Phylogeny and Molecular Species Delimitation of Long-Nosed Armadillos (Dasypus: Cingulata) Supports Morphology-Based Taxonomy

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Zoological Journal of the Linnean Society, 2019, XX, 1–13. With 3 figures. Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zly091/5298137 by University of Kansas user on 20 January 2019

Phylogeny and molecular species delimitation of long-nosed armadillos (Dasypus: Cingulata) supports morphology-based taxonomy

ANDERSON FEIJÓ1,2,3*, , JÚLIO F. VILELA3,4,5, JILONG CHENG1, , MARCO ANTÔNIO A. SCHETINO6, RAPHAEL T. F. COIMBRA6, CIBELE R. BONVICINO7,8, FABRÍCIO R. SANTOS6,9, BRUCE D. PATTERSON3 and PEDRO CORDEIRO-ESTRELA2,10

1Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing, China 2Laboratório de Mamíferos, Departamento de Sistemática e Ecologia, Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba, Campus I, João Pessoa, PB, Brazil 3Integrative Research Center, Field Museum of Natural History, Chicago, IL, USA 4Coordenação de Identificação e Planejamento de Ações para Conservação – COPAN, Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio, Brasília, DF, Brazil 5Campus Amilcar Ferreira Sobral, Universidade Federal do Piauí, Floriano, PI, Brazil 6Laboratório de Biodiversidade e Evolução Molecular, Departamento de Biologia Geral, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 7Programa de Genética, Instituto Nacional de Câncer, Rio de Janeiro, Brazil 8Laboratório de Biologia e Parasitologia de Mamíferos Reservatórios Silvestres, Pavilhão Lauro Travassos, Instituto Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil 9Pós-Graduação em Zoologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 10Programa de Pós-Graduação em Ciências Biológicas (Zoologia), Departamento de Sistemática e Ecologia, Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba, Campus I, João Pessoa, PB, Brazil

Received 19 August 2018; revised 17 October 2018; accepted for publication 26 November 2018

The armadillo genus Dasypus is the most species-rich and widely distributed genus of the order Cingulata and it has a dynamic taxonomic history. Recent morphology-based studies have proposed new taxonomic arrangements, but these were not yet assessed with molecular data. The two comprehensive phylogenetic hypotheses available for the genus are conflicting and were each based on a subset of taxa, hampering a proper evaluation of species boundaries. Using a multilocus molecular dataset, based on the broadest geographic sampling of Dasypus to date, we inferred the phylogenetic relationships of all species of the genus, including the recently reinstated D. beniensis and D. pastasae. We tested recent taxonomic hypotheses using several species-delimitation approaches. Our phylogeny recovered three main lineages of long-nosed armadillos that we treat as subgenera (Hyperoambon, Muletia and Dasypus) and identified the majority of its diversification as having occurred during the Pliocene. Molecular species delimitation supported morphological evidence in assigning D. hybridus as a subspecies of D. septemcinctus and confirming the split of the D. kappleri complex into three species. Our results strongly support the recognition of Guiana Shield populations formerly assigned to D. novemcinctus as a distinct species. The phylogenetic positions of D. mazzai and D. sabanicola remain uncertain. Further investigation using faster-evolving genes and additional samples may help to clarify the relationships of these young species.

ADDITIONAL KEYWORDS: Dasypodidae – Guiana Shield – species delimitation – subgenera – Xenarthra.

*Corresponding author. E-mail: [email protected]

