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Fungal communities of West African ectomycorrhizal woodlands

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(1)Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1987. Fungal communities of West African ectomycorrhizal woodlands BRENDAN R. FURNEAUX. ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2020. ISSN 1651-6214 ISBN 978-91-513-1064-0 urn:nbn:se:uu:diva-425732.

(2) Dissertation presented at Uppsala University to be publicly examined in Ekmansalen, Evolutionary Biology Center (EBC), Norbyvägen 14, Uppsala, Friday, 15 January 2021 at 14:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Assistant Professor Matthew Smith (University of Florida). Abstract Furneaux, B. R. 2020. Fungal communities of West African ectomycorrhizal woodlands. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1987. 58 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1064-0. Ectomycorrhizal symbiosis is a widespread mutualist relationship between fungi and plants, often trees. It is most well-known in temperate and boreal forests, but also occurs in a variety of tropical ecosystems, including Sudanian savanna woodlands and gallery forests in West Africa. In addition to their important role in nutrient cycling, many ectomycorrhizal fungi also produce edible mushrooms as their fruit-bodies. In this thesis, I explore spatial patterns of aboveground and below-ground diversity in West African ectomycorrhizal communities, as well as the use of wild edible fungi by the local human population. In Papers I and II, I used soil DNA metabarcoding to investigate fungal communities in their vegetative state. Paper I used measurements of the scale of spatial autocorrelation within the fungal community in Sudanian woodlands in Benin as a test to compare different high-throughput DNA sequencing strategies, including short (≈350 bp) amplicon barcoding using the Illumina, Ion Torrent, and PacBio sequencing platforms, as well as long (≈1550 bp) amplicon barcoding using PacBio. There were some differences in species richness and community composition recovered by the two amplicon lengths, attributable to known primer biases for the short amplicons, but these did not lead to different ecological results. Additionally, Paper I introduced new software packages for analysis of long-amplicon metabarcoding data and integrating phylogenetic information into sequence-based taxonomic identification. In Paper II we sampled both Sudanian woodland and gallery forest sites in five countries across West Africa using long-amplicon metabarcoding with PacBio. We found significant differences in fungal community composition between Sudanian woodland and gallery forest sites, but not between different tree species within each vegetation type. Papers III and IV focused above-ground to study the natural production and human use of the mushrooms themselves. In Paper III we exhaustively collected mushrooms from nine Sudanian woodland plots in Benin during three consecutive rainy seasons. We measured the total biomass produced of each morphospecies, as well as environmental variables related to microclimate, host tree availability, and soil chemistry. Mushroom production and diversity were negatively correlated with soil nitrogen levels, and positively correlated with soil phosphorus levels. Although there were no clear differences in the fungal communities associated with the four host trees present in our plots, greater host tree diversity was associated with greater fungal diversity and productivity. Finally, Paper IV combined interviews with local people from four ethnic groups in five villages near the study