Dr Andrew R Barclay
email: a.barclay@clinmed.gla.ac.uk

Lynne M Beattie

Lawrence T Weaver

David C Wilson

Systematic Review: Medical and Nutritional Interventions for the Management of Intestinal Failure and its Resultant Complications in Children Systematic review: Management of Paediatric IF

Keywords: CSBLT-combined small bowel liver transplant, EL-evidence level, EN-enteral nutrition, IF-intestinal failure, IFALD-intestinal failure associated liver disease, PN-parenteral nutrition, SBS-short bowel syndrome, UDCAursodeoxycholic short bowel syndrome, parenteral nutrition, parenteral lipid, probiotics

Background and Aims: Intestinal failure (IF) affects a growing number of children due to increasing numbers of preterm infants surviving intestinal resection for necrotising enterocolitis (NEC) and improving surgical techniques for congenital gut anomalies. Parenteral nutrition (PN) is the mainstay of therapy; enteral nutrition (EN) may have trophic effects on the gut. We aimed to systematically review evidence for the effectiveness of medical and nutritional interventions in the treatment of IF in children. Methods: Retrieval of data from studies of patients aged <18yrs and receiving >28 days PN. Outcome measures: improvement in intestinal function; intestinal adaptation; growth; prevention and treatment of IF-associated liver disease; mortality.

BACKGROUND AND OBJECTIVES

Intestinal failure (IF) is defined as a reduction of functional gut mass below that needed for digestion and absorption of fluid and nutrients for weight maintenance in adults and growth in children [1][2][3] . In developed countries IF, in children, is most commonly the result of congenital or early-life intestinal diseases, which can be separated into three main groups by pathogenesis 4,5 : 1. Short bowel syndrome (SBS) caused by congenital intestinal atresias or secondary to resection following necrotising enterocolitis (NEC) 6 ; 2. Neuromuscular disorders of the gastrointestinal tract, including long segment aganglionosis (Hirschsprung's Disease) and congenital intestinal pseudo-obstruction syndromes (CIPOS) [START_REF] Rudolph | Diagnosis and treatment of chronic intestinal pseudoobstruction in children: report of consensus workshop[END_REF] ; 3. Congenital enterocyte disorders, such as microvillus inclusion disorder and intestinal epithelial dysplasia [START_REF] Goulet | Intractable diarrhea of infancy with epithelial and basement membrane abnormalities[END_REF][START_REF] Phillips | Congenital microvillous atrophy: specific diagnostic features[END_REF] .

The prevalence of IF is increasing mainly due to the survival of greater numbers of infants following intestinal resection in the newborn period [START_REF] Gupte | Improved outcome of referrals for intestinal transplantation in the UK[END_REF] . Deficit in functional gut mass can improve over time through the process of bowel adaptation. Adaptation occurs by a combination of mucosal hypertrophy and hyperplasia of small bowel tissue [START_REF] Sacks | Early proliferative events following intestinal resection in the rat[END_REF] . The adaptation process can take several days to many years depending on the length of bowel resected, the length and condition of the remaining unresected small bowel, the presence of the ileocaecal valve and the nutritional status of the affected patient [START_REF] Sukhotnik | Intestinal adaptation in short-bowel syndrome in infants and children: a collective review[END_REF] .

The introduction of parenteral nutrition (PN) to support infants with IF while bowel adaptation occurs has dramatically improved the historically dismal prognosis 1,5,[START_REF] Goulet | Neonatal short bowel syndrome[END_REF] . However significant complications of management, such as central venous line sepsis, IF associated liver disease (IFALD) and growth failure, have contributed to long-term morbidity and mortality in IF 1,5,[START_REF] Kelly | Intestinal failure-associated liver disease: what do we know today?[END_REF] . The failure of medical and nutritional treatments for IF invariably leads to a substantial minority of patients progressing to assessment for, and, sometimes, combined small-bowel liver transplantation (CBSLT) 1,[START_REF] Gupte | Improved outcome of referrals for intestinal transplantation in the UK[END_REF] .

Children may die before referral to CBSLT centres [START_REF] Barclay | Regionally acquired intestinal failure data suggest an underestimate in national service requirements[END_REF] .

A first group of therapeutic interventions have been postulated to accelerate bowel adaptation such as the use of enteral trophic factors, manipulation of dietary protein, fat and carbohydrate and manipulation of the gut microbiota 1,[START_REF] Nightingale | Guidelines for management of patients with a short bowel[END_REF][START_REF] Quigley | Small intestinal bacterial overgrowth: roles of antibiotics, prebiotics, and probiotics[END_REF] . A second group of and PN. To identify further potential studies, the reference lists on all identified potential published studies and major reviews by identified experts in this field were reviewed. In addition, citation searches of potential studies were made on the Web of Science database. A search of the 'grey literature' was performed by hand, which included the published proceedings for the past ten years of the European Society of Paediatric Gastroenterology Hepatology and Nutrition, the North American Society of Pediatric Gastroenterology Hepatology and Nutrition. This search aimed to identify studies published in abstract, but were only included in this review if they were subsequently published in full in a peer review journal. The authors' personal collections of publications were also searched.

Two authors (ARB, DCW) constructed the search strategy and identified potential studies for inclusion. Evidence based guidelines were assessed for applicability using the AGREE tool [START_REF]Development and validation of an international appraisal instrument for assessing the quality of clinical practice guidelines: the AGREE project[END_REF] . For individual studies two authors (ARB, LMB) who were masked to study outcomes independently graded the level of evidence (EL) presented using the SIGN methodology [START_REF] Harbour | A new system for grading recommendations in evidence based guidelines[END_REF] . A third author (DCW) reviewed all studies and also acted as an arbiter in cases of disagreement between the first two. All authors were involved in the writing of the manuscript. The grading assignment of EL is summarised in table 1 (also www.sign.ac.uk). Because our aim was to review original data, rather than to include expert opinion (EL 4), only publications of an EL of 1, 2 or 3 were permitted inclusion in our review.

F

RESULTS

The aforementioned keywords and subject headings yielded 1607620 hits. The application of combinations of 'child' with the other search terms limited the search to 720 abstracts which were reviewed online. Although the search term 'PN' generated additional hits it demonstrated redundancy in terms of potential studies. A total of 61 potential studies were reviewed in full. Twenty seven of these were discarded because they did not concern children, it was impossible to separate paediatric data from adult data, or were available in abstract form only. The 34 remaining studies included one consensus-based guideline for the use of long term PN in children which was judged to be relevant, but not limited to, our key question [START_REF] Koletzko | Guidelines on Paediatric Parenteral Nutrition of the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the European Society for Clinical Nutrition and Metabolism (ESPEN), Supported by the European Society of Paediatric Research (ESPR)[END_REF] .

Therefore, 33 original papers were included in our review

PN Manipulations

Ten studies were identified that described manipulation of PN, with four studies of higher EL [START_REF] Goulet | Longterm efficacy and safety of a new olive oil-based intravenous fat emulsion in pediatric patients: a double-blind randomized study[END_REF][START_REF] Gura | Safety and efficacy of a fish-oilbased fat emulsion in the treatment of parenteral nutrition-associated liver disease[END_REF][START_REF] Lee | Title: The Impact of Fish Oil-Based Lipid Emulsion on Serum Triglyceride, Bilirubin, and Albumin Levels in Children with Parenteral Nutrition-Associated Liver Disease[END_REF][START_REF] Puder | Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury[END_REF] . Goulet et al [START_REF] Goulet | Longterm efficacy and safety of a new olive oil-based intravenous fat emulsion in pediatric patients: a double-blind randomized study[END_REF] (EL 1-) investigated the use of an olive oil-based lipid formulation (ClinOleic 20%; Baxter Clintec, Mississauga, Canada) in comparison to a standard treatment of soy bean lipid (Intralipid 20%; Upjohn-Pharmacia, Bridgwater, New Jersey, USA) in 18 infants receiving long term PN.

Patients were prospectively randomised into the treatment (n = 9) and control (n = 9)

groups. The authors found no significant difference in serum bilirubin, ALT, and AST between the two but noted a significant treatment effect in terms of reduction of total cholesterol and low-density lipoprotein cholesterol. The authors did not state their methods of randomisation or concealment or describe the use of power calculations that lead to the numbers recruited and did not describe concurrent administration of confounding treatments. As a result this study was allocated an EL 1-.

Gura et al [START_REF] Gura | Safety and efficacy of a fish-oilbased fat emulsion in the treatment of parenteral nutrition-associated liver disease[END_REF] 18 infants from the same fish oil cohort in comparison to the 59 infants in the historical cohort. They reported a significant reduction in triglyceride levels in the fish oil group at the end of treatment (40mg/dL vs. 140mg/dL p<0.001). The previous limitations as described [START_REF] Puder | Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury[END_REF] apply and as such this paper was also rated EL 2-.

Lower level evidence studies included three case series describing fish oil lipid to reverse IFALD [START_REF] Diamond | Changing the paradigm: omegaven for the treatment of liver failure in pediatric short bowel syndrome[END_REF][START_REF] Gura | The role of an intravenous fat emulsion composed of fish oil in a parenteral nutritiondependent patient with hypertriglyceridemia[END_REF][START_REF] Gura | Reversal of parenteral nutrition-associated liver disease in two infants with short bowel syndrome using parenteral fish oil: implications for future management[END_REF] , two case series describing cycling of PN administration to reverse IFALD in 57 eligible infants [START_REF] Collier | Use of cyclic parenteral nutrition in infants less than 6 months of age[END_REF][START_REF] Meehan | Prevention of liver failure in parenteral nutritiondependent children with short bowel syndrome[END_REF] and a single case report of supplementation of PN with glutamine to encourage intestinal adaptation in an infant with SBS [START_REF] Allen | Glutamine-supplemented parenteral nutrition in a child with short bowel syndrome[END_REF] .

