TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range Plant diversification in the Espinhaço Range: Insights from the biogeography of Minaria (Apocynaceae) Patricia Luz Ribeiro,1,2 Alessandro Rapini,2 Leilton S. Damascena2 & Cássio van den Berg2 1 Universidade Federal do Recôncavo da Bahia, Centro de Ciências Agrárias, Ambientais e Biológicas, Cruz das Almas, Bahia, Brazil 2 Universidade Estadual de Feira de Santana, Departamento de Ciências Biológicas, Laboratório de Sistemática Molecular de Plantas, Feira de Santana, Bahia, Brazil Author for correspondence: Patricia Luz Ribeiro, patyluzribeiro@yahoo.com.br ORCID (http://orcid.org): PLR, 0000-0003-2614-2712; AR, 0000-0002-8758-9326; CvdB, 0000-0001-5028-0686 DOI http://dx.doi.org/10.12705/636.16 Abstract The Espinhaço Range, eastern Brazil, is a region with remarkable floristic diversity and endemism, which are mainly concentrated in the campo rupestre. Minaria (Apocynaceae) is a genus with 21 species, most of which are endemic to the Espinhaço Range. In the present study, we investigated the biogeography of Minaria as the basis for understanding the origin and maintenance of plant diversity and endemism in the campo rupestre of the Espinhaço Range. We assessed the ecological divergence between clades, reconstructed the historical biogeography and dated the phylogeny of Minaria based on plastid and nuclear DNA. According to our estimates, Minaria arose in the Espinhaço Range during the Neogene. Its distribution is postulated to have been driven by a trend toward long-term retraction, interrupted by a few episodes of expansion. Ecologically, Minaria species do not present any obvious innovations that could explain their diversification by adaptive radiation. Apparently, the higher-altitude rocky savannas in the Espinhaço Range have offered stable environments in which dry seasons and fire regimes are less intense than in savannas at lower altitudes. Isolated on rocky outcrops, lineages would be more likely to differentiate by non-adaptive radiation, which may result in high plant diversity and endemism. Keywords Asclepiadoideae; campo rupestre; endemism; molecular dating; niche conservatism; Pleistocene climatic changes Supplementary Material Electronic Supplement (Tables S1–S3; Figs. S1–S3) and alignment are available in the Supplementary Data section of the online version of this article at http://www.ingentaconnect.com/content/iapt/tax INTRODUCTION The accumulation of Neotropical plant group phylogenies is revealing that patterns of diversification have differed among biomes; however, data and understanding are still lacking for several major biomes in South America (Hughes & al., 2013). The campo rupestre (rocky field), despite their high species richness and endemism, especially in the Espinhaço Range (e.g., Rapini & al., 2008), eastern Brazil, represents one of these neglected biomes. To date, only a few phylogenetic studies of lineages that are concentrated in the campo rupestre have been published: Microlicieae (Melastomataceae; Fritsch & al., 2004), Eriocaulaceae (Andrade & al., 2010; Trovó & al., 2013), Hoffmannseggella H.G.Jones (Orchidaceae; Antonelli & al., 2010), Minaria T.U.P.Konno & Rapini (Apocynaceae; Ribeiro & al., 2012a,b). These phylogenies are generally poorly resolved. The Espinhaço Range is a 1000 km long S-shaped mountain range with high rates of endemism and is well known for its rich and unique flora (Harley, 1988, 1995; Giulietti & al., 1997; Rapini & al., 2008). At the intersection of three different phytogeographic domains (sensu Fiaschi & Pirani, 2009), it occupies an area of approximately 120,000 km2 in the centre of Minas Gerais and Bahia states. The southern Espinhaço Range lies between two South American biodiversity hotspots (Meyer & al., 2000): the Atlantic forest (a tropical rain forest) to the east and the cerrado (savanna vegetation) to the west; the northern part is nested in the Brazilian caatinga (a seasonally dry forest). However, most of the Espinhaço Range is covered by campo rupestre. This savanna-like vegetation is closely associated with the quartzite and ironstone outcrops that usually appear at altitudes above 900 m and comprise a mosaic of communities dependent on differential soil depth (Conceição & Pirani, 2005) and water availability (Assis & al., 2011). The origin of the Espinhaço Range dates from the end of the Paleoproterozoic (1752 Ma), but its formation culminated only with flexures and overlaps caused by East-West pressure at the end of Neoproterozoic, around 900 Ma (Almeida-Abreu & Pflug, 1994). Tectonic activities occurred during the entire process; however, geologic stability was reached long ago, and more recently, the relief has been moulded mainly by erosive processes (Saadi, 1995; Pedreira, 1997). The soils are nutrient-poor and extremely acidic, often forming sand deposits (Harley, 1995). The region’s weather can be classified as Cwb (temperate highland tropical climate with dry winter; Köppen, Received: 8 Mar 2014 | returned for first revision: 8 May 2014 | last revision received: 27 Sep 2014 | accepted: 28 Sep 2014 | published online ahead of inclusion in print and online issues: 3 Dec 2014 || © International Association for Plant Taxonomy (IAPT) 2014 Version of Record (identical to print version). 1253 TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range 1931). Soil conditions, temperature and wind variation, water stress and fire regimes have favoured the presence of plants with several convergent morphological characteristics, and the vegetation appears scleromorphic (Giulietti & al., 1997). Although the Espinhaço Range represents less than 1.5% of the Brazilian territory, it harbours approximately 10% of the entire angiosperm flora reported for Brazil (Ribeiro & al., 2012b), and 30% of the Espinhaço Range flora is endemic (Giulietti & al., 1987). Endemics are concentrated in particular groups (e.g., Eriocaulaceae and Velloziaceae) and are distributed mainly in the campo rupestre, which are isolated by valleys and major depressions on mountain tops. The endemics have different ranges and are genetically subdivided by small-scale disjunctions caused by microhabitat heterogeneity (e.g., Borba & al., 2001; Jesus & al., 2001, 2009; Franceschinelli & al., 2006; Lambert & al., 2006; Pereira & al., 2007; Ribeiro & al., 2008). The principal hypothesis to explain the plant diversity in the Espinhaço Range is similar to the “evolutionary pump” model proposed by Morton (1972) for West African tropical mountain vegetation and postulates successive expansionretraction events in the campo rupestre