INTRODUCTION et al., 2016) datasets. However, these phylogenetic

hypotheses were incongruent and were based on Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zly091/5298137 by University of Kansas user on 20 January 2019 Xenarthra is one of the early diverging clades of relatively few individuals per species. Understanding placental mammals (Meredith et al., 2011) and includes of species boundaries, especially those of widely the surviving representatives of an ancient South distributed taxa, depends on adequate intraspecific American mammalian radiation (Patterson & Pascual, sampling across the range (Wiens & Penkrot, 2002; 1968). The group originated around 70 Mya (Gibb et al., Lim et al., 2012; Watanabe, 2016). In this study, 2016) and reached a peak of diversification with over using the broadest geographic coverage of Dasypus 150 genera during the Tertiary, when South America to date, we investigate the phylogenetic relationships was isolated (McKenna & Bell, 1997). Currently, only of all species of the genus based on mitochondrial a remnant of this diversity remains, with 14 genera and nuclear DNA sequences, including the recently and 38 species of armadillos, tree sloths and anteaters revalidated Dasypus beniensis Lönnberg, 1942 and (Wetzel et al., 2008; Abba et al., 2015; Miranda et al., Dasypus pastasae (Thomas, 1901). Additionally, 2017; Feijó et al., 2018) through multiple species-delimitation algorithms, we Among armadillos, the genus Dasypus Linnaeus, test the recently proposed morphological classification 1758, with eight extant species, is the most species-rich, using molecular data. and it exhibits the broadest distribution of all extant and fossil xenarthrans, covering an area of 19 100 000 km2 from Argentina to the United States (Feijó et al., 2018). Long-nosed armadillos are one of the few South MATERIAL AND METHODS American mammalian lineages that have successfully invaded the Nearctic, and they are the only vertebrates Sampling, amplification and sequencing to reproduce via obligatory polyembryony (Loughry & Eighteen specimens of the genus Dasypus from Brazil, Mcdonough, 2013). They are of particular interest to Uruguay and Argentina were sequenced, representing biomedical research, because it is the only non-human five of the eight currently recognized species mammal group known to be susceptible to leprosy in (Supporting Information, Table S1; Fig. 1). Because nature (Truman, 2005; Loughry & Mcdonough, 2013) most of these individuals were associated with and may also host other human pathogens, such as vouchered material, their identification was based on Trypanosoma cruzi and Leishmania spp. (Lainson diagnostic morphological characters described by Feijó et al., 1982; Morocoima et al., 2012). et al. (2018). We amplified two mitochondrial markers: Since Systema naturae (Linnaeus, 1758), up to 26 cytochrome b (Cytb) and cytochrome oxidase subunit species have been described in the genus Dasypus. I (COI), and one nuclear marker: exon 28 of the von Broad morphology-based studies in the first half of Willebrand factor (vWF). We also obtained 12 sequences the 20th century showed that many supposed species of Cytb, 13 sequences of COI and two sequences of vWF were described based on traits with high intraspecific from GenBank (Supporting Information, Table S1). In variability (Lönnberg, 1928; Hamlett, 1939; Lönnberg, total, the dataset encompassed 2127 bp (723 bp for 1942). As a result, only six species were recognized by Cytb, 681 bp for COI and 723 bp for vWF) of sequences Cabrera (1958) and Wetzel & Mondolfi (1979). After a that were obtained from 29, 37 and 19 individuals, period of only minor taxonomic changes, recent studies respectively. have uncovered previously overlooked diversity Total genomic DNA was extracted using the phenol- and proposed novel taxonomic arrangements. For chloroform method (modified from Sambrook & Russel, instance, Feijó & Cordeiro-Estrela (2016) proposed 2001). Polymerase chain reaction (PCR) was carried splitting the Amazonian greater long-nosed armadillo out using Platinum Taq DNA Polymerase kit following (Dasypus kappleri Krauss, 1862) into three species. the manufacturer’s protocol and using specific primers One year later, Billet et al. (2017) and Hautier et al. and annealing temperatures (Supporting Information, (2017) recognized morphological groups within the Table S2). All sequences newly reported in this study nine-banded armadillo, suggesting it might comprise were deposited in GenBank (Supporting Information, a species complex, rather than a single broadly Table S1). distributed species. Feijó et al. (2018) treated Dasypus In addition to the Dasypus sequences, we also hybridus (Desmarest, 1804) as a subspecies of Dasypus included sequences from other extant genera of septemcinctus Linnaeus, 1758. All these works relied armadillos as outgroups (Chlamyphorus Harlan, 1825, exclusively on morphology in determining species Cabassous McMurtrie, 1981, Chaetophractus Fitzinger, limits. 1871, Euphractus Wagler, 1830, Priodontes F. Cuvier, Until now, only two studies have proposed a 1825, Tolypeutes Illiger, 1811 and Zaedyus Ameghino, phylogeny of the entire genus Dasypus, based on either 1889) obtained from GenBank. All sequences were morphological (Castro et al., 2015) or molecular (Gibb manually edited and assembled using SeaView

