Mitochondrial genomes of African pangolins and insights into evolutionary patterns and phylogeny of the family Manidae
© The Author(s). 2017
Received: 22 February 2017
Accepted: 14 September 2017
Published: 21 September 2017
This study used next generation sequencing to generate the mitogenomes of four African pangolin species; Temminck’s ground pangolin (Smutsia temminckii), giant ground pangolin (S. gigantea), white-bellied pangolin (Phataginus tricuspis) and black-bellied pangolin (P. tetradactyla).
The results indicate that the mitogenomes of the African pangolins are 16,558 bp for S. temminckii, 16,540 bp for S. gigantea, 16,649 bp for P. tetradactyla and 16,565 bp for P. tricuspis. Phylogenetic comparisons of the African pangolins indicated two lineages with high posterior probabilities providing evidence to support the classification of two genera; Smutsia and Phataginus. The total GC content between African pangolins was observed to be similar between species (36.5% – 37.3%). The most frequent codon was found to be A or C at the 3rd codon position. Significant variations in GC-content and codon usage were observed for several regions between African and Asian pangolin species which may be attributed to mutation pressure and/or natural selection. Lastly, a total of two insertions of 80 bp and 28 bp in size respectively was observed in the control region of the black-bellied pangolin which were absent in the other African pangolin species.
The current study presents reference mitogenomes of all four African pangolin species and thus expands on the current set of reference genomes available for six of the eight extant pangolin species globally and represents the first phylogenetic analysis with six pangolin species using full mitochondrial genomes. Knowledge of full mitochondrial DNA genomes will assist in providing a better understanding on the evolution of pangolins which will be essential for conservation genetic studies.
Worldwide, the eight extant pangolin species are classified in the order Pholidota which consists of one family, Manidae (Class: Mammalia). The four African species includes Temminck’s ground pangolin (Smutsia temminckii), giant ground pangolin (S. gigantea), white-bellied pangolin (Phataginus tricuspis) and black-bellied pangolin (P. tetradactyla) [1–3]. The four Asian species include Philippine pangolin (Manis culionensis), Indian pangolin (M. crassicaudata), Chinese pangolin (M. pentadactyla) and Malayan pangolin (M. javanica). All African pangolin species are listed as Vulnerable on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species [4–7]. Of the Asian species, two are listed as Critically Endangered (Chinese and Malayan pangolin) [8, 9] and two are listed as Endangered (Philippine and Indian pangolin) [10, 11]. Pangolins face numerous threats, including habitat destruction [12–14], electrocution [15–17] as well as poaching and illegal trade [18–23]. In 2016, the IUCN voted in support of transferring all eight pangolin species from Appendix II to Appendix I at the Convention on International Trade in Endangered Species of Fauna and Flora (CITES), which was approved at the 17th Conference of Parties (COP17). The listing has resulted in worldwide commercial trade in pangolins being banned as from January 2017 [24, 25]. The taxonomy of pangolins is still under debate, with disagreement regarding the number of genera due to lack of molecular phylogenetic analysis [1, 3, 26–29]. These species have been placed into six genera by Pocock . Other authors have classified all eight extant species of pangolins into a single genus; Manis [3, 29, 30]. Corbet and Hill  suggested two genera; Manis (Asian pangolins) and Phataginus (African pangolins) while Koeningswald  and Gaudin and Wible  proposed three genera; Manis (Asian pangolins), Phataginus (African tree pangolins) and Smutsia (African ground pangolins). Based on osteological characteristics from the entire skeleton , three genera were supported, with the first two genera (Phataginus and Smutsia) forming a monophyletic African clade in the subfamily Smutsiinae . Lastly, four genera have been proposed by McKenna and Bell  and Kingdon  namely Manis, Smutsia, Phataginus and Uromanis. Several authors follow the single genus classification [35–38], however an in-depth taxonomic study of pangolin genera is required in order to clarify this issue.
