ORIGINAL ARTICLE Phylogeographic patterning in a freshwater crab species (Decapoda: Potamonautidae: Potamonautes) reveals the signature of historical climatic oscillations Savel R. Daniels1,2*, Gavin Gouws1,3 and Keith A. Crandall2 1Evolutionary Genomics Group, Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa, 2Department of Integrative Biology, 401 Widstoe Building, Brigham Young University, Provo, UT 84602-5181, USA and 3Centre of Excellence in Natural Resource Management, University of Western Australia, 444 Albany Highway, Albany, WA 6330, Australia *Correspondence: Savel R. Daniels, Evolutionary Genomics Group, Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa. E-mail: srd@sun.ac.za ABSTRACT Aim The phylogeographic relationships among populations of the common Cape River crab, Potamonautes perlatus, are examined to investigate whether the contemporary population genetic structure is congruent with the hypothesized hydrographic evolution of drainage systems established during the Pliocene, or whether it reflects an older Miocene climatic amelioration. Location 139 samples of P. perlatus were collected from 31 populations distributed among the five major perennial drainage systems and a number of smaller catchments in the Western and Eastern Cape, South Africa. Methods Phylogeographic analysis using parsimony, maximum likelihood, minimum evolution and Bayesian inferences was employed for the 16S rRNA mtDNA gene region, while bootstrapping and posterior probabilities were used to assess the robustness of clades. In addition, nested clade analysis was performed in an attempt to disentangle the contemporary and historical factors that have sculpted genealogical relationships among conspecific populations of P. perlatus. Results Phylogenetic topologies were congruent irrespective of the evolutionary method employed. Two highly distinct reciprocally monophyletic clades characterized by marked levels of corrected sequence divergence were present, with no shared haplotypes between the two major phylogroups. Phylogroup one comprises the populations of the westward-flowing drainages (mainly the Berg and Olifants drainages), and phylogroup two comprises all of the southward- flowing drainages and can further be divided into two subclades ��� one containing the Breede River populations, and the other containing the Gamtoos and Gourits drainage systems. The nested clade analysis demonstrated restricted gene flow and long-distance dispersal for a number of higher clade levels. The higher-level groups and results for the total cladogram suggest either fragmentation or isolation by distance. Main conclusions Freshwater crabs are generally highly philopatric, and dispersal, although not common, has occurred historically. The westward-flowing drainages (Berg, Olifants, Eerste, Liesbeeck and Tokai) are isolated from the southward-flowing drainages by the Cape Fold Mountains, while the southward- flowing drainages have a number of tributaries that extend into the low-lying regions, allowing for gene flow between these three major drainages systems (Breede, Gamtoos and Gourits). Among the westward-flowing drainages, a more intensive sampling regime is required to understand evolutionary relationships. Our molecular results suggest that the observed patterns pre-date the formation of contemporary hydrographic patterns in the Cape. This suggests that an older Late Miocene event has severely impacted the contemporary population structure Journal of Biogeography (J. Biogeogr.) (2006) 33, 1538���1549 1538 www.blackwellpublishing.com/jbi �� 2006 The Authors doi:10.1111/j.1365-2699.2006.01537.x Journal compilation �� 2006 Blackwell Publishing Ltd

INTRODUCTION Ubiquitous philopatric inland aquatic taxa incapable of navigating marine or terrestrial barriers and lacking active dispersal life-history stages, such as freshwater crabs (which produce a small number of lecithotrophic eggs, lack planktonic larval stages, and exhibit direct development followed by maternal care), provide an excellent opportunity to examine relationships between historical and contemporary physiogra- phy and hydrology. Freshwater crabs are generally perceived to be highly amphibious, since they are frequently observed on foraging excursions considerable distances from freshwater and are assumed to be capable of dispersing across terrestrial barriers. Banarescu (1990) argues that this renders them unsuitable for the inference of patterns of drainage evolution and demography through the examination of relationships among conspecific populations. Phylogenetic analyses of the southern African freshwater crab species (Daniels et al., 2002) have, however, revealed clear biogeographical patterning, negating Banarescu���s (1990) conclusion. More recently, a phylogeographic study, using allozymes and limited 16S rRNA mtDNA sequencing, undertaken by Daniels (2003) on the widely distributed common Cape freshwater crab, Potamonautes perlatus revealed allozymic invariance contrasted with marked sequence divergence. This study revealed two markedly distinct clades, which comprised western and southward-flowing rivers, respectively, with the Cape Fold Mountains potentially being the barrier to gene flow. These results suggest that this species is highly philopatric and is incapable of dispersing over large geographic distances. More importantly, it appears that the contemporary genetic structure observed has been affected by historical drainage isolation and climatic changes. Limited geographic sampling (37 specimens from 10 localities) as well as the absence of samples from key drainages (such as the Gourits River) in the earlier study (Daniels, 2003) precluded the author from drawing robust conclusions with respect to intraspecific evolutionary relationships and associated processes leading to population structure among the crabs of the various drainage systems in the region. The area encompassed by the Western and Eastern Cape province has undergone large-scale Miocene climatic and environmental changes. These have included several severe marine transgressions, coupled with the devel- opment of the cold-water proto-Benguela current (Siesser, 1978, 1980), which led to considerable habitat alterations that impacted taxa, including freshwater taxa, in the region (Daniels et al., 2004). The modern arrangement of drainages in the Western and Eastern Cape was established at the end of tectonic activity during the Late Pliocene (5.3 Ma) (Deacon, 1983 Partridge & Maud, 1987). The model of drainage evolution suggested by Partridge & Maud (1987) points towards a close geomorphological link between the Berg and Olifants rivers, while a close relationship is also suggested between the Breede, Gamtoos and Gourits river systems, potentially explaining faunal similarities between the drai- nages. It remains unclear whether the earlier Miocene climatic oscillations or the recent establishment of hydrographic boundaries during the Pliocene represent the more significant episodes influencing the evolutionary history of conspecific populations of P. perlatus. In the present study, a comprehensive geographic samp- ling regime of P. perlatus populations is undertaken among the five major perennial drainages in the Cape to test whether the evolutionary relationships among allopatric populations reflect the dramatic Miocene climatic events, or whether they reflect contemporary hydrographic patterns established during the Pliocene. In the present study, we apply both phylogenetic analyses and nested clade analysis (NCA) in an attempt to understand the demographic history among conspecific populations of this taxon. NCA is a powerful statistical method that allows for a rigorous assessment of the relationship between geography and haplotype distribution and discriminates between processes that have influenced population structure (Templeton et al., 1995 Templeton, 2004). MATERIALS AND METHODS Sample collection The five major perennial rivers in the region are the Olifants, Berg, Breede, Gourits and Gamtoos systems. The Olifants and Berg rivers flow towards the west coast, the Breede River drains the intermountain fault basin of the Hex and Langeberg Mountains, while the Gourits River drains the Karoo Plateau and Agulhas Plain, and the Gamtoos drains the Baviaanskloof and Groot Swartberg mountains. A total of 139 P. perlatus samples were collected from 31 localities throughout the in this species, as recent Pliocene hydrographic boundaries do not correspond to the phylogeographic pattern observed. Conservation efforts for aquatic taxa should clearly be directed at the catchments, in an attempt to conserve biological diversity. Keywords Climate change, drainage, freshwater crabs, Miocene, nested clade analysis, palaeogeography, phylogeography, Potamonautes perlatus, South Africa, 16S rRNA mtDNA. Phylogeography of a freshwater crab species Journal of Biogeography 33, 1538���1549 1539 �� 2006 The Authors. Journal compilation �� 2006 Blackwell Publishing Ltd