sites from Paper III about their knowledge and preferences for edible mushrooms with DNA barcoding of specimens. Knowledge and preferences for different mushrooms varied between ethnic groups, but people living in a village where their ethnic group is a minority tended to absorb knowledge about mushrooms from the majority group in the village. Women over age 35 were the most knowledgeable about mushrooms, but the degree of gender difference varied between groups. We compiled a list of the most choice edible mushrooms in the area, which can inform the possibility for commercial trade in wild mushrooms to supplement the income of rural people and encourage sustainable forest management. Keywords: ectomycorrhizal fungi, West Africa, metabarcoding, Sudanian woodlands, community ecology, ethnomycology, edible mushrooms, fungi Brendan R. Furneaux, Department of Organismal Biology, Systematic Biology, Norbyv. 18 D, Uppsala University, SE-75236 Uppsala, Sweden. © Brendan R. Furneaux 2020 ISSN 1651-6214 ISBN 978-91-513-1064-0 urn:nbn:se:uu:diva-425732 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-425732).

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(26) short amplicon  300bp ITS1. SSU. gITS7 ITS1. 5.8S. ITS4 ITS2 LSU1. D1. LSU LSU2. D2. LR5. D3 LSU3 LSU4. long amplicon  1550bp 6B;m`2 jX S`iBH KT Q7 `.L b?QrBM; i?2 8X3a `.L- T`iBH aal M/ Gal `.L- M/ BMi2`MHHv i`Mb+`B#2/ bT+2` UAhaV `2;BQMbX .RĜj `2T`2b2Mi i?2 }`bi i?`22 p`B#H2 `2;BQMb BM Gal- r?BH2 GalRĜ9 `2T`2b2Mi i?2 +QMb2`p2/ `2;BQMbX S`BK2` bBi2b mb2/ BM i?2 BM+Hm/2/ bim/B2b `2 BM/B+i2/ BM `2/ U7Q`r`/ T`BK2`bV M/ #Hm2 U`2p2`b2 T`BK2`bV- M/ i?2 `2bmHiBM; KTHB+QMb `2 b?QrM rBi? ;`22M #`+2bX _2bmHib #b2/ QM HQM; M/ b?Q`i KTHB+QMb r2`2 +QKT`2/ BM ST2` Ac QMHv HQM; KTHB+QMb r2`2 b2[m2M+2/ BM ST2` AAX 6B;m`2 `2T`Q/m+2/ 7`QK ST2` AX. bKHH bm#mMBib UGal- aalV Q7 i?2 `B#QbQKH QT2`QM U6B;m`2 jV- Bb mb2/ 7Q` #`+Q/BM; Bb i?i i?2 `B#QbQKH QT2`QM Bb `2T2i2/ KMv iBK2b BM 2m@ F`vQiB+ ;2MQK2b Ua+?Q+? 2i HX kyRkVX h?Bb K2Mb i?i Bi KF2b mT  H`;2` 7`+iBQM Q7 i?2 .L i?M Mv BM/BpB/mH T`Qi2BM +Q/BM; ;2M2X h?2 2tBbi2M+2 Q7 KmHiBTH2 +QTB2b HbQ H2/b iQ +QM+2`i2/ 2pQHmiBQM i?`Qm;? ;2M2 +QMp2`bBQM- r?B+? QT2`i2b iQ ?QKQ;2MBx2 Aha rBi?BM b2tmHHv `2T`Q/m+@ BM; TQTmHiBQMb- /2bTBi2 Bib ;2M2`HHv ?B;? `i2 Q7 KmiiBQM U"`QrM 2i HX RNdkVX >Qr2p2`- p`BiBQM BM +QTv MmK#2` #2ir22M bT2+B2b M/ 2p2M BM@ /BpB/mHb BMi`Q/m+2b MQi?2` bQm`+2 Q7 2``Q` iQ i?2 `2HiBQMb?BT #2ir22M +iBp2 #BQKbb M/ b2[m2M+2 #mM/M+2X "H/`BM 2i HX UkyRjV 7QmM/ i?i p`BiBQM BM Aha b2[m2M+2 #mM/M+2 T2` #BQKbb- bb2bb2/ mbBM; [mMiBiiBp2 S*_ U[S*_V QM bKTH2b 7`QK 7`mBi#Q/B2b- rb ;`2i2` i?M i?2 p`BiBQM BM 2`;Qbi2`QH-  +QKTQM2Mi Q7 i?2 7mM;H +2HH rHH r?B+? Bb mb2/ b  MQM@bT2+B2b@bT2+B}+ K`F2` 7Q` 7mM;H #BQKbb Ua2Bix RNdNVX AM //BiBQM iQ i?2b2 #BQHQ;B+H bQm`+2b Q7 #Bb- //BiBQMH i2+?MQHQ;B+H #Bb2b `2 BMi`Q/m+2/ /m`BM; S*_ M/ b2[m2M+BM;X P`;MBbKb rBi? H`;2 KBbKi+?2b iQ i?2 S*_ T`BK2`b miBHBx2/ Kv #2 +QKTH2i2Hv #b2Mi 7`QK b2[m2M+BM; `2bmHib- #mi Q`;MBbKb rBi? bKHH KBbKi+?2b Kv #2 BM2{@ +B2MiHv KTHB}2/ /m2 iQ +QKT2iBiBQM 7Q` T`BK2`b- H2/BM; iQ mM/2``2T`2@ b2MiiBQM UEM;r kyyjc EHH2 2i HX kyR9VX q?2M KmHiBTH2 T`BK2`b `2 mb2/ BM +QK#BMiBQM- iQ pQB/ bm+? KBbKi+?2b- T`BK2`b r?B+? }i KQbi bT2+B2b BM i?2 bKTH2 Kv #2+QK2 /2TH2i2/ 2`Hv BM i?2 S*_- H2/BM; bT2+B2b r?B+? Ki+? H2bb +QKKQM T`BK2`b iQ BMbi2/ #2 Qp2``2T`2b2Mi2/ UGBM/?H 2i HX kyRjVX "Qi? S*_ M/ b2[m2M+BM; bi2Tb +M HbQ #2 #Bb2/ BM 7pQ` Q7 b?Q`i2` b2[m2M+2bX h?Bb +M #2 K2HBQ`i2/ BM S*_ #v mbBM; HQM; 2ti2MbBQM iBK2b- M/ rb 7QmM/ iQ #2  KBMQ` 2z2+i #2ir22M /Bz2`@ 2Mi b2[m2M+BM; i2+?MQHQ;B2b BM ST2` AX 6Q` bQ@+HH2/ dzb?Q`i@`2/Ǵ >ha b2[m2M+BM; i2+?MQHQ;B2b- KTHB+QMb HQM;2` i?M #Qmi 9yy #T `2 bi`QM;Hv kj.

