Evolution of Blind Beetles in Isolated Aquifers: A Test of Alternative Modes of Speciation Remko Leijs 1,2,3 *, Egbert H. van Nes 4 , Chris H. Watts 1 , Steven J. B. Cooper 1,2 , William F. Humphreys 2,5,6 , Katja Hogendoorn 7 1 South Australian Museum, Adelaide, South Australia,Australia, 2 School of Earth and Environmental Sciences, University of Adelaide, Adelaide, South Australia, Australia, 3 School of Biological Sciences, Flinders University of South Australia, Adelaide, South Australia, Australia, 4 Department of Aquatic Ecology and Water Quality Management, Wageningen University, Wageningen, The Netherlands, 5 Western Australian Museum, Welshpool, Western Australia, Australia, 6 School of Animal Biology, University of Western Australia, Nedlands, Western Australia, Australia, 7 School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, South Australia, Australia Abstract Evidence is growing that not only allopatric but also sympatric speciation can be important in the evolution of species. Sympatric speciation has most convincingly been demonstrated in laboratory experiments with bacteria, but field-based evidence is limited to a few cases. The recently discovered plethora of subterranean diving beetle species in isolated aquifers in the arid interior of Australia offers a unique opportunity to evaluate alternative modes of speciation. This naturally replicated evolutionary experiment started 10-5 million years ago, when climate change forced the surface species to occupy geographically isolated subterranean aquifers. Using phylogenetic analysis, we determine the frequency of aquifers containing closely related sister species. By comparing observed frequencies with predictions from different statistical models, we show that it is very unlikely that the high number of sympatrically occurring sister species can be explained by a combination of allopatric evolution and repeated colonisations alone. Thus, diversification has occurred within the aquifers and likely involved sympatric, parapatric and/or microallopatric speciation. Citation: Leijs R, van Nes EH, Watts CH, Cooper SJB, Humphreys WF, et al. (2012) Evolution of Blind Beetles in Isolated Aquifers: A Test of Alternative Modes of Speciation. PLoS ONE 7(3): e34260. doi:10.1371/journal.pone.0034260 Editor: Bengt Hansson, Lund University, Sweden Received November 11, 2011; Accepted February 24, 2012; Published March 30, 2012 Copyright: ß 2012 Leijs et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by Australian Research Council grants to SJBC, CHW and WFH (A00106441 and DP0663675) and RL (DP0346583), and by Australian Biological Resources Study grants to CHW and WFH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: remko.leijs@samuseum.sa.gov.au Introduction Strong evidence for sympatric speciation has recently been provided in vitro [1], and the concept is well supported by theoretical analyses [2–7]. Often cited examples from natural systems involve the evolution of new species in relatively closed systems such as crater lakes (e.g. cichlid fishes [7,8]), and islands (e.g. Anolis lizards [9], palms [6,10,11] Hawaiian spiders [12]) where there is evidence for colonisation by a single ancestral species and subsequent niche partitioning. However, even after the presence of sympatric, closely related sister species has been established, it remains uncertain whether the co-occurring species pairs have evolved in sympatry or whether the divergence of the species occurred in isolation and involved multiple invasions [13–16]. To distinguish between these modes of speciation a statistical approach is needed, which requires the presence of multiple sympatrically occurring species pairs. Such data sets have hitherto been lacking. The recent discovery of communities of invertebrates in Australian subterranean aquifers that have evolved in isolation for millions of years provides a unique opportunity to evaluate the occurrence of sympatric versus allopatric modes of speciation in a natural environment. In the Late Miocene – Pliocene, (10-5 million years ago, Mya), the interior of Australia underwent aridification [17]. During this process hundreds of subterranean aquifers in calcrete limestone deposited along palaeo-drainage systems became biologically isolated [18–22] (Figure 1). Phylogenetic studies [21] revealed that surface species of diving beetles (Coleoptera, Dytiscidae) took refuge in these subterranean aquifers during one or more periods of extreme aridity. This resulted in the evolution of one to three species of blind, wingless (apterous), de-pigmented (stygobitic) endemic species per aquifer. The aquifers all provide similar, very stable ecological conditions [23], and thus the colonisation events can be viewed as a repeated natural speciation experiment. The massive radiation generated by this natural experiment has only recently been uncovered. In the last 12 years 99 new stygobitic beetle species have been described from 52 isolated aquifers [24]. These species now represent, by far, the world’s most diverse subterranean diving beetle fauna [25]. Each beetle species is restricted to a single aquifer, indicating the complete isolation of the system. The coexisting species all differ markedly in size and morphology ([24] and references therein), which points to the possibility that they occupy distinct niches. In most aquifers, the co-existing species appear to be descendants from distantly related ancestral lineages [21], suggesting an allopatric process of speciation. However, eleven of the studied aquifers contain sister species. PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34260