Whole-Genome Sequencing of Procyonids Reveals Distinct Demographic Histories in Kinkajou (Potos flavus) and Northern Raccoon (Procyon lotor).

Here, we present the initial comparison of the nuclear genomes of the North American raccoon (Procyon lotor) and the kinkajou (Potos flavus) based on draft assemblies. These two species encompass almost 21 Myr of evolutionary history within Procyonidae. Because assemblies greatly impact downstream results, such as gene prediction and annotation, we tested three de novo assembly strategies (implemented in ALLPATHS-LG, MaSuRCA, and Platanus), some of which are optimized for highly heterozygous genomes. We discovered significant variation in contig and scaffold N50 and L50 statistics and genome completeness depending on the de novo assembler used. We compared the performance of these three assembly algorithms in hopes that this study will aid others looking to improve the quality of existing draft genome assemblies even without additional sequence data. We also estimate the demographic histories of raccoons and kinkajous using the Pairwise Sequentially Markovian Coalescent and discuss the variation in population sizes with respect to climatic change during the Pleistocene, as well as aspects of their ecology and taxonomy. Our goal is to achieve a better understanding of the evolutionary history of procyonids and to create robust genomic resources for future studies regarding adaptive divergence and selection.

Phylogeographic and diversification patterns of the white-nosed coati (Nasua narica): Evidence for south-to-north colonization of North America.

White-nosed coatis (Nasua narica) are widely distributed throughout North, Central, and South America, but the patterns of temporal and spatial diversification that have contributed to this distribution are unknown. In addition, the biogeographic history of procyonid species in the Americas remains contentious. Using sequences from three mitochondrial loci (Cytochrome b, NAHD5 and 16S rRNA; 2201 bp) and genotypes from 11 microsatellite loci, we analyzed genetic diversity to determine phylogeographic patterns, genetic structure, divergence times, and gene flow among Nasua narica populations throughout the majority of the species' range. We also estimated the ancestral geographic range of N. narica and other procyonid species. We found a high degree of genetic structure and divergence among populations that conform to five evolutionarily significant units. The most southerly distributed population (Panama) branched off much earlier (∼3.8 million years ago) than the northern populations (<1.2 million years ago). Estimated gene flow among populations was low and mostly northwards and westwards. The phylogeographic patterns within N. narica are associated with geographic barriers and habitat shifts likely caused by Pliocene-Pleistocene climate oscillations. Significantly, our findings suggest the dispersal of N. narica was south-to-north beginning in the Pliocene, not in the opposite direction during the Pleistocene as suggested by the fossil record, and that the most recent common ancestor for coati species was most likely distributed in South or Central America six million years ago. Our study implies the possibility that the diversification of Nasua species, and other extant procyonid lineages, may have occurred in South America.

Comparative chromosome painting in Carnivora and Pholidota.

The order of Carnivora has been very well characterized with over 50 species analyzed by chromosome painting and with painting probe sets made for 9 Carnivora species. Representatives of almost all families have been studied with few exceptions (Otariidae, Odobenidae, Nandiniidae, Prionodontidae). The patterns of chromosome evolution in Carnivora are discussed here. Overall, many Carnivora species retained karyotypes that only slightly differ from the ancestral carnivore karyotype. However, there are at least 3 families in which the ancestral carnivore karyotype has been severely rearranged - Canidae, Ursidae and Mephitidae. Here we report chromosome painting of yet another Carnivora species with a highly rearranged karyotype, Genetta pardina. Recurrent rearrangements make it difficult to define the ancestral chromosomal arrangement in several instances. Only 2 species of pangolins (Pholidota), a sister order of Carnivora, have been studied by chromosome painting. Future use of whole-genome sequencing data is discussed in the context of solving the questions that are beyond resolution of conventional banding techniques and chromosome painting.

Kinship shapes affiliative social networks but not aggression in ring-tailed coatis.

