Current Status and Conservation Genetics of Alder Buckthorn Frangula Alnus in Northern Ireland

Current status and conservation genetics of Alder buckthorn Frangula alnus in Northern Ireland

Bradley, C., Preston, S., Provan, J., & Reid, N. (2009). Current status and conservation genetics of Alder buckthorn Frangula alnus in Northern Ireland. Northern Ireland Environment Agency.

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Download date:30. Sep. 2021

Natural Heritage Research Partnership Current status and conservation genetics of Alder buckthorn Frangula alnus in Northern Ireland Quercus Project QU08-03

Prepared for the

Northern Ireland Environment Agency (NIEA)

Drs. Caroline Bradley, Jane Preston, Jim Provan & Neil Reid, Natural Heritage Research Partnership, Quercus

This report should be cited as: Bradley, C., Preston, S.J., Provan, J. & Reid, N. (2009) Current status and conservation genetics of Alder buckthorn Frangula alnus in Northern Ireland. Report prepared by the Natural Heritage Research Partnership, Quercus for the Northern Ireland Environment Agency, Northern Ireland, UK.

Quercus project QU08-03 Quercus hosts the Natural Heritage Research Partnership between the Northern Ireland Environment Agency and Queen's University Belfast. www.quercus.ac.uk ii

Executive Summary

1. Alder buckthorn (Frangula alnus) is one of Ireland’s rarest tree species and is a Priority Species for Conservation Action in Northern Ireland and protected under the Wildlife (NI) Order (1985).

2. Surveys were carried out during summer 2007 at sites formerly occupied by the species in Northern Ireland to ascertain its current distribution and status.

3. Only one remaining population of Alder buckthorn was found in Northern Ireland at Peatlands Park, County Armagh.

4. A complete inventory of the Peatlands Park population was undertaken during summer 2008. A total of 139 individual trees were found, georeferenced, tagged and a sample of leaf and seed tissue taken for genetic analysis.

5. There was no evidence that the Peatlands Park population had ever undergone a genetic bottleneck.

6. Levels of genetic diversity were lower than those previously reported for populations elsewhere e.g. Spain, but observed levels of heterozygosity were nevertheless high suggesting that biparental inbreeding is not a major threat.

7. Allele frequencies at all eight loci examined were similar in adult trees and their seeds suggesting that seeds are a largely representative repository of genetic diversity for the next generation.

8. There was no broad-scale genetic structuring indicating a general lack of barriers to pollen dispersal with frequent long-distance cross pollination events (up to 850m). Fine- scale genetic structuring was present and probably due to limited seed dispersal.

9. High incidence of fruiting in Peatlands Park is indicative of high levels of cross- pollination and perhaps shorter generation times making the population more similar to central European populations than those of the Mediterranean or Iberia which tend to have lower incidence of fruit and higher incidence of ovule loss due to limited cross- pollination.

10. Despite low abundance and restricted distribution, the Alder buckthorn population at Peatlands Park contains a notably high level of genetic diversity. iii

11. We make two recommendations for further potential action:

i. To preserve existing high levels of genetic diversity and ensure that rare alleles are not lost from the Peatlands Park population through genetic drift, consideration should be given to artificially seeding plants. We have identified 55 individual trees that possess rare alleles (present in less than 5% of the total population). It should be relatively simple, and not particularly costly, to harvest seed from specific trees to artificially plant areas within Peatlands Park close to the parental population.

ii. Rare and isolated populations are at risk from stochastic extinction events. Consideration should be given to resurveying sites formerly occupied by Alder buckthorn pre-1980 to confirm the restricted nature of the species’ distribution. To safeguard the future survival of the species in Northern Ireland further consideration should be given to establishing populations at sites other than Peatlands Park, for example, repatriating the species on formerly occupied sites.

