Phylogenetic relationships, character evolution and biogeography of southern African members of Zygophyllum (Zygophyllaceae) based on three plastid regions

Dirk Bellstedt

Zygophyllaceae (s.l.) are a heterogeneous family with disagreement about the systematic status of some groups within the taxon. By using UPGMA method, the phenogram produced from numerical analysis of 81 morphological characters of the 44 species belonging to eight genera of Zygophyllaceae, Nitrariaceae and Peganaceae in Egypt; resulted only one tree with three major phenons, two phenons including Tribuloideae (Tribulus) and Zygophylloideae (Zygophyllum and Fagonia), with the ultimate phenon including three monospecific genera, Peganum, Nitraria and Seetzenia which are closely allied to the zygophylloid phenon. The position of these genera has been disputed. Additionally, Balanites and Tetradiclis split off as two outgroups, and these genera have been recognized as distinct families (Balanitaceae; Tetradiclidaceae) or at least subfamilies (Balanitoideae; Tetradiclidoideae). Two keys were constructed using the DELTA software, one for identification and a second as a confirmatory key. These keys were designed to facilitate the identification of the Egyptian taxa.

Patterns in traits and trait combinations reflect how organisms cope with their environment. Owing to different degrees of variability, the performance of traits varies during adaption to the changing environment. In this study, we focused on a taxon dominant in arid regions – the subfamily Zygophylloideae. We analyzed the evolutionary patterns of functional traits to clarify the impact of trait attributes on niche shifts. The results of phylogenetic signal analysis of traits revealed that quantitative traits, such as plant height, were not evolutionarily conserved. Phylogenetic regression pointed out that there are synergistic changes in environmental factors and in some traits within a phylogenetic context. These traits can meet the requirements of different environments more easily, possibly owing to their high variability. As a result, species in the subfamily Zygophylloideae showed clustering in some phenotypic spaces. Thus, the adaptive evolution of traits reduced niche restri...

Zygophyllum L., the largest genus of Zygophyllaceae comprises about 100 species known from the Mediterranean to Central Asia, more than eleven species growing in Saudi Arabia specially in desert and saline habitats. This study deals with taxonomy of Zygophyllum species growing in Saudi Arabia depending on anatomical characters of leaves and petioles to eleven investigated species. Anatomical features of leaves and petiole of the eleven investigated Zygophyllum species show characters of major importance such as stem outline, leaf outline, the arrangement of leaf vascular tissue, in addition to the number of vascular bundles in the inner leaf whorl, and characters of minor importance such as leaf mesophyll, and the branching of the main vascular bundle in the petiole Those characters enable us to separate Z. simplex from the other ten species by its cup shape transverse section in stem. Leaf vascular tissue is arranged either in straight line, or in two whorls . Petiole vascular bundles tissue arranged in two whorls the outer with number of vascular bundles, the inner whorl with two type: one main central vascular bundle and one main central vascular bundle associated with two peripheral vascular bundles. According to all those anatomical characters an artificial key explain the difference between the eleven investigated Zygophyllum species.

Molecular Phylogenetics and Evolution 47 (2008) 932–949 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Phylogenetic relationships, character evolution and biogeography of southern African members of Zygophyllum (Zygophyllaceae) based on three plastid regions D.U. Bellstedt a,*, L. van Zyl b, E.M. Marais c, B. Bytebier a, C.A. de Villiers a, A.M. Makwarela b, L.L. Dreyer b a Department of Biochemistry, University of Stellenbosch, Stellenbosch, Private Bag X1, Matieland 7602, South Africa Department of Botany and Zoology, University of Stellenbosch, Stellenbosch, South Africa c Department of Forest and Wood Science, University of Stellenbosch, Stellenbosch, South Africa b a r t i c l e i n f o Article history: Received 8 June 2007 Revised 19 February 2008 Accepted 23 February 2008 Available online 29 February 2008 Keywords: Zygophyllaceae Zygophylloideae Augea Fagonia Tetraena Zygophyllum trnLF rbcL Phylogeny a b s t r a c t The plastid coding rbcL and non-coding trnLF regions of 53 of 55 southern African Zygophyllum species were sequenced and used to evaluate the phylogenetic relationships within the southern African representatives of the genus. Published sequences of the same gene regions of Australian, Asian and North African Zygophyllum species were included to assess the relationships of the species from these regions to the southern African species. The addition of Z. stapffii from Namibia, found to be conspecific with Z. orbiculatum from Angola, lead to a greatly resolved tree. The molecular results were largely congruent with a recent sectional classification of the southern African species and supported their subdivision into subgenera Agrophyllum and Zygophyllum. Reconstruction of the character evolution of capsule dehiscence, seed attachment and seed mucilage showed that these characters allowed a division of southern African species into the two subgenera but that this could not be applied to species occurring elsewhere. Other morphological characters were found to vary and unique character combinations, rather than unique characters, were found to be of systematic value in sectional delimitation. The study suggests that repeated radiations from the horn of Africa to southern Africa and Asia and back lead to the present distribution of the taxa in the subfamily Zygophylloideae. Although this study supports some of the recent taxonomic changes in the group, the unresolved relationships between the proposed genera Tetraena and Roepera and those retained as Zygophyllum species suggest that changes to the taxonomy may have been premature. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction The genus Zygophyllum L. is widely distributed in the arid and semi-arid areas of southern, northern and north-eastern Africa (White, 1983), the Mediterranean region, central Asia and Australia (Retief, 2000). In southern Africa it ranges from the southern parts of the Eastern Cape, through the Western Cape and Northern Cape Provinces to Namibia, southwestern Botswana and as far north as southern Angola. It occurs in seven of the eight biomes defined by Low and Rebelo (1996), with the greatest number of species present in the Nama and Succulent Karoo biomes (Van Zyl, 2000). Together with the genus Salsola L. (Chenopodiaceae), it ranks as one of the most drought resistant plant genera in these arid areas. As a result, it is a vital food source for small stock and game in the very arid areas of the Northern Cape Province of South Africa and southern Namibia. Various authors have attempted an infrageneric classification for the genus Zygophyllum. Endlicher (1841) subdivided the genus * Corresponding author. Fax: +27 21 8085863. E-mail address: dub@sun.ac.za (D.U. Bellstedt). 1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.02.019 into subgenera Zygophyllum and Agrophyllum Endl. based on fruit dehiscence, while Sonder (1860; South African species) and Engler (1931, all species) ignored this character in their attempts to define a sectional classification. Engler (1931) recognized 17 sections worldwide. Van Huyssteen (1937) again recognized the taxonomic significance of flower and fruit characters and circumscribed the subgenera Zygophyllotypus Huysst. (referred to as subgenus Zygophyllum by Van Zyl, 2000) and Agrophyllum based on fruit dehiscence. The sectional classification of southern African members of the genus according to Van Huyssteen (1937) is summarized in Table 1. Outside of southern Africa, she recognized the sections Fabago Tourn. ex Adans. (22 species from Asia including the type species, Z. fabago L.) and Sarcozygium (Bunge) Engl. (one species from Mongolia, Z. xanthoxylum Engl.) in subgenus Zygophyllotypus and the sections Mediterranea Engl. (11 species from northern and eastern Africa) and Roepera Engl. (one species, Z. fruticulosum DC. from Australia) in subgenus Agrophyllum. In her revision of southern African Zygophyllum, Van Zyl (2000) recognized 54 species of which 17 were new, five of these have been published (Van Zyl and Marais, 1997, 1999), D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 933 Table 1 A comparison of the classifications of southern African Zygophyllum of Van Huyssteen (1937) and Van Zyl (2000) Van Huyssteen (1937) Subgenus Agrophyllum Endl. § Alata Huysst. subsection Angustialata Huysst. § Alata subsections Morgsana Huysst. § Bipartita Huysst. § Cinerea Huysst. Van Zyl (2000) 6 species § Alata 3 species (3 species reduced to synonomy) § Annua Engl. § Bipartita Z. simplex L. (transferred from § Alata) and 2 new species 10 species (3 species reduced to synonomy, Z. simplex L. (transferred to § Alata), Z. chrysopteron Retief and 3 new species Z. giessii Merxm. & A.Schreib., Z. longicapsulare Schinz Z. morgsana L. 10 species including Z. simplex L. Z. cinereum Schinz = Z. longicapsulare Schinz § Cinerea § Grandifolia Engl. § Prismatica Van Zyl Subgenus Zygophyllotypus (referred to as subgenus Zygophyllum by Van Zyl (2000) § Capensia Engl. 24 species § Capensia § Morgsana (Huysst.) Van Zyl § Paradoxa Huysst. Z. paradoxum Schinz, Z. cordifolium L.f., § Paradoxa Z. orbiculatum Welw. ex Oliv. § Grandifolia Z. stapffii Schinz and 12 remain to be published (Table 2). Based on a comprehensive analysis of morphological characters, she divided these species into the two subgenera, Zygophyllum and Agrophyllum on the basis