re-evaluation of species limits and taxonomy of the

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

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RE-EVALUATION OF SPECIES LIMITS AND TAXONOMY OF THE ENDEMIC GENUS STEMONOPORUS THW.

(DIPTEROCARPACEAE) 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 Stemonoporus Thw. is an endemic genus that

belongs to the family Dipterocarpaceae whose origin dates back to the Gondwana in the early Cretaceous period and thought to have migrated to present day South-east Asia through the Deccan plate. Blume in 1825 recognized the Dipterocarpaceae, which forms a very closely-knit family, and considered it to be related to Tiliaceae and Clusiaceae (Kostermans, 1992). The family Dipterocarpaceae consists of three subfamilies distributed widely in the tropics. According to the recent molecular phylogenetic classification, the family is nested within the Rosids, in the Eurosids II clade, under the order Malvales close to Thymelaeaceae and Cistaceae (APG II, 2003). Based on the most recent classification of the family (Ashton, 1982), approximately 470 species in 13 genera are recognized in the Asian subfamily Dipterocarpoideae, 39 species in two African

genera and a monotypic South American genus in the subfamily Monotoideae, and one species of one genus in the South American subfamily Pakaraimoideae (Kajita et al., 1998). However, the disjunct distribution of closely related taxa both in Sri Lanka and Malaysia suggests that dipterocarps must have already diverged to generic or even infrageneric sections before they entered the Laurasian plate from the Deccan plate (Dayanandan et al., 1999).

In Sri Lanka the family Dipterocarpaceae is

represented by 9 genera with 58 species of which Doona and Stemonoporus are endemic. More interestingly all of the Sri Lankan dipterocarp species are endemic (Kostermans, 1992). Sri Lankan dipterocarp studies, apart from a note on Vateria by Linnaeus in his Flora Zeylanica (1747), start with Thwaites’s book Enumaratio Plantarum zeylaniae (1864). Thwaites 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

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Stemonoporus is the most species-rich endemic dipterocarp genus in Sri Lanka with up to 26 species (Kostermans, 1992). All its members are placed under the highly threatened or threatened category in the IUCN red data book (IUCN Sri Lanka, 2003). The entire genus is confined to the per-humid forests in the wet zone of the island, from the Knuckles region south and westwards, and eastwards to Rakwana, with the exception of S. acuminatus, which occurs in Badulla district in the intermediate zone (Ashton, 1980). Several species reach a height of about 1800 m, the highest altitude recorded for any dipterocarp species in Sri Lanka (Ashton, 1980). Ashton (1980) described their ecology as

‘growing especially as small or large gregarious groups in the understorey or on river banks in the lowlands’, or as ‘frequently common to sub-dominant trees in the mid-mountain forests at 1000-1600 m’. Further, ‘each species has its own well-defined habit, geographical and ecological range’ (Ashton, 1980). Kostermans (1992), in his account of the genus describes the ecology as ‘always in the wettest parts, many along streams and streamlets in the wet zone’. ‘Occurrence is independent of the depth of soil or kind of soil and often they occur in small populations far apart and only a few reach timber size’ (Kostermans, 1992) (Figure 1).

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 resinous wood, rarely reaching timber size. Leaves simple, spirally arranged, usually elliptic to oblong, very variable in shape and size. Inflorescence axillary and extra-axillary, principally panicles, showing reduction to racemes and single flowers, but their paniculate origin is always indicated. Flowers bisexual, sepals and petals are five-merous. Stamens 5 or 15 (10 -13) in number, in one or two whorls, 10 external, 5 internal slightly shorter. The distinctive feature of Stemonoporus is the chrome-yellow stamens, which form a very conspicuous cone-like structure around the style in the centre. No connective appendage, anthers opening by a pseudo-pore. Two types of fruits are recognized in Stemonoporus: globose and ovoid, pointed (Figure 2).

Taxonomic history

Stemonoporus was first recognized by Thwaites in 1854. Since its inception, it has moved through Vateria and Vatica and has

currently been re-established as a proper genus. Thwaites originally recognized 11 species under Stemonoporus and 3 under the Monoporandra.