in response to Pleistocene climatic oscillations (Harley, 1988, 1995; Alves & Kolbek, 1994; Giulietti & al., 1997; Rapini & al., 2008). According to this hypothesis, the Espinhaço highlands represented interglacial refugia for the campo rupestre flora. During the warmer and moister interglacial periods, the campo rupestre would have been fragmented and isolated, favouring divergence among vicariant populations, whereas during cooler and drier glacial periods, areas dominated by forests would have retracted, allowing expansion of the campo rupestre from mountain tops to lower altitudes. Neotropical diversification seems to have been influenced by a complex set of events that occurred mainly during the last 10 Ma (Rull, 2011; Hughes & al., 2013). Neogene tectonic and palaeogeographic changes and Pleistocene climatic changes almost certainly affected the diversification of most lineages, but their importance for the accumulation of extant species differed among groups. For plants, extant species have mostly originated in the Quaternary (Rull, 2008); however, diversification patterns and drivers have differed in different biomes (Pennington & al., 2006b; Hoorn & al., 2010; Hughes & al., 2013). The savannas in central South America were assembled from numerous independent plant lineages from different biomes, mostly during the last 5–4 million years, indicating a recent origin of the modern cerrado, coinciding with C4 flammable grass dominance and suggesting that plants recurrently colonized fire-prone savannas from fire-free biomes (Pennington & al., 2006a; Simon & al., 2009; Simon & Pennington, 2012). Because of being fire-prone, their open vegetation and geographical distribution, the campo rupestre have usually been included in the cerrado s.l. (e.g., Fritsch & al., 2004; Simon & al., 2009; Trovó & al., 2013), masking their floristic differences and historical relationships. To date, the only attempt to specifically investigate plant diversification in the campo rupestre using a dated phylogeny and ancestral range reconstruction was made in Hoffmannseggella, a genus of orchids that is rich in rupicolous 1254 species endemic to the Espinhaço Range. According to the scenario postulated in that study (Antonelli & al., 2010), the diversification of Hoffmannseggella was most likely caused by the expansion of the campo rupestre in parallel with global cooling, promoting radiation through hybridisation between populations that were previously isolated. Furthermore, considering that several clades of these orchids radiated before 4 Ma, with most species arising more than 10 Ma, the study also suggested that diversification in the campo rupestre pre-dated the expansion of the modern cerrado. Nevertheless, it is clear that investigations of other lineages from the campo rupestre are needed to better understand the historical relationships between these two biomes. In the present study, we investigate the biogeography of Minaria, a genus predominantly endemic to the campo rupestre, to evaluate the evolutionary pump model often used to explain diversification and endemism in the Espinhaço Range. We reconstructed and dated the ancestral distribution of Minaria motivated by two initial questions: Where and when did Minaria diversify? If the diversification of Minaria is shown to have occurred in the Espinhaço Range during the Pleistocene, as suggested by the pump model, then it is also important to verify whether the diversification pattern coincides with Quaternary climatic oscillations and whether an adaptive radiation caused by the occupation of new habitats would have favoured its diversification. MATERIALS AND METHODS Minaria comprises predominantly erect shrubs with small leaves, usually up to 2 cm long, except Minaria volubilis Rapini & U.C.S.Silva, which has a twining habit and linear leaves, up to 3.5 cm long. The flowers in the group can vary in size, form and indumentum of the corolla, and the corona can be simple, double, or absent. Like most asclepiads, the follicaria—follicular monocarps of a bicarpellary gynoecium (Spjut, 1994)—usually produce several comose seeds, but some species produce only one or two seeds per follicarium and lack a coma. The genus was delimited based on phylogenetic studies from plastid DNA (Rapini & al., 2006). Originally, it included 19 species, all represented in the Espinhaço Range of Minas Gerais, and most narrowly endemic (Konno & al., 2006). However, more comprehensive phylogenetic analyses (Silva & al., 2012; see also Ribeiro & al., 2012a) have shown that Minaria also should have included two narrowly endemic species from the Espinhaço Range of Bahia. Currently, the genus comprises 21 species, 75% of which are endemic to the Espinhaço Range (Ribeiro & al., 2012b), but the distribution range of a few species reaches as far as Argentina, Bolivia, northern Brazil and the Atlantic coast (Konno & al., 2006). The genus occurs in savanna-like vegetation types (campo rupestre, cerrado), usually above 900 m, among rocks and on sandy or stony soils. Spatial analyses. — For the biogeographic analyses, we used a phylogenetic tree of Minaria based on molecular and morphological data (Ribeiro & al., 2012b), in which Minaria bifurcata (Rapini) T.U.P.Konno & Rapini, M. inconspicua Version of Record (identical to print version). TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range (Rapini) Rapini and M. monocoronata (Rapini) T.U.P.Konno & Rapini were represented exclusively by morphological data. We based species distributions on 762 herbarium collections; only specimens with geographic coordinates or accurate locality data were considered. Localities were confirmed or coordinates were recovered with Google Earth or during our expeditions to the Espinhaço Range, Central Brazil and the mountainous regions in southern and northeastern Brazil. Because Minaria mainly occurs above 900 m, its occurrence was indicated in a hypsometric map showing relief discontinuities. To link species distribution and relief, we used a hydrographic basin map Otto-codified for details of levels 3 and 4 (National Agency of Waters; ANA, 2011). Based on this information, and considering biotic and abiotic aspects such as geomorphology, microbasins, geographic connectivity and species endemism, we established 10 biogeographic units for Minaria (Fig. 1A) listed and described below. Rio de Contas (A). – Rio de Contas and Serra das Almas ranges, southwest Chapada Diamantina, in the Rio de Contas Basin, separated from the following area (B) by the Rio de Contas Valley (15–30 km wide). Sincorá (B). – Sincorá Mountain Range, eastern