CHAPTER 6 PHYLOGENETIC STUDIES OF DIPLAZIUM FROM WEST MALESIA: EVIDENCE FROM MORPHOLOGY

Transcription

1 CHAPTER 6 PHYLOGENETIC STUDIES OF DIPLAZIUM FROM WEST MALESIA: EVIDENCE FROM MORPHOLOGY 6.1. Introduction Most of pteridologist thought that Diplazium are quite distinct and should be separated from Athyrium (See Ching 1940; van Alderwerelt van Rosenburgh 1908; Alston 1956; Sledge 1962; Holttum 1940, 1966; Kato 1977, 1995; Edie 1978; Tagawa & Iwatsuki 1988; Andrews 1990; Kramer et al 1990). Strictly Kato (1977) showed the differences between Diplazium and Athyrium. In Athyrium, the stipes bases on ascending to erect rhizome swollen with pneumatophores, frond axes V-shaped in transaction, acroscopic basal pinnules larger than others, laminar margin cartilaginous or not, spines present adaxially at the junction of costules or not, sori horseshoe- or J-shaped, or linear, scales entire. Whereas in Diplazium, the stipes bases neither swollen nor bearing pneumatophores, frond axes U-shaped with flat base in most species, acroscopic basal pinnules equal or smaller, laminar margin not cartilaginous, spine absent, sori linear, scales toothed or entire. Moreover the separation of Diplazium from Athyrium has been supported by both cytological evidence and molecular data. The different in the basic chromosome number of Athyrium (x= 40) and Diplazium (x=41) is useful diagnostic character (Tryon & Tryon 1982). Preliminary phylogenetic study of Diplazium conducted by Sano et al (2000) based on chloroplast rbcl gene sequences showed monophyletic of this genus. By using evidence from chloroplast trnl-f region sequences Wang et al (2003) also supported the monophyletic of Diplazium clade that include Callipteris Bory, Allantoidea R. Br. Emend. Ching, and Diplaziopsis C. Chr. Morphological variation among species of Diplazium are very diverse. But, a natural subdivision of this genus has not been given yet. Van Alderwereld van Rosenburgh (1908) tried to divide Malayan Diplazium, include those of the Malay Peninsula,The Philippines and New Guinea, into two sections based on only the venation type: (1) Eudiplazium for Diplazium species having free veins and (2) Anisogonium for Diplazium species that possess anastomousing veins. 83

2 Based on the characters such as scales, stipe, lamina and venations, Kato (1977) recognized Japanese Diplazium that consisting of five groups, namely: (1) Diplazium dilatatum group that includes member with groove generally U-shaped with a flat base, acroscopic basal pinnules or segments equal to or smaller than the basiscopic or subsequent ones, scales entire or toothed, but not clathrate; (2) Diplazium wichurae group that includes species member with groove U-shaped, acroscopic base of pinna auricled, adaxial surface of lamina concave along veins, scales entire, sometimes subclathrate; (3) Diplazium mesosorum group that having characters groove U-shaped with a flat base, acroscopic basal pinnules or segments equal to or slightly larger than the basiscopic or subsequent ones, scales entire and subclathrate; (4) Diplazium javanicum group in which includes species with groove V-shaped, frond pinnate or imparipinnate, laminar margin entire or undulate, veins sagenoid-reticulate and scales entire; and (5) Diplazium longicarpun group that includes species with frond pinnate, acroscopic base of pinna truncate, basiscopic cuneate, adaxial surface of lamina not concave along veins, scales entire. However the naturalness of the subdivision of Diplazium circumcribed by van Alderwereld van Rosenburgh (1908) and Kato (1977) have never been tested and analyzed phylogenetically using morphological datasets. Therefore phylogenetic studies on Diplazium using morphological datasets should be introduced. Most grouping on the tree of life were first inferred using morphological characters, which have been widely used since Darwin s time. Most of the million-plus described species are know only from morphology, and their inferred phylogenetic positions are based on these characters; with between 90 and 99 percent of life still to be formally recognized, usually initially from morphological features alone (Lee 2004). Although the pool of morphological characters is much smaller than molecular characters (See Chapter 8), and often insufficient for robust phylogentic resolution, is a common view (e.g. Hillis 1987; Givnish & Sytsma 1997), however morphological datasets often contain as much relevant phylogenetic signal as typical molecular datasets that have orders of magnitude more characters (Lee 2004). 84

3 This chapter presents phylogenetic study of Malesian Diplazium by using morphological datasets generated from sixty nine species from West Malesia. All data on the characters analyzed here are cited from this present work (Chapter 9). This study was conduct to address question of phylogenetic relationships among species within Diplazium. The objectives of this study were to: (1) to reconstruct the phylogenetic relationships among the species in the genus Diplazium; (2) identify monophyletic species groups within Diplazium, (3) establish sister-group relationships among these monophyletic groups Character Selection and Construction Character Selection Cladistic analysis is the most common method currently used to reconstruct phylogenetic trees, even it is the best method (See Bremer & Wanntorp 1978, Estabrook 1978, Wiley 1980). For generating a set of trees, it involves two basis phase-exploration of characters (including selection and examination), followed by analysis of the data (Thiele 1993). Thiele (1993) described an ideal morphological character as one in which the character states vary between terminal units in the analysis but not in other members of the unit (e.g. conspecific individuals) being represented Character Type In the morphological systematic studies, there are two types of characters used: qualitative and quantitative. Qualitative characters are mostly obtained by examination without measurement. These characters can be divided into binary characters and multistate characters. Binary characters comprises two character states, such as scales margin (without thickening black strands or with thickening black strands, in character 14, Table 6.1.). Multistate characters consist of more than two character states such as lamina division that comprise 7 character states (simple, pinnatifid, imparipinnate, bipinnatifid, bipinnate, tripinnatified, and tripinnate, in character 23, Table 6.1.). Generally, qualitative characters are more 85

