The Arabian Peninsula possesses a unique assemblage of plant and animal species that result from the dynamic geologic history and shifting climate of this region. The flora and fauna range from localized temperate endemics occurring in the disjunct extensions of the Horn of Africa Hotspot and the Eastern Afromontane Hotspot [1, 2] in the southwest regions (Fig. 1), to more broadly distributed arid-adapted clades occurring throughout much of the Arabian Peninsula [3–13]. Many large-scale biogeographic patterns for Arabian taxa have been profoundly shaped by the complex tectonic history of the Arabian Peninsula [14–22]. The tectonic activity ultimately responsible for the separation of the Arabian Plate from Africa began approximately 30 Ma, and rifting of the southern Red Sea occurred in the early Miocene (27 Ma), and a connection to the Neotethys Sea and was completed by 23 Ma [23, 24]. The formation of the Red Sea represented a major vicariance event for taxa with formerly continuous Afro-Arabian distributions. The isolated Arabian plate collided with the Anatolian plate approximately 18–16 Ma, forming the temporary Gomphotherium bridge [25, 26]. A more permanent land bridge was established after the Arabian plate collided with Eurasia approximately 15 Ma [24], which allowed regular faunal exchanges between North Africa, the Arabian Peninsula, and Eurasia. These land bridges also created opportunities for trans-continental dispersals using the Arabian Peninsula as a stepping-stone. Sea levels dropped throughout the Red Sea around 10 Ma, providing evidence for the closing of the Strait of Bab el Mandeb and establishment of a southern land bridge between Africa and Arabia [24]. Pliocene marine sediment deposits formed around 5.3 Ma, indicating this land bridge was subsequently lost, and although cyclical Pleistocene land bridges related to glacial cycles have been proposed [27], more recent evidence does not support their existence [28]. Conversely, there is evidence the Persian Gulf was reduced to a series of freshwater lakes [29] or completely waterless [30] during the late Pleistocene, providing a connection between the Arabian Peninsula and Southwest Asia. Plio-Pleistocene climate change is hypothesized to have driven speciation in the southwest montane regions of the Arabian Peninsula [3, 11], and is linked to aridification in the Neogene [31, 32]. This may explain additional biogeographic patterns, but on a much more localized scale.

Fig. 1 The Arabian Peninsula. A map of the Arabian Peninsula showing disjunct extensions of the Horn of Africa Hotspot (medium grey) and the Eastern Afromontane Hotspot (dark grey), along with pertinent seas and country borders. Shape files for hotspots were obtained through the “Biodiversity Hotspots” package produced by Conservation International (2011). Full size image

The geologic activity associated with the Arabian Peninsula altered the historical connectivity of this landmass to surrounding continents, and as a result opportunities for faunal exchanges with particular continents occurred in temporally distinct periods. For example, taxa exhibiting disjunct Afro-Arabian distributions either exhibit early Miocene divergences associated with vicariance resulting from the Red Sea formation (30–23 Ma), or dispersed more recently from the Horn of Africa via a southern landbridge or overwater dispersal (10–5.3 Ma or 5.3 Ma to present, respectively) (reviewed in [18]). If the phylogenetic placement of an Arabian focal group is recovered, predictions can be made concerning the timing of speciation and tested in a temporally explicit phylogenetic framework to infer historical biogeographic patterns. The limited phylogenetic studies focusing on or including Arabian species have demonstrated concordant divergence timings for taxa with similar biogeographic origins [16–20], however these biogeographic patterns remain largely understudied for most Arabian taxa.

Although overall levels of endemism among vertebrate groups across the Arabian Peninsula are moderate, Arabian amphibians exhibit a high degree of endemism [15, 33]. Of the nine described Arabian anuran species, six of these species are true toads belonging to the family Bufonidae, a clade that contains close to 600 described species and which exhibits a nearly worldwide distribution [34]. The dispersal ability of toads is limited by their reliance on freshwater habitat for breeding and their intolerance to saltwater, making them an interesting system for investigating historical biogeography in this region. Several broad-scale phylogenetic studies of bufonids have revealed the existence of largely discrete continental clades [35–39] that are now accepted as distinct genera or subgenera [34, 40]. Three such genera are currently distributed in geographic regions directly surrounding the Arabian Peninsula, including Amietophrynus (Africa), Bufotes (Northern Eurasia and North Africa), and Duttaphrynus (Southwest and South Asia), allowing the possibility of multiple biogeographic origins for the Arabian species.

