Cey. J. Sci. (Bio. Sci.) 36 (1): 17 - 34, 2007 17

RE-EVALUATION OF SPECIES LIMITS AND OF THE ENDEMIC STEMONOPORUS THW. () USING MORPHOLOGICAL DATA

S.C.K. Rubasinghe1*, D.M.D. Yakandawala1 and D.S.A. Wijesundara2 1Department of Botany, University of Peradeniya, Peradeniya, Sri Lanka 2Royal Botanic Gardens, Peradeniya, Sri Lanka Accepted 14 May 2007

ABSTRACT Stemonoporus Thw. is the most species-rich endemic dipterocarp genus in Sri Lanka and all its members are categorized as highly threatened or threatened in the IUCN red data book. Species limits within this important taxon are ill-defined, with some authors recognizing only a few variable species, and others recognizing a number of separate morphologically circumscribed species. These controvasies regarding the number of species and the species limits of Stemonoporus Thw. are a hindrance to implementing management measures to conserve this taxon. The main aim of the present study was to evaluate the species limits of the endemic genus Stemonoporus using morphological characters.

Numerical and cladistic analyses were performed based on morphological data obtained from specimens collected from different geographical locations and herbarium specimens. Cluster analysis and Cladistic analysis on 170 specimens, divided the genus into 27 different clusters. These clusters corresponded to the 26 species recognized by Kostermans (1992) and in additional the present study identified a new morphologically distinct species, making the species limit of the genus 27, further strengthening its position as the most species rich endemic dipterocarp genus in Sri Lanka. The examination of 73 variables by cluster analysis and cladistic analysis revealed a number of clear differences in morphological features between the currently recognized species. In addition to the previously recognized characters, many important characters of leaves, flowers and especially of the leaf venation were recognized to define species limits. The study has revealed the long standing ambiguity regarding the species limits of Stemonoporus and further has recognized a new species to the genus.

Key words: Stemonoporus, Dipterocarpaceae, cladistics, phenetics, species limits, morphological data.

INTRODUCTION genera and a monotypic South American genus in Stemonoporus Thw. is an endemic genus that the subfamily Monotoideae, and one species of belongs to the family Dipterocarpaceae whose one genus in the South American subfamily origin dates back to the Gondwana in the early Pakaraimoideae (Kajita et al., 1998). However, Cretaceous period and thought to have migrated the disjunct distribution of closely related taxa to present day South-east Asia through the both in Sri Lanka and Malaysia suggests that Deccan plate. Blume in 1825 recognized the dipterocarps must have already diverged to Dipterocarpaceae, which forms a very closely- generic or even infrageneric sections before they knit family, and considered it to be related to entered the Laurasian plate from the Deccan plate Tiliaceae and Clusiaceae (Kostermans, 1992). (Dayanandan et al., 1999). The family Dipterocarpaceae consists of three subfamilies distributed widely in the tropics. In Sri Lanka the family Dipterocarpaceae is According to the recent molecular phylogenetic represented by 9 genera with 58 species of which classification, the family is nested within the Doona and Stemonoporus are endemic. More , in the Eurosids II clade, under the order interestingly all of the Sri Lankan dipterocarp close to Thymelaeaceae and species are endemic (Kostermans, 1992). Sri (APG II, 2003). Based on the most recent Lankan dipterocarp studies, apart from a note on classification of the family (Ashton, 1982), Vateria by Linnaeus in his Flora Zeylanica approximately 470 species in 13 genera are (1747), start with Thwaites’s book Enumaratio recognized in the Asian subfamily Plantarum zeylaniae (1864). Thwaites Dipterocarpoideae, 39 species in two African recognized three endemic genera Doona, Stemonoporus and Monoporandra. *Corresponding author’s e-mail: [email protected]

S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 18

Stemonoporus is the most species-rich ‘growing especially as small or large gregarious endemic dipterocarp genus in Sri Lanka with up groups in the understorey or on river banks in the to 26 species (Kostermans, 1992). All its lowlands’, or as ‘frequently common to sub- members are placed under the highly threatened dominant trees in the mid-mountain forests at or threatened category in the IUCN red data book 1000-1600 m’. Further, ‘each species has its own (IUCN Sri Lanka, 2003). The entire genus is well-defined habit, geographical and ecological confined to the per-humid forests in the wet zone range’ (Ashton, 1980). Kostermans (1992), in his of the island, from the Knuckles region south and account of the genus describes the ecology as westwards, and eastwards to Rakwana, with the ‘always in the wettest parts, many along streams exception of S. acuminatus, which occurs in and streamlets in the wet zone’. ‘Occurrence is Badulla district in the intermediate zone (Ashton, independent of the depth of soil or kind of soil 1980). Several species reach a height of about and often they occur in small populations far 1800 m, the highest altitude recorded for any apart and only a few reach timber size’ dipterocarp species in Sri Lanka (Ashton, 1980). (Kostermans, 1992) (Figure 1). Ashton (1980) described their ecology as

Figure 1. Ecology of Stemonoporus Thw. A- S. petiolaris, occurs near a large rock on a mountain top close to Kitulgala/Yatiyantota area. B - S. gracilis is only known from an area along a tributary of the Kelani River, in the Kitulgala region. C - S. scalarinervis at Gilimale Forest Reserve. D - S. oblongifolius at Wewaltalawa near Yatiyantota.