Considering the three main classification

systems; Trimen (1893) in his treatment on the Dipterocarpaceae in volume I (1893) of his Handbook to the Flora of Ceylon, separated Stemonoporus from Vateria. He described 13 species in Stemonoporus and 2 in the reinstated genus Monoporandra while Ashton (1980) in his treatment of the family in the Revised Handbook to the Flora of Ceylon has recognized 15 species. The most recent treatment of the family Dipterocarpaceae by Kostermans (1992) recognized 26 species of Stemonoporus, where the genus is broadly divided into two sub-genera (Monoporandra and Stemonoporus) based on the number of stamens. A summary of the different taxonomic treatments is given in Table 1 and a comparison of major taxonomic treatments of the genus is given in Table 2.

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

Taxonomic treatment

Number of species recognized

Thwaites (1854) 11 species of Stemonoporus Thw. and 3 of Monoporandra Thw

Thwaites (1864) Referred Stemonoporus Thw.to Vateria L. and added 3 to Stemonoporus Thw.

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

Trimen (1893)

Separated Stemonoporus Thw.from Vater and recognized 13 species of Stemonoporus Thw. and 2 of Monoporandra Thw.

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

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

Ashton (1980)

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

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


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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).


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

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

revision of the Sri Lankan Flora (1980), several ambiguities still exist with the species limitations in this genus Stemonoporus. All the existing classifications of the genus including the revision of the Sri Lankan Flora are based on traditional

taxonomic methods using a few vegetative characters. Even the most recent treatment of the family Dipterocarpaceae by Kostermans (1992) is based on morphological data using conventional methods which are mostly based on intuition, and therefore are not reproducible.

S. nitidus


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This could be a reason why these treatments are not in agreement with each other, thus enforcing several ambiguities in the species limitations of this threatened endemic genus. Even though the very purpose of listing a taxon as threatened is to stimulate interest in conservation issues to preempt its decline towards extinction, controversial ideas on the number of species stand as a barrier to their implementation.

The aim of the present study was to determine

the species limits of the genus Stemonoporus, using morphological data based on empirical methods. The re-evaluation of the species delimitations within the genus Stemonoporus, will not only be highly valuable for furthering our knowledge in the taxonomy and evolution of the genus, but also important for the conservation and management of this taxon.

Morphological studies: External morphological characters currently provide almost all the characters used in field identification and many of those have been used for hypothesizing phylogenetic relationships. These features have been used for a longer time than anatomical or molecular evidence and have constituted the primary source of taxonomic evidence since the beginnings of plant systematics (Judd et al., 1999). Plant morphological characters are still the most popular type of characters used in taxonomic studies and they play a major role in taxonomic keys, Floras and also in field taxonomy. They will probably continue to be the main discriminatory feature for both the identification and grouping of plants mainly due to the fact that they are readily recognizable and easily described. Determining species limits: The literature on species concepts is enormous and currently there are two major schools of thought regarding species limit determination, the school of thought that states that species must first be determined outside a cladistic framework and then within a cladistic framework to discover their relationships to other species and the other, the theory of determining the species limits entirely within a cladistic framework (Davis and Goldman, 1993; Baum and Donoghue, 1995). Both phenetic and cladistic approaches have been adopted during the present species limits study. During phenetic analysis, individuals are grouped (clustered) according to total similarity while in cladistics monophyletic clades are recovered based on homologous characters and synapomorphies.

From the time when numerical taxonomy or phenetics was introduced, a number of articles have been published on the classification of the flowering plants, employing numerical taxonomic approaches based on morphological characters (Kelleher et al., 2004; Henderson, 2005; Castro et al., 2005). Cluster analysis groups objects based on total similarity into respective categories. During cladistic analysis, monophyletic groups are recovered and further groups with synapomorphic character combinations will occupy the clades. MATERIALS AND METHODS Materials: Specimens of all representative taxa from all possible locations (Sinharaja, Kanneliya, Hiniduma, Suriyakanda, Morningside, Peak Wilderness Sanctuary, Gilimale, Bambarabotuwa, Knuckles, Nelluwa-Pelawatta, Kitulgala, Bulathsinhala) were collected. Each specimen was labeled with a code number. A minimum of six individuals per taxon was sampled. Several attempts made to collect specimens of S. nitidus and S. scaphifolius during the study period were unsuccessful. These species were studied from herbarium specimens in the Royal Botanic Gardens, Peradeniya, Sri Lanka. The table 3 gives the specimens collected, their localities with the allocated code number. Methods Coding of morphological characters: Specime -ns collected were surveyed for vegetative and reproductive characters. Characters were selected by reviewing previous work and searching for variations that had not been previously analyzed. Leaves were analyzed in detail for leaf architectural characters. Flowers were preserved in 70% alcohol and their sepals, petals, stamens, ovary, style and stigma were observed under the light-microscope (Olympus, BH 2) and stereomicroscope (Leica, 10446322, 2xWD). Twigs with leaves were pressed between papers, treated with alcohol and oven-dried in a folder and studied under the microscope.

