Are the genera Hallea and mitragyna (Rubiaceae-coptosapelteae) pollen morpholically distinct?

BLUMEA 39 (1994) 321-340

Are the genera Hallea and Mitragyna

(Rubiaceae-Coptosapelteae)pollen morphologically distinct?

S. Huysmans E. Robbrecht & E. Smets

Summary

Recent literature is controversial asregardsthesegregationof Hallea andMitragyna,andpretends that thetwogenerashowpollen morphologicaldifferences. In thepresent studythepollen morphol-ogyof all tenspeciesof thecomplexis describedonthe basisoflightandscanningelectron micro-scopy(includingexamination of broken grains,whichwereobtained with atechniqueneverapplied inpalynology,viz.shakingwith glassbeads).

Thetwogenerahave_{3-zonocolporate grains}withcompound apertures(endoaperturesare_always H-shaped,sometimes_incompletelyso).While Hallea showedtobe_{stenopalynous}(sexinealways tectate-perforate), Mitragynaismorevariable (sexinemicroreticulateortectate-perforate),and several of its_specieshave_pollensimilar tothat of Hallea.

Numerical _analysiswasusedtoevaluate the_{palynological}observations in the_lightof the macro-morphologicalvariation in the_complex.It is concluded that both Halleaand _Mitragynadeserve generic recognition,butare notfullydistinctpollenmorphologically.

Introduction

L _{Laboratoryof Plant Systematics,} Katholieke UniversiteitLeuven, Kardinaal Mercierlaan 92, B-3001 Heverlee,Belgium.

2) _{Nationale Plantentuin} van_Belgie,DomeinvanBouchout,B-1860Meise,Belgium.

Mitragyna (s.l.)

is amedium-sized

palaeotropical

genus

occurring

in Africa

(4 spp.)

and Asia

₍₆

spp.,from India and Sri Lankato Vietnam and southwards

through

the

Malay Archipelago

toNew

Guinea);

it is absent from

_Madagascar.

_Mitragyna

isone

of the rubiaceous generaoncetransferredtothe

Naucleaceae,

whicharecharacterized

by congested, spherical,

head-likeinflorescences;this

_family

isnow

generally

accept-edtobe

_polyphyletic

and included in the Rubiaceae in all

_present-day

_systems

_(see

Robbrecht,

1993a:

_{20). Mitragyna (s.l.) placed}

in thesubtribe

Mitragyninae

Havil.

was transferredto the tribe Cinchoneae

by

Ridsdale

_(1978).

Andersson & Persson

(1991)

emended the old concepts of the tribe Cinchoneae

placing

the

_Mitragyninae

andsome other genera in the tribe

Coptosapelteae

Bremek.ex Darwin emend L. Anderss. & C. Perss. Forcomments on this delimitation of the

_{Coptosapelteae,}

_see Robbrecht

_(1993b:

_175).

Recently,

the genus

Mitragyna

has received considerable attention.

_{Leroy (1975)}

segregated

the genus Hallea,

including

three of the four African

_species.

However,

he was notfollowed

_by

Ridsdale

_(1978),

who madea worldwide revision of the group,inter alia because the variationwithin therelated genus Uncaria isgreaterthan

322 BLUMEA Vol. 39,No. 1/2, 1994

When the first ofus decidedtoundertakea

global palynological investigation

of

the

_{Coptosapelteae (sensu}

Andersson&

_Persson,

_1991),

basedon anexaminationof

representative species

of all the genera of this tribe,the

_{Mitragyna/Hallea problem}

wasconsidered

_{interesting enough}

fora

profound palynological study including

all

ten

_species.

The_presentpaperintendsto

give

afull

pollen morphological

documentationof

the

species

of

Mitragyna

s.s. andHallea.Thetaxonomic value ofthese data is then

assessed

_by

anumerical

analysis

in which

_{palynological}

data_arecombined with

macro-morphological

features.

MATERIAL AND METHODS

The

present

pollen morphological study

is basedonherbarium material ofallten spe-cies of

_Mitragyna

s.l. The collections examinedarelistedbelow,with referenceto the illustrations. The synonymy

given

is restricted tonamesunder Hallea and Mitra-gyna,exceptfor Hallea

_ledermannii;

H.ledermannii

published by Leroy (1985),

a few months before Verdcourt's combination

(1985),

is notvalid because of

incom-plete basionym

reference. For full synonymy,seeRidsdale

(1978).

