Characteristics of the complete mitochondrial genome of the monotypic genus Arctictis (Family: Viverridae) and its phylogenetic implications

The binturong (Arctictis binturong) is classified as a member of the subfamily Paradoxurinae within the family Viverridae (Carnivora: Mammalia) and comprises nine subspecies spread across Southern and Southeast Asia. Here, we describe the complete mitochondrial genome of the Indian subspecies A. b. albifrons using next-generation sequencing methods. The total length of the A. b. albifrons mitogenome was 16,642 bp. Phylogenetic analyses based on 13 mitochondrial protein-coding genes placed the binturong as a sister taxon to Paguma larvata within the Paradoxurinae and supported the clustering of Genettinae and Viverrinae and the monophyly of Viverridae and six other families of feliforms, consistent with previous studies. Divergence time estimates suggest that the Viverridae diversified during the Miocene (22.62 Mya: 95% CI [20.78–24.54] Mya) and that Arctictis and Paguma split 12.57 Mya (95% CI [8.66–15.67] Mya). Further molecular studies are required to test the distinctiveness and diversity of the nine putative subspecies of binturong.


INTRODUCTION
Arctictis binturong (Raffles, 1822), commonly called binturong or bearcat, is the largest known member of the Viverridae (Carnivora: Mammalia) and is characterized by coarse, black fur and a prehensile tail (Pocock, 1933). In forest ecosystems of Southeast Asia, the frugivorous binturong has co-evolved with fig trees to form a keystone relationship, wherein the animal facilitates and propagates seed germination while the fig tree provides a stable dietary source (Kinnaird & O'Brien, 2007). Binturongs are presently being poached for their meat, traditional medicines and the pet trade, and alongside habitat destruction, these factors have contributed to decreasing the numbers of binturong to a few geographical pockets across the species' former range (Willcox et al., 2016). As a result of these increasing pressures, the binturong is listed as 'Vulnerable' on the IUCN Red List of Threatened Species (Willcox et al., 2016).
Nine subspecies of A. binturong have been described primarily on the basis of regionspecific variations in fur color (Pocock, 1933;Cosson et al., 2006). In addition, shared morphological similarities with other viverrids like perineal scent glands and syndactyly of the third and fourth digits of the hind foot, along with the unique features of completely naked soles of the hind feet and a prehensile tail, have helped determine the phylogenetic position of binturong within the viverrid subfamily Paradoxurinae (Pocock, 1933;Gregory & Hellman, 1939;Veron, 2007). More recent molecular phylogenetic studies indicate that this subfamily also includes Paguma, Arctogalidia and Macrogalidia (Albert, 2001;Cosson et al., 2006;Patou et al., 2008;Nyakatura & Bininda-Emonds, 2012;Zhou, Wang & Ma, 2017). However, the genetic structure of the binturong has not been studied in detail (but see Cosson et al., 2006), which will be essential to validate the evolutionary and conservation genetic implications of the existence of nine geographically and morphologically disparate subspecies.
Mohd Salleh et al. (2017) reported the first mitochondrial genome of a binturong as part of a larger study to generate an expanded reference mitogenome dataset of mammals from Southeast Asia that could be applied for monitoring mammalian biodiversity using environmental DNA approaches. However, the sequence reported in that study came from an animal at the Tier Park Berlin Zoo of unknown provenance. Moreover, the sequence contains many missing nucleotides (Ns) and is therefore incomplete. To rectify this, we generated a complete (gapless) mitochondrial genome sequence from a wild-caught binturong of known provenance belonging to the Indian subspecies, Arctictis binturong albifrons. The aims of our study were to: (a) characterize the Arctictis mitogenome in comparison with other viverrids and feliforms, and (b) provide the first molecular phylogenetic and divergence dating analysis of the Arctictis in the context of Viverridae and other feliform families based on whole mitochondrial genomes.

Sampling, Extraction and PCR amplification
A blood sample of an individual identified as A. b. albifrons was collected and forwarded by the Veterinary Assistant Surgeon of the Sepahijala Zoo, Tripura (Vide Letter No. F5(D)VD/Sep/Sl No. 100-102, dated 19/07/2008) for DNA analysis, and deposited in the Genome Bank at the Laboratory for Conservation of Endangered Species, CCMB, Hyderabad, India. Genomic DNA was isolated from the blood sample by the phenolchloroform-isoamyl alcohol method (Sambrook, Fritsch & Maniatis, 1989) and the DNA integrity was checked electrophoretically in a 0.8% agarose gel. The mitochondrial genome was amplified by long range PCR using the TaKaRa LA PCR kit v2 (Takara Bio Inc, USA) following the manufacturer's recommendations. Three PCR products of 4.1 kbp, 8.8 kbp and 4.5 kbp were generated using three sets of primers (Table S1).