(27) bmT`2bb2/ U*biƢQ 2i HX kykyVX Hi?Qm;? i?Bb +QmH/ H2/ iQ bm#biMiBH #Bb- bQ`;MBbKb rBi? BMb2`iBQMb BM i?2 `2;BQM Q7 BMi2`2bi #2BM; +QKTH2i2Hv #b2Mi 7`QK b2[m2M+BM; `2bmHib- i?Bb Kv #2 Q7 KBMQ` BKTQ`iM+2 BM T`+@ iB+2X LQ bm+? Q`;MBbKb r2`2 /2i2+i2/ BM  Mim`H bvbi2K BM ST2` A/2bTBi2 T`HH2H b2[m2M+BM; rBi?  dzHQM;@`2/Ǵ i2+?MQHQ;vX L2r2` b2[m2M+BM; i2+?MQHQ;B2b 7`QK S+B}+ "BQb+B2M+2b US+"BQV M/ Pt@ 7Q`/ LMQTQ`2 +M T`Q/m+2 Km+? HQM;2` b2[m2M+2b- rBi? H2bb H2M;i? #Bb i?M 2`HB2` ?B;? i?`Qm;?Tmi b2[m2M+BM; i2+?MQHQ;B2bX h?2b2 ?p2 #22M TTHB2/ BM K2i#`+Q/BM; iQ b2[m2M+2 HQM;2` `2;BQMb- BM+Hm/BM; TQ`iBQMb Q7 aal M/fQ` Gal BM //BiBQM iQ i?2 7mHH Aha Uh2/2`bQQ 2i HX kyR3V Q` 2p2M 7mHH `2T2ib Q7 i?2 `B#QbQKH QT2`QM Uqm`x#+?2` 2i HX kyRNVHHQrBM; i?2 mb2 Q7 T`BK2`b rBi? KQ`2 +QKTH2i2 T?vHQ;2M2iB+ +Qp2`;2b r2HH b BKT`Qp2/ TH+2K2Mi Q7 MQp2H it rBi?Qmi +HQb2 `2HiBp2b BM b2[m2M+2 /i#b2bX >Qr2p2`- i?2b2 K2i?Q/b `2 +m``2MiHv Km+? KQ`2 2tT2MbBp2 T2` b2[m2M+2/ #b2 i?M AQM hQ``2Mi M/ 2bT2+BHHv AHHmKBMbQ i?2B` #2M2}ib Kmbi #2 #HM+2/ ;BMbi `2/m+2/ b2[m2M+BM; /2Ti? M/ 72r2` bKTH2b T2` bim/v UE2MM2/v 2i HX kyR3VX KTHB}+iBQM Q7 i?2b2 HQM; `2;BQMb r?B+? BM+Hm/2 Hi2`MiBM; +QMb2`p2/ M/ p`B#H2 `2;BQMb HbQ BM+`2b2b i?2 `BbF Q7 +?BK2` 7Q`KiBQM /m`BM; S*_- Hi?Qm;? i?Bb +M #2 +QMi`QHH2/ iQ bQK2 2ti2Mi #v mbBM; HQM; 2ti2MbBQM iBK2b M/ HQr +v+H2 MmK#2`b UEHH2 2i HX kyR9VX aQBH .L K2i#`+Q/BM; rBi? S+"BQ Bb mb2/ BM ST2` A M/ ST2` AA iQ K2bm`2 i?2 /Bp2`bBiv M/ bTiBH p`BiBQM BM i?2 #2HQr;QmM/ 7mM;H +QK@ KmMBivX //BiBQMHHv- ST2` A i2bib /Bz2`2Mi b2[m2M+BM; bi`i2;B2b 7Q` #`+Q/BM;- BM+Hm/BM; Hi2`MiBp2 >ha i2+?MQHQ;B2b M/ /Bz2`2Mi +?QB+2b Q7 KTHB+QM H2M;i? U6B;m`2 jV- M/ }M/b i?i i?2v T`Q/m+2 2[mBpH2Mi 2+QHQ;B+H `2bmHibX ST2` A HbQ T`2b2Mib M2r H;Q`Bi?Kb BM i?2 T+F@ ;2b Galt M/ ix` r?B+? //`2bb i?2 /2MQBbBM; Q7 HQM; KTHB+QMb- b /2b+`B#2/ BM i?2 bmTTH2K2Mi`v K2i?Q/b Q7 i?i TT2`X. htQMQKB+ B/2MiB}+iBQM MQi?2` i?`2/ r?B+? `mMb i?`Qm;? Kv rQ`F BM i?Bb i?2bBb Bb itQMQKB+ B/2MiB}+iBQMX ++m`i2 itQMQKB+ B/2MiB}+iBQM Bb +H2`Hv Q7 T`BK`v BKTQ`iM+2 BM bim/B2b 7Q+mbBM; QM /Bp2`bBiv- #mi Bi HbQ ;Bp2b BKTQ`iMi +Hm2b iQ //`2bb 2+QHQ;B+H 7mM+iBQMX JMv 7mM+iBQMH i`Bib `2 b?`2/ #2ir22M +HQb2Hv `2Hi2/ bT2+B2b- M/ /i#b2b bm+? b 6lL:mBH/ UL;mv2M 2i HX kyReV M/ 6mM6mM UwMM2 2i HX kykyV +QKTBH2 FMQrM 7mM+iBQMH i`Bi /i 7Q` itQMQKB+ ;`QmTb- ;2M2`HHv 7`QK i?2 bT2+B2b iQ i?2 7KBHv H2p2HX P7 T`iB+mH` `2H2pM+2 iQ Kv `2b2`+?- i?2 KQ`2 i?M 3y 7mM;H HBM2;2b r?B+? ?p2 /2p2HQT2/ i?2 +T+Biv iQ 7Q`K 1*J ivTB+HHv +Q``2bTQM/ k9.

