The Ohio River is the largest tributary by volume of the Mississippi River. It is approximately 981 miles (1,579 km) long an is located in the eastern United States. The river had great significance in the history of the Native Americans. It was a primary transportation route during the westward expansion of the early U.S. It flows through or along the border of six states, and its drainage basin encompasses 14 states, including many of the states of the southeastern U.S. through its largest tributary, the Tennessee River. During the nineteenth century, it was the southern boundary of the Northwest Territory, thus serving as the border between free and slave territory. It is sometimes referred to as the "Mason-Dixon line" as it is commonly acknowledged as the western natural extension of the original Mason-Dixon line that divided Pennsylvania and Delaware from Maryland and West Virginia (and at one time part of Virginia) thus being the unofficial, and at times disputed, border between the Northern United States and the American South or upland South. The Ohio River is a climatic transition area as its water runs along the periphery of the humid subtropical climate and humid continental climate thereby being inhabited by fauna and flora of both climates.