Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 August 2022 | 14(8): 21696–21703
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
https://doi.org/10.11609/jott.7454.14.8.21696-21703
#7454 | Received 28 May 2021 | Final received
08 June 2022 | Finally accepted 20 July 2022
Morphological
assessment and partial genome sequencing inferred from matK
and rbcL genes of the plant Tacca
chantrieri
P.C. Lalbiaknii 1 , F. Lalnunmawia
2, Vanlalhruaii Ralte
3, P.C. Vanlalnunpuia 4, Elizabeth Vanlalruati Ngamlai 5 & Joney Lalnunpuii Pachuau 6
1,2,5 Department of Botany,
Mizoram University (Central University), Aizawl, Mizoram 796001, India.
3 Department of Botany,
Pachhunga University College, Mizoram University,
Aizawl, Mizoram 796007, India.
4 Department of
Environmental Science, Mizoram University (Central University), Aizawl, Mizoram
796004, India.
6 Department of Botany,
Government Champhai College, Champhai
796321, India.
1 biakniipachuau1@gmail.com,
2 fmawia@rediffmail.com (corresponding author), 3 apuii_r@yahoo.com,
4 nunpuia0615@gmail.com,
5 elizabethruati19@gmail.com, 6 joney.lnpi@gmail.com
Editor: Mandar Paingankar,
Government Science College Gadchiroli, Maharashtra,
India. Date
of publication: 26 August 2022 (online & print)
Citation: Lalbiaknii, P.C., F. Lalnunmawia, V. Ralte, P.C. Vanlalnunpuia, E.V. Ngamlai &
J.L. Pachuau (2022). Morphological
assessment and partial genome sequencing inferred from matK
and rbcL genes of the plant Tacca
chantrieri. Journal of Threatened
Taxa 14(8): 21696–21703. https://doi.org/10.11609/jott.7454.14.8.21696-21703
Copyright: © Lalbiaknii et al. 2022. Creative Commons
Attribution 4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: None.
Competing interests: The authors
declare no competing interests.
Author details: P.C. Lalbiaknii completed her master
degree from Mizoram University, Aizawl and currently pursues her research works
in the field of forest ecology, rare, endangered and threatened plant species.
Also, on phenology and phytochemical analysis of selected ethnomedicinal plants. Dr. F. Lalnunmawia Professor (corresponding author) from
Department of Botany, Mizoram University, Aizawl who teaches in the field of
ecology, agroforestry and sustainable landuse
system. Dr. Vanlalhruaii Ralte
is an Associate Professor from Department of Botany, Pachhunga
University College, Aizawl who teaches the field of ecology. P.C. Vanlalnunpuia
conducted his research on biodiversity of tree species and the anthropogenic
influence. Elizabeth Vanlalruati Ngamlai
conducted her research in diuretic effect of Hedyotis
scandens in Albino rats. Joney Lalnunpuii Pachuau is an Associate Professor from Department of
Botany, Government Champhai College, Champhai Mizoram who teaches molecular biology.
Author contributons: PCL and EVN conducted field studies, collected
data, and wrote the manuscript. FL, VR, and JLP all gave valuable inputs. PCV analysed the data.
Acknowledgements: The authors are
thankful to Institutional Advanced-level Biotech-Hub, Department of Zoology, Pachhunga University College, Aizawl, Mizoram and Mr. Zohmangaiha for their laboratory assistance.
Abstract: Tacca chantrieri is a monotypic
perennial plant belonging to the family Taccaceae. It
is listed as an endangered species by different authors. The plant was found in
Thorangtlang Wildlife Sanctuary, a protected area in Lunglei District, Mizoram. Although there is a record of
its existence from the forests of Mizoram, there are no detailed studies based
on morphology, partial or whole genome sequencing. Plant samples collected from
Thorangtlang Wildlife Sanctuary were used for
morphological assessment and partial genome sequencing of matK
and rbcL genes. This study provides
information useful in making conservation decisions.
