Diversification into novel habitats in the Africa clade of Dioscorea (Dioscoreaceae): erect habit and elephant’s foot tubers

RESEARCH ARTICLE Open Access Diversification into novel habitats in the Africa clade of Dioscorea (Dioscoreaceae): erect habit and elephant’s foot tubers Olivier Maurin 1,2 , A. Muthama Muasya 3* , Pilar Catalan 4,5 , Eugene Z. Shongwe 1 , Juan Viruel 6,7 , Paul Wilkin 2 and Michelle van der Bank 1 Abstract Background: Dioscorea is a widely distributed and highly diversified genus in tropical regions where it is represented by ten main clades, one of which diversified exclusively in Africa. In southern Africa it is characterised by a distinct group of species with a pachycaul or “elephant’sfoot”structure that is partially to fully exposed above the substrate. In contrast to African representatives of the genus from other clades, occurring mainly in forest or woodland, the pachycaul taxa and their southern African relatives occur in diverse habitats ranging from woodland to open vegetation. Here we investigate patterns of diversification in the African clade, time of transition from forest to more open habitat, and morphological traits associated with each habitat and evaluate if such transitions have led to modification of reproductive organs and mode of dispersal. Results: The Africa clade originated in the Oligocene and comprises four subclades. The Dioscorea buchananii subclade (southeastern tropical Africa and South Africa) is sister to the East African subclade, which is respectively sister to the recently evolved sister South African (e. g., Cape and Pachycaul) subclades. The Cape and Pachycaul subclades diversified in the east of the Cape Peninsula in the mid Miocene, in an area with complex geomorphology and climate, where the fynbos, thicket, succulent karoo and forest biomes meet. Conclusions: Diversification out of forest is associated with major shifts in morphology of the perennial tuber (specifically an increase in size and orientation which presumably led them to become pachycaul) and rotation of stem (from twining to non-twining). The iconic elephant's foot morphology, observed in grasslands and thicket biomes, where its corky bark may offer protection against fire and herbivory, evolved since mid Miocene. A shift in pollination trait is observed within the forest, but entry into open habitat does not show association with reproductive morphology, except in the seed wing, whichhasswitchedfromwingedallroundtheseedmargin to just at the base or at the apex of it, or has been even replaced by an elaiosome. Keywords: Biogeography, Dioscoreales, “elephant’s foot”, Fire adaptation, Habitat transition, Pachycaul, Southern Africa, Yams Background Dioscorea L. is a monocotyledonous plant genus that is highly diverse in many tropical regions of the world, with comparatively few taxa found in temperate latitudes. It comprises over 600 species, almost all of which have perennating organs (rhizome and/or tuber). These organs give rise to herbaceous, usually twining stems bearing leaves with basal and apical petiolar pulvinii and campylodromous venation. Most species are dioecious, with relatively small, typically monocotyledonous trimerous flowers in spicate or racemose (partial) inflorescences, with female plants usually containing up to six (usually) winged seeds in each inferior ovary. The highest species diversity per unit area is found in tropical areas, for example, southern Brazil, parts of Mexico, the Greater Antilles, western Madagascar and Asia from southern * Correspondence: [email protected] 3 Department of Biological Sciences, University of Cape Town, Rondebosch, Western Cape 7701, South Africa Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Maurin et al. BMC Evolutionary Biology (2016) 16:238 DOI 10.1186/s12862-016-0812-z China to the Isthmus of Kra in Thailand [1–5]. These are largely areas with seasonal climates supporting open, deciduous forests that allow these light-demanding plants to thrive. Wilkin et al. [6] established the broad phylogenetic outline of Dioscorea, which comprises 10 main clades. The same tree topology has been supported through significantly increased sampling and a further plastid marker [7] as well as was with additional data from the nuclear region Xdh, (Viruel, personal communication). The first branching group, the Stenophora clade (Fig. 3), is rhizomatous, with its highest diversity in subtropical Asia, followed by two large clades endemic to the Neotropics. The remaining clades comprise smaller units of diversity from the Mediterranean and Africa plus the principal reservoirs of species numbers in the Caribbean, Madagascar and the