More than 350 wildlife sites, including nature reserves, ancient woodlands and wetlands which are home to some of Britain's rarest species, are threatened by the high-speed rail link, an investigation by The Independent on Sunday has established.

The HS2 project in both its phases could affect wildlife in an area the size of Dorset. Among the sites that could be directly damaged or indirectly affected are a national nature reserve, 10 county wildlife trust reserves, about 50 ancient woods, 30 river corridors, 24 Sites of Special Scientific Interest (SSSIs, the highest level of protection) and hundreds of other wildlife habitats. Among the rare or scarce species at risk are the small blue butterfly, long-eared owl, stag beetle, great crested newt, purple hairstreak butterfly and Bechstein's bat.

Hilary Wharf, the director of HS2 Action Alliance, said: "The Government has done everything it can to pretend that HS2 will not have a huge negative environmental impact. But we will lose for ever ancient woodlands, wildlife and nature reserves, not to mention an area of outstanding natural beauty."

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Paul Wilkinson, head of living landscape for the Wildlife Trusts, said: "The HS2 proposals could have devastating impacts on hundreds of wildlife-rich places previously saved over decades. Nottinghamshire Wildlife Trust, for example, spent 20 years trying to save an area from becoming landfill only for it to be carved up by HS2."

Fierce campaigns against the impact of phase one to Birmingham are already being fought. The outcomes of five judicial reviews are expected soon, and there are sure to be more when the struggles get going over the phase two route to Manchester and Leeds. Time is short. The route must be decided by 2016.

The cost of the project is £33bn, but it is liable to exceed that. Phase one is due to start construction in 2017, and be finished in 2026; two is due for completion in 2032. The Government says it will create 100,000 jobs and cut journey times from London to Manchester to 68 minutes, about half what it is now. But Stephanie Hilborne, chief executive of the Wildlife Trusts, said: "This scheme is being driven forward in the name of progress, but what kind of progress is it that goes backwards on the protection of the natural environment?"

Our calculations about the impact of HS2 are based on the detailed assessment done by environmental organisations on phase one, and preliminary studies of phase two. The total mileage of the proposed lines is 330 miles, and the width of the track and associated lineside varies between 27m and 57m. In an estimated 165 cases, the line goes straight through a wildlife site. But in many other instances it will threaten a site and its species, either through construction, disrupting watercourses, pollution, or creating a barrier between sites and isolating them.

The Wildlife Trusts have identified sites where the line goes through or right by them, and those within 500m of the centre of the planned track. Many of the trusts' concerns are detailed on our map. They include:

• The line going through the middle of Water Haigh Woodland Park near Woodlesford, formerly an area of spoil heaps where enormous amounts of work had been done to return it to nature.

• Bernwood Forest in Buckinghamshire, where the line would cut through the middle of the territory of the rare Bechstein's bat, a tiny species about the size of a matchbox.

• Ancient woodland right across the country, a habitat that has taken at least 400 years of non-disturbance to create, and is already depleted.

• Nature reserves such as Norbriggs Flash in Derbyshire cut in half.

A powerful lobby is building against the routes, or even against the idea of such a hugely expensive scheme. The Countryside Alliance's executive chairman, Barney White-Spunner, said: "The costs of HS2 – both to the environment and the public purse – are simply too great to justify."

Nick Reeves, executive director of the Chartered Institute of Water and Environmental Management, said: "It's another example of unsustainable development that exposes the myth of the 'greenest government ever'. The coalition has sanctioned the building of unnecessary infrastructure in the name of economic growth at a huge cost to the environment and some of Britain's most precious landscapes. Romantics see this as a Victorian-style grand project that is more about Britain's prestige and trumping the French and the Germans, and ensuring that large, lucrative contracts flow in response to the wailing of powerful lobbyists. With £33bn at stake, the priceless beauty of the countryside cannot compete."

Sue Holden, chief executive of the Woodland Trust, said: "Transport that destroys ancient woodland cannot be called 'green'. This irreplaceable habitat covers just 2 per cent of the UK. The unique, undisturbed soils and ecosystems found in ancient woodland form our richest land habitat and support a host of rare, protected and threatened wildlife – species that are slow to react to change, find it difficult to adapt and are not mobile enough to move to other locations to survive. Ancient woodland can never be replaced."

