European Journal of Taxonomy 54: 1-21

http://dx.doi.org/lQ.5852/ejt.2013.54

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ISSN 2118-9773

WWW. europeanj oumaloftaxonomy. eu

2013 • Tanawat Chaowasku & Paul J.A. Kehler

This work is licensed under a Creative Commons Attribution 3.0 License.

Research article

Phylogeny of Miliusa (Magnoliales: Annonaceae: Malmeoideae:

Miliuseae), with descriptions of two new species from Malesia

Tanawat CHAOWASKU' & Paul J.A. KEBLER^

^ Naturalis Biodiversity Center (seetion NHN), Leiden University,

P.O. Box 9514, 2300 RA Leiden, the Netherlands

E-mail: [email protected] (eorresponding author)

^Hortus botanieus Leiden, P.O. Box 9516, 2300 RA Leiden, the Netherlands

Abstract. The moleeular phylogeny of Miliusa (Annonaeeae) is reeonstrueted, with 27 (of ea. 50)

speeies ineluded, using a eombination of seven plastid markers (rbcL exon, tmL intron, trnL-F spaeer,

matK exon, ndhF exon, psbA-trnH spaeer, and ycfl exon) eonstituting ea. 7 kb. In addition, two new

speeies of Miliusa are deseribed from the Malesian area: M. butonensis sp. nov. from Buton Island,

Indonesia and M. viridiflora sp. nov. from Papua New Guinea. The former is ineluded in the moleeular

phylogenetie analysis. The reeonstrueted phylogeny eorresponds well to the informal morphologieal

grouping proposed earlier. A revised key to 13 Austro-Malesian speeies of Miliusa is provided.

Keywords. Annonaeeae, Buton Island, identifieation key, Papua New Guinea, moleeular phylogeny.

Chaowasku T. & KeBler P.J.A. 2013. Phylogeny of Miliusa (Magnoliales: Annonaceae: Malmeoideae: Miliuseae),

with descriptions of two new species from Malesia. European Journal of Taxonomy 54: 1-21. http://dx.doi.

org/lQ.5852/eit.2013.54

Introduction

The genus Miliusa Leseh. ex A.DC. (de Candolle 1832) (Annonaeeae) eomprises approximately 50

speeies of shrubs, or small to large trees, distributed from the Indian subeontinent, southern China and

mainland Southeast Asia to Southeast Asian islands. New Guinea (ineluding D’Entreeasteaux Islands

and Louisiade Arehipelago) and northern Australia (Chaowasku & KeBler 2006). It belongs to the tribe

Miliuseae of the subfamily Malmeoideae (Chatrou et al. 2012). Members of the tribe Miliuseae are

almost exelusively Asian (ineluding New Guinea, Australia, and the western Paeifie islands). Only two

elades within this tribe eonsist of non-Asian members: one elade of four Neotropieal genera and another

elade of Afro-Madagasean speeies whieh are part of the reeently deseribed genus sister to Miliusa.

Huber a Chaowasku (Chaowasku et al. 2012).

Aeeording to Chaowasku & KeBler (2006), Miliusa is eireumseribed by having (1) equally-sized

sepals and outer petals, both of whieh are mueh smaller than the inner petals; (2) a densely hairy torus;

(3) miliusoid stamens (sensu Mols & KeBler 2003a), i.e. stamens that are loosely arranged without

eonspieuously dilated eonneetive tissue eovering the theeae; and (4) four-part-lamellate ruminations of

the endosperm. Ten speeies were reeognized aeeording to the revision of the genus in the Austro-Malesian

area (Mols & KeBler 2003b). One additional speeies, M. lanceolata Chaowasku & Kessler (Chaowasku

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European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

& KeBler 2006), was later described from D’Entrecasteaux Islands and Louisiade Archipelago, southeast

off Papua New Guinea. New Miliusa species from southwestern India (Narayanan et al. 2010, 2012)

as well as the Indian eastern Himalaya (Chaowasku 2013) have recently been described. Further, seven

new species from Thailand are being added to this medium-sized genus by Chaowasku & Kebler (in

press) who use floral/inflorescence morphology to elaborate the four informal groups within Miliusa

first introduced by Chaowasku & Kebler (2006). These four morphological groups were the starting

point to systematically study this genus further. In order to obtain additional evidence supporting the

four mentioned groups, their pollen was investigated in detail (Chaowasku et al. 2008). The aim of the

present study is to test whether each of the four morphological groups of Miliusa is monophyletic by

means of molecular phylogenetic analysis.

