How and why overcome the impediments to resolution: lessons from rhinolophid and hipposiderid bats

Abstract

The phylogenetic and taxonomic relationships among the Old World leaf-nosed bats (Hipposideridae) and the closely related horseshoe bats (Rhinolophidae) remain unresolved. In this study, we generated a novel approximately 10-kb molecular data set of 19 nuclear exon and intron gene fragments for 40 bat species to elucidate the phylogenetic relationships within the families Rhinolophidae and Hipposideridae. We estimated divergence times and explored potential reasons for any incongruent phylogenetic signal. We demonstrated the effects of outlier taxa and genes on phylogenetic reconstructions and compared the relative performance of intron and exon data to resolve phylogenetic relationships. Phylogenetic analyses produced a well-resolved phylogeny, supporting the familial status of Hipposideridae and demonstrated the paraphyly of the largest genus, Hipposideros. A fossil-calibrated timetree and biogeographical analyses estimated that Rhinolophidae and Hipposideridae diverged in Africa during the Eocene approximately 42 Ma. The phylogram, the timetree, and a unique retrotransposon insertion supported the elevation of the subtribe Rhinonycterina to family level and which is diagnosed herein. Comparative analysis of diversification rates showed that the speciose genera Rhinolophus and Hipposideros underwent diversification during the Mid-Miocene Climatic Optimum. The intron versus exon analyses demonstrated the improved nodal support provided by introns for our optimal tree, an important finding for large-scale phylogenomic studies, which typically rely on exon data alone. With the recent outbreak of Middle East respiratory syndrome, caused by a novel coronavirus, the study of these species is urgent as they are considered the natural reservoir for emergent severe acute respiratory syndrome (SARS)-like coronaviruses. It has been shown that host phylogeny is the primary factor that determines a virus's persistence, replicative ability, and can act as a predictor of new emerging disease. Therefore, this newly resolved phylogeny can be used to direct future assessments of viral diversity and to elucidate the origin and development of SARS-like coronaviruses in mammals.

Keywords: Rhinonycteridae; biogeography; exon versus intron; mammals; phylogenetics; virus.

Fig. 1.

(a) Tree derived from Parsimony analysis of morphological discrete state data using Nelson-like consensus cladogram from Bogdanowicz and Owen (1998). (b) Consensus tree from Parsimony analysis of unordered morphological characters on 30 taxa common to this study described in (a) from Hand and Kirsch (1998). (c) Single ML tree derived from PAUP* analysis of intron supermatrix from Eick et al. (2005). (d) ML tree derived from PAUP* analysis of ND2 and RAG1 from Murray et al. (2012).

Fig. 2.

Phylogram inferred from Bayesian Analysis in BEAST on the exon+intron-outliers removed data set, 10,420 bp comprising 12 nuclear exons and 7 nuclear introns, under a fully partitioned model. Nodal support for the exon+intron-outliers removed data set is summarized on the tree for all four analyses—RaxML, BEAST, MrBayes, and PhyloBayes. All numeric support values are shown as percentages and refer to each analysis in the order listed above. Black squares denote highly supported nodes all of which received support >99 BSS or 0.99 PP across all four analyses. A “-” indicates that this relationship was not supported by the analysis. See Systematic Summary for full description of the newly elevated family Rhinonycteridae. Frontal views of nose leaves of representatives of the major clades are shown as follows: Rhinolophidae—Rhinolophus pearsoni and Hipposideridae—Hipposideros spp. (photo credit—Sébastien J. Puechmaille) and Rhinonycteridae—Triaenops (photo credit—Paul Webala).

Fig. 3.

Bayesian Tree derived from BEAST analysis of 1,223 bp comprising three nuclear introns—STAT5A, PRKC1, and THY under GTR+G substitution model, highlighting the position of Anthops ornatus.

Fig. 4.

Molecular time scale resulting from MCMCTREE analysis in PAML using the BEAST topology shown in figure 2, four fossil calibrations (as described in Materials and Methods using stratigraphic bounding), and a root prior of 64–65 Ma. Numbers at nodes are divergence time estimates in millions of years and the 95% confidence interval for each estimate is denoted by a blue shaded bar. Biogeographic reconstructions resulting from ML analysis in Lagrange under the same topology are shown as letters at each node. Areas are coded as follows: A—Europe, B—South America, C—South East Asia, D—India, E—Middle East, F—East Asia, G—Australia, and H—Africa.

Fig. 5.

LTT plot showing the diversification rate of the genera Rhinolophus and Hipposideros for Cyt b and Cox1, where time is represented by arbitrary values with 0.0 representing the present.

Fig. 6.

Showing the relative difference in nodal support between intron and jack-knifed exon data sets of equal size, where colored dots indicate the proportion by which nodes are better resolved by either intron (red) or exon (green) data for (a) intron topology and (b) the exon topology. Species names are as in figure 2.