Due to a lack of molecular sampling, the assignment of the Arabian bufonid species to genera, and therefore biogeographic origin, has long been problematic. One Arabian toad species, Bufotes cf. variabilis, is attributable to the Western Eurasian Bufotes viridis species complex on the basis of morphology [33, 41]. However, the affinities of the other Arabian toads are unresolved in part due to the similar morphology among the many species of toads occurring in regions surrounding the Arabian Peninsula. Based on molecular evidence the Arabian endemic Duttaphrynus dhufarensis was recently determined to be of Asian origin, with its closest relatives occurring on the Indian subcontinent [37], although several taxa occurring across Southwest Asia were not included in the analysis. This discovery, and the assignment of D. dodsoni (a species distributed throughout the Horn of Africa) to the genus [37, 40], implies the Arabian Peninsula has acted as a stepping-stone for Asian-derived bufonid species to colonize mainland Africa. This scenario remains to be tested in a phylogenetic framework, but has important biogeographic implications. Other than B. cf. variabilis and D. dhufarensis, the remaining Arabian toads could not be allocated to genera, and remain in a non-taxon Bufo that is currently polyphyletic and is denoted as “Bufo”. These species include “Bufo” arabicus, “Bufo” tihamicus, “Bufo” hadramautinus, and “Bufo” scorteccii. Both “Bufo” arabicus and D. dhufarensis possess the largest geographic distribution of the Arabian toads, whereas “Bufo” tihamicus is only distributed along the coastal Red Sea region. Although “Bufo” tihamicus is thought to be a close relative of the Sahelian distributed African taxon “Bufo” pentoni, the latter species is also currently not assigned to a genus, and therefore the phylogenetic relationships of this complex remains convoluted. The species “Bufo” hadramautinus and scorteccii are restricted to one or two localities, and their validity has been questioned as they may represent isolated phenotypically variable populations of the wider ranging “Bufo” arabicus and D. dhufarensis, respectively [33]. As a result of these hypotheses “Bufo” scorteccii has been tentatively assigned to the genus Duttaphrynus [40], although this taxonomic assignment is certainly premature, and some authors have regarded “Bufo” hadramautinus as a synonym of “Bufo” arabicus [42].

The evolution of Arabian bufonids and their close relatives remains a challenging biogeographic and taxonomic problem, and it is unknown if the unassigned Arabian toads are derived from African, Southwest Asian, or Western Eurasian lineages, or are the result of in-situ diversification on the Peninsula. In addition, the biogeographic patterns of the genus Duttaphrynus have not been investigated yet have important implications concerning whether amphibians successfully colonized the Horn of Africa using the Arabian Peninsula as a stepping-stone. A similar problem persists for “Bufo” tihamicus and pentoni, which could be derived from African, Southwest Asian, or Western Eurasian clades, each of which has implies different biogeographic scenarios for explaining the current geographic distributions of these two species.

Through several years of fieldwork, TJP obtained key samples of Arabian taxa (including “Bufo” arabicus, “Bufo” tihamicus, D. dhufarensis) as well as many biogeographically important species surrounding the Arabian Peninsula (including “Bufo” pentoni and D. dodsoni), finally allowing an assessment of their evolutionary relationships using a molecular phylogenetic framework. We seek to untangle the various biogeographic scenarios resulting in the evolution of the Arabian bufonids and their close relatives. We aim to identify the closest relatives of “Bufo” tihamicus and “Bufo” arabicus to determine if they are derived from African, Western Eurasian, or South Asian lineages. If they are African in origin, we predict they will have diverged from their closest mainland relatives either 1) following the formation of the Red Sea (23 Ma), or 2) as a result of dispersal across a southern Afro-Arabian land bridge (10–5.3 Ma). Alternatively, if they are Western Eurasian or South Asian in origin, we predict they will have colonized the Arabian Peninsula following establishment of Eurasian land bridge connections (18–15 Ma). We also seek to test the stepping-stone colonization of the Horn of Africa by the genus Duttaphrynus, and predict a sister-taxon relationship between D. dodsoni and D. dhufarensis, with a divergence time following permanent Eurasian land bridge connections (15 Ma). We use newly generated multi-locus molecular data and a comprehensive data set of published bufonid sequences to reconstruct phylogenetic relationships to identify the origins of the Arabian taxa. We use these molecular data to estimate divergence times to identify key biogeographic events that could have led to the formation of these lineages. We examine our results in the context of prior studies that have investigated historical biogeographic patterns in other Arabian taxa, and highlight areas requiring further study.