In this study we examined how cutthroat trout diversity may be organized by isolation into major geographic boundaries. Our data indicate that watershed boundaries do organize primary phylogenetic divisions in cutthroat trout, but historical connections that are no longer evident also appear to have influenced the pattern of diversification. Our range-wide analysis of cutthroat trout diversification provides an additional line of evidence for historic watershed connections that have long been suggested by geologists and are of interest to biologists as a means of explaining the current distribution of fish fauna across a landscape [8, 16, 18, 43].
Intraspecific taxonomic evaluations of cutthroat trout have described up to 14 different subspecies of cutthroat trout, two of which are now considered to be extinct [20, 24]. Of the subspecies we evaluated, the Bonneville-Yellowstone, Colorado River, Rio Grande, greenback, and Lahontan lineages are delimited at least in part by the height of land defining major portions of watersheds. Based on the mtDNA sequence data we compared and the partitioning of genetic variance, cutthroat trout can be divided into eight major lineages that correspond with six of the primary subspecies of cutthroat trout commonly recognized. Hence, watershed boundaries and the intraspecific taxonomic categories that have been used to describe this diversity have been relatively successful in capturing a significant component of the evolutionary diversification in cutthroat trout.
If a molecular clock is applied to the sequence divergence, we estimate an initial divergence from a common ancestor approximately 2 mya. This primary divergence led to one major clade that includes the Bonneville-Yellowstone, Lahontan, westslope, and coastal lineages. The second branch includes the greenback, Rio Grande, Great Basin, and Colorado River lineages. The pattern of haplotype diversification indicates that the lineage leading to the Bonneville-Yellowstone clade was first to diverge from the common ancestor of all cutthroat trout, which may have first colonized the ancestral Snake River as well as areas that are now part of Colorado River watershed. Although the pattern of colonization can never be known with exact certainty, drainage patterns have changed significantly over the period of time estimated from the divergence of the ancestral cutthroat trout. The greatest divergence of cutthroat trout between the Snake and Colorado River drainages indicates a former connection between the two drainages that has since isolated these two main lineages for the longest period of time. Geologic evidence does point to connections between areas that are now in the upper Colorado River watershed but were once part of the Bear River of Idaho, Utah, and Wyoming .
Within each of the main lineages of cutthroat trout, the pattern of relatedness also indicates how several sub-basins may have been connected to each other. The next closest associations within the Bonneville-Yellowstone group are those from coastal cutthroat trout populations followed by a split between populations from the Lahontan basin (and adjacent areas) and westslope cutthroat trout from the Columbia River as well as populations from eastern drainages across the Continental Divide. Downstream colonization and origin of coastal cutthroat trout could have occurred if the ancestral Snake River was connected to the Columbia watershed or other rivers draining to the Pacific Ocean, but the association of the coastal cutthroat trout with westslope cutthroat trout found in Columbia drainages suggests that the Snake River was part of the Columbia watershed when the lineages emerged. Despite the proximity of some parts of the Lahontan basin to areas of the Colorado River watershed, Hubbs and Miller  noted the similarity of the Lahontan fish fauna with that of Columbia, Sacramento, and Death Valley watersheds, indicating that the Lahontan basin was at one time connected to one of these systems and not the Colorado River watershed. Our sequence data on cutthroat trout again point to a connection with the Columbia watershed based on similarity of lineages also found between the watersheds. Finally, the associations of the greenback, Rio Grande, and Great Basin lineages with that of the Colorado lineage all indicate that they were derived in an earlier form of the Colorado River basin. Based on the evolutionary history of the ND2 gene, our data indicate a complex history of isolation and evolutionary divergence of the ancestral Colorado lineage of cutthroat trout. This divergence led to the current Colorado lineage as well as the Rio Grande and Great Basin lineages. The lineage that led to the Rio Grande also appears to have given rise to the greenback lineage, which subsequently colonized its current distribution. The presence of greenback cutthroat trout in eastern draining rivers, or rivers draining to the south in the case of Rio Grande cutthroat, indicate transfers of populations through headwater connections and subsequent isolation. As Minckley and others  note, fish species that commonly penetrate into headwater streams often have representative populations across drainage divides presumably by finding connections, even if they occur infrequently, and are further isolated over longer time periods by the elevation of land.
Two subspecies that did not form monophyletic lineages were the Bonneville and Yellowstone cutthroat trout. The core distribution of Bonneville cutthroat trout is thought to occur in the Bear River, a watershed that originates in the Uinta Mountains of northeastern Utah and flows northward into Wyoming and Idaho before turning sharply back southward towards Utah and the Great Salt Lake (Figure 5). Such a dramatic change of direction is probably representative of stream capture, and in fact the Bear River is thought to have been a tributary to the upper Snake River . Between 600 000 and 50 000 years ago, lava flows crossed the Soda Springs area of the Bear River valley and forced the path of the river to change direction southward . A past connection between the Bear River and upper Snake River would explain the intermixing of the primary lineages of cutthroat trout found in these two watersheds, as well as the morphological similarities observed between these two subspecies [24, 45]. The presence of the two main cutthroat trout lineages in the upper Snake River, but only one in the Bear River would suggest that it originated in the Bear River and subsequently colonized downstream to the Snake River, whereas the second lineage was unable to colonize upstream to the Bear River.
