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Descriptions of two new species of Nepenthes (Nepenthaceae)
from Thailand and their phylogenetic analysis based on AFLP
technique species confirmation
Sunya Nuanlaong
1
, Maruay Mekanawakul
2
& Potjamarn Suraninpong
1
Summary. Two new species of Nepenthes (Nepenthaceae) from Southern Thailand, N. bracteosa sp. nov. and
N. hirtella sp. nov. are described and illustrated. Descriptions of the species are given compared to their related
species, N. krabiensis and N. kerrii, collected in Thailand. They are separated from N. krabiensis and N. kerrii by
characteristics of leaf shape, pitcher shape, indumentum, and bracteole. Confirmation of these two new species
was determined by phylogenetic relationships using the Amplified Fragment Length Polymorphism (AFLP)
technique.
Key Words. monkey cup, phylogenetic relationship, pitcher plant, taxonomy.
Introduction
The genus Nepenthes, known as the tropical pitcher
plant or monkey cup, is in the family Nepenthaceae
(order Caryophyllales). The first encounter with
Nepenthes in the mid-17
th
century focused on its
taxonomy, diversity and distribution. The Nepenthes
flora of Indochina and the Philippines, in particular,
have attracted much recent attention (McPherson
2009; McPherson 2012). Nepenthes comprises approxi-
mately 160 species, with the greatest diversity with
many endemic species, in the Philippines and Borneo.
Several taxa have been recently described from
Indochina: N. bokorensis Mey (Mey 2009), N. thai Cheek
(Cheek & Jebb 2009), N. holdenii Mey (Mey et al. 2010),
N. mirabilis var. globosa M.Catal. (Catalano 2010a),
N. suratensis M.Catal. (Catalano 2010b), N. andamana
M.Catal. (Catalano 2010c), N. kerrii M.Catal. & Kruetr.
(Catalano 2010d), N. chang M.Catal. (Catalano 2010e),
N. rosea M.Catal. & Kruetr. (Catalano 2014),
N. kongkandana M.Catal. & Kruetr. (Catalano 2015),
N. krabiensis Nuanlaong, Onsanit, Chusangr. & Sura.
(Nuanlaong et al. 2016), N. malayensis A.Amin,
M.N.Faizal & Dome (Tamizi et al. 2020), N. latiffiana
M.N.Faizal, A.Amin & Dome and N. domei M.N.Faizal,
A.Amin & A.Latif (Ghazalli et al. 2020). In Thailand,
14 species and one variety of Nepenthes are currently
recognised. Of these, nine species including our new
species are considered to be closely related and they
have been attributed to section Pyrophytae Cheek &
Jebb (Cheek & Jebb 2016). Seven species in section
Pyrophytae are found on the summit areas of moun-
tains, namely, N. chang (300 m a.s.l.), N. kerrii (500 m
a.s.l.), N. krabiensis (500 –600 m a.s.l.), N. rosea (450 –
520 m a.s.l.), N. smilesii Hemsl. (16 –1,500 m a.s.l.),
N. bracteosa sp. nov. (710 –760 m a.s.l.) and N. hirtella
sp. nov. (348 –355 m a.s.l.). While four species,
N. andamana,N. kampotiana Lecomte, N. kongkandana
and N. suratensis are found at sea level. Generally,
plants in section Pyrophytae have tuberous rootstocks,
which is an adaptation for fire, and are found in
seasonal habitats. Members of section Pyrophytae lack a
climbing stem and the upper pitchers (which arise
after the inflorescence is first produced) are borne on
stems with short internodes. The tendril of the upper
pitchers lacks a coil, and the partial-peduncles are 1-
flowered, with short pedicels/partial-peduncles
(Cheek & Jebb 2016). During the dry season, Nepenthes
decrease leaf, flower, and pitcher production. The
tuberous rootstock is likely to act as a water and
nutrient store for survival during periods of unsuitable
environmental conditions (Mey et al. 2010).
However, the pyrophytic species are found to be
closely related to section Montanae Danser (Danser
1928), which share most of their characteristics. Plants
from section Montanae lack fleshy tubers, have
climbing stems, coiled tendrils, and longer pedicels
(Cheek & Jebb 2009). In southern Thailand near the
Malaysian border, two species namely, N. sanguinea
Lindl. and N. thai Cheek, have been found, which are
relatives of Penisular Montanae, such as N. benstonei
C.Clarke, N. gracillima Ridl., N. macfarlanei Hemsl. and
N. ramispina Ridl. in highland Malaysia (Cheek & Jebb
2009). However, the two new species are similar in
overall appearance and share many characteristics
Accepted for publication 10 August 2021.
1
School of Agricultural Technology, Walailak University, Nakhon Si Thammarat, 80160, Thailand. e-mail: potjamas@hotmail.com
2
School of Science, Walailak University, Nakhon Si Thammarat, 80160, Thailand.
KEW BULLETIN
DOI 10.1007/S12225-021-09997-6
ISSN: 0075-5974 (print)
ISSN: 1874-933X (electronic)
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
with pyrophytic species such as: long racemose inflo-
rescence, flowering at the rosette stage, seed with
reduced filiform appendages, coriaceous narrow
leaves, decurrent leaf attachment, tuberous rootstock,
and pyrophytic habitat. As a result, the two new species
could not be classified as members of section
Montanae.
