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Plant Systematics and Evolution ISSN 0378-2697Volume 299Number 5 Plant Syst Evol (2013) 299:865-871DOI 10.1007/s00606-013-0768-z

Floral size variation causes differentiationof pollinators and genetic parameters inAlpinia nieuwenhuizii, a flexistylous ginger(Zingiberaceae)

Atsuko Takano, Johnny Gisil & MonicaSuleiman

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ORIGINAL ARTICLE

Floral size variation causes differentiation of pollinatorsand genetic parameters in Alpinia nieuwenhuizii, a flexistylousginger (Zingiberaceae)

Atsuko Takano • Johnny Gisil • Monica Suleiman

Received: 30 September 2012 / Accepted: 2 February 2013 / Published online: 24 February 2013

� Springer-Verlag Wien 2013

Abstract Floral size dimorphism, pollination, and

genetic variation of Alpinia nieuwenhuizii (Zingiberaceae),

a flexitylous ginger, were studied. This study revealed that

floral size differed among habitats (i.e., roadsides/river-

sides vs. forest floors). The effective pollinators of small-

flowered populations of the species on a forest floor were

different from those of large-flowered populations along

roadsides/riversides. Using inter-simple sequence repeat

(ISSR) PCR, considerable genetic differentiation was

detected between small- and large-flowered populations.

These results indicate that reproductive isolation in A.

nieuwenhuizii owing to the differentiation of pollen vectors

between two floral size morphs may lead to genetic dif-

ferentiation between the two morphs.

Keywords Alpinia � Flexistyly � ISSR � Genetic analysis �Pollinator ecotype � Zingiberaceae

Introduction

Speciation in plants can result from the formation of eco-

types adapted to the environment of a local pollinator (Grant

and Grant 1962; Stebbins 1970), and several pollination

ecotypes have been reported, e.g., in Gilia splendens

Douglas ex H. Mason and A.D. Grant (Grant and Grant

1965), Campanula punctata L. (Inoue and Amano 1986),

Platanthera ciliaris Lindl. (Robertson and Wyatt 1990),

Isodon umbrosus (Maxim.) H. Hara and Isodon shikokianus

(Makino) H. Hara (Suzuki 1992), and Satyrium hallackii

Bolus (Johnson 1997). These studies have provided evidence

that floral differentiation (e.g., size or length of spur) occurs

among populations and is induced by pollinator differences

between floral types. Pollinator differences may lead to

genetic differentiation between floral types because the gene

flow between types is reduced. There are no published

studies that provide data regarding genetic variations among

pollinator ecotypes, and it remains to be established whether

there is genetic differentiation between ecotypes.

Alpinia nieuwenhuizii Val. is endemic to Borneo and is

found in various habitats, including riversides, roadsides,

and montane forest floors (Smith 1985). This species is

easily distinguished from other Bornean Alpinia species by

its strongly paniculate inflorescence and small ligule

(maximum of 1.5 cm; Smith 1985). It is also known to

exhibit flexistyly, a form of unique stylar polymorphism

combining reciprocal herkogamy and dichogamy, which

enhances outcrossing (Cui et al. 1995, 1996; Li et al. 2001,

2002; Zhang et al. 2003; Takano et al. 2005; Sun et al.

2007). Takano et al. (2005) observed visitors to flowers of

A. nieuwenhuizii growing in a freshwater swamp forest

(altitude, 15 m) near the mouth of the Segama River,

Sabah, Malaysia and concluded that two species of large

carpenter bees, Xylocopa (Koptorthosoma) latipes and

Xylocopa (Zonohirsuta) collaris alboxantha, were the

effective pollinators. However, floral size differentiation

has been reported in A. nieuwenhuizii, with flowers

appearing to be smaller in highland populations than in

lowland populations (Poulsen 2006). This suggests that

pollinators may differ between highland and lowland

populations of A. nieuwenhuizii.

A. Takano (&)

Museum of Nature and Human Activities, Hyogo, Sanda,

Hyogo, Japan

e-mail: takano@hitohaku.jp

J. Gisil � M. Suleiman

Institute for Tropical Biology and Conservation, University

Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu,

Sabah, Malaysia

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Plant Syst Evol (2013) 299:865–871

DOI 10.1007/s00606-013-0768-z

Author's personal copy

This study aimed to: (1) confirm floral size differentia-

tion among populations of A. nieuwenhuizii, (2) identify

the effective pollinators of montane populations of A.

nieuwenhuizii, (3) clarify genetic variations within and

among A. nieuwenhuizii populations using inter-simple

sequence repeat (ISSR) PCR, and (4) evaluate genetic

differences between populations with different floral sizes.