Hallea ledermannii

_{(K. Krause)}

Verde.

_[

Adina ledermannii K.Krause;

_Mitragyna

ledermannii

(K. Krause)

Ridsd.; Halleaciliata (Aubrev. &

_{Pellegr.) Leroy;}

Mitra-gynaciliata Aubrev. &

Pellegr.]: Liberia,

Bos 2645

(BR) (Fig. le); Ivory

Coast,

Leeuwenberg

2639

_{(L) (Figs.}

Id, f, g;

6a; 7a).

Hallea

_{rubrostipulata (K. Schum.) Leroy}

_{[Mitragyna rubrostipulata (K.Schum.)}

Havil.]:

Rwanda,Bridson 185(BR)

(Figs,

la, b;

7b).

Hallea

_{stipulosa (DC.) Leroy [Mitragyna stipulosa (DC.) Kuntze]:}

Zaire,

4660

Breyne

(BR) (Fig.

lc).

Mitragyna diversifolia (Wall,

ex

G.Don)

Havil.:

Thailand,

Maxwell 85-827

_(L)

(Figs.

2a-c;

6b).

Mitragyna

hirsuta Havil.:

Thailand,

Smitinand10887 BKF 6243

_{(L) (Figs.}

2d-f;

6d; 7c).

Mitragyna

inermis

_(Willd.)

Kuntze:

_Togo,

Warnecke 247

_{(BR) (Figs. 3a-c;}

6e;

7d).

Mitragyna parvifolia (Roxb.)

Korth.: India, collector unknown

_{(L) (Figs.}

3d-f;

6f).

Mitragyna rotundifolia (Roxb.)

Kuntze: Thailand, Maxwell 88-1145

_{(L) (Figs.}

4a-c; 6g).

Mitragyna speciosa

(Korth.)

Havil.: Borneo,Kostermans 7693

_{(L) (Figs.}

4d-f;

7e).

Mitragyna

tubulosa Havil.: South India, Ridsdale 110

_{(L) (Figs. 5a-d;}

_6c;

_7f).

generic

statusofHallea,

mainly

ontreearchitectural criteria. A few yearslater,Hallea

was

adopted

in the Rubiaceae instalment in the 'Flora of

_tropical

East Africa'

(Verd-court, 1988:

_447),

because "the

_separation

of Halleaonrather small but constant floral characters is

_{supported by palynology,}

wood and leaf_anatomy,and inflores-cence

development",

and because "therearealsosomedifferences in thespectrum of

alkaloids." The

_{palynological}

evidencewas basedona

light

and

_scanning

electron

microscopic study by Leroy (1975)

of

_only

two

species:

the African

_species

S._Huysmans,E. Robbrecht & E. Smets: Pollen _{morphology of}Halleaand_Mitragyna 323

All

_samples

were

acetolysed according

toReitsma's

_{(1969) 'wetting agent'}

method.

Pollen

_descriptions

arebasedon

light microscopy (LM)

and

_scanning

electron micro-scopy

(SEM).

The

glycerine jelly

slides have been observed withaLeitz Dialux 20.

Acetolysed grains

for SEM have been

_suspended

inethanol,air dried_onastub and

coated with

_{gold, using}

a Balzers SCD 020_sputter coater, and observed with a

Philips

SEM 501.

Measurements of the

_length

of the

_{polar (P)}

and

_equatorial

axis

_(E)

were made in aboutten

fully developed grains

per

specimen

under oil immersionat a x 1000

magnification.

All othermeasurements weremadeon

scanning

electron

micrographs.

Hallea H. _{rubrostipulata;} H. _stipulosa; H. _{ledermannii).}

Fig. 1. —_{a, c:}polarview;

b,f:_ectocolpus; d:equatorialview; e,g:apocolpium.—Scale baron a=5_µm; scale baronb=

2_µm,also for e—g; scale baronc=5_µm,also for d.

324 BLUMEA Vol. 39,No. 1/2, 1994

Inour

opinion,

characters atthe innersurfaceof the exinehave,atleast in

Rubia-ceae,a_great

systematic

value. For thisreasonbroken

_{pollen grains}

of all

_investigated

species

wereobserved with SEM. To obtain broken

_grains

we

applied

a

technique

that,asfaras weknow,wasneverdescribed in

_{palynological}

_literature,

viz.