Despite previous research, evolution within the
genus Nepenthes is still poorly understood. Currently,
research in Nepenthes has been focused on their actual
diversity. These include biogeographical and environ-
mental processes, and animal-plant interactions that
are facilitated mostly by characteristics of pitchers and
flowers (Ellison & Adamec 2018). Nepenthes is a cross-
pollinated plant, in which the wide differential archi-
tectures of the phenotypic traits differ resulting from
both genetic and environmental factors found in their
habitat (Rowe & Speck 2005). Genetic analyses have
revolutionised systematics and provided important
insights into the understanding plant evolution. The
complete genome of Nepenthes has not yet been
published, resulting in a limited number of molecular
markers available for characterisation of Nephethes
taxa. AFLPs, a PCR-based method that selectively
amplifies a subset of digested DNA fragments to
produce distinctive fingerprints, is the common mo-
lecular marker used for comparing genetic differences
(Paun & Schönswetter 2012). The effectiveness of
AFLP analysis is derived from its capacity to rapidly
produce large numbers of marker fragments for any
organism without the requirement of understanding
any genomic sequence. The AFLP technique has been
used for various applications, including genetic map-
ping, genealogical studies among closely related
individuals, quantification of genetic diversity within
and among species, and phylogenetic studies of closely
related species (Savelkoul et al. 1999). Thus, in this
study, the two new Nepenthes found on the mountain
tops of Khao Wang Hip (Nakhon Si Thammarat
Province) and Khao Shawaplab (Krabi Province) are
described and classified through their phylogenetic
relationships using AFLP techniques.
Materials and Methods
Morphological analysis
Study samples of Nepenthes were collected during a
field investigation from Mt Khao Wang Hip, Nakhon
Si Thammarat Province, 8°16'06.80"S, 99°40'11.99"E,
and Mt Khao Shawaplab, Krabi Province, 7°50'29.53"S,
99°16'35.45"E, Southern Thailand in 2017 –2018
(Map 1). The extent of occurrence (EOO) and area
of occurrence (AOO) of the Nepenthes populations was
calculated to determine the conservation status using
Geocat (Bachman et al. 2011). All data and dimensions
were either collected in the field from live plant
specimens, or by a more thorough examination using
a Nikon SMZ-U Stereo Zoom Microscope with a Nikon
DXM1200F digital camera and ACT-1 software (ver-
Map 1. Sample collecting sites for the Nepenthes species in this study in Southern Thailand. Locality 1N. bracteosa and locality 2
N. hirtella. Map was modified from Google Maps.
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sion 2.70). Morphological terminology applied for the
descriptive studies follows Cheek & Jebb (2009) and
Nuanlaong et al. (2016).
AFLP analysis and phylogenetic relationships
To determine the putative phylogenetic relationships
of the new Nepenthes species studied, they were
compared with five Nepenthes species distributed on
mountain tops, specifically N. chang,N. kerrii,
N. krabiensis,N. rosea, and N. smilesii. In addition,
N. thorelii and N. mirabilis were used as an ingroup and
an outgroup, respectively (Table 1). Each species
included five replications (random plants and sex).
Total genomic DNA was isolated from fresh pitcher
tissues using the methods of Suraninpong et al. (2015).
Before analysis, DNA from five replicates was pooled
in equal quantities. The AFLP analysis was conducted
as described by Vos et al. (1995) with some modifica-
tions. Initial/first genomic DNA (300 ng) was digested
with 10 units of EcoRI (Roche, USA) and 5 units of
Tru9I (Roche, USA) in 1X SuRE/Cut™Buffer A
(Roche, USA) to a final volume of 30 μl for 24 h at
37°C. The mixture was added to 10 μl of ligation mix
containing 50 pmol adapters of EcoRI and Tru9I, 1
unit of T4-DNA ligase (Roche, USA), and 1x ligation
buffer. The ligation reaction was performed at 37°C
for 12 h. Then, 5X DNA template (digestion-ligation
mixture) dilution was prepared for pre-amplification
(PCR I) by adding 2 μl 5X DNA template dilution with
0.25 μM of primer (1 selective base of EcoRI and
Tru9I), 1X TopTaq Master Mix Kit (QIAGEN, USA)
and dH
2
O, in a final volume of 10 μl. The thermal
conditions for PCR I were 20 cycles of 30 s at 95°C,
1 min at 56°C, 1 min at 72°C with a final extension of
7 min at 72°C (T100 Thermal Cycler Bio Red, USA).
Then, the PCR I product was diluted to 10X (1 μl PCR
I plus with 9 μldH
2
O) and was mixed with 0.25 μMof
primer (2 or 3 selective bases of EcoRI and Tru9I), 1X
TopTaq Master Mix Kit and dH
2
Otoafinal volume of
10 μl. The PCR II was conducted with the annealing
temperature reduced by 1°C every cycle. It started with
10 cycles of 30 s at 95°C, then 30 s at 65°C, and 1 min
at 72°C. Continuously, 30 cycles of 30 s at 95°C, 30 s at
56°C, 1 min at 72°C, 7 min at 72°C and 4°C until the
reaction was complete. Then, the reaction was stopped
by adding 10 μl of stop-loading dye (10 mM EDTA pH
8.0, 98% formaldehyde, Bromophenol Blue &
Xylenecyanol). The PCR product was separated on
5% (w/v) polyacrylamide gel electrophoresis. The
DNA bands were visualised via silver staining and were
scored manually.
The NTSYSpc (version 2.20j N) program was used
for cluster analysis based on a similarity matrix.