To our knowledge, this is the first study of genetic variation

in a flexistylous ginger and of genetic differentiation

between pollination ecotypes.

Materials and methods

All five study sites (Kimanis, Trail4, Mahua, Ranau, and

Segama) were located in Sabah, Eastern Malaysia

(Table 1). The first three populations were located on the

forest floor of lower montane forests in Crocker Range

Park (CRP), western Sabah. The remaining two popula-

tions were located in very different habitats: the Ranau

population was situated along the side of a trunk road from

Kota Kinabalu through Ranau, and the Segama population

was located on the banks of the Segama River, which runs

through a lowland area (Takano et al. 2005).

Measurement of floral morphology

To describe the floral size, five floral characteristics were

selected and measured: floral tube length, labellum length

and width, filament length, and anther length (Fig. 1). The

number of flowers examined is shown in Table 2. Data

collected from the measurements of each floral part were

analyzed by analysis of variance (ANOVA). When differ-

ences were significant, a multiple comparison was per-

formed using the Tukey–Kramer HSP multiple comparison

test. All statistical tests were performed using Statview J.

ver. 5 (SAS Institute Inc., Cary, NC USA).

Observations of flower visitors in the lower montane

forest

Visitors to flowers of A. nieuwenhuizii in the montane

forest were observed directly at Kimanis during the periods

of 13–16 November 2008 and 15–18 September 2009. We

found two flowering individuals (one cataflexistylous and

one that started to release pollen in the morning, but no

stigma movement occurred in the afternoon) in 2008 and

three (one analflexistylous and two cataflexistylous) in

2009. All observations of flower visitors were made

between 8:00 and 17:00 (a total of 12.5 h in 2008 and

37.9 h in 2009). Flower visitors were only recorded when

they touched the anther or stigma. Some visitors were

captured for identification. Vouchers of flower visitors

were deposited at the Institute for Tropical Biology and

Conservation (ITBC), University Malaysia Sabah (BORH).

ISSR-PCR analysis

Background

Inter simple sequence repeat (ISSR) markers are generated

from single-primer PCRs where the primer is designed

from di- or trinucleotide repeat motifs with a 50 or 30

anchoring sequence of one to three nucleotides (Gupta

Table 1 Populations of Alpinia nieuwenhuizii studying floral morphology, ISSR variation, and flower visitors

Population Locality Latitude and Longitude alt. (m) Habitat

Kimanis Ulu Kimanis, Papar 5� 300 16.80 0 N, 116� 470 000 0 E 555 below montane forest

Trail4 In Crocker Range, Papar 5� 290 19.40 0 N, 116� 010 07.80 0 E 700 below montane forest

Mahua Crocker Range, Tambunan 5� 470 50.00 0 N, 116� 260 000 0 E 1,200 below montane forest

Ranau Along route A4, Ranau 5� 560 35.80 0 N, 116� 390 00.10 0 E 737 road side

Segama Tidong village, kinabatangan 5� 240 48.80 0 N, 118� 430 35.60 0 E 15 below freshwater swamp forest

All sites are located in Sabah E Malaysia, Borneo

Fig. 1 Measured parts of an Alpinia nieuwenhuizii flower: floral tube

length, labellum length and width, filament length, and anther length

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et al. 1994; Zietkiewicz et al. 1994). Recent ISSR studies

of natural populations have demonstrated the hyper-vari-

able nature of these markers and their potential for popu-

lation-level studies (Wolfe et al. 1998; Camacho and

Liston 2001; Ge et al. 2005; Gupta et al. 2008).One limi-

tation of the ISSR technique, which is the same as for

Random Amplification of Polymorphic DNA (RAPD;

Williams et al. 1990), is that bands are scored as dominant

markers and genetic diversity estimates are based on

diallelic characters (Ge et al. 2005).

