Biodiversity Taxonomy, Genetics and Ecology of Sub-Arctic Willow Scrub
A collaborative research project by the Scottish Montane Willow Research Group Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Contents Introduction 3 sub-arctic willows 4 sub-arctic willow scrub 6 the situation in Scotland today 7 research questions 8 study sites 9
Taxonomy 10 Patterns of hybridization among sub-arctic willows in Scotland 11 Patterns of hybridization between S. arbuscula and S. lapponum 12 Dynamics of hybridization between S. arbuscula and S. lapponum 13
Genetics 14 Sexual versus asexual reproduction 15 Genetic diversity within willow populations 16 Genetic differentiation between willow populations 17
Mycology 18 Ectomycorrhizal fungi associated with S. herbacea 19 Sub-arctic willows and rust fungi (Melampsora spp.) 20 Ectomycorrhizal colonization potential of upland soils 21
Ecology 22 Herbivory and seed production 23 Predicting the potential distribution of S. arbuscula 24 Herbivory and seedling establishment 25
Conservation implications 26 Taxonomy 26 Genetics 26 Mycology 28 Ecology 29
Further information 30
All photographic images included in this report were taken by members of the project during the course of the research.
The species distribution maps on pages 4 - 5 are reproduced from: Preston, C.D., Pearman, D.A. and Dines, T.D. (Eds.) (2002). New Atlas of the British and Irish Flora. Oxford University Press. © Crown, 2002.
Citation: Scottish Montane Willow Research Group (2005). Biodiversity: taxonomy, genetics and ecology of sub-arctic willow scrub. Royal Botanic Garden Edinburgh.
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Introduction
Salix lapponum
Sub-arctic willow scrub is one of the rarest and most endangered habitats in the UK. It is almost entirely restricted to the Scottish mountains where it forms a component of the montane scrub occurring above the natural tree line.
Remaining sub-arctic willow scrub exists as isolated populations that are considered to be remnants of a formerly more widespread post-glacial vegetation type. These surviving fragments are the subjects of acute conservation concern and, without intervention, many populations are in danger of terminal decline.
The rare and threatened status of this vegetation type is recognized by its inclusion in Annex I of the European Habitats Directive and subsequent prioritization for conservation within the UK. One of the species included in this vegetation type, S. lanata, is listed in the British Red Data Book of Vascular Plants and is the subject of a Species Action Plan that sets out targets for its conservation.
This booklet presents a summary of the findings of a large collaborative research project that ran from 2002 - 2005 and aimed to provide the underpinning scientific research required for determining the most appropriate strategies for management and species recovery programmes for sub-arctic willows. Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b Sub-arctic willows Willows (Salix spp.) belong to the family Salicaceae that consists of dioecious (separate sexes), catkin-bearing trees and shrubs with simple, alternate leaves. Salix species are believed to be primarily entomophilous (insect-pollinated) and partially anemophilous (wind-pollinated). The small seeds are characteristically enveloped in long, soft, silky hairs and dispersed by wind.
Montane willow scrub communities in Scotland consist of mixtures of species which, elsewhere, have arctic-subarctic-alpine distributions (S. arbuscula, S. lanata, S. lapponum, S. myrsinites, S. reticulata) and willow species of general northerly distribution (S. herbacea, S. myrsinifolia, S. phylicifolia).
Five species of sub-arctic willow and one of the more common ‘northerly’ willows were included in this research project.
Salix arbuscula (Mountain willow) Recorded in 32 10 x 10 km squares Conservation status: SCARCE. since 1970, all in Scotland.
Salix herbacea (Dwarf willow) Recorded in 335 10 x 10 km Conservation status: COMMON. squares in Great Britain since 1970.
Map key 1987 - 1999 Native 1970 - 1986 Native Pre-1970 Native Salix lapponum (Downy willow) Recorded in 83 10 x 10 km squares Conservation status: SCARCE. in Great Britain since 1970. A co l l ab o r at i v e r e s e a r c h p r o j e c t Sub-arctic willows
Salix myrsinites (Whortle-leaved willow) Recorded in 59 10 x 10 km squares Conservation status: SCARCE. since 1970, all in Scotland.
Salix reticulata (Net-leaved willow) Recorded in 18 10 x 10 km squares Conservation status: SCARCE. since 1970, all in Scotland.
S. lanata Species Action Plan
S. lanata is included in the British Red Data Book of Vascular Plants. It is extant at only 14 localities in Scotland and only five of these populations are currently considered to be viable. The Species Action Plan for S. lanata includes the following targets:
ensure that all populations are restored to a state capable of regenerating by 2008
ensure that each population will consist of at least 50 plants (or the sustainable maximum for the site) by 2008
reintroduce the species to two Salix lanata (Woolly willow) Recorded in 13 10 x 10 km former sites by 2003. Conservation status: RARE & VULNERABLE. squares since 1970, all in Scotland. Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Sub-arctic willows Under favourable conditions, sub-arctic willows can form extensive scrub communities. In Great Britain these have been classified under the National Vegetation Classification as W20 – Sub-arctic Salix lapponum – Luzula sylvatica scrub. This tends to occur on moist, relatively base-rich soils
willow scrub in rocky situations with a north to east aspect, generally at altitudes from 600 m to over 900 m. A degree of shelter may be favourable to scrub development and there may be a positive association with late snow-lie which offers some protection from spring frosts and browsing.