Genome sequencing, assembly and annotation
Next-generation sequencing libraries were constructed in three steps: enzymatic shearing, adapter ligation and fragment size selection. PCR product quality was first assessed using the Qubit R 2.0 Fluorometer (Life Technologies, USA). Amplified products were then subjected to enzymatic fragmentation to an average of 550 bp fragments using the Covaris M220 system (Covaris, USA). Libraries were prepared with the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer's recommendations. The High Sensitivity DNA Analysis Kit (Agilent Technologies, USA) was used for quantification and size estimation of the libraries generated on a 2100 Bioanalyzer (Agilent Technologies, USA). The libraries were standardized to 1.5pM and sequenced using the NextSeq 500 sequencer (Illumina, USA). The paired end reads generated on Illumina were used for reference assembly. Raw sequences were extracted in the FASTQ format and checked for quality using the CLC Genomics Workbench v 9.0 software (https://www.qiagenbioinformatics.com/). Raw sequences filtering was performed by trimming adaptors. Nucleotides and sequence reads showing ambiguity or low quality scores (<Q20) were excluded from further analysis. High quality data obtained after filtering was assembled and annotated using CLC Genomic Work bench v 9.0. Mauve version 2.4.0 (Darling et al., 2004) was used for the comparative reference assembly. The binturong mitogenome sequence was analyzed using CLC Genomic Workbench v 9.0. to identify the mitochondrial gene locations, their order, and start and end points.

Genome analysis
The circular map of mitogenome was created using Geneious R 10.1 (Kearse et al., 2012). Sequences of the 13 protein-coding genes were translated into amino acid sequences using ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) to verify orthology with other feliform taxa and exclude the potential presence of NUMTs (nuclear-mitochondrial paralogues). The control region was extracted and scanned for the presence of palindromes and other repeats using the EMBOSS and REPFIND tools (Rice, Longden & Bleasby, 2000;Betley et al., 2002). We evaluated the base composition of the binturong mitogenome and those of 11 other feliform species using Geneious R 10.1 (Table S2).

Phylogenetic analysis and estimation of divergence times
The phylogenetic relationships of the binturong within Viverridae and Feliformia were reconstructed by aligning its 13 protein-coding gene sequences with those of 22 feliform species comprising five viverrids, 11 felids, two hyaenids, and one species each from Nandiniidae, Herpestidae, Eupleridae and Prionodontidae (Table S2). The Cuon alpinus mitogenome (Canidae, NCBI Accession No. NC_013445.1) was also included as outgroup to root the feliform tree. Sequences were aligned using MEGA 6.06 with the default parameters of CLUSTALW (Thompson, Higgins & Gibson, 1994) and then concatenated, resulting in an 11,313 bp alignment. Maximum likelihood phylogenetic analysis was conducted using raxmlGUI v1.3 (Silvestro & Michalak, 2012). Support for different nodes was estimated using 1,000 bootstrap replicates (ML + bootstrap option) under the GTR+I+G model, as estimated with jModeltest 2.1.5 (Darriba et al., 2012) and Bayesian Inference (BI) method using Mr. Bayes v.3.2.5.
We jointly estimated the phylogeny and divergence times between species within a Bayesian inference framework using the program BEAST v1.7.5 (Drummond & Rambaut, 2007). The 13 protein-coding genes were partitioned and the appropriate model of sequence evolution was determined by the BIC criteria in jModeltest 2.1.5 (Darriba et al., 2012). Substitution rates were estimated under a relaxed uncorrelated clock model [14] for 1×10 8 million generations, sampling every 1,000th generation to allow for adequate mixing. Base frequencies were set to ''All equal'' and the number of gamma categories was set to 4. Four fossil priors with a uniform probability distribution were used for calibration: (a) Minimum and maximum ages of split between Caniformia and Feliformia were set at 43 Mya and 63.8 Mya, respectively (Benton & Donoghue, 2007) (b) Minimum age of origin of Viverridae was set at 23 Mya (Hunt, 1991;Hunt Jr, 1996) (c) Minimum age of the split between Crocuta and Hyaena was set 9.5 Mya (Wozencraft, 2005); and (d) Minimum and maximum ages of origin of Felidae were set at 5.3 Mya and 23 Mya (McKenna & Bell, 1997). The first 25% of MCMC iterations were removed from the posterior sample as burn-in. Convergence was monitored in Tracer (Ver. 1.4) to determine if effective sampling sizes (ESS) were adequate (>200). Trees were summarized with Tree Annotator and represented as the maximum clade credibility tree.

