Molecular phylogenetics of the African horseshoe bats (Chiroptera: Rhinolophidae): expanded geographic and taxonomic sampling of the Afrotropics

Abstract

Background: The Old World insectivorous bat genus Rhinolophus is highly speciose. Over the last 15 years, the number of its recognized species has grown from 77 to 106, but knowledge of their interrelationships has not kept pace. Species limits and phylogenetic relationships of this morphologically conservative group remain problematic due both to poor sampling across the Afrotropics and to repeated instances of mitochondrial-nuclear discordance. Recent intensive surveys in East Africa and neighboring regions, coupled with parallel studies by others in West Africa and in Southern Africa, offer a new basis for understanding its evolutionary history.

Results: We investigated phylogenetic relationships and intraspecific genetic variation in the Afro-Palearctic clade of Rhinolophidae using broad sampling. We sequenced mitochondrial cytochrome-b (1140 bp) and four independent and informative nuclear introns (2611 bp) for 213 individuals and incorporated sequence data from 210 additional individuals on GenBank that together represent 24 of the 33 currently recognized Afrotropical Rhinolophus species. We addressed the widespread occurrence of mito-nuclear discordance in Rhinolophus by inferring concatenated and species tree phylogenies using only the nuclear data. Well resolved mitochondrial, concatenated nuclear, and species trees revealed phylogenetic relationships and population structure of the Afrotropical species and species groups.

Conclusions: Multiple well-supported and deeply divergent lineages were resolved in each of the six African Rhinolophus species groups analyzed, suggesting as many as 12 undescribed cryptic species; these include several instances of sympatry among close relatives. Coalescent lineage delimitation offered support for new undescribed lineages in four of the six African groups in this study. On the other hand, two to five currently recognized species may be invalid based on combined mitochondrial and/or nuclear phylogenetic analyses. Validation of these cryptic lineages as species and formal relegation of current names to synonymy will require integrative taxonomic assessments involving morphology, ecology, acoustics, distribution, and behavior. The resulting phylogenetic framework offers a powerful basis for addressing questions regarding their ecology and evolution.

Keywords: Afrotropical biodiversity; East Africa; Introgression; Introns; Phylogeny; Rhinolophus; Species tree; Taxonomy.

Fig. 1

Type localities for recognized species of Rhinolophus (black circles), as well as subspecies and synonyms (white circles); label names represent the specific epithets of currently recognized species. Biomes of Africa and neighboring regions indicated by color shading, dark yellow: Tropical and subtropical moist broadleaf forests; orange: Flooded grasslands and savannas; gray: Tropical and subtropical grasslands, savannas, and shrublands; olive brown: Deserts and xeric shrublands; gray-green: Tropical and subtropical moist broadleaf forests; peach: Mangroves; ochre: Mediterranean forests, woodlands, and shrub; dark tan: Tropical and subtropical dry broadleaf forests [14]

Fig. 2

Geographic sampling for the genetic analyses presented by this study. The white circles denote the locations of one or more specimens represented solely by mitochondrial sequences (cyt-b), the half-filled circles indicate specimens or groups represented only by nuclear intron sequences, and the black circles identify locations where samples with both mitochondrial and nuclear sequences were acquired

Fig. 3

Maximum likelihood phylogeny of mitochondrial cytochrome-b sequences of Rhinolophus. The phylogeny was inferred in IQ-TREE and its topology was very similar to the Bayesian phylogeny calculated in MRBAYES. Filled black circles on nodes denote bootstrap values (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.95, left-half-filled circles indicate BS ≥ 70% and PP < 0.95, right-half-filled circles indicate BS < 70% and PP ≥ 0.95, and unmarked nodes indicate BS < 70% and PP < 0.95. Specific epithets in parentheses following clade names indicate sequences from specimens used in recent species descriptions that were not supported as monophyletic and are subordinate to other clades and would render them paraphyletic. Branch colors indicate individual clade membership; species groups are from [13]

Fig. 4

a Bayesian phylogeny of Rhinolophus based on four nuclear introns. The phylogeny was inferred in MRBAYES and its topology closely resembled the maximum likelihood phylogeny calculated in IQ-TREE. Filled black circles on nodes denote bootstrap values (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.95, left-half-filled black circles indicate BS ≥ 70% and PP < 0.95, and unmarked nodes indicate BS < 70% and PP < 0.95. b–d enlarged sections of the complete nuclear intron tree showing individual relationships. Branch colors indicate individual clade membership; species groups are from [13]. Specimen localities include counties for Kenya. Museum acronyms are defined in Additional file 1

Fig. 5

Species tree estimated in StarBEAST2 using the four nuclear intron dataset. Numbers adjacent to nodes indicate posterior probabilities. Terminal tips in the tree that are statistically well-supported (PP ≥ 0.95) from BPP are indicated by “*” preceding the clade name, and terminal tips that had PP < 0.95 are indicated by “?” preceding the clade name. Species groups are from [13]