Leaf architecture (venation): the pattern made

by the primary vein (mid vein) and secondary, tertiary and quaternary veins, were coded by clearing leaves using 5% NaOH, staining and observing their pattern. Quantitative characters were counted or measured with a ruler or protractor.


Data Matrix: A total of 170 specimens of Stemonoporus was studied and searched for both qualitative and quantitative characters. Characters were scored to the extent possible from all the collected specimens and also from herbarium specimens based in the collection at the Royal Botanic Gardens, Peradeniya. Characters that are linked have been coded by coding the linked character as a separate independent character as explained under the method ‘C’ in Kitching et al. (1998).

Determining species limits: Multivariate methods of analysis – cluster analysis (CA) and UPGMA were carried out using the statistical packages PCORD 4 version and PAUP * 4d55 for Macintosh respectively. Groups of specimens defined by unique combinations of character states were examined for internal consistency by studying character states within the group. For cladistic analysis, the morphological data matrix was constructed using MacClade version 3.04. Cladistic analysis was performed using PAUP * 4d55 for Macintosh to assure recovery of the most parsimonious tree or trees. Alternative tree topologies and resultant changes in tree lengths were explored using MacClade 3.04. For all analyses, heuristic searches were performed initially in the unordered and equal weighting

criteria of Fitch parsimony with 100 replicates, random sequence additions, tree bisection-reconnection (TBR) branch swapping and MULPARS in effect, steepest descent on. Ten trees were held for each step. Strict consensus, 50% majority rule and Adams consensus trees were obtained and branch lengths and tree scores were calculated using ACCTRAN (accelerated transformation optimization). The initial trees found with equal (Fitch) weights were used as the basis for successive weighting. Successive weighting was carried out using the retention index. Reweighting was continued until the same tree length was obtained in two successive rounds. Bootstrapping was carried out to evaluate the robustness of the clades. Bootstrap analysis employed 1000 replicates of full heuristic search, searching with the initially weighted trees and successive weighted trees. Successive weighting was performed to “improve” the matrix; in effect this procedure optimizes the fit of the most consistent characters on the tree such that more changes are forced into the characters found to be least consistent in the initial round of analysis. The effect is generally the reduction of tree number as those created by characters that change frequently are eliminated, as they are less parsimonious (Kitching et al., 1998).

Table 3. Specimens collected and their localities


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RESULTS

The morphological data matrix consisted of a total of 73 morphological characters comprising several discrete vegetative and reproductive

characters. The data set included 18 quantitative variables and 55 qualitative variables and is presented in Table 4.

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.


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


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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.


Phenetic analysis: The data were analyzed by different clustering methods using the total number of OUT’s (i.e.170 specimens) and 73 variables. All analyses resulted in 27 distinct clusters (Figures not shown).

Cladistic analysis: Heuristic search under the Fitch criterion yielded 100 most parsimonious trees (MPTs) of 357.674 steps, CI = 0.330 and RI = 0.991 (Figures not shown). The successive weighting resulted in a single most parsimonious tree with a Length of 117.984, Consistency Index of 0.321 and a Retention Index of 0.910. The tree was rooted under the option of midpoint rooting. This resulting cladogram identifies 27 different clades supporting the results of the cluster analysis.