ISSR-PCR analysis

Leaves from 92 individuals of A. nieuwenhuizii were col-

lected from the five populations (Table 3). To minimize the

possibility of sampling from the same genets, shoots that

were at least 5–6 m apart from each other were selected.

Plant material was stored in silica gel immediately after

collection and kept at -20 �C prior to DNA extraction.

Total DNA was isolated from 0.5 to 1.0 g of silica gel-

dried leaves, using a modified version of the 19 cetyltri-

methylammonium bromide extraction protocol from Doyle

and Doyle (1987). We screened 17 ISSR primers for the

814.1 markers (50 ? 30): [(CT)8-TG], M1 [CAA-(GA)5],

ISSR-2 [(AC)8-TA], ISSR-4 [(AG)8-TA], ISSR-5 [(CA)8-

G], ISSR-6 [(CT)8-GC], UBC-808 [(AG)8-C], UBC-820

[(GT)8-TC], UBC-827 [(AC)8-CG], UBC-830 [(TG)8-GG],

UBC-831 [(AT)8-CA], UBC-843 [(CT)8-TGA], UBC-852

[(TC)8-CGA], UBC-864 [(ATG)6], UBC-868 [(GAA)6],

UBC-870 [(TGC)6], and UBC-874 [(CCCT)4]. We asses-

sed the reproducibility of the bands by running duplicate

reactions on different days. Nine of the primers (ISSR-2,

ISSR-4, ISSR-6, UBC-808, UBC-820, UBC-827, UBC-

852, UBC-864, and UBC-868) produced 67 strong, clear,

and reproducible polymorphic bands, which were used for

further analysis. PCRs were carried out in a total volume of

25 ll containing: 2.5 ll of 109 PCR buffer, 2.5 mM

MgCl2, 2.5 mM dNTPs, 0.4 lM of primer, 1.25 U of Taq

polymerase (TaKaRa Biotechnology, Otsu Japan), and

50 ng of genomic DNA. The ISSR-PCR amplifications

were conducted with a PCR Thermal Cycler (MP) (Takara

Bio Inc., Japan) under the following conditions: initial

denaturation for 1 min at 94 �C followed by 36 cycles of

40 s at 94 �C, 45 s at the annealing temperature of each

primer (46–52 �C), and 90 s at 72 �C, with a final exten-

sion for 5 min at 72 �C. The amplification products were

separated by electrophoresis in 2 % (w/v) agarose gels in

19 TAE buffer at 100 V for 2 h. A 100 bp ladder was used

as a size reference. The gel was stained with ethidium

bromide and photographed using a gel imaging system

(ImageSaver AE-6905C; ATTO, Tokyo, Japan). The

banding patterns were scored manually.

Table 2 Floral characters of Alpinia nieuwenhuizii in each population

Population Flower

number

examined

Floral tube length

(mm, mean ± SD)

Filament length

(mm, mean ± SD)

Labellum length

(mm, mean ± SD)

Labellum width

(mm, mean ± SD)

Anther length

(mm, mean ± SD)

Kimanis 28 8.01 ± 0.79a 10.2 ± 0.95a 12.8 ± 0.93a 13.2 ± 3.5a 4.94 ± 0.19a

Trail4 2 7.90 ± 0.61a 11.7 ± 2.90a 12.8 ± 0.15a 15.0 ± 0.16‘ 4.81 ± 0.10a

Mahua 6 8.44 ± 0.41a 6.92 ± 0.86b 13.6 ± 0.38a 12.2 ± 1.31a 5.05 ± 0.02a

Ranau 26 14.1 ± 1.45b 16.6 ± 2.31c 19.3 ± 3.10b 18.5 ± 3.48b 7.00 ± 0.22b

Segama 16 12.5 ± 1.21b 15.8 ± 1.85c 19.3 ± 1.38b 17.5 ± 6.53b 6.65 ± 0.21b

Different superscripts mean significantly different at the 5 % level (Turkey-Kremer HSP multiple comparison test)

Table 3 Population size (N), effective number of alleles, ISSR diversity, and Shannon’s index, percent of polymorphic loci of Alpinianieuwenhuizii