Well developed willow scrub at Corrie Sharroch, Glen Doll
Developed scrub communities tend to be dominated by S. lapponum which is the commonest and most widely distributed of the sub-arctic willows but can include some or all of the other species as well as hybrids. Associated vascular plants include sub-shrubs such as Vaccinium myrtillus, V. vitis-idaea and Empetrum nigrum; tall herbs such as Alchemilla glabra, Rhodiola rosea, Oxyria digyna and Saussurea alpina; small herbs such as Alchemilla alpina, Thalictrum alpinum and Polygonum viviparum.
S. lanata with Oxyria digyna S. arbuscula with Saxifraga aizoides A co l l ab o r at i v e r e s e a r c h p r o j e c t Sub-arctic willows Based on comparisons with Scandinavia and the distribution of suitable terrain, it is generally considered that sub-arctic willow scrub was once more widespread in Scotland than it is today. Cutting for firewood and charcoal-burning coupled with more recent intensification The situation in of grazing patterns in upland areas has resulted in a situation where montane willow Scotland today communities are now largely restricted to inaccessible ledges. Consequently, willow populations are typically small and isolated and of acute conservation concern.
Remaining willow scrub is confined largely to inaccessible ledges
Sampling inaccessible willows Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b Research questions Conservation action requires underpinning scientific research. At the outset of this project it was apparent that there are a number of knowledge gaps that need to be addressed if conservation targets set for sub-arctic willow in Scotland are to be achieved. Examples of these are as follows.
Willows are notorious for their taxonomic complexity and numerous hybrid combinations have been recorded in the UK. Do sub-arctic willows exist as separate well- defined taxonomic entities for which conservation plans can be devised or is hybridization so extensive that these willows should effectively be treated as one large aggregate?
Small and isolated plant populations are often found to be low in genetic diversity. This tends to limit their capacity to respond to both acute and chronic environmental change. Furthermore, small numbers of individuals in populations can lead to problems through inbreeding depression. Therefore, has the fragmentation of willow scrub resulted in genetically depauperate populations that may be lacking in vigour and ill-equipped to respond to future conditions?
In attempting to restore willow scrub, is enough known about associated organisms such as fungi? For example, it is known that species of Salicaceae form mycorrhizal associations. Is this something that should be taken into account when willows are being planted?
Grazing of willow seedlings and saplings by herbivores inhibits natural regeneration of willow scrub. However, herbivores may affect the reproductive capacity of willows in other ways. For example, how does removal of shoots by sheep and red deer influence catkin production, seed set and hence population viability?
Research aims: Our research was carried out 4O CLARIFY under four broad headings: SPECIES AND HYBRID taxonomy, genetics, DEFINITIONS mycology and ecology.
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4O INVESTIGATE FACTORS 4O ASSESS PATTERNS CURRENTLY LIMITING %#/,/'9 '%.%4)#3 OF INTRASPECIFIC WILLOW REGENERATION GENETIC VARIABILITY
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4O EXAMINE DIVERSITY OF MUTUALISTIC AND PARASITIC FUNGI A co l l ab o r at i v e r e s e a r c h p r o j e c t
Study sites Samples were collected from many sites representing the full range of sub-arctic willow distribution across Scotland. However, intensive cross-cutting research was carried out at four focal study sites.
Meall Ghaordiadh Assessment of clonal growth, ectomycorrhizal colonization and insect herbivory of S. herbacea on the ridge-top. Hybridization studies of S. lapponum and S. arbuscula on the crags below.
Corrie Sharroch Assessment of clonal growth of S. lanata and S. lapponum, assessments of taxonomic status, rust infection, insect herbivory and phytochemistry of S. lanata, S. lapponum and S. myrsinites.
Ben Lawers Effect of large and small mammal herbivory on reproductive biology of S. arbuscula, seed dispersal, potential establishment sites, ectomycorrhizal colonization potential of soils.
Meall nan Gabhar, Ben Lui Hybridization studies of S. lapponum and S. arbuscula. 10 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Taxonomy Willows are well-known for their tendency to hybridise. There are currently 64 willow hybrid combinations recorded in the UK but we do not know the extent to which hybridization occurs within natural populations. Do willow species limits remain well-defined with only occasional hybridization events or is hybridization in fact much more frequent and widespread? Under what circumstances does hybridization occur?
This is important from a conservation perspective as the development of appropriate strategies and targets requires a sound understanding of the taxonomic status of the plants we wish to protect. For example, if one strategy for conservation is to augment existing populations or establish new ones by planting, should efforts be made to avoid inclusion of hybrids among planting stock? Or are hybrids in fact so common in nature that inclusion of hybrids in restoration programs is inevitable?
As the range of phenotypic variation in willow hybrids is poorly understood we developed molecular tools that would enable us to discriminate between species and detect hybrids. To provide complementary data we also employed phytochemical techniques that were found to successfully distinguish species. We then used these tools together with morphological methods to investigate three topics.
1. Patterns of hybridisation among sub-arctic willows in Scotland. 2. Patterns of hybridization between S. lapponum and S. arbuscula. 3. Dynamics of hybridization between S. lapponum and S. arbuscula.
Leaf morphology of S. lapponum, S. arbuscula and their hybrid, S. x pseudoglauca
S. lapponum
S. x pseudoglauca
S. arbuscula
Sampling S. lapponum at Drumochter Pass A co l l ab o r at i v e r e s e a r c h p r o j e c t 11
Taxonomy What can molecular studies tell us about the patterns of hybridization among sub-arctic willows in Scotland? Is hybridization common or rare? Patterns of Many samples of eleven species of willow were collected from sites across Scotland as well hybridization as examples of hybrids identified by their morphology. We also collected samples from among sub-arctic morphological hybrid specimens from an extensive ex-situ collection of native willows. DNA-fingerprinting techniques were applied and data analysed to detect patterns of willows in Scotland hybridization.