Animal groups typically contain individuals with varying degrees of genetic relatedness, and this variation in kinship has a major influence on patterns of aggression and affiliative behaviors. This link between kinship and social behavior underlies socioecological models which have been developed to explain how and why different types of animal societies evolve. We tested if kinship and age-sex class homophily in two groups of ring-tailed coatis (Nasua nasua) predicted the network structure of three different social behaviors: 1) association, 2) grooming, and 3) aggression. Each group was studied during two consecutive years, resulting in four group-years available for analysis (total of 65 individuals). Association patterns were heavily influenced by agonistic interactions which typically occurred during feeding competition. Grooming networks were shaped by mother-offspring bonds, female-female social relationships, and a strong social attraction to adult males. Mother-offspring pairs were more likely to associate and groom each other, but relatedness had no effect on patterns of aggressive behavior. Additionally, kinship had little to no effect on coalitionary support during agonistic interactions. Adult females commonly came to the aid of juveniles during fights with other group members, but females often supported juveniles who were not their offspring (57% of coalitionary interactions). These patterns did not conform to predictions from socioecological models.

The ancestral carnivore karyotype (2n = 38) lives today in ringtails.

Chromosome painting was used to investigate the conservation of high-resolution longitudinal 4',6-diamidino-2-phenylindole (DAPI)/G bands in Carnivore chromosomes. Cat (Felis catus) and raccoon dog (Nyctereutes procyonoides) painting probes were hybridized to the ringtail (Bassaricus astutus), dwarf mongoose (Helogale parvula), and Malagasy civet (Fossa fossa) to identify homologous chromosome elements. The patterns of chromosome segment homology among Carnivore species allowed us to reconstruct and propose the disposition of a high-resolution banded ancestral carnivore karyotype (ACK). Three bi-armed chromosomes consistently found among Caniformia species are represented as 6 homologous acrocentric chromosomes among Feliformia species of Carnivora. However, reexamination of the most basal of Feliformia species, the African palm civet Nandinia, revealed the presence of the 3 heretofore Caniformia bi-armed chromosomes. Because these 3 bi-armed chromosomes are found in both Caniformia and Feliformia lineages, they are presumed ancestral for all Carnivora, suggesting that the ACK chromosome number would be 38, rather than the previously supposed 42. Banded chromosomes of the ACK are used to evaluate the consistency between recently determined molecular phylogenetic relationships and postulated cytogenetic dynamics in the same Carnivore species.

Phylogeny of the Procyonidae (Mammalia: Carnivora): molecules, morphology and the Great American Interchange.

The Procyonidae (Mammalia: Carnivora) have played a central role in resolving the controversial systematics of the giant and red pandas, but phylogenetic relationships of species within the family itself have received much less attention. Cladistic analyses of morphological characters conducted during the last two decades have resulted in topologies that group ecologically and morphologically similar taxa together. Specifically, the highly arboreal and frugivorous kinkajou (Potos flavus) and olingos (Bassaricyon) define one clade, whereas the more terrestrial and omnivorous coatis (Nasua), raccoons (Procyon), and ringtails (Bassariscus) define another clade, with the similar-sized Nasua and Procyon joined as sister taxa in this latter group. These relationships, however, have not been tested with molecular sequence data. We examined procyonid phylogenetics based on combined data from nine nuclear and two mitochondrial gene segments totaling 6534bp. We were able to fully resolve relationships within the family with strongly supported and congruent results from maximum parsimony, maximum likelihood, minimum evolution, and Bayesian analyses. We identified three distinct lineages within the family: a (Nasua, Bassaricyon) clade, a (Bassariscus, Procyon) clade, and a Potos lineage, the last of which is sister to the other two clades. These findings, which are in strong disagreement with prior fossil and morphology-based assessments of procyonid relationships, reemphasize the morphological and ecological flexibility of these taxa. In particular, morphological similarities between unrelated genera possibly reflect convergence associated with similar lifestyles and diets rather than ancestry. Furthermore, incongruence between the molecular supermatrix and a morphological character matrix comprised mostly of dental characters [Baskin, J.A., 2004. Bassariscus and Probassariscus (Mammalia, Carnivora, Procyonidae) from the early Barstovian (Middle Miocene). J. Vert. Paleo. 24, 709-720] may be due to non-independence among atomized dental characters that does not take into account the high developmental genetic correlation of these characters. Finally, molecular divergence dating analyses using a relaxed molecular clock approach suggest that intergeneric and intrageneric splits in the Procyonidae mostly occurred in the Miocene. The inferred divergence times for intrageneric splits for several genera whose ranges are bisected by the Panamanian Isthmus is significant because they suggest diversification well precedes the Great American Interchange, which has long been considered a primary underlying mechanism for procyonid evolution.