4 acceptable and seem to be unambiguous in cladistic analysis because their states are considered to be clearly defined and no overlapping (Kitching et al 1998). Quantitative characters are obtained by measurement. Generally quantitative characters are continuously variable. Continuously variable should only be exclude if the cladistic analysis cannot handle such data or if it can be shown empirically that those characters convey no information of phylogenetic signal relative to other characters in the data matrix (Kitching et al. 1998). In this study quantitative characters are included in the analysis because they convey information of phylogenetic signal relative to other characters in data matrix. Another argument for inclusion of quantitative characters is that some qualitative characters may be a collection or transformation of quantitative characters. For example. Leaf shape (a qualitative characters) can be defined by ration of leaf length to leaf width, which is quantitative caharcters (Thiele 1993.). Characters number 7, 8, 16, 17, 25, 26, 37, 38, 49, 50, and 56 in the Table 6.1. are quantitative characters that are important and having tight correlation with the qualitative characters examined. Excluding these characters would give illogical relationships among the species. Therefore these characters were included in analyis Character Coding The crucial point in the phylogenetic analysis using morphological datasets is how features might be usefully coded so as to reflect accurately our observations for particular scale problem (Kiching et al 1998). Character coding is the link between observation and explanation (Strong & Lipscomb 1999). Therefore, ability, or inability, of a coding method to reflect the evidential significant of observations should be the primary concern in considering alternative methods of coding. Basing on the philosophy above, in cladistic analysis of morphological characters in Diplazium, a conservative approach of composite characters coding (Strong & Lipscomb 1999) was employed. Composite coding refers to the creation of single multistate character from several potentially dependent character (method A in Kitching et al 1998) that designed to minimize the effect 86

5 of character linkage. Kitching et al (1998) stated that the more that characters become linked, i.e. dependent on other characters, the greater is the departure from independence and consequently the risk that one false homology can obscure the topologies of true homologies decreased. Composite coding applied in this study was also designed to reduce the error that extensive amounts of missing data (a character state scored as? ) can create in a parsimony analysis (Maddison 1993). The fronds architecture of Diplazium are varying, from simple to quadripinnate. Consequently taxa with simple, pinnate, bipinnate fronds do not posses any organ that possessed by those with pinnate, bipinnate and tripinnate fronds, respectively. Character 36. (Table 6.1) is an example of composite characters in this study. It was created from the combination of two characters: 1) Lateral pinnae exist/not exist and 2) Shape of lateral pinnae oblong / oblong lanceolate / elliptical / liniery triangular / oblong subtringular / lanceolate / ovate. These two characters in which the latter is dependent on the former, were combined into a single character with eight character states (lateral pinna oblong / oblong lanceolate / elliptical / liniery triangular / oblong subtringular / lanceolate / ovate/ organ not exist). This method reduced the numbers of linked characters and limited the influence of multiple missing characters states on the analysis for previously coded characters lateral pinnae oblong / oblong lanceolate / elliptical / liniery triangular / oblong subtringular / lanceolate / ovate in those taxa which have no lateral pinnae. Maddison (1993) stated that missing character states can cause difficulties for parsimony analysis if the missing state for the character occurs in more than one clade Character Variation within West Malesian Diplazium Previous pteridologist worked on Diplazium have summarized the principal characters of the genus Diplazium and identified the important morphological features in identification and classification. Characters those particularly variable across the taxa and which may be significant in an evolutionary sense are discussed as follow. 87

6 a. Rhizome Most of West Malesian species posses erect rhizome. The appearances of the erect rhizome are varying. The very short and small rhizome are seen in D. crenatoserratum and D. tomentosum. The stout and medium rhizome are found in D. dilatatum, D.kunstlerii, D. megasegmentum, D. polypodioides, and D. subpolypodioides. The stout long rhizome is present in D. esculentum. Some species have sreeping rhizome, such as D. donianum, D. procumbens, and D. subvirescens Mickel (1974) presumed that the main lines of the ferns begun with probably creeping rhizome. Thus the erect rhizome is regarded as derived. Figure 6.1. showed the rhizome patterns of Diplazium. b. Scales and Hairs Scales in Diplazium are varying. As showed by Holttum (1940, 1966), Kato (1977, 1995), and Tagawa & Iwatsuki (1988), scales are very important characters in determining species in Diplazium (Figure 6.2.). The scales mainly attach on upper of the rhizome and stipes. On some species scales are also present on the rachis or the costa, but in smaller size than those on the rhizome and stipes. Scales are generally linear lanceolate to obong lanceolate. Rounded or ovate scales are present on the projections of a muricate stipe such as in D. megasegmentum and D. profluens (new species proposed, See Chapter 9), D. kunstlerii, and D. latisquamatum. Rounded or ovate scales which is initially having a function as an covered organ of spines are seen on the stipe of D. spiniferum. Scales are generally attached with a basal point. Some species with rounded or ovate scales, such as in D. kunstlerii, D. latisquamatum, D. megasegmentum and D. spiniferum, have scales with a subbasal point attachment (Figure 6.2.g. in this Chapter; Chapter 9). Scales with a subbasal point attachment may be derived. Dickason (1946) presumed that a scales with a basal point of attachment may be more easily derived from a hair than one with broad base or one peltately attached. Diplazium scales are entire or toothed, with tooth consisting of two upturned ends of adjacent marginal cells. The marginal may be with thickening brown-black strand or not. Toothed scales may show sharp and blunt teeth, 88

