A peer-reviewed open-access journal
PhytoKeys 65: 35–46 (2016)
Flora of Bokor National Park V: Two new species of Machilus (Lauraceae)...
doi: 10.3897/phytokeys.65.7403
RESEARCH ARTICLE
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Launched to accelerate biodiversity research
Flora of Bokor National Park V: Two new species
of Machilus (Lauraceae), M. bokorensis
and M. brevipaniculata
Tetsukazu Yahara1, Shuichiro Tagane1, Keiko Mase1,
Phourin Chhang2, Hironori Toyama1
1 Center for Asian Conservation Ecology, Kyushu University, 744 Motooka, Fukuoka, 819-0395, Japan
2 Institute of Forest and Wildlife Research and Development, Forestry Administration, 40 Preah Norodom
Blvd, Phnom Phen, Cambodia
Corresponding author: Shuichiro Tagane (stagane29@gmail.com)
Academic editor: Pavel Stoev | Received 1 April 2016 | Accepted 6 June 2016 | Published 15 June 2016
Citation: Yahara T, Tagane S, Mase K, Chhang P, Toyama H (2016) Flora of Bokor National Park V: Two new species
of Machilus (Lauraceae), M. bokorensis and M. brevipaniculatae. PhytoKeys 65: 35–46. doi: 10.3897/phytokeys.65.7403
Abstract
Two new species, Machilus bokorensis Yahara & Tagane and M. brevipaniculata Yahara & Tagane (Lauraceae) are described from Bokor National Park, Cambodia with their illustrations and DNA barcodes of
the two plastid regions of rbcL and matK and the nuclear region of ITS.
Keywords
Bokor National Park, Cambodia, Lauraceae, Machilus, new species
Introduction
Lauraceae is a large family containing 54 genera and 2500–3500 species distributed
from tropical to temperate areas in the world (Rohwer 1993). In Southeast Asia, species of Lauraceae are found from lowlands to high elevations, and are often dominant
components of tropical and subtropical evergreen forests. Thus, some efforts have been
made to elucidate species taxonomy of Lauraceae in Southeast Asia (e.g., Lecomte
Copyright Tetsukazu Yahara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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1914; Liou 1934; Kostermans 1974, 1988; Kochummen 1989), including regional
revisions for Beilschmiedia of Borneo (Nishida 2008), Cinnamomum of Borneo (WuuKuang et al. 2011), Cryptocarya of Thailand and Indochina (de Kok et al. 2015) and
Litsea of Thailand (Ngernsaengsuruay et al. 2011). However, no volumes on Lauraceae
have been published in Flore du Cambodge du Laos et du Vietnam, Flora Malesiana,
or Flora of Thailand. Thus, species of Lauraceae still remain difficult to identify in
many areas of Southeast Asia.
The genus Machilus Nees is a member of the monophyletic Persea group consisting of Alseodaphne Nees, Apollonias Nees, Dehaasia Blume, Machilus, Nothaphoebe
Blume, Persea Mill., and Phoebe Nees (Rohwer et al. 2009; Li et al. 2011). Although
Kostermans (1990) merged Machilus into Persea, recent molecular evidence showed
that Persea of the New World may be polyphyletic and Machilus of Asia is well differentiated from the two clades including the majority of the Neotropical Persea species
(Rohwer et al. 2009; Li et al. 2011). Machilus comprises about 100 species in tropical and subtropical S and SE Asia (Wei and van der Werff 2008). Twelve Machilus
species (including M. balansae (Airy Shaw) F.N.Wei & S.C.Tang, M. cochinchinensis
Lecomte, and M. velutina Champ. ex Benth., all treated as Persea) are recorded from
Vietnam (Hô 1999), three species from Laos (Newman et al. 2007), eight species from
Malaysia (treated as Persea, Kochummen 1989), and 82 species from China (Wei and
van der Werff 2008). In Cambodia, only one species, Machilus odoratissima Nees, has
been reported based on specimens (Lecomte 1914; Liou 1934; Phon 2000); three additional species, M. gamblei King ex Hook.f., M. salicina Hance, and M. velutina, were
recorded from Cambodia in Flora of China (Wei and van der Werff 2008), but we
could not find any specimens supporting those records.