In the course of selecting specimens for DNA extraction, we came across two collections, one from

Buton Island (Indonesia) and another from Papua New Guinea, which are different from other described

species in the Austro-Malesian area. After a thorough examination and comparison with similar species,

we have concluded that they both represent undescribed species of Miliusa, which are herein formally

described as M. butonensis sp. nov. and M. viridiflora sp. nov. The former is included in the molecular

phylogenetic analysis. The number of Miliusa species in the Austro-Malesian area is thus increased to

13 and a key to these species is provided.

Materials and Methods

Molecular phylogenetic analysis

All accessions belong to the subfamily Malmeoideae (Appendix). Twenty-seven accessions of Miliusa

covering the entire morphological variation known comprise the ingroup. The outgroups consist of

14 accessions, 11 of which were included as representatives of related genera in the tribe Miliuseae.

Seven plastid markers, i.e. rbcL exon, trnL intron, trnL-F spacer, matK exon, ndhF exon, psbA-trnH

spacer, andyc/7 exon, were amplified (see Table 1 for the number of included, variable, and parsimony

informative characters). In total, 7033 characters, including six separately coded indels were included

in the analyses. Indel coding follows Simmons & Ochoterena (2000). The reverse complement of 15

continuous nucleotides in the psbA-trnH marker for roughly half of the accessions sequenced was

present and altered into the reverse complement, following Pirie et al. (2006).

All methods of DNA extraction, amplification, and sequencing performed in Chaowasku et al. (2012)

were used in the present study. Due to a poor quality of the extracted DNA or unavailability of leaf

material, we could not produce seven markers for all accessions (see Appendix, Table 1). Sequences

were edited using the program Staden version 1.7.0 t http://staden.sourceforge.net/ ) and subsequently

manually aligned. Some sequences were obtained from previous studies (Mols et al. 2004a, 2004b;

Pirie et al. 2006; Chaowasku et al. 2012). Maximum parsimony analyses were performed in TNT

version 1.1 (Goloboff et al. 2008). All characters were equally weighted and unordered. Multiple most

parsimonious trees were generated by a heuristic search of the combined data, with 6 000 replicates

of random sequence additions, saving 10 trees per replicate, and using tree bisection and reconnection

(TBR) branch swapping algorithm. Clade support was measured by symmetric resampling (SR), which

is not affected by a distortion (resulting in incorrectly estimated percentages) as with some bootstrap

and jackknife methods (Goloboff et al. 2003). A default change probability was used. Four hundred

thousand replicates were run, each with two replicates of random sequence additions, saving one tree

per replicate. Groups with SR of > 85%, 70-84%, and < 69% were considered strongly, moderately, and

weakly supported, respectively.

Bayesian analysis was performed in MrBayes version 3.1.2 (Ronquist & Huelsenbeck 2003). Two

independent runs were simultaneously run; each run comprised four Markov-chain-Monte-Carlo

2

CHAOWASKU T. & KEBLER RJ.A., Phylogeny and new species of Miliusa (Annonaceae)

Table 1. Important descriptive values of sequence data. NA = not applicable.

DNA region

No. of included

characters

No. of accessions (out of

all 41 accessions) lacking

sequence data (%)

No. of variable

characters (%)

No. of parsimony-

informative

characters (%)

rbcL exon

1380

31 (75.6)

48 (3.5)

16(1.2)

trnL intron +

trnL-F spacer

926

0

112(12.1)

46 (5.0)

matK exon

828

20 (48.8)

78 (9.4)

31 (3.7)

ndhF exon

2033

0

225 (11.1)

105 (5.2)

psbA-trnH

spacer

431

0

90 (20.9)

51 (11.8)

ycfl exon

1429

0

220 (15.4)

95 (6.7)

Combined data

7027

NA

773 (11.0)

344 (4.9)

(MCMC) chains and was set for 10^ generations. The data matrix was divided into seven partitions [trnL

intron and trnL-F spacer were included in the same partition (= trnLF)], including a set of binary indel

coding. The most appropriate model of sequence evolution for each partition was selected by Akaike

information criterion (AIC) scores, using FindModel I http://www.hiv.lanl.gov/content/sequence/

findmodel/findmodel.html T The default model as well as the command ‘coding=variable’ were applied

for the binary indel partition. The default prior settings were used except for the ratepr [=variable] and

brlenspr [=unconstrained:exp(100)]. The latter prior setting was used to prevent the MCMC chains

from being trapped in the areas of parameter space with umealistically high values for the tree length

parameter, resulting in a false convergence or a failure to reach convergence after hundreds of millions

of generations (Marshall 2010). The temperature parameter was set to 0.1. Trees and all parameter

values were sampled every 1000^*^ generation. Convergence of the runs was checked by both the standard

deviation of split frequencies and the values for effective sample sizes (ESS) using Tracer version 1.5

(Rambaut & Drummond 2009). The 50% majority-rule consensus tree was generated from the two runs

combined, with 10% of the first trees removed as bum-in. Groups with posterior probabilities (PP) of >

0.96, 0.91-0.95, and < 0.90 were considered strongly, moderately, and weakly supported, respectively.