_shaking

a

pollen suspension

with

_glass

beads

_(Huysmans

etal.,

1993):

0.4 ml

_pollen

suspen-sion inacetoneandc. 0.5 ml

_glass

beads

₍₁

mmin

_diameter)

were

agitated together

in

asmalltesttube

_by

aVortex;50to70 seconds of

_shaking

wasfoundtobe effective.

Fig. 2. M. diversifolia; —a,d:_polarview; b,e:mesocolpium c,f:ectocolpus.—Scale bar ona=5_µm,also ford;scale baronb=2_µm,also forc, e,f.

S.Huysmans,E. Robbrecht & E. Smets:Pollen morphologyofHalleaandMitragyna 325

After

checking

the number of broken

_grains

withLM,afew

drops

of the

_suspension

were

brought

on astub for SEM observation.

For the

_{palynological terminology}

werefertoPuntetal.

(1994); shape

classesin

equatorial

view are

adopted

from Erdtman

_(1971).

The

_{interpretation}

of the

_apertural

systemfollowsLobreau-Callen

(1978).

Fig.3. M._parvifolia).—a,d:polar view;b:_{ectocolpus;e:}

326 BLUMEA Vol. 39,No. 1/2, 1994

Macromorphological

data_were

_gathered

in the first

_place

from the

_keys

and

de-scriptions by

Ridsdale

_(1978);

in thisrevision, however,

only

fourout of theten

species

weredescribed. Additional informationwasobtainedfrom

_protologues

and

floristic literature

_(Haviland,

1897;Koorders & Valeton, 1902; Pitard,

1922;

Halle,

1966; Leroy,

1975;

Verdcourt, 1988)

aswellasfrom

_personal

observations

(dissec-tions of BR

_specimens).

Mitragyna

Fig.4. (a—c:M. rotundifolia; —a:polarview; b:mesocolpium; c, f:ectocolpus; d:_apocolpium;e:equatorialview.—Scale bar ona=5_{µm, also} fore;scale bar onb =2_µm,also for_c,d,f.

S.Huysmans,E. Robbrecht & E. Smels: Pollen_{morphology of}Hallea and_Mitragyna 327

The

_data,

both

_{palynological}

and

_{macromorphological,}

wereencoded

estimating

fre-quencies

of character states, and submittedtoanumerical

analysis (SYSTAT

hierar-chical

_clustering

_{with average}

_linkage

and Pearson's correlationcoefficient;

Wilkin-son,

1988).

The acronyms used for the OTU's in tables and

figures

arethe first three

letters ofthe

_generic

namecombinedwith the first three letters of the

_specific

name.

PALYNOLOGICAL CHARACTERS

General

_morphology

Mitragyna

s.l. has

_{small, isopolar}

and

_{radially symmetrical pollen grains.}

The

polar

axis ranges from 14to22 pm, the

equatorial

axis from 15to_{25 pm. In}

equa-torialview, the

_shape

of the

_grains

varies from suboblateto

_{prolate-spheroidal (P/E}

0.75to

1.06).

The outline in

_polar

view

_{(- amb)}

is

_mostly

circular; Hallea

_stipulosa

has_a

_{subtriangular}

outline withconvexsides.

The_{aperture system} is

_{always 3-zonocolporate;}

the

compound

apertures consist

of three_parts which arelocated in different wall

layers.

The

ectoapertureisawide

colpus

witha

granular, slightly

sunken membraneand distinct

margins

which are

of-ten

_irregular.

The ends of the

colpi

areacute, obtuse orintermediate;the

apocolpium

—a:apocolpium;b:ectocolpus;c: equatorial view;d; mesocol-pium.—Scale barona=2 µm, also forb, d;scalebar on c= 5µm.

Mitragyna (M. tubulosa).

BLUMEA Vol. 39,No. 1/2, 1994 328

index varies from 0.17to0.42. The_mesoapertureis a

lolongate

porus,

mostly

sur-rounded

by

a± smooth

_aspis.

Theterm

'aspis'

is

_preferred

above themore

_general

'annulus'because thedifferentiated_area

_surrounding

_{the pore is}

_always

a

thickening

of the exine. Acosta

_(thickening

of the

_{nexine) surrounding}

the_mesoapertureatthe inside of the

grain

occurs in six

_species.

