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Background  The Caribbean offers a unique opportunity to study evolutionary dynamics in insular mammals. However, the recent extinction of most Caribbean non-volant mammals has obstructed evolutionary studies, and poor DNA preservation associated with tropical environments means that very few ancient DNA sequences are available for extinct vertebrates known from the region’s Holocene subfossil record. The endemic Caribbean eulipotyphlan family Nesophontidae (“island-shrews”) became extinct ~ 500 years ago, and the taxonomic validity of many  Nesophontes  species and their wider evolutionary dynamics remain unclear. Here we use both morphometric and palaeogenomic methods to clarify the status and evolutionary history of  Nesophontes  species from Hispaniola, the second-largest Caribbean island.    Results  Principal component analysis of 65  Nesophontes  mandibles from late Quaternary fossil sites across Hispaniola identified three non-overlapping morphometric clusters, providing statistical support for the existence of three size-differentiated Hispaniolan  Nesophontes  species. We were also able to extract and sequence ancient DNA from a ~ 750-year-old specimen of  Nesophontes zamicrus , the smallest non-volant Caribbean mammal, including a whole-mitochondrial genome and partial nuclear genes.  Nesophontes paramicrus  (39-47 g) and  N. zamicrus  (~ 10 g) diverged recently during the Middle Pleistocene (mean estimated divergence = 0.699 Ma), comparable to the youngest species splits in Eulipotyphla and other mammal groups. Pairwise genetic distance values for  N. paramicrus  and  N. zamicrus  based on mitochondrial and nuclear genes are low, but fall within the range of comparative pairwise data for extant eulipotyphlan species-pairs.    Conclusions  Our combined morphometric and palaeogenomic analyses provide evidence for multiple co-occurring species and rapid body size evolution in Hispaniolan  Nesophontes , in contrast to patterns of genetic and morphometric differentiation seen in Hispaniola’s extant non-volant land mammals. Different components of Hispaniola’s mammal fauna have therefore exhibited drastically different rates of morphological evolution. Morphological evolution in  Nesophontes  is also rapid compared to patterns across the Eulipotyphla, and our study provides an important new example of rapid body size change in a small-bodied insular vertebrate lineage. The Caribbean was a hotspot for evolutionary diversification as well as preserving ancient biodiversity, and studying the surviving representatives of its mammal fauna is insufficient to reveal the evolutionary patterns and processes that generated regional diversity.

Rapid size change associated with intra-island evolutionary radiation in extinct Caribbean “island-shrews”