Chapada Diamantina, in the Paraguaçu Basin, coinciding with M. volubilis endemism. Grão Mogol (C). – Discontinuous mountains in the northern Espinhaço Range of Minas Gerais, near the border of the state of Bahia, in the Jequitinhonha Basin. Serra do Cabral (D). – Mountain plateau in the western Espinhaço Range of Minas Gerais, in the São Francisco basin. Diamantina Plateau (E). – Plateau of quartzite of the Espinhaço Supergroup, extending from the Araçaí Basin, in the southernmost region of the Jequitinhonha Basin, and Rio das Velhas Basin, characterised by the endemism of M. bifurcata, M. campanuliflora Rapini, M. diamantinensis (Fontella) T.U.P.Konno & Rapini, M. grazielae (Fontella & Marquete) T.U.P.Konno & Rapini, M. inconspicua and M. refractifolia (K.Schum.) T.U.P.Konno & Rapini. Serra do Cipó (F). – Narrow area in the central Espinhaço Range of Minas Gerais at the confluence of the Santo Antônio Basin, a tributary of the Rio Doce, and the Rio das Velhas Basin, characterised by the endemism of M. hemipogonoides (E.Fourn.) T.U.P.Konno & Rapini, M. magisteriana (Rapini) T.U.P.Konno & Rapini, M. polygaloides (Silveira) T.U.P.Konno & Rapini and M. semirii (Fontella) T.U.P.Konno & Rapini. Southern Espinhaço Range (G). – The southernmost area of the Espinhaço Range at the end of Espinhaço Supergroup, at the confluence between the São Francisco and Rio Doce basins. Paraná Basin (H). – The Paraná Basin, a region extending from southern Minas Gerais to northern São Paulo. Cerrado of Bahia (I). – Upland region in the São Francisco Basin, western Bahia. Cerrado of Goiás (J). – Upland region in Central Brazil at the junction of the São Francisco (East), the Tocantins (North) and the Paraná (South) basins. We investigated the biogeography of Minaria using statistical dispersal-vicariance analysis (S-DIVA) in RASP v.1.107 (Nylander & al., 2008; Yu & al., 2010, 2011), across the Minaria phylogeny (Ribeiro & al., 2012b) based on a matrix of species distribution according to the 10 biogeographic units. We established a limit of four regions for the ancestral distributions and took into account phylogenetic uncertainty by running the analyses on a set of 6000 trees saved after the Bayesian phylogenetic analysis reached stationarity. Ecological analyses. — We collected 22 variables for climate and altitude from WordClim (Hijmans & al., 2005), geomorphological data from Geological Survey of Brazil (CPRM, 2014) and soil data from the Brazilian Ministry of the Environment (MMA, 2011) for the 762 Minaria collections. Environmental data were extracted for all georeferenced locations of each species using GIS. As described in Struwe & al. (2009), each variable was divided into categories and a table was prepared listing the number of collections per species for each variable and for each category within variables. This table and the Minaria phylogenetic tree (Ribeiro & al., 2012b) were imported into SEEVA v.1.01 (Heiberg, 2008) to analyse ecological divergence between sister clades. Following Struwe & al. (2011), only nodes with D > 0.75 (P < 0.0023, after Bonferroni correction for 22 terminals) were considered strongly divergent and relevant to Minaria evolution. SEEVA has already been shown to be a useful tool for statistically assessing niche conservatism and niche divergence from phylogenetic, geographical and ecological data in plants (Struwe & al., 2011), animals (Wooten & al., 2013) and fungi (Walker & al., 2013). Dating. — Because Apocynaceae are poorly represented in the fossil record, we used a secondary calibration to constrain the Minaria phylogeny based on a divergence time estimated for subtribe Metastelmatinae. For the primary age estimates, we analysed 107 sequences of trnL-F (trnL intron and trnL-F intergenic spacer; available in GenBank) representing the main clades of the Apocynaceae and a sequence from the Loganiaceae as outgroup (Appendix 1) using BEAST v.1.6.2 (Drummond & Rambaut, 2007) with a GTR substitution model and gamma distribution (GTR + G; the best-fitting model according to the MrModeltest v.2.3; Nylander, 2004), an uncorrelated lognormal relaxed clock model and a tree prior with a Yule speciation model. The Monte Carlo-Markov chains ran for 107 generations, sampling trees every 1000 generations. Fossils of the comose seeds of Apocynospermum C.Reid & M.E.J.Chandler from the Middle Eocene (47 Ma; Collinson & al., 2010) were used to constrain the minimum age of the APSA stem group (including Apocynoideae, Periplocoideae, Secamonoideae and Asclepiadoideae), and the Polyporotetradites M.Salard-Cheboldaeff pollen tetrads from the Oligocene/Miocene boundary (23 Ma; Muller, 1981) were used to set the minimum age for the Periplocoideae stem group, using lognormal prior distributions (Mean = 1.5, Log St Dev = 1.0, Offset = 47; Mean = 1.5, Log St Dev = 1.0, Offset = 23, respectively). Because tetrads can also be found in unrelated groups of the Apocynaceae (Van de Ven & Van der Ham, 2006), we considered the Periplocoideae calibration to be based on a risky fossil (sensu Sauquet & al., 2012). Therefore, a cross-validation analysis using the same settings, Version of Record (identical to print version). 1255 TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range but excluding that calibration, was also conducted to evaluate congruence between the estimates, similar to an approach used by Perret & al. (2013). The output file was visualised in Tracer v.1.5 (Rambaut & Drummound, 2007) to assess ESS values (only ESS > 200 were accepted), and the results were summarised in a maximum clade credibility tree using BEAST’s TreeAnnotator module. The chronogram was visualised and edited using FigTree v.1.2.2 (Rambaut, 2009). Divergence time estimates for Minaria are based on a matrix combining plastid (psbA, trnD-T, trnS-G, rps16) and nuclear (ITS, ETS) sequences for 41 taxa (Ribeiro & al., 2012a). Bayesian analyses performed in MrBayes v.3.2.2 (Ronquist & al., 2012) based on the dataset of plastid and nuclear sequences were used for visual inspection of topological inconsistency, as specified in Ribeiro & al. (2012a). MrBayes was used as opposed to BEAST because multiple chains can be implemented more easily in the former, allowing for better exploration of parameter space (McCormack & al., 2011). We conducted age estimates as for the Apocynaceae (above). Partitions were unlinked in the combined analysis and the bestfitting model used for partitions was as in Ribeiro & al. (2012a), calculated with MrModeltest v.2.3 (Nylander, 2004): psbAtrnH: GTR + G; trnS-trnG: HKY + equal; rps16: GTR + I + G; trnE-trnY: HKY + G; ITS: GTR + I + G; ETS: GTR + I + G. Monte Carlo-Markov chain ran for 2 × 106 generations. The secondary calibration derived from the divergence time estimates for the Metastelmatinae crown group used a normal distribution and the uncertainty from the higher level Apocynaceae chronogram. Ecological analyses. — Figure 1 shows the relevant ecological divergence for nodes of the total evidence tree (see Electr. Suppl.: Table S1 for degrees of ecological divergence of every clade and Table S2 for classification of samples per species). The vicariant lineages that are endemic to the Diamantina Plateau and Serra do Cipó (nodes 2 and 9) had the highest number of variables with significant differences, which are related to precipitation and temperature. The divergence of M. monocoronata (node 8) is related to the occupation of an area with ferruginous soil (vs. quartzite), higher annual precipitation and the most intense warm season. Type of soil and temperature variables characterised the divergence among the lineages that comprise part of the M. cordata complex (node 16), which is distributed mainly in the Espinhaço Range and the cerrado A BA I A B GO MG J C RESULTS D Spatial analyses. — The ancestral area reconstruction for Minaria (Fig. 1) suggests that the lineage had a broad ancestral distribution, although it was restricted to the Espinhaço Range, most likely occupying areas in both Minas Gerais and Bahia states or only in Minas Gerais, although this distribution is less likely (node 1). Since then, the ancestral area of Minaria was fragmented, with clades tending to show areas smaller than the ancestral ones, suggesting a general contraction of its distribution. The principal disjunction was marked by the vicariance between Bahia (node 4) and Minas Gerais (node 5) whereas the sister group (node 2) of this principal clade (node 3) occurs only in Minas Gerais. The clade of rupicolous species with seeds lacking a coma (node 6) had a likely ancestral area comprising the Serra do Cipó and Diamantina Plateau in Minas Gerais. Most species of the rupicolous clade are currently restricted to only one of these areas, although M. monocoronata dispersed to southern Espinhaço Range. Reconstruction is mostly equivocal between nodes 11 and 13 because widespread species, occurring also in areas of central Brazil outside the Espinhaço Range, allow several possible reconstructions. Apart from the M. cordata (Turcz.) T.U.P.Konno & Rapini complex, Minaria remained mainly restricted to the Espinhaço Range, with one clade (node 20) restricted to the Diamantina Plateau, from which M. acerosa (Mart.) T.U.P.Konno & Rapini and M. micromeria (Decne.) T.U.P.Konno & Rapini dispersed to other areas. 1256 E F ES G SP Version of Record (identical to print version). H RJ TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range of central Brazil. However, M. abortiva (E.Fourn.) Rapini (node 19), another lineage of the M. cordata complex, which is genetically closely related to the species with acerose leaves, is geographically isolated in southern Minas Gerais and inhabits areas with higher annual precipitation. Overall, we did not detect any ecological divergence or evolutionary trend that could be confidently assigned to environmental shifts or adaptive radiations. Ecological divergences observed within Minaria are mostly restricted to relatively recent cladogenic events and usually reflect latitudinal climatic variation in areas that are occupied by vicariant lineages (Fig. 1). Minaria inconspicua MG EF G >183 AP <183 <600 2PWQ >600 2 Dating. — Topologies produced with plastid and nuclear datasets did not reveal any supported (posterior probability ≥ 95%) conflict within Minaria (Electr. Suppl.: Fig. S1). The combined analysis resulted in more supported clades (Fig. 2) and provided the estimates that are presented and discussed hereafter. The Periplocoideae fossil calibration does not conflict with the age estimated for this node without constraining it. Therefore, we used both fossils to calibrate the Apocynaceae phylogenetic tree. According to our dating (Electr. Suppl.: Fig. S2; Table S3), the Apocynaceae originated (stem group) B E >15.6 MTDQ <15.6 Minaria polygaloides FG Minaria harleyi BA B EFG 4* Minaria volubilis AB B B EFG Minaria hemipogonoides 1 BG Minaria bifurcata E F E EF 6 BF 3* EF BE Minaria monocoronata 7 <183 AP >183 EF 8 EF MG Core Group FG EG 9 E 558-600 2PWQ >403 * G Greenstone GEO Psammitc rocks Minaria grazielae >183 <558 AP 2PWQ <183 >600 >15.6 MTQD <15.6 F 5* E Minaria magisteriana 10 Minaria parva CEFG Minaria cordata - VIR E 14 Minaria semirii F F ABGJ Minaria lourteigiae D 11* Minaria cordata 12 Minaria ditassoides 15 EJ >15.6 MTQD <15.6 16* E E 13 ABIJ 17 CEFG Minaria campanuliflora E EJ EH Minaria cordata - GO Minaria decussata J Minaria abortiva E 18* EH 19 193-207 AP >183 ACDEFG H Minaria refractifolia E E 20* Minaria acerosa E 21* E 22 Changes 403-558 MTQD >403 ABCDEFGHIJ Minaria diamantinensis E Minaria micromeria CDEFGHIJ Fig. 1. A, map of eastern Brazil showing the biogeographic regions considered in this study: A, Rio de Contas; B, Sincorá; C, Grão Mogol; D, Serra do Cabral; E, Diamantina Plateau; F, Serra do Cipó; G, Southern Espinhaço Range; H, Paraná Basin; I, Cerrado of Bahia; J, Cerrado of Goiás. Areas A–G, Espinhaço Range. States: BA, Bahia; ES, Espírito Santo; GO, Goiás; MG, Minas Gerais; RJ, Rio de Janeiro; SP, São Paulo. B, phylogram (ACCTRAN optimization) based on the majority-rule consensus tree derived from the Bayesian analysis of combined molecular and morphological data of Minaria (Ribeiro & al., 2012b) showing ancestral area reconstruction and ecological divergences. Node numbers correspond to those discussed in the text; those with posterior probability ≥ 95% are indicated with an asterisk. Pies represent frequencies of area reconstructions; node with letters only have a single area reconstruction (frequency = 100%); the black portion of pies represents the sum of area reconstructions with frequencies < 10%; clades 12, 14 and 15 show only frequency reconstructions < 10%. Grey boxes on some nodes indicate the main detected ecological divergences: AP, annual precipitation (mm); GEO, geomorphology; MTQD, mean temperature of driest quarter (°C); 2PWQ, precipitation of warmest quarter (mm). GO, Goiás; VIR, “var. virgata”. Version of Record (identical to print version). 