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DNA Barcoding and Phylogenetic Analysis of Searsia

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DNA Barcoding and Phylogenetic Analysis of Searsia Poster ID : 828 DNA barcoding and phylogenetic analysis of Searsia Makhado Tshililo, Michelle van der Bank and Sanele N. S. Shiba The African Centre for DNA Barcoding (ACDB), Department of Botany & Plant Biotechnology, University of Johannesburg, South Africa ABSTRACT Background. The family Anacardiaceae includes approximately 800 species in 82 genera. Rhus L. is the largest and most widely distributed genus in Anacardiaceae. However, controversy has surrounded the delimitation of the genus. Searsia F.A. Barkley was originally included into the Rhus complex but recent molecular studies have separated it along with six other genera from the Rhus complex and recognised them as separate genera. The genus Searsia, which is the focus of the current study, is represented by approximately 120 species and is widely distributed in Africa with only three species currently known from Asia. Here we include an extensive sampling and molecular analyses of Searsia species across its distribution range in Africa to access the relationships within the genus. Results. In total 205 taxa, representing 35 species of Searsia, were sequenced for the core barcoding regions (matK and rbcLa) along with additional markers, i.e. ITS, ETS, trnL-F and ndhF. Significance. This study contributes a first large sampling of Searsia. An infrageneric classification for the genus will be presented. Anacardium occidentale AY594459 Anacardium occidentale OM3645 Anacardium occidentale GH0152 Trichoscypha acuminata MPADN0277 Trichoscypha arborea MPADN0003Anacardium excelsum GQ9 Trichoscypha acuminata MPADN0004 Trichoscypha acuminata MPADN0001 Gluta wallichii AY594468 Trichoscypha patens Tricpate PM5076 Trichoscypha patens Tricpate PM4897 INTRODUCTION Trichoscypha preussii Tricpreu PM5027 Trichoscypha klainei Tricklai PM5051 971 1 1 9 9 Anacardiaceae also known as the “cashew family” consists of 82 1 Toxicodendron vernix AY594495 Toxicodendron sylvestre HQ4153 8 1 932 Mangifera indica AY594472 Fegimanra africana AY594489 Semecarpus australiensi AY594479 Pachycormus discolor AY594493 Dobinea vulgaris AY594466 Pegia nitida AY594473 Loxopterygium huasango AY594471 Spondias mombin AY594480 genera represented by around 800 species. The largest genus in this Lannea antiscorbutica OM2704 Lannea discolor OM3322 Lannea edulis OM Lannea edulis OM Lannea schweinfurthii OM3413 family is Rhus L. (senso lato) for which issues around its delimitation Lithrea molleoides AY594470 Lannea schweinfuthii OM1355 Cotinus coggygria HE966907 Lannea schweinfuthii OM2446 Lannea schweinfuthii OM2505 75 51 Comocladia glabra HM446677 89 Lannea shimperi OM2521 Amphipterygium adstring AY594458 1 76 9 has caused a lot of controversy among taxonomists. Searsia, a newly Dracontomelon dao AY594467 96 53 1 Pleiogynium timoriense AY594474 8 Toxicodendron rydbergii BAB 0354 97 Pleiogynium timoriense AY724340 97 Harpephyllum caffrum BS0058 accepted genus within Anacardiaceae, was originally part of the Rhus Toxicodendron radicans AP422 Harpephyllum caffrum DMP204 Thyrsodium puberulum FJ514723 85 Harpephyllum caffrum OM1555 Thyrsodium puberulum FJ514664 Tapirira bethanniana AY5944 complex but was recently removed along with five other genera. The 54 Tapirira obtusa AY594482 Schinopsis