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Figure 1. Geographical distribution of sampled localities (dots) of Dasypus (including GenBank records) used in this study. Species ranges follow Feijó et al. (2018). Question marks represent approximate locations. v.4.6.1 (Gouy et al., 2010). Sequence alignments were majority rule consensus trees and summary statistics. performed using the MUSCLE algorithm (Edgar, 2004) ML phylogenies were inferred with RAxML v.8.2.10 implemented in MEGA 6 (Tamura et al., 2013). (Stamatakis, 2014) using a GTRGAMMAI evolution model and 1000 bootstrapping replicates. The trees were then edited in FigTree 1.4.2 (available at http:// Phylogenetic and divergence time analyses tree.bio.ed.ac.uk/software/fig-tree/). To evaluate the phylogenetic relationships among We estimated divergence times of Dasypus species species of Dasypus, trees were reconstructed from using the Bayesian phylogenetic approach in BEAST the concatenated mtDNA and vWF sequences using 1.8.2 (Drummond et al., 2012). The analysis was both Bayesian inference (BI) and maximum likelihood performed using the concatenated mtDNA dataset, (ML) approaches. We concatenated the Cytb and COI because of its higher resolution and support. We used genes in ML analyses, because they are linked and soft bounds of the fossil calibration (FC) points and likely evolved under similar constraints, but all three the parameters were set to log-normal distributions loci were partitioned in the BI analyses. We used with a 95% interval boundary. Three FC points were jModelTest 2.1.7 (Darriba et al., 2012) to select the used. FC1: Kuntinaru boliviensis for the appearance best model of molecular evolution using the Bayesian of Tolypeutinae (23.0–37.8 Myr) (Billet et al., 2011) – information criterion (BIC). The following substitution we assigned a minimum age of 23.0 Myr [offset = 23.0; models were selected: GTR+I+G for COI, HKY+I+G for mean = 26.0; log (SD) = 3.6]. FC2: Chaetophractus Cytb and HKY+G for vWF. Two parallel runs of one cold sp. (5.3–11.6 Myr) (Tauber, 2005) – we assigned a and three heated Markov chain Monte Carlo (MCMC) minimum age of 5.3 Myr [offset = 5.3; mean = 4.0; log analyses were performed for 20 million generations, (SD) = 3.0]. FC3: the oldest known fossil of Dasypus with trees sampled every 1000 generations, until from the Miocene of Argentina (6.8–9.0 Myr) (Cione convergence (SD < 0.01). The first 25% of the Markov et al., 2000) – we assigned a minimum age of 6.0 chain samples (N = 20 000) were discarded as burn-in, Myr [offset = 6.0; mean = 1.0; log (SD) = 1.0]. Prior and the remaining samples were used to generate to divergence calibration, the mtDNA clock model

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, XX, 1–13 4 A. FEIJÓ ET AL. was selected based on marginal likelihood estimated than using time to estimate branching rates, as in the