N Ne H S % P GST Nm

Kimanis 16 1.41 ± 0.35 0.25 ± 0.18 0.38 ± 0.25 82.1

Trail4 10 1.36 ± 0.35 0.22 ± 0.19 0.33 ± 0.27 68.7

Mahua 17 1.43 ± 0.37 0.25 ± 0.19 0.38 ± 0.26 80.6

Ranau 21 1.36 ± 0.38 0.21 ± 0.20 0.31 ± 0.29 61.2

Segama 28 1.42 ± 0.38 0.25 ± 0.20 0.37 ± 0.27 76.1

Species level 1.50 ± 0.34 0.30 ± 0.16 0.45 ± 0.21 100 0.203 1.96

N population size, Ne Effective number of alleles, H Nei’s gene diversity, S Shannon’s index, % P percent of polymorphic loci ISSR diversity,

GST total genetic diversity residing among populations, Nm estimate of gene flow from GST or Gcs. e.g., Nm = 0.5(1 - GST)/GST

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ISSR data analysis

Amplified fragments were scored as ‘‘1’’ for the presence

and ‘‘0’’ for the absence of homologous bands. A binary

data matrix of different ISSR phenotypes was established

and analysed using the program POPGENE version 1.31

(Yeh et al. 1999). Hardy–Weinberg equilibrium was

assumed, and the following genetic diversity parameters

were estimated at the population and species levels: the

percentage of polymorphic bands (% P), the effective

number of alleles per locus (Ne), Nei’s (1973) gene

diversity (H), and Shannon’s index (S). To examine the

population genetic structure, gene differentiation (GST)

among populations was estimated. To visualize the genetic

relationships among populations, a dendrogram was con-

structed based on Nei’s genetic distance (D) and using an

unweighted pair-group cluster analysis method with arith-

metic averages (UPGMA).

Results

Measurement of floral morphology

Table 2 shows the measurements of floral morphology.

The measured size of each floral organ was consistent

within each population studied. No significant differences

were found among the three populations in the lower

montane forests, except for the filament length of the

Mahua population. The same trend was observed in both

the roadside and riverside populations. Flowers of the three

forest populations were significantly smaller than those in

Ranau and Segama (P [ 0.05, Tukey–Kramer HSP mul-

tiple comparison test), suggesting two flower size classes in

A. nieuwenhuzii.

Observations of flower visitors

Flower visitors to the small-flowered form at Kimanis

in 2008

The most frequent visitor to the small-flowered form

(hereafter SF form) of A. nieuwenhuizii at Kimanis was

Amegilla calceifera (Fig. 2a; 62 visits). The second most

common visitor was the stingless bee Tetragonilla collina

(Fig. 2b; 35 visits), followed by Trigona melanocephala (4

visits), and finally a carpenter bee, Xylocopa caerulea

(Fig. 2c; 2 visits). The most frequent visiting time for

A. calceifera and T. collina was during the early morning

(6:00–8:00), followed by a period after midday (12:00–

14:00). Amegilla calceifera descended onto the labellum,

foraged for nectar, and typically collected pollen by

scratching the anther with their forelegs. We visually

confirmed pollen attachment on the back of the bees while

they were moving on the labellum. Tetragonilla collina

descended directly onto the opened thecae and collected only

pollen. Xylocopa caerulea foraged for nectar and did not

collect pollen, although the back of the insects did touch the

thecae during visits to the flower (Fig. 2c).

Flower visitors at Kimanis in 2009

Amegilla calceifera was observed visiting the flowers of A.

nieuwenhuizii 181 times during this observation period.

The next most common visitors were another stingless-bee

(unidentified, but clearly different from T. collina and T.

melanocephala), which visited the flowers 139 times, and

X. caerulea, which visited 68 times. Visits by A. calceifera,

to both cataflexistylous and anaflexistylous flowers,

occurred most frequently between 10:00–12:00 and then

12:00–14:00. In contrast, visits by the stingless bee were

concentrated in the morning and occurred only on flowers

in the male stage. Xylocopa caerulea visited the flower

most frequently between 16:00–17:00 and then early in the

morning (6:00–8:00), with visits to both cataflexistylous

and anaflexistylous flowers.

