1. INTRODUCTION
Adansonia digitata L. belongs to the
Malvaceae family and is commonly known as the Baobab tree, native to Africa.
The African baobab is a very long-lived tree with multipurpose uses. It is said
that some species are over 1000 years old (Rahul et al., 2015). A.
digitata L. is iconic as it is emblematic, culturally significant, and essential
in traditional medicine in Africa and India (Bellary et al.,
2021). Several studies in different African countries, such as Benin, Burkina
Faso, Malawi, Mali, Nigeria, Tanzania, and South Africa, have highlighted this
deciduous stem-succulent taxon as a priority species for domestication and
enhanced utilization (Gebauer et al., 2016).
The fruit of A. digitata is also used
daily in the diet of rural communities in West Africa (Ibrahima et al., 2013).
In the Northern part of Nigeria, the leaves are relished for a local soup
called “miyan kuka”. The species contributes to rural incomes (Kamatou et al.,
2011) and has various important medicinal and food uses (Kaboré, 2011).
Traditionally, the pulp is consumed in different forms. It is also used in the
formulation and preparation of cereals and beverages. It is reported that
baobab pulps have many nutrients, including Vitamin C, riboflavin, niacin,
pectin and citric, malic, and succinic acids, while the oil also contains
Vitamins A, D, and E (Donkor et al., 2014). According to Silva et al.
(2023), ethnopharmacological uses of various plant parts of A. digitata
have been reported for hydration, antipyretic, antiparasitic, antitussive, and
sudorific properties and in the treatment of diarrhoea and dysentery in many
African countries. A. digitata has been reported to exert hypoglycemic,
hypolipidemic, antimicrobial, analgesic, anti-inflammatory, antioxidant and
antipyretic properties (Braca et al., 2018). Biochemical studies also
showed that the pulp of A. digitata is rich in dietary fibres and
carbohydrates (Eltahir & Elsayed, 2019).
Despite its wide
distribution and importance, the taxonomy and conservation of A. digitata
remain challenging due to high morphological variability, broad ecological
adaptability, and potential hybridization with other Adansonia species (Muthai et al.,
2017; Jansen et al., 2020). Traditional morphological approaches
often fall short in accurately distinguishing A. digitata from its
congeners, especially in regions where multiple species may coexist or where
phenotypic traits are influenced by environmental conditions. This taxonomic
uncertainty hinders conservation planning and the sustainable management of
genetic resources.
Molecular identification
methods, particularly DNA barcoding, offer a robust alternative for resolving
species boundaries and understanding genetic diversity within A. digitata.
DNA barcoding is a species identification tool that utilizes a short segment of
the genome, which provides a genetic signature or fingerprint of the species
(Abdulkareem et al., 2024). The main advantage of this novel technique
is that it requires a small sample of tissue (Trujillo-Argueta et al.,
2022). Single-locus barcoding approaches, however, have shown limited
resolution in species with complex evolutionary histories or low interspecific
divergence (Karabanov
et al., 2023). To overcome these limitations, a multi-loci DNA barcoding
strategy is increasingly recommended. gene regions such as nuclear ITS and
chloroplast markers rbcL, rpoC1, and psbA-trnH could be
adopted to assess their utility in accurately identifying taxa. Each of
these loci offers different levels of variability and phylogenetic resolution:
while rbcL and rpoC1 are highly conserved and functional for
higher-level taxa, ITS and psbA-trnH provide greater discriminatory
power at the species level due to their higher mutation rates and sequence
variability (Letsiou et al., 2024).
The ITS region, located in the
nuclear genome, evolves quickly and is highly variable, making it effective for
distinguishing between closely related species. However, it can sometimes be
difficult to amplify and may show variation within a single species. The
chloroplast gene rbcL is widely used in plant barcoding due to its ease
of amplification and alignment (Abdulkareem et al., 2024), though it is
relatively conserved and may not separate closely related species well. The rpoC1
region, another coding gene in the chloroplast genome, offers moderate
variability and is also considered reliable for general plant identification
(Abdulkareem et al., 2023). The psbA-trnH intergenic spacer, a
non-coding region, tends to show higher levels of variation and has been useful
in distinguishing species in several plant groups (Anakha & Hari, 2022). By
combining these nuclear and chloroplast regions, a multi-locus barcoding
approach increases the accuracy of species identification and helps overcome
the limitations of using a single DNA marker.