Keywords: Black Bat Flower,
Endangered, genetics, genomics, herb, morphology, northeastern India.
INTRODUCTION
Tacca chantrieri
Andre, or Black Bat Flower, belongs to the family Taccaceae (Fu & Jin 1992).
The bractea of this particular species is very
similar to that of bats, hence the common name Bat Flower. In local language,
Mizo, it is called ‘Thialkhasuak’. It is a perennial
herb with underground rhizomes distributed mainly in tropical regions of Asia (Drenth 1972, 1976; Ding & Larsen 2000). The family Taccaceae tends to be divergent in the number of genera and
species. According to Linn & Kuntz 2010, it is represented by two genera
and about 13 species. Taccaceae comprises 10 species
of pan-tropical distribution (Zhang & Li 2008) and comprised of one genus
and 11 species (Ding et al. 2000). They are commonly found in the forest understorey, and a majority species are rare in the wild.
Black Bat Flower has
a unique shape which mimics bats, with broad wings and numerous long dangling
filaments with rich maroon black or deep purple color (Charoensub
et al. 2008). It exhibits a low germination rate and can survive only under
specific environmental conditions. As a result of its rapidly disappearing
natural habitats and low germination rate, the species has become an endangered
plant (Fu & Jin 1992).
Globally there are 10
species representing this genus, with nine confined to the Indo-Malaysian
region. Beyond this region, only two species are found; an inclusive species: T.
leontopetaloides distributed mainly from the
Indo-Malaysian region to tropical Africa and the other species T. parkeri, the only native to South America. There are
five species presently occurring in Malaysia, viz., T. leontopetaloides,
T. integrifolia, T. palmata,
T. chantrieri, and T. bibracteata.
In both peninsular and eastern Malaysia T. leontopetaloides,
T. integrifolia, and T. palmata
are found whereas T. chantrieri is found
only in the northern parts of peninsular Malaysia while T. bibracteata , a very rare plant is only found in
Sarawak (Saw 1993).
Tacca chantrieri was first reported
from Assam in 2015 as a new record from India (Baruah et al. 2015).
Morphologically, Tacca chantrieri
resembles Tacca khanhhoaensis
which is assessed as Critically Endangered (CR) on the IUCN Red List Categories
(IUCN 2012). To date, a study on reproductive biological observation of Tacca is still lacking (Faegri
& van der Pijl 1971; Drenth
1972; Saw 1993). Mizoram is situated in the northeastern part of India along
with its sister states of Manipur, Nagaland, Tripura, Arunachal Pradesh, Assam,
and Meghalaya. It is abundantly furnished with dense forests and diverse
species of flora and fauna but many areas of several regions are unexplored.
Although a preliminary record of the plant’s existence is recorded, there are
no detailed studies based on its morphology, anatomy, and partial genomic
sequencing. Due to exploitation and destruction of forests, the habitat of this
species has diminished. Tacca chantrieri exhibits improvident floral arrangement and
a high reproductive structure investment, which leads to highly suitable of it
for out crossing thus possessing sapromyophilous
(pollination by flies where the flower mimic rotting meat) syndrome of
pollination (Drenth 1972; Saw 1993).
DNA bar coding based
techniques such as DNA sequencing are the most relevant and innovative
techniques which can analyze the genetic linkage and evolution of plants and
species identification. CBOL (Consortium for the Barcode of Life) plant-working
researchers suggest that rbcL and matK
(the 2-locus) combination is the standard plant barcode based on the sequence
attribute or trait, levels of species differentiation, and evaluation of
resiliency. A brief reflection of Maturase K Gene in plant DNA barcoding and
phylogenetics (Kar et al. 2015).
