Exon Capture Museomics Deciphers the Nine-Banded Armadillo Species Complex and Identifies a New Species Endemic to the Guiana Shield

Abstract

The nine-banded armadillo (Dasypus novemcinctus) is the most widespread xenarthran species across the Americas. Recent studies have suggested it is composed of 4 morphologically and genetically distinct lineages of uncertain taxonomic status. To address this issue, we used a museomic approach to sequence 80 complete mitogenomes and capture 997 nuclear loci for 71 Dasypus individuals sampled across the entire distribution. We carefully cleaned up potential genotyping errors and cross-contaminations that could blur species boundaries by mimicking gene flow. Our results unambiguously support 4 distinct lineages within the D. novemcinctus complex. We found cases of mito-nuclear phylogenetic discordance but only limited contemporary gene flow confined to the margins of the lineage distributions. All available evidence including the restricted gene flow, phylogenetic reconstructions based on both mitogenomes and nuclear loci, and phylogenetic delimitation methods consistently supported the 4 lineages within D. novemcinctus as 4 distinct species. Comparable genetic differentiation values to other recognized Dasypus species further reinforced their status as valid species. Considering congruent morphological results from previous studies, we provide an integrative taxonomic view to recognize 4 species within the D. novemcinctus complex: D. novemcinctus, D. fenestratus, D. mexicanus, and D. guianensis sp. nov., a new species endemic of the Guiana Shield that we describe here. The 2 available individuals of D. mazzai and D. sabanicola were consistently nested within D. novemcinctus lineage and their status remains to be assessed. The present work offers a case study illustrating the power of museomics to reveal cryptic species diversity within a widely distributed and emblematic species of mammals.

Keywords: Integrative taxonomy; mito-nuclear discordance; multilocus phylogeny; museomics; phylogeography; species delimitation.

FIGURE 1.

Phylogenetic relationships reconstructed by maximum likelihood and rooted using Dasypus kappleri from a) the mitogenomes of 81 individuals, and b) the 832 filtered nuclear loci of 62 individuals. Circles indicate bootstrap support (BS > 94) for the main nodes and node labels represent gene- and site-concordance factors (gCF/sCF). Maps represent the distribution of individuals according to their lineage and to c) mitogenomic and d) nuclear data. Individuals with discordant mito-nuclear lineages are represented by stars and D. pilosus by open circles. The D. novemcinctus distribution is highlighted. Photo credits: A. Baertschi (xenarthrans.org), K. Miller, Andresiade, A. Reed (iNaturalist.org), and Q. Martinez.

FIGURE 2.

Phylogenetic relationships reconstructed by maximum likelihood from the 837 filtered nuclear loci and rooted using Dasypus kappleri and D. septemcinctus as outgroups. The color of the diagrams represents the assignment of individuals to lineages. For phylogenetic analyses (IQ-Tree and ASTRAL) barplot represents qualitative assignment to the monophyletic lineages reconstructed. For the Admixture analysis, barplot corresponds to the assignment probability. Finally, for phylogenetic delimitation barplot represents the species support.

FIGURE 3.

Principal component analysis of genetic variance (PCA) conducted on the 3350 SNPs in 837 nuclear loci shared by all 56 individuals. a) Projection on first (PC1) and second (PC2) axes. b) Projection on first (PC1) and third (PC3) axes. The outer circles represent mitochondrial lineages and pie charts the nuclear lineages identified in admixture analyses. Individuals of D. pilosus, D. sabanicola and D. mazzai are labeled.

FIGURE 4.

Distribution map and genetic composition of individuals of the four recognized species: Dasypus mexicanus (Northern lineage), Dasypus fenestratus (Central lineage), Dasypus novemcinctus (Southern lineage), and Dasypus guianensis sp. nov. (Guianan lineage ). Outer circles represent mitochondrial lineages and pie charts the assignment probability generated by admixture analyses based on nuclear data. Open circles indicate individuals for which only mitogenomic data is available. Squares represent individuals from Hautier et al. (2017) according to their morphogroup affiliation based on a discriminant analysis of skull shape using geometric morphometrics and for which no genetic data are available. Three-dimensional reconstructions of skulls obtained using microCT scans of representative specimen vouchers are presented for each species with frontal paranasal sinuses and recesses highlighted: USNM Mammals 33867 (Dasypus mexicanus), AMNH Mammals M-32356 (Dasypus fenestratus), AMNH Mammals M-136252 (Dasypus novemcinctus), and ROM Mammals 32868 (Dasypus guianensis sp. nov.).