The extent of hybridization appears to be limited with most hybrids occurring only occasionally. An exception to this is hybridization between S. arbuscula and S. lapponum which occurs freely at some sites.
Where it is possible to distinguish morphological hybrids, hybridization in sub-arctic willows appears to follow one of three patterns A, B or C (Figures 1 and 2):
Figure 1. Three patterns of hybridization among sub-arctic willows. Distance between points approximates the relative genetic similarity of pairs of samples. See legend text for descriptions.
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Figure 1/Pattern A. Morphological hybrids are molecularly intermediate Figure 1/Pattern B. Morphological hybrids are either intermediate or are more closely allied (e.g., S. aurita x S. repens). to either of the parent species, suggesting back-crossing (e.g., S. herbacea x S. lapponum).
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Figure 1/Pattern C. Morphological hybrids always fall within the range of molecular Figure 2. Patterns of hybridization between sub-arctic and montane willow species. variation of one or both parent species (e.g., S. caprea x S. lapponum). As no genetically See Figure 1 for description of patterns. intermediate individuals occur, it is currently difficult to say whether the observed pattern is due to back-crossing or extreme morphological variation in the parent species. 12 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Taxonomy Are the patterns of hybridization that we revealed for particular hybrid combinations consistent across sites or can we detect differences between sites that might then offer some Patterns of insights into factors that influence this process? hybridization between Two sites were selected where S. arbuscula and S. lapponum grow together (Meall Ghaordiadh and Meall nan Gabhar, Ben Lui). Patterns of hybridization at the two sites S. arbuscula and were compared using a combination of morphological and molecular techniques. S. lapponum The patterns of hybridization at the two sites were different. A much higher incidence of morphological hybrids was recorded at Meall Ghaordiadh than at Meall nan Gabhar.
At Meall nan Gabhar, the morphology of the two hybrids found was clearly intermediate between the parent species. However, their molecular fingerprints clustered within the parent species (Figure 3). This indicates that these hybrid individuals are probably derived from back-crossing of hybrids to either parent with the transmission of morphological character states from the other parent.
At Meall Ghaordiadh, the morphology of most of the hybrids was also intermediate. However, the molecular data were more complex with some hybrids falling within the range of the parents and some being intermediate (Figure 3). The presence of intermediate individuals leads to more of a genetic continuum between the two species at this site.
These data indicate that patterns of hybridization between the same two species can vary between sites.
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Figure 3. Comparison of morphological (A) and molecular (B) variation in S. arbuscula and S. lapponum and hybrids between them at Meall nan Gabhar (1) and Meall Ghaordiadh (2). A co l l ab o r at i v e r e s e a r c h p r o j e c t 13
Taxonomy Having detected different patterns of hybridization at Meall Ghaordiadh and Meall nan Gabhar, we aimed to investigate factors involved in maintaining species barriers and the circumstances under which hybridization between S. lapponum and S. arbuscula occurs. Dynamics of hybridization At the two sites we counted the relative numbers of plants of the two species and recorded the between flowering time of a large number of tagged plants at weekly intervals during the flowering season. S. arbuscula and Relative abundances of the two species were different at the two sites. At Meall nan S. lapponum Gabhar, the numbers were roughly equal. However, at Meall Ghaordiadh there were many more individuals of S. arbuscula than of S. lapponum (ca. 30:1).
The assessment of flowering dates showed a clear tendency for S. lapponum to flower before S. arbuscula at both sites (Figure 4).
At Meall nan Gabhar neither of the hybrids flowered during the two years of observation. At Meall Ghaordiadh, morphological hybrids were detected that flowered at intermediate dates between the two species as well as outwith the range of overlap between the flowering times between the two species (Figure 4). This could increase the probability of introgression through back-crossing in either direction if there was no selection against hybrid genotypes.
These data are consistent with hybridization theory that predicts that where two species with the potential to hybridize co-occur in disproportionate numbers, hybridization is more likely to take place. Where fertile hybrids are generated as in the present case, introgression of genes between species may accentuate the breakdown of species barriers.
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Figure 4. Flowering date of S. arbuscula, S. lapponum and their hybrid, S. x pseudoglauca, at two sites. 14 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Genetics Fragmentation of vegetation cover into small and isolated populations can result in serious losses of genetic diversity which can hinder a species’ ability to respond to both acute (short- term) and chronic (long-term) environmental change. Small populations can also be affected by inbreeding depression due to the increased chance of mating among related individuals. Subsequent expression of deleterious recessive alleles can result in reduced vigour and fitness.
It is believed that sub-arctic willow populations in the UK are remnants of a previously more widespread vegetation type. Given the small sizes and patchy distribution of many of the remaining populations, it is possible that they consist of related individuals with low levels of genetic diversity. Such populations would be a poor source of donor material for reintroduction programmes, and may themselves be poor candidates for conservation management due to intrinsic low levels of fitness.
The amount of genetic diversity available in remaining populations is related to the amount of sexual reproduction occurring within and between them. Willows are also capable of reproducing asexually via the spread of clonal suckers and at the outset of this project we did not know whether sub-arctic willows reproduce primarily sexually or asexually. An understanding of this crucial aspect of willow biology is necessary if restoration programmes are to successfully imitate natural populations.
We developed molecular tools that would enable us to differentiate individual willow genotypes. These tools were then applied to natural populations of sub-arctic willows across Scotland to investigate three topics.