brasiliensis AY594477 75 97 Tapirira obtusa JQ626383 genus Searsia has approximately 120 species and is mainly Astronium graveolens AY594492 76 Choerospondias axillari AY594463 Choerospondias axillari HQ427341 Rhus hypoleuca HQ427342 89 Antrocaryon amazonicum AY594460 characterised by trifoliate leaves (Yang et al., 2016). It is widely 82 Cyrtocarpa procera AY594464 Rhus hirta AP 99 Pseudospondias microcarpa PM5455 Rhus copallina AY594485 distributed across Africa with 111 species occurring in southern Africa 184 1 59 8 Sclerocarya birrea AY594478 95 Rhus aromatica AY594494 89 Sclerocarya birrea subsp caffra OM0278 and 3 species occurring in Asia. The African species all belong to the Rhus aromatica AY594486 71 Sclerocarya birrea subsp caffra OM0498 Mosquitoxylum jamaicens AY594490 73 Sclerocarya birrea subsp caffra RL1117 section Gerontogeae. Recent phylogenetic studies have defined Commiphora africana OM0334 Schinus terebinthifolius PPRI 0027 65 Commiphora glandulosa RBN160 Schinus terebinthifolius OM 61 1982 Commiphora schimperi OM13 Searsia as a monophyletic group but these findings need to be 85 100 Schinus terebinthifolius DMP256 50 Searsia chirendensis OM1987 Searsia ciliata Hahn29 confirmed by conducting a broader sampling of the genus (Yang et al., Schinus terebinthifolius DMP237 100 81 50 Searsia pyroides OM713 1 Schinus terebinthifolius BS0098 Searsia tumulicola OM3907 2016). Schinus molle JG052 98 Searsia tumulicola var meeseana OM3 Schinus molle DMP231 Searsia zeyheri OM2256 62 818 Schinus molle DMP224 93 Searsia leptodictya RBN205 Searsia leptodictya RL1645 Schinus molle DMP200 The aim of this study is to investigate the phylogenetic relationships 83 88 Searsia leptodictya RL1655 Schinus molle DMP145 77 65 Searsia tenuinervis Hahn2999 among the seven genera within the Rhus complex as well as resolve Schinus areira AY594488 Searsia nebulosa Abbott Searsia pyroides var integrifolia OM2477 Rhus thouarsii AY594484 59 Searsia discolor OM3 Heeria argentea PG0016 9106 the systematics within Searsia. 80 Searsia fastigiata Abbott 88 948 1 99 Searsia lancea OM Ozoroa sphaerocarpa OM1106 9 Searsia lancea PG0001 11 Ozoroa sphaerocarpa OM0940 99 Searsia pendulina OM 90 9135 Ozoroa paniculosa OM Searsia pondoensis Burrows102421942 72 Searsia pyroides OM1236 85 Ozoroa englerii RL1277 Searsia transvaalensis OM0943 MATERIAL & METHODS 64 Searsia transvaalensis RL1427 1984 Ozoroa insignis subsp reticulataOzoroa OM3580 englerii OM1169 95 Searsia tumulicola var tumulicola OM2028 63 Searsia tumulicola var tumulicola OM3 Ozoroa englerii OM1154 56 62 Searsia wilmsii OM3 Sampling Searsia gueinzii OM0248 Searsia gueinzii OM0265 827 Searsia gueinzii OM0336 1 Searsia gueinzii OM3371 Protorhus longifolia OM1764 Searsia natalensis OM2655 Ozoroa barabetorens Burrows08069 Searsia pentheri OM0942 A total of 205 taxa were included in the study. These include Searsia pentheri OM0945 Searsia longispina AM0243 Searsia pallens OM Protorhus longifolia MWC28088 Searsia undulata OM2940 Searsia lucida MWC05809 Searsia crenata OM Searsia crenata OM3209 9 10 154 representatives of the genera: Searsia, Rhus, Lannea, Schinus, Cotinus 9 Loxostylis alata OM KEY Ozoroa obovata subsp elliptica OM2733 8 Ozoroa obovata subsp elliptica OM2511 13 Sorindeia juglandifolia PM4949 and Toxicodendron (Figure 2). 