palaeotropics as a whole. Thus the focus of research in this genus has now shifted to species forming these 10 major clades. Of those 10 major clades, three are distributed in sub-Saharan Africa. One of these is the first Dioscorea clade to be studied here via a species level phylogeny, the Africa clade of Viruel et al. [7]. It is also the only clade to have diversified exclusively in Africa and comprises 13 species, as listed in [1] with minor taxonomic changes made in [8]. Nine are South African (sub)endemic species, two extend from South Africa into southern tropical Africa (D. buchananii Benth. and D. sylvatica Ecklon) and two are disjunct in northeastern tropical Africa (D. gillettii Milne-Redh. and D. kituiensis Wilkin & Muasya). In contrast, the remaining Dioscorea clades found in sub-Saharan Africa sensu [7] are poorly represented in South Africa, with only one species in the Enantiophyllum clade (D. cotinifolia Kunth; Fig. 3) and three in the Compound Leaved clade (CL; Fig. 3). This contrasts with the substantial tropical African diversity in these lineages. The species of the Africa clade (Fig. 3) of Viruel et al. [7] possess a number of distinctive or unusual morphological traits. They include perennial tubers, some of which are large, “elephant’s foot”pachycaul structures that are partially or wholly exposed from the substrate (Fig. 1; [9, 10]). Similar structures also occur infrequently in neotropical species such as D. mexicana Scheidw.; two of the main neotropical lineages of Dioscorea also possess perennial tubers. Stems are usually sinistrorse (climbing towards the left hand, as viewed externally) but in some taxa they are non-twining [10, 11]. This trait is also encountered elsewhere, for example in the Mediterranean clade (D. pyrenaica Bubani & Bordère ex Gren. and D. chouardii Gaussen; Fig. 3) from Pyrenean France and Spain, in the Epipetrum group from Chile [12], and D. hexagona Baker from Madagascar [13]. Leaves are always alternate and blades are entire to deeply palmately lobed. Stamen number is reduced from 6 to 3 in one species [14]. Seeds in the Africa clade vary from possessing a wing all round the margin of the seed with a longer and shorter axis to being winged just at the apex [10, 15]. This is correlated with a capsular fruit that is longer than broad. Dioscorea gillettii and D. kituiensis have seeds that are wingless but possess an aril-like structure [16]. Among Dioscorea species occurring in Africa, the Africa clade is the richest source of steroidal saponins [17, 18]. Dioscorea sylvatica in particular was extracted from the wild in South Africa in the 1950s to produce synthetic human hormones for contraceptive purposes and other steroidal drugs. In contrast, some taxa of the CL clade have alkaloid chemistry [18] which is the basis of the use of D. dregeana in South African traditional medicine (e.g. [19]). The principal use of Enantiophyllum clade species is as a starch source that feeds at least 60 million people in tropical Africa [20]. The species of the CL and the Enantiophyllum clades are typical of the genus as a whole in that they mainly inhabit forest or woodland biomes, often those that are seasonal in climate. However, the Africa clade occupies an unusually broad range of vegetation types for the genus, including not only afromontane forests or forest margins and savannah woodlands but also the fynbos heathlands, succulent karoo and thicket. This observation reinforces three key questions that this research sets out to investigate. First, what are the patterns and timing of diversification in the Africa clade, especially in relation to transitions from forest to more open habitats such as thickets and karoo? Second, how are the traits associated with forest or woodland habitats modified in taxa inhabiting more open biomes, especially vegetative traits of perennating organs, stems and leaves, including their size and shape? Finally, are floral and fruit reproductive traits similarly affected by these biome shifts in addition to vegetative traits? Methods Taxon sampling Representatives of all known African perennial-tubered Dioscorea (Dioscoreaceae) were sampled (Table 1). These included five pachycaul species, three Cape species, three species of the D. buchananii subclade (as defined by Wilkin and Muasya [8]), two species of the southern African members of the CL clade, and two species from the Enantiophyllum clade as well as two species from the East Africa subclade. We also included representatives from all known Dioscorea lineages [6]: four from the Mediterranean clade, D. tentaculigera and D. prazeri from South-East Asia and two New World taxa (D. brachybotrya