1. The extent of sexual versus asexual reproduction. 2. The amount of genetic diversity within willow populations. 3. The amount of genetic differentiation between willow populations.
DNA microsatellite ‘fingerprints’ Sampling S. lapponum at Creag Megaidh A co l l ab o r at i v e r e s e a r c h p r o j e c t 15
Genetics Sexual recruitment in willow populations often appears to be very limited and we know that willows have the ability to spread vegetatively via suckers. What proportion of the individual bushes that we observe in a population is derived from sexual reproduction and what Sexual versus proportion from asexual, vegetative growth? asexual reproduction Leaf samples were collected from over 400 closely spaced bushes of S. lanata and S. lapponum at Corrie Sharroch and from S. herbacea at 90 points on an exposed ridge-top at Meall Ghaordiadh. Samples were genotyped using molecular techniques.
99% of S. lanata and 95% of S. lapponum samples represented unique genetic individuals.
28% of the S. herbacea samples represented unique genetic individuals. Samples with identical genotypes showed strong spatial aggregation with one genotype being at least 8 m across (Transect F, Figure 5).
Despite field observations of sexual reproduction in S. lanata and S. lapponum being rare, this appears to be their main mechanism of reproduction and dispersal. The pattern observed in S. herbacea suggests greater levels of clonal growth and patch formation. This matches well with the mat forming rhizomatous growth of the species and the more open habitat in which it occurs.
S. lanata and S. lapponum at Corrie Sharroch. Our results suggest that there are many genetic individuals resulting from sexual reproduction rather than few individuals resulting from clonal growth.
Figure 5. Distribution of S. herbacea genotypes along six transects of 8 m length at Meall Ghaordiadh (not to scale). Circles of the same colour are samples with the same genotype. The photograph shows flags marking one of the transects. 16 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Genetics Many of the remaining populations of sub-arctic willows in Scotland are small and isolated. Such populations can lack genetic diversity due to low numbers of individuals and Genetic diversity insufficient gene flow to ensure new genetic combinations. Are Scottish sub-arctic willow within willow populations genetically depauperate? populations Leaf samples of S. lanata (rare), S. lapponum (scarce) and S. herbacea (common) were collected from a total of 19 sites across Scotland and subjected to DNA-fingerprinting.
There was no difference between the three species in diversity levels within populations in spite of their different abundance.
The data sets showed very little correlation between population size and levels of genetic diversity. In S. lanata, the largest population contains the highest diversity (Coire Cheap), but some smaller populations also have high diversity. For example, the small population of 12 individuals at Caenlochan has the same level of diversity as the larger population of 243 individuals at Corrie Sharroch (Figure 6).
The levels of genetic diversity and the lack of correlation between population size and diversity suggest either high levels of gene flow or limited divergence since fragmentation.
Genetic diversity (A)
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Figure 6. Relationship between population size and genetic diversity (A) in S. lanata. A co l l ab o r at i v e r e s e a r c h p r o j e c t 17
Genetics In addition to genetic diversity within populations, another component of inter- and intraspecific genetic diversity is the level of genetic differentiation between populations. This is important because we need to know to what extent the full range of genetic Genetic diversity within a species is represented within any one population. This has implications differentiation for the targeting of conservation resources where the aim is to conserve genetic diversity. between willow Measuring population differentiation also allows us to assess the impact of isolation by offering important insights into levels of pollen and seed dispersal between populations. populations
Using the same DNA fingerprint data as for the analysis of genetic diversity within populations of S. lanata, S. lapponum and S. herbacea, we examined the level of genetic differentiation between populations.
All three species showed some evidence of low but significant population differentiation.
The highest population differentiation was found in S. herbacea and this was significantly greater than that found in S. lapponum (Figure 7). S. lanata had a similar level of differentiation to S. herbacea, though this was not significantly different to either of the other species. It is unclear why the common species S. herbacea shows higher population differentiation than the more scarce species S. lapponum.
What can be concluded is that the rarity of S. lanata is not associated with high population differentiation. This suggests either high gene flow or limited divergence since fragmentation. It is not possible to distinguish between the two at present, but the latter seems most likely. The longevity of willows may have led to limited generations since the populations have been fragmented and hence limited opportunities for differentiation via genetic drift.
Genetic distance (F ) st
Fst is a measure of genetic differentiation between populations (ie, the proportion of total genetic diversity attributable to between population variation). A value of 45
' 1 indicates that populations are completely different and a value of zero indicates that populations are identical.
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Figure 7. Mean percentage variation between populations for three species of sub-arctic willow. 18 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Mycology Associating with mycorrhizal fungi benefits plants by enhancing their capacity to acquire limited nutrients. One type of mycorrhizal association, the ectomycorrhiza (see box) is considered to be particularly important in upland environments where decomposition processes are slow and much of the soil nitrogen is bound in organic matter. Communities of ectomycorrhizal fungi are often very diverse. Is this diversity related to diversity of host genotypes? Understanding this relationship is important if we are to mimic natural populations in restoration programmes. Furthermore, successful establishment of planted willows may rely on the ability of young plants to form ectomycorrhizal associations. Do upland soils targeted for restoration have sufficient ectomycorrhizal inoculum?
Rust fungi in the genus Melampsora (see box) can have devastating effects on willows grown in plantations. Infection epidemics can lead to serious reductions in biomass production which may limit reproductive potential. We sought to assess levels of infection in wild populations of sub-arctic willow and to examine the distribution of Melampsora species among different willow species.
Identification of ectomycorrhizal fungi on plant roots and rust fungi on leaves is extremely difficult using morphology alone. We used molecular techniques to identify these fungi and thereby investigate the following three topics.