of capsule dehiscence, seed attachment and the presence of spiral threads in the seed mucilage. Her classification is compared to that of Van Huyssteen in Table 1. The most important changes made to Van Huyssteen’s (1937) classification were the transfer of section Grandifolia (containing only Z. stapffii), from Zygophyllum to Agrophyllum and of section Morgsana (containing only Z. morgsana) from subgenus Agrophyllum to subgenus Zygophyllum. In a study to establish the familial boundaries and intrafamilial relationships of the family Zygophyllaceae, Sheahan and Chase (1996) on the basis of sequence data from the plastid rbcL gene of all representative genera, recircumscribed the family to comprise the subfamilies Zygophylloideae, Tribuloideae, Seetzenioideae, Larreoideae and Morkillioideae. They re-defined the subfamily Zygophylloideae to include the genera Zygophyllum (ca. 150 species), Fagonia L. (30 species) and the monotypic genera Augea Thunb. and Tetraena Maxim. Subsequently, in an expanded study based on the trnLF and rbcL sequences of 15 species of Zygophyllum, three of Fagonia, and the monotypic Augea and Tetraena, Sheahan and Chase (2000) attempted to establish the relationships of these genera within the subfamily Zygophylloideae. Only six species of southern African Zygophyllum were included in this analysis, of which three belong to subgenus Agrophyllum and three to subgenus Zygophyllum sensu Van Zyl (2000). Species from subgenus Agrophyllum grouped with Asian and North African Zygophyllum species and with Tetraena; those from subgenus Zygophyllum grouped with Australian species. The genera Fagonia and Augea and two Zygophyllum species from the horn of Africa, Z. hildebrandtii Engl. and Z. robecchii Engl., formed a separate clade. The relationships between some of these clades were, however, poorly supported. Beier et al. (2003) expanded on the previous study. They used a wider sampling as well as morphological data and trnL intron sequence data to elucidate the relationships within the subfamily Zygophylloideae. In addition to the six southern African Zygophyllum species used by Sheahan and Chase (2000), they included Z. morgsana and Z. foetidum and the monotypic genera Augea and Tetraena. Beier et al. (2003) retrieved a monophyletic subfamily Zygophylloideae, but a paraphyletic genus Zygophyllum Z. stapffii Schinz Z. prismatocarpum Sond. (transferred from section Bipartita) and 2 new species 29 species Z. morgsana L. Z. cordifolium L.f., Z. fusiforme Van Zyl ined., Z. orbiculatum Welw. ex Oliv. including the genera Fagonia, Augea and Tetraena. Within the paraphyletic Zygophyllum, a number of strongly supported clades were retrieved, but relationships between these groupings were poorly supported. Their phylogeny also did not retrieve the subgenera Zygophyllum and Agrophyllum, as circumscribed by Van Huyssteen (1937) and Van Zyl (2000). They used these results to establish a new generic classification for the Zygophylloideae. The generic classification was supported by a limited number of morphological synapomorphies. In the current study, the plastid trnL intron and trnLF spacer sequences of a near complete sampling (53 out of 55 taxa) of the southern African Zygophyllum species were combined with published trnL intron and trnLF spacer sequences (Sheahan and Chase, 2000) of Zygophyllum species occurring elsewhere and the phylogenetic relationships were analyzed. To confirm the position of some species in the tree, the rbcL gene of a representative subset of species was also sequenced and the combined rbcL and trnLF sequence data were analyzed. These trees were used to assess the subgeneric and sectional boundaries defined by Van Zyl (2000). Patterns of evolution of three seed characters were traced onto the combined results. Additionally, phytogeographical trends within the subfamily Zygophylloideae were assessed based on the combined tree. Our results were then compared to the newly proposed generic classification of Beier et al. (2003). 2. Materials and methods 2.1. Sampling of taxa Fifty-two out of the 54 Zygophyllum species recognized by Van Zyl (2000) and an undescribed species, referred to as Zygophyllum spec. aff. maritimum, were sampled (Table 2). All subgenera and sections sensu Van Zyl (2000) were represented. 2.2. Outgroups Outgroup taxa were chosen on the basis of Sheahan and Chase (1996, 2000) and Beier et al. (2003). These included Tribulus macropterus as the most distant outgroup (Tribuloideae), Seetzenia lanata (Seetzenioideae), and the New World species Bulnesia arborea, Guaiacum guatemalense and Larrea tridentata (Larreoideae). 934 Table 2 Sectional classification of the southern African members of the genus Zygophyllum sensu Van Zyl (2000), geographic distribution, voucher details, collection locality and database accession number Section Species Subgenus Agrophyllum § Annua Z. simplex L. Z. spongiosum Van Zyl ined. Z. inflatum Van Zyl ined. Geographic distribution Voucher details Collection locality Database accession number, trnLF Database accession number, rbcL Africa & Asia Chase 806 (K) Bellstedt 854 (STE) HK 1573 (WIND) Northern Hemisphere Orange River bed, Aussenkehr, Namibia 27 km from Otjinungwa on Kunene River along track to Rooidrom, Kaokoveld, Kunene Region, Namibia 7.5 km from Ruacana Falls on road to Swartbooisdrift, Kunene Region, Namibia *AJ387974 EF 656004 EF 656006 *Y15031 EF655984 EF655985 EF 656005 nd NN & southern Angola NN & southern Angola HK 1490 (WIND) Z. prismatocarpum Sond. Z. patenticaule Van Zyl ined. Z. pterocaule Van Zyl SN & NC SN & NC SN & NC Bellstedt 860 (STE) Bellstedt 868 (STE) Mucina 270806/25 (STE) Orange River embankment, Rosh Pinah district, Namibia Lorelei Farm, Rosh Pinah district, Namibia Alexander Bay, Richtersveld, NC EF 656009 EF 656008 EF 656007 EF655990 EF655989 nd § Bipartita Z. applanatum Van Zyl Z. clavatum Schltr. & Diels in Schultze Z. cylindrifolium Schinz Z. segmentatum Van Zyl Z. tenue R. Glover Z. retrofractum Thunb. SN Coastal SN & NC Bellstedt 870 (STE) Bellstedt 878 20 km N of Rosh Pinah, Namibia Lüderitz Peninsula, Namibia EF 656012 EF 656010 EF655988 EF655986 NWN SN SN SN, NC & inland WC NC & inland WC Inland WC N & NC Craven 3800 (WIND) Bellstedt 861 (STE) van Zyl 4593 (STE) Marais 430 (STE) Twyfelfontein Rock Art Site, Namibia Boom Riverbed, West of Rosh Pinah, Namibia S Namibia Doring River Crossing, Ceres Karoo, South Africa *AJ387966 EF 656015 EF 656017 EF 656014 *AJ133864 EF655987 nd nd Marais 427 (STE) Bellstedt 799 (STE) van Zyl 4588 (STE) Doring River Crossing, Ceres Karoo, South Africa Prince Albert, South Africa S Namibia EF 656013 EF 656016 EF 656011 nd nd EF655991 *AJ387967 *AJ133865 Z. chrysopteron Retief Z. turbinatum Van Zyl ined. Z. decumbens Delile var. decumbens Z. decumbens Delile Horn of Africa Thulin et al. 7981 UPS § Alata Z. microcarpum Licht. ex Cham. Z. rigidum Schinz Z. longistiputalum Schinz SN, NC & WC SN & NC SN van Zyl 4591 (STE) van Zyl 4590 (STE) not collected S Namibia S Namibia Southern Namibia EF 656002 EF 656003 EF655983 EF655982 nd § Cinerea Z. longicapsulare Schinz Z. giessii Merxm. & A.Schreib. Z. stapffii Schinz SN SN Coastal NN Bellstedt 879 (STE) Bellstedt 874 (STE) Mannheimer 6577 (STE) Lüderitz Peninsula, Namibia Arimas Farm, Witputs district, Namibia Swakopmund, Namibia EF 656001 EF 656000 ** EF655981 EF655980 ** SN, NC & WC SN & NC NN & southern Angola Marais 446 (STE) Bellstedt 857 (STE) Craven 5096 (WIND) Koekenaap, South Africa Aussenkehr, Namibia 30 km west of Foz de Cunene, Southern Angola EF 656022 EF 656023 EF 655999 EF655993 nd EF655979 § Grandifolia Subgenus Zygophyllum § Paradoxa Z. cordifolium L.f. Z. fusiforme Van Zyl ined. Z. orbiculatum Welw. ex Oliver § Capensia I Z. Z. Z. Z. Z. teretifolium Schltr. botulifolium Van Zyl spinosum L. pygmaeum Eckl. & Zeyh. rogersii Compton Inland WC WC Coastal NC & WC NC & inland WC Inland WC Marais 447 (STE) Marais 451 (STE) Bellstedt 801 (STE) Marais 424 (STE) Marais 432 (STE) Douse the Glim road, Van Rhynsdorp district, WC Farm Bulslaagte, Ceres to Matjiesfontein Road, Tanqua Karoo, WC Rondeberg Nature Reserve, North of Cape Town Robertson, South Africa Hartnekskloof, Ceres Karoo, WC EF EF EF EF EF 656025 656026 656038 656046 656037 nd nd nd nd nd § Capensia II Z. Z. Z. Z. divaricatum Eckl. & Zeyh. namaquanum Van Zyl ined. sessilifolium L. spitskopense Van Zyl ined. Coastal SC & EC NC Coastal WC Coastal WC Dold 4655 (GRA) Marais 440 (STE) Marais 434 (STE) van Zyl 4606 (STE) Coega River Mouth, EC Studor’s Pass, Kamieskroon, NC Farm Neulfontein, Morreesburg district, WC Morreesburg District, WC EF EF EF EF 656031 656036 656047 656048 nd nd EF655997 nd D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 § Prismatica § Capensia III § Morgsana cuneifolium Eckl. & Zeyh. hirticaule Van Zyl calcicola Van Zyl ined. fulvum L. porphyrocaule Van Zyl ined. swartbergense Van Zyl ined. fuscatum Van Zyl flexuosum Eckl. & Zeyh. Marais 455 (STE) van Zyl 3894 (STE) Dreyer s.n. (STE) van Zyl 4605 (STE) Bellstedt 800 (STE) Bellstedt 798 (STE) Bellstedt 892 (STE) Bellstedt 794 (STE) Lutzville District, WC Witputz district, Namibia Stilbaai, South Africa Franschhoek Pass, WC Hex River Pass, South Africa Swartberg Pass, Prince Alfred district, South Africa Betty’s Bay, South Africa Langebaan, South Africa EF 656024 *AJ387972 EF 656030 EF 656044 EF 656018 EF 656043 EF 656045 EF 656032 nd *AJ133869 nd nd EF655992 EF655996 nd EF655995 Z. lichtensteinianum Cham. NC, WC & EC Van Zyl 4594 (STE) Laingsburg district, WC EF 656020 nd Z. incrustatum E.Mey. ex Sond. Z. maritimum Eckl. & Zeyh. Z. aff. maritimum Z. debile Cham. Z. cretaceum Van Zyl ined. Z. foetidum Schrad. & J.C.Wendtl. Bellstedt 509 (STE) Dold 4656 (GRA) Dold 4654 (GRA) Bellstedt 796 (STE) Bellstedt 856 (STE) Marais 423 (STE) Beaufort West, South Africa Coega River Mouth, South Africa Somerset Heights, Grahamstown, South Africa 20 km N of Oudtshoorn, South Africa Aussenkehr, Namibia