Complete mitochondrial genomes of the Eastern lowland olingo (Bassaricyon alleni), ringtail (Bassariscus astutus), white-nosed coati (Nasua narica), and crab-eating raccoon (Procyon cancrivorus) elucidate the phylogeny of the Procyonidae

Fourteen morphologically diverse small American mesocarnivores form the family Procyonidae, one of the sixteen comprising the mammalian order Carnivora (Helgen et al., 2013). Procyonids fall within the suborder Caniformia, infraorder Arctoidea, superclass Musteloidea; the latter also includes families Mephitidae (skunks and stink badgers), Ailuridae (red pandas), and Mustelidae (badgers, martens, weasels, and otters) (Hassanin et al., 2021;Wozencraft, 2005). The bestknown procyonids are the charismatic food-washing masked bandits, the raccoons (genus Procyon). Other procyonids, however, include the solitary, monochromatic, arboreal olingos (genus Bassaricyon); the ringtail and cacomistle, nocturnal, opportunistic omnivores with striking black-and-white ringed tails (genus Bassariscus); the social, ground-dwelling coatis (genus Nasua) and dwarf coatis (genus Nasuella); and the arboreal kinkajou (genus Potos) (Hunter & Barrett, 2018). According to the IUCN Red List of Threatened Species, Procyon pygmaeus and Nasuella meridensis are Critically Endangered and Endangered, respectively (Cuarón et al., 2016;Gonzalez-Maya & Arias-Alzate, 2016), while Bassaricyon neblina and Nasuella olivacea are categorized as Near Threatened (Gonzalez-Maya et al., 2016;Helgen et al., 2020). The other 10 species of procyonids are categorized as Least Concern, but except for the northern raccoon, these species' population sizes are trending downward, raising concern for their long-term conservation.During the last two decades, studies based on DNA sequences have clarified the systematics of the Procyonidae, revealing a pattern of evolutionary relationships at odds with expectations based on morphological characters (Koepfli et al., 2007). DNA-based studies consistently support a topology that groups Bassaricyon, Nasua, and Nasuella in one clade, Bassariscus and Procyon in another clade, and Potos as a distinct basal lineage, although morphology would suggest groupings of Bassaricyon + Potos and Nasua + Procyon (Eizirik et al., 2010;Hassanin et al., 2021;Koepfli et al., 2007;Law et al., 2018). The procyonids continue to undergo taxonomic revision, including the discovery of the olinguito (Bassaricyon neblina) in 2013, uncertainty about the species status of the coati found on Cozumel Island, Mexico (Jaramillo & Ruiz-García, 2022;Valenzuela-Galván et al., 2023), and repeated calls to synonymize the genera Nasua and Nasuella (Helgen et al., 2013;Jaramillo & Ruiz-García, 2022;Nigenda-Morales et al., 2019;Ruiz-García et al., 2021). Thus, more attention to evaluating phylogenetic relationships within this complex clade is appropriate.Phylogenetic relationships can be elucidated relatively quickly using mitochondrial DNA, or mtDNA. mtDNA is haploid, considerably smaller than nuclear genomes, and exhibits a high rate of DNA substitution, making it a good benchmark for inferring phylogenetic relationships among and within species (Moritz et al., 1987). Though partial or complete sequences from individual mitochondrial genes are sometimes used for phylogenetic inference, whole mitochondrial genomes, or mitogenomes, are considered more informative (Boore et al., 2005). Due to their high copy number in the mitochondria of cells, mitogenomes can be assembled relatively easily and inexpensively from low coverage (≤5x) high-throughput sequencing data, an approach known as genome skimming (Hoban et al., 2022;Trevisan et al., 2019). This approach is growing in popularity for resolving systematic relationships (e.g., Quattrini et al., 2024;Taite et al., 2023). To date, complete mitogenomes of only four procyonid species have been deposited into NCBI's GenBank database: northern raccoon, Procyon lotor (AB297804, AB291073, CM027276, MT410951); northern olingo, Bassaricyon gabbii (ON704723); South American coati, Nasua nasua (HM106331); and kinkajou, Potos flavus (MW257234). Complete mitogenomes of species