UNDERSTANDING THE STATUS, AUTECOLOGY AND TAXONOMY OF UK BIODIVERSITY ACTION PLAN SPECIES: ARABLE BRYOPHYTES FINAL REPORT MARCH 2012 Simon Conyers Christine Conyers Ruth Laybourn Food and Environment Research Agency Sand Hutton, York, YO41 1LZ Contents 1. Introduction ................................................................................................................................ 4 2. Methods ..................................................................................................................................... 5 2.1 Field survey .......................................................................................................................... 5 2.1.1 Weissia squarrosa ............................................................................................. 5 2.1.2 Weissia multicapsularis ..................................................................................... 7 2.2 pH and texture of soil ........................................................................................................... 8 2.3 Genetic studies .................................................................................................................... 8 2.3.1 Sampling of Weissia species for genetic testing ............................................... 8 2.3.2 Molecular analysis ............................................................................................. 8 2.4 Spore germination studies ................................................................................................... 9 2.4.1 Germination from sporophyte collections .......................................................... 9 2.4.2 Germination from Soil Samples ....................................................................... 11 3. Results ..................................................................................................................................... 11 3.1 Field assessments ............................................................................................................. 11 3.1.1 Weissia squarrosa ........................................................................................... 11 3.1.2 Weissia multicapsularis ................................................................................... 17 3.2 Genetic studies .................................................................................................................. 17 3.2.1 nrITS ................................................................................................................ 17 3.2.2 trnL-trnF, rbcL, and trnH-psbA ........................................................................ 20 3.3 Spore germination studies ................................................................................................. 23 3.3.1 Effect of pH ...................................................................................................... 23 3.3.2 Effect of Fertilisers ........................................................................................... 23 3.3.3 Germination from Soil Samples ....................................................................... 28 4. Discussion ................................................................................................................................ 28 5. Further Research ..................................................................................................................... 33 Appendix 1. Personnel .................................................................................................................... 37 Appendix 2. Sequences of primers ................................................................................................ 39 Appendix 3. Bryophytes recorded during survey of over-wintered stubble fields in Lincolnshire and Gloucestershire .................................................................................................... 40 Appendix 4. Results of genetic studies with nrITS region ............................................................... 47 Appendix 5. Initial samples of Weissia for genetic testing: their location, collection date, verifier, GenBank® result using nrITS region and groupings from trnL-trnF and rbcL .................... 52 Appendix 6. Samples of Weissia from trnL-trnF analysis: their location, collection date, verifier and sample code ................................................................................................................. 55 Appendix 7. Location of soil sample, their number and results of germination and subsequent growth at 20oC and 60 r.h. ........................................................................................... 60 Appendix 8: Text for advisory leaflet for the conservation of arable mosses .................................. 61 Appendix 9. Dissemination of results .............................................................................................. 62 2 List of Tables Table 1 Results of field surveys of Staffordshire, Gloucestershire and Worcestershire ...... 13 Table 2 Results of field surveys of Surrey and Sussex ........................................................ 14 Table 3 Results of over-wintered stubble field surveys of Lincolnshire ............................... 15 Table 4 Results of over-wintered stubble field surveys of Gloucestershire ......................... 16 Table 5 Results of soil sampling at sites for W. multicapsularis .......................................... 17 Table 6 Species comparisons of nrITS sequences - Number of basepair differences (number of gaps in sequence) ..................................................................................................... 18 Table 7 Initial samples of Weissia for genetic testing: their location, GenBank® comparisons using nrITS results and the species groupings from trnL-trnF and rbcL sequences ...... 19 Table 8 Results of trnL-trnF sequence analysis for Weissia species: Number of basepair differences (number of gaps in sequence) .................................................................... 21 Table 9 Results of rbcL sequence analysis: Number of basepair differences ..................... 22 Table 10 Location of soil sample, their number and number of leafy growths produced ...... 27 Table 11 Example of the results of microsatellite analysis ................................................... 33 List of Figures Figure 1 Effect of pH on the germination of the spores of Weissia multicapsularis: average number (with SEMs) of protonemata and leafy growths produced after 11 month's growth at 20oC ............................................................................................................... 24 Figure 2 Effect of pH on the germination of the spores of Weissia squarrosa: average number (with SEMs) of protonemata and leafy growths produced after 11 month's growth at 20oC ............................................................................................................... 24 Figure 3 Effect of Ammonium nitrate on the germination of the spores of Weissia multicapsularis: average number (with SEMs) of protonemata and leafy growths produced after 8 month's growth at 20oC ...................................................................... 25 Figure 4 Effect of Ammonium nitrate on the germination of the spores of Weissia squarrosa: average number (with SEMs) of protonemata and leafy growths produced after 8 month's growth at 20oC .................................................................................................. 25 Figure 5 Effect of Sodium phosphate on the germination of the spores of Weissia multicapsularis: average number (with SEMs) of leafy growths produced after 8 month's growth at 20oC ............................................................................................................... 26 Figure 6 Effect of Sodium phosphate on the germination of the spores of Weissia squarrosa: average number (with SEMs) of protonemata and leafy growths produced after 8 month's growth at 20oC .................................................................................................. 26 3 1. INTRODUCTION The UK Biodiversity Action Plan (UK BAP), published in 1994, described the UK’s biological resources and presented a detailed plan for the protection and sustainable use of these resources. It contained objectives for conserving and enhancing species and habitats as well as promoting public awareness. In 2007 as part of the BAP review process, the UK Biodiversity Reporting and Information Group (BRIG) updated the UK BAP species and habitats, highlighting the priorities for conservation action. A UK list of priority species and habitats was published together with ‘signposts’ and subsequent actions for each of the 1150 UK BAP species (Peak Ecology Ltd, 2009). Bryophytes were an important part of this conservation list. They are also an important constituent of arable plant communities and at least 17 liverworts and hornworts and 73 mosses have been recorded on arable land (Porley, 2000). Arable land is a transient habitat subject to regular disturbance due to cultivation. Arable bryophytes have compressed life cycles that are adapted to growth within this ephemeral environment. Some species also produce vegetative propagules such as tubers, bulbils or gemmae, which also allow bryophytes to persist through stressful periods such as seasonal drought and ploughing (Porley, 2008). Two species of arable mosses, Weissia squarrosa (Nees & Hornsch.) Mull.Hal. (Spreading-leaved beardless-moss)