Growth Factors

Only lower level evidence studies (EL 3) exist for the use of growth factors in paediatric IF. This includes case series of five patients receiving epidermal growth factor (EGF) [START_REF] Sigalet | A pilot study of the use of epidermal growth factor in pediatric short bowel syndrome[END_REF] , combinations of growth hormone (GH) (0.2-0.3mg/kg/wk) and insulin-like growth factors (two patients) [START_REF] Nucci | Results of growth trophic therapy in children with short bowel syndrome[END_REF] , GH (0.3mg/kg/day) and glutamine supplementation (two patients), [START_REF] Ladd | The effect of growth hormone supplementation on late nutritional independence in pediatric patients with short bowel syndrome[END_REF] and a somastatin analogue (one infant) [START_REF] Ohlbaum | Use of a longacting somatostatin analogue (SMS 201-995) in controlling a significant ileal output in a 5-year-old child[END_REF] .

Bile Acid Analogues and Anticholeretics

Two studies of higher EL were identified describing the use of bile acid analogues [START_REF] Chen | Ursodeoxycholic acid (UDCA) therapy in very-low-birth-weight infants with parenteral nutrition-associated cholestasis[END_REF][START_REF] Heubi | Tauroursodeoxycholic acid (TUDCA) in the prevention of total parenteral nutrition-associated liver disease[END_REF] . Heubi et al [START_REF] Heubi | Tauroursodeoxycholic acid (TUDCA) in the prevention of total parenteral nutrition-associated liver disease[END_REF] (EL 2-) aimed to prospectively investigate the potential of tauroursodeoxycholic acid (30mg/kg/day) to prevent or ameliorate IFALD in postsurgical preterm infants. 22 infants and 30 controls were recruited, but some patients did not fulfil the criteria of post-term corrected age and PN>28 days. There were no significant differences in serum bilirubin, AST, ALT and alkaline phosphatase between groups. However, because of poor recruitment the authors used nonconsenting controls, and the resultant patient heterogeneity, enrolment bias and lack of controlling for confounding factors meant that this study was allocated an EL 2-.

Chen et al [START_REF] Chen | Ursodeoxycholic acid (UDCA) therapy in very-low-birth-weight infants with parenteral nutrition-associated cholestasis[END_REF]

Dietary Manipulations

Six studies were identified that described dietary manipulations, although only one was of higher EL. Ksiazyk et al [START_REF] Ksiazyk | Hydrolyzed versus nonhydrolyzed protein diet in short bowel syndrome in children[END_REF] (EL 1-) prospectively investigated the role of hydrolysed formula in promoting growth and development in SBS. Ten infants were randomly assigned to receive either a hydrolysed enteral formula (Pepti Junior, Nutricia, Zoetermeer, Holland), or a non-hydrolysed mirror formula over a 60-day period (cross-over day 31). They found no significant difference between the two treatments in terms of weight gain, enteral feed tolerance and energy expenditure. Due to minimal description of blinding, patient assignment, prior treatment exposure and concurrent use of confounding treatments, this study was allocated an EL 1-. Lower level evidence included reporting the use of elemental formulae, oral pectin and a World Health Organisation solution 23,25,52 29,42 .

Bacterial Manipulations

Seven studies that describe therapeutic strategies to manipulate the gut bacteria (microbiota) were identified but only one was of higher EL. Kubota et al [START_REF] Kubota | The effect of metronidazole on TPN-associated liver dysfunction in neonates[END_REF] The control data were taken from a historical group before this treatment was commenced. The authors reported no significant difference in serum bilirubin levels between groups, but a high-dose subgroup (50mg/kg/day) had significantly lower serum glutamic oxalocetic transferase and gamma glutamyl transpeptidase (p<0.05).

With its retrospective nature, use of historical controls and heterogeneous inclusion and exclusion criteria this study was assigned an EL 2-. with SBS to reduce bacterial sepsis and encourage intestinal adaptation [START_REF] Kanamori | Combination therapy with Bifidobacterium breve, Lactobacillus casei, and galactooligosaccharides dramatically improved the intestinal function in a girl with short bowel syndrome: a novel synbiotics therapy for intestinal failure[END_REF][START_REF] Kanamori | Experience of long-term synbiotic therapy in seven short bowel patients with refractory enterocolitis[END_REF][START_REF] Uchida | Immunonutritional effects during synbiotics therapy in pediatric patients with short bowel syndrome[END_REF] .

Vanderhoof et al [START_REF] Vanderhoof | Treatment strategies for small bowel bacterial overgrowth in short bowel syndrome[END_REF] , in a single case series described the use of bisacodyl suppositories and hypertonic polyethyleglycol solution (GoLYTELY, Braintree Laboratories, Braintree, U.S.A.) for D-lactate and small bowel bacterial overgrowth, and also the use of Lactobacillus planetarium and Lactobacillus GG (CAG Nutrition, Omaha, NE, U.S.A.) for arthritis associated with small bowel bacterial overgrowth. Newall et al [START_REF] Newall | Warfarin therapy in children who require long-term total parenteral nutrition[END_REF] also reported that the prophylactic administration of oral warfarin (INR 2.0-3.0) to eight children with SBS resulted in a significant reduction in the number of central venous access device thromboses, possibly through reduction of bacterial adherence.

Mouw et al also reported reduced episodes of CVC sepsis by the administration of a 70% ethanol lock in 5 infants and children [START_REF] Mouw | Use of an ethanol lock to prevent catheter-related infections in children with short bowel syndrome[END_REF] .

DISCUSSION

In spite of the publication of an enormous body of literature describing methods for the treatment of paediatric IF, we have shown that the evidence base for the use of medical and nutritional therapies in childhood IF is clearly inadequate. Although not formally studied, we believe that the evidence base for the various potential treatment modalities for IF in adults is similarly limited.

There are few paediatric data on the clinical effects of manipulations of PN and its effect on IF and IFALD. Although a short-term adult and paediatric study suggests that the MCT:LCT ratio of intravenous intralipid may affect hepatotoxicity [START_REF] Donnell | The metabolic response to intravenous medium-chain triglycerides in infants after surgery[END_REF] , the evidence in patients with established IF is lacking. Recent studies of use in infancy of fish oil-based fats are encouraging [START_REF] Diamond | Changing the paradigm: omegaven for the treatment of liver failure in pediatric short bowel syndrome[END_REF] , and newer lipid preparations with multiple fat sources have been used effectively in adults in North America [START_REF] Mertes | Safety and efficacy of a new parenteral lipid emulsion (SMOFlipid) in surgical patients: a randomized, double-blind, multicenter study[END_REF] . Paediatric RCTs are currently underway. However new lipids, for which there is much enthusiasm, have been a source of immense speculation amongst paediatric gastroenterologists and there are concerns that we are already beyond the phase of therapeutic equipoise for these therapies, with many units already using a variety of novel lipids in their practice. [START_REF] Flynn | Paediatric parenteral nutrition and lipid usage in the UK -A pick N' mix situation?[END_REF] .

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The effective use of growth factors to stimulate adaptation has been described in both animal and adult studies [START_REF] Inoue | Growth hormone enhances amino acid uptake by the human small intestine[END_REF][START_REF] Shulman | Effects of short-term growth hormone therapy in rats undergoing 75% small intestinal resection[END_REF] . Initial data reported success in improving long term intestinal function [START_REF] Byrne | A new treatment for patients with short-bowel syndrome. Growth hormone, glutamine, and a modified diet[END_REF] . However, later studies failed to reproduce these findings [START_REF] Jeppesen | Effect of high-dose growth hormone and glutamine on body composition, urine creatinine excretion, fatty acid absorption, and essential fatty acids status in short bowel patients: a randomized, double-blind, crossover, placebo-controlled study[END_REF][START_REF] Scolapio | Effect of growth hormone, glutamine, and diet on adaptation in short-bowel syndrome: a randomized, controlled study[END_REF] . A systematic review in 2004 of adult data on the use of GH and glutamine [START_REF] Matarese | Growth hormone, glutamine, and modified diet for intestinal adaptation[END_REF] identified eleven published studies. The authors concluded that the data were too limited and contradictory (six positive studies and four negative) to draw any conclusions on the efficacies of these treatments. A recent Cochrane review again focussed on adult data only [START_REF] Wales | Human growth hormone and glutamine for patients with short bowel syndrome[END_REF] . We have demonstrated that the data from childhood studies are even more limited; the use of GH is not currently standard practice in most paediatric IF centres [START_REF] Scolapio | Short bowel syndrome[END_REF] .

UDCA is the established treatment of primary biliary cirrhosis in adults [START_REF] Poupon | Combined analysis of randomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis[END_REF] , and short term data support UDCA use in established CF liver disease [START_REF] Cheng | Ursodeoxycholic acid for cystic fibrosis-related liver disease[END_REF] . A systematic review of the use of UDCA for patients with IFALD was published in 2007 [START_REF] Luis | Ursodiol in patients with parenteral nutritionassociated cholestasis[END_REF] . The authors identified six studies (five paediatric and one adult) in which UDCA was administered in doses of 10-30mg/kg/day in children and 10-15mg/kg/day in adults. The authors concluded that UDCA appeared to improve the signs and symptoms of IFALD but that optimisation of timing, dosage and duration of UDCA administration had still to be achieved. The paediatric data are limited but appear to support short term improvement in biochemical markers of IFALD. Higher quality studies that examine long-term efficacy are required.

