South African Journal of Botany 2003, 69(2): 204–206 Copyright © NISC Pty Ltd Printed in South Africa — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299 Short Communication Phylogenetic relationships of the recently discovered species — Clivia mirabilis F Conrad1*, G Reeves1 and JP Rourke2 1 Leslie Hill Molecular Systematics Laboratory, National Botanical Institute, Private Bag X7, Claremont 7735, South Africa 2 Compton Herbarium, National Botanical Institute, Private Bag X7, Claremont 7735, South Africa * Corresponding author, email: [email protected] Received 14 October 2002, accepted in revised form 13 January 2003 A phylogenetic study of Clivia, including the newly trnC intergenic spacer, the rps16 intron, the trnL intron described species, Clivia mirabilis, recently discovered and the trnL-F intergenic spacer (the latter two collec- in the Northern Cape, was carried out to elucidate the tively known as the trnL-F region). In the combined tree relationship of this geographically isolated taxon to the C. mirabilis is placed as sister to a clade comprising the remaining four species. DNA sequence data were col- other four taxa. lected from four non-coding plastid regions: the rpoB- The genus Clivia Lindl. (Amaryllidaceae) comprises five sequencing primers. Amplification products were purified species of which C. caulescens R.A.Dyer, C. gardenii Hook., using Qiaquick (Qiagen) spin columns according to manu- C. miniata (Lindl.) Regel and C. nobilis Lindl. are best facturer’s instructions and directly sequenced on an ABI 377 known. These four species are found in coastal and inland automated sequencer using standard dye-terminator chem- Afromontane forest along the east coast of southern Africa, istry following manufacturer’s protocols (Applied Biosystems). from the Eastern Cape northwards to Limpopo Province and For assembly and editing of the complementary strands Swaziland (Rourke 2002). The recent discovery of Clivia Sequencher version 4.1 (GeneCodes) was used. mirabilis Rourke (Rourke 2002), from a semi-arid valley in A branch and bound parsimony analysis was performed the Northern Cape, has prompted questions regarding the using the parsimony algorithm of the software package phylogenetic and biogeographic relationships of this taxon to PAUP* for Macintosh (phylogenetic analysis using parsimony the other four Clivia species. v.4.0b 10, Swofford 2000). To assess internal support, 1 000 Plant material and associated voucher information used in bootstrap replicates were performed using simple taxon this study are shown in Table 1. Standard DNA extraction addition and TBR branch swapping with a tree limit of ten and PCR amplification protocols were used as described in trees per replicate. Cryptostephanus vansonii Verdoorn was Reeves et al. (2001). The following programmes for DNA designated as the outgroup taxon based upon results of a amplification were used: 1) for the rpoB-trnC spacer region: larger analysis of DNA sequence data for the first five cycles; denaturation, 94°C for one minute; anneal- Amaryllidaceae (Meerow et al. 1999). ing, 52°C for one minute (with a decrease of one degree per Of the 2 897 characters included in the combined analysis cycle); extension, 72°C for one minute; the next 25 cycles: 17 were potentially parsimony informative. Branch and denaturation, 94°C for one minute; annealing, 48°C for one bound parsimony analysis gave a single tree with length 65, minute; extension, 72°C for one minute: 2) for the trnL-F consistency index (CI) of 0.97, and retention index (RI) of region and rps16 intron: denaturation, 94°C for one minute; 0.92. In the single most parsimonious tree shown in Figure annealing, 48°C for one minute; extension, 72°C for one 1, C. mirabilis is placed as sister to a clade comprising the minute. Amplification of the rpoB-trnC intergenic spacer other four Clivia species. C. nobilis is placed as sister to a region was achieved using primers rpoB5’ and trnC5’ clade comprising C. gardenii, C. miniata and C. caulescens, (Ohsaka and Ohnishi 2000). Primers rps16F and rps162R and C. gardenii is in turn placed as a sister to the terminal (Oxelman et al. 1997) were used to amplify and sequence clade comprising C. miniata and C. caulescens. the rps16 intron. Primers ‘c’ and ‘f’ (Taberlet et al. 1991) were Previous phylogenetic analysis of Clivia using information used to amplify the adjacent trnL intron and trnL-F intergenic from the nuclear ribosomal 5S non-transcribed spacer and spacer between the trnL and trnF exons. For each of the the internal transcribed spacer (ITS) of 45S rDNA was per- above regions amplification primers were then used as formed by Ran et al. (2001). Their study included five Clivia South African Journal of Botany 2003, 69: 204–206 205 Table 1: Voucher and molecular database accessions for all taxa included in the study Voucher/accession EMBL accession number rpoB-trnC intergenic spacer trnL-F region rps16 intron Clivia gardenii Hook. Winter 434/99; NBG AJ519550 