RESEARCH ARTICLE
Sex Distribution of Paper Mulberry
(Broussonetia papyrifera) in the Pacific
Johany Peñailillo
1
, Gabriela Olivares
1
, Ximena Moncada
2
, Claudia Payacán
1
,
Chi-Shan Chang
3
, Kuo-Fang Chung
4
, Peter J. Matthews
5
, Andrea Seelenfreund
6
,
Daniela Seelenfreund
1
*
1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile, Santiago, Chile, 2 Centro de Estudios Avanzados en Zonas Áridas (CEAZA), La
Serena, Chile, 3 National Museum of Prehistory, Taitung 95060, Taiwan, 4 Biodiversity Research Center,
Academia Sinica, Nangang, Taipei 11529, Taiwan, 5 National Museum of Ethnology, Osaka, Japan,
6 Escuela de Antropología, Universidad Academia de Humanismo Cristiano, Santiago, Chile
*
dseelen@ciq.uchile.cl; daniela.seelenfreund@gmail.com
Abstract
Background
Paper mulberry (Broussonetia papyrifera (L.) L'Hér. ex Vent) is a dioecious tree native to
East Asia and mainland Southeast-Asia, introduced prehistorically to Polynesia as a source
of bark fiber by Austronesian-speaking voyagers. In Oceania, trees are coppiced and har-
vested for production of bark-cloth, so flowering is generally unknown. A survey of botanical
records of paper mulberry revealed a distributional disjunction: the tree is apparently absent
in Borneo and the Philippines. A subsequent study of chloroplast haplotypes linked paper
mulberry of Remote Oceania directly to a population in southern Taiwan, distinct from
known populations in mainland Southeast-Asia.
Methodology
We describe the optimization and use of a DNA marker designed to identify sex in paper
mulberry. We used this marker to determine the sex distribution in selected localities across
Asia, Near and Remote Oceania. We also characterized all samples using the ribosomal
internal transcribed spacer sequence (ITS) in order to relate results to a previous survey of
ITS diversity.
Results
In Near and Remote Oceania, contemporary paper mulberry plants are all female with the
exception of Hawaii, where plants of both sexes are found. In its natural range in Asia, male
and female plants are found, as expected. Male plants in Hawaii display an East Asian ITS
genotype, con sistent with modern introduction, while females in Remote Oceania share a
distinctive variant.
PLOS ONE | DOI:10.1371/journal.pone.0161148 August 16, 2016 1/19
a11111
OPEN ACCESS
Citation: Peñailillo J, Olivares G, Moncada X,
Payacán C, Chang C-S, Chung K-F, et al. (2016) Sex
Distribution of Paper Mulberry (Broussonetia
papyrifera) in the Pacific. PLoS ONE 11(8):
e0161148. doi:10.1371/journal.pone.0161148
Editor: Kenneth M Olsen, Washington University,
UNITED STATES
Received: March 28, 2016
Accepted: August 1, 2016
Published: August 16, 2016
Copyright: © 2016 Peñailillo et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This project was funded by the Fondo
Nacional de Desarrollo Científico y Tecnológico
(
www.conicyt.cl/fondecyt/) grants 1080061 and
1120175 from the Government of Chile to AS, and
National Science Council, Taiwan (
www.most.gov.tw/)
Grant NSC-102-2621-B-002-007 to KFC. The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
Conclusions
Most paper mulberry plants now present in the Pacific appear to be descended from female
clones introduc ed prehistorically. In Hawaii, the presence of male and female plants is
thought to reflect a dual origin, one a prehistoric female introduction and the other a modern
male introduction by Japanese/Chinese immigrants. If only female clones were dispersed
from a source-region in Taiwan, this may explain the absence of botanical records and
breeding popul ations in the Philippines and Borneo, and Remote Oceania.