Robertson, South Africa EF EF EF EF EF EF 656019 656035 656034 656041 656028 656039 nd nd nd nd nd nd Z. macrocarpon Retief Z. maculatum Aiton Z. schreiberanum Merxm. & Giess Z. leptopetalum E.Mey. ex Sond. Z. pubescens Schinz Z. leucocladum Diels in Schultze NC, WC & EC Coastal WC & EC Coastal WC & EC WC & EC SN & NC Inland NC, WC & EC SN & NC Inland WC SN SN, NC & WC SN & NC SN not collected Marais 433 (STE) Bellstedt 871 (STE) Marais 422 (STE) Bellstedt 881 (STE) van Zyl 4479 (STE) SN Hartnekskloof, Ceres Karoo, South Africa 20 km N of Rosh Pinah, Namibia Klawer, South Africa Tiras Farm, Aus district, Namibia Witputz district, Namibia nd EF 656033 EF 656027 EF 656040 EF 656042 EF 656029 nd nd nd nd nd nd Z. morgsana L. NC, WC & EC Bellstedt 890 (STE) Steinkopf, South Africa EF 656021 EF655994 Augea capensis Thunb. Bulnesia arborea Engl. Fagonia cretica L. Fagonia indica Burm.f. Fagonia luntii Baker Guaiacum guatemalense Planch. Rydb. & Vail Larrea tridentata Coult. Seetzenia lanata (Willd.) Bullock Inland WC New World North Africa Horn of Africa Horn of Africa New World Bellstedt 934 (STE) Chase 641 (K) Chase 3432 (K) Collenette 10/93 (K) Wieland 4504 (K) Chase 640 (K) Ceres Karroo, WC EF 655998 *AJ387947 *AJ387942 *AJ387943 *AJ387944 *AJ387948 EF655978 *Y15017 *AJ133855 *Y15018 *AJ133856 *Y15019 New World Koue Bokkeveld, SA Mongolia Africa Asia Asia Australia Australia Chase 636 (K) Herman 3964 (K) *AJ387951 *AJ387956 *Y15022 *Y15025 Sheahan 1994 (K) Collenette 3/93 (K) Chase 516 (K) Chase 1700 (K) Chase 2204 (K) SR 417 Adelaide Bot Gardens Chase 2203 (K) Thulin et al. 9012 (UPS) Thulin et al. 8428 (UPS) Ryding 1347 (K) >Thulin et al. 7977 (UPS) *AJ387959 *AJ387961 *AJ387968 *AJ387975 *AJ387970 *AJ387964 *Y15027 *Y15028 *Y15030 *AJ133872 *AJ133867 *AJ133862 *AJ387969 *AJ387971 *AJ133866 *AJ133868 *AJ387973 *AJ133870 *AJ387965 *AJ387963 *AJ133863 *AJ133861 Additional species Tetraena mongolica Maxim. Tribulus macropterus Boiss. Z. fabago L. Z. xanthoxylum Engl. Z. glaucum F.Muell. Z. billardieri DC. Z. fruticulosum DC. Melocarpum hildebrandtii (Engl.) Beier & Thulin Melocarpum robecchii (Engl.) Beier & Thulin Z. coccineum L. Z. album L.f. Australia Horn of Africa Horn of Africa Middle East Middle East D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 Coastal WC SN Coastal WC Coastal WC Inland WC Inland WC Coastal WC Coastal WC Z. Z. Z. Z. Z. Z. Z. Z. Abbreviations used: Namibia (N), Northern Namibia (NN), North Western Namibia (NWN), Southern Namibia (SN), South Africa (SA) South African provinces: Eastern Cape (EC), Northern Cape (NC), Western Cape (WC). Sequences downloaded from GenBank are indicated with an *, nd = not determined. The sequences of Z. stapffii were found to be identical to those of Z. orbiculatum and are indicated with an **. 935 936 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 2.3. Molecular data DNA was extracted using the CTAB method of Doyle and Doyle (1987). For a few taxa, the extraction was done from fresh plant material and for the remaining taxa silica gel dried material was used. Polymerase chain reaction (PCR) amplification of the trnL intron and the trnLF spacer region was achieved using the c and f primers of Taberlet et al. (1991). PCRs were performed in a Hybaid Thermal Cycler (Thermo Electron Corporation, Waltham, MA, USA) in a total volume of a 100 ll containing 2.5 mM MgCl2, 1 JMR-455 buffer (Southern Cross Biotechnology, Cape Town, RSA), 1 U of Super-Therm Taq polymerase (Southern Cross Biotechnology, Cape Town, RSA), 200 lM of each of the dNTP’s and 0.5 lM of each primer. Amplification profiles were 35 cycles with 1 min denaturation at 94 °C, 1 min annealing at 55 °C, 90 s extension at 72 °C, followed by a final extension step of 6 min at 72 °C. The amplified products were separated by agarose gel electrophoresis and purified from excised gel slices using the Wizard PCR Prep kit (Promega Corp., Madison, USA). Sequences were obtained by cycle sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, USA) using primers c and f and in some instances d and e (Taberlet et al. 1991), when polyA runs gave ambiguous base calling. Cycle sequencing was done in 10 ll reactions consisting of an estimated 100 ng of DNA, 2 ll 5 buffer (400 mM Tris–HCl, 10 mM MgCl2 at pH 9), 3.2 pmol primer, 2 ll of Terminator Ready Reaction Mix and water. The cycle sequencing profile was 35 cycles consisting of 10 s at 96 °C, 30 s at 52 °C and 4 min at 60 °C. Excess terminator dye was removed by gel filtration through Centri-Sep 96 Multi-well Filter Plates (Princeton Separation, Adelphia, USA). Sequencing reactions were subsequently analyzed on an ABI 377 sequencer (Applied Biosystems Inc., Foster City, USA). Electropherograms were edited using Chromas v1.45 (Technelysium Pty., Tewantin, Australia). Any samples giving sequencing ambiguities were resequenced until unequivocal sequences were obtained. Sequences were imported into DAPSA (Harley, 1998), combined with 21 Zygophylloideae trnLF sequences generated by Sheahan and Chase (2000) (indicated in Table 2) and aligned. The aligned data matrix was imported into BioEdit v7.0.1 (Hall, 1999), which was used to generate a Nexus file. All gaps, polyA rich areas and areas that could not be aligned unambiguously were excluded from the matrix. These were the 192 bp deletion in the trnL region identified by Sheahan and Chase (1996) which is not present in the zygophyllid clade, and four further regions, one which was in the trnL intron and was 15 bp in length, and three regions which were 67, 21 and 19 bp, respectively, in length in the trnLF spacer. The plastid rbcL region of a subset of 19 Zygophyllum species as indicated in Table 1 was amplified by PCR using the primers 1F or 32F and 1460R (Lledó et al., 1998). Polymerase chain reactions (PCR) were performed in a Hybaid Thermal Cycler (Thermo Electron Corporation, Waltham, MA, USA) in a total volume of a 100 ll containing 2.5 mM MgCl2, 1 JMR-455 buffer (Southern Cross Biotechnology, Cape Town, RSA), 1 U of Super-Therm Taq polymerase (Southern Cross Biotechnology, Cape Town, RSA), 200 lM of each of the dNTP’s and 0.5 lM of each primer. Amplification profiles were 30 cycles with 30 s denaturation at 94 °C, 30 s annealing at 50 °C, 60 s extension at 72 °C, followed by a final extension step of 6 min at 72 °C. Sequencing was performed using the primers 1F, 636F and 1460R as described above. Sequence alignment was performed using BioEdit with the rbcL sequences of 23 species which Sheahan and Chase (2000) had generated (indicated in Table 2). A reduced trnLF alignment, in which only these taxa were included, was also generated. 2.4. Phylogenetic analysis All data matrices were analyzed using the parsimony option in PAUP 4.0b10 (Swofford, 2002), and all substitutions were weighted equally. The combined trnL intron and trnLF spacer data matrix, the rbcL data matrix and the combined trnL intron, trnLF spacer and rbcL data matrices were analyzed treating all missing and gap characters as missing data. In each analysis, 1000 replicates were run using TBR branch swapping, holding 10 trees with MulTrees on and Maxtrees was set to 10,000. Clade support was calculated with 1000 bootstrap replicates using TBR branch swapping and MulTrees on. Bootstrap percentages P75% were considered as wellsupported. 2.5. Bayesian analysis MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) was used for a Bayesian approach. The two gene regions (trnLF, rbcL) were treated as separate partitions. The General Time Reversible model of nucleotide distribution with gamma shape parameter and a proportion of invariant sites (GTR + I + C) was selected for each partition with the help of MrModeltest v2.2 (Nylander, 2004). The chains were run for 2.5 million generations and sampled every 100 generations. To check that the log-likelihood distribution had become stationary, the fluctuating values of the log-likelihood were plotted. The burn in was estimated empirically on the basis of these plots. 11,000 samples (44% of the run) were discarded. Swapping among chains and acceptance of proposed changes to model parameters were monitored to ensure that efficient mixing had occurred. Under the default 0.2 temperature parameter for heating the chains, swapping of chains proved to be below the recommended value of 10% and we therefore lowered this value to 0.15 to get an acceptable rate (>10%) of chain swapping. Only posterior probabilities P95 were considered statistically significant support. 2.6. Morphological data Van Zyl (2000), in her revision of the southern African members of genus Zygophyllum, evaluated morphological attributes of stems, flowers, sepals, nectar discs, seeds and seed mucilage of all species. Since fresh material for Z. orbiculatum (native to Angola) was not available, she compiled the morphological characters for this species from descriptions by previous authors (Van Huyssteen, 1937) and the scant herbarium material of which the flowers were in a poor condition. She followed Van Huyssteen (1937) and placed Z. orbiculatum in section Paradoxa because of its unifoliolate leaves, yet concluded that the position of Z. orbiculatum was uncertain because of a lack of floral details. However, the availability of recently collected fresh material allowed us to reassess some of the morphological characters of Z. orbiculatum. Fresh material of Augea capensis was also examined, and leaf morphological characters were reassessed. The morphological character assessment of non-southern African species was based on Beier et al. (2003). Patterns of the evolution of capsule dehiscence, seed attachment and seed mucilage were investigated by ancestral character-state optimizations using the parsimony criterium in Mesquite (Maddison and Maddison, 2006). Characters were traced onto the Bayesian inference tree with the highest likelihood score of the combined trnLF and rbcL matrix. 