Cserhati BMC Genomics (2021) 22:228 https://doi.org/10.1186/s12864-021-07531-3 RESEARCH ARTICLE Open Access A tail of two pandas— whole genome k-mer signature analysis of the red panda (Ailurus fulgens) and the Giant panda (Ailuropoda melanoleuca) Matyas Cserhati Abstract Background: The red panda (Ailurus fulgens) is a riddle of morphology, making it hard to tell whether it is an ursid, a procyonid, a mustelid, or a member of its own family. Previous genetic studies have given quite contradictory results as to its phylogenetic placement. Results: A recently developed whole genome-based algorithm, the Whole Genome K-mer Signature algorithm was used to analyze the genomes of 28 species of Carnivora, including A. fulgens and several felid, ursid, mustelid, one mephitid species. This algorithm has the advantage of holistically using all the information in the genomes of these species. Being a genomics-based algorithm, it also reduces stochastic error to a minimum. Besides the whole genome, the mitochondrial DNA from 52 mustelids, mephitids, ursids, procyonids and A. fulgens were aligned to draw further phylogenetic inferences. The results from the whole genome study suggested that A. fulgens is a member of the mustelid clade (p =9·10− 97). A. fulgens also separates from the mephitid Spilogala gracilis. The giant panda, Ailuropoda melanoleuca also clusters away from A. fulgens, together with other ursids (p =1.2·10− 62). This could be due to the geographic isolation of A. fulgens from other mustelid species. However, results from the mitochondrial study as well as neighbor-joining methods based on the sequence identity matrix suggests that A. fulgens forms a monophyletic group. A Maximum Likelihood tree suggests that A. fulgens and Ursidae form a monophyletic group, although the bootstrap value is weak. Conclusions: The main conclusion that we can draw from this study is that on a whole genome level A. fulgens possibly belongs to the mustelid clade, and not an ursid or a mephitid. This despite the fact that previously some researchers classified A. fulgens and A. melanoleuca as relatives. Since the genotype determines the phenotype, molecular-based classification takes precedence over morphological classifications. This affirms the results of some previous studies, which studied smaller portions of the genome. However, mitochondrial analyses based on neighbor- joining and maximum likelihood methods suggest otherwise. Keywords: Red panda, Giant panda, Whole genome k-mer signature, Pearson correlation, Mustelid, Ursid, Procyonid, Mephitid Correspondence: [email protected] Independent Scholar, 2615C Muscatel Avenue, Rosemead, CA 91770, USA © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cserhati BMC Genomics (2021) 22:228 Page 2 of 12 Background these researchers found that A. fulgens is most closely The red panda (Ailurus fulgens) is an enigmatic animal related to procyonids based on analysis of intron 4 of and is hard to classify based on its morphology. It lives the FGB gene. But when intron 7 was analyzed, it clus- in parts of India, Nepal and China, and has a distinct tered towards ursids. Classification based on the ND2 red-white coloration, and a striped, bushy tail. It goes by gene A. fulgens clustered with mustelids, but these several nicknames, such as the ‘bear-cat’, the ‘cat-bear’, results had poor bootstrapping support. When the two the ‘lesser panda’ or the ‘fire-fox’. Some researchers think introns were combined with analysis of the genes IRBP A. fulgens is a relative of the giant panda (Ailuropoda mela- and TTR, A. fulgens was closest to mustelids [9]. noleuca) based on several physical characteristics. These in- Sato et al. analyzed a 5.5 Kbp segment of DNA coding clude an almost exclusive diet of bamboo (both species eat for five genes, AOPB, BRCA1, RAG1, RBP3, and VWF, meat on occasion), and have an enlarged radial sesamoid and found that A. fulgens clusters together with procyo- bone, which they use to process bamboo [1, 2]. nids and mustelids, and not with mephitids (skunks and Because of these similarities, the giant panda even re- stink badgers) [10]. An earlier, similar result was attained ceived its name from the red panda. According to other when studying a 3.2 Kbp segment containing the genes opinions, A. fulgens has been classified as a member of APOB, RAG1 and IRBP [11]. Genomically, A. fulgens shares the family