and D. galeottiana), respectively belonging to the New World I (NWI) and II Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 2 of 17 clades (NWII); and Tacca and Stenomeris (Dioscoreaceae) were selected as outgroups. Voucher specimen information and GenBank accession numbers are listed in Table 1 and trace files and sequences are available on the Barcode of Life Data System (BOLD; www.boldsystems.org). DNA extraction, amplification, sequencing and alignment DNA was extracted from 0.3 g silica gel dried leaves [21] using 2x CTAB method [22] with the addition of 2 % polyvinyl pyrrolidone (PVP) to reduce the effects of high polysaccharide concentration in the samples. In order to avoid problems of PCR inhibition, DNA was precipitated in 2.5 volume ethanol and purified using QIAquick PCR Purification Kit according to manufacturer’s protocol (QiIAgen Inc., Hilden, Germany). All PCR reactions were carried out using Thermo Scientific Master Mix (Thermo Fischer Scientific, Waltham, Massachusetts, USA). Amplification of rbcLa was carried out using the primers rbcLa-F and rbcLa-R described respectively by Levin et al. [23] and Kress and Erickson [24]. For matK, the following primers were used MatK-1R-Kim- F and MatK-3 F-Kim-R (Kim, unpublished; [25]). Amplification of trnL-F was carried out using primers c and f of Taberlet et al. [26], but the internal primers dandewerealsousedforseveraltaxaduetodifficulty in amplifying the region as a single piece. The trnH-psbA spacer was amplified using primers 1 F and 2R [27]. The psaA-ycf3 spacer was amplified using the PG1f and PG2r primers [28]. Finally the rpl32- trnL (UAG) intergenic spacer was amplified according to Shaw et al. [29]. Amplified products were purified using QIAquick columns (QIAgen, Germany) following the manufacturer’sprotocol. PCR amplification primers were also used as cycle sequencing primers. Cycle sequencing reactions were carried out using BigDye© V3.1 Terminator Mix (Applied Biosystems, Inc., ABI, Warrington, Cheshire, UK) and cleaned using the EtOH-NaCl method provided by ABI; they were then sequenced on an ABI 3130xl genetic analyser. Complementary strands were assembled and edited using Sequencher version 5.1 (Gene Codes Corp., Ann Arbor, Michigan, USA) and sequences were aligned manually in PAUP* (version 4.0b1; [30]) without difficulty due to low levels of insertions/deletions. Phylogenetic analyses: parsimony and Bayesian approaches Maximum parsimony (MP) usingPAUP*version4.0b1[30] was performed on the individual and combined datasets. Tree searches were conducted using 1,000 replicates of random taxon addition, retaining 10 trees at each step, with tree-bisection-reconnection (TBR) branch swapping and MulTrees in effect (saving multiple equally parsimonious trees). Support for clades in all analyses was estimated using bootstrap analysis [31] with 1000 replicates, simple sequence addition, TBR swapping, with MulTrees in effect but saving a maximum of 10 trees per replicate. Delayed transformation character optimization (DELTRAN) was used to calculate branch lengths, due to reported errors http://paup.sc.fsu.edu/paupfaq/paupans.html with accelerated transformation optimization (ACCTRAN) in PAUP v.4.0b1. Bootstrap support (BP) was classified as high Fig. 1 Mapping of habit, tuber and leaf traits on five South Africa yam lineages of the Pachycaul clade. Photographs: BJvN = Brian J van Niekerk; GG = Graham Grieve; JB = John Burrows; OM = Olivier Maurin; PW = Paul Wilkin Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 3 of 17 Table 1 Table of material and Genbank accession. Collectors references acronyms are Olivier Maurin (OM), Muthama Muasya (AMM), Sebsebe Demissew (SD), Pilar Catalán (PC), Ernesto Pérez-Collazos (EP). Genbank accessions in plain text are new to this study, accessions in bold were retrieved from www.ncbi.nlm.nih.gov Taxon Voucher (Museum) Distribution Habitat Habit rbcL matK trnL-F trnH- psbA PsaA- Ycf3 rpl32- trnL Dioscorea brachybotrya Poepp. Rudall P. s.n (K) C. & S. Chile to Argentina At medium altitude up to the timber line, and at low altitude in interior valleys Shrub climber, 2 m in height AF307469 AY956482 KM878027 KR086979 KR087124 KR088291 Dioscorea brownii Schinz Grieve 53 (K) South Africa - KwaZulu-Natal Grassland Pachycaul slightly emerging with annual solitary stems reaching 1 m in height KR087028 KR086942 KR070855 KR086980 KR087125 KR088292 Dioscorea buchananii Benth. Bingham10290 (K) Tanzania, Angola, Malawi, Mozambique, Zambia, Zimbabwe, DRC Combretum thickets and at proximity of woody outcrop Twining wine with perennial tuber and stems up to 9 m long KR087029 KR086943 KR070856 KR086981 KR087126 