MATERIALS AND METHODS
The plant sample was
collected from Thorangtlang Wildlife Sanctuary at an
elevation of 500–550 m, where necessary investigation of Tacca
chantrieri was done by field observation and
measurement of observable morphological features and the state of its
efflorescence within the natural habitat. The research analysis was conducted
between September 2017 and December 2020. Tacca
chantrieri prefers moist, shaded brushwood
habitats (Image 1A,B). Plants are 2–4 feet tall with rhizomes imperfectly
cylindrical, leaves are oblong or elliptic shape with caudate apex and
attenuate base in various sizes and are green in color. Petiole slightly dark
brown to black. Our study reveals that T. chantrieri
bears inflorescence from late April to September and by October to November
berries are ripened. T.S. and L.S. of both stems and leaves were observed under
fluorescence microscope. The exposure of the anatomical studies for exceedingly
large organs or tissues require to be dissected into tiny segments for
microscopic observations. Section cutting or sectioning is the most stereotypic
technique of studying microscopic anatomy or histology of large specimens (Karuppaiyan & Nandini 2016). Sections were stained
using the double staining method, a technique involving a mixture of two
contrasting dyes (safranin and methylene blue). These procedures can be used on
paraffin, paraplast, or historesin-embedded
free hand and microtome sections. A section of young leaves was preserved in
70% alcohol which needs to undergo further partial genome sequencing process.
Partial DNA sequencing was inferred from matK and rbcL genes. The length of DNA bands acquired from matK and rbcL genes are 635 and
675 respectively and are deposited in GenBank, NCBI with accession no MW289205
(matK) and MW289206 (rbcL).
DNA extraction, PCR
amplification and sequencing
DNA isolation of the
specimen was obtained from leaves and stems of Tacca
chantrieri following the protocol recommended by
White et al. (1990). For polymerase chain reaction (PCR) analysis, each DNA
sample was diluted to the appropriate concentrations. A total reaction volume
of 25 μl consisted of 12.5 μl
Tag Master Mix (Takara), 9.5 μl of nucleus free
water, 1 μl each of primers, and 1 μl of DNA sample. Maturase-K region was amplified using:
Forward primer matK390F: 5’-CGATCTATTCATTCAATATTTC-3’ and Reverse primer matK1326R:
5’-TCTAGCACACGAAAGTCGAAGT-3’ with the following parameters; initial
denaturation at 94 oC for 3 min, 35 cycles
of 94 oC for 30 sec, 50 oC for 30 sec, and 72 oC
for 45 sec, followed by the final elongation step at 72 oC
for 7 min. RbcL region was amplified using: forward
primer rbcL 1F: 5’-ATGTCACCACAAACAGAAAC-3’ and
reverse primer rbcL 724R: 5’-TCGCATGTACCCTGCAGTAGC-3’
with the following parameters; initial denaturation at 95 oC
for 4 min, followed by 35 cycles of 94 oC
for 30 sec, 55 oC for 1 min, 72 oC for 1 min, followed by the final elongation
step at 72 oC for 7 min (Bafeel et al. 2012).
The PCR products were
electrophoresed on 0.8% (w/v) agarose gel in 1.0 x TAE buffer [containing 1 μl Safe DNA gel stain (Invitrogen, Thermo
Fisher Scientific) per 20 ml of 10 gel] at 150 V for 20 minutes. The amplified
PCR products were sequenced by Sanger’s dideoxy method (Sanger et al. 1997) on
ABI 3730XL automated sequencer (AgriGenome Labs Pvt.
Ltd., Smart City Kochi, Kerala, India). Consensus sequences for contigs were
trimmed and aligned using Bioedit sequence alignment
editor (Hall 1999). Sequences were then compared to those in GenBank database
using the BLASTn (Altschul
et al. 1990) search tool for similarities. DNA sequence of matK
and rbcL data of the studied species have been
submitted to GenBank. The sequences were then aligned with Clustal
W (Larkin et al. 2007) and the phylogenic tree was established using maximum
likelihood in MEGAX. The bootstrap consensus tree inferred from 1,000
replicates was taken to represent the evolutionary history of the taxa
analyzed. Branches corresponding to partitions reproduced in less than 50%
bootstrap replicates were collapsed. The percentage of replicate trees in which
the associated taxa clustered together in the bootstrap test (1,000 replicates)
is shown next to the branches. Initial tree(s) for the heuristic search were
obtained automatically by applying Neighhbor-Join and
BioNJ algorithms to a matrix of pairwise distances
estimated using the Tamura-Nei model, and then
selecting the topology with superior log likelihood value. This analysis
involved 12 nucleotide sequences.