Convincing theoretical data and animal models exist for the use of dietary manipulations, suggesting that diets high in complex carbohydrate and fat are more palatable and may stimulate enteral adaptation more quickly than diets with lower energy contents [START_REF] Grimble | Effect of peptide chain length on absorption of egg protein hydrolysates in the normal human jejunum[END_REF][START_REF] Jeppesen | The influence of a preserved colon on the absorption of medium chain fat in patients with small bowel resection[END_REF][START_REF] Vanderhoof | Effect of high percentage medium-chain triglyceride diet on mucosal adaptation following massive bowel resection in rats[END_REF] . The use of hypo-osmolar formulae and continuous feeding are well described 1 , but there is a lack of data from children to support the use of specific enteral formulations or to advise on their relative constituents including carbohydrate, fat, protein, micronutrients and potential trophic factors.

Probiotics, defined as live bacteria that confer a health benefit to the host, have purported protective properties [START_REF] Agostoni | Probiotic bacteria in dietetic products for infants: a commentary by the ESPGHAN Committee on Nutrition[END_REF] . Positive results exist for probiotic use in the management of post-surgical inflammatory bowel disease [START_REF] Gionchetti | Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial[END_REF][START_REF] Mimura | Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis[END_REF] , antibiotic-associated diarrhoea [START_REF] Szajewska | Probiotics in the prevention of antibiotic-associated diarrhea in children: a meta-analysis of randomized controlled trials[END_REF] and NEC [START_REF] Barclay | Probiotics for necrotizing enterocolitis: a systematic review[END_REF] [START_REF] Kanamori | Combination therapy with Bifidobacterium breve, Lactobacillus casei, and galactooligosaccharides dramatically improved the intestinal function in a girl with short bowel syndrome: a novel synbiotics therapy for intestinal failure[END_REF][START_REF] Kanamori | Experience of long-term synbiotic therapy in seven short bowel patients with refractory enterocolitis[END_REF][START_REF] Newall | Warfarin therapy in children who require long-term total parenteral nutrition[END_REF][START_REF] Uchida | Immunonutritional effects during synbiotics therapy in pediatric patients with short bowel syndrome[END_REF] . Antibacterial locks such as Gentamicin or Taurolidine have been used to prevent CVC colonisation and sepsis and a systematic review of the efficacy of the later exists [START_REF] Bradshaw | Taurolidine and catheter-related bloodstream infection: a systematic review of the literature[END_REF] . However we found no studies for such practice that fulfilled inclusion criteria to our study. We could also find no paediatric data to support the use of cycled enteral antibiotics.

We identified several studies which outlined multidisciplinary management for paediatric patients with IF and/or the use of a formalised 'intestinal rehabilitation programme' as a treatment for IF [START_REF] Cowles | Reversal of intestinal failure-associated liver disease in infants and children on parenteral nutrition: experience with 93 patients at a referral center for intestinal rehabilitation[END_REF][START_REF] Diamond | Neonatal short bowel syndrome outcomes after the establishment of the first Canadian multidisciplinary intestinal rehabilitation program: preliminary experience[END_REF][START_REF] Koehler | Coordinated interdisciplinary management of pediatric intestinal failure: a 2-year review[END_REF][START_REF] Sigalet | Improved outcomes in paediatric intestinal failure with aggressive prevention of liver disease[END_REF][START_REF] Torres | Role of an intestinal rehabilitation program in the treatment of advanced intestinal failure[END_REF] . Although these centres describe specific tailored therapeutic interventions, novel PN strategies and surgical interventions, with historical controls, both the variety and potential number of combination of measures described plus the resultant chance of missing specific publications despite a robust search strategy precludes these studies being used in our review. We however acknowledge that referral centres of excellence play an integral role in the improvement in outcomes for this population, and that early referral to such centres is essential for patients with potentially irreversible intestinal failure. We also recognise the evolving role of non-transplant surgical bowel reconstruction [START_REF] Sudan | Advances in the nontransplant medical and surgical management of intestinal failure[END_REF] and potential for isolated liver transplantation to avoid the necessity for CBSLT for IFALD [START_REF] Dell-Olio | Isolated liver transplant in infants with short bowel syndrome: insights into outcomes and prognostic factors[END_REF] , however this subject explicitly fell out-with the scope of our systematic review.

CONCLUSIONS

The evidence base for medical and nutritional interventions in paediatric IF is limited and of poor quality (Table 3). Low patient numbers, high drop-out rates from studies and ethical considerations that limit patient enrolment, have undoubtedly hindered the commencement and successful completion of RCTs. Current guidelines for patient management are extrapolated primarily from adult evidence and expert opinion [START_REF] Beath | Review of current management practices in intestinal failure associated liver disease[END_REF][START_REF] Beattie | The future of intestinal failure services in England and Wales; A Report on behalf of BSPGHAN[END_REF] .

RCTs in children are currently ongoing for the more expensive treatment modalities, most notably PN and PN lipids [START_REF] Diamond | Omega-3 lipids for intestinal failure associated liver disease[END_REF] . However, in the absence of RCTs, enhanced multiagency communication between centres, an improved evidence base through case control and cohort research and the study of inter-centre variations is possible.

The British Intestinal Failure Survey (BIFS) in the UK (http://bspghan.org.uk/working_groups/nutrition.shtml) may facilitate such projects in the near future. The optimisation of timing and dosage of administration of UDCA, the use of novel intralipid formulations, cycling of enteral antibiotics in bacterial overgrowth, prophylactic enteral probiotics and enteral feeding strategies are five achievable shortterm goals, which might improve the evidence base for medical and nutritional therapies for IF in children. Clinical trials of these treatment modalities will likely require being undertaken as prospective, carefully planned multi-centre studies in order to be sufficiently powered to generate significant and unambiguous data. Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale.

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5

Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched.

5 Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.

5

Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis).

5

Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.

5

Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.

Risk of bias in individual studies

12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.

Dear Professor Rhodes

Thank you for your prompt and favourable response to our submission 'Systematic Review: Medical and Nutritional interventions for the Management of Intestinal Failure and its Resultant Complications in Children'. We are pleased to return a revised manuscript to you. We thank you and the reviewers for their comments and will address each in turn. We have left the editorial comments in Trebuchet text and responded in italics and Times Roman font. When quoting from the revised manuscript we have cited the page and line number.

Editors Comments for the author

Please address the reviewers' comments and in addition: please add some more specific information in the abstract eg (i) explain briefly why intestinal failure is increasing and (ii) consider adding in something along the lines of your discussion eg perhaps based on a shorter version of: "The optimisation of timing and dosage of administration of UDCA, the use of novel intralipid formulations, cycling of enteral antibiotics in bacterial overgrowth, prophylactic enteral probiotics and enteral feeding strategies are five achievable shortterm goals ... for clinical trials ... which might improve the evidence base for medical and nutritional therapies for IF in children".

Thank-you we have amended the abstract accordingly (page 2 line 2) Intestinal failure (IF) affects a growing number of children due to increasing numbers of preterm infants surviving intestinal resection for necrotising enterocolitis (NEC)

and improving surgical techniques for congenital gut anomalies. The evidence base for medical and nutritional interventions in paediatric IF is limited and of poor quality. In the absence of RCTs, this evidence base can improve through case control and cohort research, and with better multi-agency communication the study of inter-centre differences is possible. Achievable short-term goals would include the study of; optimal ursodeoxycholic usage; novel intralipid formulations; cycled enteral antibiotics; enteral probiotics and new enteral feeding strategies.

Reviewer: 1

Comments for Transmission to the Authors I enjoyed reading this paper. I have 2 major comments: 1. In the background and objectives I think it would enhance the later discussion if the authors describe the therapeutic interventions as falling into 2 main categories namely a) enhancing bowel adaptation and b) prevention of complications. They allude to the second category of intervention when describing prevention of IFALD and make a brief reference to anti-sepsis procedures, which is not followed up in the main body of the paper.

Thank you for this comment we have amended the background and objectives accordingly (page 3 line 31).

A first group of therapeutic interventions have been postulated to accelerate bowel adaptation such as the use of enteral trophic factors, manipulation of dietary protein, fat and carbohydrate and manipulation of the gut microbiota [1][2][3] . A second group of interventions are aimed at the prevention or amelioration of complications of PN such as antisepsis procedures for CVCs, or treatment of IFALD with bile analogues, choleretic agents and changes in the constituents of PN 4 .

2. The omission of a discussion of feeding catheter line locks such as gentamicin is puzzling especially as reference is made to the use of Warfarin in reducing bacterial adherence. I appreciate that the recent studies of taurolock would not have been available at the time of the electronic search but there are a number of publications and a systematic review (Bradshaw et al JPGN 2008; 47: 179-186) which support the author's contention that the evidence base for interventions in children with IF is currently very poor. I am not suggesting a repeat search, but perhaps this paper should be included the group of papers referred to at the end of the second paragraph in results section ie "relevant but not limited to our key question". Thank you for this comment, we were aware of the systematic review by Bradshaw and Puntis 5 , and it was identified in our search strategy. However none of the studies identified in this review fulfilled our inclusion criteria, the only 2 studies that involved CVC access for PN were in two adult patients. Similarly with Gentamicin antibacterial locks; although studies describing use were identified, none of them were in paediatric patients receiving PN. We have however referenced the systematic review and mentioned bacterial locks in our discussion (page 14 line 2).

Antibacterial locks such as Gentamicin or Taurolidine have been used to prevent CVC colonisation and sepsis and a systematic review of the efficacy of the latter exists 5 . However we found no studies for such practice that fulfilled inclusion criteria to our study.

3. on page 3 line 9, I would suggest that the phrase ?in children? > is inserted after ?in developed countries?, because the previous > sentence refers to both adults and children and I feel that the > authors should signal more explicitly that from this point on the > paper is now focusing on children.

Thank you, we have amended accordingly.

. on page 7 line 14 there is typo ? I think the word ?have? in > line 14 occurring in the sentence beginning ?Twenty seven of > these..?, is redundant.

Thank you; we have removed this.

Reviewer: 2

The author should define all abbreviations used in the tables in > the table legend.

