Evolutionary relationships and population genetics of the Afrotropical leaf-nosed bats (Chiroptera, Hipposideridae)

Abstract The Old World leaf-nosed bats (Hipposideridae) are aerial and gleaning insectivores that occur throughout the Paleotropics. Both their taxonomic and phylogenetic histories are confused. Until recently, the family included genera now allocated to the Rhinonycteridae and was recognized as a subfamily of Rhinolophidae. Evidence that Hipposideridae diverged from both Rhinolophidae and Rhinonycteridae in the Eocene confirmed their family rank, but their intrafamilial relationships remain poorly resolved. We examined genetic variation in the Afrotropical hipposiderids Doryrhina, Hipposideros, and Macronycteris using relatively dense taxon-sampling throughout East Africa and neighboring regions. Variation in both mitochondrial (cyt-b) and four nuclear intron sequences (ACOX2, COPS, ROGDI, STAT5) were analyzed using both maximum likelihood and Bayesian inference methods. We used intron sequences and the lineage delimitation method BPP—a multilocus, multi-species coalescent approach—on supported mitochondrial clades to identify those acting as independent evolutionary lineages. The program StarBEAST was used on the intron sequences to produce a species tree of the sampled Afrotropical hipposiderids. All genetic analyses strongly support generic monophyly, with Doryrhina and Macronycteris as Afrotropical sister genera distinct from a Paleotropical Hipposideros; mitochondrial analyses interpose the genera Aselliscus, Coelops, and Asellia between these clades. Mitochondrial analyses also suggest at least two separate colonizations of Africa by Asian groups of Hipposideros, but the actual number and direction of faunal interchanges will hinge on placement of the unsampled African-Arabian species H. megalotis. Mitochondrial sequences further identify a large number of geographically structured clades within species of all three genera. However, in sharp contrast to this pattern, the four nuclear introns fail to distinguish many of these groups and their geographic structuring disappears. Various distinctive mitochondrial clades are consolidated in the intron-based gene trees and delimitation analyses, calling into question their evolutionary independence or else indicating their very recent divergence. At the same time, there is now compelling genetic evidence in both mitochondrial and nuclear sequences for several additional unnamed species among the Afrotropical Hipposideros. Conflicting appraisals of differentiation among the Afrotropical hipposiderids based on mitochondrial and nuclear loci must be adjudicated by large-scale integrative analyses of echolocation calls, quantitative morphology, and geometric morphometrics. Integrative analyses will also help to resolve the challenging taxonomic issues posed by the diversification of the many lineages associated with H. caffer and H. ruber.


Introduction
The Old World leaf-nosed bats, family Hipposideridae, currently include seven genera and 90 species of insectivorous bats distributed over much of the Paleotropics (Monadjem 2019; Simmons and Cirranello 2019). Both the taxonomic and phylogenetic histories of this family are confused. Throughout much of its history (e.g., Koopman 1989), Hipposideridae was considered either a subfamily of the Rhinolophidae (the horseshoe bats) or as its sister family within the Rhinolophoidea. Recently, however, the "trident bats" (Cloeotis, Paratriaenops, Rhinonicteris, and Triaenops) were shown to comprise a family-ranked group, the Rhinonycteridae, which is separate from and sister to the Hipposideridae (Foley et al. 2015;Armstrong et al. 2016). Even the genus Hipposideros Gray, 1831, as it was traditionally understood, appears paraphyletic with respect to the allied genera Asellia, Aselliscus, Coelops, and Anthops (Foley et al. 2015;Amador et al. 2018). Re-validation of Macronycteris Gray, 1866 and Doryrhina Peters, 1871 for groups of Afrotropical endemic species more closely related to each other than to African and Asian members of Hipposideros sensu stricto resolved a number of those issues (Foley et al. 2017).