Species limits and taxonomy of Stemonoporus Thw. 19

Stemonoporus species are small trees with currently been re-established as a proper genus. resinous wood, rarely reaching timber size. Thwaites originally recognized 11 species under Leaves simple, spirally arranged, usually elliptic Stemonoporus and 3 under the Monoporandra. to oblong, very variable in shape and size. Inflorescence axillary and extra-axillary, Considering the three main classification principally panicles, showing reduction to systems; Trimen (1893) in his treatment on the racemes and single flowers, but their paniculate Dipterocarpaceae in volume I (1893) of his origin is always indicated. Flowers bisexual, Handbook to the Flora of Ceylon, separated sepals and petals are five-merous. Stamens 5 or Stemonoporus from Vateria. He described 13 15 (10 -13) in number, in one or two whorls, 10 species in Stemonoporus and 2 in the reinstated external, 5 internal slightly shorter. The genus Monoporandra while Ashton (1980) in his distinctive feature of Stemonoporus is the treatment of the family in the Revised Handbook chrome-yellow stamens, which form a very to the Flora of Ceylon has recognized 15 species. conspicuous cone-like structure around the style The most recent treatment of the family in the centre. No connective appendage, anthers Dipterocarpaceae by Kostermans (1992) opening by a pseudo-pore. Two types of fruits are recognized 26 species of Stemonoporus, where recognized in Stemonoporus: globose and ovoid, the genus is broadly divided into two sub-genera pointed (Figure 2). (Monoporandra and Stemonoporus) based on the number of stamens. A summary of the different Taxonomic history taxonomic treatments is given in Table 1 and a Stemonoporus was first recognized by comparison of major taxonomic treatments of the Thwaites in 1854. Since its inception, it has genus is given in Table 2. moved through Vateria and Vatica and has

Table 1. A summary of the taxonomic treatments of Stemonoporus Thw. (Adopted from Kostermans, 1992).

Taxonomic treatment Number of species recognized

11 species of Stemonoporus Thw. and 3 of Monoporandra Thwaites (1854) Thw Referred Stemonoporus Thw.to Vateria L. and added 3 to Thwaites (1864) Stemonoporus Thw.

Reduced Stemonoporus Thw.to Vatica kept Monoporandra de Candolle (1868) Thw.separate.

Separated Stemonoporus Thw.from Vater and recognized Trimen (1893) 13 species of Stemonoporus Thw. and 2 of Monoporandra Thw.

Added 2 new species of which one belonged to Vatica (16 Hooker (1900) proper Stemonoporus Thw.)

Included Monoporandra Thw. in Stemonoporus Thw. (15 Alston (1931) proper Stemonoporus)

Restored S. lancifolius(Thw) Ashton but excluded S. moonii Ashton (1980) Thw (15 proper Stemonoporus Thw.)

Recognized two sub-genera while distinguishing 26 species Kostermans (1992) of Stemonoporus Thw.

S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 20

Figure 2. Flowers and fruits of Stemonoporus Thw. A and B - The characteristic cone-like structure around the ovary formed by stamens of Stemonoporus that are 5 (b) or 10-15 (a) in number. C - The characteristic anthers of Stemonoporus, the margins of the two valves touching in one place, where the margin bulges, forming a long oblique orifice apically and a straight one below the bulges. D - a and D - b - Two types of fruit found in Stemonoporus; globose to sub-globose thin-skinned (D -a), conical, pointed, thick-skinned (D - b). Species limits and taxonomy of Stemonoporus Thw. 21

Table 2. A comparison of major taxonomic treatments of the genus Stemonoporus Thw.

Trimen (1893) Ashton (1980) Kostermans (1992)

S. nitidus S. nitidus S. nitidus S. oblongifolius S. oblongifolius S .oblongifolius S. revolutus S. revolutus S. revolutus S. rigidus S. rigidus S. rigidus S. lanceolatus S. lanceolatus S. lanceolatus S. ceylanicus S. ceylanicus S. wightii S. wightii S. elegans S. elegans S. elegans Monoporandra elegans S. laevifolius S. acuminatus S. acuminatus S. acuminatus S. affinis S. affinis S. affinis

S. cordifolius S. angustisepalum S. cordifolius Monoporandra cordifolius S. cordifolius S. marginalis S. canaliculatus S. canaliculatus S. bullatus S. canaliculatus S. gardneri S. gardneri S. gardneri S. scalaranervis S. gilimalensis S. petiolaris S. petiolaris S. petiolaris S. kanneliyensis S. reticulatus S. reticulatus S. reticulatus S. nitidus ssp. lancifolius S. lancifolius S. lancifolius S. nervosus S. gracilis

S. moonii excluded S. moonii S. levvwisianus excluded excluded S. scapifolius S. latisepalum

As stated above, notwithstanding the recent taxonomic methods using a few vegetative revision of the Sri Lankan Flora (1980), several characters. Even the most recent treatment of the ambiguities still exist with the species limitations family Dipterocarpaceae by Kostermans (1992) is in this genus Stemonoporus. All the existing based on morphological data using conventional classifications of the genus including the revision methods which are mostly based on intuition, and of the Sri Lankan Flora are based on traditional therefore are not reproducible. S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 22

This could be a reason why these treatments From the time when numerical taxonomy or are not in agreement with each other, thus phenetics was introduced, a number of articles enforcing several ambiguities in the species have been published on the classification of the limitations of this threatened endemic genus. flowering , employing numerical taxonomic Even though the very purpose of listing a taxon approaches based on morphological characters as threatened is to stimulate interest in (Kelleher et al., 2004; Henderson, 2005; Castro et conservation issues to preempt its decline al., 2005). Cluster analysis groups objects based towards extinction, controversial ideas on the on total similarity into respective categories. number of species stand as a barrier to their During cladistic analysis, monophyletic groups implementation. are recovered and further groups with synapomorphic character combinations will The aim of the present study was to determine occupy the clades. the species limits of the genus Stemonoporus, using morphological data based on empirical methods. The re-evaluation of the species MATERIALS AND METHODS delimitations within the genus Stemonoporus, will not only be highly valuable for furthering our Materials: Specimens of all representative taxa knowledge in the taxonomy and evolution of the from all possible locations (Sinharaja, Kanneliya, genus, but also important for the conservation Hiniduma, Suriyakanda, Morningside, Peak and management of this taxon. Wilderness Sanctuary, Gilimale, Bambarabotuwa, Knuckles, Nelluwa-Pelawatta, Kitulgala, Morphological studies: External morphological Bulathsinhala) were collected. Each specimen characters currently provide almost all the was labeled with a code number. A minimum of characters used in field identification and many six individuals per taxon was sampled. Several of those have been used for hypothesizing attempts made to collect specimens of S. nitidus phylogenetic relationships. These features have and S. scaphifolius during the study period were been used for a longer time than anatomical or unsuccessful. These species were studied from molecular evidence and have constituted the herbarium specimens in the Royal Botanic primary source of taxonomic evidence since the Gardens, Peradeniya, Sri Lanka. The table 3 gives beginnings of systematics (Judd et al., the specimens collected, their localities with the 1999). Plant morphological characters are still the allocated code number. most popular type of characters used in taxonomic studies and they play a major role in Methods taxonomic keys, Floras and also in field Coding of morphological characters: Specime taxonomy. They will probably continue to be the -ns collected were surveyed for vegetative and main discriminatory feature for both the reproductive characters. Characters were selected identification and grouping of plants mainly due by reviewing previous work and searching for to the fact that they are readily recognizable and variations that had not been previously analyzed. easily described. Leaves were analyzed in detail for leaf architectural characters. Flowers were preserved Determining species limits: The literature on in 70% alcohol and their sepals, petals, stamens, species concepts is enormous and currently there ovary, style and stigma were observed under the are two major schools of thought regarding light-microscope (Olympus, BH 2) and species limit determination, the school of thought stereomicroscope (Leica, 10446322, 2xWD). that states that species must first be determined Twigs with leaves were pressed between papers, outside a cladistic framework and then within a treated with alcohol and oven-dried in a folder cladistic framework to discover their and studied under the microscope. relationships to other species and the other, the theory of determining the species limits entirely Leaf architecture (venation): the pattern made within a cladistic framework (Davis and by the primary vein (mid vein) and secondary, Goldman, 1993; Baum and Donoghue, tertiary and quaternary veins, were coded by 1995). Both phenetic and cladistic approaches clearing leaves using 5% NaOH, staining and have been adopted during the present species observing their pattern. Quantitative characters limits study. During phenetic analysis, were counted or measured with a ruler or individuals are grouped (clustered) according to protractor. total similarity while in cladistics monophyletic clades are recovered based on homologous characters and synapomorphies. Species limits and taxonomy of Stemonoporus Thw. 23