Taxonomy/morphology

Measurements/observations of the new species were made from herbarium specimens of A, BRI,

CANB, E, K, E, U herbaria. The indumentum terminology follows Hewson (1988). The term ‘velvety’

is equivalent to densely hairy/with dense hairs, whereas ‘pubemlous’ is equivalent to sparsely hairy/

with sparse hairs. Morphological data of the 11 known species of Miliusa in the Austro-Malesian area

were from Mols & Kefiler (2003b), Chaowasku & Kefiler (2006), and personal observations (= all

specimens cited in Mols & Kefiler 2003b). Morphological data of other species included in the molecular

phylogenetic analysis were from Chaowasku (2013), Chaowasku & Kefiler (in press), and personal

observations (= voucher specimens for molecular phylogenetic analysis plus a few more specimens,

see Appendix). The term ‘glandular stmctures’ in the present study means that such stmctures look like

glands but further anatomical confirmation is required. When only a single measurement/observation

was made, the word ‘circa (ca.)’ was added.

Results

Molecular phylogenetic analysis

The maximum parsimony analysis of combined data sets resulted in 135 most parsimonious trees

with 980 steps (results not shown). The consistency and retention indices (Cl, RI) were 0.86 and 0.88,

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European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

100/1

Monocarpieae

100/1

Miliuseae

99/1

94/1

79/0.8

100/1

92/1

100/1

90/0,S

100/1

94/1

100/1

A

84/1

99/1

B

97/1

68/0.31

<50/0.8

96/1

70/0.89

100/1

89/0.99

89/1

99/1

D

71/0.94

99/1

94/1

54/0.83

53/0.95

-Oxandra venezuelana

-Bocageopsis canescens

-Monocarpia maingayi

-Orophea kerrii

-Orophea enterocarpa

-Mitrephora alba

-Mitrephora macrocarpa

-Alphonsea elliptica

-Alphonsea sp.

-Platymitra macrocarpa

-Platymitra sp.

-Hubera nitidissima

-Hubera cerasoides

-Hubera pendula

Malmeeae

99/1

-Mi

usa sclerocarpa (Mh)

usa velutina|Mv)

usa macrocarpa (Me)

usa montana (Me)

usa dioeca (Me)

usa thorelii (Me)

usa campanulata (Me)

usa cuneata (Me)

usa cf. balansae (Me)

usa umpangensis (Me)

usa thailandica (Me)

usa sp. 1 (Me)

usa fusca (Mm)

usa fragrans (Mm)

usa butonensis (Mm)

usa mollis (Mm)

usa amplexicaulis (Mm)

usa intermedia (Mm)

usa horsfieldii (Mh)

usa sp. 2 (Mh)

usa parviflora (Mv)

usa macropoda (Mv)

usa novoguineensis (Mv)

usa brahei (Mh)

usa lanceolata (Mv)

usa koolsii (Mh)

usa traceyi (Mv)

Fig. 1. 50% Bayesian majority-rule eonsensus eladogram of eombined seven plastid markers. Clade

support: left of slash - parsimony symmetrie resampling values eorresponding to elades revovered in

Bayesian tree; right of slash - Bayesian posterior probabilities. Me = Miliusa campanulata Pierre group;

Mh = Miliusa horsfieldii (Berm.) Baill. ex Pierre group; Mm = Miliusa mollis Pierre group; Mv =

Miliusa velutina (DC.) Hook.f & Thomson group; all groups sensu Chaowasku & Kebler (in press).

4

CHAOWASKU T. & KEBLER RJ.A., Phylogeny and new species of Miliusa (Annonaceae)

respectively. For Bayesian analysis, the substitution model was generalized time-reversible plus gamma

(GTR + G) for all partitions except the trnLF partition (= trnL intron + trnL-F spacer), which had

Tamura-Nei plus gamma (TrN + G) model. The final standard deviation of split frequencies was < 0.002

and all ESS values after discarding the bum-in were > 1200, both indicating convergence of the mns.