All

_species

show a

H-shaped

cut-awayof

Hallea leder-mannii,

Fig. 6. BrokengrainstoshowH-shaped endoaperturesand inner surfaceof nexine. —a:

M.parvifolia; b:Mitragyna diversifolia;c:M.tubulosa;d:M. hirsuta;e:M. inermis;f:

M. rotundifolia.—Scale barona=5_µm;scalebaronb=2_µm,also for_c—g.

S.Huysmans,E. Robbrecht & E. Smets:Pollen morphology ofHalleaand_Mitragyna 329

the nexine which is the

_{endoaperture .}

The downstrokes of the Hare

parallel

with the

ectocolpus;

in some

species,

the H may be

incomplete,

i.e. thehorizontal,

equatorial

connection

maybe weak

(diffuse margins)

oreven absent. In

_{Mitragyna parvifolia}

e.g.,the

equatorial

connection is

missing.

The

H-shaped endoaperture

is reducedto

a

kidney-shaped

cut-awayatbothsidesof the_{mesoaperture,}±3 times as

long

asthe

diameter of the mesoporus. The surface of the

endoapertures

is oftenmore

coarsely

scabrate than therestof the inner side of the nexine.

The sexine is

tectate-perforate

tomicroreticulate with short columellae

(observed

in thecentreof the

mesocolpium).

The luminaor

perforations

tendtobe

_larger

in the centreof the

mesocolpium

and decrease in diameter towards the

_poles

and the

ecto-Fig.7. Details of broken_grains toshowstructureof exineand inner surface of nexine. —a:Hallea

ledermannii; H _{rubrostipulata,} M.

tubu-losa.

Mitragyna hirsuta;d:M.inermis; M. _speciosa;

b: c: e: f:

S._Huysmans,E. Robbrecht& E. Smets: Pollenmorphology ofHalleaandMitragyna 331

apertures, exceptfor

_pollen

of M. hirsuta. The

_{lumina/perforations}

of M.

_diversifolia

and M. tubulosaareoften

elongate

and

_{angular; they}

_arerounded in all other

_species.

The muriare

simplicolumellate. Any supratectal

processes areabsent;the very fine

granulation,

observedin

_only

one case

(M. parvifolia),

is

interpreted

asanartifact.

The inner surface of the nexine is

_always

scabrate,but the

_density

and the size of the elements may differ

locally.

InHallea

rubrostipulata,

Mitragyna parvifolia,

and M.

tubulosa,the scabraeare more

densely spaced opposite

the

_ectocolpi.

The

_{palynological}

characters for each

_species

_are

_given

in Tables 1 and 2. Table 3

lists the

_pollen

characters and theirstatesretained forournumerical

analysis.

Table 2. Overview of

_pollen

morphologicalfeatures of Hallea. PresentationasTable 1.

Hailed Ilalrub Halsti

p 15(16.6)18 19(19.6)22 14(15.5)18

E 18 (19.3)21 21(23.0)25 17(18.7)20

P/E 0.79 (0.86)0.95 0.80 (0.85)0.92 0.75 (0.83)0.95

amb circular circular subtriangular

apocolpiumindex 0.27-0.33 0.29 0.25-0.26

widthectoaperture 2.8-3.3 3.7 3.0-3.3

marginsectoaperture diffuseto distinct-irregular distinct-irregular distinct-irregular

ends

ectoaperture obtuseto acute obtuse obtuse

widthmesoaperture 1.5-1.7 1.7-2.0 1.5-2.0

heightmesoaperture 2.2-2.5 01UJ 2.0-2.5

aspis ± smooth smooth smooth

costa — _coarse thick,_coarse

endoaperture 11-shaped H-shaped H-shaped

equatorialconnection wider than wider than widerthan ofendoaperture mesoaperture mesoaperture mesoaperture

sexine tect.-perforate tect.-perforate tect.-perforate

max.0lumina_apocolpia 0.5 dot dot

max.0luminamesocolpia 0.8 0.5 0.3

widthmuri — — —

inner surface nexine scabrate scabrate scabrate

columellae _layer 0.17 0.17 0.12

tectum 0.50 0.50 0.44

columellae _layer/tectumratio 0.34 0.34 0.27

sexine 0.67 0.67 0.57

nexine 0.50 0.50 0.44

BLUMEA Vol. 39,No. 1/2, 1994 332

Notes on

species:

Hallea ledermannii

_{(K. Krause)}

Verde.