Introduction
Prehistoric settlement of the Pacific involved the intentional transport of many plant and ani-
mal species of economic value [
1, 2, 3, 4, 5], and concluded with settlement of the islands of
eastern Polynesia approximately 1000 years before present (BP) [
6, 7, 8]. Plant species intro-
duced into Oceania included food, medicinal and other economically important plants as part
of a ‘transported landscape’ strategy for cultural reproduction in the newly settled islands [
1,
2]. It has been estimated that around 70 plant species were dispersed by prehistoric Austrone-
sian speaking voyagers into Polynesia [
9]. Not all plants reached all islands, and only a small
number of these reached islands as distant as Rapanui (Easter Island), Hawaii or New Zealand
[
4]. One of the plants introduced to Remote Oceania was paper mulberry, which appears to be
native to mainland and subtropical Southeast and East Asia, as far east as Taiwan [3; 10, 11]. In
Remote Oceania, paper mulberry is we ll known as the main source of the highest quality fiber
for making bark-cloth for mats, clothing and cordage for practical and ceremonial or ritual
purposes. The tree is one of the few prehistoric introductions into Remote Oceania that is not a
food plant.
Paper mulberry dispersal
The plant family to which paper mulberry belongs, the Moraceae, includes figs and many other
plants that provide food and fiber [
5, 12]. A northern sister-species, B. kazinoki, is the main
source of fiber for handmade paper in Japan, where B. papyrifera is known as an introduced
species [
3]. Paper mulberry is a fast growing shrub or tree, diploid (2n = 26), and dioecious
(with male and female flowers on separate individuals). The male inflorescence is a dense,
many-flowered, pendulous catkin of 4–8 cm; the female inflorescence is a dense, many-flow-
ered, globose head of about 2 cm, and the fruit is a collection of a small fleshy orange to red
druplets [
13]. The sweet druplets are highly attractive to birds. When both male and female
plants are introduced to the same area, wind pollination makes it relatively easy for breeding
populations to become established. Such establishment has been observed in diverse environ-
mental conditions, in the Phillipines, Pakistan, Japan, and the Solomon Islands. From herbar-
ium records and floristic accounts, Matthews [
3] mapped the distribution of paper mulberry in
Asia and the Pacific, and found that males, females and breeding populations were present in
East and Southeast Asia. The tree was apparently absent in the Philippines, Borneo and Micro-
nesia, while present in Indonesia, Melanesia and Polynesia. Not including known modern
introductions, the sex of trees introduced to Indonesia, Melanesia and Polynesia was unknown.
The apparent disjunction or bottleneck in island Southeast Asia was unexpected, has been con-
firmed in the process of collecting samples for later surveys of genetic variation in paper mul-
berry, and remains unexplained
Sex Distribution of Paper Mulberry in the Pacific
PLOS ONE | DOI:10.1371/journal.pone.0161148 August 16, 2016 2/19
Competing Interests: The authors have declared
that no competing interests exist.
In a survey of B. papyrifera samples from Polynesia, Southeast Asia and East Asia, we ana-
lysed non-coding internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA, and
inter-simple sequence repeat (ISSR) sequences in total DNA extracts. Diversity was found in
Asia and not in Polynesia [
14], with the sole exception of Hawaii [ 15 ]. The data suggested a
prehistoric human-mediated movement of paper mulberry from East Asia to Polynesia, by an
unknown route through island Southeast Asia, and a second, possibly historic, human-medi-
ated introduction to Hawaii [
15]. A subsequent study of chloroplast haplotypes in paper mul-
berry by Chang et al. [
11] indicated that the most common variant of paper mulberry found
from Indonesia to Melanesia and Polynesia, and the one most likely introduced by the early
colonists in Polynesia, has an origin in southern Taiwan. The fact that a single haplotype, cp-
17, is dominant across this vast region is consistent with the dispersal of plants of a single sex
to Remote Oceania, but does not prove this.
