Knowledge of DNA evolution is central to our understanding of biological history, but how fast does DNA change? Previously, pedigree and ancient DNA studies—focusing on evolution in the short term—have yielded molecular rate estimates substantially faster than those based on deeper phylogenies. It has recently been suggested that short-term, elevated molecular rates decay exponentially over 1–2 Myr to long-term, phylogenetic rates, termed “time dependency of molecular rates.” This transition has potential to confound molecular inferences of demographic parameters and dating of many important evolutionary events. Here, we employ a novel approach—geologically dated changes in river drainages and isolation of fish populations—to document rates of mitochondrial DNA change over a range of temporal scales. This method utilizes precise spatiotemporal disruptions of linear freshwater systems and hence avoids many of the limitations associated with typical DNA calibration methods involving fossil data or island formation. Studies of freshwater-limited fishes across the South Island of New Zealand have revealed that genetic relationships reflect past, rather than present, drainage connections. Here, we use this link between drainage geology and genetics to calibrate rates of molecular evolution across nine events ranging in age from 0.007 Myr (Holocene) to 5.0 Myr (Pliocene). Molecular rates of change in galaxiid fishes from calibration points younger than 200 kyr were faster than those based on older calibration points. This study provides conclusive evidence of time dependency in molecular rates as it is based on a robust calibration system that was applied to closely related taxa, and analyzed using a consistent and rigorous methodology. The time dependency observed here appears short-lived relative to previous suggestions (1–2 Myr), which has bearing on the accuracy of molecular inferences drawn from processes operating within the Quaternary and mechanisms invoked to explain the decay of rates with time.

Several recent estimates of global biodiversity have concluded that the total number of species on Earth lies near the lower end of the wide range touted in previous decades. However, none of these recent estimates formally explore the real “elephant in the room”, namely, what proportion of species are taxonomically invisible to conventional assessments, and thus, as undiagnosed cryptic species, remain uncountable until revealed by multi-gene molecular assessments. Here we explore the significance and extent of so-called “hyper-cryptic” species complexes, using the Australian freshwater fish Galaxias olidus as a proxy for any organism whose taxonomy ought to be largely finalized when compared to those in little-studied or morphologically undifferentiated groups. Our comprehensive allozyme (838 fish for 54 putative loci), mtDNA (557 fish for 605 bp of cytb), and morphological (1963–3389 vouchers for 17–58 characters) assessment of this species across its broad geographic range revealed a 1500% increase in species-level biodiversity, and suggested that additional taxa may remain undiscovered. Importantly, while all 15 candidate species were morphologically diagnosable a posteriori from one another, single-gene DNA barcoding proved largely unsuccessful as an a priori method for species identification. These results lead us to draw two strong inferences of relevance to estimates of global biodiversity. First, hyper-cryptic complexes are likely to be common in many organismal groups. Second, no assessment of species numbers can be considered “best practice” in the molecular age unless it explicitly includes estimates of the extent of cryptic and hyper-cryptic biodiversity. [Galaxiidae; global estimates; hyper-diverse; mountain galaxias; species counts; species richness.]

River capture is potentially a key geomorphological driver of range expansion and cladogenesis in freshwater-limited taxa. While previous studies of freshwater fish, in particular, have indicated strong relationships between historical river connections and phylogeographic pattern, their analyses have been restricted to single taxa and geological hypotheses were typically constructed a posteriori. Here we assess the broader significance of river capture among taxa by testing multiple species for the genetic signature of a recent river capture event in New Zealand. During the Quaternary an upper tributary of the Clarence River system was diverted into the headwaters of the Wairau River catchment. Mitochondrial DNA (control region and cytochrome b) sequencing of two native galaxiid fishes (Galaxias vulgaris and Galaxias divergens) supports headwater exchange: populations from the Clarence and Wairau Rivers are closely related sister-groups, whereas samples from the geographically intermediate Awatere River are genetically divergent. The upland bully Gobiomorphus breviceps (Eleotridae), in contrast, lacks a genetic signature of the capture event. We hypothesize that there is an increased likelihood of observing genetic signatures from river capture events when they facilitate range expansion, as is inferred for the two galaxiid taxa studied here. When river capture merely translocates genetic lineages among established populations, by contrast, we suggest that the genetic signature of capture is less likely to be retained, as might be inferred for G. breviceps. Rates of molecular evolution calibrated against this recent event were elevated relative to traditional estimates, consistent with the contribution of polymorphisms to branch lengths at shallow phylogenetic levels prior to fixation by purifying selection and drift.