The

_pollen

of H. ledermanniiwas

previously

described

_{by Leroy (1975:}

86,

pi.

12/8-12/14,

LM and

_SEM).

The material he studied

_(no

voucher

_specimen

cited

and

_maybe

not

_acetolysed)

has

_slightly

smaller

_grains

withnarrower

ectocolpi.

The

porus thatwehave called the_mesoapertureis describedasthe

_{endoaperture. Leroy}

nowhere mentioned the existence ofathird_aperture.

We observed a

slight intraspecific

variation between thetwo examined

_specimens

as

regards

the size of themesoapertureand the diameterof the lumina

_{(compare Fig.}

le with

_Fig.

_lg):

bothare

larger

in Bos 2645.

Table 3.Palynologicalcharactersand their states observed in_{Mitragyna s.l.,} with their

_coding

usedinTable 5.

Grainsize very small(10-18 pm) SISMa

small (18-25 mm) SISMb

Shape equatorialview prolate-spheroidal(1.00-1.14) SHPS

spheroidal(1.00) SHS

oblate-spheroidal(1.00-0.88) SHOS suboblate (0.88-0.75) SHSO

Shape polarview(amb) circular AMB CI

triangular AMBTR

Apocolpiumindex small(<0.35) AISM

large (> 0.35) AILR

Ectoaperturewidth(%ofE) narrow(< 14%) ECNA

wide(> 14%) ECWI

Ectoaperture margin diffuse ECDF

distinct ECDI

Endsofectoaperture acute ECAC

obtuse ECOB

Dimensions mesoaperture small(<9

pm2) MESM

large(>9 pm2) MELR

Aspis

absent ASAB

present ASSM

Costa_{surroundingmesoaperture present} MECOA

absent MECOP

Sexine tectate-perforate SEXTP

microreticulate SEXMR

Perforations smaller towardspoles yes PFPS

no PFPL

Columellae _layer/tectumratio = 1 COL

< 1 COLS

Sexine/nexine ratio < 2 WALa

S.Huysmans,E. Robbrecht& E. Smets: Pollen morphologyofHallea andMitragyna 333

Mitragyna

inermis

_(Willd.)

Kuntze

The

_pollen

of M. inermiswas

already

describedand illustrated

_{by Leroy (1975:}

84,

pi.

12/1-12/7,LM and

_{SEM). Except}

about the width of the

_ectocolpi

thesame remarkscanbe madeasfor Hallealedermannii.

Table 4.

_{Macromorphological}

characters andtheir statesobservedinMitragynas.l.: characters retained for the numerical

analysis

andtheir_coding.

Architecture _monopodial MON

sympodial SYM

Leaf-blades medium-sized (upto14x9cm) LEAMS

large(>14_x9cm) LEAL

Interfloral bracteoles linear IBL

linear-spathulate IBLS

spathulate IBS

Interfloral bracteoles_reaching well below calyxlobes IBWB

uptocalyxlobes IBSA

beyondcalyxlobes 1BA

Calyx truncate torepand CALT

with shortobtuse lobes CALO

with triangular+interstitiallobes CALT

with spathulatelobes CALS

Marginsof_calyx lobes ciliate CALC

glabrous CALG

Corolla tube _long(>2xlengthof corolla lobes) COTL short(<2xlengthof corollalobes) COTS

Corolla tube hypocrateriform COTH

narrowlyinfundibular COTI

Corolla throat hairy COTRII

glabrous COTRG

Throat hairs notprotruding TRNP

conspicuously protruding TRP

Corolla lobes withappendage COAP

withoutappendage COWAP

Corolla lobes outside hairy COLOH

glabrous COLOG

Corolla lobes inside ciliate_alongmidrib COLIC

hairy/pubescent COLIU

glabrous COL1G

Anthers _{partiallyprotruding}from corolla tube ANPP conspicuously protrudingfrom corolla tube ANP

Stigma ± isodiametric STISO

elongate STEL

Calyxonfruits persistent CALP

BLUMEA Vol. 39,No. 1/2, 1994 334

MACROMORPHOLOGICAL CHARACTERS AND THEIR CODING

The

_{palynological}

datasetis

_{fairly complete.}

On the_contrary, oursetof

macromor-phological

characters is limited. Ridsdale's

₍₁₉₇₈₎

revision of

_Mitragyna

and Uncaria

containsanextensive discussion of the architecture of these

plants,

but otherwise

hardly

describes their

_{macromorphological}

characteristics. We have tried_asmuch_as

possible

to

gather

extrainformation,

though

fromalimited number of

_specimens.

