S. 12 (Parts I--11): 139-64. ______. 1961 I). Cytological evolution of Ferns with particular reference to Himalayan Forms. Presidential Address. Proc. 4Sth Indian Sci. Congr. Ass. Part II. Section Botany, 130-53. N a y a r. B. K. 1958. Studies in Polypodiaceae. V. Cytology of Colysis peduncu- lota (Hk. et Grev.) Ching. Sci. and Cult. 24: 181-82.

P a l . s . 1961. Chromosome numbers in some genera of Polypodiaceae. Ibid. 27: 499-501.

Panigrahi, G. and Patnaik, s . N. 1961 a. Cytology of some genera of Polypodi­ aceae in Hastern India. Nature. Land. 191: 1207-08. 1963 (7. Phyiogenctic studies in Polypodiaceae (Copeland, 1947). Mem. Indian hot. Soc. 4: 8-19. ------, 1963 /). Low Percentage and Grade of euploidy in the Family Poly­ podiaceae in relation to the epiphytic habit—An hypothesis. Amer. Fern. J 5.3: 145 M8.

P a t n a i k . , S. N. and Panigrahi, G. I963r. Cytology of some genera of Poly­ podiaceae in Eastern India—II. Amer. Fern. J. 53; 40-46.

Pichi-Serm olli, R, K. G. 1959. Pteridophyte in Vi.stas in Botany. Perganion P r e s s Ltd., London. SirBBlNS, G. L. 1950. Variation and Evolution in Plants. Columbia, U.S.A.

E x p l a n a t io n o : P late I

F ig. 1. Lepisorus ussuriensis (Regei et Maack.) Ching. n = 22, a 900.

F ig . 2. Lepisorus suhconftuense Ching. n - 47, <900.

F ig . 3. Pleopeltis pseu lonuda (Ching.) Panigr. et Patn. Com i. nov. 2 a 39, X 1,150.

Fig. 4. „ „ n - \1" + 5\ x 1,150. F ig , 5. „ „ Clumps of nuclear bodies, x 1,150.

F ig . &. „ „ Massive chain of nuclear material, X 2,500.