Data Matrix: A total of 170 specimens of criteria of Fitch parsimony with 100 replicates, Stemonoporus was studied and searched for both random sequence additions, tree bisection- qualitative and quantitative characters. reconnection (TBR) branch swapping and Characters were scored to the extent possible MULPARS in effect, steepest descent on. Ten from all the collected specimens and also from trees were held for each step. Strict consensus, herbarium specimens based in the collection at 50% majority rule and Adams consensus trees the Royal Botanic Gardens, Peradeniya. were obtained and branch lengths and tree scores Characters that are linked have been coded by were calculated using ACCTRAN (accelerated coding the linked character as a separate transformation optimization). The initial trees independent character as explained under the found with equal (Fitch) weights were used as the method ‘C’ in Kitching et al. (1998). basis for successive weighting. Successive weighting was carried out using the retention Determining species limits: Multivariate index. Reweighting was continued until the same methods of analysis – cluster analysis (CA) and tree length was obtained in two successive UPGMA were carried out using the statistical rounds. Bootstrapping was carried out to packages PCORD 4 version and PAUP * 4d55 evaluate the robustness of the clades. Bootstrap for Macintosh respectively. Groups of specimens analysis employed 1000 replicates of full defined by unique combinations of character heuristic search, searching with the initially states were examined for internal consistency by weighted trees and successive weighted trees. studying character states within the group. For Successive weighting was performed to cladistic analysis, the morphological data matrix “improve” the matrix; in effect this procedure was constructed using MacClade version 3.04. optimizes the fit of the most consistent characters Cladistic analysis was performed using PAUP * on the tree such that more changes are forced into 4d55 for Macintosh to assure recovery of the the characters found to be least consistent in the most parsimonious tree or trees. Alternative tree initial round of analysis. The effect is generally topologies and resultant changes in tree lengths the reduction of tree number as those created by were explored using MacClade 3.04. For all characters that change frequently are eliminated, analyses, heuristic searches were performed as they are less parsimonious (Kitching et al., initially in the unordered and equal weighting 1998).

Table 3. Specimens collected and their localities

S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 24

RESULTS characters. The data set included 18 quantitative The morphological data matrix consisted of a variables and 55 qualitative variables and is total of 73 morphological characters comprising presented in Table 4. several discrete vegetative and reproductive

Table 4. Qualitative and quantitative characters assessed for the morphometric analysis of the genus Stemonoporus. The quantitative characters were measured in centimeters.

Character Description

Leaf characters 1. Habit: The general appearance. Varies greatly. It was found that there is considerable variation of habit within the members of the in-group. Though they had been generally categorized as trees, few of them found to be tall trees up to 20 m, reaching timber size while the others were found to be very small trees (some up to 5 m), bushy shrub-like or slender- trees. This character was scored as tall trees = 0; slender or bushy tree = 1. 2. Branchlet nature: Branches can be stout = 0; slender = 1; drooping = 2. 3. Orientation of leaves: Leaves of some specimens were characteristically held pointing up or downward. The character was scored as hanging = 0; held upwards or held downwards = 1. 4. Leaf lamina length: Length of the leaf lamina showed great variation and was measured from its point of attachment to the petiole to its apical end. The character was broadly categorized and scored as (> 36 cm) = 0; (21 – 35 cm) = 1; (10 – 20 cm) = 2; (1 – 10 cm) = 3. 5. Width of the leaf blade: Width of the leaf lamina was measured at its widest point. Lamina width was scored as (13 – 20 cm) = 0; (8.0 – 12 cm) = 1; (5 – 6 cm) = 2; (1 – 4 cm) = 3. 6. Ratio: length / width of the leaf blade: (2:1) = 0; (3:1) = 1; (4:1) = 2; (6:1) = 3; having two or more types of ratio = 4 7. Lamina shape: Determined by comparing length to width ratio. The terminology was adopted from Dilcher, 1974. Leaf shapes of the specimens collected varied greatly. Therefore only the major shapes were considered (i.e. all elliptic leaves were taken as elliptic, and subcategories narrow elliptic and wide elliptic were not considered) Shapes observed were scored as elliptic (like an ellipse, longer than width, narrow to rounded ends and widest at or about the middle) = 0; Oblong (longer than broad with the sides more or less parallel for most of the length. The length usually less than ten times the width) = 1; ovate (with an outline like that of a hen’s egg, the broadest point bellow the middle) = 2; lanceolate (lance-shaped, much longer than broad, widening above the base and tapering to the apex, broadest point bellow the middle) = 3; obovate or narrow oblanceolate (the reverse of ovate, the terminal half broader than the basal) = 4; having two or more shapes = 5. 8. Shape of the leaf apex: Shape of the leaf apex varied from acute (pointed, forming less than a right angle), acuminate (acute apex, tapering in to a long point), apiculate, obtuse (blunt, rounded, usually forming more than a right angle), retuse (with a notch at the apex), to mucronate (terminating abruptly by a short sharp point at the apex). In certain instances two or more shapes were observed in the same specimen. Therefore only the two major types were considered and scored as acuminate = 0; not acuminate = 1. 9. Presence of an acumen: Acumen; acute apex tapering to a longer point. Scored as absent = 0; present = 1. 10. Origin of leaf acumen: Scored as abruptly acuminate = 0; gradually acuminate = 1; showing both types = 2; not applicable = 3. 11. Length of the leaf acumen: A measurement from the point of origin of the acumen to its extreme end. Scored as (0.1 – 0.7 cm) = 0; (> 0.8 cm) = 1; not applicable = 2. 12. Nature of the acumen: Stout = 0; slender and drooping = 1. 13. Shape of the leaf base: Acute (forming less than a right angle) = 0; obtuse (forming more than a right angle) = 1; rounded = 2; cordate (heart-shaped with a basal notch and ovate in general outline) = 3; obtuse or rounded (showing both types) = 4; showing more than two shapes = 5. Species limits and taxonomy of Stemonoporus Thw. 25