1 Ozoroa obovata subsp elliptica OM2425 Sorindeia juglandifolia PM5106 976 1 Sorindeia juglandifolia PM4827 Searsia lucida Abbott9230 986 Searsia tomentosa SM 731 3 Clade A Clade B Searsia dissecta JL 802 4 Searsia angustifolia OM2847 Searsia nebulosa OM2285 Searsia chirindensis OM2284 Searsia incisa var incisa OM3059 Searsia acocksii Abbott Clade C Clade D Rhus Representatives Searsia F.A. Barkley Rhus L. (S .str.) Lannea Engl. Searsia dentata Rhus chinensis Lannea schweinfurthii Schinus L. Cotinus Mill. Toxicodendron Mill. FIGURE 1: Distribution map of field collected Searsia samples. DNA extraction, amplification and sequencing DNA was extracted using the 10X CTAB method of Doyle & Doyle (1987). Standard protocols were followed for PCR and sequencing using the core barcoding regions (rbcLa & matK). Data analysis A phylogenetic tree was generated using Maximum Parsimony (MP) in PAUP* (Swofford, 2002). Schinus terebinthifolia Cotinus coggygria Toxicodendron radicans FIGURE 2: Bootstrap consensus tree generated using PAUP of the combined plastid dataset RESULTS & DISCUSSION highlighting Clades A – D, representatives of genus Rhus as well as some representatives of other Figure 2 (Clade A) shows high support (99%) for the monophyly of genera included in the study. southern African Searsia species which supports the findings of Yang CONCLUSION et al. (2016). The results obtained in this study support the monophyly of African representatives of Clade B shows high support (100%) for the monophyly of the genus Searsia and illustrates the inability of core barcoding regions to provide species Schinus, having two highly supported sister clades. These findings back delimitation within this genus. Further investigation is required with additional Guala (2016) who differentiated S. terebinthifolius from S. areira and S. markers (ITS, ETS and trnL-F) to provide better delimitation and resolution within the molle by the presence of pubescent leaflets. genus. REFERENCES Figure 2 (Clade C) shows weak support (53%) for the monophyly of Doyle, J.J. & Doyle, J.L. 1987. A rapid isolation procedure for small amounts of leaf tissue. Phytochemical Bulletin, 19: 11- Lannea. There is also moderate support (80%) for the monophyly of 15. Clade D, with the African species Protorhus nestled within it. This Guala, M.S., Lapissonde, M.O., Elder, H.V., van Baren, C.M., Bandoni, A.L. & Dellacassa, E. 2006. Essential Oils in Food supports findings by Von Teichman and Van Wyk (1996) who grouped Preservation, Flavor and Safety. Swofford, D.L. 2002. PAUP* - Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Ozoroa and Protorhus due to morphological similarities. Associates, Sunderland, MA. Yang, Y., Meng, Y., Wen, J., Sun, H. & Nie, Z. 2016. Phylogentic analyses of Searsia (Anacardiaceae) from eastern Asia and Results also show no support for the monophyly for the Rhus species its biogeographic disjunction of its African relatives. South African Journal of Botany, 106: 129-136. Yi, T., Miller, A. J. & Wen, J. 2007 . Phylogeny of Rhus (Anacardiaceae) based on sequences of nuclear NIA-i3 intron and included in this study (Figure 2). In 2007, Yi et al. cited the chloroplast trnCtrnD. Systematic Botany 32: 379-391. morphological heterogenousity of Rhus sensu Barkley (1937). Von Teichman, I. & Van Wyk, A. E. 1996. Taxonomic significance of pericarp and seed structure in Heeria argentea (Thunb.) Meisn. (Anacardiaceae), including reference to pachychalazy and recalcitrance. Botanical Journal of the Linnean Society, 122: 335-352. ACKNOWLEDGEMENTS.
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