Binturong mitogenome assembly and annotation
A total of 2,726,370 high-quality reads were obtained after filtering and trimming sequences. The assembly of the binturong mitogenome resulted in a total of 162 contigs with an N50 length of 1074 bp and the length of contigs ranging from 501 bp to 16,752 bp. After trimming ends, the A. b. albifrons mitogenome was 16,642 bp in length (Fig. 1). The assembly size of the binturong mitogenome reported by Mohd Salleh et al. (2017) is 17,067 bp, 425 bp longer than our assembly. However, this increased length is due to the presence of inserted Ns (missing nucleotides) within the sequence. The A. b. albifrons mitogenome is generally shorter when compared with those of nine other feliform species (Table S2). The A. b. albifrons mitogenome sequence was deposited in NCBI GenBank (accession number KX449332).
Annotation yielded a total of 37 genes: 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs and the non-coding control (D-loop) region (Table 1). No NUMTs were detected among the 13 PCGs when they were translated into amino acids (no insertion, deletions, frame shift mutations or premature stop codons). Analysis of base composition showed a bias towards higher adenine and thymine content in the binturong mitogenome (Table 2), amounting to 64.63% and individual base proportions amounting to 32.80% A, 31.83% T, 16.46% G and 18.91% C. This pattern is consistent with the base composition of twelve other feliform species, with AT content ranging from 60.28% in Hyaena to 64.64% in P. larvata while a higher average bias was maintained within Viverridae (65.02%). The control region was 1,234 bp long, located from 15,408-16,642 bp in the binturong mitogenome (Fig. 1). The region included five mononucleotide T stretches, two of (T) 4 and three of (T) 5 . A 12 bp long palindrome sequence (5 -TATCTATAGATA-3 ) was located between

Phylogenetic relationships and divergence time estimates
Maximum likelihood and Bayesian inference analyses (Fig. S1) of the concatenated sequences of the 13 PCGs (11,313 bp) support the monophyly of Viverridae among feliforms, the monophyly of Paradoxurinae among viverrids, the clustering of Paradoxurinae and Hemigalinae, as found by Veron et al. (2017), and the clustering of Genettinae with Viverrinae as proposed by Veron (2007) and Eizirik et al. (2010). The monophyly of Viverridae was strongly supported in both the analyses, with a Bayesian Posterior Probability (bpP) of 1 and a bootstrap support (BS) of 100%. Viverrid monophyly was also observed by Patou et al. (2008) and finds morphological support as the species therein share a characteristic union of the third and fourth digits in the hind foot and a hypocarnivorous dentition (Patou et al., 2008;Veron, 2007). Within Viverridae, Paradoxurinae (A. binturong and P. larvata) and Paradoxurinae + Hemigalinae (C.   bennetti) were both monophyletic, each with bpP of 1 and BS = 100%. Finally, Genettinae and Viverrinae were found to have a sister relationship with maximum bpP support and BS = 94%. Inferred relationships among other feliform familes were well supported and congruent with previous studies (Veron & Heard, 2000;Gaubert & Veron, 2003;Yoder et al., 2003;Flynn et al., 2005;Koepfli et al., 2006;Eizirik et al., 2010). Divergence time estimation (Fig. 2) showed that Viverridae began diversifying around 22.65 Mya (95% Credibility Interval (CI) [20.78 Mya -24.54 Mya]), close to the dates estimated in earlier studies (Gaubert & Veron, 2003;Koepfli et al., 2006;Patou et al., 2008;Zhou, Wang & Ma, 2017) but more recent than that suggested in other studies (Gaubert & Cordeiro Estrela, 2006;Eizirik et al., 2010). The estimates obtained from this study and most others agree with the temporal position of the earliest evidence for Viverridae, represented by the late Oligocene-early Miocene fossil Herpestides, which has been dated at 23 Mya (Johnson et al., 2006;Morlo, Miller & El-Barkooky, 2007). The split between Arctictis and Paguma in Paradoxurinae was estimated at 12.57 Mya (95% CI [8.66 Mya -16.51 Mya]), which agrees with the date of origin of Paradoxurinae suggested to be during the Miocene (Gaubert & Cordeiro Estrela, 2006;Veron, 2007). Divergence times estimated for the origin of Feliformia (41.80 Mya,) and the feliform families are consistent with those reported in previous studies (Gaubert & Cordeiro Estrela, 2006;Koepfli et al., 2006;Eizirik et al., 2010;Zhou, Wang & Ma, 2017).

CONCLUSIONS
We have reported the first complete (gapless) mitogenome of the binturong, representing the Indian subspecies Arctictis binturong albifrons. The binturong mitogenome, along with the one previously reported by (Mohd Salleh et al., 2017), provides a starting point for further testing the distinctiveness and diversity of the nine putative subspecies of binturong and thereby provide critical information for designing conservation management plans for this vulnerable species.