As suggested by their checkered taxonomic history, phylogenetic understanding of the Hipposideridae has slowly come into focus. Doryrhina and Macronycteris are two of a dozen generic-group names that were synonymized with Hipposideros for all of the 20 th century (Miller 1907;Allen 1939;Koopman 1994). Instead of subgenera, taxonomists used the species groups delineated by Andersen (1918) and refined by Tate (1941) and Hill (1963) in their generic revisions based on morphology. Assessment of rhinolophoid relationships using an intron supermatrix (Eick et al. 2005) confirmed the early divergence of hipposiderids and rhinolophids (estimated at 41 Ma), thereby substantiating their rank as a separate families. Despite earlier suppositions that the area of origin for Hipposideridae was in Asia (Koopman 1970;Bogdanowicz and Owen 1998) or Australia Kirsch 1998), Eick et al. (2005) clearly demonstrated the ancestry of the family (and superfamily) was in Africa. A recent supermatrix analysis with the most comprehensive taxonomic sampling (42 species; Amador et al. 2018) confirmed the early divergence of hipposiderids and rhinolophids at 41.3 Ma, but this analysis questioned the validity of both Doryrhina and Macronycteris. Amador et al. attributed the paraphyly of Hipposideros sensu lato documented by Foley et al. (2015) to their limited taxonomic sampling. Amador et al. (2018) also challenged the integrity of the commersoni, cyclops, speoris, and bicolor species groups, arguing that all African species save for H. jonesi belonged in a single, exclusively African species group.
Although new species of hipposiderids are regularly discovered and described in Asia (Robinson et al. 2003;Guillen-Servent and Francis 2006;Bates et al. 2007;Douangboubpha et al. 2011;Thong et al. 2012;Murray et al. 2018), the pace of discovery has been much slower in Africa. Only one extant species has been described since the recognition of Hipposideros lamottei (Brosset 1985(Brosset ["1984), and that one was from Madagascar . Surveys of mitochondrial sequences from African hipposiderids have strongly suggested that supposedly widespread species such as Hipposideros caffer and H. ruber actually represent complexes of cryptic species (Vallo et al. 2008Monadjem et al. 2013). Phylogenetic analyses (e.g., Vallo et al. 2008) show that these named species complexes are not monophyletic, resolving clades comprised of bats identified as both H. caffer and H. ruber. These studies have characterized the clades in both morphological and genetic terms, even establishing them in sympatry (see also Vallo et al. 2011). However, the uncertain relationship of the identified clades to the many names already proposed for Afrotropical hipposiderids, many based on incomplete or formalin-preserved specimens, has precluded formally naming them. Incomplete geographic sampling and the lack of evidence from nuclear genes for these populations has also clouded interpretations of this mitochondrial diversity.

Selection of taxa and sampling
Our genetic dataset is based on 453 hipposiderid individuals, the vast majority being represented by museum vouchers. We generated original genetic data from 319 individuals collected at 102 georeferenced localities, and complemented them with 134 mitochondrial sequences from 90 localities downloaded from GenBank (we obtained new sequence data for five individuals with prior GenBank records; see Suppl. material 1: Figure S1 and Appendix I). All individuals were sequenced for Cytochrome-b (cyt-b) in order to maximize assessment of genetic diversity; however, redundant haplotypes were removed for subsequent phylogenetic analyses (see Appendix I for complete list of individuals sequenced). The bats newly sequenced for this study were obtained over several decades in the course of small mammal surveys across sub-Saharan Africa and Madagascar, with relatively dense sampling in East Africa. Initial assignment of East African individuals to species was determined using meristic, mensural, and qualitative characters published in the bat keys of Thorn et al. (2009) and Patterson and Webala (2012). Collection methods followed mammal guidelines for the use of wild mammals in research and education (Sikes and the Animal Care and Use Committee of the American Society of Mammalogists 2016) and the most recent collections were approved under Field Museum of Natural History's IACUC #2012-003. Only Gen-Bank records for cyt-b were available for records of the Arabian-North African hipposiderid Asellia, which was included for context in the phylogenetic analyses. Lacking information from nuclear introns, we draw no firm conclusions from their placement and do not discuss Asellia in this paper (see Benda et al. 2011;Bray and Benda 2016). Appendix I contains the institutions and voucher numbers, GenBank accession numbers, and locality information for our samples. The fact that museum voucher specimens were used wherever possible for the genetic analyses permits the genetic analysis to serve as a foundation for integrative taxonomic analyses of dental, cranial, and skeletal variation, using the same specimens. To avoid adding to current taxonomic confusion, we take a conservative approach in assigning names to clades in our analyses. Where a clade's taxonomic identity was ambiguous or unknown, we referred to it simply as a numbered clade. Integrative taxonomic diagnoses of the various clades supported by our analyses will be necessary to determine which, if any, existing names may apply to them. However, to relate our results to those of earlier studies of African Hipposideros (Vallo et al. 2008Monadjem et al. 2013), we cross-referenced specimens used in two or more analyses to equate the various non-binomial names that have been applied to these cryptic lineages.