3. Results 3.1. Molecular data The trnL intron and the trnLF could be sequenced without problems. Despite slippage in the sequencing signal induced by polyArich regions in the sequences of some taxa, the sequences could be fully interpreted. Thus complete sequences of these non-coding 937 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 Table 3 Tree statistics of the full trnLF data set Gene region Length of aligned matrix Phylogenetically informative characters Phylogenetically uninformative characters Tree length CI RI Nodes with P 75 bootstrap support trnL intron including the trnL 3’ exon trnLF spacer (excluding Augea) 682 126 68 390 0.679 0.882 20/72 345 105 57 320 0.741 0.884 18/71 1027 231 125 718 0.699 0.878 30/72 Full trnLF region regions could be generated for all of the taxa with the exception of A. capensis. For many species, more than one specimen was sequenced and little intraspecies sequence variation was observed even within the highly variable polyA regions (data not shown). Like Beier et al. (2003) we were also only able to sequence the trnL intron of A. capensis and not the trnLF intron. By using additional newly synthesized primers that matched downstream conserved areas, we deduced that the trnLF spacer and the trnF gene must have been deleted from the plastid genome of A. capensis. Thus in the trnLF alignment matrix, only the trnL intron sequence of A. capensis could be used for phylogenetic reconstruction, and the trnLF region was treated as missing data. The rbcL gene of the reduced taxon set could be sequenced and the electropherograms interpreted without problems. Sequence comparisons of the trnL, trnLF and rbcL gene regions of two specimens of Z. orbiculatum, collected in southern Angola, revealed that they were identical to the distinctive trnL, trnLF and rbcL sequences of Z. stapffii collected in the vicinity of Swakopmund in Namibia. Z. stapffii was described as a species with bifoliolate leaves and Z. orbiculatum as having unifoliolate leaves. Recent fieldwork has revealed that Z. orbiculatum in southern Angola discards one of each of the pairs of bifoliolate leaves during periods of drought, which gives the impression that it is unifoliolate (Craven, personal communication). Schreiber (1963) and Van Zyl (2000) expressed their doubts about the systematic position of Z. orbiculatum, and although no flowers were available in the field during January 2006, the vegetative material and gene sequences strongly suggest that Z. orbiculatum and Z. stapffii are conspecific. Since the name Z. orbiculatum (published in 1868) has priority over the name Z. stapffii (published in 1888), we will use the name Z. orbiculatum hereafter. The tree statistics of the parsimony analyses of the trnL intron (including trnL exon 2) and trnLF spacer data matrix are shown in Table 3. Although the results of these analyses are not shown, we analyzed the trnL intron data (i.e. including A. capensis) alone and the trnLF spacer region alone (i.e. excluding A. capensis because it does not possess a trnLF spacer region) and assessed the number of nodes with P75% bootstrap support in these separate analyses in comparison with the combined trnL intron and trnLF spacer region analysis. In a phylogenetic analysis of n taxa, the number of nodes in a completely resolved phylogeny is n 1. In Table 3, the number of nodes with P75% bootstrap support is therefore expressed as a fraction of the potential number of nodes in the phylogeny. This allows a comparison of the informativeness of the phylogeny based on the respective gene region and shows that the combined trnL intron and trnLF spacer region give significantly greater resolution than the trnL intron only, which has been used in previous phylogenetic analyses (Beier et al., 2003). The parsimony analysis of the combined trnL intron and trnLF spacer region sequences retrieved >10,000 trees, yet due to the tree limit that was set, only 10,000 were used to compute a strict consensus tree. An examination of the trees generated showed that the large number of trees retrieved in the heuristic search was the result of the many alternative trees generated among the members of the southern African subgenus Zygophyllum due to the short branch lengths in this clade. When compared to the parsimony strict consensus tree, the topology of the Bayesian tree resolved three more nodes, but these were not significantly supported. The strict consensus tree and one of the shortest trees of the parsimony analysis of the combined trnLF data set is shown in Fig. 1a and b, respectively. The tree statistics of and the proportion of supported nodes in the parsimony analyses of the rbcL data matrix, the corresponding reduced trnLF data matrix and the combined data matrix are shown in Table 4. The parsimony analysis of the rbcL gene retrieved 10 trees which were used to compute a strict consensus tree. When compared to the parsimony strict consensus tree, the topology of the Bayesian tree resolved one more node, but this was not significantly supported. One of the shortest trees of the parsimony analysis of the rbcL data set is shown in Fig. 2. The parsimony analysis of the reduced trnLF data matrix retrieved 28 trees which were used to compute a strict consensus tree. When compared to the parsimony strict consensus tree, the topology of the Bayesian tree resolved two more nodes, but these were not significantly supported. These trees are not shown. The parsimony analysis of the combined rbcL and trnLF dataset retrieved 37 trees which were used to compute a strict consensus tree. The Bayesian 50% majority rule consensus tree resulting from the combined rbcL and trnLF analysis and one of the shortest trees of the parsimony analysis are shown in Fig. 3a and b, respectively. The Bayesian Inference analysis for a double run (Nruns=2) of the combined rbcL and trnLF dataset gave an average standard deviation of split frequencies of 0.005967 after 2.5 million generations. When compared to the parsimony strict consensus tree, the topology of the Bayesian tree resolved the basal polytomy in the Zygophylloideae into three deeper nodes, but these were not significantly supported. A comparison of the trees generated with the complete trnLF data matrix, the reduced rbcL data matrix and the combined trnLF and rbcL data matrix, all revealed the following groupings: (a) A strongly supported monophyletic subfamily Zygophylloideae with a paraphyletic genus Zygophyllum, in which the genera, Augea and Tetraena were nested, was retrieved sister to a monophyletic subfamily Larreoideae. (b) Four strongly supported monophyletic groups comprised of (i) subgenus Agrophyllum, (ii) genus Melocarpum (Engl.) Beier & Thulin and the genus Fagonia, (iii) southern African and Australian members of subgenus Zygophyllum and (iv) A. capensis and Z. orbiculatum. The Asian members of subgenus Zygophyllum, Z. fabago and Z. xanthoxylum, were retrieved as a monophyletic group in the reduced rbcL and combined trnLF and rbcL data matrix, but were not retrieved as a monophyletic group in the trnLF analysis. When the topologies of the trees generated with the full trnLF, the reduced rbcL and the combined trnLF and rbcL data matrices were compared, different topologies of the five above mentioned groupings were apparent. These topologies were, however, not supported in any of the analyses, and hence no hard incongruences between the trnLF, the rbcL and the combined trnLF and rbcL topol- 938 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 100/100 85/65 94/100 76/68 100/100 92/100 64/60 100/100 92/100 100/100 99/100 80/100 90/100 86/100 74/88 99/100 96/100 99/100 99/100 64/80 99/100 95/100 100/100 79/73 63/94 80/100 97/100 94/100 76/100 96/100 95/100 95/100 90/100 77/100 Tribulus Seetzenia Larrea Bulnesia Guaiacum Z. xanthoxylum Z. fabago Augea capensis Z. orbiculatum M. hildebrandtii M. robecchii F. cretica F. luntii F. indica Z. giessii Z. longicapsulare Tetraena mongolica Z. microcarpum Z. rigidum Z. coccineum Z. album Z. simplex NA Z. simplex SA Z. inflatum Z. spongiosum Z. clavatum Z. decumbens NA Z. decumbens SA Z. pterocaule Z. patenticaule Z. prismatocarpum Z. applanatum Z. chrysopteron Z. retrofractum Z. segmentatum Z. turbinatum Z. cylindrifolium Z. tenue Z. porphyrocaule Z. incrustatum Z. lichtensteinianum Z. morgsana Z. hirticaule Z. schreiberanum Z. cordifolium Z. fusiforme Z. cretaceum Z. leucocladum Z. glaucum Z. billardieri Z. fruticulosum Z. botulifolium Z. cuneifolium Z. teretifolium Z. calcicola Z. divaricatum Z flexuosum Z. maculatum Z. aff. maritimum Z. maritimum Z. namaquanum Z. rogersii Z. spinosum Z. foetidum Z. leptopetalum Z. debile Z. pubescens Z. swartbergense Z. fulvum Z. fuscatum Z. pygmaeum Z. sessilifolium Z. spitskopense Tribuloideae Seetzenioideae Larreoideae Asian species SA species §Grandifolia Horn of Africa species Fagonia §Cinerea Asian species §Alata Asian species §Annua §Bipartita §Prismatica §Bipartita §Capensia §Morgsana §Capensia §Paradoxa §Capensia Australian species §Capensia Fig. 1. (a) The strict consensus tree of the parsimony analysis of the trnL intron and trnLF spacer sequence data. Bootstrap percentages followed by Bayesian posterior probabilities are shown below branches. The sections identified by Van Zyl (2000) to which the southern African species belong, the area of origin of non-southern African species or the outgroup subfamilies are indicated next to the relevant species. (b) One of the shortest trees of the parsimony analysis of the trnL intron and trnLF spacer data. Branch lengths are shown above branches. The main groupings within the subfamily Zygophylloideae are indicated: subgenus Agrophyllum; southern African (SA) and Australian subgenus Zygophyllum species; Asian subgenus Zygophyllum species; genus Melocarpum and genus Fagonia; and Z. orbiculatum and Augea capensis. NA = northern Africa, SA = southern Africa. ogies were found. However, if the proportion of the nodes supported in the reduced trnLF, the reduced rbcL and the combined trnLF and rbcL data matrix were compared, it became apparent that the combined trnLF and the rbcL sequence data did not result in a marked increase in resolution and node support of these groupings relative to one another. 