7 regular or irregular teeth construction, and forked or not forked teeth. The existence of glandular cell at the marginal scales are important in differentiating among species with entire scales or with blunt toothed scales, such as in D. tomentosum, D. pallidum and D. spiniferum. Scales with entire margins may be simpler than those with toothed margins. Because evolution never starts with complex constructions (Schölch 2000), the toothed margin scales may be derived. Figure 6.1. Rhizome appearance of Diplazium. a. short-creeping rhizome of D. donianum; b. medium-creeping rhizome of D. subvirescens; c. medium-erect rhizome of D. accedens; d. short-erect rhizome of D. halimunense; e. stout-erect rhizome of D. megasegmentum. 89

8 Scale colour are also important in species identification. Scales colour may be yellowish, pale brown, brown, dark brown to black. For example, black scales of D. meijerii will be differentiate fast this species from its related species, D. atrosquamatum, that has brown scales. The taxonomical evaluation of the articulated hairs (trichomes) in the Athyriaceae has been conducted by Kato (1973). He summarised that the features of the articulated hairs are important in the classification of Athyriaceae. In the genus Diplazium, Kato (1972) showed two kinds of articulated hairs, viz. hairs with glandular cells and without glandular cells in Monomelangium pullingeri (that has been proposed to be placed in Diplazium and become D. pullingeri) and D. tomentosum, respectively. There are only several West Malesian species that posses hairs. The hairs are without glandular cells. D. squarrosum posseses stellate hair. D. tomentosum shows simple articulate hairs densely covered stipe, rachis and costa. D. crenatoserratum posseses simple articulated hair sparsely on stipes when living and then it will be scarsely found on dried material. c. Stipe Diplazium stipe is not swollen and becomes gradually thicker downwards. The stipe is generally green. The stipe anatomy of some species of Diplazium has been studied by Tardieu-Blot (1932), Bir (1962, 1969), and Kato (1972). Leaf traces are binary. The xylem of a leaf-trace is the same hippocampus-shaped bundle in transaction (See Chapter 4). In this study the anatomical characters of Diplazium were no used in phylogenetic analysis, because most of species examined were based on only herbarium specimens. Stipe characters used in this study were only those from the gross morphology, such as the size, colour, and the existence of multicellular hairs, scales, protuberances and spine (Figure 6.4.). Most of Diplazium posses scales only. Multicellular hairs and scales are seen in D. crenatoserratum, D. silvaticum D. tomentosum. The multicellular hairs in D. crenatoserratum and D.silvaticum are usually only seen in living plants, they are fallen when dry. Whereas in D. tomentosum the multicelullar hairs can be seen both in living plants and dried materials. The existence of stellate hairs and scales on the stipes and rarchis of D. squarrosum make this species is very distinct among the West Malesian 90

9 Diplazium. Green protuberances are seen in D. accedens var accedens and D. accedens var. swartzii. Spines are present in D. accedens var. spinosum and D. spiniferum. Figure 6.2. The variation of scale shapes in Diplazium. a. D. halimunense; b. D. tomentosum; c. D.. silvaticum var. silvaticum; d.. D. esculentum; e. D. petiolare; f. D. batuayauense; g. D. spiniferum; h. D. sorzogonense; i. D. malaccense; j. D. donianum; k. D. silvaticum var. pinnae-ellipticum; l. D. angustipinna. 91

10 Figure 6.3. Margin of scales. a. entire with glandular cells and irregular thickening black strands (D. atrossuamatum); b. margin entire without glandular cells and thickening black strands (D. cumingi); c and d. margin sharply toothed with regular thickening black strands (c. D. subalternisegmentum, d. D. simplicivenium); e. margin entire with glandular cells and thickening dark brown strands (D. sorzogonense); f. margin sharply toothed irregularly with irregularly thickening black strands (D. halimunense); g. Margin with densely glandular cells without thickening black strands (D. batuayauense); h. margin entire without thickening brown or black strands (D. malaccense); i. margine toothed without thickening black strands D. petiolare. 92

11 When old or in dried specimens, stipes may glabrous, sparsely or densely scales throughout its length. Chistensen (1911) said that the best and most constant specific characters is to be found in the dermal appendages, hairs and scales. A glabrous shoot may be thought ofas more primitive than one having a potentiality for the developmental of dermal outgrowth (Dickason 2007). Figure 6.4. Stipes appearances of Diplazium. a. Stipes densely scales (D. simplicivenium); b. stipes dark green, covered sparsely by rounded-ovate scales, scales fallen when dry (D. spiniferum); c. Stipes covered by green protuberances (D.accedens var. swartzii) ; d. stipes spiny, scales fallen when old (D. accedens var. spinosum) 93