DNA extraction, amplification, and sequencing
Genomic DNA from preserved tissue samples was extracted using the Wizard SV 96 Genomic DNA Purification System (Promega Corporation, WI, USA). Fresh specimens were sequenced for mitochondrial cytochrome-b (cyt-b), using the primer pair LGL 765F and LGL 766R (Bickham et al. 1995;Bickham et al. 2004), and four unlinked autosomal nuclear introns: ACOX2 intron 3, COPS7A intron 4, ROGDI intron 7 (Salicini et al. 2011), and STAT5B (Matthee et al. 2001) for hipposiderid specimens and the sister group Triaenops afer (Rhinonycteridae; see Table 1 for primer information). PCR amplification, thermocycler conditions, and sequencing were identical to Patterson et al. (2018) and Demos et al. (2018). Sequences were assembled and edited using GENEIOUS PRO v.11.1.5 (Biomatters Ltd). Sequence alignments were made using MUSCLE (Edgar 2004) with default settings in GE-NEIOUS. Protein coding data from cyt-b were translated to amino acids to determine codon positions and confirm the absence of premature stop codons, deletions, and insertions. Several gaps were incorporated in the nuclear intron alignments, but their positions were unambiguous.
Sequence alignments used in this study have been deposited on the FIGSHARE data repository (https://doi.org/10.6084/m9.figshare.11936250). All newly generated sequences were deposited in GenBank with accession numbers MT149315-MT149893 (see also Appendix I).
Phylogenetic analyses jMODELTEST2 (Darriba et al. 2012) on CIPRES Science Gateway v. 3.3 (Miller et al. 2010) was used to determine the sequence substitution models that best fit the data using the Bayesian Information Criterion (BIC) for cyt-b and the four nuclear introns. PARTITIONFINDER2 (Lanfear et al. 2016) on CIPRES was used to determine the sequence substitution models for the concatenated alignment of four nuclear introns using the Bayesian Information Criterion (BIC) with the 'greedy' search algorithm. Uncorrected sequence divergences (p-distances) between and within species/clades were calculated for cyt-b using MEGA X v. 10.0.5 (Kumar et al. 2018). Maximum-likelihood (ML) analyses were performed using the program IQ-TREE v. 1.6. 10 (Chernomor et al. 2016;Nguyen et al. 2015) on the CIPRES portal for separate gene trees (cyt-b, ACOX2, COPS7A, ROGDI, and STAT5B) and a concatenated alignment, partitioned by gene, using the four nuclear introns. As in Hillis and Bull (1993), nodes supported by bootstrap values (BP) ≥ 70% were considered strongly supported. Gene tree analyses under a Bayesian Inference (BI) framework were inferred in MRBAYES v. 3.2.7 (Ronquist et al. 2012) on the CIPRES portal for the same set of genes as the ML analyses. Two independent runs were conducted in MrBayes, and nucleotide substitution models were unlinked across partitions for each nuclear locus in the concatenated alignment. Four Markov chains were run for 1 × 10 8 generations for individual gene trees, and 2 × 10 7 generations for the concatenated analysis, using default heating values and sampled every 1000 th generation. A conservative 25% burn-in was applied and stationarity of the MRBAYES results was assessed in Tracer v. 1.7 (Rambaut et al. 2018). Majority-rule consensus trees were constructed for each Bayesian analysis. Following Erixon et al. (2003), nodes supported by posterior probabilities (PP) ≥0.95 were considered strongly supported.