939 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 b 29 39 Tribulus Seetzenia 32 49 Larrea Bulnesia Guaiacum 14 Augea capensis 21 7 Z. orbiculatum 9 M. hildebrandtii 14 M. robecchii 19 12 F. cretica 22 14 F. luntii 10 10 F. indica 12 Z. xanthoxylum 5 Z. fabago 6 8 Z. giessii 17 3 Z. longicapsulare Tetraena mongolica 5 Z. microcarpum 11 Z. rigidum Z. coccineum 5 Z. album 9 Z. simplex NA 5 Z. spongiosum 4 Z. simplex SA 4 Z. inflatum 7 4 Z. clavatum 4 Z. decumbens NA 11 4 Z. decumbens SA 3 Z. pterocaule Z. patenticaule Z. prismatocarpum Z. turbinatum Z. segmentatum 3 3 Z. applanatum Z. chrysopteron Z. retrofractum 3 Z. cylindrifolium 7 4 Z. tenue 4 Z. glaucum 6 Z. billardieri 8 4 Z. fruticulosum Z. incrustatum 3 Z. lichtensteinianum Z. morgsana 3 Z. cordifolium Z. fusiforme Z. teretifolium Z. cuneifolium Z. botulifolium Z. hirticaule Z. schreiberanum Z. cretaceum Z. leucocladum Z. porphyrocaule 6 Z. leptopetalum 4 Z. pubescens Z. debile 3 Z. swartbergense Z. maculatum 5 Z. foetidum Z. rogersii Z. divaricatum Z. namaquanum Z. maritimum Z. aff. maritimum 4 Z. spinosum Z. calcicola Z. flexuosum Z. fuscatum 4 Z. fulvum Z. pygmaeum Z. sessilifolium Z. spitkopense 11 11 41 38 15 23 12 5 changes Fig. 1 (continued) Augea & Z. orbiculatum Melocarpum & Fagonia Asian Zygophyllum Agrophyllum SA & Australian Zygophyllum 940 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 Table 4 Tree statistics of the reduced trnLF and rbcL data set Gene region Length of aligned matrix Phylogenetically informative characters Phylogenetically uninformative characters Tree length CI RI Nodes with P 75 bootstrap support trnLF region rbcL 947 221 113 650 0.728 0.839 26/42 1364 196 81 548 0.595 0.807 18/42 Combined 2311 417 194 1211 0.658 0.816 22/42 37 34 Tribulus Seetzenia 13 31 100/100 Larrea 15 8 Bulnesia 23 Guaiacum 3 Z. rigidum * Z. microcarpum 5 7 Tetra mongolica 78/ 2 Z. album 2 100 3 * 3 Z. coccineum 6 Z. giessii 11 3 6 * Z. longicapsulare 5 Z. simplex NA 7 5 2 Z. simplex SA * * 23 89/100 4 Z. patenticaule 4 Z. prismatocarpum 6 * 4 5 * 33 100/100 2 2 16 100/100 15 97/100 5 - /62 6 71/ 100 Z. applanatum 2 Z. decumbens NA Z. decumbens SA 6 Z. cylindrifolium 4 Z. clavatum 2 Z. segmentatum 2 M. robecchii M. hildebrandtii 13 F. cretica 4 F. indica 13 * F. luntii 13 100/100 25 11 86/100 6 - /83 Agrophyllum Z. spongiosum 13 11 14 3 - /93 Augea capensis Z. orbiculatum Z. xanthoxylum Z. fabago 2 Z. porphyrocaule Melocarpum & Fagonia Augea & Z. orbiculatum Asian Zygophyllum Z. hirticaule 5 Z. swartbergense 21 100/100 Z. cordifolium Z. morgsana 2 2 Z. flexuosum * Z. sessiliflora SA & Australian Zygophyllum Z. glaucum 2 Z. billardieri 5 Z. fruticulosum 5 changes Fig. 2. One of the shortest trees of the parsimony analysis of the rbcL sequence data. Branch lengths are shown above branches. Branches that collapse in the strict consensus are indicated with an arrow. Bootstrap percentages followed by Bayesian posterior probabilities are shown below branches. Where there was insufficient space to indicate high branch support (P75% BS and P95% PP) this was indicated with an *. The main groupings within the subfamily Zygophylloideae are indicated: subgenus Agrophyllum; southern African (SA) and Australian subgenus Zygophyllum species; Asian subgenus Zygophyllum species; genus Melocarpum and genus Fagonia; and Z. orbiculatum and Augea capensis. NA = northern Africa, SA = southern Africa. The parsimony analysis of the combined trnL intron and trnLF spacer region retrieved some of the sections recognized by Van Zyl (2000) as monophyletic. In subgenus Agrophyllum, monophy- letic sections Cinerea, Alata, Annua and Bipartita were retrieved. Although section Prismatica was retrieved as monophyletic in the parsimony analysis, it nests within section Bipartita, render- 941 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 a Tribulus Seetzenia Larrea 100/100 86/99 Bulnesia Guaiacum 100/100 99/100 M. robecchii M. hildebrandtii 99/100 F. cretica 100/100 100/100 F. indica Melocarpum & Fagonia F. luntii Augea capensis 99/100 100/100 Z. orbiculatum Z. xanthoxyllum 98/100 Z. fabago Zygophyllum Z. glaucum 97/100 */87 Augea & Z. orbiculatum 99/100 */63 Z. billardieri Z. fruticulosum 100/100 Z. porphyrocaule Z. hirticaule 63/99 70/71 63/99 Z. cordifolium SA & Australian Zygophyllum Z. morgsana */70 Z. swartbergense 100/100 95/100 Z. flexuosum Z. sessilifolium 100/100 Z. rigidum Z. microcarpum 92/100 Tetreana mongolica */98 99/100 Z. album Z. coccineum 98/100 Z. giessii 100/100 Z. longcapsulare Z. simplex N 100/100 61/99 51/76 Z. simplex SA Z. spongiosum 100/100 100/100 Agrophyllum Z. patenticaule Z. prismatocarpum 100/100 Z. clavatum */96 77/97 */55 Z. decumbens N Z. decumbens SA Z. applanatum 52/95 52/93 Z. segmentatum Z. cylindrifolium Fig. 3. (a) The Bayesian 50% majority rule consensus tree of the combined trnLF and rbcL spacer sequence data. Parsimony bootstrap percentages followed by Bayesian posterior probabilities are shown below branches. Where nodes collapsed in the parsimony strict consensus tree, but were supported in the Bayesian analysis, they were indicated with an asterisk. The main groupings within the subfamily Zygophylloideae are indicated: subgenus Agrophyllum; southern African (SA) and Australian subgenus Zygophyllum species; Asian subgenus Zygophyllum species; genus Melocarpum and genus Fagonia; and Z. orbiculatum and Augea capensis. (b) One of the shortest trees of the parsimony analysis of the combined trnLF and rbcL spacer sequence data. Branch lengths are shown above branches. The areas in which the species occur are indicated with bars; NA = Northern Africa; SA = Southern Africa. ing it paraphyletic. In the Bayesian analysis, section Prismatica was retrieved in a polytomy with members of section Bipartita, thereby leaving the question of the paraphyly of section Bipartita unconfirmed. In subgenus Zygophyllum, a monophyletic section Paradoxa was retrieved but with the exclusion of Z. orbiculatum. Z. orbiculatum as the sole representative of section Grandifolia was retrieved in an isolated position sister to A. capensis supporting its sectional status. The parsimony analysis of the combined trnL intron and trnLF spacer region did not retrieve Van Zyl’s (2000) sections Capensia and Morgsana as monophyletic nor were the series she recognized within Capensia supported. The parsimony analyses of the reduced rbcL and combined trnLF and rbcL matrices, also retrieved the sections Cinerea, Alata, An- nua, Bipartita, Prismatica and Grandifolia as monophyletic, but section Prismatica was retrieved as part of a polytomy of members of section Bipartita in the combined trnLF and rbcL analysis leaving the monophyly of section Bipartita unconfirmed. Other groupings of Zygophyllum species occurring outside southern Africa which were also retrieved as monophyletic in all of these analyses were the Australian species belonging to section Roepera and the two north African and Asian species, Z. album and Z. coccineum. The grouping of Van Zyl’s section Alata with the two north African and Asian species, Z. album and Z. coccineum, and the Asian species, Tetraena mongolica, was retrieved as monophyletic in the complete trnLF analysis and the combined rbcL and trnLF analysis. 942 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 b 67 73 Tribulus Seetzenia 47 74 Bulnesia 33 Guaiacum 3 M. robecchii 27 3 M. hildebrandtii 26 F. cretica 38 14 F. indica 22 15 F. luntii 40 Augea capensis 31 19 Z. orbiculatum 23 Z. xanthoxyllum 8 21 Z. fabago 4 Z. glaucum 7 10 5 Z. billardieri 10 9 Z. fruticulosum 3 41 Z. porphyrocaule 25 64 Larrea 40 17 76 22 Horn of Africa Africa, Asia, New World SA Asian Australian Z. hirticaule 3 Z. cordifolium 6 5 Z. morgsana 8 Z. swartbergense SA 3 Z. flexuosum 4 Z. sessilifolium 21 6 Z. rigidum Z. microcarpum 6 9 Tetraena mongolica 5 Z. album 9 6 Z. coccineum 13 Z. giessii 34 9 Z. longicapsulare 7 Z. simplex NA 10 7 11 Z. simplex SA 10 17 Z. spongiosum 10 Z. clavatum 3 6 Z. decumbens NA 4 Z. decumbens SA 11 Z. patenticaule 7 Z. prismatocarpum 5 Z. applatum 3 4 3 Z. segmentatum 13 Z. cylindrifolium SA Asian SA NA SA SA NA SA 10 changes Fig. 3 (continued) 3.2. Results of the morphological assessment Van Zyl’s (2000) morphological comparison of the southern African members of subgenera Zygophyllum and Agrophyllum to that of Z. orbiculatum (=Z. stapffii) is shown in Table 5. It shows that there are a number of synapomorphies which allow the delimitation of the two subgenera in this region. Z. orbiculatum was also shown to share more characters with subgenus Agrophyllum than with subgenus Zygophyllum, which was the basis of Van Zyl’s placement of Z. orbiculatum in subgenus Agrophyllum. It does, however, also reveal that Z. orbiculatum displays a number of distinc- tive autapomorphies (as compared to both subgenera), in particular those of capsule dehiscence and seed attachment. Van Zyl (2000) considered both of these characters as taxonomically informative and used them to place Z. orbiculatum (=Z. stapffii) in section Grandifolia. Morphological characters used to define sectional boundaries of southern African Zygophyllum taxa are shown in Table 6. Within subgenus Agrophyllum, sectional boundaries were defined by synapomorphies but these were often homoplasious. Within subgenus Zygophyllum, morphological characters were much more uniform, with the exception of leaf morphology, which was used to define D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 section Paradoxa, and floral morphology, which was used to define the tetramerous section Morgsana. In all other southern African Zygophyllum species flowers were pentamerous. Apart from in section Morgsana, tetramerous flowers were also present in Australian members of subgenus Zygophyllum (Beier et al., 2003). The examination of fresh material of A. capensis revealed that many of the leaves on a single plant were not unifoliolate as previously interpreted from herbarium material (Sheahan and Chase, 1996; Beier et al., 2003) but rather were attached to the stem in asymmetric pairs to produce sessile bifoliolate rather than opposite unifoliolate leaves. This interpretation would have been difficult from dried herbarium material, but showed that A. capensis shares this character with most other members of the Zygophylloideae. 