KR088293 Dioscorea bulbifera L. OM3576 (BNRH) Native in Tropical and subtropical region from Africa, Asia and Australasia. Introduced elsewhere Forest and woodlands Twining wine with perennial tuber and stems up to 12 m long KR087030 KR086944 KR070857 KR086982 KR087127 KR088294 Dioscorea burchellii Baker AMM6650 (BOL) South Africa - Eastern Cape At medium to high Altitude in dense fynbos vegetation Tuberous perennial, shoot to 1 m height KR087032 KR086946 KR070859 KR086984 KR087129 KR088296 Dioscorea burchellii Baker AMM6704A (BOL) - - - KR087031 KR086945 KR070858 KR086983 KR087128 KR088295 Dioscorea chouardii Gaussen PC334 (JACA) Spain Limestone rock-crevices Tuberous perennial, shoot to 1 m height KM877855 KM877907 KR070860 KR086985 KR087130 KR088297 Dioscorea communis (L.) Caddick & Wilkin Chase536 (K) Europe, North African and temperate Asia Woodland and woodland hedges Herbaceous with climbing stem, up to 4 m in height KR087033 KR086947 KR070871 KR086996 KR087141 KR088308 Dioscorea cotinifolia Kunth AMM6112 (BOL) Mozambique, South Africa and Swaziland Open dry forest, forest margins, scrubby vegetation and rocky places Tuberous plant with vigorous annual twining stems reaching up to 10 m height KR087034 KR086948 KR070862 KR086987 KR087132 KR088299 Dioscorea cotinifolia Kunth AMM6158 (BOL) - - - KR087035 KR086949 KR070863 KR086988 KR087133 KR088300 Dioscorea cotinifolia Kunth OM1458 (BNRH) - - - KR087036 KR086950 KR070864 KR086989 KR087134 KR088301 Dioscorea dregeana (Kunth) T. Durand & Schinz AMM6104 (BOL) Mozambique and South Africa Forest, woodlands found among rocks and ravine Tuberous plants with annual to persistent stems reaching up to 12 m height KR087038 KR086952 KR070866 KR086991 KR087136 KR088303 Dioscorea dregeana (Kunth) T. Durand & Schinz AMM6166 (BOL) - - - KR087039 KR086953 KR070868 KR086993 KR087138 KR088305 Dioscorea dregeana (Kunth) T. Durand & Schinz OM1465 (BNRH) - - - JQ025042 JQ024957 KR070867 KR086992 KR087137 KR088304 Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 4 of 17 Table 1 Table of material and Genbank accession. Collectors references acronyms are Olivier Maurin (OM), Muthama Muasya (AMM), Sebsebe Demissew (SD), Pilar Catalán (PC), Ernesto Pérez-Collazos (EP). Genbank accessions in plain text are new to this study, accessions in bold were retrieved from www.ncbi.nlm.nih.gov (Continued) Dioscorea dregeana (Kunth) T. Durand & Schinz OM2247 (BNRH) - - - KR087037 KR086951 KR070865 KR086990 KR087135 KR088302 Dioscorea dumetorum (Kunth) Pax OM2315 (BNRH) Sub-Saharan Africa, excluding parts of Southern Africa Forest and woodlands and along riverbanks, generally at low altitude Tuberous plants with annual to persistent stems reaching up to 5 m height KR087040 KR086954 KR070869 KR086994 KR087139 KR088306 Dioscorea dumetorum (Kunth) Pax OM3953 (BNRH) - - - KR087041 KR086955 KR070870 KR086995 KR087140 KR088307 Dioscorea elephantipes (L’Hér.) Engl. AMM5225 (BOL) Namibia, South Africa - All Capes provinces From Medium to high altitude, in thorny and succulent vegetation (e.g. Thickets) Pachycaul up to 80 cm in diameter, with shoot up to 1 m height KR087042 KR086956 KR070872 KR086997 KR087142 KR088309 Dioscorea elephantipes (L’Hér.) Engl. AMM5226a (BOL) - - - KR087043 KR086957 KR070873 KR086998 KR087143 KR088310 Dioscorea elephantipes (L’Hér.) Engl. AMM6713 (BOL) - - - KR087044 KR086958 KR070874 KR086999 KR087144 KR088311 Dioscorea galeottiana Kunth Telez13090 (MEXU) Centre America, Mexico Tropical Dry Forest Perennial tuber, climber AY904796 AY956499 KM878046 KR087000 KR087145 KR088312 Dioscorea gillettii Milne-Redh. SD7051 (ETH) Southern Ethiopia Dry woodland vegetation Perennial tuber with twining stems up to 1.5 m height KR087046 KR086960 KR070876 KR087002 KR087147 KR088314 Dioscorea gillettii Milne-Redh. SD7052 (ETH) - - - KR087045 KR086959 KR070875 KR087001 KR087146 KR088313 Dioscorea hemicrypta Burkill AMM5800 (BOL) South Africa - Western Cape From Medium to high altitude, in thorny and succulent vegetation (e.g. Thickets) Pachycaul, partially emerged with annual shoot emerging from the crown KR087050 KR086964 KR070880 KR087006 KR087151 KR088318 Dioscorea hemicrypta Burkill AMM6633 (BOL) - - - KR087049 KR086963 KR070879 KR087005 KR087150 KR088317 Dioscorea hemicrypta Burkill AMM6886a (BOL) - - - KR087047 KR086961 KR070877 KR087003 KR087148 KR088315 Dioscorea hemicrypta Burkill AMM6697 (BOL) - - - KR087048 KR086962 KR070878 KR087004 KR087149 KR088316 Dioscorea kituiensis Wilkin & Muasya Mwachala 949a (K) Eastern Kenya Rocky area in dry woodlands Tuberous perennial with twining stems reaching 1.5 m height KR087051 KR086965 KR070881 KR087007 KR087152 KR088319 Dioscorea