Genetic distances were obtained using the NTSYS-pc
2.20j statistic package program according to Jaccard’s
similarity coefficient. The matrix was analysed by the
unweighted pair-group method with arithmetic mean
(UPGMA) (Rohlf 1998). The relationships between
species are illustrated as a dendrogram. The presence
andabsenceofbandswerescoredas1and0,
respectively. Bands of similar size and intensity were
assumed to be homologous. The polymorphism (P)
was calculated as P = (K/N) x 100%; where Kis the
number of polymorphic bands and Nis the total
number of amplified bands. The polymorphism infor-
mation content (PIC) value for each AFLP primer
combination was calculated based on Roldàn-Ruiz
et al. (2000) as PIC
i
=2f
i
(1 - f
i
); where PIC
i
is the PIC
of marker i,f
i
is the frequency of the marker bands
which were present, and 1 - f
i
is the frequency of
marker bands which were absent. The final PIC value
was the average of the PIC values of each marker over
the bands for each primer combination. The Jaccard’s
similarity coefficient was computed for each pair of
species (Jaccard 1908). The FreeTree program
(Pavlicek et al. 1999) was used for the bootstrap
analysis with 1,000 resampled datasets. We applied
the same levels of support to the Bootstrap values as
Richardson et al.(2000), which indicates that a value in
the range of 85 –100% means high reliability, 71 –
84% means medium reliability, and 50 –70% means
low reliability.
Table 1. Plant material examined in this study.
Species Source
Nepenthes mirabilis (Lour.) Druce Nakhon Si Thammarat Province, Thailand
Nepenthes thorelii Lecomte Tay Ninh Province, Vietnam
Nepenthes chang M.Catal. cult. Krabi Carnivores Nursery, Thailand
Nepenthes kerrii M.Catal. & Kruetr. cult. Krabi Carnivores Nursery, Thailand
Nepenthes smilesii Hemsl. Phu Kradueng National Park, Loei province, Thailand
Nepenthes krabiensis Nuanlaong, Onsanit, Chusangr.
& Suran.
Khao Pra-Bang Khram Wildlife Sanctuary, Krabi Province, Thailand
Nepenthes rosea M.Catal. & Kruetr. Noppharatthara Beach–Pee Pee Islands National Park, Krabi Province, Thailand
Nepenthes bracteosa Suran. & Nuanlaong sp. nov. Nam Tok Yong National Park, Nakhon Si Thammarat Province, Thailand
Nepenthes hirtella Nuanlaong & Suran. sp. nov. Khao Pra-Bang Khram Wildlife Sanctuary, Krabi Province, Thailand Krabi
Province, Thailand
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Results and Discussion
Morphological evidence
Several morphological characters allow the new
species, namely Nepenthes bracteosa and N. hirtella,
to be distinguished from presently recognised
species. The morphology of the two new species
differs distinctly from N. krabiensis and N. kerrii
asshowninTable2.
AFLP and phylogenetic relationship
In this study, more than 50 primer combinations from
EcoRI and Tru9I were examined, but only eleven
primer combinations were clearly classified for poly-
morphic profiling on nine Nepenthes species (Table 3).
A total of 1,415 DNA fragments scored across all
primer sets were observed, out of which 1,323
(93.30%) were polymorphic, which enabled differen-
tiation of the accessions analysed. The size of AFLP
fragments generated by different primer combinations
ranged from 200 to 800 bp. The highest number of
polymorphisms were for ER-AC/Tru-CGT (98.64%)
followed by ER-AC/Tru-CAG (94.96%) and ER-AGC/
Tru- CAG (94.44%). Conversely, the lowest polymor-
phism at 90.65% was found on ER-AT/Tru -CAG
primer combinations. Monomorphism was lower than
polymorphism. Low monomorphism indicates that
there is a high degree of variation between the species.
The PIC value ranged from 0.71 to 0.86 with an
average of 0.76 per fragment. The highest value 0.86
was obtained for the ER-AC/Tru-CGT primer combi-
nation, followed by 0.78 for ER-ATG/Tru-GAG. A
strong correlation between polymorphism and PIC
(r
2
=0.86) was revealed.
Jaccard’s similarity coefficient was found to vary
from 0.22 to 0.65, with an average of 0.42, suggesting a
high genetic diversity among the nine Nepenthes
species (Table 4). The dendrogram from the cluster
analysis, constructed from 11 AFLP markers, indicates
that the Nepenthes species can be divided into two
groups and two separate individual species at 0.46 of
similarity coefficient with a bootstrap value of 100%
(Fig. 1). This grouping was consistent with the existing
morphological and geographical classification of Ne-
penthes species and was similar to previous reports of
the relationships from geographical and genetic
distribution by Suraninpong et al. (2015).
From the results, the first group contains two
subgroups, the first of which is Nepenthes rosea, and
the second subgroup is N. krabiensis,N. bracteosa,
N. kerrii and N. hirtella. All species of this group were
distributed throughout southern Thailand but con-
fined to different places. Nepenthes bracteosa clearly
showed separation from N. krabiensis with a similarity
coefficient of 0.65 with a bootstrap value of 88%.
Nepenthes hirtella was identified in the same related
subgroup with N. krabiensis and N. bracteosa. From the
result of the low similarity coefficient (0.45 –0.65),
high genetic variation among these species was
discovered. In contrast, the species from North-East
Thailand namely, N. chang and N. smilesii were
separated into the second subgroup and were shown
to have a similarity coefficient of 0.50 with a bootstrap
value of 96%. The results published here suggest that
all Nepenthes species found in Thailand are derived
from a common ancestor, of which N. thorelii Lecomte
was the first species separated from the aggregate
group. Likewise, the phylogenetic relationship infor-
Table 2. Comparisons of geography and selected plant characteristics of Nepenthes bracteosa and N. hirtella with N. krabiensis and
N. kerrii.
N. krabiensis N. bracteosa N. kerrii N. hirtella
Geography SW Thailand,
500 –600 m a.s.l.
SE Thailand,
710 –760 m a.s.l.
Island, SW
Thailand, 500 m a.s.l.
SW Thailand,
348 –355 m a.s.l.