Study Area
Thorangtlang Wildlife Sanctuary
is situated about 245 km south of Aizawl, the state capital of Mizoram between
23.28°–23.19° North & 92.50°–92.62° East and 1,396 m at highest altitude
falling in Lunglei District (Fig 1). The Sanctuary
lies close to the Indo-Bangladesh border. It possesses both evergreen and
semi-evergreen forests and its richness in wildlife is the most distinctive
feature compared to other wildlife sanctuaries in the forests of Mizoram. Disastrous practices of events like shifting
cultivation and hunting from nine fringing villages leads to biotic pressure on
flora and fauna.
RESULTS
Morphological and
anatomical observations
Morphological
evaluation was conducted primarily in its natural habitat. The morphological
patterns of Tacca chantrieri
plant was investigated intensively from September 2017 to December 2020.
According to our observations, Tacca bears
inflorescence from late April to September, and berries ripen from October to
November. Plants are 2–4 feet
tall, rhizomes imperfectly cylindrical, leaves oblong or elliptic shape having
arcuate, reticulate, palmate, camptodromous and brochidodromous venation which measure 35–50cm x 14–20cm
(Image 1E–G) and are green in color. Petiole 45–60 cm by 3–6 mm slightly dark
brown to black (Image 1C). Inflorescence 2, up to 20–30 flowers comprising of involucral bracts (Image 1D).
Figure 2 depicts the
schematic diagram of Tacca chantrieri inflorescence bearing numerous flowers along
with its trailing-like filaments and leaves which resemble bats consequently
giving the plant the common name Black Bat Flower. The inflorescence
arrangements exhibit numerous flower stalks which spread from a common point,
thus referred to as cymose umbellate inflorescence
demarcated by the dark colored bracts and also consist of long trailing
filamentous bracteoles. The flowers are nearly black, deep maroon or purple-red
in color. The number of inflorescence per plant was two and in each of the two
inflorescences 20–30 florets with around 25–30 long trailing like filaments
were present. The inflorescence lasted for two to three weeks. The root of the
plant is extensive and rhizomatous which is imperfectly cylindrical.
T.S. of the stem
shows conductive collateral vascular bundles arranged in circular motion in
which xylem protrudes towards the inner side and phloem projects outwards (Image 1K,L). The inner
core mainly consists of the ground tissue. L.S. of stems of Tacca
shows sieve tubes and sieve plate (Image 1I) T.S. of leaf shows a single layer
of upper cuticle followed by epidermis which is transparent. Next to the
epidermis are tightly packed rod-shaped cells known as mesophyll cells. Beneath
the mesophyll cells, loosely bound spongy mesophyll cells are present. Stomatal
pores (tiny pores) are present in some regions (Image 1J). The stomata present
are anomocytic (Image 1M).
Nucleotide analysis
and Phylogeny
To construct
phylogeny of major lineages, representative taxa of members from the major
species were chosen. Table 1 comprises all the taxa analyzed herein and their
accession numbers.
The matK sequences of our specimen (MW289205) had 3
nucleotide differences with zero gap, from the two species of Tacca chantieri
(JQ733736 and MH748926). The rbcL sequences
(MW289206) of our specimen had 13 nucleotide differences with zero gap,
from the species of Tacca chantieri (KX171420 and JN850578).
The evolutionary
history was inferred using the maximum likelihood method and Tamura-Nei model base on the matK region
(Figure 3). The final positioning for the merged sequences for the two regions
(matK and rbcL) comprised
of 897 base pairs.