Each of these resulting clusters was evaluated for their morphological character combinations based on the most recent classification systems of the genus by Kostermans (1992) and Ashton (1980) and also with the herbarium specimens. Following this, each cluster was identified to its species level. Once the clusters were identified, one representative from each cluster was selected in order to carry out a second round of analysis. This approach was employed in cluster analysis and cladistic analyses as well. The resulting dendrograms from cluster analyses, UPGMA and cladistic analysis are given in Figures 3, 4 and 5 respectively.

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.

Dis

tanc

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Variables

Dis

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e


28

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.

S. canaliculatus

S. moonii

S. marginalis

S. bullatus

S .affinis

S. scalarinervis

S. petiolaris

S. gilimalensis

S. rigidus

S .lanceolatus

S. reticulatus

ST23

S. kanneliyensis

S. oblongifolius S. acuminatus

S. latisepalum

S. angustisepalum

S. scaphifolius

S. cordifolius

S. gardneri

S. wightii

S. revolutus

S. gracilis

S. lancifolius

S. elegans

S. nitidus

S. leavifolius

0

0.115

0.0250.114

0.114

0.0300.109

0.109

0.047

0.024

0.004

0.022

0.027

0.0360.095

0.095

0.013

0.0260.018

0.073

0.073

0.091

0.117

0.014

0.0100.036

0.097

0.097

0.133

0.012

0.0260.002

0.102

0.102

0.104

0.0760.055

0.055

0.179

0.0620.039

0.082

0.082

0.122

0.051

0.0500.014

0.093

0.093

0.107

0.0980.059

0.059

A

B


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.

72

S. canaliculatus

S. moonii

S. marginalis

S. bullatus

S .petiolaris

S. rigidus

S. gilimalensis

S. affinis

S. scalarinervis

S. cordifolius

S. angustisepalum

S. scaphifolius

S. gardneri

S. revolutus

S. wightii

S. latisepalum

S. reticulatus

ST23

S. oblongifolius

S. acuminatus

S. kanneliyensis

S. gracilis

S. elegans

S. lancifolius

S. nitidus

S .leavifolius

S. lanceolatus

66

76

5689

60

54

70


30

DISCUSSION There has been much debate on species limits

of Stemonoporus and the genus has also gained much attention among biologists and conservationists due to its vulnerability. The number of species that has been recognized within the genus has been constantly varying and the most recent studies of its species limits differ greatly. Trimen in 1893 recognized 13 species, Ashton (1980) described 15 species and the most recent treatment of the family Dipterocarpaceae by Kostermans (1992) recognized 26 species within the genus Stemonoporus.

The recovery of 27 distinct clusters based on

the two different approaches, i.e., phenetic and cladistic methods reveals that the genus is comprised of 27 morphologically different entities. The recognition of groups at this level of separation in the dendogram and monophyletic groups in the cladogram appears to be reasonable, as all these groups have been recognized as species by several eminent taxonomists in their taxonomic treatments of the genus (Trimen, 1893; Ashton, 1980; Kostermans, 1992).

Though 27 morphologically different clusters

have been recovered from phenetic and cladistic methods, in phenetic analysis groupings cannot be described on the basis of characters as the clustering is based on total similarity, whereas in the cladistic method of analysis the opportunity of describing a group based on shared apomorphic characters is an advantage. Therefore, the discussion will be primarily based on the strict consensus tree obtained from cladistic analysis (Figure 5). During the past studies the separations of certain taxa such as S. gracilis and S. lancifolius were based on only a few characters and also a limited number of examined specimens. But the present study gives very strong support for the recognition of these taxa, which has been controversial for a long period of time.

The cladogram identifies two major clades, A

and B, separating S. lanceolatus, S. nitidus, S. elegans, S. lancifolius and S. gracilis from the rest. Within the clade A the monophyletic clade including S. nitidus, S. elegans, S. lancifolius and S. gracilis with a strong bootstrap value of 72% has the characteristic reticulate type of venation without areoles in contrast to the percurrent venation possessed by the other clade. Stemonoporus elegans, which has been placed close to S. cordifolius because of the number of stamens in all the previous classification systems,

has clustered in different clades in the currently employed methods of analysis. Even though the number of stamens was a character that carried much weight in past groupings, in the present study several characters based on venation pattern play more important role. Distinctiveness of the venation pattern was observed to be stronger and effective in delimiting of the species of Stemonoporus. These characters have not been used in the past treatments of the genus (Rubasinghe S. C. K., Unpublished data).