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(36) amKK`v Q7 aim/v AAA aTiBH /`Bp2`b Q7 1*J +QKKmMBiv +QKTQbBiBQM M/ 7`mBi#Q/v T`Q/m+iBQM BM q2bi 7`B+M rQQ/HM/b 6m`M2mt- "X- >Qm/MQM- _X- ś;MQM- >X- "QMB- aX- *Q/DB- CX 1XGQm`Qm- :X- "?`K- JX- apM?QHK- X- _QbHBM;- X- uQ`Qm- LXaX- _v@ #2`;- JX h?Bb bim/v BMp2biB;i2b i?2 1*J +QKKmMBiB2b Q7 PmûKû amTû`B2m` 7`QK i?2 T2`bT2+iBp2 Q7 7`mBi#Q/v +QKKmMBiv +QKTQbBiBQM M/ T`Q/m+iBQM Qp2` i?`22 v2`b Q7 BMi2MbBp2 bKTHBM;- M/ `2Hi2b i?Bb iQ  rB/2 `M;2 Q7 2MpB`QMK2MiH p`B#H2bX 6`mBi#Q/B2b r2`2 2t?mbiBp2Hv +QHH2+i2/ #Br22FHv Qp2` i?`22 bm++2bbBp2 Kmb?`QQK b2bQMb UCmM2@P+iQ#2`V 7`QK MBM2 8y Ɠ 8y K THQib /BpB/2/ BM iQ Ry Ɠ Ry K bm#THQibX aT2+BK2Mb r2`2 ;`QmT2/ BMiQ KQ`T?QbT2+B2b M/ r2B;?2/ #v bm#THQi M/ KQ`T?QbT2+B2b- rBi? QM2 pQm+?2` T2` KQ`@ T?QbT2+B2b T`2b2`p2/ QM 2+? bKTHBM; /vX //BiBQMHHv  rB/2 `M;2 Q7 2MpB`QMK2MiH /i r2`2 +QHH2+i2/ 7`QK i?2 MBM2 THQib- BM+Hm/BM; KB@ +`Q+HBKi2 U;`QmM/ +Qp2`- +MQTv +Qp2`- bQBH KQBbim`2- bQBH i2KT2`im`2VbQBH +?2KBbi`v UMBi`Q;2M- T?QbT?Q`mb- Q`;MB+ +`#QM- +H+BmK- T>- ;`M@ mHQK2i`vV M/ ?Qbi pBH#BHBiv UT`2b2M+2 M/ bBx2b Q7 /Bz2`2Mi 1*J i`22 bT2+B2bVX "2+mb2 Q7 BM+QMbBbi2Mi bT2+B2b@H2p2H BM/2MiB}+iBQM Q7 bT2+BK2Mb- A M@ Hvx2/ 7mM;H +QKKmMBiv +QKTQbBiBQM i i?2 7KBHv H2p2H- `Qm;?Hv +Q``2@ bTQM/BM; iQ 1*J HBM2;2b b2Mbm h2/2`bQQ 2i HX UkyRyVX h?2 ?B;?2bi T`Q@ /m+BM; ;`QmTb r2`2 a+H2`Q/2`Ki+22- _mbbmH+22- M/ KMBi+22HH rBi? KQ`2 i?M k F; /`v Kbb T2` ?2+i`2 T2` v2`- M/ "QH2i+22rBi? yX9 F; /`v Kbb T2` ?2+i`2 T2` v2`X Hi?Qm;? HH Q7 i?2b2 ;`QmTb r2`2 HbQ /2i2+i2/ BM bQBH 7`QK PmûKû amTû`B2m` ST2` A M/ Qi?2` rQQ/@ HM/ bBi2b ST2` AA- QMHv _mbbmH+22 rb bBKBH`Hv /QKBMMi BM `2/ #mM/M+2X KMBi+22 M/ a+H2`Q/2`Ki+22 iQ;2i?2` `2T`2b2Mi2/ H2bb i?M R8W Q7 `2/b 7`QK rQQ/HM/ bBi2b BM ST2` AA- M/ "QH2i+22 H2bb i?M RWX JB+`Q+HBKi2 rb i?2 bi`QM;2bi +Q``2Hi2 Q7 p`BiBQM BM +QKKmMBiv +QK@ TQbBiBQM Qmi Q7 i?2 K2bm`2/ 2MpB`QMK2MiH T`K2i2`b- 2tTHBMBM; RRW Q7 bTiBH p`BiBQMX aQBH i2KT2`im`2- bQBH KQBbim`2- M/ H27 +Qp2` r2`2 i?2 KQbi BKTQ`iMi +QKTQM2Mib Q7 KB+`Q+HBKi2 BM 2tTHBMBM; +QKKmMBiv +QKTQbBiBQMX *MQTv +Qp2` M/ bQBH KQBbim`2- b r2HH b bQK2 H27 +Qp2`r2`2 TQbBiBp2Hv +Q``2Hi2/ rBi? 7mM;H /Bp2`bBiv M/ `B+?M2bbX >Qr2p2`+MQTv +Qp2` rb MQi bbQ+Bi2/ rBi? ;`2i2` iQiH 7`mBi#Q/v T`Q/m+iBQMM/ rb M2;iBp2Hv +Q``2Hi2/ rBi? T`Q/m+iBQM Q7 2/B#H2 bT2+B2bX "Qi? iQiH T`Q/m+iBQM M/ T`Q/m+iBQM Q7 2/B#H2 bT2+B2b r2`2 TQbBiBp2Hv +Q``2@ jj.

(37) Hi2/ rBi? #`2 bQBH- TQbbB#Hv `2~2+iBM;  T`272`2M+2 7Q` 7`mBiBM; rv 7`QK ;`QmM/ +Qp2` r?B+? KB;?i BMi2``mTi i?2 /BbT2`bH Q7 bTQ`2bX _B+?M2bb- /Bp2`bBiv M/ T`Q/m+iBpBiv Q7 7`mBi#Q/B2b r2`2 TQbBiBp2Hv +Q``2@ Hi2/ rBi? `B+?M2bb Q7 1*J i`22 7KBHB2b- #mi QMHv  bKHH 7`+iBQM URXeWV Q7 p`BiBQM BM 7`mBi#Q/v +QKKmMBiv +QKTQbBiBQM rb 2tTHBM2/ #v i?2 pBH#BHBiv Q7  T`iB+mH` 1*J i`22 bT2+B2b rBi?BM 8 K Q7  T`iB+mH` bm#THQiX h?2b2 TT`2MiHv +QMi`/B+iQ`v `2bmHib- HQM; rBi? i?2 H+F Q7  bB;MB}+Mi ?Qbi i`22 2z2+i rBi?BM THQib BM ST2` AA- bm;;2bi i?i i?2 i`22 T`iM2` +QKKmMBiv 2z2+ib 1*J 7mM;B BM i?2b2 bvbi2Kb i  H`;2` bTiBH b+H2 i?M i?2 BMi2`+iBQMb Q7  bBM;H2 Kv+2HBmK rBi?  bBM;H2 i`22X aQBH MBi`Q;2M H2p2Hb r2`2 M2;iBp2Hv +Q``2Hi2/ rBi? `B+?M2bb- /Bp2`bBivM/ T`Q/m+iBpBiv Q7 1*J 7`mBi#Q/B2b- BM ++Q`/M+2 rBi? 72`iBHBxiBQM 2tT2`BK2Mib BM i2KT2`i2 M/ #Q`2H 7Q`2bib US2i2` 2i HX kyyRc pBb 2i HX kyyjc :BHH2i 2i HX kyRyc >ƺ;#2`; 2i HX kyRyV- #mi /B/ MQi bB;MB}+MiHv 2z2+i +QKKmMBiv +QKTQbBiBQMX. j9.