12 d. Rachis and costa As showed by Kato (1977), the rachis and costa groove is usually open to admit the groove of leaf axis of lower order. D. accedens, D. crenatoserratum, D. subserratum, D. tomentosum, D. xiphophyllum have rachis that prominently raised above with shallow grooves and their grooves are U-shaped with flat base (Chapter 4., Figure 4.2.). The appearance of rachis groove would give more informative parsimony characters. The cross section of rachis are generally similar to the cross section of stipes near lamina. Unfortunately, in this study, anatomical data of the cross section of stipes near lamina were only obtained from 27 species due to the lack of the living collections in most species. Therefore grooves characters on rachis or stipes did not used in this analysis, because they would cause many missing data on many species. Generally, rachis are glabrous. Some species has fibrillose or sparsely minutely scales or spiny. Fibrillose rachis seen in D. sorzogonense and D. loerzingii. Spines are present in D. accedens var spinosum and D. spiniferum. Because the existence of fibrillose scales or spine are only in few species, in this study the rachis and costa characters were not used. e. Fronds architecture Fronds architectures in Diplazium are varying, they are simple to quadripinnate. Most of West Malesian species have pinnate to bipinnate fronds. Simple fronds is seen in D. subserratum, but in very young fronds are pinnate (Figure 6.5.). Deeply pinnatifid fronds, sometimes with one pair of free to numerous segments, are seen in D. fuliginosum, D. lomariaceum and D. porphyrophyllum. D. cordifolium has both simple and imparipinnate fronds. Imparipinate fronds are in D. aequibasale, D. bantamense, D. lobbianum D. donianum, D. halimunense (a new species proposed in Chapter 10), D. riparium, D. xiphophyllum, and D. wahauense. The intermediate fronds between pinnate and imparipinnate is seen in D. pallidum. Bipinnate-tripinnatifid fronds are present in D. umbrosum. Tripinnatifid fronds are seen in D. megasegmentum, D. melanolepis and D. subpolypodioides. In D. subalternisegmentum fronds are tripinnate. Tripinnatifid to quadripinnate frond is seen in D. moultonii. 94

13 Figure 6.5. Frond architectures of Diplazium. a. & b. D. subserratum, a. juvenile fronds, b. adult fronds; c. pinnate frond of D. silvaticum var. pinnae-ellipticum; d. imparipinnate frond of D. donianum; e. bipinnatifid frond of D. spiniferum; f. bipinnate frond of D. esculentum. 95

14 e. Venation types There are two kind of venation, viz. free venation and anastomosing venation (Figure 6.6.). Free venation type may be forked or pinnate in the lobes. Generally forked veins are present in the species with simple to simply pinnate fronds or in species that have intermediate fronds between simpli pinnate-pinnate such as D. subserratum, D. aequibasale, D. bantamense, D. lobbianum, D. riparium and D. pallidum. Veins may forked once or more. Whereas pinnate veins are seen in the species with pinnate to quadripinnate such as D. petiolare, D. silvaticum, D. malaccense, D. dilatatum, D. polypodioides and so on. Reticulate venations are seen in D. accedens, D. angustipinna, D. esculentum, D. cordifolium, D. cumingi, D. insigne, D. megasimplifolium (a new species proposed, See Chapter 9), and D.squarrosum. There are some species that generally have free venation, but occasionally show veins uniting at margin such as in D. fraxinifolium, D. riparium and D. xiphophyllum. Kato (1977) showed two types of reticulate venation in Diplazium, goniopterid venation and sagenoid venation. In West Malesian Diplazium, goniopterid venation are seen in D. esculentum, D. accedens, D. insigne and D. squarrosum.. Meanwhile the sagenoid venation are present in D. angustipinna, D. cordifolium, D. cumingii and D.megasimplifolium. Anastomosing venation in Ophioglossum is believed advanced, free venation in other genera as primitive (Kato 1987). However the development of anastomousing venation from free venation may be parallel, it may evolved in different phyletic lines. f. Sori Diplazium sori are dorsal on the vein and linear, elongated along veins with lateral indusium (Figure 6.6.). They are either single (Asplenoid) or double (Diplazioid). Asplenoid sori generally occur along the acroscopic side of a vein and Diplazioid sori are beared on both sides of the basal acroscopic vein. As stated by Bower (1928), Holttum (1947), and Kato (1977), Asplenioid sori are produced by abortion of the shorter arm of J-shaped sori along the basiscopic side of a vein, in view of all intermediate forms. Bower and Holttum interpreted that Diplazioid sori are produced by interruption of horseshoe- or J-shaped sori at the 96

15 distal end. Kato (1977) added that the interpretation of Bower and Holttum may lead to the possibility of Diplazioid sori on higher veins in addition to the basal acroscopic vein. Diplazioid sori bifurcate along the bifurcating basal acroscopic vein, as in Athyrium and Deparia. As pointed out by Kato (1977) Diplazioid sori are not regarded as a morphological unit, but each one of a pair as a mere single Asplenioid sorus facing either the costule or costa. The position of sori in Diplazium is important in species determination. Mainly, there are two type of sori position, viz. subcostular sori and medial sori. The subcostular sori can be divided into two group, viz. that running from until touching midveins and those reaching at proximal end. The medial sori may not touching midvein (inframedial) or almost perfectly medial. Most of West Malesian Diplazium posses subcostular sori. Generally sori are not impressed, but some species show impressed sori. The impressed are present in D.poiense, D. sorzogonense, and D. subserratum. The existence of impressed sori on D. sorzogonense becomes one of the diagnostic characters to differentiate this species from D. speciosum. In the past, a number of phylogenetic schemes presented for ferns was based great stress on the position of the sorus (marginal vs. dorsal) with only rare instances of changing from one to another, as in Bower s phyletic slide of Bower s scheme ( ). Now, this do not faithfully represent all the diversity within the ferns, the shift from marginal to dorsal sori occurring several times (Mickel 1974). In the case of soral position on Diplazium, It is not known whether those subcostular sori are advance or primitive. g. Indusia Most species have firm and persistent indusia. Some species posses very thin indusia and fragile, such as D. procumbens. Generally, the indusia is concolour, there is not color difference between attachment side and margin. But some species posses distinctive indusia, marginal indusia are paler than those at the attachment side, e.g. in D. cumingii and D. loerzingii (new species proposed, see Chapter 10). 97