3.3. Patterns of character evolution Patterns of evolution of the characters of seed dehiscence, seed mucilage and seed attachment compared by means of character optimization on the tree retrieved with the highest likelihood score in the Bayesian analysis are shown in Figs. 4–6, respectively. As this tree retrieved a monophyletic subgenus Zygophyllum and a monophyletic Agrophyllum, capsule dehiscence (loculicidal versus septicidal), the character used in the definition of these subgenera, was fully congruent with the tree topology. The character optimization illustrates how capsule dehiscence via dorsal and ventral sutures which occurs in Z. orbiculatum does not optimize within the two subgenera of Zygophyllum. The optimization of seed attachment revealed that seed attachment via an aril only occurred in the southern African and Australian members of subgenus Zygophyllum but not in the Asian members of subgenus Zygophyllum which exhibited seed attachment via a long funiculum that also occurred in the rest of subgenus Agrophyllum. Intermediate seed attachment via a short thick funiculum was present in Z. orbiculatum, Melocarpum hildebrandtii and Melocarpum robecchii. The optimization of seed mucilage showed that members of subgenus Zygophyllum occurring in southern African and Australia and A. capensis possessed threads of uniform width in their mucilage. Seed mucilage in Asian members of subgenus Zygophyllum, subgenus Agrophyllum and Z. orbiculatum, contained wineglass-shaped threads. 943 4. Discussion 4.1. Molecular data In this study, the trnL intron and the trnLF spacer sequences of 52 southern African Zygophyllum species were combined with those published by Sheahan and Chase (2000) to produce a tree of the southern African members of the genus. Both gene regions contributed almost equally to the number of phylogenetically informative characters and resulted in a tree in which 41% of the nodes were well-supported. The inclusion of Z. orbiculatum changed the topology considerably in comparison to the one of Sheahan and Chase (2000) and Beier et al. (2003), and also improved resolution. Poorly sampled groups i.e. those from outside of the southern African area, showed poor resolution. This analysis retrieved a monophyletic subfamily Zygophylloideae, within which the genera Fagonia, Melocarpum, Augea and Tetraena are nested rendering Zygophyllum paraphyletic. Although Van Zyl (2000) transferred Z. orbiculatum (=Z. stapffii) from subgenus Zygophyllum to subgenus Agrophyllum, it was retrieved as sister to A. capensis. The analysis revealed four main groupings: subgenus Agrophyllum; southern African and Australian members of subgenus Zygophyllum; A. capensis and Z. orbiculatum; and genus Melocarpum and genus Fagonia. However, the position of the Asian members of subgenus Zygophyllum, Z. fabago and Z. xanthoxylum, remained unresolved. The analysis supports the transfer of Z. morgsana from subgenus Agrophyllum to subgenus Zygophyllum as proposed by Van Zyl (2000). Within subgenus Agrophyllum, the molecular analysis strongly support Van Zyl’s (2000) sections Cinerea, Alata, Annua and Bipartita. Although section Prismatica was monophyletic, it nested within section Bipartita. Within subgenus Zygophyllum, section Paradoxa was also retrieved but with the exclusion of Z. orbiculatum. The monophyly of sections Morgsana and Capensia could not be confirmed. Van Zyl (2000) suggested a subdivision of section Capensia into three series based on leaf morphology. This was not supported by the present analyses. However, the molecular phylogeny does reveal a strongly supported Cape Floral Region (CFR) clade. The lack of resolution in section Bipartita and in subgenus Zygophyllum is due to a paucity of phylogenetically informative characters in these clades. Table 5 Morphological characters of southern African members of subgenera Agrophyllum and Zygophyllum and Z. orbiculatum (=Z. stapffii) Subgenus Zygophyllum Z. orbiculatum (=Z. stapffii) Druse crystals absent in mesophyll Druse crystals present in mesophyll Subgenus Agrophyllum Druse crystals present in mesophyll Flowers large (petals 8–22 mm long 3–12 mm wide), petals yellow, usually marked at base with red or brown, with short claws Flower size medium (petals 10–11 mm long 3– 4 mm wide), petals white, unmarked, obovate, with a long claw Flowers small (petals 2–10 mm long 0.5–3 mm wide), usually white, rarely light yellow or orange, never marked at base, with long claws Sepals not articulate, not succulent Sepals adnate at base, not articulate, leathery in texture Sepals articulate and usually succulent Sepals persistent Sepals persistent Sepals not persistent Nectar disc regularly angled never lobed Nectar disc regularly angled, 10-lobed, lobes small and directed out and downward, with nectaries visible as groups of darker cells Nectar disc angled and lobed, lobes arranged into pairs, variously orientated Nectar disc always papillate Nectar disc smooth Nectar disc smooth Nectar disc uniformly level, not sloping or with raised or sunken areas Nectar disc sloping slightly towards its periphery Nectar disc sloping towards its periphery, with raised and sunken areas Fruit a loculicidal capsule Fruit dehiscing along both ventral and dorsal sutures Fruit a septicidal capsule Seeds oblong, not compressed, white aril present Seeds sub-pyriform, compressed, funicula short and thick Seeds pyriform, sub-pyriform, compressed, funicula long and narrow Seed mucilage structured, with long spiral inclusions of uniform width Seed mucilage structured, with short spiral inclusions that unravel at apex Seed mucilage structured, with short spiral inclusions that unravel at apex Young stems usually flat on ventral side, with lateral ridges, or round in cross section Young stems with a flat ventral area but without ridges Young stems usually grooved 944 Table 6 Morphological character states used in the delimitation of sections of southern African Zygophyllum Morphological characters Indumentum Leaf attachment & number Leaf shape Stipules Seed mucilage Staminal scale morphology Grandifolia Glabrous Bifoliolate, petiolate Subrotund Succulent, leathery, triangular or subrotund, patent, 1 on ventral, 1 on dorsal side, caducous Structured, short spiral inclusions Simple, apex acute, upper margins lacerate Pale cream, spathulate Alata White, two armed hairs present or glabrous Bifoliolate, subpetiolate or petiolate Widely or narrowly obovate Subulate, white, stiff, 2 on ventral, 2 on dorsal side, semi-permanent Structured, short spiral inclusions Simple , alternately enfolding filament Pale cream or pink, spathulate Cinerea Silver-white, two armed trichomes Bifoliolate, petiolate Orbiculate or obovate Triangular or subulate?, reflexed and base thickened, 2 on ventral, 2 on dorsal side, caducous Structured, short spiral inclusions Simple, apex acute, upper margins lacerate Petals narrowly obovate or spathulate, cream or pink Annua Glabrous Simple, sessile Succulent, obovoid or globose Triangular, membranous, apex sometimes incused, 2 on ventral, 2 on dorsal side, semipermanent Unstructured, jelly like Biparted almost at base Petals spathulate, white, yellow or orange Bipartita Glabrous Bifoliolate, petiolate Cylindric, oblong, obovate or subrotund Widely triangular, membranous, 2 on ventral, 2 on dorsal side, semipermanent or caducous Structured, short spiral inclusions Biparted almost at base Pale cream, axillary Prismatica Glabrous Simple, sessile Suborbicular/obovate Filamentous, minute, caducous, 2 on ventral, 2 on dorsal side Structured, short spiral inclusions Biparted almost at bas Pale cream, axillary Paradoxa Glabrous Simple, with short petiole or sessile Subrotund or obovate Navicular, membranous, reflexed, 1 on ventral, 1 on dorsal side, caducous Structured, long spiral inclusions of uniform length Simple, margins lacerate Yellow marked at base Capensia I Glabrous Bifoliolate, sessile Terete or linear, adaxial groove, sometimes succulent Triangular, 1 on ventral, 1 on dorsal side, sometimes caducous Structured, long spiral inclusions of uniform length Simple, margins lacerate Yellow (in one case pink) marked at base Capensia II Glabrous Bifoliolate, sessile Obovate or cuneate Triangular or semi-circular, 1 or 2 on ventral, 1 on dorsal side, sometimes caducous Structured, long spiral inclusions of uniform length Simple, margins lacerate Yellow marked at base Capensia II Glabrous Bifoliolate (trifoliolate in Z. schreiberanum), petiolate Obovate or elliptic Triangular , 1 or 2 on ventral, 1 or 2 on dorsal side, sometimes caducous (in Z. schreiberanum leaflike) Structured, long spiral inclusions of uniform length Simple, margins lacerate, sometimes bordered with papillae Yellow marked at base Morgsana Glabrous Bifoliolate, petiolate Obovate or subrotund, apex rounded, base narrow or obtuse Triangular, membranous, reflexed, 1 on ventral, 1 on dorsal side, caducous Structured, long spiral inclusions of uniform length Simple oblong or obovate, margins lacerate Yellow, marked at base, 4-merous D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 Section 945 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 (a) Fruit of Z. simplex, subgenus Agrophyllum; dehiscence septicidal (b) Fruit of Z. porphyrocaule, subgenus Zygophyllum; dehiscence loculicidal Tribulus Seetzenia Bulnesia Guaiacum Larrea Augea capensis Z orbiculatum Z xanthoxyllum