such as white-nosed coati (Nasua narica) and western mountain coati (Nasuella olivacea) have, however, been reported in several studies but not deposited into public sequence databases (Hassanin et al., 2021;Jaramillo & Ruiz-García, 2022;Tsuchiya, 2017).To address this paucity of publicly available procyonid mitogenomes, we report new annotated mitochondrial genome assemblies generated with genome skimming for the following species: eastern lowland olingo (Bassaricyon alleni), ringtail (Bassariscus astutus), white-nosed coati (Nasua narica), and crab-eating raccoon (Procyon cancrivorus). We also assembled and annotated complete mitogenomes of the olinguito (Bassaricyon neblina) and the little-known cacomistle (Bassariscus sumichrasti) using short-read sequencing data generated by Hawkins et al. (2016). These mitogenomes were used to develop an updated phylogeny, which was supplemented by a broader phylogenetic analysis of just the cytochrome b gene. Using the newly annotated complete mitogenomes, we were also able to infer sequence distances and codon usage bias in relation to previously available mitogenomes of procyonid species.Frozen tissue samples used to generate the new mitogenomes reported in this study were obtained from vouchered specimens deposited at the Museum of Southwestern Biology, University of New Mexico, New Mexico, USA (Nasua narica, Procyon cancrivorus) and the Museum of Vertebrate Zoology, University of California, Berkeley, California, USA (Bassaricyon alleni, Bassariscus astutus). Details about the sampled specimens, including their geographic origin, are found in Table S1.DNA extraction, library preparation, and sequencing were conducted at Psomagen, Inc. (Rockville, MD). The Mag-Bind Blood and Tissue Kit (Omega Bio-Tek Inc., Norcross, GA) was used for genomic DNA extraction, after which concentration was estimated with the Picogreen and Victor X2 fluorometry assay (Life Technologies, Carlsbad, CA) and quality was checked with an Agilent 4200 Tapestation (Agilent Technologies, Santa Clara, CA) and 1% agarose gel electrophoresis. A Covaris S220 ultrasonicator (Woburn, MA) was used to shear genomic DNA; fragments of ~350bp were enriched, and the TruSeq DNA PCR-free library kit (Illumina, San Diego, CA) was used to prepare a genomic library for each sample. Libraries were quality checked using an Agilent 4200 Tapestation and a Lightcycler quantitative PCR assay (Roche Life Science, St. Louis, MO). An Illumina NovaSeq 6000 instrument was used to paired-end sequence (2 x 150 bp) each library to a depth of 5x.The FASTQ files from Bassaricyon alleni, Nasua narica, Procyon cancrivorus, Bassaricyon neblina (SRR2103277), and Bassariscus sumichrasti (SRR2104917) were trimmed with AdapterRemoval (Lindgreen, 2012) using default settings. The mitogenomes were assembled with GetOrganelle v1.7.7.1 (Jin et al., 2020) using the following parameter settings: 10 extending rounds, the `animal_mt` reference database, SPAdes assembler (Prjibelski et al., 2020), k-mer values 21, 45, 65, 85, and 105, and automatic word size estimation. Bassaricyon neblina and Bassariscus sumichrasti were assembled with seed sequence Bassaricyon gabbii (ON704723) and Procyon lotor (AB291073), respectively. Reads from Bassariscus astutus were cleaned with fastp v0.22.0 `detect_adapter_for_pe -l 30 -q 20 --overrepresentation_analysis` (Chen, 2023) and assembled with GetOrganelle v1.7.7.1, using k-mer values 21, 55, 85, and 115 and seed sequences Procyon lotor (NC_009126.1), Potos flavus (NC_053977.1, CM027365.1), Bassaricyon gabbii (NC_066722.1), and Nasua nasua (HM106331.1). All mitogenomes were annotated with MITOS2 (Donath et al., 2019) using the RefSeq 89 Metazoa database and genetic code 2.The six mitogenomes assembled were aligned with four previously published procyonid mitogenomes downloaded from the NCBI GenBank database: ON704723 (Bassaricyon gabbii), AB291073 (Procyon lotor), HM106331 (Nasua nasua), and MW257234 (Potos flavus). For outgroups, we included one species from Mephitidae, Mephitis mephitis (HM106332), one species from Ailuridae, Ailurus fulgens (MK886830), and two species from Mustelidae, Mellivora