16 Figure 6.6. Venation types of Diplazium. a-c. Free venation, a. vein pinnate in the lobus, veinlets simple, D. batuayaense; b. vein once forked, D. pallidum var. montanum; c. vein forked 3-4 times, D. donianum; d-e. vein anastomousing, d. goniopteris venation, D.accedens; e. sagenoid venation, D. megasimplifolium. 98

17 Figure 6.7. Sori variation in Diplazium. a. D. densisquamatum; b. D. cordifolium; c. D. accedens; d. D. xiphophyllum; e. D. megasegmentum; f. D. subvirescens; g. D. asymmetricum; h. D. esculentum. 99

18 Indusia may be entire or not. It would be one of the important characters in species determination. Most of Diplazium have entire indusia. Generally, entire indusia are seen in species with simple to pinnate fronds, such as D. suberratum, D bantamense, D. cordifolium, D. lobbianum, D. pallidum, D. riparium, D. silvaticum, and D. wahauense. Whereas non entire indusia (toothed or fringed or lacerate) are found in species with bipinnate fronds, such as D. atrosquamosum, D. beamanii, D. betimusense, D. esculentum, D. Kunstlerii, D. laevipes, and D. meijerii Materials and Methods Taxon Sampling All species of Diplazium described in the Chapter 9 (69 species) were included in this study (Table 6.1.). Athyrium anisopterum was chosen as outgroup. The description of A. anisopterum taken from Holttum (1966). Athyrium was chosen as an outgroup because this genus was considered as the closest relatives of the ingroup (See Kato 1977) Character Examination of Diplazium Morphological characters used in this investigation were obtained mainly from the observations of dried specimens. All characters used by Holttum (1940, 1966), Kato (1995) and Tagawa & Iwatsuki (1988) were assessed. Table 6.1. showed characters, character states, and coding for 88 character utilized in construction of morphological dataset of Diplazium. A list of taxa studied and the scores for characters states are provided in Appendix 1. The morphological datasets matrix was constructed by scoring character states of the characters chosen from the species descriptions (Chapter 10). All qualitative characters were coded as binary and multistate. When characters states could not be defined due to incomplete specimens examined, it was noted as?. Missing character states were marked as -. Quantitative characters are coded by using gap-weigting method (Thiele 1993). The steps are as follows: (1) The raw data are initially ranked as an 100

19 ordered set of states, arranged according to the values of the means; (2) The data are then range standardized: x s = (x- min) / max min) n where, x = the raw datum, x s = the standardized datum, n = the maximum number ordered states allowed by the cladistic computer program, in this study n = 9; (3) The value is coded as the rounded integer of the standardized values; (4) The characters are treated as an ordered multistate for analysis Phylogenetic Analysis The data matrix was run using PAUP Version 4.0 beta (Swofford, 1998). Due to the complexity of the dataset, Heuristic searching was employed. All characters are of type unordered and have equal weight. Gaps are treated as "missing. Multistate taxa interpreted as uncertainty. Starting tree(s) obtained via stepwise addition. Number of trees held at each step during stepwise addition = 1. Branch-swapping algorithm used tree-bisection-reconnection (TBR). Initial 'MaxTrees' setting = 100. The number of rearrangements tried were unlimited. 101

20 Table 6.1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium. Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 1. Rhizome Length Short Long 2.Rhizome habit Erect Suberect Creeping 3.Rhizome appearance 4. Occurrence of scales on stipes Slender Medium Stout Sparsely at base Sparsely throughout its length Basal Densely at base Densely at base to middle Densely throughout its length 5. Attachment side of Subbasal scales 6. Scales shape Linear Oblong Lanceolate Narrowly Subulate subtriangular triangular 7. Scales Length (mm) < x< x< x < Scales Width (mm) < x < x < Scales colour Yellowish Light brown Pale brown 10. Scales thickeness Thin Thick 11. Existence of teeth entire bluntly sharply at scales margin toohed toodhed teeth forked 12. Type of teeth on scale marginal teeth simple (not forked 2.00 x <2.75 Brown Oblong ovate Ovate Rounded x<13.75) x< x< x< x x< x< x< x < x < x <6 6 Dark brown Black 13. Scales (existence of margin glands) Without glandulars cells With glandulars cell 102

21 Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium. Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 14. Scales margin thickness Without thickening black strands With thickening black strands 15. Stipe appearance Slender Medium Stout 16. Stipe (length, mm) < x < Stipe thickness (near < x base, mm) < Stipe surface Smooth Protuberance Prickly x < x < x < x < 4 x < x < x < x < x x < < x < x < x < x Hair existence on Glabrous Glabrescent Tomentose stipe 20. Existence of Glabrescent Scales scales on upper part of stipe 21. Existence of scales on stipe base Glabrescent Sparsely scales Densely scales 22. Stramineous dark pale yellowish pale dark.black stramineous olivaceous brown brown 23. Lamina (division) Simple Pinnatifid Imparipinnate Pinnate Bipinnatifid Bipinnate Tripinnatified Tripinnate 24.Lamina shape Oblong Oblong lanceolate 25. Lamina length (cm) 13.50< x <30.75 Oblong subtriangular x <47.25 Oblong ovate x < Lamina width < x x x (cm) <17.25 <27.75 < Lamina incision Entire Undulate Crenate Divided Oblong subdeltoid x < x <49.75 Oblong ovoid x < x <57.75 Elliptic Lanceolate Ovate subdeltoid x x x < < < x < x < x <88.25 Deltoid < 103