Contents

Executive Summary ...... iii Contents ...... v

1.0 Introduction ...... 1

2.0 Methods ...... 3 2.1 Plant surveys and sampling ...... 3 2.3 Assessment of intraspecific genetic diversity ...... 3 2.4 Statistical Analyses ...... 3

3.0 Results ...... 5 3.1 Current distribution and status of Alder buckthorn ...... 5 3.2 Levels and patterns of genetic diversity ...... 6 3.3 Levels of pollen flow between population fragments ...... 9

4.0 Discussion ...... 11

5.0 Recommendations ...... 12

6.0 Acknowledgements ...... 12

7.0 References ...... 13

Appendix 1 ...... 14

Appendix 2 ...... 15

Appendix 3 ...... 16

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1.0 Introduction

By definition, populations of rare species have low abundance and are often isolated from other similar populations making them particularly vulnerable to stochastic extinction. Rare populations usually have low levels of genetic variation due to increased genetic drift and a higher risk of inbreeding (Hedrick & Kalinowski, 2000; Cole, 2003). Genetically depauperate populations have reduced evolutionary potential, which further increases their risk of extinction (Hansson & Westerberg, 2002; Frankham, 2005). Consequently, knowledge of the levels and patterns of genetic diversity within populations of rare or threatened species is vital in the formulation of rational, sustainable conservation plans (Hedrick 2004).

Alder buckthorn (Frangula alnus) is a small deciduous shrub up to 4-5m in height, with wide-spreading branches, inconspicuous greeny-white flowers and round fruits up to 1cm in diameter which ripen in October or November. It prefers moist acid soils along riverbanks and does well on peat.

Although widespread throughout temperate Europe, it is one of Ireland’s rarest trees (Fig. 1). In Northern Ireland, the species is protected under the Wildlife (NI) Order (1985) and is a Priority Species for Conservation Action.

Fig. 1 Alder buckthorn distribution throughout Ireland. Source: NBN Gateway.

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Alder buckthorn is exclusively sexual (Medan, 1994) with hermaphroditic flowers being pollinated by a wide variety of insect species including Hymenoptera, Diptera, Coleoptera and occasional Lepidoptera (Hampe, 2005). In controlled pollination studies, almost no selfing or geitinogamy (pollination from a flower on the same plant) occurred, indicating the existence of self-incompatibility mechanisms (Medan, 1994). Field studies suggest that limited fruiting is the result of low levels of cross- pollination (Medan, 1994; Hampe, 2005).

Due the highly restricted nature of the distribution of Alder buckthorn in Northern Ireland and the importance of gene flow in fragmented populations the aims of the current project were to:

• Determine the current distribution of Alder buckthorn in NI;

• Determine levels and patterns of interspecific genetic diversity, including the potential for inbreeding;

• Identify levels of pollen dispersal between remaining population fragments, and;

• Formulate a rational conservation plan with the aim of maintaining existing genetic diversity.

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2.0 Methods

2.1 Plant surveys and sampling

A total of 46 historical records for Alder buckthorn distribution were collated from 1832 to 2004 using the Centre for Environmental Data and Recording (CEDaR) and the NBN Gateway. To assess the species current distribution those sites where the species was known to occur after 1980, that also possessed a grid reference of at least 6-figures, were surveyed during summer 2007 to ascertain the current distribution and status of the species (Appendix 1).

During summer 2008, all individual adult trees located at Peatlands Park, Co. Armagh, were numerically tagged, georeferenced and leaves were taken for genetic analyses. Seeds were also collected from between one and six berries from selected adult plants (Appendix 2).

2.2 Assessment of intraspecific genetic diversity

All individual adult plants and a selection of seeds were genotyped at eight microsatellite loci: FaA3, FaA7, FaA104, FaA110, FaB7, FaB9, FaB101 and FaB106 (Riguiero et al. 2009). PCR was carried out in a total volume of 10 μl containing 100 ng genomic DNA, 4 - 12 pmol of dye-labelled forward primer (IRD700 or IRD800), 4 - 8 pmol of tailed forward primer, 5 - 20 pmol reverse primer, 1x PCR reaction buffer,

200 μM each dNTP, 2.5 mM MgCl2 and 0.25 U GoTaq Flexi DNA polymerase (Promega). PCR conditions are as described in Riguiero et al. (2009). Genotyping was carried out on 6% polyacrylamide gels using a LI-COR 4200 IR2 gel electrophoresis system. Allele sizes were scored using a 20 bp ladder and were checked by comparison with previously sized control samples.