multiloba Kunth AMM6167 (BOL) South Africa - Eastern Cape, KwaZulu-Natal and Swaziland High altitude forests vegetation. Tuberous perennial with twining stems reaching 2 m in height KR087052 KR086966 KR070882 KR087008 KR087153 KR088320 Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 5 of 17 Table 1 Table of material and Genbank accession. Collectors references acronyms are Olivier Maurin (OM), Muthama Muasya (AMM), Sebsebe Demissew (SD), Pilar Catalán (PC), Ernesto Pérez-Collazos (EP). Genbank accessions in plain text are new to this study, accessions in bold were retrieved from www.ncbi.nlm.nih.gov (Continued) Dioscorea mundii Baker AMM6641 (BOL) South Africa - Western Cape Coastal forest vegetation Perennial with underground tuber from which stems arise (or from the bases of old stems) that climb to at least 5 m in height in surrounding vegetation KR087054 KR086968 KR070884 KR087010 KR087155 KR088322 Dioscorea mundii Baker AMM6642 (BOL) - - - KR087053 KR086967 KR070883 KR087009 KR087154 KR088321 Dioscorea orientalis (J. Thiébaut) Caddick & Wilkin Tori 1 (HUJ) Temperate western Asia Mediterranean Woodlands and Shrublands Geophyte, climber KM877858 KM877911 KM878066 KR087011 KR087156 KR088323 Dioscorea prazeri Prain & Burkill Wilkin1075 (K) Asia Open vegetation in mixed forests Rhizome, with climbing stem up to 5 m height AY973485 KM877871 KM878019 KR087012 KR087157 KR088324 Dioscorea pyrenaica Bubani & Bordère ex Gren. EP1038 (JACA) France, Spain (Pyrenees mountain range) On limestone rocks Tuberculous plant with short annual stems reaching 40 cm height KM877859 KM877912 KM878067 KR087013 KR087158 n.a. Dioscorea rupicola Kunth AMM3676 (BOL) South Africa - Eastern Cape, KwaZulu-Natal Occurring in open areas at high altitude in shady temperate and humid forest Perennial tuber with twining stems growing on surrounding vegetation KR087055 KR086969 KR070885 KR087014 KR087159 KR088325 Dioscorea sansibarensis Pax OM2421 (BNRH) Sub-Saharan Africa, including Madagascar but excluding Southern Africa Humid forest, low altitude, along riverine Large tuberous species with vigorous stems up to 30 m in length climbing and trailing on surrounding vegetation KR087056 KR086970 KR070886 KR087015 KR087160 KR088326 Dioscorea schimperana Hochst. ex Kunth OM2372 (BNRH) Tropical Africa, including Zambia, Zimbabwe, Malawi and Mozambique Open vegetation, on Rocks, termites’mounts and along riverbanks Annual tuber, producing vigorous shoots reaching 8 m height KR087058 KR086972 KR070888 KR087017 KR087162 KR088328 Dioscorea schimperana Hochst. ex Kunth OM3532 (BNRH) - - - KR087057 KR086971 KR070887 KR087016 KR087161 KR088327 Dioscorea stipulosa Uline ex R. Knuth AMM6748 (BOL) South Africa - Eastern Cape In fynbos, in moist rich soils Perennial tuber with annual shoot reaching up to 3 m in length KR087060 KR086974 KR070890 KR087019 KR087164 KR088330 Dioscorea stipulosa Uline ex R. Knuth AMM6800 (BOL) - - - KR087059 KR086973 KR070889 KR087018 KR087163 KR088329 Dioscorea strydomiana Wilkin AMM6124 (BOL) South Africa, Mpumalanga Open woodland Pachycaul up to 80 cm in diameter, with shoot up to 90 cm height KF147467 KF147390 KR070892 KR087021 KR087166 KR088332 Dioscorea strydomiana Wilkin Burrows 10627 (BNRH) - - - KR087061 KR086975 KR070891 KR087020 KR087165 KR088331 Dioscorea sylvatica Eckl. Burrows 12487 (BNRH) Southern Africa Found from low to high altitude in a variable range of KR087062 KR086976 KR070893 KR087022 KR087167 KR088333 Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 6 of 17 Table 1 Table of material and Genbank accession. Collectors references acronyms are Olivier Maurin (OM), Muthama Muasya (AMM), Sebsebe Demissew (SD), Pilar Catalán (PC), Ernesto Pérez-Collazos (EP). Genbank accessions in plain text are new to this study, accessions in bold were retrieved from www.ncbi.nlm.nih.gov (Continued) vegetation from Dunes, to rocky outcrop and open woodland vegetation. Perennial tuber, with Herbaceous annual stem reaching 4 m in length Dioscorea sylvatica Eckl. OM1433 (BNRH) - - - KR087063 KR086977 KR070894 KR087023 KR087168 KR088334 Dioscorea sylvatica Eckl. f. glauca Burrows 12477 (BNRH) - - - KR087064 KR086978 KR070895 KR087024 KR087169 KR088335 Dioscorea tentaculigera Prain & Burkill Thapyai 436 South Central Chine, Myanmar and Thailand Evergreen forest from medium to high altitude Perennial tuber, with climbing stems up to 4 m in length AY972828 AY939886 KM878070 KR087025 KR087170 KR088336 Tamus edulis Lowe (combination in Dioscorea pending) Chase 3425 (K) Mediterranean region Woodland and woodland hedges Herbaceous with climbing stem AY939891 AY973843 KR070861 KR086986 KR087131 KR088298 Outgroups Stenomeris