Leaf shape lanceolate linear to lanceolate obovate oblanceolate
Leaf apex acute acute to acuminate acuminate acuminate
Lid of lower pitcher broadly ovate broadly ovate to round round ovate
Spur filiform filiform with branches simple or branches filiform with branches
Male inflorescence 2-flowered, partial
peduncles
1-flowered 1-flowered 1-flowered or rarely 2-
flowered, partial
peduncles
Androphore length
(mm)
4 2.0 –2.5 1.5 1.0 –2.0
Bracts male inflorescence:
present at base or
lower half of the
pedicel
female inflorescence:
absent
present all flowers of
both male and
female inflorescences
absent present at base of rachis
(1 –2flowers) both
male and female
inflorescences
Indumentum lacking on stem, lamina,
and leaf margin
lacking on stem, lamina,
leaf margin, and
shoot
covering leaf
axil and
inflorescence
covering all vegetative
parts
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mation explains the genetic link between the two
newly discovered Nepenthes species, N. bracteosa and
N. hirtella.Nepenthes bracteosa and N. hirtella are separated
from N. krabiensis by their genetic background, even
though they are found in close proximity.
Taxonomic Treatment
1. Nepenthes bracteosa Suran. & Nuanlaong sp. nov.
Type: Thailand, Nakhon Si Thammarat Province,
Thung Song Distr., Nam Tok Yong National Park, Mt
Khao Wang Hip, 710 –760 m a.s.l., 8°16'06.80"S,
99°40'11.99"E, ♂fl. lower and upper pitchers, 29
August 2019, Nuanlaong BKF197454 (holotype BKF!;
isotype PSU!).
http://www.ipni.org/urn:lsid:ipni.org:names:77234369-1
Scandent, glossy climbing herb. Root branched, fleshy
tubers. Stem terete, 0.5 –1.0 m long, 0.3 –0.5 cm
diam.; internodes 1.3 –2.7 cm long; axillary bud
present in 2 –3 axils in the upper part of the plant
when flowering; upper part green to red and lower
part brown. Leaves pseudo-petiolate, coriaceous, linear
to lanceolate; 14.0 –24.0 × 2.0 –3.9 cm, longitudinally
folded to form a V-shape; blade apex acute to
acuminate, base attenuate, entire, dilating at the node,
projecting along the stem as a wing, clasping the stem
by three-quarters of its circumference, decurrent for
0.8 –2.8 cm of its length, obscure longitudinal nerves
on each side of the midrib at the edge of lamina, as
leaves are thickened, inconspicuous reticulated pin-
nate venation; midrib conspicuous; tendrils straight,
terete, 15.0 –26.0 cm long in lower pitchers, upper
pitchers 10.0 –15.0 cm long, coiling; glossy light green
when young, green when mature, emerging from an
orange–amber, triangular lateral bud at the base of
the midrib near the leaf attachment. Lower pitcher c.
10.5 –19.0 × 3.4 –5.6 cm; ovate in the lower part with
digestive glands on the inner surface, cylindrical to
narrowing in the upper part; conspicuous midsection
hip; with two fringed wings, 0.6 –1.3 cm wide, along
the length of the pitcher, 6.5 –12.0 cm from pitcher
mouth to tendril, fringed elements 0.3 –1.2 cm long, 4
–8 fringed elements per cm; mouth ovate, concave;
peristome cylindrical, undulate, revolute when pitcher
mature, 0.7 –1.9 cm wide, inner edge with teeth 0.6 –
1.7 mm long, ridges approximately 0.4 –0.5 mm apart;
Table 3. Description of AFLP results from seven Nepenthes species and two new species, N. bracteosa and N. hirtella in Thailand:
PB polymorphic bands; MB monomorphic bands; Ppercentage of polymorphism; Mpercentage of monomorphism; PIC
polymorphism information content.
Primer pairs Total no. of bands PB MB PMPIC
ER-AG/Tru-CAG 100 92 8 92.00 8.00 0.74
ER-AG/Tru-CGT 101 93 8 92.08 7.92 0.71
ER-AT/Tru-CAG 107 97 10 90.65 9.35 0.71
ER-AC/Tru-CAG 139 132 7 94.96 5.04 0.74
ER-AC/Tru-CGT 147 145 2 98.64 1.36 0.86
ER-AGA/Tru-CGA 113 106 7 93.81 6.19 0.76
ER-AGA/Tru-CCA 136 127 9 93.38 6.62 0.76
ER-AGA/Tru-CAG 119 108 11 90.76 9.24 0.72
ER-AAG/Tru-CAG 149 139 10 93.29 6.71 0.75
ER-AGC/Tru-CAG 162 153 9 94.44 5.56 0.79
ER-ATG/Tru-GAG 142 131 11 92.25 7.75 0.78
Total 1415 1323 92 1026.26 73.74 8.32
Mean 128.64 120.27 8.36 93.30 6.70 0.76
Table 4. Percentage similarity matrix for nine Nepenthes accessions, generated from Jaccard’s similarity coefficient (Jaccard 1908).
S1 N. rosea;S2 N. krabiensis;S3 N. bracteosa;S4 N. hirtella;S5 N. smilesii;S6 N. kerrii;S7 N. chang;S8 N. thorelii;S9 N. mirabilis.
