Divergence time and biogeography
The Cape Peninsula (clade A) is an isolated mountainous area surrounded by the Atlantic Ocean in the west and the Cape Flats to the east, thus isolating the velvet worm fauna. This conforms to the mainland–island metapopulation pattern where, as a result, genetic differentiation among populations is low in comparison with the two remaining clades (B and C) (Mosblech et al., 2011). In contrast, genetic divergence (FST) among localities in the latter clades is high, indicating considerable genetic structure, and provides further evidence for low dispersal among localities (Mosblech et al., 2011). Peripatopsis capensis is generally restricted to forested palaeorefugia occupying sheltered ravines and gorges on the Cape Peninsula. Contemporary and historical forest habitat in this region could be the main driver of the observed phylogeographic pattern. Regionally, the prevailing climate during the Pliocene/Pleistocene would have marked a dynamic period for the Cape Flats–Cape Peninsula boundary. Divergences within the Cape Peninsula clade ranging between approximately 600 000 and 10 000 b.p. suggest a response to fluctuating climatic patterns. Climatic fluctuations periodically created islands of differing vegetation types, leading to repeated periods of habitat expansions, followed by allopatric diversification (Tolley et al., 2006). Schalke (1973) describes at least two extensions of Podocarpus forest on the Cape Flats between 50 000 and 20 000 b.p. Sea-level dynamics played a large role in the onshore conditions of the CFR. Linder (2003) suggests that low sea levels may have led to a loss of rainfall at mountain regions. These marine regressions are associated with lower absorption of incoming solar radiation due to a decrease in the area covered by the oceans. This results in decreased evaporation and, consequently, decreased precipitation with an overall effect of increased aridity on land (Theron, 1983). Lower temperatures further detract from past (Miocene) tropical conditions (Deacon, 1983a; Daniels et al., 2001). Forest habitat loss was further exacerbated by prehistoric pastoralists (1500–2000 b.p) who used fire to manage the vegetation (Deacon, 1983a,b; Brain & Sillen, 1988). Hence, the increasingly limited forest cover has a reduced capacity to maintain high levels of genetic diversity within the P. capensis Cape Peninsula clade.
Biogeographically, the Cape Peninsula (clade A) is separated from the Theewaterskloof-Overstrand region (clade C) by a phylogeographic barrier of approximately 60 km, the Cape Flats. The Cape Flats is assumed to have emerged after the closure of the ‘Cape Strait’, which once united Table Bay and False Bay (Schalke, 1973). The Cape Flats is characterized by the deposition of aeolian sands of marine origin (Walker, 1952; Siesser & Dingle, 1981; Adelana et al., 2010). Thus, the Cape Flats is characterized by nutrient-poor and calcareous alkaline soils supporting the shrub-dominated Cape Flats Dune Strandveld (Mucina & Rutherford, 2006). Several phylogeographic studies on mountain-living invertebrates have identified the Cape Flats as a barrier to gene flow between the Cape Peninsula and the Theewaterskloof-Overstrand region (Daniels et al., 2001; Wishart & Hughes, 2001, 2003; Gouws et al., 2004, 2010). Interestingly, the Rondevlei locality falls within the Cape Flats phylogeographic break. Yet, the Rondevlei specimen clustered with the Theewaterskloof-Overstrand clade despite being geographically closer to the Cape Peninsula. More specifically, the phylogenetic trees retrieved indicated that it has a closer genetic affinity with Jonkershoek taxa, suggesting that a historical corridor of suitable habitat may have connected these localities in the past. This can be explained by the Pleniglacial periods between 33 000 and 45 000 b.p during the Pleistocene, where mixed Podocarpus forests were present in the central Cape Flats region (Schalke, 1973; Chase & Meadows, 2007).