borneensis Oliv. Brun19174 (K) Tropical Asia - - AF307475 AY973836 ---- Tacca plantaginea (Hance) Drenth ZL002 (n.a.) Tropical Asia - - JF944619 JF956650 ---- Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 7 of 17 (85–100 %), moderate (75–84 %) or low (50–74 %). Bootstrap values are provided in Fig. 3. All data sets were analyzed separately, and the individual bootstrap consensus trees examined by eye to identify topological conflicts, i.e. moderate to high support for different placement of taxa. In order to test for significant conflicts between the independent DNA data matrices, a partition homogeneity test was performed [32–34]. The Incongruence Length Difference (ILD) test of Farris et al. [32] implemented in PAUP* 4.0 b10 [30] was performed through 1000 random-order-entry replicates to estimate if the six datasets were significantly different from random partitions of the same size. Nonsignificant results indicated that the six data sets were not heterogeneous. Highly congruent contrasted topologies (see Results) also supported the merging of the four data matrices into a single concatenated data set that was used for subsequent phylogenetic analyses. Bayesian analysis (BI; [35, 36]) was performed using MRBAYES v. 3.1.2. For each matrix rbcLa,matK,trnL-F, trnH-psbA,psaA-ycf3 and rpl32-trnL the most appropriate model was selected using MODELTEST v. 3.06 [37]. For matK and trnL-F the model TVM + G was selected, then for rbcLa,trnH-psbA,psaA-ycf3 and rpl32-trnL, the following model were selected, respectively TVM + I, HKY + G, HKY + I + G and GTR + G. The analysis was run on the CIPRES cluster [38] using a MCMC of 10 million generations with a sample frequency of 500, imposing the closest nst = 6 rates = gamma model available in the program. The resulting trees were plotted against their likelihoods to determine the point where likelihoods converged on a maximum value, and all the trees before the convergence were discarded as ‘burn-in’ (5000 trees). All remaining trees were imported into PAUP 4.0b10, and a majority-rule consensus tree was produced showing frequencies (i.e. posterior probabilities or PP) of all observed bi-partitions. The following scale was used to evaluate the PPs values: below 0.95, weakly supported; 0.95-1.00, well supported. Divergence time estimation Divergence times were estimated using a Bayesian MCMC approach implemented in BEAST (v. 1.4.8; [39]), which allows simultaneous estimation of the topology, substitution rates and node ages [39]. The GTR + I + G implemented model of sequence evolution for each partition based on the Akaike information criterion (AIC) scores for substitution models evaluated using MrModeltest (version 2.3; [40]) with a gamma-distribution with four rate categories. A speciation model following a Yule process was selected as the tree prior, with an uncorrelated lognormal (UCLN) model for rate variation among branches. For this analysis, we used a single representative per species since the Yule speciation model forces the analysis to “create”speciation events at every node and therefore makes the estimation of splits older within a species. First, the Bayesian consensus tree topology was used as a starting tree and adjusted so that branch lengths satisfied all fossil prior constraints, using PATHd8 v.1.0 [41]. Fossil dates or calibration points were used to constrain specific nodes to minimum, maximum or fixed ages. The crown node age of Dioscoreaceae was calibrated at 80 mya according to Jansen & Bremer [42]. A first fossil, Dioscorea lyelli (Wat.) Fritel, was used to calibrate the node of Dioscorea prazeri Prain & Burkill, (representative of the Stenophora clade). The fossil was discovered in the Cuisian stage of the Ypresian age at the Paris basin [43] and provided a minimum constraint of 48.2 ± 1.0 mya (LogNormal Prior mean = 48.2, SD 0.008) for the stem node of Stenophora. A second fossil, D. wilkinii Pan, attributed to the node of the Compound Leaved clade that comprises D. dregeana - D. dumetorum, provided a minimum constraint of 27.2 ± 0.1 mya (LogNormal Prior mean = 27.2, SD 0.002) for that node [44]. We performed four independent runs of MCMC, each for 100 million generations, sampling every 1000 generations. We assessed the MCMC log files for convergence using the effective sample size (ESS) statistics in Tracer v.1.5 [39]. The BEAST analysis reported ESS values > 200, indicating that the posterior estimates were not unduly influenced by autocorrelation. The resulting tree files from the four runs were then combined using LogCombiner v.1.7.5 [39], discarding the first 25 % trees as burn-in. The maximum clade credibility consensus tree, with