Mitochondrial DNA (mtDNA) accounts for 1-2% of total DNA content found in mammalian species  and is circular, double-stranded and between 14 and 19 kb in length . The vertebrate mitochondrial genome generally consists of 37 genes, specifying 13 proteins, two ribosomal RNAs, 22 tRNAs, and a control region . The control region is non-coding and contains elements that may regulate replication and transcription . Mitochondrial DNA is generally suitable for evolutionary studies due to its high mutation rate, well-structured genome with restricted non-coding DNA sequences and lack of recombination. Several studies have used portions of the mitochondrial genome including the control region (D-loop) [43, 44], cytochrome c oxidase I (CoxI) [44, 45], cytochrome B (Cob) [44, 46, 47] and 16S ribosomal RNA (16S rRNA)  for traceability of confiscated pangolin scales. Whole mitochondrial DNA genomes will however be more informative for phylogenetic analysis [48–53]. To date, full mitochondrial genomes of five pangolin species have been determined including M. pentadactyla, M. javanica, S. temminckii, P. tetradactyla and P. tricuspis [54–59]. However, two mitogenomes include misidentified Genbank records incorrectly accessioned as M. pentadactyla and P. tetradactyla that were noted in subsequent studies [58, 60]. Several techniques have been reported to generate whole mitochondrial genomes, however modern techniques such as next generation sequencing (NGS) using 454, Illumina and Ion Torrent technology have simplified and made sequencing mitogenomes from any eukaryotic DNA easier, quicker and more affordable compared to Sanger-based methods [61–63]. The vast suite of Bioinformatics software currently available facilitates the annotation and aids in analyses of large datasets .
In general, a quarter of the reads generated by RNA/DNA sequence experiments are from mitochondrial genomes [61, 64–66] which may be attributed to their high copy numbers as well as their high expression levels. Due to the AT richness of mtDNA, as well as it being polyadenylated it can contribute to an increase in poly-A RNA selection . Assembling mitochondrial genomes are significantly less complex than their nuclear genome counterparts as they are smaller in size, and harbour fewer genes . The mitogenomes of two Asian pangolin species (M. pentadactyla and M. javanica) have been assembled using Illumina HiSeq technology, whereby the authors extracted mitochondrial sequences from nuclear data obtained from NGS techniques [57, 59].
Current phylogenetic assessments of pangolins have been conducted using only two of the four African pangolin species namely; Temminck’s ground pangolin and white-bellied pangolin [56, 58]. In addition, the current genus-level classification of pangolins is still under debate. Thus, in this study we performed next generation sequencing for all four African pangolins using the Illumina HiSeq 2500 in order to reconstruct complete mitochondrial genomes. Here we present the first whole mitochondrial DNA genomes of two of the African pangolin species; the black-bellied pangolin (P. tetradactyla) and the giant ground pangolin (S. gigantea). In addition, we describe the mitochondrial genome features in order to understand the evolutionary forces shaping the mitochondrial genomes of African pangolins. Lastly, we conduct a phylogenetic assessment in order to provide a genus-level classification of African pangolins.
Sample collection and DNA isolation
This study used six deceased individuals, sampled by the African Pangolin Working Group (APWG) and representing the four African pangolin species. Tissue samples were placed in absolute ethanol and were stored at the National Biobank, National Zoological Gardens of South Africa (NZG), at −80 °C until analysis. The samples were from one black-bellied pangolin (P. tetradactyla; MF509825), one white-bellied pangolin (P. tricuspis; MF536683), both from Ghana ; and three Temminck’s ground pangolins (S. temminckii; MF536685–MF536687) from South Africa. In addition, a giant ground pangolin (S. gigantea; MF536684) scale sample was included from an illegal seizure. The species identity of samples used in this study was confirmed with Sanger sequencing of the CoxI and Cob loci which were compared to chain-of-custody voucher specimens available from the NZG species reference database  (see http://www.barcodeofwildlife.org). All voucher specimens were verified and identified by an acknowledged authority (Raymond Jansen; African Pangolin Working Group). DNA was isolated using the QIAamp Micro Kit (QIAGEN, Novato, CA, USA) and the respective manufacturers’ protocol for tissue was followed. DNA was quantified on the Qubit 3.0 Fluorometer (Thermo Scientific, Massachusetts, USA). Polymerase Chain Reaction (PCR) amplification and sequencing, to verify species identity, were performed as outlined in Mwale .