During our field surveys of vascular plants in Bokor National Park, Kampot Province, Southern Cambodia conducted from 2011 to 2013, we collected five species of the
genus Machilus among which two fertile species which differ from all known congeners.
Here we describe them as new, M. bokorensis Yahara & Tagane and M. brevipaniculata
Yahara & Tagane, with illustrations and DNA barcodes of the two plastid regions of
rbcL and matK (CBOL Plant Working Group 2009), and the nuclear ITS region.
The specimens examined are deposited in the herbarium of Forest Administration
of Cambodia, Phnom Penh (not in Index Herbariorum, abbreviated ‘PNP’ here), the
Herbarium of the Museum of Kyushu University (FU) and partly in the Herbarium
of the Kyoto University Museum (KYO) and the Forest Herbarium, Bangkok (BKF).
Materials and methods
Morphological observations
We have made morphological observations on the specimens that we had collected in
Bokor National Park, Cambodia. We have surveyed the diagnostic features of all the
known species in Cambodia and its neighboring regions by the means of a thorough
Flora of Bokor National Park V: Two new species of Machilus (Lauraceae)...
37
literature review and by morphological observations of the dry specimens, kept in the
herbaria BKF, BO, FU, HN, K, KYO, L, MBK, P, RAF, SAR and VNM and specimen
images available on the web [e.g. JSTOR Global Plants (https://plants.jstor.org/)].
DNA barcoding
Pieces of leaves were collected and desiccated with silica gel in the field. DNA was extracted from these samples using a modified CTAB method. Before DNA extraction, we
ground silica gel-dried leaves with a TissueLyser (QIAGEN) into the powder that were
subsequently washed at least four times with 1 mL buffer (0.1 M HEPES, pH 8.0; 2%
mercaptoetanol; 1% PVP; 0.05 M ascorbic acid). We determined the partial sequences
of DNA regions encoding the large subunit of ribulose-1,5-bisphosphate carboxylase
oxygenase (rbcL) and maturase K (matK), following the published protocols (Kress et
al. 2009; Dunning and Savolainen 2010). Additionally, we determined the sequences
of the internal transcribed spacer (ITS) region using the primers of ITS18-F (5’-GTCCACTGAACCTTATCATTTAGAGG-3’) and ITS26-R (5’-GCCGTTACTAAGGGAATCCTTGTTAG-3’) according to the published protocol (Rohwer et al. 2009)
with slight modification using Tks GflexTM DNA Polymerase (TAKARA, Japan).
Taxonomic treatments
Machilus bokorensis Yahara & Tagane, sp. nov.
urn:lsid:ipni.org:names:77155497-1
Figs 1, 2
Machilus odoratissima (non Nees) sensu H.Liou, Laurac. Chine & Indochine: 53
(1932), for the specimen collected from Nord de Kampot (P, no. 14625).
Diagnosis. Similar to M. odoratissima Nees in elliptic-lanceolate or oblanceolate, glabrous leaves and subterminal inflorescences, but differing in having sericeous inflorescence rachis (Fig. 2c; vs. glabrous in M. odoratissima), perianth lobes pubescent on
both surfaces (Fig. 2c; vs. outside almost glabrous), ca. 1 mm long staminodes (vs. ca. 2
mm long), and twigs drying blackish grey (vs. reddish). Similar to M. parviflora Meisn.
among Indochinese species in having large terminal buds, but differing in bud scales
densely puberulent outside (vs. densely golden hairy outside in M. parviflora).
Type. CAMBODIA. Kampot Province, Bokor National Park, dense evergreen
forest on the plateau of Mt. Bokor, 10°39'19.5"N, 104°03'36.6"E, alt. 933 m, 16 Feb.
2013, with flowers, Tagane S., Toyama H., Wachi N., Ichihasi R., Mase K., Zhu M.,
Chhnang P. 5475 (holotype KYO!, isotypes BKF!, FU!, K, P, PNP!).