The

following

survey is

especially

intendedtointroduce the characters and theirstates

re-tainedinournumerical

analysis;

thesearesummarized in Table 4.

Vegetative

characteristics

Mitragyna species

are

(often large)

treesor more

rarely

shrubs with medium-sized

to

large

leaves and veryapparentfoliaceous

_{interpetiolar stipules.}

Tree architecturewas

thoroughly

discussed

_{by Leroy (1975)}

who

_argued

thatiMi

tragynas.s.

fundamentally

differsfromHalleain

_having

theinflorescences terminal onlateral

twigs;

the

_{flowering twig}

has maintained its

_{vegetative capacity,}

asits

axil-lary

buds sometimes

_develop

intonewbranches

(sympodial growth).

In

_Hallea,

on the contrary, the inflorescencesare

axillary

onlateral

twigs

and thearchitectureis

monopodial.

Ridsdale

₍₁₉₇₈₎

studied the architecture of the Asiatic

_{species (}

Mitra-gynatubulosain the

field);

he

_compared

these observations with herbarium material of the African Hallea and concluded that all

_species

of

_Mitragyna

haveasimilar ra-mification of the

_{plagiotropic branches,}

_including

those

_segregated

into Hallea

_by

Leroy.

In 1985

_{Leroy reported}

field observations_onthe architecture of Hallea

leder-mannii;he

_convincingly

corroborated the absence of

_{sympodial growth}

in its

plagio-tropic

branches and thus confirmed the architectural differences between Hallea and

Mitragyna.

Inflorescences

Inflorescencesarecompact

perfectly spherical

heads. _{The number of heads per} branch

_strongly

varies, from 1 to15

_(30),

butso

gradually

thatwecouldnotretain it for the numerical

_analysis.

Each flower is surrounded

by

numerous

(> 10) hairy

interfloral bracteoles which

mostly

have_a characteristic

_{spathulate shape.}

In a few

_species,

thebracteoles are

linear_orhave_atransitional

_shape.

The relative

_length

of the bracteoles varies

_greatly;

they

arewell visible when

they

reach

_beyond

the

_{calyx lobes,}

_{but may also be hidden} between the ovaries.

Flowers

Calyx

and corollaarepentamerousand

_{morphologically}

_{very variable}

_(Leroy,

1975:

pi. 1). They provide

the features

_generally

usedto

_distinguish

between the

_species.

The

_calyx

is truncate to

_repand

or

provided

withdistinct lobes. In Hallea

rubro-stipulata,

the

_calyx

lobesare

_{narrowly triangular}

and alternate with five much smaller

interstitiallobes1.

1' _{Erroneouslycalled}

S.Huysmans,E. Robbrecht& E. Smets:Pollen morphology ofHallea andMitragyna 335

The corollais infundibuliform_or

_{hypocrateriform,}

with short

_triangular

lobes with

valvate-induplicate

aestivation. In the

_{species placed}

inHallea,the

_lobe-tips

are

some-times

_provided

with characteristic shorttolinear

_appendages,

somewhat reminiscent to those observed in the related genera

Pausinystalia

and

_{Corynanthe (Halle,}

1966:

pi.

9,

10).

The

gynoecium

is

_{bicarpellate.}

The

_style

ends ina

mitre-shaped stigma

of very

variable size and

_shape,

from ± isodiametricto

strongly elongated.

In Hallea

,

the whole exterior _partof the

_stigma

is

_papillary,

while in

_Mitragyna

s.s. the

_papillary

partsarelimitedtothe

_tip

and sometimestothe base of the 'mitre'. The

_cylindrical

disk is

_deeply

sunken. Placentation is characteristic of the

_{Coptosapelteae:}

each lo-cule is

_provided

witha

pendulous placenta

covered withnumerous

ascendingly

im-bricate ovules.

Fruitsandseeds

The ovaries_are

_completely

freeonthe

receptacles

of the heads and

_develop

into

±

_{elongated capsules dehiscing}

into 4 valves

_adhering

atthe base. Each locule

con-tains_numerousseeds

_provided

withan

apically triangular

and

_basally

forked

_wing.