(38) amKK`v Q7 aim/v Ao *QKT`BbQM Q7 rBH/ Kmb?`QQK mb2 #v 2i?MB+ ;`QmTb bm``QmM/BM; i?2 lTT2` PmûKû 6Q`2bi _2b2`p2 BM "2MBM- q2bi 7`B+ 6m`M2mt- "X - o2H/KM- aX - _B;;B- GX- "QMB- aX- apM?QHK- X- _v#2`;JX- uQ`Qm- LX aX b?`2/ }`bi mi?Q`b?BT. h?Bb bim/v +QK#BM2/ +QHH2+iBQM M/ B/2MiB}+iBQM Q7 7mM;H bT2+BK2Mb rBi? BMi2`pB2rb Q7 i?2 BM?#BiMib Q7 pBHH;2b /D+2Mi iQ PmûKû amTû`B2m` #Qmi i?2 Kmb?`QQKb i?2v FMQr- i?2 MK2b i?2v mb2 7Q` i?2K- M/ r?B+? QM2b i?2v +QMbB/2` iQ #2 /2bB`#H2 2/B#H2bX q2 mb2/ biiBbiB+H K2i?Q/b 7`QK +QKKmMBiv 2+QHQ;v iQ BMp2biB;i2 ?Qr T2QTH2Ƕb FMQrH2/;2 Q7 M/ T`272`2M+2b 7Q` /Bz2`2Mi bT2+B2b Q7 Kmb?`QQKb p`B2/ #v ;2- ;2M/2`- 2i?@ MB+ ;`QmT- M/ pBHH;2X h?2 T`BK`v /i 7Q` i?Bb bim/v +K2 7`QK bBt 7Q+mb ;`QmTb M/ dy BM/B@ pB/mH BMi2`pB2rb +QM/m+i2/ rBi? i?2 BM?#BiMib Q7 }p2 pBHH;2b /D+2Mi iQ PmûKû amTû`B2m`X h?2 BMi2`pB2r22b `M;2/ BM ;2 7`QK Rd iQ Qp2` dy- M/ BM+Hm/2/ K2M M/ rQK2M KQbiHv #2HQM;BM; iQ 7Qm` HQ+H 2i?MB+ ;`QmTb, i?2 "`B#- :M/Q- GQFT- M/ uQK R - 2+? Q7 r?B+? bTQF2 i?2B` QrM HM;m;2X i #Qi? 7Q+mb ;`QmTb M/ BMi2`pB2rb- T`iB+BTMib r2`2 b?QrM T?QiQb Q7 HQ+H Kmb?`QQK bT2+B2b- b r2HH b 7`2b? bT2+BK2Mb r?2M pBH#H2- M/ bF2/ iQ bv r?2i?2` i?2v `2+Q;MBx2/ BiX A7 i?2v /B/- i?2v r2`2 7m`i?2` bF2/ i?2 MK2 Q7 i?2 Kmb?`QQK BM i?2B` HM;m;2- r?2i?2` i?2v +QMbB/2`2/ Bi 2/B#H2- M/ B7 bQ iQ `i2 ?Qr r2HH i?2v HBF2/ Bi QM  b+H2 Q7 yĜ9X AM ++Q`/M+2 rBi? T`2pBQmb 2i?MQKv+QHQ;B+H bim/B2b BM q2bi 7`B+ U2X;XuQ`Qm M/ .2 E2b2H kyyRc 6/2vB 2i HX kyRdV- r2 7QmM/ i?i T2QTH2 Qp2` j8 M/ rQK2M `2+Q;MBx2/ KQ`2 /Bz2`2Mi bT2+B2b i?M vQmM;2` T2QTH2 M/ K2MX >Qr2p2`- r2 7QmM/ i?i i?2 2z2+i Q7 ;2M/2` p`B2/ #2ir22M 2i?MB+ ;`QmTb- rBi? K2M M/ rQK2M M2`Hv 2[mH BM i?2B` Kv+QHQ;B+H FMQr@ ?Qr KQM; i?2 uQKX h?2`2 rb HbQ bB;MB}+Mi p`BiBQM BM i?2 iQiH MmK#2` Q7 bT2+B2b `2+Q;MBx2/ #v K2K#2`b Q7 i?2 /Bz2`2Mi 2i?MB+ ;`QmTb#mi +QMi``v iQ Qm` 2tT2+iiBQMb i?Bb /B/ MQi b22K iQ #2 `2Hi2/ iQ i?2 H2M;i? Q7 iBK2 2+? ;`QmT ?/ #22M T`2b2Mi BM i?2 `2X AM //BiBQM iQ /Bz2`2M+2b BM i?2 MmK#2` Q7 bT2+B2b `2+Q;MBx2/- r2 HbQ 7QmM/ p`BiBQM #2ir22M 2i?MB+ ;`QmTb BM r?B+? bT2+B2b r2`2 FMQrM M/ R. AMi2`pB2rb r2`2 HbQ +QM/m+i2/ rBi?  72r T2QTH2 #2HQM;BM; iQ i?2 L;Qi M/ S2mH? 2i?MB+ ;`QmTb- #mi i?2`2 r2`2 MQi 2MQm;? Q7 i?2b2 BMi2`pB2rb iQ BM+Hm/2 i?2K BM KQbi Q7 i?2 MHvbBbX. j8.