S1 S2 S3 S4 S5 S6 S7 S8 S9
S1 1.00
S2 0.62 1.00
S3 0.49 0.65 1.00
S4 0.45 0.56 0.53 1.00
S5 0.42 0.48 0.45 0.43 1.00
S6 0.48 0.60 0.54 0.52 0.46 1.00
S7 0.44 0.51 0.47 0.45 0.50 0.50 1.00
S8 0.36 0.39 0.38 0.34 0.35 0.37 0.39 1.00
S9 0.22 0.24 0.24 0.23 0.24 0.24 0.25 0.22 1.00
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lid broadly ovate to round, 2.8 –4.4 × 3.3 –5.0 cm,
larger than mouth, sometimes vaulted or bent towards
the mouth, base cordate 1.0 –4.0 mm, apex occasion-
ally emarginate, indurate, like appendage on the lower
surface, nectar glands numerous, elliptic, 0.16 –
0.26 mm diam. along the midrib, and with crateriform
glands 0.12 –0.18 mm diam. dispersed over the lower
surface of the lid; spur filiform, 0.2 –0.8 cm long, with
branches; green to red on lower part and red on
upper part with red speckled outside, light green to
red with red blotches over the inside, absent in
digestive zone, peristome green to dark red, lid green
to red. Upper pitcher tubulose, with a conspicuous hip
one third from the base; 10.0 –19.0 cm long, 2.0 –
3.4 cm wide; two fringed wings, 0.1 –0.3 cm wide,
along the length of the pitcher, 9.0 –16.0 cm long,
fringe elements 0.3 –0.7 cm long, 3 –6 per cm;
pitcher mouth similar to lower pitcher; peristome
cylindrical, revolute when pitcher mature, 0.4 –1.0 cm
wide, inner edge with teeth 0.29 –0.43 mm long,
ridges approximately 0.4 –0.5 mm apart; lid ovate to
round, 2.1 –3.4 × 2.1 –4.4 cm, base cordate 1.0 –3.0
mm, apex emarginate similar to lower pitcher, elliptic
nectar gland along the midrib on the lower surface of
lid, numerous near base, 0.1 –0.2 mm diam. and with
crateriform glands 0.05 –0.1 mm diam. scattered over
the lower surface of the lid; spur filiform, 3.0 –7.0 mm
long, with branches; pitchers green or green to red,
sometimes red-speckled outside, light green with red
blotches over the inner surface, absent in digestive
zone, peristome and lid light green. Male inflorescence a
raceme, 70.0 –85.0 cm long, peduncle 47.0 –55.0 cm
long, 0.2 –0.5 cm diam., rachis 13.0 –32.0 cm long,
flowers 84 –135, solitary, pedicels 3.0 –5.0 mm long,
with bracts at the base or lower half of pedicel 0.8 –
1.0 mm long, present on all flowers; tepals 4, ovate,
apex obtuse, 4.0 –5.0 × 3.0 –4.0 mm, reflexed in
mature flower, densely covered with orbicular to
elliptic nectar glands 0.20 –0.36 mm diam.;
androphore 2.0 –2.5 mm long, anther head globular
2.1 –2.5 mm diam., anther basifixed, synandrium, 8 –
13 anther sacs, longitudinal dehiscence; tepals light
green when young, red when old. Female inflorescence a
raceme, 50.0 –86.0 cm long, 0.2 –0.4 cm diam.,
peduncle 42.0 –74.0 cm long, rachis 9.0 –16.0 cm
long, flowers 20 –42, solitary, pedicels 0.5 –1.7 cm
long, with bracts 1.0 –1.5 mm long, inserted at one
third of pedicel height, conspicuous on upper part of
rachis, present on all flowers; tepals 4, ovate to elliptic,
3.8 –4.0 × 2.0 –2.2 mm, nectar glands similar to male
inflorescence, 0.20 –0.28 mm diam., green when
young, green to brown when old;. ovary superior, with
four syncarpous carpels, oblong, 5.0 –6.0 mm long,
2.3 –2.5 mm diam., stigma head 1.0 –1.5 mm diam.
Infructescence similar to female inflorescence; tepals
persistent; four valves, 1.0 –1.2 cm long with septicidal
capsule; seeds linear, 6.0 –6.6 mm long c. 98 –125
seeds per pod. Indumentum conspicuous, inflorescence
and outer pitcher pubescent with simple brown hairs;
tendril with simple and branched hairs; wing fringed
Fig. 1. UPGMA-based dendrogram showing genetic relationships among the nine Nepenthes species used in this study. The
dendrogram is based on the genetic distance calculated according to Jaccard’s similarity coefficient with 1000 bootstrap.
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elements puberulent, with branched brown hairs; leaf
midrib puberulent with inconspicuous, simple, dark
green hairs; absent on stem, leaf axils, lamina, leaf
margin, and shoot. Figs 2–4.
RECOGNITION.Nepenthes bracteosa belongs to section
Pyrophytae, a section distributed in the strongly season-
al region of Indochina. It is closely related to
N. krabiensis, but differs in its shorter stem 0.5 –1.0
m (vs 1.5 –2.5 m), spur filiform with branches (vs
filiform with no branches), axillary bud present in 2 –
3 axils in the upper part of plant when flowering (vs a
single axillary bud), fringed wing present in the upper
pitcher (vs absent), occasionally emarginate and
indurate lid apex (vs a blunt apex and not indurate),
solitary flowers both male and female inflorescences
(vs 2-flowered partial peduncles in male inflorescence
and solitary flower in female inflorescence), bracts
present on all flowers of both male and female
inflorescences (vs present at the base or lower half of
the pedicel in male inflorescence but absent in female
inflorescence), short androphore (2.0 –2.5 mm vs 4.0
mm), and 8 –13 anther sacs (vs 15 –22 anther sacs).
DISTRIBUTION. Nakhon Si Thammarat Province, Thai-
land (Map 1).
SPECIMENS EXAMINED.THAILAND. Thung Song Distr.,
Yong Waterfall National Park, Mt Khao Wang Hip,
8°16'06.80"S, 99°40'11.99"E, ♂fl. upper and lower
pitchers, 29 August 2019, Nuanlaong BKF197454
(holotype BKF; isotype PSU).
HABITAT. On summit areas, in sandy soil, generally
growing in open forest and limestone rock crevices,
710 –760 m a.s.l.