(MLE) from stepping stone and path sampling (Baele GMYC model, the PTP directly uses the number of Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zly091/5298137 by University of Kansas user on 20 January 2019 et al., 2012) with 50 path steps, 500 000 iterations and substitutions. We implemented the PTP method in the samples every 500 generations; the procedure was run majority consensus tree from the above-concatenated twice to ensure convergence. According to the MLE Bayesian analysis of the final dataset in the bPTP analysis, a strict clock was chosen for the mtDNA web server (http://species.h-its.org/ptp) for 500 000 dataset. The MCMC chains were run for 20 million generations, with thinning set to 100 and burn-in generations, with sampling every 1000 generations. to 10%. The bPTP web server runs the original ML The convergence of the MCMC chains was examined version of the PTP, as well as an updated version, in Tracer 1.6 (Rambaut et al., 2014) and the first which adds Bayesian support to delimited species 25% of runs were discarded as burn-in. The trees and in the input tree (Bayesian implementation of the posteriors were displayed and edited in FigTree 1.4.2. PTP model or bPTP). Lastly, we used the Automatic Barcode Gap Discovery (ABGD) method (Puillandre et al., 2012) to calculate all pairwise distances in the Species delimitation dataset,to evaluate interspecific divergences and to To test species hypotheses for the genus Dasypus, sort the terminals into candidate species based on we used five different species discovery methods the calculated P values. We performed the ABGD and one species validation method to delimit species analyses online (http://wwwabi.snv.jussieu.fr/public/ boundaries (Carstens et al., 2013; Razkin et al., 2017). abgd/) using three different distance metrics: Jukes- Cantor (JC69) (Jukes et al., 1969), K2P (Kimura, 1980) and simple distance (p-distance; Nei & Kumar, 2000). Molecular species discovery We analysed the data using Pmin: 0.001, Pmax: 0.1, As both genetic distances and PID (Liberal) require a Steps: 10, X (relative gap width): 1.5, with the other priori species designation, we set 17 putative ‘species’ parameters set to default values. (including outgroups), based on a combination of phylogenetic topologies from the gene trees (BI and ML trees) and the recent taxonomic arrangement Molecular species validation proposed by Feijó et al. (2018). To analyse genetic We used the program *BEAST (Heled & Drummond, distance, we examined the mean interspecific Kimura 2010) in BEAST 1.8.2 to validate the candidate two-parameter (K2P) distance and the p-distance, species discovered with mtDNA sequence data and calculated for each candidate species in MEGA 6.04 the topology was obtained from BI and ML trees. (Tamura et al., 2013). The species delimitation plugin The substitution models were unlinked and the (Masters et al., 2011) in GENEIOUS 9.1.4 (Kearse substitution parameters were set according to the et al., 2012) was used to obtain PID (Liberal) statistical jModeltest results. We chose the Yule process species values, which are based on the putative species tree priors in a piecewise linear fashion and a constant resulting from BI trees and used to calculate the mean root population size model. The uncorrelated relaxed probability of the interspecific genetic distance ratios log-normal clock was set for all loci. The MCMC chains for these candidate species (Xu et al., 2015). were run for 100 million generations with sampling The other three methods do not require a priori every 2000 generations. The convergence of the MCMC assignments regarding putative species. The chains was examined in Tracer 1.6, and the first 25% Generalized Mixed Yule Coalescent method (GMYC) were discarded as burn-in. (Pons et al., 2006) uses a ML framework to delimit species based on ultrametric trees and estimates a transition point on a tree before which all nodes reflect RESULTS species diversification events and after which all nodes represent a population-coalescent process (Pons Phylogeny and divergence time et al., 2006; Razkin et al., 2017). The ultrametric guide The monophyly of the genus Dasypus is well supported tree resulted from the previously obtained divergence both in ML and BI analyses. This is in accordance with time analysis. The GMYC tests were run using the previous studies (Delsuc et al., 2003; Gibb et al., 2016). web server (http://species.h-its.org/gmyc) under the All trees of the mtDNA genes recovered three main single-threshold model, because prior studies have clades (PP > 1.00, BS > 92) (Fig. 2). The first clade shown that the output of the multiple-threshold model includes the three species of the D. kappleri complex offers no improvement (Fujisawa & Barraclough, (D. kappleri, D. beniensis and D. pastasae) with strong 2013). The Poisson tree process (PTP) method (Zhang support (PP = 1.00, BS = 100). The second clade et al., 2013) seeks to identify significant changes in the includes individuals attributed to D. septemcinctus rate of branching in a phylogenetic tree, but rather septemcinctus and D. septemcinctus hybridus. This

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Figure 2. Phylogenetic tree for Dasypus species based on mtDNA; the subgenera Hyperoambon, Muletia and Dasypus are highlighted (see Discussion). Nodal support values are Bayesian posterior probabilities. Blue bars on nodes indicate the 95% highest posterior density (HPD). NCA = North and Central America; SA = South America. clade is sister to a clade that includes D. novemcinctus, the late Miocene [7.83–11.99 Myr, 95% highest posterior D. pilosus (Fitzinger, 1856), D. mazzai Yepes, 1933 density (HPD)] (Fig. 2). In the D. kappleri complex, and D. sabanicola Mondolfi, 1968. In this last clade, D. beniensis diverged from D. kappleri and D. pastasae a lineage of D. aff. novemcinctus (hereafter named at approximately 4.88 Myr (3.68–6.29 Myr, 95% HPD). Dasypus sp.) from French Guiana (FG) is the first Dasypus septemcinctus and the D. novemcinctus species to diverge, followed by D. pilosus, a clade including complex diverged at approximately 5.14 Myr (4.08–6.45 D. novemcinctus from North and Central America Myr, 95% HPD). Subsequent divergence events separated (NCA) and another clade with samples of D. sabanicola, Dasypus sp. from French Guiana at 3.95 Myr (3.11–4.98 D. mazzai and D. novemcinctus from South America Myr, 95% HPD), D. pilosus at approximately 3.28 Myr (SA). The concatenated mitochondrial and nuclear tree and D. novemcinctus NCA from the D. sabanicola, also recovered the three main groups, albeit with lower D. mazzai and D. novemcinctus SA at approximately support (Supporting Information, Fig. S1). 2.90 Myr (Fig. 2). Pleistocene divergence events The divergence time data indicated that the most include splits leading to D. pastasae and D. kappleri (at recent common ancestor of the extant species of approximately 1.15 Myr) and to D. mazzai, D. sabanicola Dasypus diverged at approximately 9.85 Myr during and D. novemcinctus from South America.