1257 TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range * * * * HER * * * * * * 4 1 * * 3 6 * 9 10 * 5 Core Group * 14 * Wide distribution Savanna-like Cerrado Campo rupestre Atlantic forest Dry forest 12 15 16 * 13 17 18 * 20 21 * 22 8.0 7.0 6.0 5.0 4.0 Pliocene Miocene Neogene 3.0 2.0 Blepharodon ampliflorum Hemipogon acerosus Barjonia chlorifolia Barjonia cymosa Barjonia erecta Nephradenia acerosa Petalostelma martianum Hemipogon sprucei Blepharodon pictum Nephradenia filipes Hemipogon luteus Hemipogon carassensis Hemipogon hemipogonoides Hemipogon abietoides Hemipogon hatschbachii Ditassa fasciculata Ditassa burchellii Ditassa banksii Ditassa hispida Nephradenia asparagoides Ditassa capillaris Minaria polygaloides Minaria harleyi Minaria volubilis Minaria hemipogonoides Minaria grazielae Minaria semirii Minaria magisteriana Minaria parva Minaria cordata - VIR Minaria lourteigiae Minaria cordata Minaria campanuliflora Minaria cordata - GO Minaria ditassoides Minaria abortiva Minaria decussata Minaria refractifolia Minaria acerosa Minaria diamantinensis Minaria micromeria BA MG 1.0 Pleistocene Quaternary 43 Ka cycles 100 Ka cycles 2 0°C -2 -4 -6 -8 Fig. 2. Chronogram (in Ma) based on molecular data (Ribeiro & al., 2012a) showing a biogeographic scenario for the Metastelmatinae diversification. Grey bars on nodes represent 95% confidence intervals of ages. The vertical bar denotes the predominant distribution of Minaria, considering the two blocks: BA, Bahia State; MG, Minas Gerais State. The graph below represents temperature oscillations in the last 5 Ma (modified from Lisiecki & Raymo, 2005) and shows the predominant cycle periods. The bar at the top represents putative periods of retraction (light) and expansion (dark) of Minaria based on Fig. 1B. Ancestral vegetation reconstruction is based on Bayesian analysis performed in RASP (Electr. Suppl.: Fig. S3). Savanna-like vegetation comprises the modern cerrado and campos rupestres. Arrows indicate terminals from campos rupestres outside the Espinhaço Range. Node numbers in Minaria correspond to those in Fig. 1. Asterisks indicate clades with posterior probability ≥ 95%. Geological periods were converted from absolute ages based on the International Chronostratigraphic Chart (Cohen & al., 2013, updated). HER = Hemipogon from the Espinhaço Range. GO, Goiás; VIR, “var. virgata”. 1258 Version of Record (identical to print version). TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range 77.55 (58.07–101.1; 95% confidence interval) Ma and diversified (crown group) 68.9 (84.9–54.85) Ma, and the Asclepiadoideae originated 34.61 (28.87–40.51) and diversified 31.6 (37.35– 25.9) Ma. We estimated that the Metastelmatinae diversified 11.21 (16.3–6.9) Ma, which was used as secondary calibration to constrain the Minaria dating analysis. Given the current distribution of the sampled Mestastelmatinae, the reconstruction of the Minaria distribution in the biogeographic units (Fig. 1) and vegetation types (Electr. Suppl.: Fig. S3) in S-DIVA, and the age estimated by focusing on the Metastelmatinae, a biogeographic scenario on the maximum clade credibility chronogram is presented in Fig. 2. The subtribe started to diversify in the Late Miocene ca. 7 (9.5–4.9) Ma in savanna-like vegetation, in central South America. The lineage expanded to become more widespread in the Pleistocene, when it diversified into seasonally dry tropical forests in northeastern Brazil (Ditassa capillaris E.Fourn., Nephradenia asparagoides E.Fourn.), and Atlantic rain forest and Restinga in eastern Brazil (Ditassa banksii Roem. & Schult., D. burchellii Hook. & Arn.). Lineages predominantly restricted to the Espinhaço Range (campo rupestre) have appeared more than once independently during the evolution of the Metastelmatinae (Fig. 2). Minaria most likely arose between the Late Miocene and Early Pliocene, at 5.6 (6.91–4.45) Ma, but the core group, where most of the species diversity resides, started to diversify only during the Pleistocene, 1.96 (2.6–1.4) Ma, chronologically coinciding with the diversification of the “Hemipogon from the Espinhaço Range” clade (node HER), which is also predominantly distributed in the campo rupestre (Fig. 2). DISCUSSION Biogeography of Minaria. — The estimated age for the Apocynaceae crown group between the Upper Cretaceous and the Tertiary (Paleocene-Eocene boundary) in this study is older than that used to calibrate this node in the dating analysis by Rapini & al. (2007), but the age estimates for other clades are younger. These differences were possibly caused by our sampling being more equally distributed across the entire Apocynaceae and because the Apocynaceae tree in Rapini & al. (2007) was dated using nonparametric rate smoothing (NPRS), which does not take phylogenetic uncertainties into account. Moreover, NPRS tends to overfit the data, allowing rapid fluctuations in rates particularly in areas of the tree with short branches (Sanderson, 2003). According to our results, the Neotropical clade MOOG (including subtribes Metastelmatinae, Oxypetalinae, Orthosiinae and Gonolobinae) most likely originated in the Early Miocene, with most of the diversification occurring in the last 10 Ma (Fig. 2). The Metastelmatinae is estimated to have arisen and begun diversifying in the Late Miocene, most likely in open savanna-like vegetation, but most extant species appear to have originated during the Pleistocene, possibly associated with the acquisition of the twining habit and occupation of new habitats, such as Atlantic and Dry Forests (Silva & al., 2012; Electr. Suppl.: Fig. S2). This result is consistent with the overall pattern found in Neotropical plants, in which the Neogene, mostly since 10 Ma, has been shown to be a pivotal time for diversification, although many clades attained their present biodiversity mainly during the Quaternary, since 2.6 Ma (Rull, 2008, 2011; Hughes & al., 2013). The campo rupestre lineages diverged from ancestors with widespread distributions in savanna-like vegetation, suggesting specialisation to the highlands, followed by general biome conservatism (Fig. 2), potentially marking a dichotomy between the cerrado and campo rupestre floras between 8 and 4 Ma, which coincides with the establishment and expansion of fire regimes in savanna-like vegetation during the Late Miocene–Pliocene (Cerling & al., 1997; Jacobs & al., 1999; Beerling & Osborne, 2006; Simon & al., 2009; Edwards & al., 2010; Strömberg, 2011). Minaria apparently arose in the campo rupestre of the Espinhaço Range, between the Late Miocene and the Early Pliocene (Figs. 1–2). An initial cladogenesis of Minaria gave rise to two lineages, both of them broadly distributed in the Espinhaço Range. During the Pliocene, populations of Minaria from the Bahia and Minas Gerais states most likely became isolated from one