The other major group, clade B has been

divided again into two main monophyletic clades. This division separates S. canaliculatus, S. moonii, S. marginalis, S. bullatus, S. petiolaris, S. rigidus and S. gilimalensis from the others. In all these species, the flowers are developed as clusters of 1-4 and have the characteristic consistent bracts and bracteoles covering their peduncles. This group is further divided separating S. canaliculatus, S. marginalis, S. bullatus and S. moonii from the rest. Stemonoporus canaliculatus Thw. was recognized by both Trimen (1893) and Ashton (1980) in their treatments but the species was split into three; S. canaliculatus Thw., S. marginalis Kosterm. and S. bullatus Kosterm. by Kostermans (1992) based primarily on the leaf size. The present study also showed the three species to be closely related. The clade including S. moonii gains strong support from several unique morphological features within each species and a bootstrap support of 70%. All the species have a marginal vein with impressed primary and secondary veins on the upper leaf surface. Presence of inter-secondary veins, angle of loop forming branches, pattern of tertiary veins and the arrangement of areoles are characters that supported the clade and that were not recognized by previous workers.

S. moonii Thw. was recognized by Trimen

(1893) in his treatment but was excluded by Ashton (1980) during the revision, where he considered the sterile specimens with long stipules he observed to be related to Tiliaceae, Bombacaceae, or Sterculiaceae but not to be a member of the genus Stemonoporus. Kostermans (1992) has resurrected the species in his work. The present study identified the species as a member of the genus possessing all the generic characters and as closely related to S. canaliculatus, S. bullatus and S. marginalis. In addition to the above-mentioned characters S. moonii has very long slender persistent stipules and long linear hairy bracts as autapomorphies.


Further, S. petiolaris Thw. is another taxon that has been recognized by both Trimen (1893) and Ashton (1980) in their treatments but the species was split into two; S. petiolaris Thw. and S. gilimalensis Kosterm., by Kostermans (1992), by basing his S. petiolaris on its thin, smaller leaves with long slender acumen and very slender petioles in contrast to the thicker and larger leaves with short stout acumen and stout petioles of S. gilimalensis. During the present study S. petiolaris and S. gilimalensis were well separated. Stemonoporus petiolaris Thw. could be easily distinguished from S. gilimalensis by the presence of drooping leaves and clusters of flowers with reflexed petals and maroon colored bracts. Although Ashton (1980) highlighted some of these characters, he did not consider them as adequate to separate the two species. However, the present study identifies characters that have not been previously employed in delimiting the two species and yielding strong support to recognize S. petiolaris and S. gilimalensis as two distinct species.

Stemonoporus rigidus Thw. has been

recognized in all major treatments. It can be characterized by extremely rigid, obtuse leaves borne in clusters, with closely placed secondary veins, and areoles with veinlets.

The remaining cluster is comprised of

S. affinis, S. scalarinervis, S. cordifolius, S. angustisepalum, S. scaphifolius, S. gardneri, S. revolutus, S. wightii, S. latisepalum, S. reticulatus, ST 23, S. oblongifolius, S. acuminatus and S. kanneliyensis.

Trimen (1893) and Ashton (1980), in their

treatments recognized Stemonoporus affinis (Thw.), but the species has been split into Stemonoporus affinis (Thw.) and S. angustisepalum Kosterm. by Kostermans (1992). S. affinis with prominently acuminate leaf lamina pointing downwards with areoles of distinct shape and prominulous primary and secondary veins on the upper leaf surface has formed a monophyletic group with S. scalarinervis, whereas S. angustisepalum forms a monophyletic group with S. cordifolius and S. scaphifolius.

Stemonoporus scaphifolius, S.angustisepalum

and S. cordifolius, with 5 stamens, have been clustered in a single clade as closely similar but three distinct species. The clade comprising S. scaphifolius, S. angustisepalum and S. cordifolius is supported by 59% bootstrap value. All of these taxa have 5 stamens and this character has been playing an important role in

past systems, where these three species including S. elegans are included in the subgenus Monoporandra by Kostermans due to the fact that they all possess 5 stamens in contrast to the rest of the species which have 10 -15 stamens. But in the present analysis S. elegans is separated from the rest since it has a distinctly different pattern of venation.