Species delimitation and recursive partitions vary between four and five. Molecular species discovery We consider this result to indicate that there are four Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zly091/5298137 by University of Kansas user on 20 January 2019 accurate groups (P = 0.0129/0.0159). In Figure 3, The three main clades are approximately 10% we compare the results of our molecular species distant in mtDNA sequences. The smallest distance delimitations with the most recent morphology- between analysed samples is 0.6/0.6% (K2P/ based classification (Feijó et al., 2018). Consonantly, uncorrected p-distance) between D. septemcinctus the *BEAST species tree presents a similar topology hybridus and D. septemcinctus septemcinctus, as the gene tree, recognizing eight species with whereas the highest distance is 12.1/10.9% between relatively high support (PP > 0.9) for most of the D. beniensis and D. septemcinctus (Table 1). The nodes (Fig. 3). results based on the BI tree show high PID (Liberal) values of ≥0.95 for D. beniensis, D. pastasae, Dasypus sp. French Guiana and D. novemcinctus SA (Table 2). Dasypus septemcinctus hybridus and DISCUSSION D. septemcinctus septemcinctus are separated by a This is the first work to explore the phylogenetic very low distance (1.3%), whereas D. pastasae and relationships of Dasypus using a broad geographic D. kappleri are more distant (2.1%). It is noteworthy sampling and all currently recognized species. We that the average distance among specimens of provide a detailed time-calibrated phylogenetic D. novemcinctus from North and Central America is framework for Dasypus with a quantitative very high (2.9%). assessment of interspecific genetic divergences. The single-threshold GMYC model resulted We uncovered a novel lineage of Dasypus from the in eight clusters and 18 entities with confidence Guiana Shield, provided strong molecular support intervals of 7–10 and 15–18, respectively for D. beniensis and D. pastasae, and rejected the (Supporting Information, Table S3). The ML reciprocal monophyly of D. septemcinctus and obtained with the GMYC model is significantly D. hybridus. We discuss these changes in the light higher than the likelihood observed in the null of previous phylogenetic works. In addition, we model: GYMC model = 59.37; null model = 52.94; define subgeneric names for the three main clades of likelihood ratio = 12.86; and LR test < 0.001. The long-nosed armadillos and discuss the implications ML and Bayesian solution PTPs identified 11 of divergence time estimates for the historical clusters as ‘species’, eight of which included only one biogeography of the group. or two specimens (Fig. 3). The ABGD results using different parameter combinations and the initial partition of 7–25 ‘species’ (Supporting Information, Dasypus phylogeny and species limits Table S4) agreed on four species (i.e. D. beniensis, Our phylogeny provides three important advances D. pastasae/D. kappleri, D. septemcinctus and compared to the previous molecular phylogeny (Gibb D. novemcinctus; Fig. 3). Different numbers of groups et al., 2016), mainly due to our more comprehensive are obtained for the recursive partition with differing geographic sampling. First, we confirm that the prior maximal distances (P), but the stable initial nine-banded armadillo D. novemcinctus from most of

Table 1. Pairwise mtDNA genetic distances using the Kimura two-parameter (K2P) distance (bottom left) and the p-distance (top right) between nine clades of Dasypus. NCA = North and Central America; SA = South America; FG = French Guiana

Species Distance between species (%)

Dben Dpas Dkap Dssep Dshyb DspFG Dpil DnovNCA DnovSA

D. beniensis - 6.5 5.7 10.9 10.9 9.9 10.4 10.7 10.3 D. pastasae 6.9 - 1.6 9.3 9.4 9 8.7 8.8 8.6 D. kappleri 6 1.6 - 9.2 9.3 8.8 8.8 8.7 8.5 D. sept. septemcinctus 12.1 10.3 10.1 - 0.6 6.5 6.8 7.3 6.1 D. sept. hybridus 12.1 10.3 10.2 0.6 - 6.3 6.7 7.2 6.4 D. sp. FG 10.8 9.9 9.6 6.9 6.7 - 4.5 4.9 3.9 D. pilosus 11.5 9.5 9.5 7.3 7.2 4.7 - 4.6 3.6 D. novemcinctus NCA 11.8 9.6 9.5 7.3 7.7 5.2 4.8 - 3.5 D. novemcinctus SA 11.4 9.4 9.3 6.4 6.8 4.1 3.8 3.6 -