Description. Small tree, 10 m tall. Branchlets glabrous, blackish when dry, sparsely lenticellate, old branches blackish grey or blackish grey brown. Terminal buds 3–7
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Figure 1. Machilus bokorensis Yahara & Tagane. a flowering branch, b abaxial lower surface of lamina
c flowers d buds e holotype: Tagane et al. 5475 (KYO), scale bar = 10 cm f inflorescence, scale bar = 1 cm.
Photographs: a–c by S. Tagane, d by K. Fuse.
Flora of Bokor National Park V: Two new species of Machilus (Lauraceae)...
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Figure 2. Machilus bokorensis Yahara & Tagane. a flowering branch b top of branch c flowers d stamen
in 1st whorl e stamen in 2nd whorl with staminode f stamen in 3rd whorl with glands g pistil h outer
perianth lobes i inner perianth lobe j, k bud scales. Materials from Tagane et al. 5475 (KYO). Drawn by
S. Tagane.
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mm long, bud scales broadly ovate to oblanceolate, densely puberulent outside, glabrous or very sparsely pubescent inside. Leaves alternate, blades elliptic-lanceolate,
oblanceolate, (3–)7.5–11.5(–18.5) × (1.2–)2.5–4.0(–5.4) cm, thickly leathery, base
cuneate, margin entire, slightly revolute, apex usually acute to obtuse, occasionally
acuminate or rotund, developing leaves glabrous adaxially, sericeous abaxially, glabrous and densely foveolate on both sides when old, midrib concave adaxially, elevated
abaxially, lateral veins 9–12 pairs, flat adaxially, slightly prominent abaxially, tertiary
veins reticulate to scalariform-reticulate; petioles (0.5–)0.8–1.3(–2.5) cm long, glabrous. Inflorescences paniculate, subterminal, usually arising from near base of newly
sprouted branchlets, (4.0–)6.0–13.0 cm long, sericeous, with 5–8 lateral branches.
Pedicels 3–5 mm long, sericeous, bracts linear, 1–4 mm long, abaxially yellowish
brown pubescent, adaxially glabrous, caducous. Flowers yellowish green; perianth
lobes 6, oblong-elliptic, slightly unequal, outer ones, ca. 6.8 × 2.4 mm, inner ones ca.
7 × 2.5 mm, minutely pubescent on both surfaces. Stamens 9, subequal, 3.5–4 mm
long, hairy at base, glands of 3rd series stipitate, ca. 0.4 mm long; staminodes 3, ca. 1
mm long, stipitate, glabrous. Ovary subglobose ca. 1.5 mm in diam., glabrous. Style
2.5–3 mm long, glabrous. Fruits subglobose, 7–8 mm in diam., glabrous, blackish
when dry, perianth lobes spreading.
Ohter specimens examined. Cambodia, Bokor National Park [alt. 1043 m,
10°37'16.8"N, 104°01'52.3"E, 10 Dec. 2013, Toyama et al. 6258 (BKF, FU, PNP); alt.
1042 m, 10°38"N, 104°01.4’E, 9 Mar. 2001, with fruits, Middleton & Monyrak 643 (P);
alt. 1014 m, 10°38'12.6"N, 104°02'06.4"E, 4 Dec. 2011, Toyama et al. 1556 (FU, PNP);
ibidem, Toyama et al. 1575 (FU, PNP); alt. 1011 m, 10°38'11.2"N, 104°02'09.0"E,
Toyama et al. 1725 (FU, PNP); alt. 928 m, 10°39'19.5"N, 104°03'36.6"E, Toyama et
al. 2754 (FU, PNP); alt. 903 m, 10°39'35.4"N, 104°03'03.1"E, Toyama et al. 3167
(FU, PNP); alt. 871 m, 10°39'48.8"N, 104°02'53.3"E, 17 Dec. 2013, with flowers,
Fuse et al. 6227 (BKF, FU, PNP)].
Distribution. Cambodia (endemic to Mt. Bokor).