2.3 Statistical analyses

Levels of polymorphism measured as observed and expected heterozygosity (HO /

HE) were calculated using the POPGENE software package (V1.32; Yeh et al., 1997). 3

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To test for the occurrence of a genetic bottleneck, a Wilcoxon test for heterozygote excess was performed using the program BOTTLENECK (V1.2; Piry et al. 1999).

To investigate potential genetic sub-structuring within the population, Bayesian model-based clustering of individuals was carried out on data from adult plants using BAPS (V3.2; Corander et al., 2003). Spatial genetic structure was also examined by carrying out a spatial autocorrelation analysis in GenAlEx (Peakall and Smouse 2006). Finally, paternity analysis was carried out on seeds to determine levels of

pollen flow using CERVUS software (V3.0; Kalinowski et al. 2007).

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3.0 Results

3.1 Current distribution and status of Alder buckthorn

Surveys of sites where Alder buckthorn had been recorded historically found that the species is now restricted to a single population; Peatlands Park, Co. Armagh. The sole remaining population is highly fragmented, with five discrete clusters of plants numbering between 3 - 98 individuals each, separated by between 100 - 1,200m (Fig. 2). The total population is unlikely to number no more than ca. 140 individuals.

39-136 12-16

137-139 18-38

1-11

Fig. 2 Location of individual Alder buckthorn Frangula alnus trees ( numbered 1-139) in five discrete clusters within and outside Peatlands Park ASSI and SAC ( ).

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3.2 Levels and patterns of genetic diversity

Adult trees and seeds within Peatlands Park did not differ significantly in their levels of genetic diversity measured as expected heterozygosity; adult trees = 0.388 (range 0.008 - 0.727) and seeds = 0.354 (≤0.001 - 0.705). Allele frequencies at all eight loci studied were also similar in adult trees and seeds (Fig. 4).

1.0 Parents 0.9 Seeds 0.8

0.7

0.6

0.5 0.4 0.3 heterozygosity Expected 0.2 0.1 0.0 FaA110 FaB7 FaB101 FaA7 FaB106 FaB9 FaA3 FaA104 Mean Loci

Fig. 3 Levels of genetic diversity did not differ significantly between adult trees and their seeds measured at 8 microsatellite loci.

The population at Peatlands Park showed lower genetic diversity than three comparable populations in Spain at the same microsatellite loci (Fig. 5), however, there was no evidence of a genetic bottleneck found.

Bayesian analysis of population structure identified two groups of genetically similar individuals. However, these groups were not spatially distinct; with members of both groups occurring together (Fig. 6). Spatial autocorrelation analysis suggested that there was some fine-scale spatial genetic structuring, with the relatedness coefficient being significantly higher than zero at the smallest scale (<100 m; Fig. 7).

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100% 100% 100% 100%

80% 80% 80% 80%

60% 60% 60% 60%

40% 40% 40% 40%

Allele frequency 20% 20% 20% 20%

0% 0% 0% 0% Parents Seeds Parents Seeds Parents Seeds Parents Seeds FaA3 FaA7 FaA104 FaA110

100% 100% 100% 100% 90% 80% 80% 80% 80% 70% 60% 60% 60% 60% 50% 40% 40% 40% 40% 30% 20% 20%

Allele frequency Allele 20% 20% 10% 0% 0% 0% 0% Parents Seeds Parents Seeds Parents Seeds Parents Seeds FaB7 FaB9 FaB101 FaB106

Fig. 4 Allele frequencies at eight microsatellite loci did not differ between adult trees and their seeds within Peatlands Park.

0.70

0.60

0.50

0.40

0.30

0.20

Expected heterozygosity Expected

0.10

0.00 Peatlands Park Aljibe Medio Puerto Oscuro Mean

Spanish populations

Fig 5 The Peatlands Park population of Alder buckthorn had lower levels of genetic diversity (measured from 8 microsatellite loci) than three similar populations in Spain. 7

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Fig 6 Voronoi tessellations delineated by Bayesian analysis suggest that two genetic clusters exist within the Peatlands Park population (red and green) but these are not spatially distinct.

p≤0.05

Fig 7 Spatial organisation of individual relatedness (r) was significant only at distances less than 100m.