Thus the present study, while not supporting

the traditional division of the genus into two subgenera based on the number of stamens, yields much information for use in the identification of the species.

Kostermans recognized S. cordifolius auct.

non (Thw.) Alston and S. affinis auct. non Thwaites under Stemonoporus angustisepalum Kosterm. The present study agrees with the separation of S. angustisepalum, possessing larger leaves and a different type of inflorescence from S. cordifolius and S. affinis. The results suggest that S. angustisepalum is more close to S. cordifolius than to S. affinis. The number of stamens and similar venation patterns of the two species are characters supporting the clade. Further according to the present analysis S. scaphifolius Kosterm. and S. angustisepalum Kosterm. occur as sister groups. Though its leaves resemble those of S. bullatus and S. canaliculatus, S. angustisepalum can easily be distinguished from the number of stamens (5) and the paniculate inflorescence.

Trimen (1893) recognized Monoporandra

cordifola Thw. in his treatment and M. cordifolia was brought under Stemonoporu as S. cordifolius (Thw.) Alston by Alston in 1931. Kostermans (1992) in his treatment in addition to S. cordifolius (Thw.) Alston, recognized S. angustisepalum Kosterm. Characters for field identification of S. cordifolius recognized during the present study were the very small flowers borne on paniculate inflorescences, flowers with 5 stamens and leaves with long slender acumen, and the percurrent venation with distinct areoles.

S. gardneri Thw. was recognized by both

Trimen (1893) and Ashton (1980) in their treatments but the species was split into two; S. gardneri Thw. and S. scalarinervis Kosterm. by Kostermans (1992). The two species were observed to differ greatly with respect to the development of flowers, size of leaves and the pattern of venation.

During the present analysis S. gardneri,

S. revolutus, S. wightii and S. latisepalum with


32

paniculate of inflorescences occur in a monophyletic clade.

S. reticulatus Thw. was recognized by both

Trimen (1893) and Ashton (1980) in their treatments but the species was split into two; S. reticulatus Thw. and S. kanneliyensis Kosterm. by Kostermans (1992). During the present study the two species have separated well with stronger characters which had not been used previously. Two distinct groups were identified during the present analysis; a group with distinctly reticulate under surface, conical shaped fruit and few flowered inflorescence corresponding to S. reticulatus against a group with not distinctly reticulate, acute pointed fruit and many-flowered inflorescence corresponding to S. kanneliyensis. Stemonoporus reticulatus auct. non Thw. was recognized by Kostermans in 1992 as S. kanneliyensis Kosterm.

The group of plants with ST 23 code had

characters which did not match with the presently recognized species of Stemonoporus. Therefore it is considered as a new morphologically unique taxon and is placed in the clade containing S. reticulatus and S. oblongifolius more closely linked to S. reticulatus with a bootstrap support of 54%. The group of plants posses hanging, glabrous, narrow-elliptic to narrow-oblong leaves attached to a slender geniculate petiole. Flowers developed on axillary racemes bearing 4 – 6 flowers. Fifteen stamens in two rows form the characteristic cone-like structure around the ovary formed by stamens of Stemonoporus. Considering the fruits of Stemonoporus, two types of fruit have been described by previous workers; globose to subglobose and conical or pointed. Conical and pointed fruits have been observed only in S. kanneliyensis, S. wightii and S. reticulatus. The fruits of ST 23 are cone-shaped and pointed. The clade containing ST 23 does not belong to any of these species with respect to the floral, fruit and leaf characteristics and has come up in a separate clade.