Thank you we have amended the figure legends in tables 2 and 3 accordingly (pages 8 and 16).

. On page 12, lines 52-57, it is written that ?there are concerns > that we are already beyond the phase of therapeutic equipoise for Thank you. We have amended accordingly in the text (page 12 line 29). However new lipids, for which there is much enthusiasm, have been a source of immense speculation amongst paediatric gastroenterologists and there are concerns that we are already beyond the phase of therapeutic equipoise for these therapies, with many units already using a variety of novel lipids in their practice. 6 .

We thank you and the reviewers again for their useful comments and hope that this improved manuscript will now be considered for publication.

Yours Sincerely

METHODS

  at the prevention or amelioration of complications of PN such as antisepsis procedures for CVCs, or treatment of IFALD with bile analogues, choleretic agents and changes in the constituents of PN 18 . Numbers of patients with IF are low, even in large tertiary medical centres, limiting the scope for controlled trials of interventions to improve outcomes in IF. The aim of this review is to critically appraise the existing data on medical and nutritional interventions for the treatment of IF, and effect of strategies to ameliorate or prevent the complications of long term PN in children (namely sepsis and IFALD). In addition we wish to suggest how future studies may be developed to expand this evidence base. We devised a publication search strategy to answer our key question which was to determine which medical and nutritional interventions are of value in the treatment of paediatric patients (<18 years) with IF (defined as requirement for PN ≥ 28 days, while post-term age equivalent). Outcome measures sought were: improvement in intestinal function; intestinal adaptation; growth; prevention of IFALD; treatment of IFALD and mortality. We did not attempt to evaluate surgical interventions (transplant or non-transplant procedures). Electronic searches of the Medline (January 1950-November 2009) and CINAHL (January 1982-November 2009) databases were performed using combinations of the keyword and subject heading (MeSH) 'child' with: IF; short bowel syndrome (SBS);

  (EL 2-) retrospectively analysed the effects of prophylactic oral metronidazole (25-50mg/kg/day) on the development of IFALD in 21 infants receiving prolonged PN.

  Lower level evidence includes three case series describing the combination use of Bifidobacter breve, Lactobacillus casei and galacto-oligosaccharides in 12 patients

  of hepatic inflammation and bilirubin in short-term. No controlled data on long term outcomes. 2-(2), 3 (4) Clinical observation to optimise administration and dosage of UDCA Dietary manipulation No controlled benefits of hydrolysed formulae, Uncontrolled support of hypo-osmolar elemental feeds 1-(1), 3 (5) RCTs of newer formulae, in particular MCT/LCT fats PN manipulations Some controlled evidence of benefits of olive oil and FISH oil based lipids, uncontrolled evidence to support cycling of PN. 1-(1), 2-(3) 3 (6) Multi-centre RCT of intralipids underway in North America Bacterial Manipulations Data does not support routine use of metronidazole for the prevention of IFALD, some uncontrolled data describing probiotic use to prevent bacterial overgrowth 2-(1), 3 (6) Clinical studies of the efficacy of cyclical enteral antibiotics and prophylactic oral probiotics.

>

  these therapies.? The author should clarify what is meant by this > statement.

Table 1 :

 1 The revised SIGN grading system[START_REF] Harbour | A new system for grading recommendations in evidence based guidelines[END_REF]

	o r
	P
	e
	e r
	R
	e
	v i e
	w

P e e r R e v i e w 8 Author Population Intervention(s) Cases Control Data Controls Study Rating Outcomes reported

	Infants with SBS Infants on long-term PN Post-surgical Infants TUDCA Hydrolysed vs. non-hydrolysed formula Olive oil lipid vs. Soy bean Lipid F o r Ksiazyk 2002 37 Goulet 1999 30 Heubi 2002 34 (30mg/kg/day)	10 9 22	Cross-over patients Randomised eligible infants Non-consenting infants	10 9 30	1-1-2-	No significant difference in enteral intake or energy expenditure. No significant difference in bilirubin, ALT, AST. Significant treatment effect on total cholesterol and LDL. No significant difference in bilirubin or ALT, AST, Alk Phos.
	Chen 2004 24	Preterm infants	UDCA (10-	12	Historical non-treated	18	2-	Shorter duration of cholestasis in treatment
			30mg/kg/day)		infants			group (62.8 vs. 92.4 days p=0.006).
	Gura 2008 33	Infants with SBS	Fish Oil lipid in	18	Historical Cohort	21	2-	Significantly shorter duration cholestasis in
			established IFALD					treatment group. Non-significant trend
					. There were no EL			towards lower mortality.
	1++, EL1+, EL 2++ or EL 2+ studies. There were two EL 1-, six EL 2-and 25 EL 3 Kubota 1990 38 Post-surgical Metronidazole (25-21 Historical Cohort 21	2-	Significantly lower GGT in high dose
	studies. The higher-level evidence studies (EL 1 and EL 2) are summarised in Table 2 Neonates 50mg/kg/day)		treatment group during PN.
	and are grouped from the point of view of therapeutic modality. Limitations of each Puder 2009 48 Infants with IF Fish oil lipid in 42 Historical Cohort 49	2-	Significantly reduced mortality, reversal of
	study are discussed within the text. Lee 2009 40 Infants with IF	established IFALD Fish oil lipid in	18	Historical Cohort	59	2-	IFALD and improved cholesterol profile. Reduction in serum triglyceride and bilirubin
			established IFALD					levels, and improvement in albumin.

Table 2 : Studies of higher evidence level as appraised by the SIGN method 20

 2

	SBS-short bowel syndrome; PN-parenteral nutrition; ALT-alanine aminotransferase; AST-aspartate aminotransferase; LDL-low density
	lipoprotein; TUDCA-tauroursodeoxycholic acid; Alk Phos-alkaline phosphatase; UDCA-ursodeoxycholic acid; IFALD-intestinal failure
	associated liver disease; IF-intestinal failure.

Table 3 : Current evidence for the use of medical and nutritional therapies in paediatric IF and potential future studies

 3

	GH-growth hormone; UDCA-ursodeoxycholic acid; RCT-randomised controlled trial; MCT-medium chain triglycerides; LCT-long chain
	triglycerides; PN-parenteral nutrition; IFALD-intestinal failure associated liver disease.

5

METHODS

Protocol and registration

5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number.

6

  Summary measures13 State the principal summary measures (e.g., risk ratio, difference in means).Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies).Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12).From: Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(6): e1000097.

				Page 1 of 2 Department of Child Health
	Section/topic	# Checklist item	University of Glasgow Royal Hospital for Sick Children	Reported on page #
				Dalnair St, Yorkhill Glasgow	6
	Additional analyses	16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating U.K. G3 8SJ which were pre-specified. 19 th October 2010	none
	RESULTS
	Study selection	17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at Jonathon M Rhodes MD each stage, ideally with a flow diagram. Assistant Editor	7
	Study characteristics Alimentary Pharmacology & Therapeutics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations. 49 Carvers Croft, Woolmer Green	8
	Herts SG3 6LX Results of individual studies Synthesis of results Risk of bias across studies UK.	20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency. 22 Present results of any assessment of risk of bias across studies (see Item 15). r o F	8 inaprop inaprop
	Additional analysis DISCUSSION Summary of evidence Limitations	23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]). P 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to e key groups (e.g., healthcare providers, users, and policy makers). 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). e r	none 8-12 8-12
	Conclusions FUNDING Funding		26 Provide a general interpretation of the results in the context of other evidence, and implications for future research. R 27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review. e v i e	12-13 1
	Synthesis of results	14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I 2 ) for each meta-analysis. Page 2 of 2 w	none

doi:10.1371/journal.pmed1000097

For more information, visit: www.prisma-statement.org.

Alimentary Pharmacology & Therapeutic


Lotta Ström
email: lotta.strom@emg.umu.se

Roland Jansson

Christer Nilsson

MATS E Johansson

Shaojun Xiong

Hydrologic effects on riparian vegetation in a boreal river: an experiment testing climate change predictions

Keywords: Biomass, Climate change, Flooding, Productivity, Reciprocal transplant experiment, River banks, Species composition, Species richness biomass, climate change, flooding, productivity, reciprocal transplant experiment, river banks, species composition, species richness, water table, wetlands

Climate change is expected to alter the magnitude and variation of flow in streams and rivers, hence providing new conditions for riverine communities. We evaluated plant ecological responses to climate change by transplanting turfs of riparian vegetation to new elevations in the riparian zone, thus simulating expected changes in water-level variation, and monitored the results over 6 years. Turfs moved to higher elevations decreased in biomass and increased in species richness, whereas turfs transplanted to lower elevations gained biomass but lost species. Transplanted plant communities were inert in their response to new hydrologic conditions. After 6 years, biomass of transplanted turfs did not differ statistically from target level controls, but species richness and species composition of transplants were intermediate between original and target levels. By using projections of future stream flow according to IPCC climate change scenarios, we predict likely changes to riparian vegetation in boreal rivers. Climate-driven hydrologic changes are predicted to result in narrower riparian zones along the studied Vindel River in northern Sweden towards the end of the 21st century. Present riparian plant communities are projected to be replaced by terrestrial communities at high elevations as a result of lower-magnitude spring floods, and by amphibious or aquatic 1

communities at low elevations as a result of higher autumn and winter flows. Less area available to riparian plants is expected to result in fewer species, even if no species lose their hydrologic niches. Changes to riparian vegetation may be larger in other boreal climate regions: snow-melt fed spring floods are predicted to disappear in southern parts of the boreal zone, which would result in considerable loss of riparian habitat. Our study emphasizes the importance of long-term ecological field experiments given that plant communities often respond slowly and in a non-linear fashion to external pressures. Models of climate change predict that temperature will continue to increase during the 21 st century. The highest warming will take place at high northern latitudes (IPCC 2007), calling for studies of how northern ecosystems will respond. At the regional scale, species range shifts are predicted [START_REF] Saetersdal | Predicting changes in Fennoscandian vascularplant species richness as a result of future climatic change[END_REF][START_REF] Thuiller | Climate change threats to plant diversity in Europe[END_REF][START_REF] Post | Ecological dynamics across the Arctic associated with recent climate change[END_REF]), but to understand ecosystem-level responses, changes in local environmental conditions need also to be considered.