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3.3 Levels of pollen flow between population fragments

Paternity analysis identified pollen donors for 30 of the 275 seeds (ca. 10.9%) genotyped at more than 4 loci (Table 1). A total of 14 out of 30 pollination events (ca. 46.6%) were inter-fragment pollination events (Fig. 8). The greatest pollination distance was 845m and occurred between trees #1 and #16.

Table 1 Identification of pollen donors (putative fathers) for selected seeds. Bold text indicates inter-fragment pollination events.

Seed ID Father ID Distance (m) 1-3-2 16 845 2-2-1 122 836 22-1-2 21 2 25-1-2 79 241 29-1-3 25 10 29-2-1 119 302 31-2-2 60 178 33-1-3 61 179 34-1-1 50 153 35-1-2 54 163 35-2-1 13 145 38-1-3 50 158 40-4-1 25 118 45-3-1 62 48 47-2-3 123 135 49-1-3 71 67 52-1-1 50 6 52-1-2 1 760 55-3-1 50 9 69-1-2 14 312 77-2-1 65 26 78-1-2 40 137 78-3-1 40 137 82-1-2 54 137 104-1-3 50 245 106-2-1 121 107 121-1-1 50 133 123-2-1 80 32 131-1-1 80 19 133-1-2 3 791 Mean 214

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Fig. 8 The spatial relationship of inter-fragment pollination events shown in Table 1.

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4.0 Discussion

Anecdotal reports suggested that the Peatlands Park population of Alder buckthorn was founded by a relatively small number of individuals e.g. ≤6 (Georgina Thurgate, pers. comms.). However, the current study found no evidence that the population has ever undergone a genetic bottleneck.

Levels of genetic diversity were lower than those previously reported for populations elsewhere e.g. Spain (Riguiero et al., 2009), but there was no evidence of excessive levels of inbreeding. Furthermore, allele frequencies at all loci examined were similar in adult trees and seeds suggesting that the seeds were a representative repository of genetic diversity for the next generation. Consequently, for a population of its size and limited distribution the Alder buckthorn population at Peatlands Park has a notably high level of genetic diversity.

The absence of broad-scale spatial structuring of genetic variation indicates a general lack of barriers to pollen dispersal, most probably habitat type and structure (e.g. woodland interspersed with bog). The spatial autocorrelation analysis suggested some fine-scale genetic structure, but the extensive pollen flow indicated by the paternity analysis means that this is probably due to limited seed dispersal.

Spanish populations of Alder buckthorn suffer from low levels of fruiting and substantial ovule loss. Medan (1994) suggested that less than 3% of open-pollinated flowers set fruit, while Hampe (2005) suggested that the majority of ovule losses were due to limited cross-pollen and extensive geitonogamy (pollination from a flower on the same plant) with the majority of seed output being limited to a few mature trees. In contrast, the high incidence of fruiting at Peatlands Park suggests high levels of cross-pollination and perhaps shorter generation times. These characteristics are likely to make the Peatlands Park population more similar to central European populations than those of the Mediterranean or Iberia (Hampe & Bairlein, 2000).

The current study suggests that the Peatlands park population of Alder buckthorn is reasonably ‘healthy’ in genetic terms. 11

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5.0 Recommendations

We make two recommendations for further potential action:

i. To preserve existing high levels of genetic diversity and ensure that rare alleles are not lost from the Peatlands Park population through genetic drift, consideration should be given to artificially seeding plants. Appendix 2 identifies 55 individual trees that possess rare alleles (present in less than 5% of the total population). All trees were tagged, georeferenced and the majority were observed to be in fruit during the 2008 survey. It should be relatively simple, and not particularly costly, to harvest seed from specific trees to artificially plant areas within Peatlands Park close to the parental population.

6.0 Acknowledgements

This project was funded by the Northern Ireland Environment Agency (NIEA) through the Natural Heritage Research Partnership with Quercus, Queen’s University Belfast. Thnaks to Dr. Peter McEvoy for conducted field surveys during summer 2007 and Dr. Georgina Thurgate, Dr. Kathryn Turner and Tommy McDermott for assisting leaf and seed sampling during summer 2008.

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7.0 References

Cole TC (2003) Genetic variation in rare and common plants. Annual Review of Ecology, Evolution and Systematics 34, 213-237.