22 Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 28. Lamina base Cuneate Subcordate Cordate 29. Lamina apex Acuminate Attenuate Caudate 30. Number of lateral Absent less than 5 pair 5-10 pairs pairs pairs or more pinnae 31. Position of most Absent Patent Ascending lateral pinnae to the rachise 32. Deflection of lowest pinnae Absent Not deflexed Quite distinctly deflexed 33. Reduction of lowest pinnae not reduced slightly reduced much reduced 34. Stalk existence of Absent Sessile Adnate Stalked lower lateral pinnae 35. Stalk existence of lower lateral pinnae Absent Sessile Adnate Stalked 36. Shape of lateral pinnae Absent Oblong Oblong lanceolate Elliptical Liniery triangular Oblong subtringular Lanceolate Ovate 37. Length of the largest lateral pinnae (mm) 38. Width of the largest lateral pinnae (cm) 39. Upper base of lower lateral pinnae 40. Lower base of lower lateral pinnae (<8) x< x< x< x<36.90) < x < x < x x <14.75 <18.25 Absent Truncate Subtruncate Cuneate x <43.1) x < x <50.9) x < x < x <30.75 Absent Truncate Subtruncate Cuneate Subcordate Cordate Rounded Auricled 57.1 x < x < < x

23 Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 41. Margin of lower lateral pinnae 42. Margin of upper lateral pinnae 43. Incision of lateral pinnae Absent Entire Subentire Undulate Crenulate Crenate Serrate Toothed Lobed or divided Absent Entire Serrate Toothed Slightly Lobed crenate Absent Less than ¼ 1/4 ½ way 2/3 - ¾ way 4/5 5/6 6/7 way to divided way to costa to costa to costa way to costa costa to within 1 mm of costa Absent Rounded Acute Acuminate Caudate Attenuate 44. Apex of lateral pinnae 45. Pinnulae number Absent less than pairs more than 20 pairs pairs 46. The existence of Absent Shortly stalked Subsessile Sessile stalk on lower pinnuae 47. The existence of stalk on upper pinnulae Absent Shortly stalked Subsessile Sessile 48. Pinnulae shape Absent Lineary Oblong Oblong subtriangular Oblong lanceolate 49. Length of largest < x < x < x < x < pinnule (cm) Width of largest pinnule (cm) 51. Pinnule base Absent Truncate Subtruncate Broadly Subcordate cuneate 52. Pinnule margin Absent Entire Serrate Crenulate Lobed Oblong elliptical 9.75 x <11.75 Oblong subdeltoid x <12.25 Hastate x < x <17.25 (< x < x < x< x <3.0) 3.0 x < x < x < x < )

24 Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium. Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 53. Pinnule incision Absent less than ¼ way to costa 1/4 ½ way to costa 2/3 - ¾ way to costa 4/5 5/6 way to costa 54. Pinnule (apex) Absent rounded acute acuminate attenuate 6/7 way to costa to within 1 mm of costa divided = 7 (forming segments 55. Shape balance of pinnulae or segment lobes 56. The width of lobes 57. Form of lobes apice 58. Margine of lobes 59. Form of terminal pinnae 60. Texture of lamina or pinnae or pinnulae 61. Surface depression Absent not oblique slightly oblique Absent less than 5 5 mm or more mm wide wide Absent Truncate Obtuse Rounded Acute Absent Entire Subentire Slightly serrate Serrate Absent Similar to Deltoid with Without lateral ones lobes at base distinct terminal pinna Herbaceous Subherbaceous Subcoriaceous Coriaceous Shoftly chartaceous Surface not depressed below sori Surface depressed below sori 62. Rachis glabrous minutely hairy minutely scaly densely hairy densely scales Slightly toodhed Chartaceous Crenate Papyraceous Lobed 106

25 Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium. Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 63. Rachise (viviparous or not) Not gemmiferous Gemmiferous 64. Costae Glabrous Tomentose/ hairy 65. Vein reticulation type 66. Vein branching type 67. Veinlet number on pinnated vein Free Pinnate less than 4 pairs Anastomousing Forked Sparsely minute scales 4-6 pairs 7 pairs or more Densely scales 68. Veinlet branching on pinnated veins) 69. Vein (uniting with the opposite ones forming excurrent veinlets or not) Simple Forked 1 3 times uniting with opposite ones not uniting with opposite ones Forked more than 3 times 70. Vein (forking type) Forked 1-2 times Forked more then 2 times 107

26 Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 71. Vein (part of anastomousing) 72. Veins in small groups at an angle about... to the midrib or costa occasionally anastomousing near margim but never copiously anastomousing anastomousing ¼ from the margin anastomousing 1/3 from the margin about 45º about 50º about 60ºor more anastomousing 1/2 from the margin anastomousing 2/3 from the margin anastomousing more than 43/4 from the margin 73. Sori position near basal of veins 74. Sori covering on the veins or veinlets 75. sori existence on veinlets 76. Sori number on each vein group 77. Sori (on basal Less than 1/3 of veinlets length middle of veins or 1/3 of vein or veinlet length from near basal of veins or veinlet almost to reach margin 1/2 of vein or veinlet length On part of On all veinlets veinlets or more acroscopic vein) sometimes diplazioid usually diplazioid 78. Indusia form reniform/hooked crescentic linear 79. Indusia Width Narrow Broad always diplazioid 2/3 ¾ Nearly the whole length of veinlets 108

27 Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium. Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) 80. Indusia appearance Hardly evident in mature sori 81. Indusia opening opening before mature 82. Indusia curling Not rolled back when old 83. Indusia colour pale 84. Colour continuity of indusia brown Concolour 85. Indusia margin entire when opening 86. Indusia existence Not persistent 87. Indusia Fragile strengtheness Evident in matrure sori opening when mature Rolled back when old brown Attachments side darker subentire Persistent Robust dark brown toothed or fringed or lacerate or crisped 88. Spines at the junction of costules Absent Present 109