S. acuminatus is a species which has

undergone much controversy during the past. Trimen (1893) in his treatment has recognized two forms; form a and form b. Form a is based on the character leaves with prominent, oblique nerves, and b on hardly elevated spreading nerves. Ashton (1991) recognizes the two forms but doubts if this difference is not based on their location. Kostermans (1992) during his study elevated the forms to species rank by recognizing form a as S. acuminatus (Thw.) Beddome and

form b as S. laevifolius Kosterm. During the present study specimens with code numbers, ST19 and ST 31 have shown contrasting features and have clustered in two separate clusters. ST19 corresponding to S. acuminatus had more erect, more arcuate and less numerous secondary veins which are prominent on the lower leaf surface in contrast to ST 31 corresponding to S. leavifolius with less conspicuous, dense, smooth reticulate secondary veins numerous on the glossy lower leaf surface. This gives additional support for the splitting.

Trimen (1893) in his study recognized

S. nitidus ssp. lancifolius Dyer and S. nervosus Thw. as two distinct species. But Ashton (1980) elevated S. nitidus ssp. lancifolius Dyer to S. lancifolius (Thw.) Ashton and reduced S. nervosus Thw. as a synonym. Kostermans (1992) in his work recognized S. lancifolius. Further, S. lancifolius auct. non Alston was recognized as S. gracilis Kosterm. According to Ashton (1980) S. lancifolius is a variable species, where the few herbarium collections available all differ in certain leaf characters. The specimen number C.P. 3885 (PDA) has relatively more prominent nerves on the undersurface while the specimens from the Kitulgala collection are with a depressed midrib above. According to Kostermans (1992), Ashton has mixed two species, which are entirely different, and which could be recognized even in the sterile condition. He has used the following character combinations in defining the two species: S. gracilis, which has been reduced to S. lancifolius by some authors, has a separate cluster of its own in the present analysis. It can be readily distinguished by the deeply channeled midrib on the upper surface (midrib prominulous in S. lancifolius) and by the absence of areoles (areoles present in S. lancifolius).

Stemonoporus rigidus Thw., S. oblongifolius

Thw., S. revolutus Trimen ex Hooker f. and S. lanceolatus Thw., recognized by all major treatments, were recognized as distinct morphological entities, reflecting their morphological consistency within the genus.

According to the analysis, specimens

collected from different geographical locations have clustered according to their character combinations giving rise to 27 different species. This highlights the fact that these OUTs could be identified as distinct species with stable morphological characters that are not affected by


the environment or habitat. This confirms the species limits recognized by previous taxonomic treatments and also in addition recognizes a new species of Stemonoporus.

The present morphometric analysis of

Stemonoporus does not agree with the traditional division of the genus into two major groups (subgenera: Monoporandra and Stemonoporus) based on the number of stamens. Instead, another set of characters based on the number of flowers on the inflorescence, development of flowers and details in the venation pattern gives a more satisfactory basis for the separation of species. The stamen character appears to be a very good field character for identifying the genus, but it does not give much support in identifying the species within the genus.

CONCLUSIONS Long-standing ambiguity regarding the

species limits of Stemonoporus was completely resolved with well-supported entities with distinct character combinations defining species within the genus. The study identifies all 26 species recognized by Kostermans (1992) supporting his treatment of the genus. Further, in addition to the recognized taxa a new species has been recognized, indicated by the code number ST 23. Moreover, both methods of analysis here employed to determine species limits have resulted in similar grouping proving that both methods could be used in determining species limits.

The study confirms that the genus

Stemonoporus has 27 morphologically distinct taxa, further strengthening its position as the most species-rich endemic dipterocarp genus Sri Lanka.

ACKNOWLEDGEMENTS The authors wish to thank the National

Science Foundation of Sri Lanka and the International Foundation for Science, Sweden for financial assistance.

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Kelleher, C. T., Kelly, D. L. and Hodkinson, T. R. (2004). Species status, hybridization and geographic distribution of Irish populations of Quercus petraea (Matt.) Liebl. And Q. robur L. Watsonia 25 : 83-97. Kitching, I. J., Forey, P. L., Humphries, C. J. and Williams, D. M. (1998). Cladistics: The theory 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.

Thwaites, G. H. K. (1864). Enumeration Plantarum Zeylaniae: an Enumeration of Ceylon Plants. Dulau London, Pp 11-12. Trimen, H. (1893). A Handbook to the Flora of Ceylon, iii. Bishen Singh Mahendra Pal Singh, New Connaught Place, Dehra Dun. Periodical, Pp. 436-437.

re-evaluation of species limits and taxonomy of the

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