1. The relationship between S. herbacea genotype diversity and the ectomycorrhizal fungi that colonize their roots.
2. The relationship between willow species and the diversity of Melampsora rust fungi that infect their leaves.
3. The ectomycorrhizal colonization potential of soils targeted for planting of sub-arctic willows for restoration.
Melampsora rust fungi Ectomycorrhizas Melampsora rust fungi are Ectomycorrhizas are mutually beneficial relationships between plants and soil fungi that pathogenic Basidiomycetes form a sheath around root tips. The fungi obtain carbon as simple sugars from the plants that infect plants. They have and in return provide plants with nutrients absorbed from the soil through extensive complicated life cycles that networks of thread-like cells (hyphae). involve up to five different spore stages.
Melampsora rust infecting S. myrsinites Ectomycorrhizas of a Cortinarius species with S. herbacea A co l l ab o r at i v e r e s e a r c h p r o j e c t 19
Mycology What is the ectomycorrhizal fungal diversity associated with montane willow roots? Does willow genetic diversity influence ectomycorrhizal fungal diversity? Ectomycorrhizal We used DNA fingerprinting techniques to identify S. herbacea genotypes and the fungi associated ectomycorrhizal fungi associated with their roots at 90 sampling points on the ridge-top with S. herbacea at Meall Ghaordiadh.
We found 20 species of ectomycorrhizal fungi on the roots of S. herbacea at Meall Ghaordiadh (Figure 8) and an additional 13 species were collected there as fruitbodies.
By comparing the distribution of willow genotypes and fungi we found that over short distances (1 - 2 m) there was a slightly higher probability that root tips from the same willow genotype would be colonized by the same fungal species compared to root tips from different willow genotypes.
Ectomycorrhizal fungal diversity appears to be high given the unproductive nature of the habitat. This diversity may be promoted by willow genetic diversity at small spatial scales.
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Figure 8. Percentage of soil cores in which ectomycorrhizal fungi were recorded on root tips of S. herbacea (n = 90).
Montane ectomycorrhizal fungi in Scotland The total number of ectomycorrhizal fungi recorded in association with sub-arctic and montane willow species in Great Britain now stands at 105. Of these, 24 species are considered to be arctic-alpine specialists found only in this habitat. The rest can be found in lowland situations as well. During this project we discovered two new British records (Cortinarius phaeopygmaeus and Cortinarius inconspicuous) and one new Scottish record (Cortinarius subtorvus). Amanita nivalis Russula nana 20 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Mycology
Sub-arctic willows Willows are prone to infection by pathogenic rust fungi in the genus Melampsora. These can have devastating effects in uniclonal plantations of willow grown for fuel. Are natural and rust fungi populations of sub-arctic willows also affected? Are all species equally susceptible to infection? (Melampsora spp.) What is the relationship between sub-arctic willow species diversity and diversity of rust fungi?
We scored a number of willow species from one site for rust infection on a ten-point scale. We also collected rust samples from a wider set of willow species from across Scotland for molecular identification.
Willow species were found to vary in their susceptibility to infection by rust fungi in the genus Melampsora (Figure 9). S. lanata was much less susceptible than S. lapponum and S. myrsinites. Susceptibility to infection varied greatly among individuals of S. lapponum and S. myrsinites at the same site (Figure 9). This suggests that there may be a strong genetic influence on rust susceptibility.
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Figure 9. Rust infection scores recorded at Corrie Sharroch in September 2003. The boxes in the graph represent the interquartile range containing 50% of the values. The horizontal line in the boxes is the median value. The whiskers extend to the maxima and minima.
3ALIX LANATA We found that different rust DNA 3ALIX ARBUSCULA sequences tended to cluster on different 3ALIX X TETRAPLA species or groups of species of willows (Figure 10).
These data provide an important framework with which to carry forward our understanding of the taxonomy and ecology 3ALIX LAPPONUM of this group of pathogens in natural 3ALIX MYRSINITES 3ALIX MYRSINIFOLIA populations of willows. 3ALIX ARBUSCULA 3ALIX AURITA 3ALIX RETICULATA 3ALIX X OBTUSIFOLIA 3ALIX X PUNCTATA 3ALIX X TETRAPLA
3ALIX HERBACEA
Figure 10. Phylogenetic tree showing the relationship between rust DNA sequences and host identity. Each terminal point on the tree represents a unique rust DNA sequence. Lines between terminal points represent genetic distance between sequences. A co l l ab o r at i v e r e s e a r c h p r o j e c t 21
Mycology
It is known that willows form ectomycorrhizas and numerous laboratory trials have shown Ectomycorrhizal that ectomycorrhizas are beneficial to plants. So when willows are planted during restoration projects is there sufficient ectomycorrhizal inoculum available in soils or should willows be colonization inoculated before planting? potential of upland soils To assess the ectomycorrhizal colonization potential of upland soils we used a baiting technique in which uncolonized S. lapponum cuttings were planted into upland soils and dug up after 14 months to assess their ectomycorrhizal colonization. We compared the colonization potential associated with two different upland plant communities: one dominated by grass and the other by Vaccinium myrtillus.
70% of cuttings were colonized to some extent but an average of only 20% of root tips per cutting was colonized.
Only five taxa of ectomycorrhizal fungi were identified.
The two plant communities differed in the composition of inocula (Figure 11). The grass community was dominated by Laccaria proxima and an unidentified species in the order Pezizales while the Vaccinium community was dominated by Thelephora terrestris which was entirely absent from the grass community. There was no difference in willow performance in terms of leaf biomass production between the two plant communities.