Figures 1 (cladogram) and 2 (phylogram) show 50% majority-mle consensus trees derived from the

Bayesian analysis, with support values indicated in Fig. 1. Miliusa is monophyletic with maximum

support (SR 100%; PP 1.00). It is sister to Hubera with strong support (SR 90%; PP 0.98). Within

Miliusa, four strongly supported (SR > 85%; PP > 0.96) clades have been identified (Fig. 1: clades A, B,

C, D). Glade A is sister to clade B whereas clade C is sister to clade D, both with strong support. A clade

comprising clades A and B is sister to a clade consisting of clades C and D.

Taxonomy

Order Magnoliales Juss. ex Bercht. & J.Presl (Berchtold & Presl 1820)

Family Annonaceae Juss. (Jussieu 1789), nom. cons.

Tribe Miliuseae Hook.f & Thomson (Hooker & Thomson 1855)

Genus M/7/fr^a Fesch. exA.DC. (de Candolle 1832)

Miliusa butonensis Chaowasku & Kessler, sp. nov.

um:lsid:ipni.org:names:77131155-1

Figs 3^

Diagnosis

Miliusa butonensis sp. nov. belongs to clade C. It is best characterized by (I) dense hairs on the apical

half inside the inner petals and (2) glandular stmctures that cover almost the whole basal half of the inner

side of the inner petals.

0.003

Fig. 2. 50% Bayesian majority-mle consensus phylogram of combined seven plastid markers, showing

branch length proportional to amount of lineage sequence divergence. Scale bar unit: substitution per

site.

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European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

Fig. 3. Miliusa butonensis sp. nov. a. Habit, b. Outside (abaxial surface) of a persistent inner petal, c.

Inside (adaxial surface) of a persistent inner petal, d. Fruit. (All from Coode 6279). Drawn by E. Winkel.

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CHAOWASKU T. & KEBLER RJ.A., Phylogeny and new species of Miliusa (Annonaceae)

Etymology

Named after the Buton Island (Indonesia) where this species is endemic.

Type

INDONESIA: Southeast of Sulawesi, North Buton Is., Jismil camp inland from Eabuan Tobelo, Nov.

1989, Coode 6279 [holo-: U; iso-: A, K, E], in fruit (with a few persistent inner petals).

Description

Trees, ca. 8 m tall, ca. 10 cm in diameter. Young twigs appressed-puberulous. Petioles 2.0-3.0 mm long,

appressed-puberulous. Eeaves elliptic to slightly ovate, 4.7-11.1 x 2.2^.5 cm, base (broadly) cuneate,

sometimes slightly unequal, apex acute; lamina glabrous both sides; midrib usually slightly raised

to flat above, sometimes slightly sunken, glabrous, raised below, appressed-puberulous; secondary

veins 15-18 pairs, angle with midrib 42°-50°. Flowers partially known; fruiting peduncles or fruiting

pedicels axillary; fruiting peduncles up to 0.4 cm long; fruiting pedicels 0.9-2.1 cm long; sepals and

sometimes outer petals persistent in tfuit, both ovate-triangular. Inner petals a few persistent ones fallen

on herbarium sheet near the torus bearing monocarps seen, ovate-triangular, ca. 6.0 x 4.0 mm; outside

appressed-puberulous, mostly on the middle part, margin puberulous, inside (curly-)velvety on the apical

half; glandular structures observed on almost the whole basal half, slightly raised, between crescent¬

shaped and semicircular, slightly finely warty; base not saccate. Monocarps 3-21, subglobose-ellipsoid,

0.7-0.9 X 0.6-0.8 cm; surface smooth, glabrous; apex not apiculate; stipe ca. 6.0 mm long, glabrous.

Seed(s) 1 (rarely 2), subglobose(-eflipsoid), 0.6-0.8 x 0.4-0.7 cm.

Fig. 4. Distribution of Miliusa butonensis sp. nov. (fllled square) and Miliusa viridiflora sp. nov. (fllled

circle).

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European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

Distribution, habitat and phenology

Indonesia (Buton Island, Fig. 4), occurring in forests on flat ridge-tops, with slightly broken canopy due

to rocky terrain of raised coralline limestone. Elevation: ca. 300 m. Fruiting: November.

Field notes

Branches horizontal and foliage in flat sprays. Bark grey-brown, ca. 4 mm thick overall, ± smooth

with flne vertical cracks and rows of low lenticels, wood straw-colored, cut bark and wood pleasantly

aromatic. Young leaves ± yellow-green, mature leaves mid green on both sides.

Miliusa viridiflora Chaowasku & Kessler, sp. nov.

um:lsid:ipni.org:names:77131156-1

Figs 4-5

Diagnosis

Miliusa viridiflora sp. nov. is most morphologically similar to the recently described M. lanceolata. The

new species chiefly differs in having much sparser indumentum on young twigs and lower surface of the

leaf midrib, usually (broadly) elliptic leaves (narrowly ovate in M. lanceolata), and greater number of

stamens (ca. 56 vs. ca. 32 inM lanceolata) and carpels (ca. 28 vs. ca. 14 inM. lanceolata) per flower.