Phylogeny of the caniform carnivora: evidence from multiple genes.

The monophyletic group Caniformia in the order Carnivora currently comprises seven families whose relationships remain contentious. The phylogenetic positions of the two panda species within the Caniformia have also been evolutionary puzzles over the past decades, especially for Ailurus fulgens (the red panda). Here, new nuclear sequences from two introns of the beta-fibrinogen gene (beta-fibrinogen introns 4 and 7) and a complete mitochondrial (mt) gene (ND2) from 17 caniform representatives were explored for their utilities in resolving higher-level relationships in the Caniformia. In addition, two previously available nuclear (IRBP exon 1 and TTR intron 1) data sets were also combined and analyzed simultaneously with the newly obtained sequence data in this study. Combined analyses of four nuclear and one mt genes (4417 bp) recover a branching order in which almost all nodes were strongly supported. The present analyses provide evidence in favor of Ailurus fulgens as the closest taxon to the procyonid-mustelid (i.e., Musteloidea sensu stricto) clade, followed by pinnipeds (i.e., Otariidae and Phocidae), Ursidae (including Ailuropoda melanoleuca), and Canidae, the most basal lineage in the Caniformia. The potential utilities of different genes in the context of caniform phylogeny were also evaluated, with special attention to the previously unexplored beta-fibrinogen intron 4 and 7 genes.

Phylogenetic inference and comparative evolution of a complex microsatellite and its flanking regions in carnivores.

We sequenced locus Mel 08, with complex short repetitive motifs, in 24 carnivore species belonging to five different families in order to explore mutational changes in the region in the context of locus and species evolution. This non-coding locus includes up to four different parts or repetitive motifs showing size variability. The variability consists of repeat additions and deletions; substitutions, insertions and/or deletions creating interruptions in the repeat; and substitutions, insertions and deletions in the flanking regions. The locus has different repeat expansions in different carnivore subfamilies. We hypothesize that the complexity of this locus is due to a high mutation rate at an ancestral DNA sequence and, thus, prompts the emergence of repeats at mutational hotspots. High levels of homoplasy were evident, with nine electromorphs representing 28 haplotypes never shared across species. The variability in flanking regions was informative for phylogenetic inference and their evolutionary content. Tree topologies were congruent with relevant hypotheses on current conflicts in carnivore phylogenies, such as: (i) the monophyly of Lutrinae, (ii) the paraphyly of Mustelinae, (iii) the basal position of the Eurasian badger, Meles meles , in the Mustelidae, (iv) the classification of skunks as a separate family, Mephitidae, and (v) the placement of the red panda, Ailurus fulgens , as a monotypic family, Ailuridae, at a basal position in the Musteloidea.

Mitochondrial phylogeography and subspecific variation in the red panda (Ailurus fulgens): implications for conservation.

The red panda (Ailurus fulgens) is an endangered species and its present distribution is restricted to isolated mountain ranges in western China (Sichuan, Yunnan, and Tibet provinces) and the Himalayan Mountains chain of Nepal, India, Bhutan, and Burma. To examine the evolutionary history across its current range, and to assess the genetic divergence among current subspecies and population structure among different geographic locations, we sequenced mitochondrial DNA from the control region (CR) and cytochrome (cyt) b gene for 41 individuals in Sichuan, Yunnan, Tibet of China, and Burma. 25 CR haplotypes (10 for cyt b) were identified from 11 geographic locations. Only three haplotypes were shared among sample localities, including one among current subspecies. Nine haplotypes were shared with the study of Su et al. [Mol. Biol. Evol. 18 (2001) 1070]. CR haplotype diversity was high (0.95+/-0.02) and nucleotide diversity among all haplotypes was relatively low (0.018+/-0.009). Phylogenetic confirmed trees show a shallow pattern with very little structure or statistical robustness. The application of two coalescent-based tests for population growth allowed us to interpret this phylogeny as the result of a recent population expansion. Analysis of molecular variance and nested clade analysis failed to detect significant geographic structure in both data sets. The lack of significant differentiation between subspecies does not indicate the presence of evolutionary significant units. We suggest that the present population structure has resulted from habitat fragmentation and expansion from glacial refugia. Due to its habitat requirements it is likely that the red panda has undergone bottlenecks and population expansions several times in the recent past. The present population may exhibit a pattern reminiscent of a relatively recent population expansion.