These data suggest that ectomycorrhizal inocula, probably spores, are widespread in these upland soils but low in diversity (e.g., compare the five taxa of EM fungi here with Figure 8) and abundance. The differences in the species of fungi present in the two plant communities may be influenced by the vegetation itself or the edaphic conditions of the soil.
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0EZIZALES 0EZIZALES (EBELOMA SP (EBELOMA SP 4HELEPHORACEAE ,ACCARIA PROXIMA 4HELEPHORACEAE ,ACCARIA PROXIMA 4HELEPHORA TERRESTRIS 4HELEPHORA TERRESTRIS
Figure 11. Frequency (percentage of cuttings colonized) and abundance (average percentage of roots per cutting) of ectomycorrhizal fungi in grass-dominated (G) and Vaccinium-dominated (V) habitats. Error bars for abundance = standard error where n > 2. 22 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Ecology
The ecological factors leading to the current restricted distribution of many montane willow species need to be assessed if the downward trend in range and numbers is to be prevented or reversed through restoration projects. This requires information on all aspects of the life cycle from growth and reproduction to seed dispersal and seedling establishment.
A particular emphasis of this area of research was an investigation of the role of herbivory on key stages in the life-cycle of montane willows. The role of sheep and deer in preventing regeneration of woody perennials by removing seedlings has been well documented. However, large herbivores may impact willows in other ways. For example to what extent does the browsing of willow shoots limit the resources available for seed production?
One of the problems associated with removing large herbivores altogether is that a dense sward of rank grass can develop. This may reduce the number of suitable germination and establishment sites for regenerating plants. When suitable microsites are limited, then seed production and dispersal, sufficient to ensure that they are exploited, becomes crucial. Development of tall grass may also encourage growth of populations of small herbivores such as voles and slugs. To what extent is willow regeneration limited by dispersal, suitable establishment sites and seedling losses to small herbivores?
Understanding these relationships between herbivores and willow growth and reproduction may help us in developing strategies for the management of large herbivores in order to encourage regeneration.
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S. lapponum Seedling establishment A co l l ab o r at i v e r e s e a r c h p r o j e c t 23
Ecology
As willows are dioecious, successful transfer of pollen between male and female plants is Herbivory and crucial. Pollen transfer is carried out primarily by insects that are attracted by flowers. Browsing seed production by large herbivores has the potential to remove large proportions of flowering shoots. How does this affect the reproductive potential of the willows both in terms of the ability to attract insects and in the effect of the resulting resource limitation on viable fruit production?
We compared individuals of S. arbuscula that were protected from browsing in exclosures with unprotected individuals. Catkins from some individuals in each treatment were provided with supplemental pollen. Plants were scored for number of catkins produced, number of ripe and unripe fruits per catkin and number of seeds per ripe fruit.
We found that, irrespective of browsing, seed production varied significantly from year to year probably due to variation in the weather (Figure 12).
Browsing reduced the amount of seed produced per plot, particularly in years when the weather was bad (Figure 12).
Overall, hand pollination resulted in a greater proportion of ripe fruit compared to naturally pollinated controls. This effect was greater in browsed individuals than unbrowsed individuals suggesting that browsed plants are more pollen limited than unbrowsed plants (Figure 13).
The data suggest that browsing of adult plants has a negative impact on the reproductive success of S. arbuscula through a reduction in attractiveness to pollinating insects.
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4FFEMJOHFTUBCMJTINFOU 8IBUBSFUIFGBDUPST 3%%$,).' BõFDUJOHTFFEHFSNJOBUJPO BOEFBSMZTFFEMJOHTVSWJWBM S. arbuscula male S. arbuscula female 24 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Ecology
Predicting the We aimed to assess the possibility of using information on associated species and environmental conditions to predict the potential distribution of S. arbuscula at the larger potential distribution scales within a site as a way of assessing the potential for population expansion. of S. arbuscula Vegetation and environmental variables such as altitude, aspect, slope, sward height and soil depth were recorded in over 200 quadrats in the Lochan na Lairige catchment at Ben Lawers. Some quadrats were located close to or within existing S. arbuscula stands and others randomly distributed within the catchment. These data were then used to model the existing distribution and predict potential distribution.
The distribution of S. arbuscula is determined by the presence of open, moderately fertile areas on steep slopes.
If other reproductive factors are favourable, there is potential for the expansion of S. arbuscula due to the presence of suitable habitat within current stands and elsewhere in the Lochan na Lairige catchment (Figure 14).
Figure 14. Predicted distribution of S. arbuscula in one catchment at Ben Lawers. ‘Predicted’ refers to quadrats presently without S. arbuscula where conditions are suitable. ‘Willow’ refers to quadrats currently with S. arbuscula, ‘Not willow’ refers to quadrats where no willow currently grows and are unlikely to be suitable for willow establishment. Map reproduced from Ordnance Survey data by permission of Ordnance Survey, © Crown copyright. MLURI GD27237X 2005.
Seed dispersal Population expansion will only be possible where seed is dispersed away from parent plants to suitable sites for germination, establishment and growth. We aimed to quantify the amount of S. arbuscula seed being dispersed away from parent plants.
In both 2003 and 2004, over 300 sticky traps were systematically arranged within and up to 80 m away from a stand of S. arbuscula at Ben Lawers, and left in place for 2 months from mid- June to mid-August.
In spite of abundant seed production by the stand of S. arbuscula over the two years, only 11 seeds were caught, all on one trap at 20 m away from the stand in 2003. Observations suggest that much of the fertile seed remained within the bush and was washed into the ground before the silky hairs that aid dispersal were fully dried out.