Corander J, Waldmann P, Sillanpää MJ (2003) Bayesian analysis of genetic differentiation between populations. Genetics 163, 367-374.

Frankham R (2005) Genetics and extinction. Biological Conservation 126, 131-140.

Hampe A (2005) Fecundity limits in Frangula alnus (Rhamnaceae) relict populations at the species’ southern range margin. Oecologia 143, 377-386.

Hampe A, Bairlein F (2000) Modified dispersal-related traits in disjunct populations of bird-dispersed Frangula alnus (Rhamnaceae): a result of its Quaternary distribution shifts? Ecography 23, 603-613.

Hampe A, Arroyo J, Jordano P, Petit RJ (2003) Rangewide phylogeography of a bird-dispersed Eurasian shrub: contrasting Mediterranean and temperate glacial refugia. Molecular Ecology 12, 3415-3426.

Hansson B, Westerberg I (2002) On the correlation between heterozygosity and fitness in natural populations. Molecular Ecology 11, 2467-2474.

Hedrick PW (2004) Recent developments in conservation genetics. Forest Ecology and Management 197, 3-19

Hedrick PW, Kalinowski ST (2000) Inbreeding depression and conservation biology. Annual Review of Ecology and Systematics 31, 139-162.

Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16, 1099-1106.

Medan D (1994) Reproductive biology of Frangula alnus (Rhamnaceae) in southern Spain. Plant Systematics and Evolution 193, 173-186.

Peakall R, Smouse PE (2006) GenAlEx 6: Genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6, 288-295.

Piry A, Luikart G, Cornuet JM (1999) BOTTLENECK: a computer program for detecting recent reductions in the effective population size using allele frequency data. Journal of Heredity 90, 502- 503.

Provan J, Beatty GE, Hunter AM, McDonald RA, McLaughlin E, Preston SJ, Wilson S (2008) Restricted gene flow in fragmented populations of a wind-pollinated tree. Conservation Genetics 9, 1521-1532.

Yeh FC, Yang R-C, Boyle T et al. (1997) POPGENE, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada. http://www.ualberta.ca/~fyeh/.

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Appendix 1 The location of Alder buckthorn (Frangula alnus) records post-1980 from the CEDaR and NBN Gateway that present a minimum of 6-figure grid references.

Year County Location Irish grid Description Recorder(s) 1987 Armagh Peatlands Park H 890,604 Stanfield, Mr K. 1989 Down Drumawhey Bog J 547,758 Strangford Lough Rippey, Mr I. 1999 Armagh Peatlands Park H 905,614 Annagarriff National Nature Reserve Rippey, Mr I. 1999 Armagh Peatlands Park H 905,614 Annagarriff National Nature Reserve Irvine, Mr R. 2000 Armagh Peatlands Park H 899,614 McNeill, Mr I. 2004 Armagh Carganamuck Quarry H 8781,4958 Woodland to the south Birch, Mr R. 2004 Down Edenderry House J 3242,6725 In gardens of house Graham, Ms C. 2004 Armagh Tynan Abbey H 7563,4200 West of Abbey in woodland Graham, Ms C.

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Appendix 2 A list of genotyped seeds including total sample sizes from each parent plant.