CONSERVATION STATUS. This species is distributed at a
single site on the summit of Mt Khao Wang Hip. A
decline in the quality of the habitat will lead to the
disturbance of the long-term survival of the remaining
population and may thus increase the extinction risk
of this species. The calculation of EOO as 0.034 km
2
and AOO as 4.00 km
2
together with the information
above suggests that this species is Critically
Endangered (CR) [B1a,b (iii) + B2a,b (iii)] (IUCN
2019).
PHENOLOGY. Flowering June to December.
ETYMOLOGY. From the Latin “bracteosa”derived from
“bracteosus”: bracts are present on the pedicels of all
flowers of both male and female inflorescences.
VERNACULAR NAME. From the Thai language “Seua–
Nakhonsi”:“Seua”means the red-speckled tiger-like
pattern on the pitcher surface and “Nakhonsi”means
Nepenthes of Nakhon Si Thammarat Province.
NOTES.Nepenthes bracteosa was first discovered from a
photograph taken by a tourist. It is only found at a
single location in Southern Thailand. It belongs to
section Pyrophytae on account of sharing several
characteristics such as long racemose inflorescence,
climbing stem, flower from the rosette stage, tuberous
rootstocks, and pyrophytic habitat. Nepenthes bracteosa is
similar to N. krabiensis, with which it shares being
glossy, climbing, its leaf shape and an inconspicuous
indumentum, but differs in many other morphological
characteristics. However, the results of the phyloge-
netic relationship revealed a similarity coefficient as
0.65 between N. bacteosa and N. krabiensis, which is also
the highest of all species pairs recorded (Table 3).
2. Nepenthes hirtella Nuanlaong & Suran. sp. nov.
Type: Thailand, Krabi Province, Khlong Thom Distr.,
Khao Pra-Bang Khram Wildlife Sanctuary, Mt Khao
Shawaplab, 348 –355 m a.s.l., 7°50'29.53"S,
99°16'35.45"E, ♂fl. lower pitchers, 29 August 2019,
Nuanlaong BKF197453 (holotype BKF!; isotype PSU!).
http://www.ipni.org/urn:lsid:ipni.org:names:77234370-1
Scandent, climbing herb. Root branched, fleshy tubers.
Stem terete, 0.8 –1.2 m long, 0.35 –0.65 cm diam.;
internode 1.5 –5.5 cm long; upper part of the stem
green, lower part of the stem brown. Leaves pseudo-
petioles, coriaceous, oblanceolate; 15.8 –29.5 × 3.5 –
5.4 cm, longitudinally folded to form a V-shape, blade
apex acuminate, base attenuate, entire, dilating at the
node, projecting along the stem as wing, clasping the
stem by three quarters of its circumference, decurrent
for 1.7 –4.3 cm of its length, leaf thickness 0.5 –1.0
mm, inconspicuous longitudinal nerves 3 –4 on each
side of the midrib in outer third of lamina, inconspic-
uous lamina reticulated pinnate venation; midrib
conspicuous; light green in a rosette when young,
green when mature, emerging from a red triangular
lateral bud at the base of the midrib near the leaf
attachment. Lower pitcher c. 14.0 –19.7 × 4.4 –5.9 cm;
ovate at the lower part with digestive zone at the inner
surface, narrowing at the upper part; hip conspicuous
above digestive zone; with two fringed wings, 0.3 –
0.9 cm wide, along the length of the pitcher, 9.0 –
14.3 cm from pitcher mouth to tendril, serrate to
double serrate, fringed elements 0.4 –0.9 cm long, 4 –
11 fringed elements per cm; mouth oblique ovate,
concave; peristome cylindrical, entire to repand,
revolute when pitcher mature, 0.9 –1.5 cm wide,
inner edge with teeth 0.64 –1.48 mm long, ridges
approximately 0.72 –1.2 mm apart; lid ovate, 3.9 –4.9
× 3.3 –4.9 cm, repand, base cordate 0.5 –3.0 mm;
absent appendage, nectar glands numerous, elliptic
0.18 –0.28 mm diam. along the midrib, with
crateriform gland 0.09 –0.28 mm diam. dispersed
over the lower surface of the lid; spur filiform, 0.1 –
0.9 cm long, with branches; tendril straight, 12.0 –
20.0 cm long, 2.0 –3.5 mm diam., approximately 10 –
30% longer than pitcher, mostly on the side of the
pitcher; light green to yellow or green to red on the
lower and green to red or red on the upper part with
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
Fig. 2. Nepenthes bracteosa.Aleaf shape and lower pitcher; Bupper pitcher; Cstem; Dmale inflorescence; Elower surface of lid
with two types of nectar glands and a ridge-like appendage at the lid apex; Fmale flower; Gfemale flower after pollination; H
peristome; Jfiliform spur with branches; Kperistome in transverse section. Drawn from live specimens. DRAWN BY CHAYAN
YODTHAMMARAT.
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
Fig. 3. Nepenthes bracteosa.Alower pitcher; Bupper pitcher and habitat; Cinfructescence; Daxillary bud present in 2 –3 axils
when flowering; E–Gmale inflorescence (immature, young, and old flowers); Hridge-like appendage under lid apex; Jbranched
hairs on fringe elements; Knectar glands on lower surface of lid. PHOTOS: POTJAMARN SURANINPONG & SUNYA NUANLAONG.
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
Fig. 4. Nepenthes bracteosa.Alower pitcher; Bupper pitcher; C–Dhabit and habitat. PHOTOS: POTJAMARN SURANINPONG & SUNYA
NUANLAONG.
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
red blotches on the outside surface, light green
present or absent red blotches on inside surface,
absent in digestive zone, peristome light green, green
to red or red peristome, lid green or green to red.