(39) T`272``2/X KQM; i?2 :M/Q BM T`iB+mH`- r2 HbQ 7QmM/ /Bz2`2M+2b BM i?2 b2i Q7 Kmb?`QQKb FMQrM #v K2M M/ rQK2MX q2 `2+Q`/2/  iQiH Q7 dy 2/B#H2 bT2+B2b- M BM+`2b2 Qp2` i?2 8R bT2+B2b T`2pBQmbHv FMQrM 7`QK "2MBM U.2 E2b2H 2i HX kyykVX h?2 KQbi ?B;?Hv T`272``2/ Kmb?`QQKb +`Qbb 2i?MB+ ;`QmTb r2`2 Sbi?v`2HH im#2`+mHiirQ bT2+B2b Q7 h2`KBiQKv+2b- *?HQ`QT?vHHmK TH2Qi`QTB+mK- J+`Q+v#2 HQ#v2MbBb- M/ G+iB~mmb 2/mHBbX. je.

(40) *QM+Hm/BM; `2K`Fb M/ 7mim`2 T2`bT2+iBp2b. h?2 TT2`b T`2b2Mi2/ BM i?Bb i?2bBb 2tTHQ`2 i?2 /Bp2`Biv Q7 7mM;B BM q2bi 7`B+M rQQ/HM/b i b+H2b `M;BM; 7`QK K2i2`b iQ i?QmbM/b Q7 FBHQK2@ i2`bX h?2v 2tKBM2/ i?2b2 bvbi2Kb #BQHQ;B+HHv 7`QK #Qi? #2HQr;`QmM/ M/ #Qp2;`QmM/ T2`bT2+iBp2b- b r2HH b 7`QK i?2 pB2rTQBMi Q7 i?2 ?m@ KM BM?#BiMib Q7 i?2 `2;BQMX //BiBQMHHv i?2 /i +QHH2+i2/ 7`QK i?2b2 bvbi2Kb r2`2 mb2/ iQ /pM+2 K2i?Q/QHQ;v 7Q` .L K2i#`+Q/BM; mbBM; HQM; KTHB+QMb +Qp2`BM; KmHiBTH2 `2;BQMb Q7 i?2 `.LX ST2` AA M/ ST2` AAA +QM}`K2/ `2bmHib 7`QK Qi?2` bm#b?`M 7`B+M 1*J bvbi2Kb- r?B+? }M/ HBiiH2 +QMbBbi2Mi p`BiBQM BM 1*J 7mM;H +QK@ KmMBiv +QKTQbBiBQM bbQ+Bi2/ rBi? /Bz2`2Mi +Q@Q+m``BM; T`iM2` i`22 bT2+B2b- r?BH2 biBHH }M/BM; bB;MB}+Mi +?M;2b #2ir22M /Bz2`2Mi p2;2iiBQM ivT2b BM ST2` AAX ST2` AA HbQ 7QmM/ i?i /Bp2`bBiv Q7 i`22 T`iM2`b rb TQbBiBp2Hv +Q``2Hi2/ rBi? /Bp2`bBiv Q7 1*J 7mM;B- BKTHvBM; i H2bi r2F T`iM2` T`272`2M+2X ST2` A 7QmM/ 2pB/2M+2 7Q` bTiBH +Q``2HiBQMb i b+H2b i H2bi +HQb2 iQ i?2 bBx2 Q7 i?2 8y Ɠ 8y K THQib mb2/ BM ST2` AA M/ ST2` AAA- M/ +2`iBMHv H`;2` i?M i?2 Ry Ɠ Ry K bm#THQib BM ST2` AAAX aBKBH`Hv- #Qi? ST2` AA M/ ST2` AAA 7QmM/ i?i THQi@H2p2H 2z2+ib r2`2 Km+? bi`QM;2` i?M Mv i2bi2/ rBi?BM@THQi 2z2+ibX h?Bb bm;;2bib i?i- Hi?Qm;? i?2b2 THQib `2 bmBi#H2 7Q` MHvbBb b BM/BpB/mH bKTHBM; mMBib- b BM ST2` AA7mim`2 bim/B2b Q7 HQ+H@b+H2 bTiBH T`Q+2bb2b BM i?2b2 2+Qbvbi2Kb b?QmH/ B/2HHv +Qp2` H`;2` bTiBH /BbiM+2b- 7Q` BMbiM+2 #v `2``M;BM; i?2 Ry Ɠ Ry K bm#THQib BMiQ  k8y Ɠ Ry K i`Mb2+iX am+? /2bB;Mb rQmH/ HbQ #2 HBF2Hv iQ mM+Qp2` ;`2i2` bT2+B2b `B+?M2bb 7Q`  bBKBH` KQmMi Q7 bKTHBM; 2zQ`iX h?2`2 `2 KmHiBTH2 p2Mm2b pBH#H2 iQ 2tTM/ mTQM i?Bb rQ`F- BM `2;`/b iQ i?2 bim/v Q7 i?2 T`iB+mH` bvbi2Kb A BMp2biB;i2/- iQ i?2 bim/v Q7 q2bi 7`B+M 7mM;B KQ`2 #`Q/Hv- M/ iQ i?2 K2i?Q/QHQ;v Q7 KQH2+mH` 7mM;H 2+QHQ;v BM ;2M2`HX. //BiBQMH /i 7`QK PmûKû amTû`B2m` aQBH bKTH2b 7Q` K2i#`+Q/BM; 7Q` i?2 MBM2 THQib i PmûKû amTû`B2m` /2b+`B#2/ BM ST2` AAA r2`2 +QHH2+i2/ 7`QK i?2 bK2 HQ+iBQMb b i?2 jd.