Table 2. Summary of the mean intraspecific distance, nearest species, mean distance to nearest species, and probability with the 95% confidence interval for the nine candidate ‘’species’. NCA = North and Central America; SA = South America Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zly091/5298137 by University of Kansas user on 20 January 2019

Species Mean Intraspecific Nearest Species Mean distance to P ID(Liberal) Distance nearest species

D. beniensis 0.004 D. kappleri 0.078 0.95 (0.80, 1.0) D. pastasae 0.001 D. kappleri 0.021 0.95 (0.80, 1.0) D. kappleri 0.004 D. pastasae 0.021 0.89 (0.75, 1.0) D. sept. septemcinctus 0.009 D. sept. hybridus 0.013 0.77 (0.67, 0.87) D. sept. hybridus 0.003 D. sept. septemcinctus 0.013 0.87 (0.72, 1.0) D. sp. French Guiana - D. pilosus 0.063 0.96 (0.83, 1.0) D. pilosus 0.012 D. novemcinctus SA 0.057 0.85 (0.70, 1.0) D. novemcinctus NCA 0.029 D. novemcinctus SA 0.063 0.83 (0.73, 0.94) D. novemcinctus SA 0.012 D. pilosus 0.057 0.96 (0.90, 1.0)

South America is a distinct lineage from populations geographic coverage in their analysis, which might from French Guyana and from North and Central result in inaccurate phylogenetic inference (Wiens America. Based on the phylogenetic position, genetic & Servedio, 1997; Lohse, 2009; Bergsten et al., 2012; divergence and the species delimitation analyses, Lim et al., 2012; Avendano et al., 2017). Samples from Dasypus specimens from French Guyana should be both D. mazzai and D. sabanicola used by Gibb et al. recognized as a species distinct from D. novemcinctus, (2016) clustered with geographically distant samples as was also suggested by Gibb et al. (2016). Guiana of D. novemcinctus. Dasypus mazzai from Argentina Shield specimens are known to exhibit pronounced clustered with D. novemcinctus from Paraíba state, molecular (Huchon et al., 1999; Gibb et al., 2016) and north-eastern Brazil, whereas D. sabanicola from the morphological (Billet et al., 2017; Hautier et al., 2017; savannas of Venezuela clustered with D. novemcinctus Feijó et al., 2018) disparity from other D. novemcinctus from Amazonia of western Brazil. Recent speciation, populations. On the other hand, the relationships of a limited number of informative sites, ancestral D. novemcinctus from North and Central America polymorphism, hybridization and introgression, are still uncertain. Because the type locality of all common attributes of young species (Kekkonen D. novemcinctus lies in Pernambuco, Brazil, specimens et al., 2015; Gibb et al., 2016), might have generated from South America, excluding those from the Guiana those patterns. Despite the inclusion of D. mazzai Shield, should keep that name (for nomenclatural and D. sabanicola within D. novemcinctus and their discussion, see: Feijó et al., 2018). Therefore, Central conspicuous morphometric overlap, three datasets and North American populations could be (1) suggest their distinctness. Feijó et al. (2018) found D. novemcintus based on monophyly, gene flow between consistent diagnostic carapace traits for the two Central and South America (Arteaga et al., 2012) taxa, Abba et al. (2018) showed distinctions in 212 bp and the lack of consistent morphological distinctions rRNA between the holotype of D. mazzai and both (Feijó et al., 2018); or (2) a distinct and cryptic species D. novemcinctus and D. septemcinctus hybridus, and, based on the high intraspecific divergence (~ 2.9%), finally, the widely disjunct geographic distributions paranasal sinus morphology (Billet et al., 2017) and of D. sabanicola and D. mazzai precludes any gene 3D skull shape (Hautier et al., 2017). flow. Thus, we advocate recognizing these two taxa as Despite those advances, the monophyly of Dasypus distinct species until there is further evidence from novemcinctus remains uncertain, because D. mazzai faster-evolving genes and increased sample size for and D. sabanicola are found nested among South both species. American specimens of D. novemcinctus. In the Gibb A second main finding of this work is genetic et al. (2016) phylogeny, D. mazzai and D. sabanicola support for the three species of the Dasypus kappleri were sister to D. novemcinctus from the United complex. Phylogenetic trees provide well-supported States and D. pilosus. Our phylogeny recovered reciprocal monophyly for D. kappleri, D. beniensis and D. pilosus as the sister lineage to a clade that includes D. pastasae. Tree-based delimitation (GYMC and PTP) D. novemcinctus (non-Guiana specimens), D. mazzai and recovery analyses also recovered the three species. and D. sabanicola. This conflict might be due to the In contrast, the ABGD clustering method recognized shorter gene sequences used in our analysis compared D. beniensis, the older species with a Miocene/ to the complete mitogenomes of Gibb et al. (2016). Pliocene origin and D. pastasae+D. kappleri, which However, it seems more likely attributable to the diverged during the Pleistocene. Previous studies poorer sampling of intraspecific variability and have demonstrated the impaired performance of