The proper identification and evaluation of biologically
relevant plant taxa play a crucial role in understanding phylogeny of many
important plant’s species; these methods enable researchers to find similarity
and differences among different plant families. Several genes that are applied
or used for DNA barcode studies include rbcL, matK, trnH-psbA and ITS
separately or in combination. The availability of the sequences of barcoding
genes in the databases is expected to rapidly increase thereby increasing their
utilization in the identification of plant species subsequently. Therefore,
establishing a local barcode database will be valuable for a broad range of potential
ecological applications, including the building of community phylogenies, as
opined by (Gostel & Kress, 2022).
Despite the potential of DNA
barcoding, several barriers limit its application in the identification and
conservation of A. digitata. These include inadequate representation in
reference barcode databases, molecular infrastructure, and technical capacity in
many countries. Addressing these challenges is essential for the development of
integrative conservation strategies. Although baobabs are widely known, the readily
available genetic information on the species, such as DNA barcodes of the
species in Africa and particularly Nigeria, is inadequate. Thus, studies on DNA
barcoding, especially using the multi-loci approach and investigating the
phylogeny of A. digitata, are necessary.
2.
MATERIALS
AND METHODS
Collection of
Plant Samples:
Fresh leaf
samples of Adansonia digitata L. were collected in the early morning at
approximately 6:40 AM from a mature tree located within the University of
Ilorin campus, Ilorin, Kwara State, Nigeria (Latitude: 8.4799° N, Longitude:
4.5418° E). The sampling site was selected due to its accessibility, secure
environment for repeated access if necessary, and the confirmed presence of
morphologically typical A. digitata specimens, which represent the
species within the region.
The plant
species was identified based on morphological characteristics in accordance
with the Flora of West Tropical Africa and verified by a plant
taxonomist at the Department of Plant Biology, University of Ilorin. A voucher
specimen (Voucher No: UIH/PLB/AD003) was prepared and deposited in the
University of Ilorin Herbarium for future reference. The collected leaves were
immediately placed in sterile zip-lock bags.
Genomic DNA
Extraction:
One gram of fresh leaf tissue of Adansonia digitata was frozen with
liquid nitrogen and ground into a fine powder. Total genomic DNA was isolated
from the sample using the DNeasy Plant Mini Kit (Qiagen, USA). The DNA samples
were stored at -40 °C prior to PCR analysis.
PCR
Amplification and Sequencing Protocols:
PCR amplification was performed with the barcode markers
as shown in Table 1. Primers used were synthesized by Inqaba Biotec. South
Africa. The PCR was carried out with a total reaction volume of 30µl in a
thermocycler (Eppendorf, Germany) containing the following components: 23.0
μL of Milli-Q water, 1.0 μL of template DNA, 3.0 μL of 10× Taq
buffer containing MgCl₂, 1.0 μL of 5 pmol/μL forward and
reverse primers, 1.0 μL of 2.5 mM dNTPs, and 1.0 μL of Taq DNA
polymerase (1 U/μL).
The PCR setup for barcode amplification and cycle sequencing reaction PCR
amplification was performed in a thermal cycler under the following conditions:
an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of
denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and
extension at 72°C for 1 minute. A final extension was performed at 72°C for 5
minutes to complete the reaction.
The cycle sequencing reactions were conducted using purified PCR products and
performed in a 30-cycle protocol with the following thermal profile: an initial
denaturation at 96°C for 1 minute, followed by 30 cycles of denaturation at
96°C for 10 seconds, annealing at 50°C for 5 seconds, and extension at 60°C for
4 minutes. A final extension step was carried out at 60°C for 7 minutes to
ensure complete product formation. The product was then purified.
The various alleles were sequenced using the 3130xl Genetic Analyser (Applied
Biosystems, CA, USA). The editing of the sequences obtained was manually
carried out using Sequence Scanner software v1.0 Applied Biosystems, CA, USA),
and the full-length sequences were assembled using a local alignment algorithm,
Codon Code Aligner version 4.24 (Codon Code Corporation).
4. DISCUSSION
DNA barcoding is a molecular technique for species identification is relatively
simpler and more accurate than most conventional methods of species
identification. DNA barcoding utilizes one or more standardized short DNA
regions for taxon identification (Antil et al., 2023). Due to the
limitations of conventional methods, molecular techniques are used to
investigate the problems related to the identification and classification of
species (Sarwar et al., 2019). This study proved that the molecular
method of species identification should be recommended as it gives more
accurate results in species identification and even explaining the evolution of
species.