Etymology

The epithet refers to the light green flowers (probably also at anthesis).

Type

PAPUA NEW GUINEA: Central District, Port Moresby Subdistrict, on ridge below Boridi Village, Oct.

1973, Foreman & Vinas LAE 60222 [holo-: E; iso-: A, BRI, CANB, E], in flower and fruit.

Description

Trees. Young twigs (almost glabrous to) appressed-puberulous. Petioles ca. 1.0 mm long, almost

glabrous to appressed-puberulous. Eeaves usually (broadly) elliptic, sometimes slightly ovate,

4.5-13.3 X 2.1^.9 cm, base (broadly) cuneate to obtuse, often slightly unequal, apex acute to acuminate;

lamina glabrous above, (glabrous to) appressed-puberulous below; midrib flat to slightly sunken

above, almost glabrous, raised below, almost glabrous; secondary veins 11-15 pairs, angle with midrib

48°-65°. Flowers usually in > 7-flowered inflorescences, terminal developing to intemodal; peduncles

2.3^.4 cm long, glabrous; rachis 2.3^.5 cm long, glabrous; pedicels 5.0-13.0 cm long, glabrous, basal

articulation usually observed; bracts of peduncles and inflorescence axes triangular, number depending

on the number of flowers per inflorescence; pedicel bract 1 for each flower, triangular. Sepals (broadly)

triangular, 0.6-0.7 x 0.7-0.8 mm, persistent in fruit; both sides glabrous, margin puberulous. Outer

petals triangular, ca. 1.3 x 1.0 mm; both sides glabrous, margin puberulous. Inner petals ovate, 7.0-8.0

X 5.0-6.0 mm; both sides glabrous, margin puberulous; surface of the inner side somewhat raised on

the basal half compared to the apical half; base slightly saccate. Torus shortly cylindrical. Stamens

ca. 56, 1.0-1.4 mm long. Carpels ca. 28, ca. 1.3 mm long; stigmas capitate-globose; ovaries almost

glabrous; ovules 2, lateral, uniseriate. Monocarps 2-7, subglobose-ellipsoid to slightly irregular-shaped,

0.9-1.2 X 0.8-1.1 cm, slightly constricted between seeds when two seeds present in the monocarps;

surface slightly verruculose, glabrous; apex not apiculate; stipe 10.0-17.0 mm long, glabrous, obliquely

attached to the monocarps. Seed(s) 1-2, subglobose(-eflipsoid), 0.8 x 0.4-0.7 cm.

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CHAOWASKU T. & KEBLER P.J.A., Phylogeny and new species of Miliusa (Annonaceae)

Fig. 5. Miliusa viridiflora sp. nov. a. Habit, b. Flowering twig. c. Flower bud. d. Flower with one

inner petal, stamens, and earpels removed, e. Stamen, abaxial view. f. Stamen, side view. g. Carpel,

h. Monoearp. (All from Foreman & Vinas LAE 60222). Drawn by E. Winkel.

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European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

Distribution, habitat and phenology

Papua New Guinea (Central Provinee, Fig. 4), oeeurring in seeondary forests; on ridges. Elevation: ea.

1220 m. Flowering and fruiting: Oetober.

Field notes

A small tree. Leaves mid green. Flowers light green.

Key to the Austro-Malesian species of Miliusa

Notes

In some eouplets differenees are small, therefore when there is any ambiguity, it is advised to eonsult

the deseriptions of relevant speeies.

1. Inner petals with ereseent-shaped to semieireular glandular struetures inside, at base or eovering

almost the whole basal half.2

- Inner petals without pronouneed glandular struetures inside or with narrow glandular struetures

running inside along their bilateral midline.3

2. Leaves 4.7-11.1 x 2.2^.5 em, base (broadly) euneate, sometimes only slightly unequal,

not elasping the twigs. Inner petals with dense hairs inside on the apieal half; glandular

struetures inside between ereseent-shaped and semieireular, loeated on almost the whole

basal half BUTON ISLAND (INDONESIA). Miliusa butonensis sp. nov.

- Leaves 9.3-26.0 x 4.0-11.0 em, base (sub-)eordate, always (moderately to notably) unequal,

± elasping the twigs. Inner petals glabrous inside; glandular struetures inside ± ereseent-shaped,

loeated at base. THAILAND, PENINSULAR MALAYSIA.