14. Variability of leaf base: Certain specimens showed varied shapes in their leaf bases (more than one shape) while in the others the shape was distinct and not variable. The character was scored as not-variable = 0; variable = 1. 15. Shape of the leaf margin: Entire = 0; slightly revolute = 1. 16. Leaf texture: Coriaceous (leathery, thick) = 0; chartaceous (opaque and like paper) or other = 1. 17. Lamina nature: Normal (without any specialization) = 0; sub – bullate or corrugate (lamina impressed along the mid vein) = 1; bullate (lamina strongly impressed along veins showing shallow marks) = 2; revolute = 3. 18. Petiole width: Thick = 0; narrow = 1. 19. Petiole apex: The point of attachment of the leaf blade to the petiole, Inflated (petiole is thickened or swollen at the point of attachment to the leaf blade) = 1; normal (petiole not swollen or thickened) = 0. 20. Petiole length: Measured from the point of attachment of the petiole to the main axis to the point of attachment to the base of the leaf blade. Scored as (< 0.9 cm) = 0; (> 0.9 cm) = 1. 21. Petiole nature: Stout = 0; slender or drooping = 1. 22. Presence of persistent stipules: Normally the stipules were found to be small and caducous except for a few OTUs which had numerous persistent stipules on the apical branches and was scored as absent = 0; present = 1. 23. Presence of an intra-marginal vein: The vein formed by the connection of secondary veins, running along the margin of the blade. Absent = 0; present = 1. 24. Nature of the intra-marginal vein: Moderate = 0; prominent = 1; discontinuous = 2 not applicable = 3 25. Number of secondary veins: Number of secondary veins on the leaf lamina 26. Type of venation: Careful observation showed two distinct types of venation. Camptodromous – eucamptodromous (secondary veins upturned and gradually diminishing apically inside the margin, connected to the superadjacent secondaries by a series of cross veins without forming prominent marginal loops) = 0; camptodromous – brochidodromous (secondary veins not terminating at the margin but joined together in a series of prominent arches) = 1; having both types of venation in the same leaf = 2. 27. Width of the primary vein: The size of the primary vein (the thickest vein of the leaf) was determined midway between the leaf apex and base as to the ratio of vein width (vw) to leaf width (Lw); vw/Lw x 100 = size. Scored as stout = 0; slender or weak = 1. 28. Course of the primary vein: Straight = 0; markedly curved = 1 straight or markedly curved = 2. 29. Elevation of the primary vein on upper leaf surface: Prominulous (slightly elevated in a groove) = 0; deeply impressed = 1. 30. Angle of origin of secondary veins: Measured above the point of branching. Acute – narrow and acute - moderate (< 450 and 450 - 650 in the same leaf) = 0; acute - moderate and acute - wide (450 - 650 and 650 - 800 in the same leaf) = 1; right angle (800-1000) = 2; acute – narrow and acute - wide (<450 and 650 - 800 in the same leaf) = 4. 31. Variation in angle of origin of secondary veins: Lowest pair more acute that the pairs above = 0; upper more acute than lower = 1; upper more obtuse than lower = 2; irregular = 3; more acute on one side of the leaf = 4. 32. Relative thickness of secondary veins: A measure of the width of the middle secondary veins compared to those of the primary and tertiary orders. Scored as thick (proportionally wide, in relation to the primary and tertiary orders or to the secondary veins in other leaves of similar size) = 0; moderate to fine or hair like (proportionally narrow, in relation to the primary and tertiary orders or to the secondary veins in other leaves of similar size) = 1. 33. Course of secondary veins: The nature of the secondary vein curvature at the leaf margin. Curved uniformly = 0; curved abruptly = 1; both types present = 1. 34. Elevation of secondary veins on upper leaf surface: Whether the secondary veins are impressed or prominulous. Scored as improved =0; Prominulous = 1 35. Presence of loop forming branches: Absent = 0; present = 1. 36. Behavior of loop forming branches: Joining the super-adjacent secondary at right angles = 0; joining the super-adjacent secondary at obtuse = 1; not applicable = 2 37. Presence of inter-secondary veins: Thickness intermediate between that of the second and third order veins; generally originating from primary vein, interspersed among the secondary veins. Absent = 0; present = 1 S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 26