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Figure 3. Summary of the results of the species delimitation analyses (including discovery and validation approaches) on the Bayesian phylogenetic tree from the concatenated mtDNA dataset. Coloured boxes represent resultant candidate species based on GMYC, PTP, ABGD and morphology approaches (Feijó et al., 2018). The coalescent species tree was estimated by *BEAST and posterior probabilities are shown at the nodes. NCA = North and Central America; SA = South America.

ABGD relative to tree-based models, such as PTP and delimitation analyses failed to recover any partition in GMYC, in situations with poor intraspecific sampling D. septemcinctus (including hybridus). Feijó et al. (2018) (Puillandre et al., 2012; Talavera et al., 2013; Zhang synonymized D. hybridus with D. septemcinctus due to a et al., 2013; Kekkonen et al., 2015). lack of clear diagnostic characteristics but maintained The third major finding of this work is to reject its subspecific status based on partial diagnosability by the reciprocal monophyly of D. septemcinctus and morphometric traits and biogeographical distribution. D. hybridus on molecular grounds. All species Gibb et al. (2016) reported almost identical

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, XX, 1–13 PHYLOGENY AND SPECIES DELIMITATION OF DASYPUS 9 mitogenome sequences involving one specimen from siblings. Lastly, the third lineage includes mid-sized

Argentina (coded in our tree as DhybAR) and one from Dasypus with 7–11 movable bands and they generate Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zly091/5298137 by University of Kansas user on 20 January 2019 Uruguay (DhybUR). Both specimens were clustered four genetically identical offspring. They have diverse together with a third sequence from Rio Grande do ecologies, inhabiting native forests, open vegetation Sul, southern Brazil, and this clade is separated from and human-disturbed environments. To denote these a clade with specimens from central and northern distinctive monophyletic lineages in Dasypus, and to Brazil in our concatenated mitochondrial and nuclear better reflect its phylogenetic structure and distinctive tree (Supporting Information, Fig. S1). Following species biology, we recognize three previously named Feijó et al. (2018), the former clade represents the subgenera: (1) Hyperoambon Peters, 1864 comprising subspecies D. septemcinctus hybridus distributed in D. beniensis, D. pastasae and D. kappleri; (2) Muletia southern Brazil, Uruguay and eastern, central and Gray, 1874 containing only D. septemcinctus; and (3) northern Argentina, and the second clade represents Dasypus Linnaeus, 1758 including D. novemcinctus, D. s. septemcinctus, occurring in Brazil, eastern D. pilosus, D. mazzai, D. sabanicola and Dasypus sp. Paraguay, Bolivia and northern Argentina. from the Guiana Shield (Fig. 2).