. Miliusa amplexicaulis Rid\. (Ridley 1910)

3. Inner petals reflexed at anthesis, stamens > 125 per flower. BORNEO.

. Miliusa macropoda Miq. (Miquel 1865)

- Inner petals not reflexed at anthesis but sometimes the apex/apieal half reeurved at anthesis,

stamens < 65 per flower.4

4. Stamens < 9 per flower.5

- Stamens >18 per flower.6

5. Sepals and outer petals > 2.0 mm long, staminodes 4-5 per flower, earpels 12-14 per flower.

AUSTRALIA. Miliusa traceyi Jessup (Jessup 1988)

- Sepals and outer petals <1.5 mm long, staminode(s) up to 3 per flower, earpels 3-11 per flower.

NEW GUINEA. Miliusa novoguineensis Mols & Kessler (Mols & Kefller 2003b)

6. Ovules > 3 per ovary, biseriate.7

- Ovules 2(-3) per ovary, uniseriate.11

7. Inner petals without pronouneed glandular struetures inside. ANDAMAN AND NICOBAR

ISLANDS (INDIA), THAILAND, PENINSULAR MALAYSIA, SUMATRA.

. Miliusaparviflora Kid\. (Ridley 1911)

- Inner petals with narrow glandular struetures running inside along their bilateral midline.8

10

CHAOWASKU T. & KEBLER RJ.A., Phylogeny and new species of Miliusa (Annonaceae)

8. Inner petals 17.0^0.0 mm long. CHINA, EAOS, THAIEAND, MYANMAR, ANDAMAN AND

NICOBAR ISEANDS (INDIA), PENINSEn:.AR MAEAYSIA, SOUTHEAST ASIAN ISEANDS

(INCEUDING NEW GUINEA), AUSTRAEIA.

. Miliusa horsfieldii (Benn.) Baill. ex Pierre (Pierre 1881; basionym: Bennett 1840)

- Inner petals <15.0 mm long.9

9. Carpels 21-27 per flower, ovules 5-7 per ovary. AUSTRAEIA.

. Miliusa brahei (F.Muell.) Jessup (Jessup 1986; basionym: Mueller 1874)

- Carpels <18 per flower, ovules > 8 per ovary.10

10. Eeaf base usually equal. Pedicels 0.9-2.5(-4.5) cm long. Monocarps (1.3-)2.0-3.2 cm wide, stipe

(11.0-)16.0-29.0 mm long, seeds 1.1-1.7 x 0.7-1.5 cm. NEW GUINEA.

. Miliusa koolsii (Kosterm.) J.Sinclair (Sinclair 1955; basionym: Kostermans 1952)

- Eeaf base unequal. Pedicels 2.9-14.5(-21.5) cm long. Monocarps 0.6-1.3 cm wide, stipe (4.0-)

10.0-16.0(-32.0) mm long, seeds 0.6-0.8 x 0.3-0.5 cm. THE PHIEIPPINES.

. Miliusa vidalii J.Sinclair (Sinclair 1955)

11. Flowers and/or infloresences axillary, inner petals usually tightly appressed from the base to ± the

midpoint at anthesis. THAIEAND, PENINSUEAR MAEAYSIA, SUMATRA, JAVA.

. Miliusa longipes King (King 1892)

- Flowers and/or inflorescences terminal developing to intemodal, inner petals completely open at

anthesis.12

12. Young twigs with dense hairs (visible by naked eye). Eeaves narrowly ovate, lower surface of midrib

with dense hairs. Stamens ca. 32 per flower, carpels ca. 14 per flower. D’ENTRECASTEAUX

ISEANDS AND EOUISIADE ARCHIPEEAGO (PAPUA NEW GUINEA).

. Miliusa lanceolata Chaowasku & Kessler (Chaowasku & Kefller 2006)

- Young twigs (almost glabrous to) sparsely hairy (visible only under a binocular). Eeaves usually

(broadly) elliptic, lower surface of midrib almost glabrous. Stamens ca. 56 per flower, carpels ca. 28

per flower. PAPUA NEW GUINEA. Miliusa viridiflora sp. nov.

Discussion

Two clades, B and C, recovered in the phylogenetic trees (Figs 1, 2) correspond to Miliusa campanulata

Pierre (Pierre 1881) and M. mollis Pierre (Pierre 1881) groups sensu Chaowasku & Kefller (in press),

respectively. The important features characterizing the M. campanulata group: inner petals that are

usually tightly appressed from the base to ± the midpoint at anthesis (Fig. 6D-F), and the M. mollis

group: inner petals with crescent-shaped to semicircular glandular structures inside at the base or (a bit)

higher (Figs 3 c; 6G-I), are thus the synapomorphies of clades B and C, respectively. In contrast, M.

horsfieldii (Benn.) Baill. ex Pierre (Pierre 1881; basionym: Bennett 1840) andM. velutina (DC.) Hook.f

& Thomson (Hooker & Thomson 1855; basionym: Dunal 1817) groups sensu Chaowasku & Kefller (in

press) each does not form a clade; clades A and D each consists of members of both groups (see Fig. 1).