ISSR-PCR analysis

The nine selected ISSR primers generated 67 bands (loci)

that ranged from 300 to 3,000 base pairs (bp) across all 92

individuals of the five populations of A. nieuwenhuizii. The

primers yielded 6–9 bands each, with an average of 7.4

bands per primer. All 67 bands were polymorphic. The

mean percentage of polymorphic bands (% P) at the pop-

ulation level was 73.7 %, with a range of 61.2 % (Ranau)

to 82.1 % (Kimanis) (Table 3). The mean effective number

of alleles per locus (Ne) was 1.36 at the population level

and 1.50 at the species level. By assuming Hardy–Wein-

berg equilibrium, the mean gene diversity was estimated to

be 0.24 (H) within populations and 0.30 at the species level

(Table 3). Shannon’s index had a mean of 0.36 within

populations and 0.45 at the species level.

Population genetic structure

Statistically significant genetic differentiation among the

populations of A. nieuwenhuizii was observed as follows.

The GST was estimated to be 0.203 (Table 3), which means

that 20.3 % of the genetic variability was distributed

among populations. The overall level of inferred gene flow

(Nm) was estimated to be 1.96 individuals per generation

among populations (Table 3), indicating a moderate gene

flow via pollen and seed dispersal between populations.

Nei’s genetic distance (D) among populations varied from

0.044 to 0.148 (Table 4). Genetic distances among the

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Kimanis, Trail4, and Mahua populations for the SF form

ranged between 0.044 and 0.066. The genetic distance

between the Mahua and Ranau populations, with the large-

flowered form (hereafter LF form), was 0.120, which is two

to three times the distances between SF form populations in

CRP, despite their closer geographical distance (29.1 km)

compared with the distances between Mahua-Kimanis

(56.4 km) and Mahua-Trail4 (57 km; Table 4). The three

SF form populations formed a cluster and the two LF form

populations formed another cluster on a phenogram based

on Nei’s genetic distances (Fig. 3).

Discussion

Differences in the effective pollinator

among populations of the SF and LF forms

This study confirmed the existence of floral size dimor-

phism in A. nieuwenhuizii. All three populations with the

SF form were located under a montane forest canopy at

high altitudes, ranging from 555 to 1,200 m, in CRP. In

contrast, the LF form was established in open areas along a

trunk road (Ranau, ca. 700 m altitude) and along a river

Fig. 2 Flower visitors foraging pollen and/or nectar of Alpinianieuwenhuizii. a, d Amegilla calceifera collected and became dusted

with pollen, in addition to sucking nectar, when visiting the small

flower type (a, at Kimanis), but only nectar consumption, with no

pollen attachment on the body, was observed on the large flower type

(d, at Ranau). b Tetragonilla collina landed directly on the thecae and

foraged for pollen; no pollen attachment was observed. c Xylocopacaerulea landed on the labellum, sucked nectar, and was dusted with

pollen on its back whenever it came to a flower in the male stage

Table 4 Geographic distance (km, above diagonal) and Nei’s genetic

distance (below diagonal) of Alpinia nieuwenhuizii

Kimanis Trail4 Mahua Ranau Lsegama

Kimanis 1.9 56.4 85.6 300.9

Trail4 0.044 57 86.1 300.2

Mahua 0.066 0.059 29.1 258.2

Ranau 0.139 0.147 0.12 237.7

Lsegama 0.092 0.117 0.099 0.148

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bank (Segama, ca. 15 m altitude). Therefore, floral size

difference did not correspond to differences in altitude as

suggested by Poulsen (2006). Instead, we suggest that floral

dimorphism may be induced by environmental differences,

because floral, as well as vegetative, traits can exhibit

phenotypic plasticity in response to spatially variable

environments (Herrera et al. 2006). Individuals of A. nie-

uwenhuizii in the CRP populations were scattered on the

forest floor of montane rainforest where limited sunlight is

available. Many species of ground herbs, grasses and

sedges grow together, and A. nieuwenhuizii never becomes

dominant there. In contrast, populations are found along

riverside and riversides/roadsides that are sunny, and where

there is no upper layer of tall trees. Species composition is

very simple, and A. nieuwenhuizii is often dominant.

Examinations of herbarium sheets at the University

Malaysia Sabah (BORH), the Museum of Nature and

Human Activities, Hyogo (HYO), the Forest Research

Centre (SAN), and Sabah Parks (SNP) also supported our

idea: the specimens collected from montane forest floors

had smaller flowers, and those collected from roadsides and

riversides had larger flowers.