Habitat and ecology. Machilus bokorensis is commonly found in moist evergreen
forest on the top plateau of Mt. Bokor. Flowering specimens were collected in December and February and fruiting specimens in March.
GenBank accession No. Toyama et al. 1575: AB987667 (rbcL), AB987666
(matK), AB987665 (ITS).
Preliminary conservation assessment. Machilus bokorensis is commonly found
in higher elevations in Mt. Bokor. Since many mature individuals were found in the
protected areas, we suggest the status for this species as Least Concern (LC) according
to IUCN Red List criteria (IUCN 2012).
Note. Among five species we collected in Mt. Bokor, this species is most common
and agrees with the description of “M. odoratissima” in the key of Liou (1934) who
recorded “M. odoratissima” from Nord de Kampot, corresponding to Mt. Bokor.
Thus, we consider that this species is “M. odoratissima” recorded by Liou (1934),
although we could not examine the specimen of Poilane 14625 (P) cited by Liou
(1934) because it is currently on loan. In Indo-china, Machilus plants similar to this
Flora of Bokor National Park V: Two new species of Machilus (Lauraceae)...
41
species have been identified as M. odoratissima for a long time (Lecomte 1914; Liou
1934; Phon 2000). However, M. bokorensis is distinct from M. odoratissima distributed in Nepal, E Himalaya, Assam-Burma in sericeous inflorescence rachis (vs. glabrous in M. odoratissima; Pendry 2012), perianth lobes pubescent on both surfaces
(vs. outside almost glabrous), ca. 1 mm staminodes (vs. ca. 2 mm), and blackish grey
twigs (vs. reddish). In Cambodia, we collected sterile specimens of Machilus spp.
morphologically similar to M. bokorensis in Central Cardamon and Seima protection
forests but those are morphologically distinguishable and not sister to M. bokorensis
in our unpublished phylogenetic tree constructed by rbcL, matK and ITS sequences.
“M. odoratissima” is recorded from Mulu-prey, Preah Vihear (Lecomte 1914). The
specimens from Mulu-prey (Harmand s.n., P) are, however, different from M. bokorensis in having naked terminal buds and scalariform tertiary veins. Lecomte (1914)
and Liou (1934) recorded “M. odoratissima” also from Laos and Vietnam, but we
have not found specimens from Laos and Vietnam identical with M. odoratissima. As
far as we know, M. odoratissima is not distributed in Cambodia, Laos, Thailand and
Vietnam. The relationship between M. bokorensis and the specimens of Mulu-prey,
Laos and Vietnam remains to be further examined. At present, we consider that M.
bokorensis is endemic to Bokor, Cambodia. According to our unpublished ITS tree,
this species is sister to Machilus rimosa (Blume) Blume distributed in Indonesia, but
distinct from M. rimosa in thickly leathery leaves densely foveolate on both sides (vs.
thinner leaves not densely foveolate in M. rimosa) and perianths larger (6.8–7 mm
long vs. ca. 3.5 mm long).
Machilus brevipaniculata Yahara & Tagane, sp. nov.
urn:lsid:ipni.org:names:77155498-1
Figs 3, 4
Diagnosis. Similar to M. kingii Hook.f. in leaf shape and size, and short panicles less
than 5.5 cm long, but distinct from M. kingii by its naked bud (vs. bud covered with
scales in M. kingii), lamina foveolate on both surfaces (vs. obscure on lower surface),
fewer flowers per panicle (3–5 vs. 4–9), and smaller perianth lobes (2 mm vs. 3–3.5
mm long).
Type. CAMBODIA. Kampot Province, semi-evergreen secondary forest at the
bottom of Mt. Bokor, 10°35'35.6"N, 103°58'43.1"E, alt. 65 m, 7 Dec. 2013, Tagane
S., Toyama H., Fuse K., Iwanaga F., Rueangruea S., Suddee S., Kanemitsu H., Zhang M.,
Kim W., Loth M. 6011 (holotype KYO!, isotypes BKF!, FU!, PNP!).