AJ519540 AJ519545 Clivia caulescens R.A.Dyer Winter 573/99; NBG AJ519549 AJ519539 AJ519544 Clivia nobilis Lindl. Winter 735/96; NBG AJ519547 AJ519537 AJ519542 Clivia miniata (Lindl.) Regel Winter 442/99; NBG AJ519548 AJ519538 AJ519546 Clivia mirabilis J.P. Rourke J.P.Rourke 2220; NBG AJ519551 AJ519541 AJ519543 Cryptostephanus vansonii Verdoorn Ex hort Graham Duncan AJ535635 AJ535633 AJ535634 7 (Winter 1999). From this it can be concluded that the phy- Clivia nobilis logeny supports a geographical speciation model for the tubular-flowered species. Clivia mirabilis is the only species in this genus to be found in the winter rainfall region of South 5 3 Clivia caulescens Africa (Rourke 2002), and it is placed here as the sister to all 86 5 of the summer rainfall species. This may imply that the 96 2 genus formerly occupied a wider range spanning the 8 Clivia miniata Eastern and Western Cape. Subsequent fragmentation of 93 this range may have been precipitated by the increase in aridity experienced in the Northern and Western Cape dur- 57 5 Clivia gardenii ing the late Miocene, causing the range of Clivia to contract to suitable habitats on the east coast since the progressive retreat of the subtropical forest elements from Pliocene 20 10 Clivia mirabilis times. This vicariance may account for only one lineage, now represented by C. mirabilis, occupying an arid habitat in the north-western Cape. If indeed this explains current dis- tribution patterns in Clivia, it leaves open the question of why Cryptostephanus vansonii Clivia is not found in intervening favourable habitats between the eastern and north western Cape such as the Figure 1: The most parsimonious tree found from combined analy- Knysna forests and at the foot of the Outeniqua mountains. sis of all four DNA sequence matrices. Bootstrap percentages are Interpretation of morphological character evolution in light shown beneath the branches of the phylogenetic tree would suggest that the upright flow- ers found in C. miniata are derived since C. miniata is placed in the most terminal clade in the phylogenetic tree. All other taxa (C. nobilis, C. caulescens, C. miniata, C. gardenii and taxa display pendulous flowers. Observations suggest that ‘Robust Gardenii’) and focused on analysing patterns of the pendulous-flowered species are pollinated by sunbirds chromosome evolution. Our phylogenetic reconstruction is (Nectarinia spp.) whereas C. miniata is adapted to pollina- not congruent with the separate analyses of the ITS and 5S tion by swallowtail butterflies (Papilio spp.) (J Manning and spacer regions of Ran et al. (2001) with respect to the place- C Paterson-Jones pers. comm.). This indicates that butterfly ment of C. gardenii and C.caulescens. Our plastid data pollination is derived in the genus from an ancestral state of placed C. gardenii as sister to the terminal clade (93% boot- sunbird pollination. strap support) which was composed of C. caulescens and C. miniata (together receiving 96% bootstrap support). Ran et References al. (2001) placed C. caulescens as sister to a clade com- prising C. miniata, C. gardenii and ‘Robust Gardenii’. Meerow AW, Fay MF, Guy CL, Li QB, Zaman FQ, Chase MW (1999) However, bootstrap percentages in the neighbour-joining Systematics of Amaryllidaceae based on cladistic analysis of analysis for both regions were low with respect to the posi- plastid rbcL and trnL-F sequence data. American Journal of tion of C. caulescens to the other taxa in their analysis (57% Botany 86: 1325–1345 and 70% bootstrap support for the ITS and 5S spacer data Ohsaka T, Ohnishi O (2000) Intra- and interspecific phylogeny of sets respectively). Support for the position of C. gardenii and wild Fagopyrum (Polygonaceae) species based on nucleotide sequences of noncoding regions in chloroplast DNA. American C. miniata as sister taxa was high only in the ITS analysis Journal of Botany 87: 573–582 (92%) receiving only 59% support in the 5S spacer analysis. Oxelman B, Lidén M, Berglund D (1997) Chloroplast rps16 intron Biogeography: Clivia nobilis occurs in the Eastern Cape phylogeny of the tribe Sileneae (Caryophyllaceae). Plant between the Zuurberg and Dwessa, C. gardenii extends Systematics and Evolution 206: 393–410 from Port St. Johns to northern KwaZulu-Natal and C. Ran Y, Hammet KRW, Murray BG (2001) Phylogenetic analysis and caulescens extends from Swaziland and Mpumalanga karyotype evolution in the genus Clivia (Amaryllidaceae). Annals northwards to Limpopo Province. The distribution of C. mini- of Botany 87: 823–830 ata is extensive throughout the east coast where its range Reeves G, Chase MW, Goldblatt P, Rudall P, Fay MF, Cox AV, overlaps with C. nobilis, C. gardenii and C. caulescens Lejeune B, Souza-Chies T (2001) Molecular Systematics of 206 Conrad, Reeves and Rourke Iridaceae: Evidence from four Plastid DNA Regions. American Swofford DL (2000) PAUP*. Phylogenetic Analysis using Parsimony Journal of Botany 88: 2074–2087 (*and other methods) Version 4.0b 10.