It is generally considered that flower size is strongly

associated with pollinator size (e.g., Inoue and Amano

1986; Inoue and Amano 1986), and the results obtained in

this study indicate that a difference in flower size is asso-

ciated with the differentiation of the effective pollinator.

The frequent flower visitors observed at Kimanis were

A. calceifera, stingless bees (T. collina and an unidentified

species), and a carpenter bee (X. caerulea). Among these

species, stingless bees cannot serve as pollinators for A.

nieuwenhuizii because they collect only pollen (at least for

this species) and visit flowers in the male stage only. In

flexistylous flowers, the stigma is behind the thecae when

the flower is in the male stage; thus, pollen-collecting bees

do not touch the stigma. In contrast, A. calcelfera and X.

caerulea are both honey suckers, and pollen was confirmed

to attach to their backs. By visiting the flowers during both

the male and female stages, they are effective pollinators

for the SF form (Fig. 2a, c). Takano et al. (2005) studied

flower visitors to the LF form of A. nieuwenhuizii at Se-

gama and reported that two large carpenter bees (X. latipes

and X. alboxantha) were the effective pollinators. Visits of

X. latipes to the flowers of A. nieuwenhuizii were also

observed at Ranau in 2004 (Takano, personal observation).

However, large carpenter bees were not observed visiting

the SF form at Kimanis, despite the wide distribution of

both bee species in Southeast Asia. Although there were

frequent observations of A. caleifera (=37 visits during

11.8 h observation, Takano unpublished data) and the

unidentified stingless bee on the LF form at Ranau,

A. calceifera did not touch the anther because the flower

was too large (Fig. 2d). This suggests that even

though A. calceifera visits both forms of the flower, it can

be an effective pollinator for only the SF form of

A. nieuwenhuizii.

Genetic differentiation among populations of the SF

and LF forms

Flexistyly is believed to be a mechanism for promoting

outcrossing (e.g., Li et al. 2001, 2002; Zhang et al. 2003).

The present study provides evidence that A. nieuwenhuizii

maintains high genetic diversity as well as genetic differ-

entiation among populations similar to that of other

outcrossing species. The observed mean gene diversity

within populations of A. nieuwenhuizii (H = 0.296) was

higher than the average reported for outcrossing species

(H = 0.260) (Nybom and Bartish 2000; Nybom 2004), and

the value of genetic differentiation among populations of

A. nieuwenhuizii (GST = 0.20) was slightly lower than the

values for other species (GST = 0.23, Nybom and Bartish

2000).

However, there may be a barrier preventing gene flow

between populations of the LF and SF forms. The genetic

distances between the Ranau and CRP populations

(GD = 0.119–0.147) were much larger than those among

the three CRP populations (GD = 0.044–0.066). The UP-

GMA phenogram indicated that the Ranau population

formed a cluster with the Segama population, which both

consisted of individuals of the LF form, but not with the

Fig. 3 An unrooted UPGMA phenogram based on Nei’s (1973)

genetic distance among five populations of Alpinia nieuwenhuizii.Population names follow those in Table 1

870 A. Takano et al.

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CRP population. This indicates that gene flow has fre-

quently occurred among the CRP populations, but has been

limited between Ranau and the CRP populations. The

barrier to gene flow could be the effective pollinator, which

is a consequence of the size of the flowers. The amegilla

bee (A. calceifera) is an effective pollinator of CRP pop-

ulations, but not the Ranau population. Although it is still

unknown which genetic or plastic variations induce the

difference in flower size, this change may result in the

differentiation of effective pollinators, leading to genetic

differentiation among different floral size forms.

Acknowledgments We sincerely thank the Economic Planning

Unit, Prime Minister’s Department, for giving us permission to

undertake research in Malaysia (UPE: 40/200/19/1523). Sabah Parks

allowed us to use their facilities in the CRP. We are grateful to the

curators of BORH, HYO, SAN, and SNP for allowing us to examine

their facilities and collections. Prof. Dr. Makoto Kato (Kyoto Univ.)

kindly identified flower visitors. Drs. H. Okada and T. Denda pro-

vided critical comments on an earlier version of the manuscript. We

also thank two anonymous reviewers for their constructive comments.

This study was partly supported by a Grant-in-Aid for young scien-

tists (B) (no. 18770075 to A.T.) from the Japanese Ministry of

Education, Culture, Sports, Science, and Technology.

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