Z fabago Z billardieri Z fruticulosum Z glaucum Z porphyrocaule Z cordifolium Z morgsana Z hirticaule Z flexuosum Z sessilifolium Z swartbergense Z giessii Z longicapsulare Z simplex NA Z simplex SA Z spongiosum Z clavatum Z decumbens NA Z decumbens SA Z segmentatum Z cylindrifolium Z applanatum Z patenticaule Z prismatocarpum Tetraena mongolica Z album Z coccineum Z rigidum Z microcarpum M robecchii M hildebrandtii F cretica F indica F luntii Augea & Z. orbiculatum Asian Zygophyllum SA & Australian Zygophyllum Agrophyllum Melocarpum & Fagonia (c) Fruit of Z. orbiculatum; dehiscence via dorsal and ventral sutures Fig. 4. Character evolution of fruit dehiscence as reconstructed on the tree retrieved with the highest likelihood score in the Bayesian analysis of the combined analysis of rbcL and trnLF using Mesquite (Maddison and Maddison, 2006). Insert (a) illustrates the fruit of Z. simplex, typical of subgenus Agrophyllum, and a detached mericarp after septicidal dehiscence. Insert (b) illustrates the fruit of Z. porphyrocaule ined. which dehiscences loculicidally. Insert (c) illustrates the fruit of Z. orbiculatum, which dehiscences via dorsal and ventral sutures during dry weather conditions. The alternating exocarp and endocarp sections are shown with seeds still attached to the endocarp. The rbcL phylogeny also retrieved the four main groups found in the analysis of the trnL intron and trnLF spacer sequences and added a fifth group of Asian members of subgenus Zygophyllum, Z. fabago and Z. xanthoxylum. Although the relationships between these groups were resolved, these were not supported, as was also found by Sheahan and Chase (2000). The sister relationship between A. capensis and Z. orbiculatum was, however, confirmed, which was important, as the trnLF spacer is absent in this taxon, and therefore only the trnL data contributed to the combined trnLF data set. The parsimony statistics for the rbcL results in comparison to those of the trnLF phylogeny, revealed that homoplasy was high- er in the rbcL matrix. The number of phylogenetically informative characters in the rbcL matrix (196) was also lower than in the reduced trnLF data matrix (221), which increased to 231 if all taxa were included. Although it may have been expected that the combined analysis could have retrieved a greater number of supported nodes than the single matrices upon which it was based, this was not the case and therefore reflects the increased amount of homoplasy in the combined analysis. However, the combined trnLF and rbcL phylogeny also retrieved the five main groupings in the subfamily Zygophylloideae but the relationships between 946 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 (a) aril attached to an oblong seed (b) Long funicle attached to pyriform seed (c) Short, thick funicle attached to subpyriform seed Tribulus Seetzenia Bulnesia Guaiacum Larrea Augea capensis Z orbiculatum Z xanthoxyllum Z fabago Z billardieri Z fruticulosum Z glaucum Z porphyrocaule Z cordifolium Z morgsana Z hirticaule Z flexuosum Z sessilifolium Z swartbergense Z giessii Z longicapsulare Z simplex NA Z simplex SA Z spongiosum Z clavatum Z decumbens NA Z decumbens SA Z segmentatum Z cylindrifolium Z applanatum Z patenticaule Z prismatocarpum Tetraena mongolica Z album Z coccineum Z rigidum Z microcarpum M robecchii M hildebrandtii F cretica F indica F luntii Augea & Z. orbiculatum Asian Zygophyllum SA & Australian Zygophyllum Agrophyllum Melocarpum & Fagonia Fig. 5. Character evolution of seed attachment as reconstructed on the tree retrieved with the highest likelihood score in the Bayesian analysis of the combined analysis of rbcL and trnLF using Mesquite (Maddison and Maddison, 2006). Insert (a) illustrates seed attachment via a long funiculum to a pyriform seed and insert (b) illustrates seed attachment via an aril to an oblong seed. The third character state (c) of seed with short thick funicles attached to a sub-pyriform seed (in Z. orbiculatum, Melocarpum hildebrandtii and Melocarpum robecchii) is not illustrated. these were poorly supported. Thus the trnLF and rbcL data will have to be augmented by additional sequence data from other genes to establish the true relationships within the subfamily Zygophylloideae. 4.2. Morphological assessment and character evolution The morphological analysis performed by Van Zyl (2000) supported the division of southern African members of the genus Zygophyllum into the two subgenera Agrophyllum and Zygophyllum based on capsule dehiscence, a classification which was contended by previous authors (Endlicher, 1841; Van Huyssteen, 1937). The dehiscence of the fruit of Z. orbiculatum (=Z. stapffii) is neither loculicidal as in southern African members of the subgenus Zygophyllum, nor septicidal as in southern African members of the subgenus Agrophyllum, but dehisces via ventral and dorsal sutures. Based on capsule dehiscence, it therefore appears that Z. orbiculatum occupies an intermediate position, although on the basis of other characters it seems to have closer affinities with subgenus Agrophyllum. This was then also the reason why Van Zyl (2000) transferred it from subgenus Zygophyllum to subgenus Agrophyllum. In this study, a resolved, but not supported tree in which a monophyletic subgenus Agrophyllum and a monophyletic subgenus Zygophyllum were retrieved, was used 947 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 (a) long, uniform width spirals in seed mucilage (b) Short, unravelling Wineglass-shaped spirals in seed mucilage (c) No threads in seed mucilage (d) Seed mucilage under outer layer Tribulus Seetzenia Bulnesia Guaiacum Larrea Augea capensis Z orbiculatum Z xanthoxyllum Z fabago Z billardieri Z fruticulosum Z glaucum Z porphyrocaule Z cordifolium Z morgsana Z hirticaule Z flexuosum Z sessilifolium Z swartbergense Z giessii Z longicapsulare Z simplex NA Z simplex SA Z spongiosum Z clavatum Z decumbens NA Z decumbens SA Z segmentatum Z cylindrifolium Z applanatum Z patenticaule Z prismatocarpum Tetraena mongolica Z album Z coccineum Z rigidum Z microcarpum M robecchii M hildebrandtii F cretica F indica F luntii Augea & Z. orbiculatum Asian Zygophyllum SA & Australian Zygophyllum Agrophyllum Melocarpum & Fagonia Fig. 6. Character evolution of seed mucilage as reconstructed on the tree retrieved with the highest likelihood score in the Bayesian analysis of the combined analysis of rbcL and trnLF using Mesquite (Maddison and Maddison, 2006). Insert (a) shows a photograph of the long, spirals of uniform width found in the mucilage and insert (b) shows a photograph of short, unraveling wineglass-shaped spirals found in the mucilage. The third character state (c) exhibits mucilage in which no threads occur. The fourth character (d) state exhibits seed mucilage under the outer cell layer which occurs only in Seetzenia. Mucilage characters were not scored for Tribulus. to illustrate the evolution of fruit and seed characters within the subfamily Zygophylloideae. As fruit dehiscence was used to define the subgenera it was therefore logical that the character optimization of loculicidal and septicidal fruit dehiscence should fit perfectly onto the monophyletic subgenera in this tree. All of the molecular phylogenies retrieve Z. orbiculatum as sister to A. capensis and not in subgenus Agrophyllum. Thus the character optimization of fruit dehiscence of Z. orbiculatum also illustrates the unique fruit dehiscence of Z. orbiculatum, supporting the suggestion that it should be placed in neither subgenus Agrophyllum nor subgenus Zygophyllum. Character optimization revealed that seeds attached via an aril covering the hilum to an oblong seed only occur in the clade containing southern African and Australian members of subge- 948 D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 nus Zygophyllum but not in the clade consisting of the Asian members of subgenus Zygophyllum, which rather share seed attachment via a long funiculum with the rest of the recognized subgenus Agrophyllum. A. capensis, genus Fagonia and the rest of subgenus Agrophyllum possess a seed attachment via a long funiculum to a pyriform seed. Van Zyl (2000) described the seed attachment of Z. orbiculatum as a short and thick funiculum and the seed shape as sub-pyriform. Beier et al. (2003), who examined the morphology of the two species from the horn of Africa, M. hildebrandtii and M. robecchii, found that they also possess sub-pyriform seeds attached via short funicula. Seed attachment is therefore variable in these clades. Van Zyl (2000) suggested that the short, thick funiculum of Z. orbiculatum may be an adaption to drought conditions. It would ensure attachment to the xerochastic fruit capsule, which is dispersed by wind when dry and which, when it becomes wet, retains moisture for seed germination. As Z. orbiculatum, M. hildebrandtii and M. robecchii occupy similar habitats, this may be a plausible explanation. In contrast, the aril present in southern African and Australian members of subgenus Zygophyllum suggests myrmecochorous seed dispersal, which is a typical mechanism of seed dispersal in the CFR (Bond and Slingsby, 1983). Character optimization of seed mucilage revealed that short, funnel-shaped, spiral inclusions are found in Z. orbiculatum (Van Zyl 2000) and members of subgenus Agrophyllum (with the exception of section Annua which has mucilage that lack these structures). In subgenus Zygophyllum and A. capensis, mucilage has long rod-shaped spiral inclusions, while in M. hildebrandtii and M. robecchii and Fagonia the mucilage has no structure (Beier et al., 2003). The sister clades of the Zygophylloideae, i.e. the Larriodeae, exhibited a seed mucilage state without threads which appears to be the plesiomorphic state and to which section Annua in subgenus Agrophyllum apparently reverted to. The members of the Z. orbiculatum and A. capensis clade and the Fagonia and Melocarpum clade therefore share different