Haplotype networks for cyt-b were inferred using the median-joining network algorithm in PopArt v. 1.7 (Leigh and Bryant 2015). Separate analyses were carried out for the following clades, each consisting of four subclades: Hipposiderid taxa included in the species tree analyses were assigned to either species or numbered clades based on clade support in the ML and BI gene-tree analyses of the cyt-b dataset. This in turn identified populations to be used as 'candidate species' in a coalescent-based species-tree approach implemented in StarBEAST2 (Ogilvie et al. 2017), an extension of BEAST v. 2.5.1 (Drummond et al. 2012;Bouckaert et al. 2014). Species tree analysis was conducted using the four nuclear intron alignments. Substitution, clock, and tree models were unlinked across all loci. A lognormal relaxed-clock model was applied to each locus under a Yule tree prior and a linear with constant root population size model. Four independent replicates were run with random starting seeds, and chain lengths of 1 × 10 8 generations and parameters were sampled every 5,000 steps. For the StarBEAST2 analyses, evidence of convergence and stationarity of posterior distributions of model parameters was assessed based on ESS values >200 and examination of trace files in Tracer v. 1.7. The burn-in was set at 10% and separate runs were assembled using LOGCOMBINER v. 2.5.1 and TREEANNOTATOR v. 2.5.1 (Rambaut et al. 2018).

Coalescent lineage delimitation
Based on the well supported clades obtained in the cyt-b gene tree analyses and available intron samples, a lineage delimitation scenario with 18 candidate species was tested. We inferred the evolutionary isolation of their gene pools using the phased nuclear DNA dataset (ACOX2, COPS7A, ROGDI, and STAT5A; 104 individuals) for joint independent lineage delimitation and species-tree estimation evaluated under the multi-species coalescent model using the program BPP v. 3.3 (Yang and Rannala 2014;Rannala and Yang 2017). This analysis was carried out to guide future investigations of the species status of evolutionarily isolated lineages inferred here. Supported lineages will be examined using an integrative species taxonomic approach, including morphological, morphometric, and acoustic characters, as well as ectoparasite associations and distributional data. Species/clade memberships for BPP were identical to individuals assigned to lineages in the species tree analyses. The validity of our assignment of individuals to populations was tested using the guide-tree-free algorithm (A11) in BPP. Because the probability of delimitation by BPP is sensitive to selected parameters (Leaché and Fujita 2010;Yang 2015), we evaluated two independent runs for each of four different combinations of divergence depth and effective population sizes priors (τ and θ, respectively; Table 2). Two independent MCMC chains were run for 5 × 10 4 generations. The burn-in was 20% and samples drawn every 50 th generation. In total, eight BPP runs were carried out using four phased nuclear intron alignments. Lineages were considered to be statistically well supported when the delimitation posterior probabilities generated were ≥0.95 under all four combinations of priors.

Results
In terms of cyt-b sequence divergence, clades within Doryrhina are separated by 3.0-5.7% genetic distances, whereas less than 3% separates the four recognized species of Table 2. Primer information and chosen substitution models for regions amplified in this study. Substitution models before "/" are the best-supported models inferred by jMODELTEST2 and models after "/" indicate those inferred by PARTITIONFINDER2 for the concatenated intron alignment.

Primer name Sequence Primer publication Substitution model
Macronycteris. Between Afrotropical Hipposideros, the greatest distances separate H. jonesi from other lineages (13.4-16.1%). The various numbered clades allied to Hipposideros caffer differ from one another in cyt-b sequences by 2.5-10.3% and clades allied to H. ruber differ by 3.0-8.2% (Table 4) Figure 5; they are not recovered as monophyletic units and the geographic structure evident in mtDNA analyses disappears. Table 3. Prior Schemes (PS) used in BPP analyses. Prior distributions on τ represent two relative divergence depths (deep and shallow) and on θ represent two relative effective population sizes (large and small) scaling mutation rates.     A species tree generated using StarBEAST from the four introns appears in Figure  6. It depicts well-supported relationships among the various clades allied with H. caffer, H. ruber, and H. beatus. Remarkably, and in contrast with the concatenated analyses,

Overall genetic variability
The three Afrotropical hipposiderid genera differ substantially in terms of their internal genetic differentiation. Clades of Hipposideros are separated by cyt-b p-distances averaging 9.7% (2.5-16.1%), whereas Doryrhina clades average p-distances of 4.8% (3.0-5.7%) and Macronycteris clades 2.7% (2.6-2.9%). Distance values for these gen-era tend to fall at the lower end of values obtained with similar sampling intensity for species-ranked clades in other Afrotropical bat genera: 2.5% for Otomops (Patterson et al. 2018), 9.3% for Miniopterus (Demos et al. 2020, 10% for Scotophilus and Rhinolophus (Demos et al. 2018(Demos et al. , 2019a, 13.5% for Myotis , and 17% for Nycteris (Demos et al. 2019b). Fewer cyt-b substitutions on average for these hipposiderids does not limit support for individual clades, and because distances do not approach those characteristic of substitutional saturation, the cyt-b tree recovers much of the deeper phylogenetic structure evident with nuclear intron sequences (compare Figs 2, 5).