Warmer climates will likely result in more precipitation in general and less precipitation falling as snow in high northern latitudes, altering the hydrology of streams and rivers [START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF]IPCC 2007;Woo et al. 2007). Stream-flow regimes in the boreal zone are strongly seasonally variable. In natural conditions they are characterized by a spring flood peak with the highest levels of the year driven by snowmelt, followed by lower summer discharge (Woo et al. 2007). Regionally, summer and autumn rains might create flood peaks but during winter the flow is low, primarily fed by groundwater discharge. A warmer climate will generally increase winter discharge in all types of boreal rivers since more precipitation will fall as rain [START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF]Woo et al. 2007). Earlier snowmelt will also result in earlier spring floods of lower amplitude. In accordance with these expectations, annual stream flow in high northern rivers has increased in recent decades [START_REF] Peterson | Increasing river discharge to the Arctic Ocean[END_REF].

The seasonal timing and amount of flow variation vary with latitude, degree of climate continentality, topography and influence of buffering waterbodies, and thus the response to climate change will vary among rivers and regions (Woo et al. 2007). In Sweden the temperature is expected to increase by an average of 2.5 to 4.6 ºC by the year 2100 [START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF]. Hydrologic models predict mean annual runoff to increase by up to 24 % in the boreal part of Sweden with decreased spring flood peaks, increased frequency of high flow events during autumn, and higher winter flows [START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF]. In Arctic river systems, where prolonged periods with below-zero temperatures will still occur, the increased precipitation might even lead to deeper snow, creating higher but earlier spring flood peaks [START_REF] Dankers | River discharge and freshwater runoff to the Barents Sea under present and future climate conditions[END_REF]. In areas with continental climate, summer flows are predicted to decrease as a result of increased evapotranspiration, but in other regions summer flows will increase due to higher precipitation (Woo et al. 2007). Rivers draining glaciers are also likely to experience increasing discharge of summer melt water.

Climate-driven hydrologic changes are expected to change riverine communities since they are controlled by the magnitude and variation in flow [START_REF] Décamps | River margins and environmental change[END_REF][START_REF] Naiman | The ecology of interfaces: riparian zones[END_REF][START_REF] Poff | The natural flow regime[END_REF]. We focused on riparian ecosystems, which are unusually complex, dynamic, and diverse, and possess numerous economic, societal, and biological values [START_REF] Sharitz | Integrating ecological concepts with natural resource management of southern forests[END_REF][START_REF] Naiman | Legitimizing fluvial ecosystems as users of water: an overview[END_REF]. These qualities are all maintained by regular floods, and might thus change as a result of climate alterations. Riparian plant communities are structured by differences in the hydrologic niches of species [START_REF] Silvertown | Hydrologically defined niches reveal a basis for species richness in plant communities[END_REF].

Substantial changes in riparian vegetation may take place without altering the mean annual flow, as riparian vegetation is especially sensitive to changes in minimum and maximum flows [START_REF] Auble | Relating riparian vegetation to present and future streamflows[END_REF]. In boreal rivers, riparian vegetation is vertically zoned according to the flooding tolerance and moisture requirements of species [START_REF] Auble | Relating riparian vegetation to present and future streamflows[END_REF][START_REF] Johansson | Responses of riparian plants to flooding in free-flowing and regulated boreal rivers: an experimental study[END_REF]. Floods pose stresses and disturbances to riparian plants by affecting physiological processes such as photosynthesis and respiration [START_REF] Kozlowski | Responses of woody plants to flooding[END_REF]Vervuren et al. 2003;[START_REF] Van Eck | Is tolerance to summer flooding correlated with distribution patterns in river floodplains? A comparative study of 20 terrestrial grassland species[END_REF][START_REF] Mommer | Underwater photosynthesis in flooded terrestrial plants: a matter of leaf plasticity[END_REF][START_REF] Renöfält | Connecting variation in vegetation and stream flow: the role of geomorphic context in vegetation response to large floods along boreal rivers[END_REF], by removing plant biomass [START_REF] Bendix | Stream power influence on southern Californian riparian vegetation[END_REF] or burying plants under deposits of sediment or organic matter Nilsson 1997). These processes may enhance species richness by suppressing dominants and enhancing the colonization and survival of competitively subordinate species (Day et al. 1989[START_REF] Pollock | Plant species richness in riparian wetlands: a test of biodiversity theory[END_REF]. However, along gradients of increasing stress and disturbance, species richness may decline as the challenges of flooding exceed the capacity of species. Local species richness peaks in the mid-elevation riparian shrub zone [START_REF] Pollock | Plant species richness in riparian wetlands: a test of biodiversity theory[END_REF], but the pool of riparian species is largest close to the annual high-water level [START_REF] Nilsson | Frequency distributions of vascular plants in the geolittoral vegetation along two rivers in northern Sweden[END_REF], where flood duration is short and species lacking physiological adaptations to flooding may grow. Plant productivity also varies predictably with flood frequency and duration, with biomass production generally increasing from spring-flood maintained communities to sedgedominated communities experiencing prolonged periods of inundation [START_REF] Dwire | Plant biomass and species composition along an environmental gradient in montane riparian meadows[END_REF]. To be able to predict how riparian vegetation will respond to climate change we addressed the following questions:

F o r R e v i e w O n l y 5 (Xiong &
(1) How will changes in flood duration and frequency affect riparian species richness?

According to hydrological models the upper part of the riparian zone will be flooded less frequently while the lower part will experience longer periods of inundation. Changes in flood disturbance can both increase and decrease species richness depending on where the community is situated along the disturbance continuum (e.g. [START_REF] Pollock | Plant species richness in riparian wetlands: a test of biodiversity theory[END_REF]. To be able to predict changes in species richness experiments are necessary.

(2) Similarly, how will the hydrologic changes alter biomass production? Biomass is expected to decrease in high to mid elevation riparian forest and shrub communities as a result of lower duration of flooding, but increase at lower elevations dominated by graminoids, where flood duration is expected to increase. This does not take into account that a warmer climate may increase plant biomass production as a result of warmer and longer growing seasons, potentially resulting in more plant litter accumulation, if not limited by water availability or flooding. However, experimentally manipulation flow in streams and rivers to test scientific hypotheses are usually not an option. As an alternative, we assessed the effects of climate change on productivity and species richness of riparian plant communities by performing a reciprocal transplant experiment, moving vegetation turfs between different elevations in the riparian zone. This simulated changes in the frequency and duration of floods, and we monitored the responses over 6 years. Reciprocal transplantation is a powerful method to assess the effects of contrasting environments on species, but it has only recently been used to assess responses of plant communities in wetland ecosystems [START_REF] Toogood | Response of floodplain grassland plant communities to altered water regimes[END_REF]. The flow of most rivers is affected by dams and diversions [START_REF] Nilsson | Fragmentation and flow regulation of the world's large river systems[END_REF], making it difficult to isolate the effects of climate change on hydrology and ecosystems from effects of land-use. Therefore, we chose to work along the seventh-order Vindel River in northern Sweden, a free-flowing tributary in the Ume River catchment. Our objective was to investigate how floodplain meadow plant communities respond to manipulations in hydrology and litter inputs simulating those expected as a result of future climate change. The experimental design allowed asking how long it will take for plant communities to adjust to new hydrologic conditions. The results will improve our ability to predict how and at what rate vegetation succession will occur on floodplains subject to changes in hydrology and litter inputs as a consequence of climate change.

Methods

Study area

The experiment was conducted on a floodplain meadow along the lower reaches of the freeflowing Vindel River in northern Sweden (N 64˚22', E 19˚22'). The Vindel River originates in the Scandes mountain range and joins the Ume River about 30 km from the coast of the Gulf of Bothnia. The catchment area is 12 654 km 2 of which 5 % are lakes. The river is 455 km long and the mean annual discharge is 200 m 3 s -1 at the confluence with the Ume River. The discharge is unaffected by dams and diversions and varies between 16 and 1787 m 3 s -1 [mean annual minimum and maximum flows during 1911-2000; data from the Swedish Meteorological and Hydrological Institute (SMHI), Norrköping, Sweden]. The hydrologic regime is characterized by a spring flood peak due to snowmelt in May or June followed by progressively lower water levels during summer and winter. In some years summer and autumn rains and reduced evapotranspiration towards the end of the growing season result in additional flood events.

The river-margin vegetation is distinctly vertically zoned along the entire river. The width and plant zonation and width of vegetation belts can be defined by hydrologic criteria, the upper end of riparian vegetation being equivalent to the spring flood peak level, whereas the lower end of graminoid vegetation is synchronous with average summer low water levels (Wassén 1966;[START_REF] Nilsson | Rivers and streams[END_REF]. Up to the 1950s and 1960s, most riparian zones along tranquil reaches with fine-grained soils were used for haymaking, favouring plant communities rich in graminoids and herbs. Following abandonment, riparian tree and shrub species have colonized higher elevations of these meadows. The riparian vegetation of the catchment is primarily composed of native species, only a few occurrences of exotic species are known and invasive species are absent [START_REF] Nilsson | Rivers and streams[END_REF][START_REF] Dynesius | Intercontinental similarities in riparian-plant diversity and sensitivity to river regulation[END_REF]. The experimental site was located about 70 km upstream of the confluence between the Vindel and Ume Rivers at 220-230 m a.s.l., and was formerly used for haymaking. The site was selected since it was a fairly large, homogenous riparian meadow with uniform soil grain size consisting of silt, located 2.5 km upstream of a stream flow gauge. The vegetation at the site was zoned, going from communities dominated by graminoids and herbs with some older birches (Betula pubescens) at the top, to mid elevations dominated by Salix shrubs and graminoids such as Calamagrostis canescens, followed by herbaceous communities dominated by Carex acuta, Calamagrostis canescens and various amphibious species such as Ranunculus reptans at the bottom. The annual growing season (days where the average temperature exceeds +5°C) is about 150 days (period 1961(period -1990, SMHI), SMHI).