capensis (MW257239) and Lutra lutra (MW573979), for a total alignment of 14 mitogenomes from distinct species. We aligned these sequences using the MAFFT v7.450 plugin in Geneious Prime 2023.1.1 with default settings: algorithm "Auto", scoring matrix PAM200 with k = 2, gap open penalty = 1.53, and offset value = 0.123 (Katoh & Standley, 2013). The control region was trimmed due to poor alignment within the tandem repeat regions, resulting in a final alignment length of 15,541bp, which included sequences of 13 protein-coding genes (PCGs), 2 rRNAs and 22 tRNAs. We constructed a maximum-likelihood phylogeny with the RAxML version 8.2.11 (Stamatakis, 2014) plugin in Geneious Prime 2023.1.1 using the rapid hill-climbing algorithm, applying 1000 bootstrap replicates to evaluate node support. For both analyses, we used the GTR+GAMMA nucleotide substitution model, as this is one of the four applicable models in RAxML, and because our alignment contains a relatively small number of taxa, such that the alpha parameter of the gamma distribution sufficiently accounts for amongsite rate heterogeneity (Yang, 2006).We also constructed a Bayesian phylogeny using the MrBayes 3.2.6 (Huelsenbeck & Ronquist, 2001) plugin in Geneious Prime 2024.0.7 with default settings (chain length = 10 7 , subsampling frequency = 10 3 , heated chains = 4, heated chain temperature = 0.2, burn-in length = 10 3 ), the GTR+GAMMA substitution model, and designating Ailurus fulgens as the outgroup in the full mitogenome alignment.To incorporate a more complete taxon set, we generated an additional phylogeny using only sequences of the cytochrome b (CYTB) gene. We extracted these sequences from all the complete mitogenomes used in our initial phylogeny except for A. fulgens and M. mephitis, which were removed to prevent error from long branch attraction. Additionally, we downloaded 25 CYTB sequences from GenBank, representing all procyonid species in our complete mitogenome phylogeny as well as Nasuella olivacea, Bassaricyon medius, and Bassaricyon beddardi, which has now been synonymized with Bassaricyon alleni (Helgen et al., 2013). Ultimately, our sample set for this analysis included one sample each for two outgroup species (M. capensis and L. lutra), B. neblina, B. neblina neblina, B. beddardi, two samples each for B. astutus, B. sumichrasti, N. olivacea, B. gabbii, B. alleni, and B. medius medius, three samples each for P. cancrivorus, P. lotor, N. narica, and N. nasua, and four samples each for P. flavus and B. medius orinomus, for a total of 37 sequences. We aligned these sequences using the same methods as the complete mitogenome and trimmed the resulting alignment to 1,140 bp. We then used the same RAxML settings, including the GTR+GAMMA substitution model, to generate a maximum-likelihood phylogeny from this alignment and evaluated node support with 1000 bootstrap replicates.We estimated the absolute number of pairwise differences (p-distances) between procyonid taxa in the trimmed mitogenome alignment with MEGA 11.0.10 (Tamura et al., 2021) using the following settings: Variance Estimation Method: None, Substitutions Type: Nucleotide, Model/Method: No. of differences, Substitutions to Include: d: Transitions + Transversions, Rates among Sites: Uniform Rates, Pattern among Lineages: Same (Homogeneous), Gaps/Missing Data Treatment: Pairwise deletion. We repeated the analysis with a Kimura twoparameter model (Kimura, 1980), which accounts for different substitution rates between transitions and transversions. Settings were identical to those described above, except the Model/Method setting was changed to "Kimura 2-parameter model."To test for differences in codon usage in the 13 PCGs, protein-coding sequences from each procyonid mitogenome (except Bassariscus sumichrasti, which was done by hand due to technical difficulties) were concatenated with a homemade Python script, which is available upon request. Each concatenated sequence was analyzed with the Codon Usage calculator in the Sequence Manipulation Suite (Stothard, 2000) under the vertebrate mitochondrial genetic code.We assembled and annotated full mitogenomes for