Upper pitcher tubulose, with a conspicuous hip one
third from the base or middle of pitcher; 13.0 –
21.0 cm long, 0.9 –2.8 cm wide; two fringed wings, 1.5
–8.0 × 0.1 –0.2 cm, which run down the ventral
exterior surface from pitcher mouth to the middle or
tendril of the pitcher, serrate or double serrate,
fringed elements 0.5 –2.0 mm long, 4 –8 per cm;
mouth oblique ovate; peristome cylindrical, revolute
when pitcher mature, 0.3 –1.0 cm wide, inner edge
with teeth 0.48 –0.84 mm long, ridges approximately
0.92 –1.40 mm apart; lid ovate, 2.1 –4.3 × 2.0 –4.3
cm, base cordate 0.5 –1.0 mm, nectar gland similar to
lower pitcher; spur filiform, 2.0 –7.0 mm long; tendril
straight, 7.0 –15.0 cm long, 1.0 –3.0 mm diam.,
coiling, shorter than pitcher; green or green to orange
outside surface with or without red blotches, green
with red blotches over inner surface, absent in
digestive zone, peristome light green sometimes red
stripes present, lid green. Male inflorescence a raceme,
65.0 –105.0 cm long, 0.3 –0.4 cm diam., peduncle
44.0 –56.0 cm long, rachis 22.5 –47.0 cm long, flowers
68 –194, solitary flowers or rarely two-flowered partial
peduncle, pedicels 0.5 –0.8 cm long, bracts present at
base of peduncle 0.5 –3.0 mm long, absent on upper
half; tepals 4, elliptic to obovate, 2.0 –4.0 × 1.0 –2.0
mm, explanate and not reflexed, cruciferous, densely
covered with orbicular to elliptic nectar glands 0.35 –
0.70 mm diam.; androphore 1.0 –2.0 mm long, anther
head globular, 1.0 –2.0 mm diam., anther basifixed,
synandrium, 8 –12 anther sacs, longitudinal dehis-
cence; tepals light green when young, green to red
when old. Female inflorescence a raceme, 65.0 –75.0 cm
long, 0.30 –0.35 mm diam., peduncle 40.0 –58.5 cm
long, rachis 12.0 –20.0 cm long, bracts similar to male
inflorescence; flowers 26 –57, solitary, pedicels 0.2 –
1.0 mm long; tepals 4, elliptic to ovate, 2.0 –3.0 × 1.0 –
2.0 mm, nectar glands similar to male inflorescence,
0.35 –0.60 mm diam.; tepals green when young, green
to red when old; ovary superior, with four syncarpous
carpels, oblong, 3.0 –5.0 mm long, 2.0 –2.5 mm
diam., stigma head 1.5 –2.0 mm diam. Infructescence
similar to female inflorescence; tepals persistent; four
valves, 1.5 –1.6 cm long with septicidal capsule; seed
linear, 4.0 –8.0 mm long c. 73 –109 seeds per pod.
Indumentum conspicuous, simple brown hairs, pubes-
cent, 0.17 –0.30 mm long, present on all vegetative
parts; fringed elements puberulent. Figs 5–7.
RECOGNITION.Nepenthes hirtella belongs to section
Pyrophytae, a section distributed in strongly seasonal
Indochina. It is closely related to N. kerrii which grows
in Tarutao Marine Park, Satun Province, Thailand.
Nepenthes hirtella differs from N. kerrii in having an
oblanceolate leaf shape (vs obovate), tendrils longer
than pitcher approximately 10 –30% (vs longer than
pitcher approximately 40 –50%), tendrils displaced to
the side of lower pitcher (vs tendril centrally placed at
base of lower pitcher), longer lower pitcher (14.0 –
19.7 cm vs 6.0 –14.0 cm), lid ovate (vs round), spur
filiform (vs simple or branched), solitary flowers or
rarely two-flowered partial peduncle (vs solitary
flowers), and indumentum covering all the vegetative
parts (vs covering leaf axil and inflorescence).
DISTRIBUTION. Krabi Province, Thailand (Map 1).
SPECIMENS EXAMINED.THAILAND. Khlong Thom Distr.,
Khao Pra-Bang Khram Wildlife Sanctuary, Mt Khao
Shawaplab, 348 –355 m a.s.l., 7°50'29.53"S,
99°16'35.45"E, ♂fl. lower pitchers, 29 August 2019,
Nuanlaong BKF197453 (holotype BKF!; isotype PSU!).
HABITAT. On summit areas, in sandy soil, generally
growing in open forest, savannas and scrubland, at 348
–355 m a.s.l.
CONSERVATION STATUS.Nepenthes hirtella is found only
on a single site on the summit of Mt Khao Shawaplab.
A decline in the quality of the habitat will lead to the
disturbance of the long-term survival of the remaining
population. It is easy to get to the habitat, which might
put this species at risk of extinction. The calculation of
EOO as 0.053 km
2
, AOO as 8.00 km
2
together with the
information above suggests that this species is
Critically Endangered (CR) [B1a,b (iii) + B2a,b (iii)]
(IUCN 2019).
PHENOLOGY. Flowering June to December.
ETYMOLOGY. From the Latin “hirtella”derived from
“hirtellus”: short hairs are present on all the vegetative
parts of the plant and rather hairy.
VERNACULAR NAME. From the Thai language “Seua-
Amphon”:“Seua”means the red-speckled tiger-like
pattern on the pitcher surface and “Amphon”is the
name of the first collector, Mr Amphon Dumkliang
who found them in 2014 –2015.
NOTES.Nepenthes hirtella was first discovered in 2014 –
2015 by a Nepenthes collector/seller and was known
as “Seua-Amphon”.Nepenthes hirtella is a member of
section Pyrophytae like N. bracteosa and is only found in
Mt Khao Shawaplab of Krabi Province, Thailand.