The highest levels of genetic variation were observed within the Overberg clade (B). High genetic variation and a lack of shared haplotypes (Fig. 4) suggest that dispersal is limited among localities at the Overberg. However, within clade B, two specimens from Marloth were nested within the Grootvadersbosch subgroup, suggesting the possibility of a historical connection between the two localities. Both these localities occur along the sheltered slopes at the middle and upper reaches of the west–east-trending southern Langeberg mountain range (McDonald et al., 1996). These areas receive between 600 and 1200 mm of rainfall annually. According to McDonald & Cowling (1995), the latter lies within the nonseasonal rainfall zone of the CFR where rainfall is associated with circumpolar westerly fronts and post-frontal conditions related to the advection of cool moist air above the warm Indian Ocean. Gene flow between Potberg-De Hoop and the former localities is limited by an area colloquially known as the ‘Rûens’, which is an undulating landscape straddled by the Potberg-De Hoop Mountain in the south and the Langeberg Mountains in the north. The Rûens is characterized by a highly dissected landscape, relatively low rainfall and shallow.
Soils supporting Rûens Silcrete Renosterveld (Schloms et al., 1983). Divergence within clade B is dated between 0.75 and 0.92 Mya in the mid-Pleistocene. The late Pleistocene period was characterized by lower sea levels (100–160 m below current sea levels) and a drier climate (Deacon, 1983a,b; Cowling & Lombard, 2002).
Clades B and C are separated by the Breede River Valley Basin. According to Theron (1983), the elevated coastal topography in the southern Cape facilitated the establishment of larger rivers being deeply entrenched in steep-sided valleys as a result of marine regressions. The Breede River Valley lies between the Riviersonderend Mountains in the south and the Langeberg Mountains in the north (Kirchner et al., 1997). The valley displays unique abiotic characteristics relative to the latter montane regions. It is characterized as semi-arid and receives approximately 270 mm of rainfall per annum. The valley comprises a variety of soil types (sandy, aeolian, acidic, alluvial, clay and loam) supporting different variants of fynbos (Mucina & Rutherford, 2006). These abiotic factors are unsuitable for the establishment of forests.
Clade C (Theewaterskloof-Overstrand) represents an area that forms the boundary between the winter rainfall zone (WRZ) of the westernmost CFR and the year-round rainfall zone (YRZ) in the south-east. In addition to high climatic variability, the Theewaterskloof-Overstrand subregion also forms a large part of the Cape Fold Mountains and hence represents a landscape characterized by high topographic heterogeneity. As a result, clade C displayed three unique haplotypes and had the highest haplotypic (h) and nucleotide (πn) diversity. These findings suggest that clade C has had a long evolutionary history in large, stable populations (Fitzpatrick, 2009).
Evolutionary and taxonomic considerations
The biogeographical conclusions of this study have important implications for the taxonomy of the Cape velvet worm species, Peripatopsis capensis. Multiple lines of independent evidence exist for the recognition of three evolutionary units within the P. capensis. All three clades are geographically discrete and exclusive, with no dispersal between them. Furthermore, all three clades were characterized by marked sequence divergence values, > 7% for the COI locus. This value is higher than what has been reported for sister velvet worm species. For example, Hebert et al. (1991) employed a 3.3% sequence divergence in the Jamaican velvet worm species, Plicatoperipatus jamaicensis, which comprised two distinct lineages. Similarly, Rockman et al. (2001) obtained sequence divergence values ranging from 1.1 to 11.6% for morphologically distinct Planipapillus species. Despite the comparably high divergence values retrieved for P. capensis, we exercise caution before basing our classification on a single molecular marker. The inclusion of the nuclear 18S rDNA marker to create a combined topology was congruent with the COI data, providing additional corroborative evidence for the genetic distinction of the three clades. We prefer to use the phylogenetic species concept as a starting hypothesis for species description (Cracraft, 1989). Preliminary gross morphological and scanning electron microscopy (SEM) analyses have revealed several potential diagnostic characters. Collectively, these results provide evidence for the recognition of two novel lineages within P. capensis because the species was originally described from the Cape Peninsula. A study is currently underway to describe these two novel lineages. The present study shows that velvet worm diversity associated with CFR forests was underestimated. The use of Peripatopsis capensis as a model organism to make biogeographical extrapolations of its habitat adds to a growing body of literature that demonstrates the utility of invertebrate habitat specialists in understanding forest biogeographic patterning and highlights the conservation value of these forest patches because our study further suggests the presence of undocumented biodiversity (Boyer et al., 2007; Garrick et al., 2007; Álvarez-Presas et al., 2011). Our study yielded significant novel insight into the biogeographic patterning of Afromontane forest habitat and provided a unique glimpse into the phylogeographic structure of an enigmatic endemic invertebrate group.