another, a pattern also detected in other groups, such as Eriocaulaceae (Trovó & al., 2013). One of the two lineages (node 2) probably became extinct in Bahia and is currently represented only by two species from Minas Gerais. The lineage of Minaria in Bahia (node 4; Figs. 1–2) became restricted to the Chapada Diamantina, isolated in highlands surrounded by seasonally dry forests. The divergence time of the two species in this clade is older than the radiation of the genus in Minas Gerais, which explains their discrepant morphologies (Silva & al., 2012) and the high phylogenetic endemism of Minaria in Chapada Diamantina (Ribeiro & al., 2012b). In contrast, the Minaria core group (node 5) in Minas Gerais diversified exclusively during the Pleistocene and comprises 17 of the 21 species of the genus. It can be divided in a clade with seeds lacking coma (node 6) and a clade with comose seeds (node 11). The clade lacking a coma (node 6) is composed of typically rupicolous plants and produces fruits with only one or two seeds. The absence of a coma on the seeds appears to have limited dispersal and the lineage diversified exclusively within the Espinhaço Range of Minas Gerais. Seeds in this group tend to remain close to nearby rocky areas and are not lost in soils where they would not be recruited or competitive. Islands of vegetation in rock outcrops usually occur on higher portions of the Espinhaço Range, where the aridity of the dry season is minimised by the orographic humidity, and the amount of flammable material is lower. Together, these factors help prevent spread of fire, favouring lineages that are less tolerant to aridity and fire disturbances, many of which are currently narrowly endemic (Neves & Conceição, 2010; Ribeiro & al., 2012b; Bitencourt & Rapini, 2013). The other (comose) clade of the Minaria core group (node 11) reached areas outside the Espinhaço Range for the first time in the history of Minaria. The expansion of this lineage was most likely followed by a retraction, particularly to the Diamantina Plateau (node 20) in the Espinhaço Range. More recently, however, two species (M. acerosa and M. micromeria) Version of Record (identical to print version). 1259 TAXON 63 (6) • December 2014: 1253–1264 Ribeiro & al. • Plant diversification in the Espinhaço Range dispersed from the Diamantina Plateau and became widespread and, currently, the clade is represented by species that are narrowly distributed or that have discontinuous distributions. According to our interpretation of the spatial, ecological and dating analyses (Figs. 1–2), the distribution of Minaria seems to be mainly governed by an overall retraction (larger ancestral distributions resulting in smaller, disjunct distributions), in parallel with a general trend of decreasing temperatures and increasing aridity through the Quaternary (Zachos & al., 2001). Nevertheless, it is possible that the ecological scale of variables used in our analysis is not sufficiently detailed to detect important changes in microhabitats. The uncertain phylogenetic relationships and area reconstructions during the diversification of Minaria at approximately 1.5 Ma (nodes 11–13; chronologically coincident with the divergences of M. bifurcata and M. monocoronata in the sister clade: nodes 7 and 8 in Fig. 1 and between nodes 6 and 9 in Fig. 2) is interpreted as a rapid expansion of the range of Minaria, followed by retraction and multiple geographic isolations. Some isolated lineages kept most of the ancestral features and form cryptic species defined by symplesiomorphies, as postulated by Ribeiro & al. (2012b) to explain the intricate taxonomy of the M. cordata complex. In contrast, other isolated lineages, particularly in the Espinhaço Range, are recognised as distinct species (Rapini, 2010), suggesting that morphological (and even genetic) differentiation can be more intense in this region. Genetic drift is likely an important mechanism for the differentiation of these narrowly distributed, isolated lineages and for the non-adaptive radiation of the group. Plant diversification in the Espinhaço Range. — Diversification of many Neotropical plant groups studied so far occurred mainly during the Pleistocene (Rull, 2008), and climatic oscillations are usually used to explain the high levels of taxonomic richness and endemism in several tropical regions (e.g., Haffer, 1969; Fjeldsa & Lovett, 1997; López-Pujol & al., 2011; Schnitzler & al., 2011). Areas that are rich in endemism, especially mountain regions, represent potential refugia for species during global cooling and the climatic instability that characterises the Quaternary. During periods of retraction, stable regions can house older species with lower capacities of adaptation and dispersal, and also recent species that originated after the geographic isolation of ancestral lineages that had broader distributions in favourable periods. Because the general retraction of the range of Minaria occurred in parallel with global cooling, it is unlikely that the highlands represented refugia during interglacial periods. According to Ribeiro & al. (2012b), the phylogenetic conservatism of Minaria to the campo rupestre seems to be associated with aridity and fire regime, which also affected tropical environments more markedly at the end of the Miocene and became more intense according to the extension and intensity of the region’s seasonality. Compared to savannas at lower altitudes, the highlands in the Espinhaço Range are mild environments. The orographic moisture throughout the entire year makes the dry seasons less intense, and the dominance of rocky outcrops, along with the lower abundance of C4 grasses that fuel fire in savannas, prevent fires from spreading over large areas in 1260 the campo rupestre. Therefore, islands of vegetation in rocky outcrops are refugia for species that are less tolerant to long periods of dryness and/or more sensitive to fire; consequently, these species remained restricted to these areas. The pattern postulated for Minaria diversification contrasts with that suggested by Antonelli & al. (2010) for Hoffmannseggella, according to which diversification was caused mainly by hybridisation (vs. geographic isolation in Minaria) after the expansion (vs. retraction) of the campo rupestre. In that study, Antonelli & al. (2010) also concluded that patches of campo rupestre may have existed prior to the expansion of the cerrado. Our results suggest specialisation from widespread ancestors from generalist open vegetation, such as a type of proto-savanna before fire regimes became fully established, or from