Next-generation sequencing and assembly
The products were run on an Illumina HiSeq 2500 (Illumina Incorporated, San Diego, CA, USA) using a rapid run and the TruSeq DNA LT Sample Prep Kit (Illumina Incorporated, San Diego, CA, USA). Data quality was evaluated using FastQC v0.11.2  software, and trimmed and edited through Trimmomatic v0.36  to remove the adapters and poor quality sections. Mitogenomes were assembled in CLC Genomics Workbench v6 (https://www.qiagenbioinformatics.com; CLC Bio, Aarhus, Denmark) using De Novo alignment, with paired reads. Sequence identity of contigs was validated by performing a BLAST search on the National Centre for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Mitogenome annotation and phylogenetic analysis
The mitogenomes were annotated with MITOS v806  and a circular alignment between the six available pangolin species were drawn in Circos v0.69 . The GC content of the four African pangolin mitogenomes were calculated using GPMiner  with a sliding window of 300 bp. Arlequin v3.5.1  was used to validate the GC scores obtained for the four mitogenomes using ANOVA analysis and the diagrams were plotted in R v3.3.1 .
List of 17 mitogenomes used in the study presented here
Genbank Accession Number
Brown Fur Seal
Temminck’s Ground Pangolin
Temminck’s Ground Pangolin
Giant Ground Pangolin
Temminck’s Ground Pangolin
Temminck’s Ground Pangolin
Temminck’s Ground Pangolin
Codon usage analysis for African and Asian species
The Relative Synonymous Codon Usage (RSCU) values for mitochondrial genes were established using the Mega v7  software. This was performed on the four African pangolin species evaluated in this study as well as for the previously published M. pentadactyla (KT445978.1) and M. javanica (KT445979.1). The Principle Component Analysis (PCAs) generated from this data was performed using the FactoMineR package in R . Codon usage bias (AT3 and GC3 content) was calculated using Mega v7, for each of the protein coding genes, where the A and T values at the third base were summed for the AT3 value. The same was performed with G and C for the GC3 content. The ratios were reported as percentages.
Confirmation of insertions observed in the control region of the pangolin mitogenome
Sanger sequencing of the control region of the mitochondrial genome was performed using five additional samples from each of the African pangolin species. The white-bellied and black-bellied pangolin samples were from Ghana and the Temminck’s ground pangolin samples from South Africa and Tanzania . The giant ground pangolin samples were obtained from the collection of the Zoological Museum, University of Copenhagen. The protocol and cycle conditions outlined in Du Toit  were used for all the samples. Sequencing was conducted in order to verify the presence of insertions in the D-loop observed in the mitogenomes obtained from next-generation sequencing during this study. A sequence fragment of around 500 bp was targeted using the primer pair: PNG_Dloop forward 5′-CGTTCCTCTTAAATAAGACATCTCG-3′ and reverse 5′-TCTTGCTTTTGGGGTTTGAC-3′ for verification.