28 6.5. Results and Discussion The eighty eight morphological characters (Table 6.1. and Appendix 1.) were analysed to determine relationship among the 69 species of West Malesian Diplazium. The eighty eight morphological characters comprises eighty five parsimony-informative characters, one constant character, and two parsimonyuninformative characters. The eight equally most parsimonious trees of 1366 steps produce a Consistency Index (CI) = 0.24, Retention Index (RI) = 0.48, RC = 0.12, and Homoplasy Index (HI) = Figure 6.8. showed the topology of a strict consensus tree of the eight equally most parsimonious trees of 1366 steps without the high level of Bootstrap value. Due to the low level of Bootstrap support, it is very difficult to state any statement with confidence about the relationships within West Malesian Diplazium generated from morphological date derived from unweighted maximum parsimony analysis. The out group (Athyrium anisopterum) is separated from the in group without Bootstrap value. The relationships within Diplazium were also unresolved, without high Bootstrap values but clade that comprised D. silvaticum and D. petiolare and clade that include D. lomariaceum, D. megasimplifolium, D. subserratum and D. prophyrorachis with Bootstrap support 65% and 82%, repectively. The lack of support of the monophyly of the genus Diplazium due to the high proportion of homoplastic characters (HI = 0.75). Homoplasy the independent origin or loss of one or more traits in different organism can distort the inference of phylogenetic relationship, tying together similar but unrelated taxa (Givnish & Sytsma 1997). Although the phylogenetic tree derived from the maximum parsimony was lack of or weak support, however, it does not indicate that the pattern observed is incorrect. Triono (2006) stated that the lack of or weak support for a phylogeny does limit the amount of confidence that can be placed in the relationships between taxa and also the conclusions that can be drawn from the inferred phylogeny. Therefore some clades generated from the phylogenetic analysis of Diplazium that seems formed from the closely related taxa were discussed below. 110

29 Figure 6.8. Strict concensus of 8 trees of length 1366 from unweighted morphological dataset comprises 88 morphological characters. Bootstrap support values between are given above line. Only support value above 50% are shown. 111

30 Two major clades were identified in the morphological parsimony analysis without well-supported Bootstrap value: Clade I and Clade II (Figure 6.8.). Clade I consist of D. fuliginosum, D. lomariceum, D. porphyrorachis, D. megasimplifolium and D. subserratum in which D. megasimplifolium and D. subserratum are the most closely related. In this clade D. fuliginosum is at the base clade and diverse into four species, viz. D. lomariceum, D. porphyrorachis, D. megasimplifolum and D. subserratum, respectively. Clade II included most of West Malesian species and splited into two clades with very low-supported Bootstrap value, viz. D. tomentosum alone (Clade II.1.) and all the remaining species (Clade II.2.). In the Clade II.2. D. crenatoserratum is positioned at the basal clade and diverse into two subclade, the small clade in which composed of D. velutinum, D.silvaticum and D. petiolare (Clade II.2.A.) and the large clade (Clade II.2.B.) The affinity of D. fuliginosum, D. lamariceum and D. porphyrorachis. In the topological tree (Figure 8.6.) D. fuliginosum, D. lomariceeum, D. megasimplifolium, D. suseratum and D. porphyrorachis form a separated clade from the other West Malesian species and the wo species, D. subserratum and D. megasimplifolium (a new species proposed, see Chapter 9), are the most closely related. The affinity of the two species will be discussed separately below. The affinity of D. fuliginosum, D. lomariceum and D. porphyrorachis are supported by the share characters as follow: rhizome short-erect, wiry black roots; fronds narrowly elliptic, deeply pinnatifid or pinnate, apex coadnate, segments numerous, usually very dark green when living; scales abundant on stipe and rachis, narrow, usually dark and shiny; veins free. Price (1983) stated that D. fuliginosum is one of the most unusual of all diplazia, was not transferred to the genus Diplazium until recently. This species is strickingly peculiar by the smooth rachis channel without raised sides, uninterrupted by the insertion of pinna-costa. In small fronds, and distally on large fronds, the rachis is almost flat above. Other features unusual to Diplazium are sori informally extending from costa to margin, and thin translucent pinna margin. D. lomariaceum is very closely related to D. porphyrorachis and until Price (1983) distinguished it from D. porphyrorachis, the name seems to have been ignored since Christ himself who described is as 112