The very high speeds of the trains will make getting changes to the routes all the more difficult, as Natalie Bennett, leader of the Green Party, explained: "The design speed is 250mph. This should be reduced to 186mph or 200mph, similar to HS1. The higher design speed requires straighter alignments, which are more difficult to fit into the landscape. A lower design speed would enable it to follow existing transport corridors and contours more closely."

Many in the conservation world are also concerned about the line ruining "living landscapes", where much has been done to join up wildlife sites. Liz Ballard, chief executive of the Wildlife Trust for Sheffield and Rotherham, said: "With so many areas potentially destroyed or damaged, our vision for a connected network for nature and all the work we've done over the past few decades will be under threat." Alister Hayes, living landscapes manager at the London Wildlife Trust, said: "Further landscape fragmentation as a result of HS2 will isolate species, leading to their decline. We are looking at significant environmental damage here when the economic benefits for HS2 have not yet been proven satisfactorily."

Some influential MPs are now among those dismayed at the potential impact. Cheryl Gillan, the Conservative MP for Chesham and Amersham, said: "I've always objected to it since 2009. I'm not sure that this is the right project at the right time in the right place. If it is going to help kick-start the economy in the North, why not start it in the North and do the connectivity between the northern cities? My constituency is an area of outstanding natural beauty. Why designate something an AONB and then run a major thing right through the middle of it?"

Even supporters have concerns. Angela Smith MP said: "I support HS2, because we do need more capacity. But that shouldn't come at a disproportionate price paid by wildlife."

The toll on individual animals will be great. A Spanish study found that high-speed trains kill about 36 animals per kilometre. If that was applied to both phases of HS2, this would result in some 20,000 mammals, reptiles and amphibians being killed on the line every year.

An HS2 Ltd spokesman, said: "Wherever practicable the proposed routes for HS2 have been designed to minimise potential impacts on protected habitats, wildlife, historic sites, waterways and rivers. We have carried out extensive environmental surveys and have set up regular meetings with environmental organisations to talk about ways in which to minimise the environmental impact."

Sites under threat

Buckinghamshire

Bernwood Forest, where the line would cut through territory of the rare Bechstein's bat and bite into several other key sites in the Chilterns region, including Weedon Hill Woods, Lotts Wood and Pipers Wood near Amersham.

Sheffield

Twelve sites including three ancient woodlands at Smithy Wood and Hesley Wood, sites at Holbrook Marsh, Woodhouse Washlands and Treeton Dyke.

Birmingham

Six hundred metres of viaduct will carve up Park Hall, a nature reserve at Water Orton (home to rare moths and birds), and cut through ancient woodland.

Staffordshire

The line would go through ancient woodlands near Lichfield, and bisect a wetland with botanically rich grassland near the village of Hints.

Cheshire

Two ancient woodlands will be bisected, a serious loss in England's least wooded county.

Lancashire

Lightshaw Meadows reserve near Wigan, and Abram Flash SSSI, important for rare birds. Also crosses 7,000-year-old Chat Moss peat bog and splits two SSSIs.

Yorkshire

Water Haigh Woodland Park, near Woodlesford, and Wombwell Wood, near Barnsley, an ancient woodland home to skylarks and woodpeckers, will be destroyed, claim campaigners.

Nottinghamshire

SSSI at Bogs Farm Quarry, home to rare frog orchid, would be cut in two. There would also be damage to the county's unimproved grassland, nearly 99 per cent of which has been lost since 1930.

Derbyshire

Norbriggs Flash nature reserve, mix of pasture and wetland and home to snipe, will be cut in half.

Warwickshire

Up to 80 sites of county importance are vulnerable.

Middlesex and Hertfordshire

Broadwater Lake Nature Reserve, part of the Mid-Colne Valley SSSI, and home to one of the most important wetland breeding bird communities in Greater London.

London

Eighteen wild sites affected, including Perivale Wood, the capital's oldest nature reserve and home to stag beetle, great crested newt and bluebells, an important indicator of undisturbed and ancient woodland.