Layout of plots and treatments

A total of 96 plots were distributed at three elevations (Fig. 1), corresponding to the riparian belts of forest, shrub and graminoids [START_REF] Nilsson | Frequency distributions of vascular plants in the geolittoral vegetation along two rivers in northern Sweden[END_REF]. The vertical difference in elevation was 0.55 m between high and middle plots, and 0.90 m between middle and low plots. To simulate changes in the flood regime, we transplanted vegetation between elevations in June 2000.

Each plot was 70x100 cm in size and was excavated to a depth greater than the rooted zone. Excavated turfs were either carefully put on metal cradles with handles to allow transport to one of the other two elevations with minimal disturbance to the soil, or rotated 180° and put back into their original positions to evaluate the effect of excavation. The remaining plots were left intact serving as controls. The meadow was divided into four blocks, with two replicates of each treatment per block. Thus, eight replicates that each consisted of 12 plots, i.e. three elevations with the four treatments each (Fig. 1). To simulate increased litter production as a consequence of potential increase in plant productivity due to longer and of the plots, one of the replicates in each block. The litter consisted of graminoids and forbs and tree leaves, collected from the riparian zone in nearby reaches of the Vindel River during spring flooding, and dried at room temperature. To keep the applied litter in place a 5 cm high boundary marker of chicken net was placed around each transplanted plot. To control for an eventual effect on vegetation of this net, it was applied also to plots without added litter. At each level a pressure transducer data logger (Divers, van Essen Instruments Limited) was placed in a well of polystyrene plastic inserted into the ground to measure water level. The difference in ground surface elevation between the loggers was measured using a total station [Geodolite ® 506, Geotronics (Trimble), Sweden].

Sampling

We recorded the frequency of all vascular plant species in each plot using the point touch method (16 points in a 50x50cm area, 10 cm from the edge of the plots, to minimize edge effects; Fig. 1) in August each of the years 2000-2003, 2005 and 2006. Every species touching the stick was recorded once at each point, i.e. the maximum score was 16 per plot. In addition, the presence of any additional species in the plot was recorded. Plant biomass was harvested from subplots (15x20 cm) nested within each plot (Fig. 1) every year species richness was recorded. After harvest, biomass was dried at 60˚C for 72 h and weighed. A subset of 24 plots, representing all treatments, was used for soil sampling for chemical The definition of species follows the taxonomy in [START_REF] Krok | Svensk Flora. Fanerogamer och Ormbunksväxter[END_REF]. In the following cases two or more species were treated as one taxon: Carex juncella + C. nigra, Hieracium spp., Hierochloë hirta + H. odorata, Luzula multiflora + L. sudetica, Poa spp., Salix borealis + S. myrsinifolia + S. phylicifolia, and Taraxacum spp.

Data analysis

Flood duration and frequency for the entire sampling period were quantified by correlating the logger data for the first 4 years with water level measurements made by the SMHI at the gauge in Granåker 2.5 km downstream in the same tranquil reach; there were no intervening steps or rapids. By comparing instrumental water level records from the Granåker gauge and the experimental site date by date, we constructed a calibration curve by which the gauge data could be used to generate water-level data for each elevation at the experimental site in Simulations of future streamflow for the period 2071-2100 at the nearby gauge were obtained from SMHI [START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF]. They were calculated using the Rossby Centre Regional Atmosphere-Ocean Model (RCAO) and the runoff model HBV developed by SMHI, using data from two general circulation models: HadAM3H from the Hadley Centre of the UK Meteorological Office and ECHAM4/OPYC3 from the Max-Planck Institute for Meteorology in Hamburg. The general circulation models were run with data according to the A2 and B2 IPCC emission scenarios [START_REF] Nakicenovic | Special Report on Emissions Scenarios[END_REF], where the A2 scenario results in larger increases in greenhouse gas emissions than the B2 scenario (for detailed description of the simulation models, see [START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF]). We used the same method as above to interpolate projected future water-level records at the gauge to water levels at the study site. To evaluate the effects of plot trenching, such as root damage and soil disturbance, we compared plots that were excavated, removed and put back into their original position after rotating 180°, with control plots at the same elevation. We used mixed-effects ANOVAs to analyze variation in biomass and species richness depending on trenching (excavated or not) and elevation (treated as fixed factors), with experimental block as a random factor. The effects of litter addition were evaluated with factorial ANOVA with original and target elevations and litter addition as fixed factors. The tests were performed separately for each year of vegetation sampling. The effect of blocking the experiment was found to be weak (i.e. variation within blocks was similar to between-block variation), and hence we did not include block as a factor in these and subsequent ANOVAs.

To test the effect of changed frequency and duration of flooding on species richness and biomass, we compared turfs that were transplanted to a new elevation with trenched turfs remaining in their original position at both the original and target levels of the transplanted turfs. To evaluate the effect of transplantation, we compared the relative changes in biomass and species richness between 2000 and 2006, as well as differences between treatments the final study year (2006), using one-way ANOVA (with treatment as a factor) and evaluated significant differences among treatments using Tukey's post-hoc tests of honest significant difference (α = 0.05). The treatment groups were transplanted turfs and excavated control plots at the origin and target levels of transplants. We made separate tests for each exchange of transplants between levels (i.e. between high and low levels, high and mid levels, and mid and low levels; Fig. 1). When litter addition was included as a factor in these ANOVAs, it did not significantly (P>0.05) affect biomass and species richness, and was dropped during model simplification. To analyze whether transplanted turfs resembled the species composition of turfs at the original elevation, or had attained a species composition similar to the target levels, we tested for differences among treatments in the first axis site scores of the DCAs, using one-way ANOVAs with treatment (transplantation vs. excavated controls at the original and target levels) as a factor. We used untransformed species frequency data from the first and final years of the experiment in the analyses, detrended by segments and downweighting rare species. Significant differences among treatments were evaluated using Tukey's post-hoc tests. We made separate tests for each exchange of transplants between levels as above. We also tested for differences in soil moisture between elevations (high, mid and low) in the riparian zone with a one-way

Results

Present and projected future hydrologic regimes

During the experiment, the low elevation was flooded every year, the mid elevation was flooded in 5 of the 7 years, and the high elevation was only flooded in 2004 and 2005 (Table 1). Flood duration also varied among years (Fig. 2). In 2001, flood duration was twice as long at the low elevation well compared to the other years, or 81 days compared to 16-40 days the other years. Both duration and frequency during the experimental period (2000)(2001)(2002)(2003)(2004)(2005)(2006) were lower compared to the reference period 1961-1990 (Table 1), which is the period used by SMHI as a baseline to calculate future scenarios. The largest difference was at the low elevation well, with 20 days shorter mean flood duration per year during the experiment (Table 1). The projections of future hydrologic conditions predicted shorter flood duration and longer flood return times at the high riparian elevation in 2071-2100 compared to the normal period 1961-1990 (from 2.1 to up to 7.5; Table 1). The mid elevation will also be flooded less often and with less duration, but the changes are smaller. The low elevation is projected to still be flooded every year with similar or longer durations compared to 1961-1990 (Table 1). Soil moisture increased towards lower elevation in the riparian zone (mean ± 1SE, high 54.9 ± 2.2, mid 61.8 ± 1.0, low 67.9 ± 1.8; F = 8.5, P = 0.002, one-way ANOVA with elevation as a factor).

Responses of riparian vegetation to changes in hydrology

The only significant effect of trenching was found on biomass in 2001. This year, biomass in the trenched plots at low elevations was significantly lower than in the controls (178±35 g compared to 554±84 g, P < 0.0001, mixed-effects ANOVA), and the trenched plots at high elevation had significantly more biomass compared to the controls (388±66 g compared to 219±42 g, P = 0.024). We found no significant differences between trenched and control plots 14 in species richness for any year or elevation (P > 0.05). Thus, after an initial biomass response, trenching per se did not significantly affect the vegetation features. In the remaining analyses, we used the trenched plots as controls for the transplantation treatments. Litter addition significantly reduced biomass in 2002, which was the second and last year litter was applied (Table 2), and significantly reduced species richness in 2003. Litter addition reduced biomass and species richness at all elevations in the riparian zone. No statistically significant effects of litter addition were detected in any of the following years.

The overall effect of the transplantation experiment was that turfs transplanted to higher elevations decreased in biomass and increased in species richness, whereas turfs transplanted to lower elevations gained in biomass but lost in species richness (Figs 3-5), although not all changes following transplantation were significant. Turfs transplanted from high to low elevations increased in biomass compared to the high elevation controls and turfs transplanted to mid elevations (P < 0.05, one-way ANOVA; Fig. 3a). Comparing biomass between years shows that both transplanted turfs and the low-elevation controls decreased substantially in biomass from 2000 to 2001 (Fig. 4a). The low-elevation controls and the turfs transplanted to low elevations successively increased in biomass thereafter, while the turfs transplanted from low to high elevations remained low during the remainder of the experiment. The final year of the experiment, the plots transplanted between high and low elevations did not differ in biomass compared to the target elevation controls, but differed significantly from their original values (Fig. 4a). The relative change in biomass did not differ among plots transplanted between mid and low elevations (Fig. 3c ande; P > 0.05), and there were no significant differences in biomass between them the last year (Fig. 4b andc). We found between four and five times as many species on high and mid elevation controls compared to low elevation controls (P < 0.05, one-way ANOVAs and Tukey's tests; Fig. 5a andc). In contrast, we found no difference in species richness between high and mid elevations, and turfs transplanted between mid and high elevations did not change in species richness (P > 0.05; Figs 3b andd and5b). Turfs from high elevations lost species when transplanted to low elevations (P = 0.002, one-way ANOVA and Tukey's tests), but did not differ from high elevation controls if transplanted to mid elevations (P > 0.05; Fig. 3b).

