Out of India, thrice: diversification of Asian forest scorpions reveals three colonizations of Southeast Asia

The "Out of India" hypothesis proposes that Southeast Asian taxa originated in Gondwana, diverged when India rifted, and colonized Southeast Asia upon its Eurasian collision. However, evidence suggests this process was more complex. This study focuses on Asian forest scorpions (Scorpionidae: Heterometrinae) to quantitatively reconstruct their biogeography, testing the "Out of India" hypothesis and refining our understanding of India's interaction with Eurasia. Understanding the biogeography of this group is crucial because it provides insights into the complex interplay of geological events and biological diversification in a geologically dynamic region. Previous studies on various taxa (plants, fish, amphibians, birds, lizards, and arthropods) have offered support for the "Out of India" hypothesis, but the details of the dispersal events remain debated. The timing and manner of India's collision with Eurasia is also a subject of ongoing discussion, with hypotheses ranging from an early collision around 55-50 Ma to a series of contacts spanning millions of years. This research aims to add to this discussion by providing a detailed biogeographical analysis of Heterometrinae, providing a more precise timeline of their dispersal and diversification, integrating geological and paleogeographic data.

Previous studies on the biogeography of Heterometrinae have been largely qualitative. Couzijn's hypothesis, for example, suggested that the ancestor of Heterometrinae and Pandininae originated in eastern Gondwana during the Triassic, with subsequent divergence and dispersal across the Indian and Indochinese landmasses. However, this hypothesis doesn't align with recent geological and biogeographical data. Prendini's phylogenetic work, based on morphological and molecular data, supported an 'Out of India' origin for Asian scorpionids, indicating a divergence from African relatives after India separated from Africa. This study aims to build upon these prior analyses using a more rigorous quantitative approach, incorporating recent paleogeographical models and advanced analytical techniques to refine the understanding of Heterometrinae's evolutionary history.

The study used a reduced dataset from a larger phylogenetic analysis of Heterometrinae, focusing on one terminal per species (35 terminals representing all seven genera and 31 species), plus an outgroup (three genera of Scorpionidae from Africa and the Middle East). DNA was extracted from muscle tissue of preserved specimens and sequenced for three mitochondrial (COI, 16S, 12S) and two nuclear (18S, 28S) gene loci. Sequences were aligned using MAFFT and divergence time estimation was performed in BEAST using a relaxed lognormal molecular clock. Ancestral range reconstruction was conducted using BioGeoBEARS with six defined biogeographical areas (Africa, Western Ghats & Sri Lanka, Greater Indian Subcontinent, Indochina, Sundaland, Philippines). The most suitable model was determined using model selection based on likelihood scores. Diversification analyses were performed using LASER and BAMM to determine the best-fitting model and infer speciation and extinction rates. Phylogenetic diversity and evolutionary distinctiveness were calculated using Picante.

Phylogenetic analyses confirmed the monophyly of Asian Heterometrinae and their sister group relationship with African Pandininae, indicating an African origin for Scorpionidae. The divergence time between Asian and African scorpionids was estimated to be approximately 113 Ma, consistent with earlier biological connectivity between Africa and India. The analysis revealed that after the divergence from African relatives, Asian scorpionids diverged into two major clades around 92 Ma (Late Cretaceous). The KT mass extinction event (around 66 million years ago), potentially exacerbated by Deccan volcanism, caused a range contraction, confining the ancestor of one clade to the Western Ghats of India and the Central Highlands of Sri Lanka. Three independent colonization events of Southeast Asia were identified, corresponding to the periods when India’s northern edge was adjacent to Sumatra (55 Ma), eastern India was near Sumatra (45 Ma), and India made final contact with Eurasia (35 Ma). After the final dispersal event, Heterometrus exhibited a northwest to southeast dispersal pattern in Southeast Asia, with a rapid increase in lineage diversification. The analysis also showed a northward dispersal and diversification of Heterometrinae across the Indian subcontinent during the Miocene, associated with environmental changes linked to the uplift of the Tibetan Plateau and the onset of monsoonal climate, enabling these scorpions to exploit newly available arid habitats. Diversification analyses supported a pattern of fast speciation, predominantly early in the phylogeny.

The findings provide compelling evidence for the "Out of India" hypothesis but demonstrate the complexity of this biogeographical process. The three independent dispersal events to Southeast Asia highlight the dynamic nature of India's interaction with Eurasia and the multiple opportunities for faunal exchange. The diversification in new Southeast Asian habitats was likely driven by the availability of new resources and niches. The subsequent northward diversification in India suggests adaptive radiation in response to changing climatic conditions and habitat types. These results are congruent with biogeographical patterns observed in other taxa and contribute to a more nuanced understanding of India's role in the biogeographical history of South and Southeast Asia. The study refutes previous hypotheses, such as Couzijn's model, by integrating recent geological findings and using sophisticated phylogenetic and biogeographical methods.

This study provides the first comprehensive quantitative biogeographic analysis of Asian forest scorpions, significantly advancing our understanding of their evolutionary history. It confirms an African origin for Scorpionidae and the "Out of India" dispersal of Heterometrinae, with three distinct colonization events in Southeast Asia. The study highlights the influence of both major extinction events and long-term environmental changes on the diversification patterns of this group. Future research could focus on expanding the taxon sampling, incorporating additional molecular markers, and investigating the detailed mechanisms of adaptation to various habitats. Analyzing the evolution of specific traits could also provide further insights into the diversification process.

The study relied on a reduced taxon sampling compared to the original phylogenetic analysis. The absence of fossil data for Heterometrinae required the use of a relaxed molecular clock, introducing uncertainty into divergence time estimations. Incomplete sampling might influence the diversification analyses, although a Monte Carlo Constant Rates test partially addressed this. The biogeographical model used made assumptions about dispersal probabilities between regions.