Differences between

_species

aresmall andconcern

especially

the

_persistency

of

the

_calyx

and the size of the fruit

_{(length varying}

fromc.5 toc. 15

mm).

NUMERICAL ANALYSIS

Ourobservations established 14

_{pollen morphological}

characters

_{(30 states)}

within

the

_{Mitragyna/Ha llea-complex.}

On the other handwe wereabletodocument

varia-tionin 16

macromorphological

characters

_{(37 states).}

The

following

cluster

analyses

were

performed: 1)

with

_{palynological}

characters,

2)

with

_{macromorphological}

characters,and

₃₎

with both

_{macromorphological}

and

palynological

characters. In thetwolattercases,the number of

species

wasrestricted

to

eight

because

_{Mitragyna speciosa}

and M.

diversifolia

were

insufficiently

document-ed

_{macromorphologically.}

The

_{purely palynological clustering (Fig.}

8

_A)

resulted inadistinct

separation

ofone

species, Mitragyna speciosa;

this

_species

has indeed several

_unique

statesas

regards

the relative thickness of its exine

_layers,

columellae/tectumand sexine/nexine ratio

(Table 5);

in fact thesestatesareall relatedto the

_large

absolute thickness of the

co-lumellae

_layer.

The

separated position

of M.

speciosa

should thusnotbeoverrated.

The

_remaining

_species

are divided intotwoclusters. The first cluster groups the

African

_species

ofHallea, withone Asiatic

_species

of

_Mitragyna

s.s.

(M. tubulosa);

these

_species

have

_{larger pollen}

with

_larger

_{mesoapertures,}twofeatures

mostly

but

not

_always

associated witha tectum

_perforatum

and blunt

_{ectoapertural}

ends. The

second cluster contains the five

_{remaining species}

of

_Mitragyna

s.s.; amongthese

species

M.

parvifolia occupies

arather isolated

position,

because it is the

only

one

withmore

prolate pollen

and without

_aspides.

The

_{macromorphological clustering}

and the combined

BLUMEA Vol. 39,No. 1/2, 1994 336

Mitragyna

s.s. and Hallea

(Fig. 8B).

This is ofcourse not

astonishing

and results

from the

strongcorrelationbetween certain

(especially floral)

charactersasdiscussed

by Leroy (1975).

The

weight

of this

_{macromorphological}

evidence is so

high

that

distinction between Hallea and

_Mitragyna

s. s. is

equally

corroborated

_by

the com-bined

_analysis.

DISCUSSION AND CONCLUSION

Leroy (1975)

concluded

_(from

_anexamination of_a

_{single species}

from each

_genus)

that

_Mitragyna

_s.s. and Halleacanbe

distinguished palynologically,

viz.

by

reticulate

pollen

with_athicker ectexine_versus

_{tectate-perforate pollen}

with_athinner ectexine.

We have observed both microreticulate and

_{tectate-perforate grains}

in

_Mitragyna

s.s., and found that the sexine thickness of

_Mitragyna

s.s.

continuously

varies from 0.5 to 1.0 gm. This range includes the sexine thicknesses

(0.57-0.67 gm)

that we ob-served in Hallea. Itis thus clear that

_Leroy's

statementis a

simplification,

which

illustrates the

_danger

of

_{studying single representatives.}

Fig.8. Hierarchical clustering (usingaverage linkageand 1-Pearson’s correlationcoefficient)of Hal-leaand_{Mitragyna species}(Africanspeciesmarked withanasterisk, otheronesfrom_tropicalAsia): A,using palynologicalcharacters; B, usingpalynologicalandmacromorphologicalcharacters. Data matricesin Tables 5 and 6. Distancesare a measureofphenetic similaritybetween _speciesor

S.Huysmans,E. Robbrecht & E. Smets: Pollenmorphology ofHalleaandMitragyna 337

Hallea

_pollen

has

_always

a

perforate

tectumand is in

general larger,

while

Mitra-gynas.s.

mostly

possesses smaller microreticulate

grains;

however,several

Mitra-gynas.s.

species

have

_pollen

similartothat of Hallea

,

viz. M. hirsuta, M.

_parvifolia

and M. tubulosa.The differentiationofthe

pollen

of the

Mitragyna/Hallea-complex

seems to_{be very low. This is inagreement}with the remainderof the

_{Coptasapelteae}

(pers. obs.).