Limited seed dispersal may be a factor inhibiting population expansion. A co l l ab o r at i v e r e s e a r c h p r o j e c t 25
Ecology
For seed that does manage to get dispersed, what are the factors that determine the Herbivory successful establishment of new seedlings? Is seed and early seedling mortality a problem and if so what is causing it? and seedling establishment An experiment was carried out in which small mammals were excluded from some plots but not others and in which sub-plots were either mown (clipped to 5 cm), disturbed (all vegetation removed to leave bare ground) or left as controls. Some of the disturbed plots were protected from slugs with pellets. Seed of S. arbuscula and S. lapponum was set out in the plots and their germination and survival was followed for 90 days. In a separate experiment one-year-old seedlings were planted into plots with the same treatments.
Disturbance strongly favoured germination and survival of both species. No seedlings survived in the control plots and only very few in the mown plots (Figure 15).
Slugs strongly reduced survival during the early stage of establishment (Figure 16).
In spite of an abundance of bank voles (Clethrionomys glareolus) in the area, there was no effect of small mammals during the early stage of establishment. However, we found that they caused significant damage to 40% of one-year-old seedlings.
Successful regeneration of willow seedlings was influenced by slug grazing and availability of suitable microsites with bare ground for seeds. Small mammals reduce growth of one-year-old seedlings.
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Figure 15. Effect of microsite treatment on proportion of S. arbuscula seeds surviving as seedlings (error bars = 95% confidence intervals).
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Figure 16. Effect of slugs on proportion of S. arbuscula seedlings surviving in disturbed microsites (error bars = 95% confidence intervals). 26 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Conservation implications The overarching aim of this project was to provide scientific underpinning for the conservation of sub-arctic willows. Here we seek to summarise the specific Taxonomy implications of our results for the conservation of willows.
Despite their formidable reputation for taxonomic complexity, sub-arctic willows appear to hybridize infrequently. In general, while hybridization can be viewed as a process that does occur within the genus, in the majority of cases species barriers are maintained. One exception to this is seen in patterns of hybridization between S. arbuscula and S. lapponum which occurs freely at some sites.
When considering the conservation of a given species, care should be taken to avoid planting material derived from hybridizing populations. To this end, collection of material for planting should be targeted where possible to sites at which parent species are growing alone or with few other willow species.
Hybridization is particularly likely to occur where there are serious imbalances in species abundance. Due to the fragmentary nature of montane willow distribution in Scotland, differences in relative abundance might be more pronounced between certain pairs of species than might have been the case in the past when willow scrub and the species within it were more widespread and more numerous. Further acceleration of this process should be discouraged by avoiding the creation of extreme imbalances of species abundance in restoration programmes.
Evidence suggests that flowering hybrids may encourage further hybridization between the parent species in certain circumstances, and as such, known hybrids should not be deliberately included in re-introduction programmes. If hybrid lineages are to be actively increased in frequency, this should be done ex-situ where no effect on the natural population balance would occur.
S. x obtusifolia (S. lapponum x S. aurita) A co l l ab o r at i v e r e s e a r c h p r o j e c t 27 Conservation implications Despite their obvious abilities for clonal growth and the lack of observed seedling recruitment, montane willow species like S. lanata and S. lapponum reproduce predominantly by sexual reproduction. Thus restoration programmes should focus on large numbers of Genetics different genotypes if they are to match natural populations (rather than multiple replicates of vegetatively propagated individuals).
Management to facilitate sexual reproduction (e.g., by avoiding excessive large mammal grazing) and seedling recruitment (e.g., creating suitable areas/conditions for establishment) is a necessary component of willow conservation. Willow populations are unlikely to expand via vegetative growth and dispersal.
Even small willow populations show high levels of genetic diversity and low levels of population differentiation. There is no evidence of them being affected by major genetic bottlenecks and high levels of genetic drift. Although gene flow between fragmented populations is possible, it is unlikely. More likely is the possibility that the genetic consequences of reduced population sizes and physical isolation have been limited by the longevity of willows and a limited number of generations since fragmentation.
The positive aspect of this is that even the smallest fragments represent a useful genetic resource. These patches should not be considered ‘dead-ends’ beyond hope. They represent both potentially useful sources as donors for new populations/ex situ collections, as well as reservoirs of diversity for the expansion of existing sites. In this respect, it is not too late to restore and expand populations of these species. However, those fragments with population sizes of less than 50 are of conservation concern and it would take few generations or environmental catastrophes to lose diversity or whole plants. Thus conservation action in the near to mid term is appropriate.
S. lapponum 28 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b
Conservation implications S. herbacea supports a high diversity of ectomycorrhizal fungi. Many of these are arctic/ alpine specialists found only in these habitats. S. herbacea should therefore be viewed as an Mycology important component of Scottish biodiversity.
The below-ground ECM community associated with S. herbacea was dominated by species that either do not produce fruiting structures above-ground or else produce fruiting structures that are cryptic. This highlights the importance of looking below-ground in order to properly assess fungal biodiversity.
There was some evidence to suggest that over short distances in S. herbacea beds the composition of the ECM community may be influenced by the genotype of the host. Although it is not possible to extrapolate directly to other willow species, these data tend to support the principle of maintaining genetic diversity within willow populations in order to maintain diversity of associated organisms.
Inocula of ECM fungi available to planted S. lapponum were frequent in the upland soils studied. However, the diversity of inocula and the colonization potential were low compared to other studies of montane habitats. Trials to assess the benefits of inoculum enhancement for willow establishment should be carried out.