Procyonidae (raccoons). Yet others put the several apomorphic chromosome fusions with mustelids, red panda into its own family (Ailuridae) [3]. A. fulgens namelyF2+C1pandA1p+C1q[12]. However, A. fulgens also has some unique characteristics: a large zygomatic differs in several other chromosomal rearrangements arch, a powerful jaw, and complex cheek teeth, following indicating that it diverged early from other mustelids. aP2–3 pattern [1]. Interestingly, several genes have been found in both According to new genetic evidence, there are two species which show convergent development. For species of red panda, the Himalayan red panda (A. ful- example, changes in the amino acid composition of the gens), and the Chinese red panda (A. styani)[4]. Due to DYNC2H1 and PCNT proteins lead to polydactyly in reduced numbers, the red panda is an endangered spe- humans and mice, but to the pseudo-thumb in the giant cies. Previous studies based on different combinations of and red pandas. Three other convergent genes (PRSS1, nuclear and mitochondrial genes have given contradict- PRSS36, and CPB1) are responsible for more efficient ory results as to the taxonomic relationship of A. fulgens uptake of nutrients from bamboo, which makes up a with other carnivores. This may be because only several large part of their diet as well. Four other genes, mitochondrial and/or nuclear genes were analyzed, and ADH1C, CYP3A5, CYP4F2, and GIF also enable the not the entire whole genome sequence (WGS). more effective utilization in the giant and red pandas of The red panda’s classification as a procyonid or pro- vitamins A and B12 as well as arachidonic acid, which cyonid-relative is based on immunological, DNA-DNA are absent or very low in bamboo [2]. hybridization, and isozyme evidence [5]. A phylogenetic Intron analysis is useful, since these sequences are not tree based on Bayesian analysis of cytochrome-b put under selection pressure. An analysis of 22 Kbp of nu- A. fulgens next to Canidae [6]. clear intron sequences from 16 carnivore species groups For example, Peng et al. classify A. fulgens either as a A. fulgens with Musteloidea sensu stricto (Mustelidae+ mustelid, placing them next to the American marten Procyonidae) to the exclusion of mephitids [13]. These (Martes americana), or as a mephitid, next to the striped results, however, contradict results coming from mtDNA skunk (Mephitis mephitis). This was based on the analyses [14]. analysis of 13 concatenated mitochondrial proteins, Since morphology-based classification of A. fulgens is based on neighbor-joining (NJ) and maximum likelihood ambiguous, it would be helpful to determine the precise (ML) phylogenetic methods, respectively [7]. In a study taxonomic status of this species based on a whole gen- of three mtDNA genes (12S rRNA, 16S rRNA and cyto- ome-based algorithm. To this end, the Whole Genome K- chrome b) and intron 1 of the nuclear transthyretin mer Signature (WGKS) algorithm [15]isusedtoanalyze gene, Flynn et al. also found that A. fulgens is neither an the genomes of five bear species, eleven cat species and ursid, nor a procyonid, nor a mephitid, but a mustelid ten species from the family Mustelidae (weasels, otters, [1]. Another study including three mitochondrial and martens, and badgers), Spilogala gracilis, a mephitid spe- three nuclear genes by Fulton and Strobeck, based on 16 cies, as well as A. fulgens, making 28 species in total. arctoid species, with Canis lupus as an outlier, placed The advantages of using a genomics-based algorithm A. fulgens in close relationship to M. mephitis [8]. to analyze the WGS of these organisms is that it takes Yu and Zhang studied introns 4 and 7 from the all the information present in the WGS, as opposed to nuclear gene ß-fibrinogen (FGB) as well as the mito- just a handful of genes, utilized in gene studies. Deciding chondrial gene NADH dehydrogenase subunit 2 (ND2) which genes are important is subjective and may vary in 17 species from the order Carnivora. In their results, between investigators. Whole genome-based algorithms Cserhati BMC Genomics (2021) 22:228 Page 3 of 12 also have the advantage that they greatly reduce stochas- Additional File 1 online. The Hopkins statistic is 0.9, tic error, due to the vast number of characters (DNA which means that the data set is of very good quality for bases) that they analyze [16]. Using this algorithm can clustering.