38. Pattern of tertiary veins: Purcurrent (tertiaries from the opposite secondary veins joining) = 0; ramified (tertiary veins branching into higher orders without rejoining the secondary veins) = 1; having both types = 2. 39. Angle of origin of tertiary veins: The combination obtained when the predominant angle of tertiary origin on the exmedial (lower) side of the secondary veins is compared with that on the admedial (upper) side of the secondary veins. Right angles-right angles (RR) = 0; acute- right angles (AR) = 1; not applicable = 2. 40. Course of tertiary veins: Simple and straight = 0; simple and convex, concave or retroflexed = 1; not applicable = 2. 41. Relationship to mid vein: Oblique = 0; oblique and right angles = 1; not applicable = 2. 42. Variation in angle of tertiary veins: Decreases upward = 0; constant = 1; not applicable = 2. 43. Arrangement of tertiary veins: Predominantly opposite = 0; predominantly alternate = 1; opposite and alternate in about equal proportions = 2. 44. Highest vein order of the leaf: (5 -6) = 0; (7 – 8) = 1. 45. Marginal ultimate venation: 46. Presence of areoles: The smallest areas of the leaf tissue surrounded by veins which taken together form a contiguous field over most of the area of the leaf. Scored as well developed (meshes of relatively consistent size and shape) = 0; imperfect (meshes of irregular shape, more or less variable in size) = 1. 47. Arrangement of areoles: Oriented = 0; random = 1; not applicable = 2. 48. Shape of areoles: Definite (triangular, quadrangular) = 0; definite and irregular = 1; not applicable = 2. 49. Size of the areoles: (< 2 mm) = 0; (> 2 mm) = 1; not applicable = 2. 50. Presence of vein-lets: Absent = 0; present = 1; not applicable = 2.

Characters of flowers and inflorescences 51. Nature of flowering: Way in which the flowers are borne whether solitary, in clusters or on inflorescences. The character was scored as not congested = 0; congested = 1. 52. Inflorescence type: Panicle (where a raceme is branched) = 0; raceme (a simple elongated indeterminate inflorescence with stalked flowers) = 1; cluster = 2; complex structure = 3. 53. Length of the inflorescence: Measured from the point of attachment of the peduncle to the main axis to the terminal flower. (25 – 30) cm = 0; (10 – 18) cm = 1; (5 – 7) cm = 2; (1 – 4) cm = 3. 54. Width of the peduncle: Thick = 0; narrow = 1. 55. Length of the peduncle: Measured from the point of attachment to the stem to the point of attachment of the bract of the first flower. (> 0.8) cm = 0; (0.1 – 0.7) cm = 1. 56. Width of the pedicel: Thick = 0; narrow = 1. 57. Length of the pedicel: Length of the flower axis from the point of the attachment of the bract to the point of attachment of the flower. (> 0.7) cm = 0; (0.1 – 0.6) cm = 1. 58. Pedicel nature: stout = 0; slender or drooping = 1 59. Color of the pedicel: Brown to green in color = 0; maroon in color = 1. 60. Presence of bracteoles covering the pedicel: Absent = 0; present = 1. 61. Nature of bracts: Prominent = 0; not prominent = 1 62. Number of bracts: (1-5) = 0; (10-14) = 1; not applicable = 2. 63. Number of flowers: (1-4) = 0; (5-14) = 1; (>15) = 2 64. Diameter of the flower: (1.0 – 1.5) = 0; (>1.5) = 1 65. Color of flowers: white = 0; yellow or orange = 1 66. Aestivation of sepals: Arrangement of calyx in bud. Quincuncially imbricate = 0; valvate = 1. 67. Variability of sepal shape: Two shapes of sepals were observed in the same flower in some taxa but in others sepals of the flowers were of the same shape. The character was scored as sepal shape variable = 0; sepal shape not variable = 1 68. Shape of sepals: Ovate = 0; oblong = 1; ovate and oblong = 2; elliptic and oblong = 3; lanceolate = 4; other = 5. 69. Color of sepals: Maroon = 0; greenish to yellowish = 1. 70. Length of the sepal: Measured from the point of attachment to the hypanthium to the apex of the sepal. (> 0.7) cm = 0; (0.5 – 0.7) cm = 1; (0.3 – 0.4) = 2. 71. Number of stamens: (5) = 0; (10 –15) = 1 72. Number of staminal rows: one = 0; two = 1 73. Shape of the fruit: globose to sub-globose = 0; cone-shaped or pointed = 1 74. Texture of the fruit: smooth = 0; longitudinally furrowed = 1. Species limits and taxonomy of Stemonoporus Thw. 27

Phenetic analysis: The data were analyzed by Each of these resulting clusters was evaluated different clustering methods using the total for their morphological character combinations number of OUT’s (i.e.170 specimens) and 73 based on the most recent classification systems of variables. All analyses resulted in 27 distinct the genus by Kostermans (1992) and Ashton clusters (Figures not shown). (1980) and also with the herbarium specimens. Following this, each cluster was identified to its Cladistic analysis: Heuristic search under the species level. Once the clusters were identified, Fitch criterion yielded 100 most parsimonious one representative from each cluster was selected trees (MPTs) of 357.674 steps, CI = 0.330 and RI in order to carry out a second round of analysis. = 0.991 (Figures not shown). The successive This approach was employed in cluster analysis weighting resulted in a single most parsimonious and cladistic analyses as well. The resulting tree with a Length of 117.984, Consistency Index dendrograms from cluster analyses, UPGMA and of 0.321 and a Retention Index of 0.910. The tree cladistic analysis are given in Figures 3, 4 and 5 was rooted under the option of midpoint rooting. respectively. This resulting cladogram identifies 27 different clades supporting the results of the cluster analysis.

Distance Distance

Variables

Figure 3. Dendrogram obtained from cluster analysis with Minitab 13.2, clustering method with Euclidean Distance Measure with Group Average Linkage method performed with 27 species of Stemonoporus Thw., and on the basis of the full set of 73 variables.

S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 28

0.114 0.025 S. canaliculatus 0.114 S. moonii 0.115 0.109 A 0.030 S. marginalis 0.109 S. bullatus 0.095 0.036 S .affinis 0.095 S. scalarinervis 0.073 0.027 0.018 S. petiolaris 0.026 0.073 S. gilimalensis 0.091 0.013 S. rigidus 0.117 S .lanceolatus 0.022 0.097 0.036 S. reticulatus 0 0.010 0.097 ST23 0.133 S. kanneliyensis 0.102 0.014 0.002 S. oblongifolius 0.004 0.026 0.102 S. acuminatus 0.104 0.012 S. latisepalum 0.055 S. angustisepalum 0.076 0.055 0.024 S. scaphifolius 0.179 S. cordifolius 0.082 0.039 S. gardneri 0.062 0.082 S. wightii 0.047 0.122 B S. revolutus 0.093 0.014 S. gracilis 0.050 0.093 S. lancifolius 0.107 0.051 S. elegans 0.059 0.098 S. nitidus 0.059 S. leavifolius

Figure 4. Phenogram for Stemonoporus constructed by the UPGMA clustering method based on 27 species and 73 variables. Data are equally weighted. Numbers on each branch are numbers of steps separating each node in the tree. The colored boxes indicate the major monophyletic groups.