The maximum likelihood phylogenetic analyses revealed a strong correlation
between the consensus; sample UIL Adansonia digitata 1B and several Adansonia
species including Adansonia kilima, Adansonia rubrostipa, Adansonia
gregorii, Adansonia madagascariensis and a particularity stronger
affinity to Adansonia digitata underscoring an effective species
identification ability of the primers used. The maximum likelihood is the most
used inference technique, especially for estimating evolutionary relatedness
between isolates, possibly owing to its computational efficiency and robustness
while theoretically relatively simple (Dhar & Minin, 2016).
The multi-loci approach of using more than a single primer gave more insight
into species identification. In the work of Larrain et al. (2019),
multi-locus strategy outperformed the mono-locus methods for the molecular
identification of the Mytilus taxa. Also, in a study by Promputtha et
al. (2005), it was suggested that some degree of inaccuracy was associated
with using the ITS region alone for the identification of species and thus,
recommends using multiple primers for effective species identification.
adopting a multi-loci approach, the consensus; Sample UIL Adansonia digitata
1B clustered with Adansonia digitata in all but one of the four
phylogenetic trees constructed using sequences obtained from ITS, rbcL, psbA-trnH
and rpoC1 markers, consistency of the clustering of consensus and A.
digitata species in all the phylogeny except that of rpoC1 cannot be
coincidental.
Considering the BLAST (Basic Local Alignment Search tool) analyses conducted on
the NCBI database, the consensus; sample UIL Adansonia digitata 1B
scored strong similarity with Adansonia digitata in all primers used for
the analyses except rpoC1 primer this further corroborates the efficacy
of the primers in species level identification of plants.
The consensus sample; UIL Adansonia digitata 1B obtained from the psbA-trnH
primer, showed a tremendous 100% similarity with Adansonia digitata isolate
259 of accession number JN400287 obtained from the work of Pettigrew et al.
(2012), who claimed the sample was obtained from west Africa. This study
further narrows down the origin of the species as our results suggests that the
sample might have been have obtained or similar to the species available in
Nigeria. It also showed a 99.55% percentage identity with another isolate of Adansonia
digitata. High percentage identity was also recorded with other species
from the genus Adansonia notably Adansonia rubrostipa with 98.83%
identity, Adansonia gregorii with 98.20% identity, and Adansonia
kilima with 99.55% percentage identity. It is noteworthy that these species
did not persist in other primers except Adansonia kilima which showed up
with the ITS primer.
Inferences
from the DNA barcodes obtained from the Internal Transcribed Spacer (ITS)
primer showed similarity with several samples of Adansonia digitata with
an approximate percentage identity of 98% this result is indicative of the
efficacy of the primer and this point to the fact that the DNA barcodes could
be relevant for subsequent species identification. It also showed similarity
with Adansonia kilima with approximately 97% identity. This is
corroborated with the findings of Pettigrew et al. (2012) where it was
reported that the ITS phylogeny demonstrated a
genetic similarity between A. digitata and A. Kilima. Also, the
variation in floral and pollen characters and chromosome
number was examined in specimens from Africa and identified a new diploid
baobab species, it was found that Adansonia kilima sp., co-existed with Adansonia
digitata in Africa. Adansonia kilima is superficially similar to A.
digitata but can be differentiated on the basis of floral morphology,
pollen, and chromosome number (Pettigrew et al., 2012). From the
phylogeny tree, the bootstrap value of the clade containing the consensus;
sample UIL Adansonia digitata 1B which clustered with Adansonia digitata
has a value of 88%. Although, it has been pointed out that ITS primer only
cannot be completely effective for species and genus identification, So, it can
be said that from this study that ITS primer is the best primer for DNA
barcoding of Adansonia digitata on the species level.
The BLAST analysis with the A. digitata DNA
barcode from the rpoC1 primer was rather noteworthy as there was no
similar sequence of the species on the NCBI database, and our submission was
the first on the database for the rpoC1 region. This result can be
attributed to the observation that no or not many DNA barcoding works have been
done on Adansonia digitata using the rpoC1 primer. However, rpoC1
primers have been used for effective DNA barcoding of plant species such as Medicago
sativa by El-Sherif and Ibrahim (2020) and Chen et al. (2022), who
reported that rpoC1 showed excellent power in identifying 18
of 21 Fritillaria species except three closely related species,
including Fritillaria cirrhosa, F. dajinensis,
and F. omeiensis. Abdulkareem et al. (2023) indicated that rpoC1
produced higher identification to species level in identification of Vernonia
amygdalina. However, in this study, rpoC1 was able to identify members in the same family as A.
digitata including Reevesia spp, Theobroma spp, Ochroma spp,
Ceiba spp., Tilia spp.