In Oceania, most traditional crops, except for coconuts and a few others, are propagated
asexually [16, 17, 18, 19]. Very few dioecious plants were introduced prehistorically into
Remote Oceania such as the narcotic plant kava (Piper methystichum) a well-studied dioecious
domesticate that was introduced from Vanuatu into Polynesia [
16], and the greater yam (Dios-
corea alata)[
20]. Sexual reproduction of these species is dependent on the presence of both
mature female and male plants in some proximity, for pollination and seed production. Paper
mulberry naturally suckers from the root system, both in the wild and in cultivation, and is eas-
ily propagated by the use of cuttings [
3]. In the absence of pollination and seed production,
long-distance dispersal must depend entirely on humans. In Remote Oceania, paper mulberry
stems are usually cut approximately every two years when they reach about 2 to 2.5 meters
height for harvesting the bark to obtain fibers, so the plants do not reach maturity, and rarely
develop flowers [
21]. As a result, the sex of paper mulberry plants is usually not seen, and when
botanical specimens are collected for herbaria, floral parts are generally lacking [
3].
A relict population of paper mulberry has long been known to exist in a crater on Rapanui
[
22, 23], and was found to produce female flowers (Fig 1), but the sex of paper mulberry in
Remote Oceania generally remained unknown.
The question of sex of paper mulberry in the Pacific
Matthews [
3] noted that the pop ulation of paper mulberry plants now present in Polynesia
could be desc ended from one individual of either sex brought by the original human co loniz-
ers or from individuals of both sexes which may or may not have been able to reach the same
islands and form breeding populations. Whistler and Elevitch [
9] asserted that all speci mens
in Polynesia might be/ are male clones, but we have not found evidence to support this, in
either botani cal or palaeobotanical reports. One reason to suggest that only one sex of paper
mulberry was introduced into the Pacifi c is its non-invasive natu re there. When both sexes
are introduced into non-native areas, paper mulberry quickly disrupts the native habita t,
becoming highly invasive, choking out native flo ra in its com petition for nutr ients, space and
light [3, 24, 25, 26].
Male B. papyrifera plants produce large quantities of pollen, as attested in India and Paki-
stan [27; 28, 29] where the airborne pollen of large numbers of male plants have become a pub-
lic health problem. Since paper mulberry in Polynesia generally is cut before it flowers, it seems
unlikely that pollen or seeds will be found in archaeological sites [
3]. No large amounts of pol-
len belonging to Moraceae have ever been identified in archaeological sites in Polynesia, and
those found are likely to represent native Moraceae and/or Urticaceae species (see also [
30]).
Pollen types are not very distinct among different species and genera of Moraceae, making the
identification of Broussonetia species difficult. The absence of verified paper mulberry pollen
Sex Distribution of Paper Mulberry in the Pacific
PLOS ONE | DOI:10.1371/journal.pone.0161148 August 16, 2016 3/19
in palaeoecological contexts does not disprove the past presence of male plants, or prove the
presence of only female plants in an area where the tree is present today.
We are interested in the sex distribution of paper mulberry in Oceania because the question
of sex has direct bearing on whether or not the plant can naturalize, form breeding populations,
and survive without human assistance.
In dioecious plants sex determination genes and pathways are not universal. No master sex
determination gene has been identified, and many genes that affect sex determination have
been found [
31]. In some plants, the development into female or male is controlled by epige-
netic signals or diverse environmental stimuli [
31]. Very little is known at the molecular level
of sex-specific DNA sequences and the mechanisms of sex differentiation in plant s. Recently
Wang et al. [
32] identified a molecular sex marker for B. papyrifera. A comparison of gene
sequences for Eucommia ulmoides Oliv. [
33], Trichosanthes dioica [34], Carica papaya [35],
Pistacia vera [
36] and paper mulberry [32] does not show common sex-defining sequences in
these different species and genera.
In the present paper, we have identified the sex of paper mulberry in Near and Remote Oce-
ania in order to determine if one or both sexes are present, and to reconsider the possible
mechanisms of dispersal and survival of the tree in the light of this information.