(41) bQBH +?2KBbi`v bKTH2bX aKTHBM; M/ b2[m2M+BM; Q7 #Qi? HQM; M/ b?Q`i KTHB+QMb Ub BM ST2` AV rBi? S+"BQ a2[m2H M/ AHHmKBM JBa2[- `2@ bT2+iBp2Hv- `2 H`2/v +QKTH2i2- #mi MHvbBb rb MQi `2/v BM iBK2 7Q` BM+HmbBQM BM i?Bb i?2bBbX MHvbBb Q7 i?Bb /i- 2Bi?2` T`HH2H iQ Q` BM +QK@ #BMiBQM rBi? i?2 7`mBi#Q/v /i- rQmH/ T`QpB/2  KQ`2 +QKT`2?2MbBp2 TB+im`2 Q7 i?2 1*J +QKKmMBiv BM PmûKû amTû`B2m`X h?`22 v2`b Q7 /i `2 MHvx2/ BM ST2` AAA- #mi 7`mBi#Q/v bm`p2vb +QMiBMm2/ i i?2 THQib BM PmûKû amTû`B2m` 7Q` M //BiBQMH irQ v2`bX //BiBQMHHv- i?2 MHvbBb BM ST2` AAA /Q2b MQi +QMbB/2` i2KTQ`H /v@ MKB+b- BMbi2/ TQQHBM; Q#b2`piBQMb +`Qbb iBK2 iQ 7Q+mb QM bTiBH T`Q@ +2bb2bX AM+Q`TQ`iBQM Q7 i?2b2 /i iQ 2tKBM2 b2bQMH M/ BMi2`MMmH /vMKB+b Q7 7`mBi#Q/v T`Q/m+iBQM rQmH/ BM+`2b2 Qm` mM/2`biM/BM; Q7 i?2 BKT+ib Q7 +HBKi2 2p2Mib- M/ mHiBKi2Hv +HBKi2 +?M;2- QM i?2 TQ@ i2MiBH 7Q` 7mim`2 Kmb?`QQK ?`p2bib- b r2HH b QM q2bi 7`B+M 1*J rQQ/HM/ 2+Qbvbi2Kb KQ`2 ;2M2`HHvX. S?vHQ;2M2iB+b BM K2i#`+Q/BM; "Qi? ST2` A M/ ST2` AA miBHBx2/ i?2 HQM;@`2/ +T#BHBiB2b Q7 S+"BQ iQ ;2M2`i2 T?vHQ;2M2iB+ i`22b 7`QK K2i#`+Q/BM; /i- M/ mb2/ i?2b2 7Q` T?vHQ;2M2iB+HHv BM7Q`K2/ itQMQKB+ bbB;MK2Mib UST2` A M/ S@ T2` AAV- bT2+B2b ?vTQi?2b2b UST2` AAV- M/ 2+QHQ;B+H /BbiM+2 K2bm`2b UST2` AVX h?2 mb2 Q7 ;mBH/@ Q` i`Bi@MMQii2/ `272`2M+2 b2[m2M+2b BM  K2i#`+Q/BM;@#b2/ T?vHQ;2Mv +QmH/ HbQ HHQr bbB;MK2Mi Q7 7mM+@ iBQM iQ b2[m2M+2b mbBM;  KQ/B}+iBQM Q7 S>uGPhs rBi?Qmi TbbBM; i?`Qm;?  itQMQKB+ MMQiiBQM bi2TX h?Bb rQmH/ #2 2bT2+BHHv mb27mH BM +b2b r?2`2 i?2 T`2b2M+2 Q7  T?vHQ;2M2iB+HHv +Hmbi2`2/ i`Bi bm+? b 1*J@7Q`KiBQM /Q2b MQi +Q``2bTQM/ 2t+iHv iQ  ;`QmT Q++mTvBM; QM2 Q7 i?2 KBM GBMM2M `MFb- `2bmHiBM; BM 2``Q`b mbBM; i?2 itQMQKB+ T@ T`Q+? Q7 MMQiiBQM mb2/ #v 6lL:mBH/ M/ 6mM6mM UL;mv2M 2i HX kyRec wMM2 2i HX kykyVX 6Q` 2tKTH2- i?2 ;2Mmb KMBi- r?B+? Bb M+2bi`HHv bT`Qi`QT?B+ #mi +QMiBMb  H`;2 1*J +H/2 UqQH72 2i HX kyRkV- Bb MMQ@ ii2/ b 1*J rBi? i?2 ?B;?2bi +QM}/2M+2 `MFBM; BM 6lL:mBH/X h?2`2 `2 HbQ b2p2`H 1*J HBM2;2b r?B+? `2 FMQrM QMHv 7`QK b2[m2M+BM; Q7 1*J `QQi iBTb M/ ?p2 MQ itQMQKB+HHv /2b+`B#2/ K2K#2`b- M/ bQ +MMQi #2 `2+Q;MBx2/ itQMQKB+HHv Uh2/2`bQQ 2i HX kyRyc h2/2`bQQ M/ aKBi? kyRdVX Hi?Qm;? MMQiiBQMb BM lMBi2 KF2 Bi TQbbB#H2 iQ B/2MiB7v i?2b2 HBM2;2b i?`Qm;? b2[m2M+2 bBKBH`Biv Uh2/2`bQQ M/ aKBi? kyRdV-  T?vHQ;2M2iB+ TT`Q+? rQmH/ #2 KQ`2 `B;Q`QmbX j3.