Transplants originating from the mid elevation did not differ in species from mid-elevation controls, but there was a difference in species-richness change between the ones moved to high vs. low elevations (P = 0.014), since turfs moved up gained and turfs moved down lost species (Fig. 3d). Turfs transplanted from low to mid and high elevations gained significantly more species compared to low elevation controls (P < 0.01; Fig 3f).

The composition of the transplanted plant communities adjusted slowly to the hydrologic conditions at the target elevations: Only the turfs transplanted from high to low elevations were statistically indistinguishable in species richness and composition from low elevation controls 6 years after transplantation (Figs 5a and6a). In all other cases, species richness or composition still differed significantly. Changes in species richness occurred gradually during the experiment (Fig. 5): Turfs transplanted from high to low elevation lost species successively and did not differ in species richness from the low elevation controls at the end of the experiment (P = 0.282; Fig. 5a). Although turfs transplanted from low to high gained species over time, they still had fewer species compared to high elevation controls at the end of the experiment (P = 0.031; Fig. 5a). Turfs transplanted from mid to low elevations were intermediate in species richness between the low and mid elevation controls (P = 0.040, Fig. 5c). Turfs transplanted from low to mid elevation gained species over time and had more No species other than those already growing in the meadow at the start of the experiment invaded the turfs during the 6 years; exotic species were absent. In all ordinations of the site scores based on species data, the first axis was strong with eigenvalues ranging between 0.50 and 0.87 (Fig. 6), mainly representing the riparian elevation gradient. The control plots of the three different elevations were all significantly different in community composition from each other, both the first and last year of the experiment (P < 0.05, one-way ANOVA; Fig. 6).

Although the site scores of the transplants had become more similar to the target elevation during the course of the experiment, only the transplantation from high to low was statistically indistinguishable from the community composition of control plots at the new elevation (P > 0.05, one-way ANOVA; Fig. 6a). In the remaining cases, species composition of the transplants was intermediate between original and target levels (Fig. 6b, d ande), or was statistically similar to the original levels (Fig. 6c andf).

Discussion

Our experiment demonstrates that riparian-plant communities will change in response to the hydrologic alterations expected as a result of climate change. This study also shows the importance of conducting long-term field experiments [START_REF] Milchunas | Inertia in plant community structure: state changes after cessation of nutrient enrichment stress[END_REF]. Plant communities had not fully adjusted to the new elevation 6 years after transplantation.

Extrapolating from our results suggests that full adjustment to the new hydrological regime will take at least 10 years. However, rare events such as unusually large floods or droughts may result in non-linear vegetation responses [START_REF] Jentsch | A new generation of climate-change experiments: events, not trends[END_REF].

Hydrology

Flood duration during the experiment for the mid and high elevation plots was in many respects intermediate between the normal period 1961-1990 and the projections for 2071-2100, implying that the observed warming during the last decades has led to hydrological trends going in the direction of the changes expected at the end of the century as a result of warming [START_REF] Peterson | Increasing river discharge to the Arctic Ocean[END_REF]. We conducted the experiment during a period which was drier and with shorter floods at all elevations compared to 1961-1990 (Table 1).

Although inundation duration at the low elevation in 2001 was long in comparison with other years during the experiment, lasting from May to July, it was close to the average for .

Effects of trenching

The fact that the only significant effect of trenching was on biomass the second year suggests that excavation had limited effects and that excavated plots could be used as realistic controls for transplantation effects. The effect of trenching on biomass was probably caused by damage to below-ground plant parts. The fact that high-elevation trenched turfs had significantly more biomass than the controls in 2001, may be due to reduced soil competition from neighbouring plants outside the plots. [START_REF] Coomes | Impacts of root competition in forests and woodlands: a theoretical framework and review of experiments[END_REF] suggested that root trenching would enhance growth when nutrients and/or water are limiting, and found support for this hypothesis in a review of experiments. In contrast, where soils are relatively rich and droughts are uncommon, trenching effects were weak or absent [START_REF] Coomes | Impacts of root competition in forests and woodlands: a theoretical framework and review of experiments[END_REF]. The reduced biomass on the low-elevation transplants was likely a response to the combined effect of long duration of inundation in 2001 and lower tolerance to flooding after root damage.

Complete submergence slows down growth even in inundation-tolerant species [START_REF] Johansson | Responses of riparian plants to flooding in free-flowing and regulated boreal rivers: an experimental study[END_REF]Voesenek et al. 2006) and water entrance into root aerenchyma following trenching might have further reduced growth. Also the turfs transplanted down from the high and mid elevations decreased substantially in biomass the same year (Fig. 5).

Effects of hydrologic changes

Why did biomass generally increase when turfs were transplanted to lower elevations? Growth at low elevations might have been enhanced by environmental conditions, such as higher soil moisture, or species on transplanted turfs were replaced with species that grow larger. In general, above-ground biomass of herbs and graminoids is high in low-lying riparian and other wetland plant communities where light availability is high and water does not limit growth (strongly nutrient limited communities excepted). The fact that biomass increased successively from 2001 to 2006 in plots transplanted from high to low elevations suggests that successive species replacement was the cause. In 2005 these plots were dominated by Calamagrostis canescens and Carex acuta (on average 96% of the total biomass). Both of these species were absent before transplantation. In contrast, the reason why turfs transplanted from low to high elevations decreased in biomass the first year after transplantation and remained low thereafter is better explained by the change in external conditions. The two species dominating turfs of low origin, Calamagrostis canescens and Carex acuta, remained high in frequency at high elevations, but decreased in biomass production, dropping from 206 g to 74 g and 260 g to 17 g, respectively, between 2002 and 2005, potentially as a result of drought stress. In the long run, they are likely to be outcompeted by more drought-tolerant species, given the existence of a trade-off between flooding and drought tolerances [START_REF] Luo | Trade-off between tolerance to drought and tolerance to flooding in three wetland plants[END_REF].

The slow adjustment in species composition following transplantation was probably caused by the ability of many species to tolerate hydrologic conditions distinct from those in unmanipulated systems [START_REF] Van Eck | Is tolerance to summer flooding correlated with distribution patterns in river floodplains? A comparative study of 20 terrestrial grassland species[END_REF]). The persistence of many species likely delayed or inhibited establishment of species better adapted to the conditions at the target elevations.

Even in the case of transplanting from high to low elevations, where one might expect that flood duration would exceed the tolerance of high-elevation species, species losses occurred gradually over many years (Fig. 5a). Thus, long-term experiments are needed to evaluate the potential effects of hydrologic change on riparian ecosystems. Most ecological field experiments rarely last more than 3 years, which may be inadequate to draw conclusions about mechanisms controlling natural ecosystems, and treatment effects may change with time [START_REF] Milchunas | Inertia in plant community structure: state changes after cessation of nutrient enrichment stress[END_REF]. Even our 6-year study was too short for plant communities to fully adjust to the new conditions imposed by the experiment.

Catastrophic events, such as hurricanes or floods, can trigger permanent changes to ecosystems by causing widespread mortality, allowing new species to establish. Flooding events of exceptional duration have been shown to severely decrease species richness in riparian zones [START_REF] Renöfält | Connecting variation in vegetation and stream flow: the role of geomorphic context in vegetation response to large floods along boreal rivers[END_REF]) and alter limits of riparian vegetation belts (Vervuren et al. 2003;[START_REF] Van Eck | Is tolerance to summer flooding correlated with distribution patterns in river floodplains? A comparative study of 20 terrestrial grassland species[END_REF]). If such an event had occurred during our experiment, rapid [START_REF] Jentsch | A new generation of climate-change experiments: events, not trends[END_REF].

The future of riparian vegetation in the Vindel River

Combining the results of the transplant experiment and projections of future stream flows, we may predict likely responses of riparian vegetation to climate-driven hydrologic changes. The high riparian elevations in the experiment are projected to experience less frequent flooding (Table 1) and are expected to shift into terrestrial forest communities likely to lose most riparian species with time. Differences in species richness between riparian and terrestrial plant communities in the region are dramatic, with forest understories being dominated by a few dwarf-shrub species [START_REF] Nilsson | Floristic differences between riparian corridors of regulated and free-flowing boreal rivers[END_REF]. The boundary between riparian and terrestrial plant communities is generally sharp, occurring over a few decimetres in elevation and coinciding with the limit of floods occurring every second year [START_REF] Nilsson | Frequency distributions of vascular plants in the geolittoral vegetation along two rivers in northern Sweden[END_REF]. For the middle-elevation communities, the projected frequency of flooding resembles present ones at the high elevation. Hence, the transplantation of turfs from middle to high elevation is relevant to predict likely effects of climate change to mid-elevation plant communities. There were no significant responses in biomass or species richness of mid-to-high transplants, implying that mid-elevations will mainly change in species composition to constitute the highest part of the regularly flooded riparian zone, representing a decline in the area of species-rich spring-flood communities.

All four climate-change scenarios predict an overall increase in river runoff in the boreal part of Sweden between 1961-1990and 2071-2100[START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF]). The largest flow increases will occur outside the growing season when water levels are below summer low water-levels coinciding with the lower limit of continuous Carex vegetation (Fig. 7), and are thus expected to primarily impact amphibious plant communities. However, low-elevation Carex communities may become flooded any time of the year in the future, with effects difficult to predict. For example, ice disturbance during winter floods may become common.