The M. velutina group seems to have been characterized principally by a likely symplesiomorphy: an

absence of conspicuous glandular structures inside the inner petals (Fig. 6B, K), whereas the main

feature characterizing the M. horsfieldii group [inner petals with narrow glandular structures running

inside along their bilateral midline (Fig. 6A, J, E)] seems to have evolved multiple times in Miliusa.

Detailed ancestral character reconstructions in combination with a denser taxon sampling, however, are

needed before any solid conclusion on character evolution occurred in Miliusa can be drawn.

Besides the inner petal morphology, flower and/or inflorescence position also corresponds to the

phylogenetic results, i.e. all species recovered in clade C and most species recovered in clade B possess

11

European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

Fig. 6. Flowers/inner petals of (representatives of) Miliusa speeies reeovered in eaeh elade. A-C. Clade

A. A. Miliusa sclerocarpa (A.DC.) Kurz. B, C. Miliusa velutina (DC.) Hook.f & Thomson. D-F. Clade

B. D. Miliusa campanulata Pierre. E. Miliusa thorelii Finet & Gagnep. F. Miliusa thailandica Chaowasku

& Kessler. G-I. Clade C. G. Miliusa amplexicaulis Ridl. H. Miliusa intermedia Chaowasku & Kessler.

I. Miliusa mollis Pierre. J-L. Clade D. J. Miliusa brahei (F.Muell.) Jessup. K. Miliusaparviflora Ridl.

L. Miliusa sp. 2. (Photographs taken by: A: P. Kehler; B, G, I, K: T. Chaowasku; C: A. Rodphitak;

D, L: B. Siriphiphat; E: S. Poungeharean; E: S. Thanapathomsinehai; H: S. Gardner; J: L. Jessup).

12

CHAOWASKU T. & KEBLER RJ.A., Phylogeny and new species of Miliusa (Annonaceae)

axillary flowers and/or inflorescences, while all species recovered in clades A and D possess terminal

(developing to intemodal) flowers and/or inflorescences. This correlation shows predictive signiflcance,

e.g. species exhibiting completely open inner petals at anthesis and terminal (developing to intemodal)

flowers and/or inflorescences (= belonging to the M. horsfieldii or M. velutina group; see Chaowasku &

Kefller in press), but having not been included in the phylogenetic analyses are likely to belong to clade

Aor D.

After a thorough examination on the floral morphology of Miliusa velutina, a member of clade A

(Fig. 1), peculiar stmctures have been observed. At the base inside the inner petals, there are thickened

stmctures (Fig. 6C) hidden at female anthesis, but as male anthesis begins and continues, these stmctures

are gradually becoming exposed. These stmctures seem to be non-glandular; however, anatomical

comparisons with the glandular stmctures observed in the species of clade C (Figs 3c; 6G-I) are likely

to shed light on whether they are homologous.

Miliusa butonensis sp. nov. is only known from the type specimens. Results of the molecular phylogenetic

analysis, nonetheless, assure the new species status. Miliusa butonensis sp. nov. is sister to a clade

comprising three mainland Asian species (Fig. 1). These three species share one remarkable feature:

(sub-)cordate leaf base, whereas M. butonensis sp. nov., and the remaining species of clade C, M. fusca

Pierre (Pierre 1881) and M. fragrans Chaowasku & Kessler (Chaowasku & Kefller in press), do not.

The occurrence of this species on Buton Island is unexpected since all other species (expected to be

part) of clade C thus far known occur on mainland Asia only. Phy to chemically, it is worthwhile to note

that neolignans have been found as principal secondary metabolites in two species of clade C: M. mollis

(Sawasdee et al. 2010, 2013a) and M. fragrans (Sawasdee et al. 2013b), but have not been reported to

occur in any species (expected to be part) of clade A, B or D so far investigated (see Sawasdee et al.

2010). If it is eventually proved that neolignans really occur only in the species (expected to be part)

of clade C, including M butonensis sp. nov., this class of natural product could be developed as a

chemotaxonomic marker.