Species limits and taxonomy of Stemonoporus Thw. 29

S. canaliculatus

S. moonii

70 S. marginalis S. bullatus

S .petiolaris

S. rigidus

S. gilimalensis

S. affinis

S. scalarinervis

S. cordifolius 56 89 S. angustisepalum S. scaphifolius

S. gardneri

60 S. revolutus

S. wightii

S. latisepalum

54 S. reticulatus

ST23

S. oblongifolius

S. acuminatus

S. kanneliyensis

66 S. gracilis S. elegans 76 S. lancifolius 72 S. nitidus

S .leavifolius

S. lanceolatus

Figure 5. Strict consensus of 100 equally most parsimonious trees of 163 steps for Stemonoporus by cladistic analysis of morphological characters. Numbers above branches are the bootstrap percentages (of over 50%). The tree had a CI of 0.571 and a RI of 0.604. S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 30

DISCUSSION has clustered in different clades in the currently There has been much debate on species limits employed methods of analysis. Even though the of Stemonoporus and the genus has also gained number of stamens was a character that carried much attention among biologists and much weight in past groupings, in the present conservationists due to its vulnerability. The study several characters based on venation pattern number of species that has been recognized play more important role. Distinctiveness of the within the genus has been constantly varying and venation pattern was observed to be stronger and the most recent studies of its species limits differ effective in delimiting of the species of greatly. Trimen in 1893 recognized 13 species, Stemonoporus. These characters have not been Ashton (1980) described 15 species and the most used in the past treatments of the genus recent treatment of the family Dipterocarpaceae (Rubasinghe S. C. K., Unpublished data). by Kostermans (1992) recognized 26 species within the genus Stemonoporus. The other major group, clade B has been divided again into two main monophyletic clades. The recovery of 27 distinct clusters based on This division separates S. canaliculatus, the two different approaches, i.e., phenetic and S. moonii, S. marginalis, S. bullatus, S. petiolaris, cladistic methods reveals that the genus is S. rigidus and S. gilimalensis from the others. In comprised of 27 morphologically different all these species, the flowers are developed as entities. The recognition of groups at this level of clusters of 1-4 and have the characteristic separation in the dendogram and monophyletic consistent bracts and bracteoles covering their groups in the cladogram appears to be reasonable, peduncles. This group is further divided as all these groups have been recognized as separating S. canaliculatus, S. marginalis, species by several eminent taxonomists in their S. bullatus and S. moonii from the rest. taxonomic treatments of the genus (Trimen, Stemonoporus canaliculatus Thw. was 1893; Ashton, 1980; Kostermans, 1992). recognized by both Trimen (1893) and Ashton (1980) in their treatments but the species was Though 27 morphologically different clusters split into three; S. canaliculatus Thw., have been recovered from phenetic and cladistic S. marginalis Kosterm. and S. bullatus Kosterm. methods, in phenetic analysis groupings cannot by Kostermans (1992) based primarily on the leaf be described on the basis of characters as the size. The present study also showed the three clustering is based on total similarity, whereas in species to be closely related. The clade including the cladistic method of analysis the opportunity S. moonii gains strong support from several of describing a group based on shared unique morphological features within each apomorphic characters is an advantage. species and a bootstrap support of 70%. All the Therefore, the discussion will be primarily based species have a marginal vein with impressed on the strict consensus tree obtained from primary and secondary veins on the upper leaf cladistic analysis (Figure 5). During the past surface. Presence of inter-secondary veins, angle studies the separations of certain taxa such as of loop forming branches, pattern of tertiary veins S. gracilis and S. lancifolius were based on only a and the arrangement of areoles are characters that few characters and also a limited number of supported the clade and that were not recognized examined specimens. But the present study gives by previous workers. very strong support for the recognition of these taxa, which has been controversial for a long S. moonii Thw. was recognized by Trimen period of time. (1893) in his treatment but was excluded by Ashton (1980) during the revision, where he The cladogram identifies two major clades, A considered the sterile specimens with long and B, separating S. lanceolatus, S. nitidus, stipules he observed to be related to Tiliaceae, S. elegans, S. lancifolius and S. gracilis from the Bombacaceae, or Sterculiaceae but not to be a rest. Within the clade A the monophyletic clade member of the genus Stemonoporus. Kostermans including S. nitidus, S. elegans, S. lancifolius and (1992) has resurrected the species in his work. S. gracilis with a strong bootstrap value of 72% The present study identified the species as a has the characteristic reticulate type of venation member of the genus possessing all the generic without areoles in contrast to the percurrent characters and as closely related to S. venation possessed by the other clade. canaliculatus, S. bullatus and S. marginalis. In Stemonoporus elegans, which has been placed addition to the above-mentioned characters close to S. cordifolius because of the number of S. moonii has very long slender persistent stipules stamens in all the previous classification systems, and long linear hairy bracts as autapomorphies. Species limits and taxonomy of Stemonoporus Thw. 31