Thetribeis in

_{general stenopalynous;}

the

_grains

are

3-colporate,

except

for

_{Coptosapelta,}

which has

_{3-pororate grains. Only}

afew generaare

pollen

morpho-logically

distinct. Uncaria hasacharacteristic striate-reticulate

sexine, Greeniopsis

and

Mussaendopsis

have

_{colpus-shaped}

_{mesoapertures}and

_endocinguli,

while Luculia

shows_areticulate sexine with

_long

columellae.

Table 5.Matrixoffrequencies of statesofpalynologicalcharactersused inthe cluster

analysis. Symbols

ofstatesexplainedinTable 3.

Hailed Halrub Halsti Mitdiv Mithii Mitine Milpar Mitrot Mitspe Mittub

338 BLUMEA Vol. 39, No. 1/2, 1994

Table 6. Matrixoffrequenciesofstatesofmacromorphologicalcharacters usedinthe cluster

analysis. Symbols

ofstatesexplainedinTable 4.

Hailed Halrub Halsti Mithir Mitine _Mitpar Mitrot Mittub

S.Huysmans,E. Robbrecht & E. Smets:Pollen morphology ofHallea andMitragyna 339

Leroy's generic segregation

of Hallea from

_Mitragyna

was basedon

convincing

macromorphological

evidence. It istruethat thesetof

_supporting

characters is limited:

architecture

(summarized above)

and_some floral features

_(summarized

in

_Leroy,

1975:

_{pi. 1).}

The correlation of advanced characters

_supporting

Hallea is_strong, however. As the

generic

distinction is also confirmed

_{by chorology (}

Halleais

_strictly

limitedtothe African rain forestareawhile the sole

_{representative}

of

_Mitragyna

s. s. in Africa is

_Soudanian),

we areinclinedtoacceptthe

_segregation

of thetwo_genera. Ridsdale's

_{(1978: 57)}

statementthat the "variation within Uncaria

_[is]

_greaterthan that found between Hallea and

_Mitragyna

s.s." is

_hardly

anargument

against

this,

butmore an

expression

of the

_general

fact that

_larger

_genera

₍

Uncaria isarather

large

and

_{widespread palaeotropical}

genus with 35

species)

tendtobemorevariable

(com-parewith

Tricalysia,

GardeniaorRothmannia in the

Rubiaceae-Gardenieae;

Rob-brecht &Puff, 1986:

_131).

Leroy

offeredtwo

phylogenetic explanations:

in 1975 heconsidered

Mitragyna

s.s. andHalleaas two branches

_diverging

fromacommonancestor, while in 1985 he rather believed Halleatobe the descent ofanextinct African

Mitragyna.

Is there

any

palynological

evidenceto supportthese

_hypotheses?

It is

plausible

toconsider

the microreticulate

_pollen

of

_Mitragyna

s.s. asderived

_compared

to the

tectate-per-forate

_pollen

of other

_{Mitragyna species}

and Hallea

_(Walker

&

_Doyle,

1975:

_684;

Keddam-Malplanche,

1985:

_{30). Consequently,}

the ancestral stock of the

_complex

probably possessed

tectate-perforate

grains,

which were

'replaced'

by

the more

advanced microreticulate

_pollen

inarestricted number of

_species

of

Mitragyna

s. s. This scenario is in_agreementwith both

_hypotheses.

Inconclusion,weacceptthe

_{generic recognition}

of Hallea and

_Mitragyna

s.s.on thebasis oftreearchitectural and flower

_{morphological}

features

_presented

in the_past, but found

_hardly

_any

_{convincing palynological}

evidenceto_supportit. The

_presumed

evolution of the ornamentation of the

pollen

wall corroborates the

_phylogenetic

sce-narios offered

_{by Leroy.}

ACKNOWLEDGEMENTS

The first author is much indebted toDr. W.Puntand P. Hoenfor the kindguidanceduringa short trainingperiodatthe_Laboratoryof_Paleobotanyand_Palynology(Utrecht).We thank the director of L for_permissiontoremove_pollenfrom herbarium _specimens and Dr. R.W. J.M.vander Ham for the _practicalassistance duringsampling.WeategratefultoMr. M._Verhaegenfor_preparingthe photographsfor_{reproduction.}

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