Fig 1. Female flowering of B. papyrifera, Rapanui, 2014. Tree at left, small branch with female inflorescence at right. The stigmas are exerted but no
male tree is nearby for wind pollination. This crater population is isolated and not frequently harvested.
doi:10.1371/journal.pone.0161148.g001
Sex Distribution of Paper Mulberry in the Pacific
PLOS ONE | DOI:10.1371/journal.pone.0161148 August 16, 2016 4/19
In work aimed at helping plant breeders create new cultivars, Wang et al. [30] identified a
short male-specific DNA sequence in B. papyrifera. To distinguish the sex of these plants
more effectively, we developed a duplex PCR protocol based on the male-specific sequence
recorded in GenBank (HQ202152.1) [
32]. We then characterized the sex distribution of liv-
ing plants on 25 islands in 13 archipelagos or island groups across a broad region of Near and
Remote Oceania. We also located a small number of flowering paper mulberry specimens in
herbarium collections from the Pacific. For the purposes of interpretation, we compare the
geographical sex distribution with the distributions of nuclear ribosomal DNA ITS-1 variants
and chloroplast haplotypes. In our sample set, female trees predominate across the intro-
duced range from island Southeast Asia to Polynesia. Exceptions are few and are thought to
reflect known or likely modern introduction. The results allow us to solve the long-standing
question of the sex distribution of this dioecious species in Remote Oceania and suggest a
new explanation for the absence of botanical records of paper mulberry in Borneo and the
Philippines, reconfirm the dependence of this crop on human transmission outside the natu-
ral range, and point to a strategy for avoiding the spread of invasive breeding populations
outside the natural range.
Materials and Methods
The Corporación Nacional Forestal (CONAF) from Chile issued a research permit for collect-
ing inside the National Park on Easter Island. The Département de la Récherche, French Poly-
nesia issued permits for collecting in the Marquesas, Tahiti and Austral Islands; the Province
Museum of Central Sulawesi aided in sampling in Sulawesi, Indonesia. The Herbarium Pacifi-
cum (B. P. Bishop Museum, Hawaii), the Auckland Herbarium (Auckland Institute and
Museum, New Zealand) and the US National Herbarium (Smithsonian Institute, USA) issued
permits for herbarium sampling.
Archival research
Plant specimens were examined directly or online at the following locations: (1) B.P. Bishop
Museum, Honolulu, USA; (2) Herbarium Bogoriense, Bogor, Indonesia; (3) National Museum
of Natural History, Santiago, Chile; (4) Auck land Herbarium, Auckland Institute and War
Memorial Museum, New Zealand, (5) Landcare NZ, Lincoln, New Zealand, (6) National Her-
barium, Smithsonian Institution, Washington D.C., USA., and (7) National Museum of Natu-
ral History, Paris, France.
Field areas, sampling of plant material and DNA extractions
Young leaves were collected in New Caledonia, Western Polynesia (Fiji, Samoa, Wallis and
Tonga) and Eastern Polynesia (Marquesas archipelago [Ua Pou, Nuku Hiva, Fatu Hiva,
Tahuata], Society Islands [Raiatea, Tahiti], Hawaii [Oahu, Big Island], Austral islands [Rapa],
Pitcairn and Rapanui) between 2008 and 2014. Leaf samples collected in 2008 in Fiji, Samoa,
Tonga, Marquesas, Tahiti, Pitcairn, Raiatea and Rapanui were preserved at -20°C in the labora-
tory [10]. Leaf samples collected later in Sulawesi, New Caledonia, Wallis, Fiji, Tonga, Hawaii,
Rapanui, Rapa and Marquesas were dried with silica gel and stored at room temperature. Leaf
samples collected in Taiwan, China, Japan and Vietnam were dried in silica gel. Samples from
Rapa and New Caledonia were kindly provided by J.-Y. Meyer and A. Brianchon, respectively
and samples from Pitcairn were collected by C. Walter. Additionally five herbarium samples
from New Guinea and the Solomon islands were included in the study.
The origin of all 340 contemporary and the 12 herbarium samples are shown in
Table 1,
and include 33 samples from Asia, 26 (18 contemporary and eight herbarium) samples from
Sex Distribution of Paper Mulberry in the Pacific
PLOS ONE | DOI:10.1371/journal.pone.0161148 August 16, 2016 5/19