Further, S. petiolaris Thw. is another taxon that past systems, where these three species including has been recognized by both Trimen (1893) and S. elegans are included in the subgenus Ashton (1980) in their treatments but the species Monoporandra by Kostermans due to the fact was split into two; S. petiolaris Thw. and that they all possess 5 stamens in contrast to the S. gilimalensis Kosterm., by Kostermans (1992), rest of the species which have 10 -15 stamens. by basing his S. petiolaris on its thin, smaller But in the present analysis S. elegans is separated leaves with long slender acumen and very slender from the rest since it has a distinctly different petioles in contrast to the thicker and larger pattern of venation. leaves with short stout acumen and stout petioles of S. gilimalensis. During the present study Thus the present study, while not supporting S. petiolaris and S. gilimalensis were well the traditional division of the genus into two separated. Stemonoporus petiolaris Thw. could subgenera based on the number of stamens, be easily distinguished from S. gilimalensis by yields much information for use in the the presence of drooping leaves and clusters of identification of the species. flowers with reflexed petals and maroon colored bracts. Although Ashton (1980) highlighted some Kostermans recognized S. cordifolius auct. of these characters, he did not consider them as non (Thw.) Alston and S. affinis auct. non adequate to separate the two species. However, Thwaites under Stemonoporus angustisepalum the present study identifies characters that have Kosterm. The present study agrees with the not been previously employed in delimiting the separation of S. angustisepalum, possessing two species and yielding strong support to larger leaves and a different type of inflorescence recognize S. petiolaris and S. gilimalensis as two from S. cordifolius and S. affinis. The results distinct species. suggest that S. angustisepalum is more close to S. cordifolius than to S. affinis. The number of Stemonoporus rigidus Thw. has been stamens and similar venation patterns of the two recognized in all major treatments. It can be species are characters supporting the clade. characterized by extremely rigid, obtuse leaves Further according to the present analysis borne in clusters, with closely placed secondary S. scaphifolius Kosterm. and S. angustisepalum veins, and areoles with veinlets. Kosterm. occur as sister groups. Though its leaves resemble those of S. bullatus and The remaining cluster is comprised of S. canaliculatus, S. angustisepalum can easily be S. affinis, S. scalarinervis, S. cordifolius, distinguished from the number of stamens (5) and S. angustisepalum, S. scaphifolius, S. gardneri, the paniculate inflorescence. S. revolutus, S. wightii, S. latisepalum, S. reticulatus, ST 23, S. oblongifolius, Trimen (1893) recognized Monoporandra S. acuminatus and S. kanneliyensis. cordifola Thw. in his treatment and M. cordifolia was brought under Stemonoporu as S. cordifolius Trimen (1893) and Ashton (1980), in their (Thw.) Alston by Alston in 1931. Kostermans treatments recognized Stemonoporus affinis (1992) in his treatment in addition to (Thw.), but the species has been split into S. cordifolius (Thw.) Alston, recognized Stemonoporus affinis (Thw.) and S. S. angustisepalum Kosterm. Characters for field angustisepalum Kosterm. by Kostermans (1992). identification of S. cordifolius recognized during S. affinis with prominently acuminate leaf lamina the present study were the very small flowers pointing downwards with areoles of distinct borne on paniculate inflorescences, flowers with shape and prominulous primary and secondary 5 stamens and leaves with long slender acumen, veins on the upper leaf surface has formed a and the percurrent venation with distinct areoles. monophyletic group with S. scalarinervis, whereas S. angustisepalum forms a monophyletic S. gardneri Thw. was recognized by both group with S. cordifolius and S. scaphifolius. Trimen (1893) and Ashton (1980) in their treatments but the species was split into two; Stemonoporus scaphifolius, S.angustisepalum S. gardneri Thw. and S. scalarinervis Kosterm. and S. cordifolius, with 5 stamens, have been by Kostermans (1992). The two species were clustered in a single clade as closely similar but observed to differ greatly with respect to the three distinct species. The clade comprising development of flowers, size of leaves and the S. scaphifolius, S. angustisepalum and pattern of venation. S. cordifolius is supported by 59% bootstrap value. All of these taxa have 5 stamens and this During the present analysis S. gardneri, character has been playing an important role in S. revolutus, S. wightii and S. latisepalum with S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 32

paniculate of inflorescences occur in a mono- form b as S. laevifolius Kosterm. During the phyletic clade. present study specimens with code numbers, ST19 and ST 31 have shown contrasting features S. reticulatus Thw. was recognized by both and have clustered in two separate clusters. ST19 Trimen (1893) and Ashton (1980) in their corresponding to S. acuminatus had more erect, treatments but the species was split into two; more arcuate and less numerous secondary veins S. reticulatus Thw. and S. kanneliyensis which are prominent on the lower leaf surface in Kosterm. by Kostermans (1992). During the contrast to ST 31 corresponding to S. leavifolius present study the two species have separated well with less conspicuous, dense, smooth reticulate with stronger characters which had not been used secondary veins numerous on the glossy lower previously. Two distinct groups were identified leaf surface. This gives additional support for the during the present analysis; a group with splitting. distinctly reticulate under surface, conical shaped fruit and few flowered inflorescence Trimen (1893) in his study recognized corresponding to S. reticulatus against a group S. nitidus ssp. lancifolius Dyer and S. nervosus with not distinctly reticulate, acute pointed fruit Thw. as two distinct species. But Ashton (1980) and many-flowered inflorescence corresponding elevated S. nitidus ssp. lancifolius Dyer to to S. kanneliyensis. Stemonoporus reticulatus S. lancifolius (Thw.) Ashton and reduced auct. non Thw. was recognized by Kostermans in S. nervosus Thw. as a synonym. Kostermans 1992 as S. kanneliyensis Kosterm. (1992) in his work recognized S. lancifolius. Further, S. lancifolius auct. non Alston was The group of plants with ST 23 code had recognized as S. gracilis Kosterm. According to characters which did not match with the presently Ashton (1980) S. lancifolius is a variable species, recognized species of Stemonoporus. Therefore it where the few herbarium collections available all is considered as a new morphologically unique differ in certain leaf characters. The specimen taxon and is placed in the clade containing number C.P. 3885 (PDA) has relatively more S. reticulatus and S. oblongifolius more closely prominent nerves on the undersurface while the linked to S. reticulatus with a bootstrap support specimens from the Kitulgala collection are with of 54%. The group of plants posses hanging, a depressed midrib above. According to glabrous, narrow-elliptic to narrow-oblong leaves Kostermans (1992), Ashton has mixed two attached to a slender geniculate petiole. Flowers species, which are entirely different, and which developed on axillary racemes bearing 4 – 6 could be recognized even in the sterile condition. flowers. Fifteen stamens in two rows form the He has used the following character combinations characteristic cone-like structure around the in defining the two species: S. gracilis, which has ovary formed by stamens of Stemonoporus. been reduced to S. lancifolius by some authors, Considering the fruits of Stemonoporus, two has a separate cluster of its own in the present types of fruit have been described by previous analysis. It can be readily distinguished by the workers; globose to subglobose and conical or deeply channeled midrib on the upper surface pointed. Conical and pointed fruits have been (midrib prominulous in S. lancifolius) and by the observed only in S. kanneliyensis, S. wightii and absence of areoles (areoles present in S. reticulatus. The fruits of ST 23 are cone- S. lancifolius). shaped and pointed. The clade containing ST 23 does not belong to any of these species with Stemonoporus rigidus Thw., S. oblongifolius respect to the floral, fruit and leaf characteristics Thw., S. revolutus Trimen ex Hooker f. and and has come up in a separate clade. S. lanceolatus Thw., recognized by all major treatments, were recognized as distinct S. acuminatus is a species which has morphological entities, reflecting their undergone much controversy during the past. morphological consistency within the genus. Trimen (1893) in his treatment has recognized two forms; form a and form b. Form a is based According to the analysis, specimens on the character leaves with prominent, oblique collected from different geographical locations nerves, and b on hardly elevated spreading have clustered according to their character nerves. Ashton (1991) recognizes the two forms combinations giving rise to 27 different species. but doubts if this difference is not based on their This highlights the fact that these OUTs could be location. Kostermans (1992) during his study identified as distinct species with stable elevated the forms to species rank by recognizing morphological characters that are not affected by form a as S. acuminatus (Thw.) Beddome and Species limits and taxonomy of Stemonoporus Thw. 33