(42) >B;? i?`Qm;?Tmi .L #`+Q/BM; Q7 bT2+BK2Mb UBM 7`B+5V AMBiBH bT2+BK2M #`+Q/BM; mbBM; aM;2` b2[m2M+BM; `2p2H2/ i?i i?2 bT2+B2b MK2b bbB;M2/ BM i?2 }2H/ iQ i?2 9yyyY +QHH2+iBQMb BM ST2` AAA r2`2 MQi +QMbBbi2Mi #2ir22M BM/BpB/mH +QHH2+iQ`b M/ bKTHBM; 2p2MibX h?Bb Bb MQi bm`T`BbBM; ;Bp2M i?2 ?B;? /2;`22 Q7 2tT2`iBb2 `2[mB`2/ iQ ++m@ `i2Hv B/2MiB7v Kmb?`QQKb iQ bT2+B2b- 2bT2+BHHv BM  `2;BQM r?2`2- /2bTBi2 QM;QBM; 2zQ`ib- i?2`2 Bb biBHH Km+? rQ`F iQ #2 /QM2 +iHQ;mBM; i?2 2t@ BbiBM; /Bp2`bBiv UuQ`Qm 2i HX kyR9c SB2T2M#`BM; 2i HX kykyV M/ i`mHv +QKT`2?2MbBp2 `272`2M+2 rQ`Fb `2 H+FBM;X >Qr2p2`- i?Bb T`Qp2/ T`Q#@ H2KiB+ 7Q` 2+QHQ;B+H MHvbBb Q7 i?2 /ib2iX AM i?2 +m``2Mi KMmb+`BTiQ#b2`piBQMb `2 +Hmbi2`2/ i i?2 7KBHv H2p2H- r?B+? Bb `2HiBp2Hv `2HB#H2 #mi H2p2b Km+? iQ #2 /2bB`2/ BM i2`Kb Q7 `2bQHmiBQMX .L #`+Q/BM; Q7 HH i?2 `2KBMBM; pQm+?2` bT2+BK2Mb rQmH/ HHQr i?2K iQ #2 B/2MiB}2/ iQ bT2+B2b r?2`2 TTHB+#H2 `272`2M+2 b2[m2M+2b 2tBbi- Q` i H2bi /BpB/2/ BMiQ +QMbBbi2Mi- bT2+B2b@H2p2H PhlbX h?Bb rQmH/ HbQ 7+BHBii2 B/2MiB}+iBQM Q7 TQi2MiBH M2r bT2+B2bX a2p2`H ;`QmTb ?p2 T@ THB2/ >ha iQ #`+Q/BM; H`;2 bT2+BK2M +QHH2+iBQMb Ua?QF`HH 2i HX kyR9c qm`x#+?2` 2i HX kyRNc GQBi 2i HX kyRNVX h?Bb T`QKBb2b iQ #2 2bT2+BHHv +Qbi@2z2+iBp2 mbBM; i?2 dz6HQM;H2Ǵ /Ti2` 7Q` Pt7Q`/ LMQTQ`2 /2pB+2b U2X;X- 1iQM 2i HX kykyV- r?B+? `2/m+2b i?2 T`B+2 TQBMi 7Q`  bBM;H2 b2@ [m2M+BM; `mM iQ QMHv ī3y THmb HB#``v T`2TX h?2 bKHH bBx2 Q7 i?2 Pt7Q`/ LMQTQ`2 /2pB+2b M/ pBH#BHBiv Q7 `27`B;2`iBQM@7`22 HB#``v T`2T FBib HbQ KF2 i?2K TQi2MiBHHv bmBi#H2 7Q` mb2 /m`BM; }2H/ 2tT2/BiBQMbX q2 bm++2bb7mHHv i2bi2/ T`QiQ+QHb mbBM; i?2 KBMBS*_ bvbi2K UKBMBS*_ #BQ- *K#`B/;2- Jbb+?mb2iibV 7Q` .L 2ti`+iBQM M/ S*_ 7`QK 7`2b? 7`mBi#Q/B2b QM i?2 LiBQMH :2Q;`T?B+ 2tT2/BiBQM r?2`2 bQBH bKTH2b 7Q` ST2` AA r2`2 +QHH2+i2/X >Qr2p2`QM i?i 2tT2/BiBQM r2 b2Mi Qm` S*_ T`Q/m+ib iQ M +QKK2`+BH H# BM 1m`QT2 7Q` aM;2` b2[m2M+BM;X .2p2HQTBM; zQ`/#H2 M/ `2HB#H2 T`QiQ+QHb 7Q` #`+Q/BM; rBi? i?2 KBMBS*_ THmb 6HQM;H2- M/ i`BMBM; Qm` +QHH2;m2b BM 7`B+ iQ mb2 i?2K- rQmH/ BKT`Qp2 i?2 /2p2HQTK2Mi Q7 HQ+H b2[m2M+BM; +T+Biv M/ `2b2`+? BM/2T2M/2M+2- b r2HH b 2Mbm`BM; ?B;? [mHBiv /i 7Q` 7mim`2 +QHH#Q`iBQMbX. jN.

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