Phylogenetics
Both cyt-b and intron analyses securely recovered Doryrhina, Macronycteris, and Hipposideros as monophyletic. Doryrhina + Macronycteris are sister to the remaining hipposiderids. However, only the cyt-b analysis included the hipposiderid genera Aselliscus, Coelops, and Asellia alongside Hipposideros. That analysis recovered all four genera as monophyletic with strong support. Aselliscus and Coelops were recovered as sister to Hipposideros, with Asellia joining later, but these relationships lacked confident support.
Using a supermatrix approach on exemplars of 46 species of hipposiderids, Amador et al. (2018) found Hipposideros sensu stricto to be paraphyletic. They recovered a mostly Asian group of Hipposideros as sister to two subclades, Coelops + Aselliscus and Asellia + African hipposiderids excluding H. jonesi, which was recovered with the Asian taxa. Paraphyly in this molecular analysis echoed earlier indications of Hipposideros paraphyly from morphology (Bogdanowicz and Owen 1998;Kirsch 1998, 2003). In another supermatrix analysis of exemplars belonging to 49 hipposiderid species, Shi and Rabosky (2015) failed to recover Macronycteris as monophyletic; M. commersoni was sister to all remaining hipposiderids, but strangely it did not group with M. gigas. When the anomalous position of M. commersoni in their tree is ignored, their topology is highly similar to that of Figure 2, except that Asellia (Aselliscus, Coelops) become the sister of Hipposideros (Macronycteris, Doryrhina), rather than sister of just Hipposideros. Using both mitochondrial and nuclear loci, Lavery et al. (2014) found that 17 species of Asian, Oceanian and Australasian Hipposideros were monophyletic with respect to the genera Aselliscus, Coelops, and Anthops. Clearly, missing data and missing taxa compromise all of these phylogenetic appraisals, so that the question of hipposiderid and Hipposideros monophyly remains open. However, subject to its sampling limitations, there is clear support in our analyses of monophyly for Doryrhina, Macronycteris, and Hipposideros as we apply these names.
Despite employing different mitochondrial and nuclear loci and using different sets of taxa, the phylogeny recovered by Lavery et al. (2014) is largely congruent with that in Figure 5. Their earliest diverging species group of Hipposideros is the calcaratus group, not represented in our tree unless H. obscurus is a member (Table 1). Their next diverging unit is the diadema group, which is also positioned near the base of our tree. and H. cervinus, which are listed in different groups but were both considered members of the galeritus unit by Tate (1941); and the erstwhile bicolor group (sensu Hill 1963), which was subdivided into the ater subgroup (for Asian, Oceanian, and Australasian species) and the ruber subgroup (for Atrotropical ones) by Monadjem (2019).
The ater subgroup members included in our mitochondrial analysis (Fig. 2) Figure 2A.
Parsimony, topological position, and the strong support of branching relationships in the mitochondrial and intron trees ( Fig. 5; also Lavery et al. 2014) make it clear that the Afrotropical ruber group represents a comparatively recent colonization event from Asian ancestors-the ruber group is sister to the ater group and this pair has Asian sisters. However, although the basal dichotomy within Hipposideros includes an all Asian clade, lack of support for its sister(s) clouds the phylogenetic position of the H. jonesi-H. marisae clade-possibly sister to all sampled Hipposideros but more likely sandwiched between Asian clades. In any case, Figure 2 suggests that the H. jonesi-H. marisae clade resulted from an earlier African-Asian colonization event.