In the phylogenetic
tree (Figure 3), as expected, a close relationship between the specimens
examined (MW289205 Voucher BMZU) and the two species of Tacca
chantieri (JQ733736 and MH748926) was observed.
The two species of Tacca chantieri (along with the specimen examined MW289205),
form a distinct clade with a high support bootstrap value of 96 (Figure 3).
Assessments of the two selected loci culminated in a well-supported
phylogenetic tree. T. leontopetaloides and T.
maculata formed the sister clade to all other Tacca species. T. palmata, T. plantaginea and T. bracteata
form a clade with low support values (Figure 3). Section Tacca
has been well supported based on the phylogeny shown by Zhang et al. (2001).
This section is distinguished by its geophytic behavior, perennial leaves with
decompound foliar blades, a long ascending peduncle, substantially more
inflorescences, more than two inner segments, many threadlike floral
bracteoles, and a low number of ovules per fruit. According to Tanaka (1954)
and Li & Li (1997), the contemporary genetic diversity dispersal patterns
of Tacca chantrieri
populations are believed to be the result of a hypothetical evolutionary event
involving vicariance from a single common ancestor and fragmentation of the
species’ historic geographic range. Genetic drift affects the genetic structure
and increases differentiation among populations when populations are small and
geographically and genetically distant from one another (Barrett & Kohn
1991; Ellstrand & Elam 1993). This highlights a
shortage of gene flow between groups, which may be inadequate to combat genetic
drift. Both morphological and phylogenetic analysis confirm that the specimen analysed (MW289205 Voucher BMZU) is identical to Tacca chantrieri.
DISCUSSION
The species T. chantrieri, though not included in the IUCN Red List,
is still described by many authors as an endangered species as they are rare
even in their wild habitats. T. chantrieri
consists of several dark colored or maroon inflorescence with bracts and
whisker like filiforms that makes it captivating.
According to Zhang et al. (2005), it is a shade loving plant in its own natural
habitat and florets are primarily self-pollinated and have several
characteristics that encourage autonomous self-pollination. A potential
explanation for its unusual inflorescence structure is that it aids in
photosynthesis in the shady understory while also protecting the plant from
herbivores. And due to the changes in the climatic conditions and landscape
morphology of its native habitats it can be considered a rare, endangered or
threatened species. The plant is very difficult to grow in an artificial or
controlled environment, requiring specific temperature, moisture, and shade,
and can take up to 11 to 12 months to germinate when cultivated by
agriculturists. Hence, there is a significantly larger potential for it to be
developed as ornamental plants so as to conserve it from extinction. Apart from
the species detailed, there might be many more species that are yet to be
discovered in the unexplored terrains. So, it is imperative that we protect and
conserve whatever species have been found regardless of their abundance and
scarcity. Considering that habitat loss and overharvesting have been the
primary cause of species endangerment, a central component of species recovery
has been to establish a network of conservation areas and reserves that
represent all the pertinent terrestrial and riparian natural communities.
Species delineation provided by DNA-based techniques would provide important
insights into the evolutionary biology and species diversity, but their
versatility is limited in the apparent lack of multigene phylogenetic analysis.
Future research in phylogenetic analysis will be critical in determining
relevant perception to organise and better understand
the basic similarities and differences between organisms, as well as other
emergent properties of early life.
Table 1. matK sequences.
|
Name of species |
Accession Number |
|
Tacca chantrieri |
JQ733736 |
|
Tacca chantrieri |
MH748936 |
|
Tacca reducta |
MK153216 |
|
Tacca reducta |
MK153205 |
|
Tacca palmata |
MK153192 |
|
Tacca palmate |
MK153200 |
|
Tacca bibracteata |
MK153225 |
|
Trichopus sempervirens |
KP083035 |
|
Tacca plantaginea |
AY973842 |
|
Tacca maculate |
MK153197 |
|
Tacca leontopetaloides |
MK153196 |
|
Tacca leontopetaloides |
MK153193 |
|
Tacca sumatrana |
MK153224 |
|
Tacca havilandii |
MK153210 |
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