Nepenthes hirtella is closely related to N. kerrii but differs
in several morphological details, in particular in
tendril and indumentum. Nepenthes hirtella is covered
by brown hairs throughout, while in N. kerrii the
indumentum is confined specifically to the leaf axil
and inflorescence. It is distributed on the mainland
while N. kerrii is distributed on an island. Moreover,
the phylogenetic relationship results show a low
similarity index between them (Table 4).
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
Fig. 5. Nepenthes hirtella.Aupper pitcher; Blower pitcher with leaf shape; Clower surface of lid with two types of nectar glands;
Drear view of pitcher mouth of B;Espur of lower pitcher; Fperistome of lower pitcher in transverse section; Gfemale
inflorescence after pollination; Hmale inflorescence; Jhabit. Drawn from live specimens. DRAWN BY CHAYAN YODTHAMMARAT.
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
Key to Nepenthes species in Thailand
1. Tuberousrootstock................................................................. 2
1. Non–tuberousrootstock .............................................................12
2. Inflorescence racemose with flowers all arranged singly (a 1-flowered) or rarely 2-flowered partial peduncle 3
2. Inflorescence racemose with 2-flowered partial peduncles ………… .............................10
Fig. 6. Nepenthes hirtella.Aupper pitcher and habitat; Bleaves and lower pitcher; Clower pitcher; Dmale inflorescence; Efemale
inflorescence; Fanther head; Gperistome with teeth; Hbranched hairs on fringed element; Jnectar glands on lower surface of lid;
Kstem and leaf axil; Lbrown hairs on lamina and leaf margin. PHOTOS: POTJAMARN SURANINPONG & SUNYA NUANLAONG.
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
Fig. 7. Nepenthes hirtella.Alower pitcher; Bupper pitcher; C–Dhabit and habitat. PHOTOS: POTJAMARN SURANINPONG & SUNYA
NUANLAONG.
KEW BULLETIN
© The Board of Trustees of the Royal Botanic Gardens, Kew, 2022
3. Leaves obovate or oblanceolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Leaves linear-lanceolate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4. Leaves obovate, tendril longer than pitcher approximately 40 –50%; indumentum covering leaf axil and
inflorescence ............ .................................................... N. kerrii
4. Leaves oblanceolate, tendril longer than pitcher approximately 10 –30%; indumentum covering all vegetative
parts...................................................................... N. hirtella
5. Short hairs present on all parts or some part of plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5. Stem and leaves glabrous (pitcher, inflorescence, tendril excluded) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6. Indumentum on all vegetative parts; tendril as long as or shorter than pitcher . . . . . . . . . . . . . . . N. smilesii
6. Indumentum present on some parts of the plant; tendril longer than pitcher. . . . . . . . . . . . . . . . . . . . . . 7
7. Lower pitcher narrowly ovate in the lower third or lower half and tubular above………......….N. kongkandana
7. Lower pitcher ovate in lower half and narrowing above or completely ovate . . . . . . . . . . . . . . . . . . . . . . . 8
8. Lid smaller than mouth, triangular mouth as large as 13to ½ of the lower pitcher length; flower green with red
margins.................................................................. N. suratensis
8. Lid larger than mouth, ovate mouth as large as ¼ of the lower pitcher length; flower red . . . . N. andamana
9. Pseudo-petiolate leaves; lid broadly ovate to round; bract present in all flowers in both male and female
inflorescences.............................................................. N. bracteosa
9. Sub-petiolate leaves; lid orbicular; bract present on partial peduncles . . . . . . . . . . . . . . . . . . . N. kampotiana
10. Hip present at the mid-section or absent in lower pitcher; indumentum covering all aerial parts except upper
surfaceofleaves.............................................................. N. chang
10. Hip present at the lower 13in both lower and upper pitchers; indumentum present on some parts of the
plant ...........................................................................11
11. Indumentum often covering leaf margin and midrib; lower pitcher green to light pink, with dark speckles
outside and uniformly green to dark pink within . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. rosea
11. Indumentum lacking on stem, lamina, leaf margin and shoot; lower pitcher green to red, with red speckles
outside and light green or green with blotches over the inside. . . . . . . . . . . . . . . . . . . . . . . . . N. krabiensis
12. Leaves petiolate; fimbriate leaf margin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
12. Leaves sessile or with a short, poorly defined petiole; entire leaf margin. . . . . . . . . . . . . . . . . . . . . . . . . . 14
13. Lower pitcher ovoid in the basal third, narrowing slightly to form a hip and cylindric above. . . . N. mirabilis
13. Lower pitchers round to oval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. mirabilis var. globosa
14. Producing ground pitchers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
14. Not producing ground pitchers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
15. Lower pitcher urceolate, peristome incurved; inflorescence paniculate . . . . . . . . . . . . . . . . . . N. ampullaria
15. Lower pitcher elongated and smaller, very thin peristome; inflorescence racemose. . . . . . . . . . . . N. gracilis
16. Leaf base amplexicaul, clasping the stem transversely; lid ovate, truncate at base . . . . . . . . . . . N. sanguinea
16. Leaf base decurrent, running longitudinally down the stem as wings; lid elliptic, round at base . . . . N. thai
Acknowledgements
This work was supported by The Plant Genetics
Conservation Project under the Royal Initiation of
Her Royal Highness Princess Maha Chakri Sirindhorn,
Walailak University and Walailak University Fund
(Grant No.WU_IRD61_11). We thank the Yong Wa-
terfall National Park and Khao Pra-Bang Khram
Wildlife Sanctuary for permission to conduct field-
work. We also thank Dr Sarayut Onsanit and Asst. Prof.
Dr Suraphon Thitithanakul, Prince of Songkhla Uni-
versity, Suratthani Campus for supporting the field-
work. Special thanks are given to Mrs Raumporn
Ketsarapong, Walailak Botanic Garden for her valu-
able suggestions and Mr Chayan Yodthammarat for
assistance with the illustrations and photographs.
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