Plant ID No. of No. of Total no. Plant ID No. of No. of Total no. berries seeds of seeds berries seeds of seeds 1b 3 2,3,3 8 71 1 2 2 2 2 3,2 5 72 2 2,3 5 3 - - - 73 2 3,2 5 4 2 2,3 5 74 - - - 5 - - - 75 - - - 6 2 2,2 4 76 3 3,2,2 7 7 2 3,2 5 77 2 2,2 4 8 - - - 78 3 2,2,3 7 9 - - - 79 - - - 10 - - - 80 - - - 11 - - - 81 3 4,2,3 9 12 3 2,2,2 6 82 3 2,3,3 8 13 2 3,2 5 83 2 2,2 4 14 3 2,3,2 7 84 - - - 15 - - - 85 - - - 16 3 2,2,2 6 86 1 2 2 18 2 2,3 5 87 2 3,3 6 19 2 3,2 5 88 - - - 20 2 3,3 6 89 - - - 21 3 2,2,3 7 90 - - - 22 2 2,3 5 91 - - - 23 - - - 92 - - - 24 - - - 93 - - - 25 1 2 2 94 - - - 26 2 3,3 6 95 - - - 27 2 2,2 4 96 - - - 28 2 3,3 6 97 - - - 29 2 3,3 6 98 - - - 30 - - - 99 - - - 31 2 2,3 5 100 2 2,2 4 32 - - - 101 - - - 33 3 3,2,2 7 102 1 2 2 34 3 3,3,2 8 103 - - - 35 3 3,2,2 7 104 1 3 3 36 2 2,3 5 105 - - - 37 - - - 106 2 2,2 4 38 3 3,2,2 7 107 - - - 39 2 3,3 6 108 2 2,2 4 40 4 2,3,2,2 9 109 1 3 3 41 4 3,3,3,3 12 110 - - - 42 3 2,3,2 7 111 - - - 43 2 2,2 4 112 2 2,2 4 44 2 3,3 6 113 1 3 3 45 3 2,3,3 8 114 1 2 2 46 3 3,2,3 8 115 2 2,2 4 47 3 2,3,2 7 116 - - - 48 3 2,2,2 6 117 - - - 49 2 3,2 5 118 1 2 2 50 6 1,3,2,2,2,3 13 119 1 1 1 51 3 2,2,2 6 120 - - - 52 3 2,2,3 7 121 2 3,2 5 53 4 3,3,3,3 12 122 2 2,2 4 54 - - - 123 2 2,2 4 55 3 3,2,2 7 124 2 2,3 5 56 - - - 125 1 2 2 57 2 2,2 4 126 1 3 3 58 1 2 2 127 - - - 59 3 2,3,3 8 128 - - - 60 - - - 129 1 3 3 61 - - - 130 1 2 2 62 1 3 3 131 2 3,2 5 63 - - - 132 - - - 64 - - - 133 1 2 2 65 - - - 134 - - - 66 1 3 3 135 1 3 3 67 - - - 136 - - - 68 - - - 137 3 2,3,3 8 69 2 2,2 4 138 - - - 70 2 2,2 4 139 - - - Mean 2.17 2.37 5.23 15

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Appendix 3 A list of 55 individual trees possessing rare alleles (present in less than 5% of the population). Individuals in bold text possess rare alleles at multiple loci.

Plant ID Irish grid ref Loci FaA3 FaA7 FaA104 FaA110 FaB7 FaB9 FaB101 FaB106 1 H 89768,60593 9 2 H 89767,60596 9 3 H 89767,60596 9 8 H 89765,60602 99 9 H 89766,60604 9 19 H 89622,61292 99 9 22 H 89617,61289 9 25 H 89625,61290 9 27 H 89623,61289 9 33 H 89629,61280 9 9 9 34 H 89630,61282 9 35 H 89630,61274 9 36 H 89634,61273 9 40 H 89740,61317 9 42 H 89757,61330 9 45 H 89755,61335 9 48 H 89770,61344 99 49 H 89764,61348 9 50 H 89767,61349 9 52 H 89772,61353 9 58 H 89783,61361 9 59 H 89782,61365 9 60 H 89781,61364 99 9 61 H 89783,61371 9 9 62 H 89788,61370 9 9 76 H 89821,61409 9 77 H 89816,61395 9 79 H 89823,61427 9 80 H 89842,61427 9 9 81 H 89859,61443 9 83 H 89893,61467 9 9 85 H 89897,61484 9 9 87 H 89910,61489 9 90 H 89904,61494 9 91 H 89904,61494 9 9 96 H 89910,61495 9 100 H 89909,61495 9 106 H 89950,61503 99 108 H 89900,61460 9 114 H 89894,61451 9 115 H 89887,61453 9 116 H 89891,61449 99 119 H 89883,61430 9 121 H 89875,61427 9 122 H 89875,61425 9 123 H 89874,61422 9 126 H 89864,61431 9 129 H 89847,61415 99 130 H 89844,61410 99 134 H 89809,61357 9 9 135 H 89802,61357 9 136 H 89757,61312 9 137 H 90818,61324 9 138 H 90819,61322 99 9 9 139 H 90817,61320 99 99