Description. Small tree, 8 m tall. Branchlets sericeous near the tip, soon glabrous,
brownish when dry, old branches greyish brown to reddish brown, without lenticels;
terminal buds naked. Leaves alternate; blades lanceolate to oblanceolate, 5.0–8.3 ×
1.4–3.1 cm, leathery, reddish brown when dried, glabrous and foveolate on both sides,
lustrous adaxially when dry, base cuneate, margin slightly revolute when dry, apex
acute to obtuse, midrib concave adaxially, elevated abaxially, lateral veins 8–10 pairs;
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Figure 3. Machilus brevipaniculata Yahara & Tagane. a flowering branch b flowers c abaxial lower surface of lamina d flower e holotype: Tagane et al. 6011 (KYO), scale bar = 10 cm, f portion of leaves g apical
bud. Photographs: a, b, d by S. Tagane and c by S. Rueangruea.
Flora of Bokor National Park V: Two new species of Machilus (Lauraceae)...
43
Figure 4. Machilus brevipaniculata Yahara & Tagane, a flowering branch b top of branch with abaxial
leaf surface c flower buds d flower e outer perianth lobe f inner perianth lobe g–i stamens in 1st, 2nd and
3rd whorl from left j staminode k pistil. Materials from Tagane et al. 6011 (KYO). Drawn by K. Mase.
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petioles 0.5–1.4 cm, glabrous. Inflorescences paniculate, terminal or arising as lateral
branches of newly sprouted shoots, 4–11 cm long, usually with frondose bracts subtending secondary paniculate axes, secondary axes 0.8–1.3 cm long with 3–15 flowers. Pedicels ca. 2 mm long. Flowers yellowish green. Outer perianth lobes ovate, ca.
2 × 1 mm, inner ones broadly ovate, ca. 2 × 1.3 mm, pubescent on both surfaces,
with many gland dots. Stamens 9, subequal, ca. 1.5 mm long, hairy at base, anthers
4-celled, ca. 1 mm long; glands of 3rd series stipitate, ca. 0.3 mm diam. Staminodes 3,
ca. 0.6–1 mm long, glabrous. Ovary ellipsoidal, ca. 0.8 mm in diam., glabrous. Style
ca. 0.8 mm long, glabrous. Fruits not seen.
Distribution. Cambodia. Known only from the lowland of Bokor National Park.
Habitat and ecology. This species was found in evergreen forest at the foot of Mt.
Bokor. Flowering specimens were collected in December.
GenBank accession No. Tagane et al. 6011: AB987676 (rbcL), AB987675
(matK), AB987674 (ITS).
Preliminary conservation assessment. Critically endangered (CR) (IUCN
2012). We collected only one flowering individual of this species at alt. 65 m during 7
field surveys on the southern slope and top plateau of Mt. Bokor (Tagane et al. 2015).
Further botanical inventories might enable us to find more individuals, but it seems to
be a rare species. Considering the fact that the forest is almost cleared in the foot of Mt.
Bokor (below 100 m in particular), we believe that this species should be considered
critically endangered.
Note. This species is distinct in having naked terminal buds and panicles having
frondose bracts subtending secondary paniculate axes, while most of Machilus species have scaly terminal buds and panicles without frondose bracts. According to our
unpublished ITS tree, this species is sister to Machilus coriacea A.Chev. endemic to
southern Vietnam, but can be readily distinguished from the latter by the two above
mentioned traits and also in having glabrous leaves (vs. densely hairy beneath when
young in M. coriacea).
Acknowledgement
We thank the Cambodian Ministries of the Environment and Forest Administration
for the permission for our field works, and particularly the staffs and rangers of the
Bokor National Park for their kind help. We are grateful to the curators and the staffs
of the herbaria BKF, BO, FU, HN, K, KYO, L, MBK, P, RAF, SAR and VNM for
providing us the opportunities to examine the collections. This study was supported by
the Environment Research and Technology Development Fund (S9) of the Ministry
of the Environment, Japan and also by a JSPS grant for Global Center of Excellence
Program ‘Asian Conservation Ecology as a basis of human-nature mutualism’.
Flora of Bokor National Park V: Two new species of Machilus (Lauraceae)...
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