Although there were differences between grass-dominated and Vaccinium-dominated habitats in the composition of inocula available to planted S. lapponum cuttings, no difference in willow performance was detected. Given that we do not at present understand the relative benefits of different ECM fungi to willows it is recommended that choice of planting site continues to be driven by consideration of edaphic conditions that are known to be important to willow establishment; for example soil moisture.
There was considerable variation within species in the degree of rust infection. Susceptibility to infection is likely to have a strong genetic basis and these data further support the principle of maintaining genetic diversity within willow populations, in this case to minimise the chances of catastrophic pathogen epidemics.
Willow species diversity promotes rust diversity. This tends to support the view that the diversity of organisms associated with sub-arctic willow scrub will be enhanced by having mixtures of willow species where environmental conditions allow.
Inocybe praetervisa with S. herbacea A co l l ab o r at i v e r e s e a r c h p r o j e c t 29 Conservation implications All conclusions below are for S. arbuscula. Extension of these points as generalities to other species are to be considered as tentative, but should be possible as long as species differences in ecology, and site differences, are taken into account. Ecology
The low frequencies of seed production and/or poor dispersal recorded over the course of two years suggest that regeneration from seed is likely to require collection and translocation of seed to new sites or within or close to existing sites which are already colonised.
More successful establishment from seed was recorded in areas where ground was experimentally disturbed. Scarification or mechanical ground disturbance to reveal bare soil could be used to promote establishment. This is particularly necessary in areas where a grassy or mossy mat forms the main vegetation community, and would be unnecessary where the soil or other substrate is naturally mobile, such as flushed areas on steeper slopes. It is suggested that larger (c. 1 m2) disturbed areas would reduce seedling losses due to predation as this would render a larger area unsuitable for small mammals and slugs.
Small mammals did not have a great influence on very early seedling establishment even in years of peak abundance. However, small mammal herbivory did result in greater damage of one-year-old seedlings although this did not necessarily cause seedling mortality. Managers attempting to establish willows may consider planting year-old seedlings in bare patches to reduce competition and coinciding planting with years of low vole abundance.
Exclusion of large herbivores (sheep and deer) permitted more vigorous vegetative growth of shoots, flowering and pollen and seed production. Limiting the numbers of these herbivores in areas where their impacts on the vegetation is significant would promote growth and sexual reproduction. However, this measure alone would result in little or no new establishment from seed given the other factors referred to above.
S. lapponum catkins 30 Biodiversity: ta xo n o m y, g e n e t i c s a n d e co lo g y o f s u b - a r c t i c w i l lo w s c r u b Further information Inevitably, given the nature of this publication and space constraints, the work presented here is highly summarised. If you would like to know more about this project please visit our website which has additional information, resources and photographs.
http://rbg-web2.rbge.org.uk/willow/Index.html
For additional information please contact the project co-ordinators in the first instance.
Dr. Pete Hollingsworth Dr. Glenn Iason Royal Botanic Garden Edinburgh Macaulay Institute 20A Inverleith Row Craigiebuckler Edinburgh EH3 5LR Aberdeen AB15 8QH Tel +44 131 248 2883 Tel + 44 1224 498200 Fax +44 131 248 2901 Fax +44 1224 311556 email [email protected] Email [email protected] A co l l ab o r at i v e r e s e a r c h p r o j e c t 31
Acknowledgements Scottish Montane Willow Research Group
Royal Botanic Garden Edinburgh Dr. Pete Hollingsworth Dr. Stephan Helfer Douglas McKean Alan Forrest Dr. Jeremy Milne
Macaulay Institute Dr. Glenn Iason Prof. Robin Pakeman Ros Shaw
Scottish Crop Research Institute Dr. John Brown Dr. Joanne Russell Konstantina Stamati
Biomathematics and Statistics Scotland Prof. David Elston
Scottish Agricultural College Prof. Dale Walters
University of Edinburgh Dr. Richard Ennos
University of Aberdeen Dr. Mark Young
Group members on an early reconnaissance trip to Ben Lawers
Thanks
We would like to thank the Scottish Executive Environment and Rural Affairs Department for funding this project through the Collaborative Flexible Fund (SEERAD Commission No. RBG/837/01).
Our thanks are extended to the following individuals who contributed to the project in a variety of ways: Prof. Roy Watling, Prof. Riitta Julkunen-Tiitto, David Tennant, Dr. Chris Sydes, Ken Slater, David Mardon, Andrew Warwick, Diana Gilbert, Rob Soutar, Dr. Ian Anderson, Prof. Ian Alexander, Sandie Blackie, Ros Anderson, Sven Rasmussen, Vimi Lomax, Catherine Dickison, Dr. Michelle Hollingsworth, Alex Clark, Dr. Chris Walker, Keith Watson, Lynne Torvell, David Sim, Vera Thoss, Joan Beaton, Kenny Hood, Betty Duff, Dr. Jane Squirrell.
Finally we would like to thank the numerous landowners and staff of Scottish Natural Heritage who granted us access to the willows and permission to research. This booklet summarises some of the key findings from a collaborative research project entitled “Biodiversity: taxonomy, genetics and ecology of sub- arctic willow scrub” that took place during 2002 - 2005.
The project was funded by the Scottish Executive Environment and Rural Affairs Department (SEERAD Commission No. RBG/837/01).
It involved researchers from: Royal Botanic Garden Edinburgh Macaulay Institute Scottish Crop Research Institute Biomathematics and Statistics Scotland Scottish Agricultural College University of Edinburgh University of Aberdeen.