A new perspective generated from our range wide analysis was the geographic distribution and position of what we have referred to as the Great Basin clade of cutthroat trout. Although some previous studies have detected and noted the dramatic genetic divergence of representative samples from this lineage [27, 39], our study provides a clearer picture of its geographic distribution and its position within the phylogeny of cutthroat trout. Our data indicate that despite its proximity to the Bear River and upper Snake River watersheds, it is more closely related to the major diversification of cutthroat trout that includes the Colorado River, greenback, and Rio Grande clades of cutthroat trout. Such a relationship again points to historic connections between watersheds now isolated from each other. As Smith and collaborators  note, previous studies have documented possible hydrological connections between the Bonneville basin and the Colorado River watershed. Connections between these watersheds would be congruent with the Great Basin lineage of cutthroat trout arising in an ancient Colorado River area that either invaded the Bonneville basin by a past connection or evolved within the basin when some parts drained toward the Colorado River watershed. The presence of the Great Basin cutthroat lineage in the upper Snake River may also represent past hydrological connections between the Snake River and the Bonneville basin, but were probably more recent, corresponding to well-documented Pleistocene connections . Approximately 15 000 years ago, Red Rock pass in southeast Idaho was the path of overflow of Lake Bonneville into the upper Snake River travelling through upper Marsh Creek and the Portneuf River; the same location where we detected the Great Basin cutthroat trout lineage . The second location where we observed the Great Basin lineage in the upper Snake River watershed was in the Raft River drainage in southern Idaho, about 150 km to the west of the Portneuf River (Figure 5). This area borders the Bonneville basin and our data suggest that part of the current Raft River drainage was previously in the Bonneville basin and has been captured by the Snake River watershed, isolating the Great Basin cutthroat trout in the headwater tributary streams observed today. Such captures may not be too unexpected because of the gradual subsidence of the Snake River plain that may capture streams bordering the region . As noted in phylogeographic studies of galaxiid fish in New Zealand, geographic patterns of genetic diversity can provide an additional line of evidence to infer historical changes to drainage patterns on the landscape .
Watershed boundaries and barriers to movement can organize major phylogenetic lineages for freshwater fishes in some cases but appear to be most important when populations have been isolated within them for extended periods of time. Glaciated areas often appear to have been more recently connected, allowing major lineages to disperse from glacial refugia over large geographic areas, as illustrated by examples of lake trout (Salvelinus namaycush) and lake whitefish (Coregonus clupeaformis). Each of these species is thought to have dispersed through large proglacial lakes that submerged or connected a number of contemporary watersheds in North America [49, 50]. Populations of freshwater fish that have not established themselves by dispersal into recently de-glaciated areas appear much more likely to exhibit significantly greater evolutionary diversification as a consequence of isolation within watershed boundaries [13, 15, 51]. As a widely distributed species spanning both glaciated and non-glaciated regions of North America, cutthroat trout exhibit reduced haplotype diversity at the northern periphery of its range and higher levels to the south. A reduction in genetic diversity in northern regions is concordant with a pattern of dispersal from a past southern glacial refuge, illustrating the importance of past geologic events on the phylogenetic structure of a freshwater fish species .
As a widely distributed trout species in North America, cutthroat trout have received a great deal of interest as a sport fish, but have also suffered severe declines in abundance when populations have been affected by human plans for consumption of water, land, or introductions of non-native species (see Trotter  for a review). Despite early and more recent attempts to describe the diversity of cutthroat trout [21, 24] it is somewhat surprising that there is remarkably little comparative data to support subspecific designations commonly used to manage different cutthroat trout populations . Perhaps as a result of a lack of such data, only one taxonomic ranking of cutthroat trout is currently recognized by the joint Committee on Names of Fishes, sponsored by the American Fisheries Society and the American Society of Ichthyologists and Herpetologists , and possibly because of the difficulty in diagnosing subspecies of cutthroat trout with traditional meristic characters . A number of previous studies have provided evidence for significant genetic diversification of cutthroat trout [55–58] and even phylogenetic comparisons of representative populations from different parts of the species range [27, 39, 59]. However, our study provides the first range-wide comparison attempting to identify major phylogenetic lineages over much of the geographic area occupied by this polytypic species.
Our analysis provides a better understanding of the main evolutionary lineages of cutthroat trout and their geographic distribution. Similar components have been proposed as a basis for organizing management and conservation units of species [3, 60]. Although primary evolutionary lineages of cutthroat trout may be a logical starting point for management units, even with the addition of genetic diversity at nuclear loci, they may not capture important ecological variation that can occur has as result of local adaption to specific conditions [61, 62]. Hence, in addition to measures of mtDNA and nuclear DNA divergence, ecological diversification not always apparent from molecular phylogenies should also be considered in attempts to preserve the diversity present within a species. Indeed, salmonid fishes often exhibit significant ecological variation  and cutthroat trout in particular have been observed to exhibit significant morphological variability that is associated with specific ecological conditions . Fortunately, biologists have often used the precautionary principle in arguing for protecting populations with unusual life history or ecological relationships even in the absence of direct comparative data. By combining comprehensive range-wide phylogenetic comparisons to identify the distribution of major species lineages, with an understanding of the main ecological factors shaping the phenotype of a species, biologists are likely to provide the best chance of protecting the evolutionary potential of a species in a changing landscape.