31 Asplenium lamoariaceum, reduced lomariaceum to porphyrorachis in Ann. Jard. Bot. Buitenz. 15 (1898, p.119). Examining many specimens from K, L, MICH, NY, P, PNH, UC, and US, Price (1983) showed the signs of apparent gene interchange between D. porphyrorachis and D. lomariceium. In relation of the two species with D. fuliginosum, Price (1983) believed that rather than to D. lomariceum and D. porphyrorachis, the closest relationship of D. fuliginosum is to D. cumingii (Presl.) C.Chr., with which it agrees in dark frond colour and black axis aging to greeyish; axes without cartilaginous ridges; scales on stipe abundant, dark, entire; and indusia black with very narrow pale brown margin, curling back at maturity; even though D. cumingii is very different in its conform frond apex and broadly elliptic pinnae. Price (1983) added that almost exactly the same frond form (and even margin structure) as D. fuliginosum was independently evolved in the Central American D. harrisonii (Bak.) C. Chr. which otherwise differs markdedly in scales and the architecture of the axes. The affinity of D. megasimplifolium and D. subseratum. In the Clade I the closely related between D. megasimplifolium and D. subseratum is not supported by well-bootstrap value. The two species possess similarities on characters as follow: lamina simple with cuneate bases, margin entire on lower part, apex acuminate. However the two species are very different. The lamina of D. megasimplifolium is elliptic, much wider (up to 10.5 cm wide), margin always entire; veins copiously anastomousing up to 4/5 way of margin. D. subserratum possess lamina lanceolate, less than 4 cm wide, margin entire to irregularly crenate; veins free. In juvenile stage lamina of D. subserratum is pinnate. Holttum (1940) presumed that D.subserratum is probably allied to D. lanceum of India and China but is larger, and a shorter rootstock which is more or less with tufted fronds; the scales also appear to be smaller. The relationship among D. malaccense, D. sorzogonense and D tricholepis. Of three species, D. malaccense is closest related to D. sorzognense. Ecologically the two species also grow in similar localities, species of lowland and mid mountain forest in West Malesia. As also stated by Holttum (1940), D. malaccense is differing from D. sorzogonense in glabrescent stipe and rachis.less deeply lobed pinnae, and sori not at all impressed. Whereas, D. sorzogonense 113

32 seems to have affinity with D. tricholepis, mainly its fibrillose rachis, in deeply lobed of lanceolate pinnae, oblong lobes and veinlet number in each lobe. Imparipinnate frond group. Imparipinnate frond group refer to the clade in the Figure 8.6. that comprises D. aequibasale, D. angustipinna, D. donianum, D. cordifolium, D. halimunense, D. cumingii, D. subintegrum, D. bantamense, D. lobbianum, D. fraxinifolium, D. xiphophyllum, D. hottae, D. crameri, D. riparium, and D. wahauense. The affinity of these species seems to be natural. The affinity among these species are discussed below. The affinity of Clade D. cumingii. Clade D. cumingii consists of D. cumingii, D. halimunense, D. cordifolium, D. donianum, D. angustipinna, and D. aequibasale. In this clade, two pair of species, D. aequibasale and D. angustipinna, D. cordifolium and D. halimunense, are the closest related, D. donianum to be the sister clade of D. aequibasale and D. angustipinna, and D. cumingii at the basal clade. The relationship patterns among the species of Clade D. cumingii seems unnatural. The similarities on many quantitative characters among these species that seems not to be correlated with their qualitative resulted the unnatural patterns. The closest relationship of D. aequibasale is to D. riparium or D. wahauense. Morphologically, D. aequibasale is intermediate between D. riparium and D. wahauense. The three species share in characters such as lanceolate dark brown entire scales and oblong lateral pinnae with cuneate base and margin entire. D. angustipinna and D. halimunense should to be closely related to D. cordifolium and D. donianum, respectively. D. angustipinna and D. codifolium share light brown scales, margin entire with irregularly thickening black strands, ovate-lanceolate lateral pinna, and copiously anastomousing veins. While D. donianum and D. bantamense share in irregular sharp toothed scales and ovate-lanceolate pinnae.. The relationship among D. bantamense, D. lobbinaum, D. subintegrum, D. fraxinifolium, D. xiphophyllum,d. hottae. D. crameri. The closest relation of D. lobbianum and D. bantamense and also D. xiphophyllum and D. fraxinifolium are very reasonable and natural. The two first species share in the following character combination: dark brown toothed scales; pinnae ovate-lanceolate with rounded base, margin entire or serrate near apex; vein free and forked several times. 114

33 Meanwhile D. fraxinifolum and D. xiphophylum are in the collection often mixed due to the misidentification. D. fraxinifolium differs from D. xiphophyllum in lineary lanceolate scales (less that 10 mm long), waved-crenate lateral pinnae, forking type of veins (5-7 times). D. hottae seems to have close relation to D. xiphophyllum and D. subintegrum. Tagawa (1972) stated that D. hottae is alled to D. subitegrum. D. hottae differs in: terminal pinna not lobed at the base and similar to upper lateral ones, pinnae apparently entire, venation obscure, sori narrower. D. crameri is more close to D xiphophyllum, and differs from this species mainly in characters: scales dark brown, pinnae stalked to 1.5 cm long, ovate-lanceolate, all veins free (D. xiphophyllum is occasinolly showing the uniting veins near margin) and forked to 2 times. The affinity of D. riparium and D. wahauense. As explained in Chapter 2, 5 and 9 D. riparium and D. wahauense are morphologically very similar and closely related (Kato et al 1991). Topological tree showed in Figure 8.6. also revealed that the two species are closely related. Thus the statement of Kato et al (1991) has been verified in this study. The affinity of D. pallidum and D. prescottianum. The morphological characters between the two species are very similar. Holttum (1940) presumed that D. prescottianum has close relation to D. pallidum. D. prescottianum differs from D. pallidum in large brown scales. More lobed edges to pinnae, and many more soriferous veins in each group. Relationship among D. velutinum, D. petiolare and D. silvaticum. In the topological tree inferred from morphological data the relationshop among D. velutinum, D. petiolare and D. silvaticum are not resolved because the Bootstrap value is very low (<50%), but the closest related of D. petiolare and D. sivaticum are supported (Bootstrap value 65%). Morphologically, the two species are very similar (See Chapter 9). D. petiolare differ from D. silvaticum in characters combination as follow: D. petiolare has scales linear with distantly teeth and thickening black strand; upper surface of lamina light green when living, pinnae lobed ¾ way toward costa, upper base not auricle. Whereas D. silvaticum has lanceolate scales with closely teeth without thickening black strands; upper surface of lamina dark green, pinnae lobed ¼-1/2 toward costa 115