The psbA-trnH primer showed to be a very good and efficient primer for
the barcoding of Adansonia digitata. The DNA barcode BLAST gave a result
of 100% species percentage identity with the sample. In the phylogenetic tree
constructed with the psbA-trnH sequences, the consensus sample clustered
with Adansonia digitata with a bootstrap value of 57%, a value that is
higher than the bootstrap value from the phylogenetic tree of rbcL
sequences of 39%. The relatively higher bootstrap support value however
supports the results from other primers.
One notable outcome was the variation in bootstrap support across different
gene regions. For instance, phylogenetic trees constructed using rbcL
showed relatively low bootstrap values (e.g., 39%), reflecting limited
phylogenetic resolution. Low bootstrap values indicate weak statistical support
for particular clades and may result from several factors, including short
sequence lengths, low variability within the marker, or high conservation among
closely related species. While these values do not invalidate the clustering
observed, they highlight the importance of combining multiple loci to improve
confidence in species delineation.
Bolson et al. (2015) reported ITS and trnH-psbA as efficient DNA
barcodes to identify threatened commercial woody angiosperms from Southern
Brazilian Atlantic rainforests. Loera-Sánchez et al., (2020)
opined trnH-psbA was
efficient and promising for identification of forage legumes and grasses while Hassan (2023) reported DNA Barcode of trnH-psbA is a promising candidate gene for
efficient identification of bitter and sweet almond and related species.
Concatenation all common sequences in the 3 primers; ITS, rbcL and psbA-trnH
that positively associated the consensus with A. digitata in separate
phylogenetic analysis showed that the consensus; sample UIL Adansonia
digitata 1B again clustered together with Adansonia digitata isolate
259. This gives a great level of confidence that the isolated DNA sequence can
be identified as Adansonia digitata. Thereby successfully indicating
that the sequences obtained in this study are representative DNA barcodes of A.
digitata. It is noteworthy that the inclusion of the rpoC1 markers
in the concatenation analysis yielded divergent results. While rpoC1 did
not produce BLAST hits for A. digitata possibly due to the absence of A.
digitata rpoC1 sequences in public databases. its inclusion in the
concatenated dataset significantly improved the robustness of the phylogenetic
tree, with increased bootstrap support and clearer clade formation. This occurence
could be explained by the conserved yet phylogenetically informative nature of
the rpoC1 gene. The lack of matching sequences in BLAST does not reflect
poor marker performance but rather highlights the underrepresentation of rpoC1
sequences for A. digitata in GenBank. In fact, this study contributes
the first publicly available rpoC1 sequence for A. digitata,
representing a novel resource for future phylogenetic studies. Previous studies
(El-Sherif & Ibrahim, 2020; Chen et al., 2022; Abdulkareem et al.,
2023) have reported the efficacy of rpoC1 in resolving plant taxa,
suggesting its value in multi-locus barcoding approaches, especially when
paired with more variable regions. The consistency of the results across
independent markers and phylogenetic reconstructions indicates that the
sequences generated in this study are reliable DNA barcodes for Adansonia
digitata, contributing to its growing molecular resources and providing a
foundation for future taxonomic and conservation studies.
CONCLUSION
Molecular identification is no doubt emphasized to be a more promising
technique for species barcoding and identification. From the study, it can be
concluded that ITS primer is a recommendable primer for the barcoding of Adansonia
digitata., however psbA-trnH and rbcL are credible primers
for Adansonia digitata identification. DNA barcoding of Adansonia
digitata for easier identification and classification is necessary. This
allows for insight into the related species, provides more information about
the species in the gene bank database and ultimately curbs misidentification
and interchangement of the species for another.
Acknowledgements:
The authors acknowledge the Molecular Laboratory Unit, Department
of Plant Biology, for providing technical support throughout this study.
Declaration:
All authors declare that the work presented in this manuscript is
original, has not been published elsewhere, and is not currently under
consideration for publication by any other journal.
Consent for
Publication:
All authors have read and approved the final version of the
manuscript and have given their full consent for its publication.
Competing
Interests:
The authors declare that there are no competing interests related
to this work.
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