The lack of agreement in the phylogenetic position of H. diadema and H. larvatus between the concatenated intron tree (Fig. 5) and the species tree ( Fig. 6) deserves comment, as both analyses were based on the same genetic dataset. The position of H. diadema -H. larvatus as sister to the ruber group ( Fig. 6) runs counter to both our other genetic analyses (Figs 2, 5) and morphological assessments (Hill 1963;Murray et al. 2012; Table 1). This discrepancy is likely due to the generally weaker support for deep nodes within the tree; in the absence of saturation, this is often taken as evidence of rapid evolutionary radiations (e.g., Almeida et al. 2011). Lanier and Knowles (2014) used simulated data on deep phylogenies to show that species-tree methods do account for coalescent variance at deep nodes but that mutational variance among lineages poses the primary challenge for accurate reconstruction. In either case, vastly expanded genetic sampling via NGS techniques offers the most plausible avenues to clearer resolution.
However, the highly distinctive species H. megalotis belongs to its own species group (Table 1) and has not been included in any genetic analysis. Distributed in the Horn of Africa and the Arabian Peninsula, H. megalotis is the only hipposiderid with a fold of skin joining the base of the ear pinnae. Its uniquely specialized auditory system and derived dentition (e.g., loss of anterior premolars and enlargement of outer lower incisors), led Hill (1963) to regard it as a species that diverged early from the other

Species limits
The lineage delimitation analyses indicate that a number of hipposiderid lineages are either unnamed or unidentified, and also that a number of recognized species may not be genetically and evolutionarily independent.
Previous studies had indicated that both Hipposideros caffer (Vallo et al. 2008) and H. ruber  appear to be complexes of cryptic species. The two are traditionally distinguished on the basis of size and pelage color, H. ruber being the larger and more brightly colored form, but this distinction is clouded by geographic variation in size and the presence of both reddish and gray-brown phases in both species. Our mitochondrial analyses identified four H. ruber lineages and eight H. caffer lineages in two distinct groupings among the sampled populations (Fig. 2). Four of the caffer lineages and three of the ruber clades were identified as putative species by the BPP analyses (Table 5). The large number of clades in East Africa is remarkable: Kenya and Tanzania each support four of the eight clades allied with H. caffer, and all but one of the eight clades known from throughout the continent occur in one or the other East African country. This undoubtedly reflects the region's great landscape diversity, where West and Central African rainforests reach their eastern limit, southern savannas reach their northern limits, the Sahel reaches its southern limits, and all are riven by the African Rift Valley. It also is a product of our sampling intensity (see Suppl. material 1: Fig. S1).
Because some cyt-b sequences were used in multiple studies of this group, it is possible to relate our clade labels to those used by earlier studies (Table 6). Based on attributions made on morphological grounds by Vallo et al. (2008) and Monadjem et al. (2013), some well-supported but unnamed clades in our analysis can be identified. For instance, caffer1 has a distributional range and includes specimens previously identified as Hipposideros tephrus (Appendix I), while specimens of caffer4 come from near the type locality of H. caffer Sundevall, 1846, and may well represent that species. However, no samples confidently identified as H. ruber from the vicinity of its type locality have been sequenced, leaving the application of that name to clades in any of these trees purely conjectural. Applying formal names only after integrative taxonomic assessment is a responsible course as multispecies coalescent models like BPP can lead to over-splitting of species, especially when applied to geographically variable species complexes with parapatric distributions (Chambers and Hillis 2020).