means and 95 % highest posterior density (HPD) intervals, was generated with TreeAnnotator v.1.7.5 [39]. Map preparation Distribution maps illustrated on Fig. 2 were prepared using occurrence data downloaded from http://newposa.sanbi.org and http://www.gbif.org. Distribution ranges were drawn on Adobe ® Illustrator ® CS6. Figure 2a represents the occurrence of the three major subclades occurring in South Africa, the Pachycaul, Cape and D. buchananii subclades, while Fig. 2b displays the distribution of all species belonging to the Pachycaul subclade. Results Statistics for MP analysis for the six plastid markers and combined dataset are presented in Table 2. Of all the genes used, matK and rpl32-trnL had a significantly higher number of variable sites (27.85 % and 25.17 % respectively) compared to the other regions than display percentages below 10 % (see Table 2). The number of potentially informative characters is higher for matK (12.07 %) than rpl32-trnL (10.01 %), however contribution to total of parsimony informative character (PIC) is lower for matK (26.35 %) than for rpl32-trnL (30.84 %; Table 2). Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 8 of 17 Maximum parsimony analyses MP analysis of each of the six regions resulted in trees that were similar in topology (Additional files 1, 2, 3, 4, 5 and 6), and were thus combined and treated as a single dataset. ILD test results provide support for congruence (p> 0.05). The psaA-ycf3 region is significantly different from rbcLa,matK,trnL-F,trnH-psbA, and rpl32-trnL and probably caused by the psaA-ycf3 sequence of D. galeottiana. However, the observed congruence between the trees obtained for each region separately and the Fig. 2 Distribution maps and habitat images of the southern African Dioscorea taxa. aDistribution map of the three South African subclades of the Africa clade: Pachycaul, Cape and D. buchananii.bDistribution map of the five Pachycaul subclade species in South Africa. Note that the distribution of D. elephantipes extends slightly into Namibia, and D. sylvatica extends into Mozambique, Zambia and Zimbabwe. From cto g:inorder,habitatof D. elephantipes,Dioscorea brownii,D. hemycrypta,D. strydomiana, and D. sylvatica. The habitat image of D. elephantipes (c) displays in the foreground shoots and fruits of this taxa; the habitat image for D. strydomiana has an immature or damaged specimen in the bottom right corner. All other images only show the habitat and individuals of the species are not visible. Photographs: c-g:PaulWilkin Maurin et al. BMC Evolutionary Biology (2016) 16:238 Page 9 of 17 Conclusions Diversification out of forest is associated with a major increase in perennial tuber size and change in tuber orientation from horizontal to vertical, both of which presumably underlie the development of pachycauly. There is also a shift in stem habit, from twining on supporting vegetation to erect and self-supporting. This diversification does not show association with reproductive morphology, except in the seed wing, which has switched from being winged all round the seed margin (to promote gliding flight) to only on its basal or apical side (generating spinning flight). The wing has even been completely replaced by an elaiosome in two species. The single pollinator shift event is observed within the forest biome. Although only D. brownii and D. strydomiana currently occur within typical fire driven grassland, the transition of the vegetation from closed habitat to savanna grasslands occurring during the Miocene and Pliocene, with an associated increase in fire regime and similar time of origin of other southern African savannah flora elements, suggests that this change has influenced the development of corky barks covering the above-ground pachycauls and therefore the origin of efficient fire and perhaps herbivory protection. Additional files Additional file 1: rbcLa MP tree. One of the most equally parsimonious tree generated from the Maximum Parsimony (MP) analysis using rbcLa sequence dataset. Values above branches are number of steps and values below branches are reported percentage of Bootstrap support values. Collapsing branches from the strict consensus tree obtained in the combined Maximum Parsimony (MP) analysis are illustrated with a •. (PDF 210 kb) Additional file 2: matK MP tree. One of the most equally parsimonious tree generated from the Maximum Parsimony (MP) analysis using matK sequence dataset. Values above branches are number of steps and values below branches are reported percentage of Bootstrap support values. Collapsing branches from the strict consensus tree obtained in the combined Maximum Parsimony (MP) analysis are illustrated with a •. (PDF 230 kb) Additional file 3: trnL-F MP tree. One of the most equally parsimonious trees generated from the Maximum Parsimony (MP) analysis using trnL-F sequence dataset. Values above branches are number of steps and values below branches are reported percentage of Bootstrap support values. Collapsing branches from the strict consensus tree obtained in the combined Maximum Parsimony (MP) analysis are illustrated with a •. (PDF 221 kb) Additional file 4: trnH-psbA MP tree. One of the most equally parsimonious trees generated from the Maximum Parsimony (MP) analysis using trnH-psbA sequence dataset. Values above branches are number of steps and values below branches are reported percentage of Bootstrap support values. Collapsing branches from the strict consensus tree obtained in the combined Maximum Parsimony (MP) analysis are illustrated with a •.(PDF206kb) Additional file 5: psaA-ycf3 MP tree. One of the most equally parsimonious trees generated from the Maximum Parsimony (MP) analysis using psaA-ycf3 sequence dataset. Values above branches are number of steps and values below branches are reported percentage of Bootstrap support values. Collapsing branches from the strict consensus tree obtained in the combined Maximum Parsimony (MP) analysis are illustrated with a •. (PDF 220 kb) Additional file 6: rpl32-trnL MP tree. One of the most equally parsimonious trees generated from the Maximum Parsimony (MP) analysis using rpl32-trnL sequence dataset. Values above branches are number of steps and values below branches are reported percentage of Bootstrap support values. Collapsing branches from the strict consensus tree obtained in the combined Maximum Parsimony (MP) analysis are illustrated with a •.(PDF224kb) Additional file 7: Combined MP tree. One of the most equally parsimonious trees generated from the Maximum Parsimony (MP) analysis using the combined sequence dataset. Values above branches are number of steps and values below branches are reported percentage of Bootstrap support values. Collapsing branches from the strict consensus tree obtained in the combined Maximum Parsimony (MP) analysis are illustrated with a •. (PDF 264 kb) Abbreviations CL: Compound-Leaved; CTAB: Hexadecyltrimethylammonium bromide; ILD: Incongruence Length Difference; NWI: New World I (clade); NWII: New World II (clade); Xdh (nuclear region Xdh): Newly Sequenced Nuclear Gene Acknowledgements We thank the Government of Canada through Genome Canada and the Ontario Genomics Institute (2008-OGI-ICI-03), the International Development Research Centre (IDRC), Canada and the Central Analytical Facility of the Faculty of Science, University of Johannesburg (Spectrau), the Spanish Aragon Government and the European Social Fund (PC, Bioflora research grant), and the South African National Research Foundation for financial support towards fieldwork and sequencing. Plant samples were collected with permission of land owners and respective authorities, with special thanks for assistance going to Charles Stirton, Graham Grieves, Geoffrey Mwachala, John Burrows, Sebsebe Demissew, the late Tony Abbotts. The Royal Botanic Gardens, Kew, DNA Bank, in particularly Felix Forest, Laszlo Csiba and Rhina Duque-Thues, for DNA aliquots and Bezeng S. Bezeng for assistance with preparation of distribution maps. Availability of data and materials The trace files and sequences are available on the Barcode of Life Data System (BOLD; www.boldsystems.org). All trees and the combined data matrix are available on request from the authors ([email protected]). Authors’contributions OM, AMM, PW: designed research; OM, EZS, MvdB: performed research; MvdB: contributed new reagents/analytic tools; PC, JV: provided some data and guidance; OM, JV: analyzed data; OM, AMM, PW, MvdB, PC: wrote the paper. All authors read and approved the manuscript. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Not applicable for this study. Author details 1 African Centre for DNA Barcoding, Department of Botany & Plant Biotechnology, University of Johannesburg, PO Box 524 Auckland Park, Johannesburg, Gauteng 2006, South Africa. 2 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, UK. 3 Department of Biological Sciences, University of Cape Town, Rondebosch, Western Cape 7701, South Africa. 4 Departamento de Ciencias Agrarias y del Medio Natural, Escuela Politécnica Superior de Huesca, Universidad de Zaragoza, Ctra. Cuarte km 1, Huesca 22071, Spain. 5 Institute of Biology, Tomsk State University, 36 Lenin Avenue, Tomsk 634050, Russia. 6 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Av. 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