six procyonid species: Eastern lowland olingo (Bassaricyon alleni), olinguito (Bassaricyon neblina), ringtail (Bassariscus astutus), cacomistle (Bassariscus sumichrasti), white-nosed coati (Nasua narica), and crab-eating raccoon (Procyon cancrivorus) (Figure 1). The coverage and lengths of the mitogenomes varied between 627 -6758.1x and 16,486 -16,653 bp, respectively (Table S1). These lengths were consistent with those of previously published procyonid mitogenomes, which range from 16,388 -16,600 bp.Each mitogenome contained the standard set of vertebrate mitochondrial genes, with 13 proteincoding genes, 22 tRNAs, 2 rRNAs, two origins of replication, and a control region, arranged in the order typical for therian mammals (Kocher et al., 1989).Figure 1. The six newly generated mitogenomes. Photo attributions are listed in Table S5. Black outer rings show the relative nucleotide position of each gene in the mitogenomes. Inner rings are colored according to the annotated features: protein-coding genes (green), ribosomal RNAs (red), transfer RNAs (pink), control region (orange), light strand and heavy strand origins of replication (blue). Arrowed bars indicate transcription on either the plus strand (clockwise arrows) or minus strand (counterclockwise arrows).Maximum-likelihood and Bayesian inference phylogenetic analysis of the 15,541 bp alignment yielded a tree in which Bassaricyon and Nasua, the olingos and coatis, formed one clade, while Bassariscus and Procyon, the ringtail, cacomistle, and raccoons, formed a second clade (Figure 2A and Figure S1). The kinkajou (Potos flavus) was basal to these groupings. All labeled nodes had a bootstrap and posterior probability support of 100% and 1.0, respectively. This phylogeny is generally concordant with the relationships inferred in previous studies using partitioned supermatrices of mitochondrial and nuclear DNA sequences (Koepfli et al., 2007;Eizrik et al. 2010;Helgen et al. 2013;Law et al. 2018). The same groupings appear in the maximum likelihood and Bayesian inference CYTB trees (Figure 2B and Figure S2), with two major exceptions. First, the kinkajou clade is positioned as the sister lineage to the clade containing the coatis and olingos (Nasua, Nasuella, and Bassaricyon) rather than basal to the raccoon+ringtail+cacomistle and coati+olingo clades. However, this deep node only had a 59% bootstrap and 0.89 posterior probability support, respectively. Second, the two species of Bassariscus are paraphyletic, with the cacomistle sister to the clade containing the two species of Procyon. The node uniting B. sumichrasti with P. cancrivorus+P. lotor had a bootstrap support of 42% and a posterior probability support of 0.51, suggesting this grouping is unstable. The sole deviation from previous phylogenies in the complete mitogenome tree is the placement of Bassaricyon neblina. In Helgen et al. (2013), based on a combination of the complete cytochrome b (CYTB) gene and a nuclear intron of the CHRNA1 gene, B. neblina was placed basal to all other olingo species, while our phylogeny places B. neblina sister to B. gabbii, with B. alleni basal to both. Moreover, based on Kimura 2-Parameter distances among the CYTB sequences, B. neblina showed a 9-11% sequence divergence from other olingo taxa in Helgen et al. 2013. The sequence divergence, however, between the B. neblina mitogenome assembled in this study and the B. gabbii assembled by Winter et al. (2023) is only 0.966%, while B. alleni's p-distances from B. neblina and B. gabbii are 7.44% and 7.37%, respectively. Thus, our results strongly suggest that the mitogenome of B. gabbii reported by Winter et al. (2023) instead likely represents B. neblina. Our CYTB tree supports this assertion: this B. gabbii (ON704723) groups with our assembled B. neblina sequence (PQ424047) and with EF107709, another B. neblina CYTB sequence (Figure 2B and Figure SX). These three are basal to all other olingos, including JX948744, a taxonomically verified B. gabbii sequence that occupies its own branch, separate from ON704723. The B. gabbii mitogenome from Winter et al. (2023) was derived from a cell culture with no information