combinations of capsule dehiscence, seed attachment and seed mucilage characters in comparison to the combination of these characters in the members of subgenus Agrophyllum or subgenus Zygophyllum. This can now be explained as sorting of distinctive character combinations in the major clades of the Zygophylloideae in which different combinations of these characters occur and is established here for the first time. The character optimizations also showed that the characters of seed mucilage and seed attachment were synapomorphies of the two subgenera in southern Africa, but do not hold for the members of the subgenera occurring outside this area. The taxonomic importance of fruit dehiscence, which has for long been used in the delimitation of the subgenera Zygophyllum and Agrophyllum is, however, confirmed. A number of other morphological characters (leaf morphology, stipule morphology, trichome types, etc.), were phylogenetically not informative. Leaves, for example, were bifoliolate in most members of subgenus Zygophyllum, in many members of Agrophyllum and in Z. orbiculatum, but were unifoliolate in M. hildebrandtii and M. robecchii, section Paradoxa of subgenus Zygophyllum, section Annua and section Prismatica of subgenus Agrophyllum, and uni- to trifoliolate and rarely up to 7-foliolate in genus Fagonia and trifoliolate in Z. schreiberanum of subgenus Zygophyllum. It was previously thought that A. capensis was unifoliolate, but our results have revealed that it is partially bifoliolate. Variation in leaflet number and shape appears to have been selected for as an adaptation to drought. This hypothesis is supported by the fact that species with simple leaves all occur in areas of high aridity along the west coast of southern Africa from about 150 km north of Cape Town to southern Angola, never more than 200 km from the coast. In sections Paradoxa and Prismatica these leaves were often conduplicate to additionally reduce moisture loss. This is also supported by the leaf structure in Z. qatarense Hadidi (from the Middle East), which is bifoliolate after rain but becomes unifoliolate during periods of drought (Ismael, 1983). Species of Zygophyllum with bifoliolate leaves have developed mechanisms to shed leaflets in response to drought (Sheahan and Chase, 1996; Van Zyl, 2000, see also earlier comments about Z. orbiculatum). Some species have also developed trichomes to reduce moisture loss such as in Fagonia (Beier et al., 2003) and section Cinerea (Van Zyl, 2000). This may explain why leaf shape, leaflet number and several other morphological characters have been found to be largely uninformative in phylogenetic reconstructions (Beier et al., 2003), as they indicate environmental adaptations rather than historical relationships. 4.3. Historical biogeography Climatologically, the Northern Cape Province (South Africa), Namibia and southern Angola have been exposed to a general aridification since the Miocene as a result of the establishment of the cold Benguela current (Marlow et al., 2000; Linder, 2003; de Menocal, 2004). This would have led to extinction of many plant species in this area, leaving only the most droughtresistant species, such as members of genus Zygophyllum and genus Salsola. Currently, the narrow coastal strip in Namibia, the slightly inland (±100 km) transfrontier region between South Africa and Namibia in the Richtersveld (SA), Hunsberge Mountains (Namibia) and the lower Orange River course, which receives a slightly higher precipitation than the surrounding areas, constitutes a refugium for the few plants that can survive such a harsh environment (Midgley et al., 2005). Moisture in this area is restricted to the extremely scant rainfall and coastal fog, but this is sufficient for the survival of many Zygophyllum species, as this is the center of diversity for subgenus Agrophyllum in southern Africa (Van Zyl, 2000). Drought resistance of species in subgenus Zygophyllum would have allowed these species to survive in the Northern Cape, and when the dry summers and wet winters established themselves in the CFR, this climate change appears to have favoured a radiation of the subgenus into this area. Most of the members of subgenus Zygophyllum possess excellent drought resistance mechanisms to survive the dry summers in the CFR. These include xerophytic, often terete leaves and a flowering season in the winter and early spring when moisture is still abundant. Seed attachment via a myrmecochoric aril which occurs commonly in the CFR, also appears to have developed as an adaptation to survival in this area. As southern African members of subgenus Zygophyllum occur primarily in the CFR, while the rest of the genus occurs outside of this area, its evolution serves as an interesting example of the radiation of a plant genus in the CFR. Although no attempts were made at dating our phylogeny, the long branch of the clade containing southern African and Australian members of subgenus Zygophyllum and the short terminal branches within the subgenus, support a hypothesis of relatively recent radiation of the subgenus in the CFR. By contrast, the subgenus Agrophyllum, if gauged by the more even and longer branch lengths between the nodes, is older, and has shown a more constant rate of radiation outside of the CFR. This concurs with the views of Engler (1896) and Van Huyssteen (1937) who both considered subgenus Agrophyllum to be ancestral in the genus. Interestingly, where species in section Bipartita have also speciated into the CFR, the same pattern of short branch lengths is found, possibly implying recent radiation. In a phytogeographic study of the genus Fagonia, Beier et al. (2004) concluded that this genus had evolved in the horn of Africa because it is sister to M. hildebrandtii and M. robecchii, D.U. Bellstedt et al. / Molecular Phylogenetics and Evolution 47 (2008) 932–949 and that the southern African members of genus Fagonia had migrated from the horn of Africa southward. This study reveals that an ancestor of the rest of the Zygophylloideae may have evolved in the horn of Africa and migrated from there to southern Africa where many species of both subgenera of Zygophyllum have speciated in the Northern Cape Province, southern Namibia and along the Namibian coast. Additionally the phylogeny shows evidence of repeated migrations of Zygophyllum species from southern Africa to the arid areas in the horn of Africa and Asia and back. These migrations could have occurred during repeated dry glacial periods (de Menocal, 2004) in which it is postulated that a dry corridor existed from southern Namibia to the horn of Africa (White, 1983). 4.4. Taxonomic implications Sheahan and Chase (2000) found that Augea, Fagonia and Tetraena were embedded in Zygophyllum, which therefore became a paraphyletic taxon. Accordingly, Beier et al. (2003) proposed a subdivision for the Zygophylloideae based on morphological and trnL intron data. The name Zygophyllum was retained for the clade that contains the type species, Z. fabago, whereas other Zygophyllum species were transferred to the genera Roepera and Tetraena; and Z. hildebrandtii and Z. robecchii were transferred to Melocarpum. The genera Fagonia and Augea, however, were retained. Beier et al. (2003) argued against lumping all of the above genera into Zygophyllum because the Linnean names Zygophyllum and Fagonia had been in use since 1753, Augea since 1794 and Tetraena since 1889. Our findings support their transfer of the horn of Africa species, Z. hildebrandtii and Z. robecchii to Melocarpum. However, our evidence for a sister grouping of Z. orbiculatum and A. capensis, does not support the renaming of Zygophyllum stapffii as Tetraena stapffii. Beier et al. (2003) did not examine Z. orbiculatum (=Z. stapffii) but placed Z. stapffii in the newly circumscribed Tetraena on the basis of fruit dehiscence, which they indicated as a schizocarp. On the basis of the unique morphological characters of Z. orbiculatum (=Z. stapffii), and its sister relationship to Augea, we suggest that it ought to be put in a monotypic genus (Marais, Bellstedt, Van Zyl and Craven, in prep.) being a true Kaokoveld endemic, and not, as previously classified (van Zyl, 2000), a peculiar outlier of the subgenus Zygophyllum that occurs predominantly in the CFR (see Table 2). Beier et al. (2003) advocated that the genus name Zygophyllum be retained for a poorly supported clade (56% bootstrap value in their trnL-based phylogeny) which includes Z. fabago, the type species of the genus, Z. xanthoxylum and a number of related species. The evidence found in this study places doubt on assigning all other Zygophyllum species to Tetraena and Roepera as our combined rbcL and trnLF analysis retrieves a monophyletic grouping consisting of the Asian Zygophyllum species as well as the groupings renamed Tetraena and Roepera. In our opinion, the inclusion of more taxa and more gene regions will be required to establish the proper relationships between the members of the subfamily Zygophylloideae, and until such time we advocate against changing the current taxonomy. Acknowledgments We express our sincere gratitude to Patricia Craven, Herta Kolberg, Colleen Mannheimer and Tony Dold for the collection of plant material. Cape Nature, Northern Cape Department of Nature Conservation and Environmental Conservation, and the Namibian Ministry of Environment and Tourism are thanked for permission to collect material. Funding for this research was provided by the South African National Research Foundation and the University of Stellenbosch. 949 Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev.2008.02.019. References Beier, B.A., Chase, M.W., Thulin, M., 2003. 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Phylogenetic relationships, character evolution and biogeography of southern African members of Zygophyllum (Zygophyllaceae) based on three plastid regions

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