Results and discussion
List of mitochondrial genes and loci, indicating size in base pairs from four African pangolin species, Smutsia gigantea, S. temminckii, Phataginus tricuspis and P. tetradactyla
(Giant ground pangolin)
(Temminck’s ground pangolin)
12S Ribosomal RNA (rRNA)
16S Ribosomal RNA (rRNA)
NADH dehydrogenase I (NadI)
NADH dehydrogenase II (NadII)
Cytochrome c oxidase I (CoxI)
Cytochrome c oxidase II (CoxII)
ATP synthase VIII (AtpVIII)
ATP synthase VI (AtpVI)
Cytochrome c oxidase III (CoxIII)
NADH dehydrogenase III (NadIII)
NADH dehydrogenase IV-L (NadIV-L)
NADH dehydrogenase IV (NadIV)
NADH dehydrogenase V (NadV)
NADH dehydrogenase VI (NadVI)
Cytochrome b (Cob)
Control region (D-loop)
Phylogenetic analysis of African pangolins
Analysis of GC content and codon usage
Total GC content of the African pangolin species was observed to be similar between species (P. tetradactyla = 36.5%, S. gigantea = 36.9%, S. temminckii = 37.3% and P. tricuspis = 36.7%). These results confirm an AT-bias that has been reported in several other mammal species . Analysis of codon usage and pattern of mitochondrial genes; AtpVI, AtpVIII, Cob, CoxI, CoxII, NadI, NadII, NadIII, NadIV, NadIV-L, NadV and NadVI provided evidence of bias in terms of the use of codons with A and C occurring most frequently at the third codon (Additional file 1: Table S1). Variation in base compositions within and among species has been suggested to occur as a result of two evolutionary processes namely biases in the process of mutation and/or natural selection . Selective nucleotide compositional biases have been reported in chiropteran mitochondrial genomes . Uddin and Chakraborty  similarly observed A or C as the most frequent codon at the 3rd position in a study of mitochondrial AtpVI in a variety of mammalian species. The authors attributed this bias to mutational pressure that can influence codon usage bias in mitochondria.
GC content and codon usage variation between pangolin species
Mitogenome comparison between Smutsia and Phataginus
In conclusion, our research study presents the mitogenomes for the four African pangolin species. These include two new reference genomes for the black-bellied pangolin and the giant ground pangolin. This study also presents the first phylogenetic assessment of six of the eight extant pangolin species using whole mitochondrial DNA genomes. The African and Asian pangolin species are shown to separate into two distinct monophyletic clades. Within the African pangolins it was further demonstrated that there is support for classification of the species into separate genera, representing the arboreal (P. tricuspis, P. tetradactyla) and ground-dwelling (S. temminckii and S. gigantea). The availability of these reference mitogenomes will, furthermore, contribute to a better understanding of the evolutionary processes of pangolin species globally, which in turn can contribute to essential conservation genetic studies.
We thank the African Pangolin Working Group (APWG) for sharing their specimen database. We also thank the Zoological Museum, University of Copenhagen for providing us with museum samples of their pangolin specimens (Specimens CN191 and CN339).
This study was financially supported by the National Zoological Gardens of South Africa (NZG) and partially funded by the National Research Foundation (NRF) grant no: (86125).
Availability of data and materials
The reference mitogenomes generated and/or analysed during the current study are available in the GENBANK repository, [https://www.ncbi.nlm.nih.gov/].
All data generated or analysed during this study are included in this published article [and its additional information file].
Conceptualised idea for research: AK; RJ. Responsible for data collection/analysis/design: ZDT; MDP; DLD; JPG. Lead author writing up of article: ZDT. Editorial input: ZDT; MDP; DLD; JPG; RJ; AK. Postgraduate supervisor of the lead author: DLD; JPG; AK; RJ. Co-developed and executed research: ZDT. Project leader/budget owner: DLD; AK. All authors read and approved the final manuscript.
Ethical approval was obtained from the Animal Research Ethics Committee, University of the Free State, South Africa (UFS-AED2015/0070) and the NZG Research, Ethics and Scientific Committee (NZG/RES/P/001/F/02). Samples were obtained under a Section 20 permit (12/11/1/1/18) from the Department of Agriculture, Forestry and Fisheries, South Africa. Sample collection was also approved under a CPC5 permit (02437) from the Department of Agriculture and Rural Development, South Africa and a Biodiversity permit (FAUNA 714/2012) from the Department of Environment and Nature Conservation, South Africa.
Consent for publication
The authors declare that they have no competing interests.
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