about the origin or source of the culture. This misidentification emphasizes the importance of reference genome resources being tied to vouchered specimens that have been taxonomically verified (Buckner et al., 2021).In addition, the CYTB gene tree includes a sequence downloaded from GenBank (KX756273) and identified as Bassaricyon beddardi in Nascimento et al. (2017), which is nested among two B. alleni sequences, including the one from which we assembled the complete mitogenome (PP990717). However, Helgen et al. (2013) concluded that B. beddardi should be regarded as a synonym of B. alleni, based on the limited molecular and morphological differences between these taxa. Therefore, according to our data, B. alleni is a monophyletic species.Intergeneric Parameter distances between whole mitogenomes averaged 18.5%, ranging from 12.9% (B. sumichrasti versus P. cancrivorus) to 20.6% (N. narica versus P. flavus) -1785 to 2708 base pairs different (Table S3). Intrageneric distances range from 7.37-13.6%, averaging 9.7%. The highest intrageneric distance was observed between the two Nasua species at 13.6% (1864 base pairs different), while the lowest was found between Bassaricyon alleni and B. gabbii, at 7.37% (1064 base pairs different).The codon usage results show a significant bias in favor of codons ending with pyrimidines (Table S4), which is common among mammalian mitogenomes (Jia & Higgs, 2008). Although the preferred codon of a synonymous set is usually the same across the family, the magnitude of this preference varies considerably even within genera. For example, both Nasua narica and Nasua nasua prefer TGC for cysteine, but N. nasua uses this codon 75% of the time, while N. narica uses it only 58% of the time. N. narica consistently shows the least extreme bias, while B. neblina and B. gabbii frequently show the most extreme bias. These latter two mitogenomes have a highly similar codon usage profile, with an average difference of 0.6% between them. Bassaricyon spp. codon bias is 7-8% different from any other genus, while the other genera, regardless of phylogenetic distance, differ from each other by 4-6%. B. sumichrasti codon bias seems more closely correlated with that of P. cancrivorus than that of B. astutus: P. cancrivorus and B. sumichrasti have on average difference in codon while B. astutus and B. sumichrasti have on average These results show that or differences in codon bias not with species to of the evolutionary of in the and the of mitochondrial in the relationships among species in this For that on morphological and data to separate these species phylogenies that Bassaricyon with Potos arboreal species that and Bassariscus with a containing both Nasua and Procyon and & These groupings are also of as the latter and not observed in the (Koepfli et al., 2007). However, more that used both partial mitogenomes and of nuclear that the phylogeny of the more closely the topology we in Figure 2A (Eizirik et al., et al., 2007;Law et al., and not with relationships based on In the based on DNA sequence data consistently suggest that Potos was the species to the morphologically taxa Bassaricyon + Nasua and Bassariscus + Procyon each separate clades. This that and morphology should be supplemented by genetic This information not only evolutionary relationships but also phylogenetic that from poor of morphological on which to base more than the mitogenome resources available for the Although two of the six mitogenomes we assembled have been previously this study is the to this data publicly Our mitogenome analysis includes all procyonid genera except Nasuella, the mountain however, show that the Nasuella within Nasua (Helgen et al., & Ruiz-García, 2022;Nigenda-Morales et al., 2019;Ruiz-García et al., suggesting that Nasuella should be synonymized with ON704723 to from an species full mitogenomes the northern olingo (Bassaricyon gabbii), the western lowland olingo (Bassaricyon the eastern mountain coati (Nasuella the western mountain coati (Nasuella and the raccoon (Procyon (Cuarón et al., Although this our of this family and additional for and a mitogenome would our of evolutionary across