disturbed areas, to patches of the campo rupestre highlands. This pattern contrasts with that suggested for the occupation of cerrado, where many unrelated lineages have been recruited from different fire-free biomes, but mainly from forests, facilitated by adaptation to fire (Simon & al., 2009; Simon & Pennington, 2012). According to the model proposed here, the geographic expansion of lineages has initiated, and the subsequent geographic isolation has driven plant diversification in the Espinhaço Range. To some extent, this model is similar to the species pump model. However, our results question the Espinhaço Range as an interglacial refuge for the campo rupestre, to which lineages would have retracted during warmer periods. As detected for some Neotropical lineages of Gesneriaceae (Perret & al., 2013), most Minaria diversity seems to have originated by in situ diversification from a limited number of radiations. Apparently, the radiation of the Minaria core group, which comprises most of the extant species of the genus, was initiated by a few, most likely only one, Pleistocene cycle of expansion–retraction of the campo rupestre, not by successive episodes directly associated with Pleistocene climatic oscillations. Nevertheless, phylogenetic, dating and spatial uncertainties of our results permit other interpretations. An alternative model, for instance, may evoke pulses of dispersal from the Espinhaço Range of Minas Gerais, most likely from the Diamantina Plateau, rather than a single expansion as postulated here. However, such an explanation does not properly explain the biogeography of the M. cordata complex. Although responses to environmental changes can vary from group to group, we believe that many other endemics-rich plant lineages will most likely fit a diversification pattern similar to the one postulated for Minaria in the Espinhaço Range. The confirmation of this pattern for other groups will require investigations combining precise time-calibrated phylogenetic trees and detailed knowledge about the palaeogeography, palaeoclimatology and palaeoecology of central South America to detect the influential Pleistocene events that could have driven range expansions of lineages restricted to campo rupestre. Whereas the accumulation of studies along these lines may take some time, phylogeographic studies in species endemic to the Espinhaço Ranges and modelling of palaeo-distributions of the campo rupestre, simulating glacial and interglacial periods, may reveal the influence of Pleistocene climatic changes on the spatial dynamics of the Espinhaço Range endemic flora. 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RASP: Reconstruct ancestral state in phylogenies, version 1.1. http://mnh.scu.edu.cn/soft/blog/ RASP (accessed 04 Jun 2012). Zachos, J., Pagani, M., Sloan, L., Thomas, E. & Billups, K. 2001. Trends, rhytms, and aberrations in global climate 65 Ma to present. Science 292: 686–693. http://dx.doi.org/10.1126/science.1059412 Appendix 1. Apocynaceae species used for molecular dating. Taxon name: country, largest political subdivision, collector and collector number (herbarium acronym), GenBank accession numbers: trnL-F complete sequence or trnL intron, trnL-F intergenic spacer. * Outgroup. Aganosma cymosa (Roxb.) G.Don: Thailand, D.J. Middleton 243 (A), EF456144. Alafia thouarsii Roem. & Schult.: Madagascar, G. McPherson & al. 17584 (MO), EF456240. Alstonia boonei De Wild.: Ivory Coast, Leeuwenberg 10724 (BR), AF102374, AF214151. Alyxia spicata R.Br.: Australia, D.J. Middleton 701 (A), EF456092. Angadenia berteroi Miers: Cuba, S. Nilsson s.n. (Z), EF456129. Anisotoma cordifolia Fenzl: South Africa, Eastern Cape, Nicholas 2811 (UDW), AJ410017, AJ410018. Anodendron paniculatum A.DC.: Thailand, D.J. Middleton & al. 3159 (A), EF456194. Apocynum androsaemifolium L.: USA, New York, T. Livshultz 03-32c (GH, BH), AF214308, AF214154. Araujia sericifera Brot.: Argentina, Entre Rios, Liede & Conrad 3007 (ULM), AJ428793, AJ428794. Asclepias curassavica L.: Brazil, Rapini 933 (SPF), AY163664, AY163664. Aspidosperma pyrifolium Mart.: Bolivia, de Queiroz 4136 (NY), AF214318, AF214164. Astephanus triflorus R.Br.: South Africa, Cape Province, Williams 659 (MO), AJ410188, AJ410189. Baissea multiflora A.DC.: cult. Belgium, Nat. Bot. Gard. Belgium, F. Billiet S3853 (BR), EF456199. Barjonia chlorifolia Decne.: Brazil, Bahia, Rio de Contas, Rapini 1059 (HUEFS), JN701896, AJ704467. Beaumontia grandiflora Wall.: cult. Germany, Bot. Gard. Munich, G. Gerlach & J. Babczinsky 5/06 (M), EF456184. Blepharodon lineare (Decne.) Decne.: Argentina, Forzza 2027 (SPF), AJ704465, AJ704468. Blepharodon pictum (Vahl) W.D.Stevens: Brazil, São Paulo, Atibaia, Rapini 938 (SPF), AJ704468. Blyttia fruticulosum (Decne.) D.V.Field: Kenya, Baringo, Liede & Newton 2946 (UBT), AJ410194, AJ410195. Calotropis procera (Aiton) W.T.Aiton: Puerto Rico, Struwe 1095 (NY) AF214324, AF214170. Caralluma arachnoidea (P.R.O. Bally) M.G. Gilbert: Kenya, Meve 934 (UBT), AJ410038, AJ410039. Carissa carandas L.: s. loco, s. coll. 77780 (FTG), AF214327, AF214173. Catharanthus roseus (L.) G.Don: s.l., Potgieter 245 (NY), AF102392, AF214175. Ceropegia juncea Roxb.: India, Řičánek & Hanáček 92 (UBT), AJ428799, AJ428800. Couma macrocarpa Barb.Rodr.: Colombia, Cárdenas 7379 (Z), AF214339, AF214185. Cycladenia humilis Benth.: USA, California, B. Smith 822 (Z), EF456211. Cynanchum acutum L.: Portugal, BG LIS s.n. (UBT), AJ428583, AJ428584. Cynanchum laeve (Michx.) Pers.: USA, Missouri Liede s.n. (UBT), AJ428652, AJ428653. Cynanchum morrenioides Goyder: Brazil, Minas Gerais, Omlor 166 (MJG), AJ428685, AJ428686. Cynanchum roulinioides (E.Fourn.) Rapini: Bolivia, Wood & al. 13300 (K), AJ428733, AJ428734. Dewevrella cochliostema De Wild.: Gabon, G.Walters 2131a (PH), GU901314. Diplolepis geminiflora (Decne.) Liede & Rapini: Chile, Limari Province, Heyne 103 (MSUN), AJ410182, AJ410183. Ditassa banksii Roem. & Schult.: Brazil, Konno 754 (SPF), AJ704474, AY163674. Ditassa hispida (Vell.) Fontella: Brazil, Konno 779 (SPF), AJ704478, AJ7044780. Echites woodsonianus Monach.: Costa Rica, F. Morales 8810 (INB), EF456175. Eustegia minuta (L.f.) N.E.Br.: South Africa, Bruyns 4357 (K; MWC 3291), AJ410206, AJ410207. Fockea edulis K.Schum.: s.l., Potgieter 249 (NY), AF214353, AF214199. Folotsia grandiflora (Jum. & H.Perrier) Jum. & H.Perrier: Madagascar, BG Munich s.n. (UBT), AJ290855, AJ290856. Forsteronia guyanensis Müll.Arg.: French Guiana, Prevost & Feuillet 3970 (CAY), EF456134. Funastrum clausum (Jacq.) Schltr.: Mexico, Liede & Conrad 2599 (MO, MSUN), AJ290861, AJ290862. Glossonema boveanum (Decne.) Decne.: Yemem, Rowaished 3014 (ULM), AY163684, AY163685. Gomphocarpus fruticosus (L.) W.T.Aiton: Egypt, Chase 9370 (K), AY163687, AY163687. Gonolobus gonocarpos (Walter) L.M.Perry: USA, ex Wyatt (GA), AJ704277, AJ704276. Hemipogon acerosus Decne.: Bolivia, Santa Cruz, Wood & Goyder 15689 (K), AJ704289, AJ704291. Hemipogon carassensis (Malme) Rapini: Brazil, Minas Gerais, Grão Mogol, Rapini 1364 (HUEFS), JN574706, JN574635. Hemipogon sprucei E.Fourn.: Bolivia, Wood & Goyder 15719 (K), AJ704297, AJ704299. Heterostemma cuspidatum Decne.: Philippines, Luzon, Laguna, Liede 3275 (UBT), AJ574829, AJ574828. Holarrhena curtisii King & Gamble: Thailand, D.J. Middleton & al. 2042 (A), EF456122. Hoya australis R.Br. ex J.Traill: s.l., Potgieter 247 (NY), AF214367, AF214213. Hunteria umbellata K.Schum.: s.l., Endress 97-16 (Z), AF214369, AF214215. Jobinia lindbergii E.Fourn.: Brazil, Minas Gerais, São Roque de Minas, Farinaccio 194 (SPF), AY163694, AY163694. Laubertia contorta (Mart. & Galeotti) Woodson: cult. Sam Houston State University, J. Williams 2005-1 (SHST), EF456246. Leptadenia arborea (Forssk.) Schweinf.: Egypt, Asswan, Heneidak s.n. (Suez Canal Univ. Herb.), AJ574833, AJ574834. Mandevilla boliviensis (Hook.f.) Woodson: cult. Harvard University, T. Livshultz 03-35 (GH), EF456153. Marsdenia suberosa (E.Fourn.) Malme: Brazil, Minas Gerais, Diamantina, Rapini 384 (SPF) AY163697, AY163697. Matelea pedalis (E.Fourn.) Fontella & E.A.Schwarz: Brazil, Minas Gerais, Catas Altas, Rapini 714 (SPF), AY163699, AY163699. Melodinus monogynus Roxb.: s.l., Billiet 3359 (Z, BR) AF214380, AF214226. Metaplexis japonica Makino: Russia, Primorsk, ex BG Tartu s.n. (UBT), AJ428811, Version of Record (identical to print version). 1263 Ribeiro & al. • Plant diversification in the Espinhaço Range TAXON 63 (6) • December 2014: 1253–1264 Appendix 1. Continued. AJ428812. Metastelma parviflorum R.Br.: Venezuela, Liede & Meve 3328 (UBT), AJ428777, AJ428779. Metastelma schaffneri A.Gray: Mexico, Nayarit, Liede & Conrad 2962 (UBT) AJ410214, AJ4102146. Minaria grazielae (Fontella & Marquete) T.U.P.Konno & Rapini: Brazil, Minas Gerais, Omlor147 (MJG), AJ410202, AJ410204. Minaria harleyi (Fontella & Marquete) Rapini & U.C.S.Silva: Brazil, Bahia, Rapini 354 (HUEFS), JN574693, JN574619. Minaria micromeria (Decne.) T.U.P.Konno & Rapini: Brazil, Goiás, Silva & al. 2040 (NY), AJ704226, AJ704248. Minaria polygaloides (Silveira) T.U.P.Konno & Rapini: Brazil, Minas Gerais, Conceição do Mato Dentro, Ribeiro 263 (HUEFS), JN574716, JN574645. Minaria volubilis Rapini & U.C.S.Silva: Brazil, Bahia, Mucugê, Rapini 1410 (HUEFS), JN574707, JN574636. Araujia odorata (Hook. & Arn.) Fontella & Goyder: Argentina, Buenos Aires, Liede & Conrad 3009 (MO, MSUN, ULM), AJ704345, AJ704344. Nautonia nummularia Decne.: Argentina, Corrientes, Liede & Conrad 3031 (ULM), AJ410226, AJ410228. Neobracea valenzuelana Urb.: Cuba, S. Nilsson s.n. (Z), EF456139. Neoschumannia kamerunensis Schltr.: Cameroon, Mt. Cameroon, Meve & Etuge 910 (B, K, UBT), AJ410053, AJ410054. Nephradenia acerosa Decne.: Brazil, Philcox 3303 (K), AJ704497, AY163705. Orthosia urceolata E.Fourn.: Brazil, Paraná, Carrião 27500 (NY) AJ704323, AJ704325. Oxypetalum banksii R.Br. ex Schult.: Brazil, São Paulo, Ubatuba, Rapini 911 (SPF), (AY163710, AY163710. Oxypetalum capitatum Mart.: Argentina, MelloSilva 1924 (SPF), AY163711, AY163711. Oxypetalum wightianum Hook. & Arn.: Brazil, Rapini 705 (SPF), AJ704524, AJ704523. Oxystelma esculentum (L.f.) Sm.: Egypt, Elephantine Island, Shirley s.n. (cult. Bayreuth) AJ290885, AJ290887. Pachypodium baroni Costantin & Bois: cult. U.S.A., Cornell University, T. Livshultz 03-20 (BH), EF456154. Peltastes peltatus (Vell.) Woodson: cult. Belgium, Nat. Bot. Gard. Belgium, F. Billiet S3526 (BR), EF456203. Pentacyphus andinus (Ball.) Liede: Peru, Liede & Meve 3451 (UBT), AJ492150, AJ492151. Pentalinon luteum (L.) B.F.Hansen & R.P.Wunderlin: Bahamas, M. Vincent 11460 (GH), EF456180. Pentarrhinum abyssinicum Decne.: Bidgood, Mbago & Vollesen 2240 (K), AJ428817, AJ428818. Peplonia asteria (Vell.) Fontella & E.A.Schwarz: Brazil, Fontella sub Konno 773 (SPF), AJ704300, AJ704302. Pergularia daemia (Forssk.) Chiov.: Tanzania, N of Arusha, Masinde 888 (in cult. Bayreuth), AJ290892, AJ290893. Periploca graeca L.: s.l., Endress s.n. (Z, ZBG), AF102468, AF214244. Pervillaea tomentosa Decne.: Madagascar, ex hort. Palmengarten (UBT), AJ431768, AJ431769. Petalostelma sarcostemma (Lillo) Liede & Meve: Argentina, Liede & Conrad 3090 (MSUN, ULM), AJ428786, AJ428788. Philibertia lysimachioides (Wedd.) T.Mey.: Bolivia, Liede & Conrad 3139 (MSUN, ULM), AJ290901, AJ290900. Phyllanthera grayi (P.I.Forst.) Venter: Australia, P.I. Forster 24232 (BRI), EF456103. Picralima nitida T.Durand. & H.Durand: D. R. Congo, Kiss s.n. 86-0334, Billiet 3440 (BR), AF214404, AF214250. Plumeria alba L.: Puerto Rico, Struwe 1096 (NY), AF214408, AF214254. Rauvolfia serpentina (L.) Benth. ex Kurz: s.l., Potgieter 252 (NY), AF214415, AF214261. Rhabdadenia biflora Müll.Arg.: USA, Florida, S. Zona 616 (FTG), EF456150. Sarcostemma viminale (L.) R.Br.: Zimbabwe, Bulawayo, Albers, Liede & Meve 540 (MSUN, UBT), AJ290913, AJ290912. Schizostephanus alatus Hochst. ex K.Schum.: Kenya, Noltee s.n. sub IPPS 8111 (UBT), AJ410248, AJ410249. Schubertia grandiflora Mart.: Argentina, Liede & Conrad 3033 (MSUN, ULM), AJ428826, AJ428827. Secamone glaberrima K.Schum.: Madagascar, Toocmasina, Stevens 26007 (MO), AF214420, AF214266. Stapelia glanduliflora Mass.: South Africa, S of Klawer, Albers & Meve 04 (MSUN), AJ402128, AJ402151. Stipecoma peltigera Müll.Arg.: Brazil, L.S. Kinoshita s.n. (UEC), EF456193. Strychnos tomentosa Benth.*: French Guiana, Région de Saül, Mori 24166 (NY) AF214301, AF214147. Tabernaemontana undulata G.Mey: s.l., T10212, HQ634605. Tassadia obovata Decne.: Ecuador, Matezki 332 (UBT), AJ699281, AJ699283. Telosma accedens (Blume) Backer: Philippines, Luzon-Sorgoson, Schneidt 96-101 (UBT), AJ431783, AJ431784. Telosma cordata Merr.: s.l., Gilding s.n. (NY), AF214280, AF102493. Thevetia peruviana K.Schum.: s.l., Livshultz 03-34 (GH), EF456088. Toxocarpus villosus (Blume) Decne.: Thailand, D.J. Middleton & al. 1341 (A), EF456117. Tweedia brunonis Hook. & Arn.: Argentina, Mendoza, Liede & Conrad 3058 (UBT), AJ704260, AJ704258. Tylophora flexuosa R.Br.: Philippines, Luzon, Laguna Province, Liede 3252 (UBT), AJ290916, AJ290917. Wrightia coccinea (Roxb.) Sims: Thailand, D.J. Middleton & al. 897 (A)EF456165. Zygostelma benthamii Baill.: Thailand, D.J. Middleton & al. 849 (A), EF456109. 1264 Version of Record (identical to print version).