Subgeneric classification of dasypus Divergence times and speciation in dasypus Based on external and cranial characters, Wetzel Divergence times point to the Pliocene as the major & Mondolfi (1979) allocated Dasypus species into period for diversification of Dasypus. Lineages now three subgenera (Dasypus, Hyperoambon and including D. beniensis, D. septemcinctus, Dasypus sp. Cryptophractus). Recently, Castro et al. (2015) from French Guyana, D. pilosus, and D. novemcinctus proposed to resurrect Cryptophractus as a valid from North and Central America, all originated genus, based on the apparent early divergence and during this period as the Neotropics underwent major the unique morphology of its type species, D. pilosus. geographic, tectonic and ecological restructuring into However, as shown here and in Gibb et al. (2016), its modern configuration. Several of these large-scale D. pilosus is closely related to D. novemcinctus events appear to be associated with the cladogenesis of and accepting Cryptophractus as a separate genus these lineages based on their present distribution. The would render Dasypus paraphylectic. Considering expansion of grassland systems during the Miocene that Dasypus is the only extant representative of an and the cooling of the Pliocene are coincident with the ancient lineage of armadillos of Eocene origin (Gibb divergence of D. septemcinctus (Ortiz-Jaureguizar & et al., 2016), its undisputed monophyly and continuous Cladera, 2006). The final uplifting of the Andes (Ortiz- usage since Linnaeus, we advocate keeping all long- Jaureguizar & Cladera, 2006; Hoorn et al., 2010) nosed armadillos in a single genus in the interests and the recent, but radical, divergence of D. pilosus, of nomenclatural stability (Feijó & Langguth, 2013; likewise appear connected. Changes in the Amazonian Teta, 2018). On the other hand, our results recover drainage and separation of D. beniensis, and uplifting three distinctive and well-supported lineages within of the Isthmus of Panama and the divergence of Dasypus (Fig. 2). Each of these lineages shows a set D. novemcinctus from North and Central America are of unique morphological and biological features that also implicated. Our study corroborates a growing reflect their different evolutionary histories. For number of studies that identify the Pliocene as an example, the three species of the D. kappleri complex important period of diversification for South American are the only living representatives of a lineage that birds (Smith & Klicka, 2010) and mammals [Almeida diverged in the Late Miocene. They are the largest et al. (2007) for Calomys; Hassanin et al. (2012) for species of the genus and share well-developed scales South American artiodactyls; Leite et al. (2014) for at the knee, a poorly developed occipital lobe on the sigmodontine tribes and genera; Miranda et al. (2017) cephalic shield, five digits on the forefoot, marked for species of Cyclopes; and Nascimento et al. (2013) for lateral margin of the palatine, 7–8 movable bands Thrichomys]. and reproduction via two genetically identical siblings Even though no theory can encompass all (Feijó & Cordeiro-Estrela, 2016). In addition, they are diversification processes, the accumulation of historical forest dwellers and share a common biogeographic biogeographical analyses and molecular dating has history, having probably diversified in Amazonia changed the perspective that Pleistocene climatic events following major basin tectonic activity associated were the principal drivers of Neotropical speciation with Andean uplift (Feijó & Cordeiro-Estrela, 2016). (Haffer, 1969; Vanzolini & Williams, 1970). Pleistocene Dasypus septemcinctus represents the smallest species glaciations do not appear to be as significant in terms of of the genus and is typical of open-area formations. It ‘cladogenetic power’ as they have been in the Nearctic is unique in possessing a straight dorsal profile of the and Palearctic regions (Gascon et al., 2000; Rull, skull and in giving birth to 6–12 genetically identical 2008). Many extant Neotropical species predate the

Pleistocene, many considerably so [e.g. fairy armadillos Tecnológico (CNPq), and Fundação de Apoio à Pesquisa

(Delsuc et al., 2012), Cyclopes (Miranda et al., 2017), de Minas Gerais (FAPEMIG). AF is supported by Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zly091/5298137 by University of Kansas user on 20 January 2019 Potos, Nasua and Bassariscus (Helgen et al., 2013)]. Chinese Academy of Sciences President’s International Instead, Pleistocene glaciations seem to have had more Fellowship Initiative (Grant number 2018PB0040) impact on population demography (Lessa et al., 2003; and CNPq – Brazil Science without Borders Program Turchetto-Zolet et al., 2013; Leite et al., 2016). (Grant number 201129/2015-9), FRS by a CNPq fellowship, and JFV by a CNPq – Brazil Science without Borders Program (Grant number 206882/2014-9), Field Museum of Natural History’s Brown Fund for Mammal CONCLUSION Research, and ICMBio/CNPq–SET. 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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher's web-site. Table S1. Tissue sample data and molecular markers used in this study. Asterisks refer to sequences obtained from Genbank Table S2. Primers used for partial amplification of Dasypus genes, PCR annealing temperatures, and associated references Table S3. Results of General Mixed Yule-Coalescent (GMYC) analyses Table S4. Results of automatic barcode gap discovery (ABGD) analyses Figure S1. Phylogenetic tree for the Dasypus species based on concatenated mitochondrial (Cytb and COI) and nuclear (vWF) genes. Nodal support values are Bayesian posterior probabilities (BI).