Miliusa viridiflora sp. nov. is also only known from the type specimens; however, it is readily

distinguishable from its most morphologically similar species, M. lanceolota, by both vegetative and

generative features (see diagnosis). Additionally, the elevation where both species occur is considerably

different [ca. 1220 m in M. viridiflora sp. nov. vs. 2-20 m in M. lanceolata (Chaowasku & Kefller

2006)]. These differences convince us to recognize the former as new species.

So far neither macromorphological nor pollen morphological (see Chaowasku et al. 2008) features have

been found to be able to distinguish species of clade A from those of clade D. Nevertheless, Miliusa

viridiflora sp. nov. is more likely to belong to clade D because (1) clade A thus far known contains

only continental Asian species and (2) all species known to occur in New Guinea belong to clade D

(Fig. 1). The peduncles plus rachis (if present) plus pedicels of M. viridiflora sp. nov. (Fig. 5a, b) and

of M. lanceolata (Chaowasku & Kefller 2006) are notably long. This trait may be associated with bat

dispersal syndrome since the fmits eventually set will be clearly separated from the foliage, and hence

can be more easily detected (Marshall 1983). In Miliuseae, this feature has been earlier reported in some

New Guinean species of Pseuduvaria Miq. (Miquel 1858) (Su & Saunders 2006; Su et al. 2008).

It is worth conducting an anatomical investigation of the inner side of the inner petals of Miliusa to

reveal the ontogeny and possible functions of the glandular [and seemingly non-glandular (e.g. Fig. 6C,

E)] structures. Probably these differentiations are associated with the different pollination strategies. So

far there has been no detailed study on pollination biology of Miliusa', however, according to Mols &

Kefller (2003b), fruit flies were observed to visit the flowers of M. horsfieldii. A detailed pollination

biological study is required to determine if this kind of insect is potential pollinators for this species.

13

European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

Certain species of clade B exhibit ± transparent, window-like structures at the base of the inner petals

(Chaowasku & Kebler in press), e.g. M. campanulata and M. thorelii Finet & Gagnep. (Finet &

Gagnepain 1907; see Fig. 6E). Flies are likely to be the potential pollinators for these species as they are

lured to crawl inside to the stamens/stigmas by light (Dafni 1984).

There are correlations between clades and habitat preferences in Miliusa. Most species (expected to be

part) of clades A and D prefer drier habitats (e.g. deciduous/dipterocarp forests), resulting in a various

degree of deciduous lifecycle exhibited by a number of species (Mols & Kebler 2003b). In contrast, the

majority of the species (expected to be part) of clades B and C prefer more humid habitats, e.g. dry/moist

evergreen forests (Mols & Kebler 2003b; Chaowasku & Kebler in press). It is apparent that habitat shifts

occurred in Miliusa', these shifts might correspond to the paleoclimate.

More species of Miliusa, especially the Indian, Philippine, and Vietnamese ones, need to be included in

order to reconstruct a more robust molecular phylogeny, which will be the ground for the study in, for

example, biogeography/molecular dating and character evolution.

Acknowledgements

The authors would like to thank A, AAU, BRI, CANB, CNS, E, K, E, MO, NSW, U, UC, US, WAG

herbaria for the material studied, as well as two anonymous reviewers for their useful comments on the

manuscript. Esmee Winkel, Ben Kieft, and Peter van Welzen (all Naturalis Biodiversity Center, section

NHN) are kindly acknowledged for providing the beautiful line drawings, compiling the plate, and

assisting in the production of the distribution map, respectively. Aimom Rodphitak, Bullung Siriphiphat,

Eaurie Jessup, Santi Poungcharean, Simon Gardner, and Suwit Thanapathomsinchai provided useful

color photographs. The first author is grateful to the Royal Thai Government for granting a scholarship

and providing an opportunity to study plant systematics at Eeiden University.

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16

CHAOWASKU T. & KEBLER RJ.A., Phylogeny and new species of Miliusa (Annonaceae)

Manuscript received: 24 March 2013

Manuscript accepted: 26 July 2012

Published on: 29 August 2012

Topic editor: Thomas Janssen

Desk editor: Natacha Beau

Printed versions of all papers are also deposited in the libraries of the institutes that are members of

the EJT consortium: Museum National d’Histoire Naturelle, Paris, France; National Botanic Garden

of Belgium, Meise, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Natural History

Museum, Eondon, United Kingdom; Royal Belgian Institute of Natural Sciences, Brussels, Belgium;

Natural history Museum of Denmark, Copenhagen, De nm ark

17

European Journal of Taxonomy 5 A\ 1-21 ( 2013 )

Appendix

Appendix. Voucher specimens for molecular phylogenetic analysis (with GenBank accession numbers

indicated) and morphological observations.

— = sequence not available for this study.

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