the environment or habitat. This confirms the Ashton, P.S. (1982). “Flora Malesiana. Series I- species limits recognized by previous taxonomic Spermatophyta. Flowering Plants.” “Diptero- treatments and also in addition recognizes a new carpaceae. Martinus Nijhott Pub., The species of Stemonoporus. Netherlands 9 (2).

The present morphometric analysis of Ashton, P.S. (1980). In: Dassanayake, M.D. and Stemonoporus does not agree with the traditional Fosberg, F.R. (Eds.) A Revised Handbook to the division of the genus into two major groups Flora of Ceylon. Amerind Publishing New Delhi. (subgenera: Monoporandra and Stemonoporus) Vol 1: Pp. 404-418. based on the number of stamens. Instead, another set of characters based on the number of flowers Baum, D. A., and Donoghue, M. J. (1995). on the inflorescence, development of flowers and Choosing among alternative "phylogenetic" details in the venation pattern gives a more species concepts. Systematic Botany 20 (4): 560- satisfactory basis for the separation of species. 573. The stamen character appears to be a very good field character for identifying the genus, but it Castro, S., Silveira, P., Pereira, C., and does not give much support in identifying the Figueiredo, E. (2005). Systematic studies in species within the genus. Tylosema (Leguminosae). Botanical Journal of the Linnean Society 147: 99 - 115.

CONCLUSIONS Davis, J.I. and Goldman, D. H. (1993). Isozyme Long-standing ambiguity regarding the variation and species delimitation among diploid species limits of Stemonoporus was completely populations of the Puccinellia nuttalliana resolved with well-supported entities with distinct complex (Poaceae): Character fixation and the character combinations defining species within discovery of phylogenetic species. Taxon 42:585- the genus. The study identifies all 26 species 599. recognized by Kostermans (1992) supporting his treatment of the genus. Further, in addition to the Dayanandan, S., Ashton, P., Williams, S. M. and recognized taxa a new species has been Primack, R. B. (1999). Phylogeny of the tropical recognized, indicated by the code number ST 23. tree family Dipterocarpaceae based on nucleotide Moreover, both methods of analysis here sequences of the chloroplast rbcLgene. American employed to determine species limits have Journal of Botany 86 (8): 1182-1190. resulted in similar grouping proving that both methods could be used in determining species Henderson, A. (2005). A Multivariate Study of limits. Clyptrogyne (Palmae). Systematic Botany 30(1): 60 – 83. The study confirms that the genus Stemonoporus has 27 morphologically distinct IUCN (2003). The 1999 list of Threatened Fauna taxa, further strengthening its position as the most and Flora of Sri Lanka. IUCN, Colombo, Sri species-rich endemic dipterocarp genus Sri Lanka, vii+ 114. Lanka. Judd, W.S., Campbell, C.S., Kellogg, E.A. and Donoghue, M.J. (2002). Plant Systematics. A ACKNOWLEDGEMENTS Phylogenetic Approach. Sinauer Associates, Ind. The authors wish to thank the National Publishers, Sunderland, Massachussets, U.S.A. Science Foundation of Sri Lanka and the International Foundation for Science, Sweden for Kajita, T., Kamiya, K., Nakamura, K., Tachida, financial assistance. H., Wickneswari, R. Tsumura, Y., Yoshimuru, H., and Yamazaki, T. (1998). Molecular Phylogeny of Dipterocarpaceae in Southeast Asia REFERENCES Based on Nucleotide Sequences of matK, trnL APG II (2003). An update of the Angiosperm Intron, and trnL-trnF Intergenic Spacer Region in Phylogeny Group classification for the orders and Chloroplast DNA. Molecular Phylogenetics and families of flowering plants: APG II. Botanical Evolution 2: 202-209. Journal of the Linnean Society 141(4):399. S.C.K. Rubasinghe, D.M.D. Yakandawala and D.S.A. Wijesundara 34

Kelleher, C. T., Kelly, D. L. and Hodkinson, T. Thwaites, G. H. K. (1864). Enumeration R. (2004). Species status, hybridization and Plantarum Zeylaniae: an Enumeration of Ceylon geographic distribution of Irish populations of Plants. Dulau London, Pp 11-12. Quercus petraea (Matt.) Liebl. And Q. robur L. Watsonia 25 : 83-97. Trimen, H. (1893). A Handbook to the Flora of Ceylon, iii. Bishen Singh Mahendra Pal Singh, Kitching, I. J., Forey, P. L., Humphries, C. J. and New Connaught Place, Dehra Dun. Periodical, Williams, D. M. (1998). Cladistics: The theory Pp. 436-437. and practice of parsimony analysis. The Systematics Association publication No.11. Oxford.

Kostermans, A.J.G.H. (1992). A Hand Book of the Dipterocarpaceae of Sri Lanka. Wildlife Heritage Trust of Sri Lanka, Colombo.