If inundation of low-elevation, Carex-dominated communities would increase as suggested in the more extreme projections (Fig. 7), present communities may become replaced by amphibious plant communities. The low elevation was inundated on average 20 days shorter per year during the experiment compared to the period 1961-1990 (37.4 days compared to 57.0 days; Table 1), implying that the contrast between present and projected future conditions is larger than shown in Fig. 7. To conclude, we predict that climate-driven hydrologic changes will result in narrower riparian zones along the Vindel River, with present riparian-plant communities being replaced by terrestrial communities at the top and by amphibious or aquatic communities at the bottom. Given strong species-area relations, less area available to riparian plants is expected to reduce species richness, even if no species lose their hydrologic niches.

Predicting the effects of climate change in different boreal regions

Hydrologic changes caused by a warmer climate are expected to vary across boreal and Arctic regions (Woo et al. 2008) as a result of regional differences in climate change and differences in characteristics between catchments. The hydrologic response will vary depending on the proportion of precipitation expected to fall as snow in the future, the overall trend in the amount of precipitation, and the balance between precipitation and evapotranspiration (Woo et al. 2008). Most northern rivers will get a significant proportion of the precipitation as snow also in the future, either because they lie in the far north or because they drain high mountain ranges. Effects of hydrologic change on such rivers are expected to be similar to the ones The magnitude of change is expected to vary latitudinally, with the smallest changes in northernmost rivers. High Arctic rivers may even experience higher spring floods as a result of higher precipitation and more snow accumulation.

Rivers in the southern parts of the boreal zone with catchments lacking large alpine areas will experience significant decreases in the proportion of precipitation falling as snow. The qualitative hydrologic changes will be similar to the Vindel River, but be more extreme: Spring floods will have lower magnitudes or disappear, resulting in losses or reductions in the area of riparian forest communities, with subsequent losses in riparian species richness. In addition, water-levels will vary less among seasons, with higher winter flows, resulting in upward displacements of low-elevation riparian zonation, amphibious and aquatic plant communities replacing herbs and graminoids. In boreal continental-climate regions, warming is expected to lead to more extreme summer humidity deficits, resulting in decreased summer flows and more drought stress, which may reduce riparian biomass production and alter competitive relations among species. This might correspond to transplanting turfs to higher elevations, and hence result in biomass losses and local species-richness increases. However, we do not know how the effect of summer droughts will combine with other climate-change effects, such as reduced spring flooding or higher frequency of autumn floods.

Increases in the occurrence of major rain storms have been implicated as a result of warming [START_REF] Ilg | Long-term reactions of plants and macroinvertebrates to extreme floods in floodplain grasslands[END_REF]. [START_REF] Andréasson | Hydrological change: climate change impact simulations for Sweden[END_REF] predicted a general increase in the frequency of 100-year floods in autumn for northern Sweden at the end of the century. Given that extreme floods of long duration have long-lasting effects on riparian plant communities, e.g. by causing extinctions of flood-sensitive species [START_REF] Renöfält | Connecting variation in vegetation and stream flow: the role of geomorphic context in vegetation response to large floods along boreal rivers[END_REF], increases in the Riparian plant communities make up a significant proportion of regional plant diversity in boreal landscapes [START_REF] Nilsson | Floristic differences between riparian corridors of regulated and free-flowing boreal rivers[END_REF][START_REF] Renöfält | Spatial and temporal patterns of species richness in a riparian landscape[END_REF], and the predicted losses in the area of riparian zones may thus have landscape-level consequences. A number of actions may be taken to reduce the risk of riparian species loss. First, remaining species-rich areas may be managed to ensure long-term viability of component species. Second, streams and rivers degraded by dams and channelization may be restored to increase the abundance of riparian species. Third, further studies are required to identify species especially at risk, for which actions to maintain their population size may be warranted. Specifically, information about the hydrologic requirements of individual riparian plant species is needed to predict their climate-change responses. Finally, riparian zones perform many ecosystem functions important for other landscape elements, such as supplying wood and leaf litter to aquatic habitats and acting as a buffer against flows of matter and pollutants from terrestrial areas to streams [START_REF] Naiman | Riparia: Ecology, Conservation and Management of Streamside Communities[END_REF]. Elucidating how these riparian functions are affected by climate-driven hydrologic change is a priority area for research.

  temperature has increased by 0.7°C during the 20 th century (IPCC 2007).

  requires manipulating the frequency and duration of floods.

  , we applied 2000 g m -2 of riverborne litter in 2001 and 2002 to half

  analysis and for measuring soil moisture in 2000-2002. Soil moisture was measured twice each season with plaster cubes in the ground and a soil moisture meter (Model 14.22, Eijkelkamp Agrisearch Equipment, Giesbeek, The Netherlands). Water levels were measured continuously with the data loggers from 2000 to 2003.

  Strycksele. By interpolating gauge data, we estimated the flooding regime for 2004-2006 for all plots, to supplement the 2000-2003 water-level data.

  in community composition as a result of transplantation, we used Detrended Correspondence Analysis (DCA; ter Braak & Šmilauer 1998).

  ANOVA and Tukey's post-hoc test. All statistical analyses were performed in the statistical package R (R Development Core Team 2006), except for the ordination which was done in Canoco version 4 (ter Braak & Šmilauer 1998).

  low elevation controls (P = 0.015), and did not differ significantly in richness from the controls of their new elevation (P = 0.099, Fig.3c).

  composition may have resulted. The importance of events rather than gradual changes in biotic responses to climate change has recently been emphasized

  Vindel River, with reductions in spring flood height and higher winter flows.

  events may have profound effects. If more frequent than the recovery time, a reduction in average diversity is expected.

  Blom C, de Kroon H (2003) Extreme flooding events on the Rhine and the survival and distribution of riparian plant species. Journal of Ecology, 91, 135-146. Voesenek L, Colmer TD, Pierik R, Millenaar FF, Peeters AJM (2006) How plants cope with complete submergence. New Phytologist, 170, 213-226. Wassén G (1966) Gardiken. Vegetation und Flora eines Lappländischen Seeufers. Kungliga Svenska Vetenskapsakademiens Avhandlingar i Naturskyddsärenden, 22, 1-142. Woo MK, Thorne R, Szeto K, Yang DQ (2008) Streamflow hydrology in the boreal region under the influences of climate and human interference. Philosophical Transactions of the Royal Society B -Biological Sciences, 363, 2251-2260. Xiong SJ, Nilsson C (1997) Dynamics of leaf litter accumulation and it effects on riparian vegetation: a review. Botanical Review, 63, 240-264.

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

 1234567 Fig. 1 Schematic illustration of the experimental setup showing one of eight replicates along with

Table 1

 1 Flooding regime for each elevation. Values from our study and from 1961-1990 were from data loggers or a nearby gauge, whereas the four scenarios for 2071-2100 were calculated from hydrologic data presented by the Swedish Meteorological and Hydrological Institute (Norrköping, Sweden). RT = Return time, Duration = number of flooded days per year. Model abbreviations are explained in the Methods section.

					Riparian elevation
				Low		Mid		High
				Duration		Duration		Duration
	Period	Years	RT mean range	RT mean range	RT mean range
	Observed data
	2000 -2006	7	1	37.4 16-81	1.4	6.9 0-14	3.5	1.7 0-7
	1961-1990	30	1	57.0 28-100	1.3	9.7 0-26	2.1	2.8 0-12
	Models
	ECHAM4-B2	30	1	67.2 30-108	1.6	6.1 0-27	6.0	1.7 0-22
	HadAM3H-B2	30	1	58.0 28-99	2.3	5.0 0-26	7.5	1.3 0-21
	ECHAM4-A2	30	1	75.2 29-126	2.5	4.4 0-23	7.5	1.0 0-10
	HadAM3H-A2	30	1	56.0 18-95	2.7	2.9 0-24	7.5	0.9 0-10

Table 2

 2 Effects of transplantations and the litter addition of 2001 and 2002, factorial ANOVA. Df for 2002-2006 is the same as for 2001. *** P<0.001, ** P<0.01, * P<0.05.

			2000		2001	2002	2003	2005	2006
	Source of variation	df	F	df	F	F	F	F	F
	Species richness
	Origin of turf	2	61.7 ***	2	17.2 ***	24.6 ***	26.5 ***	15.9 ***	16.4 ***
	Target of turf	2	1.9	2	3.8 *	13.2 ***	12.1 ***	35.8 ***	39.9 ***
	Litter addition			1	3.2	2.9	5.1 *	1.3	0.3
	Origin x Target	4	0.9	4	0.3	0.7	0.8	1.0	1.6
	Origin x Litter			2	0.7	0.2	0.3	0.5	1.4
	Target x Litter			2	0.7	1.2	0.0	0.4	0.4
	Biomass
	Origin of turf	2	3.9 *	2	0.2	1.4	4.6 *	0.6	1.1
	Target of turf	2	5.0 **	2	8.9 ***	4.3 *	8.1 ***	7.9 ***	20.7 ***
	Litter addition			1	1.9	4.2 *	0.1	0.9	0.3
	Origin x Target	4	0.5	4	3.6 *	2.7 *	1.9	1.3	0.9
	Origin x Litter			2	0.0	0.2	0.5	1.7	0.3
	Target x Litter			2	1.2	0.1	0.7	0.2	0.7

Global Change Biology

Acknowledgments

We thank Johan Andréasson for providing us with hydrologic data, Elisabet Carlborg, Pia Hansson and Magnus Svedmark for assistance in the field and David Merritt for valuable comments on the manuscript. Funding for the study was provided by the Swedish Research Council Formas (to C. Nilsson and R. Jansson).