Doryrhina is a poorly known genus characterized morphologically by the peculiar club-shaped processes on the central and posterior nose leaves. This trait is shared by the two recognized African species, D. cyclops and D. camerunensis, which differ chiefly in size (the latter is larger, with forearm lengths >75 mm). Although D. cyclops is considered to be monotypic, mitochondrial sequences clearly separate West African populations in Liberia and Senegal (cyclops1) from Central African populations in Gabon and Central African Republic (cyclops2), and these are substantially separated from D. camerunensis and a specimen referred to that species from Tanzania (Figs 2, 3). However, both the intron analysis (Fig. 5) and the species tree (Fig. 6) show little or no geographic structure. The BPP analyses confirm that none of the mitochondrial clades is behaving as an independent evolutionary lineage (Table 5). Geographic structure in mtDNA but continent-wide admixture in the nuclear genome could result from either male-biased dispersal with female philopatry or highly structured seasonal migrations, which are known in other hipposiderids. In any case, the genetic patterns of Doryrhina are hard to reconcile with its space-use behavior; individuals appear to have very small home ranges, on the order of a few hectares (Monadjem 2019). An integrative taxo- Table 5. Lineage delimitation results from BPP based on the four intron dataset for mtDNA-supported clades of Afrotropical Hipposideridae. PS1-PS4 refer to four different prior schemes based on population size and age of divergence priors (see Table 3 for parameter details). Bold font indicates that the putative species was delimited under all parameter settings.  (1963) Table 6. Clade names and associated binomials (if used) for three analyses of cryptic lineages within the ruber species group of Hipposideros. No genetic analysis of this group has included type material; consequently, the application of binomials hinges on the robustness of ancillary morphological analyses, which were not conducted in our study. Boldfaced names denote clades supported by all four prior schemes in our BPP delimitation analyses. resolve any of the Macronycteris species, and none appear as monophyletic in the concatenated intron analyses. Our results clearly underscore the importance of using multilocus datasets to evaluate phylogenetic and phylogeographic relationships at the genus and species level in mammals. Use of a single genetic system may lead to widely divergent conclusions regarding species identity and distribution. Toews and Brelsford (2012) reviewed cases of mito-nuclear discordance in animals generally. Fully 18% of the cases they reviewed had discordant patterns of mitochondrial and nuclear DNA. In most cases, such patterns are attributable to adaptive introgression of mtDNA, demographic disparities, and sex-biased asymmetries; in some cases they found evidence for hybrid zone movement or human agency. Discordant patterns of variation between mitochondrial and nuclear DNA have been reported in at least six other families of bats (Nesi et al. 2011;Furman et al. 2014;Naidoo et al. 2016;Hassanin et al. 2018;Demos et al. 2019a;Gürün et al. 2019). Gürün et al. (2019) implicated the role of sex-biased dispersal in causing such discordance, male dispersal spreading nuclear variation farther and faster than the movement of mitochondria. This may be a more general pattern in bats (see also Demos et al. 2019b). To understand the processes responsible for these discordant patterns of genome evolution, extensive genomic sampling and far fuller knowledge of natural history will be required.

Acknowledgments
We thank Michael Bartonjo, Carl Dick, Ruth Makena, Beryl Makori, David Wechuli, Richard Yego, and Aziza Zuhura for help in obtaining specimens in the field. We acknowledge with special thanks the assistance of Simon Musila (National Museums of Kenya), Donna Dittman and Jacob Esselstyn (Louisiana State University) and Maria Eifler (University of Kansas) for loans of material. We also appreciate the efforts of curators and collection managers in all the institutions cited in Appendix I for maintaining the museum voucher specimens that enable integrative taxonomic studies to confidently name our numbered lineages. Fieldwork in eastern and southern Africa was funded by various agencies in cooperation with the Field Museum, especially the JRS Biodiversity Foundation. Field Museum's Council on Africa, Marshall Field III Fund, and Barbara E. Brown Fund for Mammal Research were critical to fieldwork and analyses, as were supporters Bud and Onnolee Trapp and Walt and Ellen Newsom. The John D. and Catherine T. MacArthur Foundation, Fulbright Program of US Department of State, Wildlife Conservation Society, and the Centers for Disease Control and Prevention sponsored and/or assisted in providing samples from DRC, Malawi, Mozambique, and Uganda. WWF Gabon supported fieldwork in Gabon, as did the Partenariat Mozambique-Réunion dans la Recherche en Santé: pour une approche intégrée d'étude des maladies infectieuses à risque épidémique (MoZaR; Fond Européen de Développement Régional, Programme Opérationnel de Coopération Territoriale) in Mozambique. We thank Ara Monadjem and Daniela Rossoni for reviews of an earlier draft of this manuscript.

Appendix I
Genetic sampling of Hipposideridae. Wherever possible, the voucher numbers associated with the genetic samples are specified. Accession numbers identify sequences downloaded from GenBank or accessioned to Genbank for this study. The designation 'redundant' indicates a cyt-b sequence that was omitted from the 303 individual alignment because of its identity to another. Institutional acronyms are as follows: