A Preliminary Phylogeny of Loasaceae Subfam. Loasoideae

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Organisms, Diversity & Evolution 4 (2004) 73–90 www.elsevier.de/ode

A preliminary phylogeny of Loasaceae subfam. Loasoideae (Angiosper- mae: Cornales) based on trnL(UAA) sequence data, with consequences for systematics and historical biogeography Maximilian Weigenda,*, Marc Gottschlinga,b, Sara Hootc, Markus Ackermanna a Institut fur. Biologie, Systematische Botanik und Pﬂanzengeographie, Freie Universitat. Berlin, Altensteinstr. 6, D-14195 Berlin, Germany b Fachbereich Geologische Wissenschaften, Fachrichtung Palaontologie,. Malteser Strasse 74-100, D-12149 Berlin, Germany c Department of Biological Sciences, Lapham Hall, P. O. Box 413, University of Wisconsin, Milwaukee, Milwaukee, WI 53201, USA Received5 May 2003; accepted11 December 2003

Abstract

The phylogeny of Loasaceae subfam. Loasoideae is investigated with sequences of the chloroplast trnL(UAA) intron, all genera and infrageneric entities are included in the analysis. Loasaceae subfam. Loasoideae is monophyletic, and the two most speciose, andmonophyletic, clades(which account for approximately 90% of the species total) are Nasa andthe so-calledSouthern AndeanLoasas ( Blumenbachia, Caiophora, Loasa s.str., Scyphanthus), but the phylogeny of the remainder is not completely resolved. The data underscore a basal position for Chichicaste, Huidobria, Kissenia, andKlaprothieae ( Xylopodia, Klaprothia, Plakothira). High bootstrap support values confirm the monophyly both of Klaprothieae and Presliophytum (when expanded to include Loasa ser. Malesherbioideae). Aosa and Blumenbachia are not resolvedas monophyletic, but have clear morphological apomorphies. Within Nasa,‘‘N. ser. Saccatae’’ is paraphyletic, and‘‘ N. ser. Carunculatae’’ is polyphyletic. However, the N. triphylla group in ‘‘N. ser. Saccatae’’ is a well-supportedmonophyletic group, as is N. ser. Grandiflorae.‘‘Loasa’’ in its traditional circumscription is paraphyletic, but Loasa s.str. (L. ser. Macrospermae, L. ser. Deserticolae, L. ser. Floribundae) is monophyletic. The remainder of ‘‘Loasa’’ (L. ser. Pinnatae, L. ser. Acaules, L. ser. Volubiles) is probably closely alliedto the essentially Patagonian-High Andean group comprising also Scyphanthus and Caiophora. These findings are congruent with morphology andphytogeography. Nasa seems to have undergone its primary radiation at moderate elevations (1500– 2500 m) in the Andes of northern Peru (Amotape-Huancabamba Zone) and subsequently diversified into high elevations (above 4000 m) of the tropical Central Andes. South Andean Loasas appear to have undergone their primary diversification in the southern temperate and mediterranean regions of Chile and Argentina, with a subsequent northwards expansion of Caiophora into the high elevations of the tropical Andes. Hummingbird pollination has evolved independently from melittophily in High Andean clades of Nasa and Caiophora. r 2004 Elsevier GmbH. All rights reserved.

Keywords: Loasaceae; High Andean clades; Floral morphology; Molecular systematics; Nasa; South Andean Loasas

Introduction

Loasaceae are medium-sized (ca. 300 spp.) and largely *Corresponding author. Tel.: +49-30-838-565-11. Neotropical plant family whose precise relationships E-mail address: [email protected] (M. Weigend). among angiosperms have been controversially discussed.

The last few years have brought enormous progress in subgenus in ‘‘Loasa’’, was re-instatedat genus rank this field, and the Loasaceae have been shown to be (Grau 1997). However, because no detailed new studies closely alliedto Hydrangeaceae andfirmly nestedin are available, the treatments of Urban andGilg (1900) Cornales (Hempel et al. 1995; Moody and Hufford have been largely followed, and only Schismocarpus 2000). Morphological studies confirm this placement, Blake (subfam. Mentzelioideae) from southern Mexico andthe similarity between some groups in Loasaceae and Plakothira Florence (subfam. Loasoideae, tribe andsome groups in Hydrangeaceae (e.g. Deutzia Klaprothieae) from the Marquesas Islands in Polynesia Thunb., Jamesia Torr. & Gray, and Philadelphus L.) is have been described since the ‘‘Monographia Loasa- indeed striking in varied character complexes such cearum’’. as seedmorphology, flower morphology, indument Weigend(1997) made an attempt to arrive at a more morphology, phytochemistry, andleaf morphology natural classification of Loasaceae subfam. Loasoideae (Weigend, 2004). (Table 1) considering a wide range of morphological The subdivision of Loasaceae has also been con- traits andcharacter polarity. This studyledto the troversially discussed (Davis andThompson 1967 ; segregation from ‘‘Loasa’’ of a total of four genera: Poston andThompson 1977 ; Weigend1997 ; Moody 1. Nasa Weigend(short for ‘‘North AndeanLoasas’’; andHufford2000 ). The mainly North American Urban andGilg’s groups Loasa ser. Grandiflorae, subfamilies Gronovioideae (Cevallia Lag., Fuertesia L. ser. Saccatae, L. ser. Carunculatae, L. ser. Alatae), Urb., Gronovia L.), Mentzelioideae (Eucnide Zucch., for by now approx. 100 spp. mainly from the Mentzelia L., Schismocarpus Blake), andPetalonychoi- northern andcentral Andes; deae (Petalonyx A.Gray) have been extensively studied, 2. Aosa Weigend, for the Brazilian and Hispaniolan but these make up only about 1/3 of the family’s species representatives of ‘‘Loasa’’ (Urban andGilg’s groups total. The subfamily that is by far the largest (over 200 L. ser. Corymbosae, L. ser. Parviflorae, L. ser. spp.) andmost diverse(morphologically, ecologically, Pusillae); andphytochemically), the Loasoideae,has been the 3. Presliophytum (Urban andGilg) Weigend(Urban & subject of very few detailed studies, and these have Gilg’s Loasa subg. Presliophytum); usually been limitedto the few commonly cultivated 4. Chichicaste Weigend, for Loasa grandis Standl. representatives, such as Blumenbachia insignis Schrad., (described after Urban & Gilg’s studies). B. hieronymi Urb., Caiophora lateritia Klotzsch, and Nasa triphylla (Juss.) Weigendsubsp. triphylla. Another systematic addition was the description of Until recently, the only comprehensive study available Xylopodia Weigendbelonging to the Klaprothieae was the ‘‘Monographia Loasacearum’’ (Urban andGilg (discovered in northern Peru in 1997), and Caiophora 1900) which was basednearly exclusively on herbarium was redefined by removing the two sections Angulatae material much of which was very poorly preserved. This and Gripidea to Blumenbachia (Weigend1997 ). study recognized a total of seven genera in Loasoideae The Loaseae were informally segregatedinto two (Blumenbachia Schrad., Caiophora C.Presl, Kissenia ‘‘grades’’: ‘‘Lower Loaseae’’, with a number of small Endl., Klaprothia Kunth, Loasa Adans., Sclerothrix genera characterizedby relatively simple andupright C.Presl, Scyphanthus D.Don), with ‘‘Loasa’’ accounting flowers without thigmonastic stamens (Chichicaste, for more than half of the species (83 of 153). The genera Huidobria, Presliophytum); andHigher Loaseae, com- were groupedinto three tribes, Kissenieae ( Kissenia;2 prising genera with more complex andusually pendu- spp.), Klaprothieae (Klaprothia, Sclerothrix; 2 spp.), and lous flowers with thigmonastic stamens (Aosa, Loaseae (Blumenbachia, Caiophora,‘‘Loasa’’, Scy- Blumenbachia, Caiophora,‘‘Loasa’’, Nasa, Scyphanthus). phanthus; 149 spp.). ‘‘Loasa’’ was circumscribedexclu- The Higher Loaseae divide into three clearly mono- sively by the presence of fruits opening with apical phyletic assemblages: Aosa, Nasa, anda complex valves, a truly plesiomorphic character also foundin informally calledSouth AndeanLoasas comprising Mentzelioideae and outside of Loasaceae in the putative Blumenbachia, Caiophora,‘‘Loasa’’, and Scyphanthus. sistergroup Hydrangeaceae. Urban andGilg (1900) Currently, South Andean Loasas remain largely un- wrote detailed studies on many aspects of morphology resolved, since they show reticulate patterns of variation andstudiedLoasaceae with enormous accuracy, but in in many characters and have not been studied in detail. their subsequent classification they made little use of the Caiophora has recently been subdivided into species numerous characters observed. groups to make the large genus more manageable At species level, their decisions have been widely (WeigendandAckermann 2003 ). ‘‘Loasa’’ still contains criticizedas being too narrow ( Darlington 1934; one highly divergent entity, L. ser. Malesherbioideae Sleumer 1956), andthe generic concepts have also been (Table 1), which lacks the derived characters of Loasa challenged: Sclerothrix was reduced to synonymy under s.str. andrather appears to be closely alliedto Klaprothia (Poston andNowicke 1990 ), and Huidobria Presliophytum. It is here treatedseparately (both species Gay, which Urban andGilg (1900) hadtreatedas a of L. ser. Malesherbioideae were available for analysis). ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 75 . 3 1 N Colombia & NW Venezuela Weigend(1997) BlBlBlGrBlAn 4 3 3 (4?) 2 3 1 Argentina–S Brazil S Chile S & Brazil Argentina LoMa 2 2 N Chile AoCo 1 — Brazil NaSc 29 (?) 11 Cordillera Mexico–Bolivia NaCa 4 2 Peru & S Ecuador NaAl 26 (?) 7 Colombia-Bolivia NaGr 38 (?) 7 N Colombia to Peru LoAc 1 1 Chile & Argentina (Andes) LoDe 2 2 N & C Chile (Costa) LoFl 4 3 N & C Chile (Costa) LoLo 2 — S Chile & Argentina LoMc 8 (?) 7 C & N Chile, Peru (Costa) LoVo 4 2 S Chile LoPi 20 (?) 4 Chile & Argentina (Andes) AoPu 2 1 Brazil AoPa 4 2 Brazil & Hispaniola b a group group CaAr 1 — S Brazil–Uruguay group CaPt 2 1 Peru group CaCh 8 2 Peru–Argentina group CaCa 5 — Peru group CaCi 2 1 N Chile–Peru group CaCr 2 — Peru–Chile group CaCo 2 — Ecuador–Peru group CaRo 1 — Peru–Argentina group CaLa 8 1 Peru–Argentina group CaCl 5 — S Bolivia–N Argentina group CaNi 2 — Argentina Blumenbachia Gripidea Angulatae Malesherbioideae Corymbosae Saccatae Carunculatae Alatae Grandiflorae Acaules Deserticolae Floribundae Loasa Macrospermae Volubiles Pinnatae Pusillae Parviflorae . ——————— 2 3 1 2 1 2 3 2 1 1 1 1 2 2 C & S America Marquesas Islands N Peru Africa Panama & Costa Rica N Chile Peru — 2 1 C Chile C. arechvaletae C. carduiifolia C. chuquitensis C. cirsiifolia C. clavata C. contorta C. coronata C. lateritia C. nivalis C. pterosperma C. rosulata sect. sect. sect. ser. ser. ser. ser. ser. N. venezuelensis ser. ser. ser. ser. ser. ser. ser. c ’’ ser. ’’ ser. Loasa Loasa . GenusKlaprothia Infrageneric entity Acronym Species total Species studied Distribution Plakothira Xylopodia Kissenia Chichicaste Huidobria Presliophytum ‘‘ Scyphanthus Caiophora ‘‘ Blumenbachia Aosa Nasa basedon molecular andmorphological data. WeigendandAckermann (2003) Loasa Weigend(1997) Synopsis of the classification of Loasaceae subfamily Loasoideae, modified from Not formalized, see Here removedfrom Informal classification, see ?not fully revised, numerous undescribed species. a b c Table 1. Loasaceae subfam. Loasoideae Tribe Klaprothieae Tribe Loaseae, ‘‘Lower Loaseae’’ Tribe Loaseae, Higher Loaseae ser. ARTICLE IN PRESS 76 M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90

Some molecular data have already been published quartet puzzling steps. Likelihoodsettings from the (matK: Moody and Hufford 2000; matKandtrnL-trnF: best-fit model were determined using the AIC criterion Huffordet al. 2003 ). While the principal results of these in Modeltest 3.06 (Posada and Crandall 1998)(Fig. 1). studies are largely congruent with the re-classification The trnL phylogeny is usedas the basis for a provided by Weigend(1997) , we here aim at providing a hypothetical phylogeny of Loasoideae with possible more complete understanding of subfamily Loasoideae morphological apomorphies mappedonto a cladogram andespecially its largest groups ( Nasa, South Andean (Fig. 7). The corresponding morphological characters Loasas). The present study, using sequences from the are illustratedin Figs. 2–6. chloroplast trnL(UAA) intron, addresses three primary aims: Firstly, molecular delimitation of the generic entities recognizedin Weigend(1997) , andrelationships among them; secondly, identification of the lineages Results within the poorly understood South Andean Loasas; thirdly, recognition of infrageneric groupings in ‘‘Loa- The aligned trnL data set was 536 bp in length. Of sa’’ and Nasa. these sites, 113 (22%) were parsimony-informative (1.5 per taxon). The heuristic search found13,284 most parsimonious trees, for which a strict consensus tree was computed( Fig. 1; L ¼ 293; CI=0.78, RI=0.91). In the Material and methods tree, bootstrap support values (BS; criterion=parsimony) andPUZZLE support values (P; criterion=like- The plant material usedin this studylargely came lihood, calculated with the best-fit model: GTR+G from our own fieldcollections over the past 10 years. model) are indicated. Species were identified on the basis of all available Within the monophyletic Loasaceae subfam. Loasoi- literature, andare voucheredin herbaria as indicatedin deae (63 BS), the analysis of trnL sequences results in a Table 2. A few taxa were only available from herbarium basal polytomy involving eight unresolvedsingle spe- collections, samples of these were taken from specimens cies, two minor clades (Klaprothieae, 90 BS; and in the herbaria B, LPB, M, andMO. Presliophytum including ‘‘Loasa’’ longiseta from L. ser. A total of 77 species assignedto Loasaceae have been Malesherbioideae, 84 BS), andtwo major andspecies- investigated( Table 2). Furthermore, 3 sequences from rich clades (South Andean Loasas, 72 BS, comprising Deutzia and Philadelphus (Hydrangeaceae) were used Blumenbachia, Scyphanthus, Caiophora, andthe major- for the user-specifiedoutgroup comparison, they were ity of ‘‘Loasa’’; and Nasa, 88 BS, 91 P). treatedas monophyletic in the molecular analysis. Only Within Klaprothieae, Xylopodia is sister to a clade a small sample of species of Caiophora was included, comprising Klaprothia and Plakothira (monophyletic: 99 since these showedminimal sequence divergence, andno BS, 67 P), andPUZZLE analysis further indicates that internal resolution couldbe obtainedwith the marker Klaprothia may be paraphyletic with respect to Pla- chosen. kothira (K. mentzelioides as sister to K. fasciculata and DNA extraction, PCR, purification andsequencing Plakothira parviflora: 62 P). The two species of followed standard protocols, which are described in Presliophytum are retrievedas sister taxa (85 BS, 55 P) detail in Gottschling andHilger (2001) . Primers usedfor andappear to be closely alliedto ‘‘ Loasa’’ longiseta (84 amplification andsequencing of the trnL(UAA) intron BS) as well as to ‘‘L.’’ malesherbioides as a second were those of Taberlet et al. (1991). The sequences were species of L. ser. Malesherbioideae. The latter relation- manually alignedusing Se-Al v2.0a72 ( Rambaut 2001). ship receives support from the PUZZLE analysis (54 P). The complete data matrix is available in NEXUS format The species remaining unresolvedin the basal on request. polytomy represent Huidobria, Chichicaste, and Kissenia Phylogenetic calculations were run on a Macintosh from the ‘‘Lower Loasoideae’’, and Aosa from the computer with the help of PAUPÃ 4.0b1 (Swofford Higher Loaseae. Neither Aosa (3 of 7 species sampled) 1998). Parsimony trees were generatedusing heuristic nor Huidobria (2 of 2) are retrievedas monophyletic, but searches, with gaps considered as informative based on their distinctness from ‘‘Loasa’’ is evident. The same is the frequent occurrence of deletions and insertions in the true for monotypical Chichicaste. trnL intron. The poly A region in the R-loop of the trnL Nasa is well supported(88 BS, 91 P) andfalls into two secondary structure (Kuhsel et al. 1990) was excluded clades comprising: (1) N. carunculata (Urb. & Gilg) since it follows no phylogenetic pattern in Loasaceae. A Weigend( N. ser. ‘‘Carunculatae’’) andsome species of bootstrap analysis (criterion=parsimony, BS) was N. ser. ‘‘Saccatae’’ (moderately supported: 63 BS); (2) estimated based on 350 replicates (addseq=random, the remainder of N. ser. ‘‘Carunculatae’’ and N. ser. nreps=10, MaxTrees=1000). A PUZZLE analysis ‘‘Saccatae’’, all of N. ser. Grandiflorae and N. ser. (criterion=likelihood, P) was performed with 1000 Alatae, andthe N. venezuelensis group (99 BS). Within ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 77 L) trn GenBank Acc. No. ( origin (M, NY, USM) Peru AY285688 (M, NY, USM) Peru AY285686 (B) Hispaniola AY388479 (BSB, HUT, M, USM) Peru AY285696 (B) Argentina AY285682 (herbarium) Country of (M) Chile AY285705 (BRCO, BSB, M, NY) Argentina AY285698 (BSB, HUT, M, USM) Chile AY388488 (BSB, HUT, M, USM) Peru AY285695 (BRCO, BSB, M, NY) Chile AY285697 (BSB, M) Chile AY388487 (BSB, HUT, M. USM) Peru AY285687 (BSB, HUT, M, USM) Peru AY285689 (BSB, HUT, M, USM) Chile AY388486 (BSB, HUT, M, USM) Chile AY285702 (BRCO, BSB, M) Argentina AY285684 (BRCO, BSB, M, NY) Argentina AY285700 (BSB, HUT, M, USM) Peru AY285707 (BSB, M) Chile AY388484 (BRCO, BSB, M) Argentina AY285683 (BRCO, BSB, M, NY)(BSB, M) Argentina AY285699 Chile(BRCO, BSB, M, NY) AY285701 Argentina AY285706 (BRCO, BSB, M) Argentina AY285679 (M) Chile AY285703 (B) Chile AY388490 (BSB, M, SGO)(BSB, M, SGO) Chile Chile AY388483 AY388485 (BSB, HUSA, M, USM) Peru AY285685 6984 5934 5362 6848 5922 5022 5188 5913 5920 6807 6880 7346 5937 6823 6846 7057 7080 6816 (B 045-13-87-10/274, BSB) China AY285690 (B 103-26-74-80/1, BSB) China AY285691 (BSB, M) Brazil AY285678 886 (M) Argentina AY285681 (B) Argentina AY285680 (M) Chile AY285704 (B) South Africa AY285694 (CORD) Argentina AY388480 (MO) Panama AY388482 (M) Brazil AY285677 (F, M) Chile AY285693 (M) Chile AY388489 et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al., Grau s.n. Ehrhardt s.n. Wagenknecht 18509 Werdermann 1342 Weigend 5615 Henning & Schneider 276 Weigend Weigend Weigend Croat 13381 Greuter 2167 Weigend Weigend 5613 Dillon 8034 Weigend Weigend Coccucci s.n. M. & K. WeigendWeigend 2000/191 Weigend M. & K. Weigend 2000/70 Ricardi Weigend Weigend Ackermann 514 Weigend Ackermann 519 Weigend Ackermann 360 Weigend Collector/collection No. Salino 3042 Weigend, s.n. Schwabe s.n. anno 1958 Weigend Weigend Weigend Weigend Weigend 7138 R.A. & E.S. Howard 9398 Weigend Sehnem 3993 CaPt (Ruiz & Pav. ex Phil. ‘‘Lower Loaseae’’ Gay BlAn Kunth Klaprothieae Poepp. BlAn (Meyen) Urb. & Gilg CaCh (Killip) CaLa Cambess. BlBl (C.Presl) Poston Klaprothieae WeigendBlGr Schrad. BlBl Gay BlAn C.Presl CaCi Phil. ‘‘Lower Loaseae’’ Poepp. LoVo (Standl.) Weigend ‘‘Lower Loaseae’’ Gay ‘‘Lower Loaseae’’ Ackermann 482 (BSB, CONC, M, SGO) Chile AY285692 Hook. & Arn. LoMc Phil. ‘‘Lower Loaseae’’ Poepp. LoPi Urb. & Gilg CaCh Lillo CaNi Gill. ex Arn. LoAc Endl. ‘‘Lower Loaseae’’ Dusen LoPi Phil. LoFl Urb. & Gilg LoVo Hemsley Hydrangeaceae Poepp. LoMc Domb. LoMc Phil. LoFl Hook. & Arn. LoDe Gay LoMc Poepp. LoPi Rehder Hydrangeaceae (Urb.) WeigendAoPa (Gardner) Weigend AoPa Phil. LoPi (Urb. & Gilg) WeigendAoPu Pterosperma Gill. ex Arn. LoFl Poepp. LoMc Desr. LoMc cf. insons malesherbioides micrantha longiseta lateritia asterias filicifolia bergii gayana nana ’’ ’’ ’’ ’’ ’’ ’’ ’’ ’’ ’’ cf. Loasa Loasa Loasa Loasa Loasa Loasa Loasa Loasa Loasa Klaprothia fasciculata Klaprothia mentzelioides Deutzia discolor Deutzia rubens Loasa acerifolia Huidobria fruticosa Loasa intricata Caiophora nivalis G.Don) Urb. & Gilg Caiophora Chichicaste grandis Kissenia capensis Huidobria chilensis Loasa Caiophora cirsiifolia Caiophora madrequisa Loasa illapelina Loasa insons Caiophora andina Caiophora chuquitensis Loasa floribunda Loasa pallida Blumenbachia sylvestris Loasa elongata Loasa heterophylla Loasa nitida Species Classification Aosa rostrata Aosa rupestris Blumenbachia exalata Blumenbachia espigneira Blumenbachia insignis Blumenbachia prietea ‘‘ ‘‘ ‘‘ Blumenbachia latifolia Aosa plumierii List of species andvouchers Ã Ã Ã Ã Ã Ã 597 13281466 ‘‘ ‘‘ 1367 ‘‘ 596 1350 ‘‘ 1348 1349 1293 1292 1340 1355 ‘‘ 1327 1392 1389 1356 1472 1364 1325 1391 1345 1357 1354 ‘‘ 1390 1394 1342 1341 1393 1386 598 1385 1464 1323 1339 590 Table 2. DNA No. 1376 1289 1384 1383 591 1387 592 ARTICLE IN PRESS 78 M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 Table L) trn GenBank Acc. No. ( origin . at Berlin. Abbreviations for infrageneric taxa see (HUT, M, NY, USM) Peru AY285711 (M, NY, USM) Peru AY285728 (HUT, M, NY, USM) Peru AY285726 (HUSA, M, NY, USM) Peru AY285734 (HUT, M, NY, USM) Peru AY285714 (M, QCNE) Ecuador AY285710 (M, USM) Peru AY285731 (F, M, USM) Peru AY285733 (F, HUT, M, USM) Peru AY285736 (F, M, USM) Peru AY285739 (BSB, HUT, M, USM) Peru AY285713 (herbarium) Country of (QCNE, M) Ecuador AY285719 (M, QCNE) Ecuador AY285716 (F, HUT, M, USM) Peru AY285709 (M) Chile AY285741 (F, M, HUT, USM) Peru AY285727 (F, M, USM) Peru AY285725 (F, HUT, M, USM) Peru AY285720 (F, M, USM) Peru AY285742 (M, USM) Peru AY285724 (BSB) Ecuador AY285722 (M) Ecuador AY285730 (BSB, HUT, M, USM) Peru AY285712 (BRCO, BSB, M, NY) Argentina AY285708 (UPS) Ecuador AJ430868 (BSB, HUT, M, USM) Peru AY285721 /816 (HUT, M, NY, USM) Peru AY285723 (BSB, CONC, M, SGO) Chile AY388492 (COL, M) Colombia AY285729 7458 98/470 6865 (F, HUT, M, USM) Peru AY285718 (F, HUT, MSB, USM) Peru AY285717 97/307 97/s.n. 97/468 98/547 5446 97/450 (COL, M) Colombia AY285732 (COL, M) Venezuela AY285735 (B 270-16-96-10, BSB) China AY285737 (BSB, HUT, M, USM) Peru AY285740 . . (BSB, M, NTBG 970008) Marquesas Isl. AY285738 orther 97/542 orther 97/848 (LPB) Bolivia AY285715 (B) Chile AY388491 et al. et al. et al. et al. et al. et al. et al., et al., et al., . uller 6596 Weigend M. & K. WeigendWeigend 2000/167 3523C Schwerdtfeger 22207 Weigend & Dostert 98/259 Weigend 3610 M. & K. Weigend 2000/208 Weigend Weigend Grant & Struwe 4063 Weigendet al. 2000 Erixon & Bremer 42 Weigend & Horn 3838 Weigend Dostert 98/161 Weigend Weigend & F M Weigend & Horn 3815 Dostert 98/154 M. & K. Weigend 2000/289 Weigend 3604 M. & K. Weigend 2000/199 Henning & Schneider, 243 Weigend Weigend & Dostert 98/261 Weigend 5614 Weigend s.n. Weigend & F Weigend Weigend & Weigend, 2000/363 Weigend 7368 Grau & Ehrhart 2-093 Weigend & Jaramillo, 3937 Weigend Ackermann, 491 Weigend Collector/collection No. Mahu, 1412 Juss.). Loasa triphylla ,as NaSc NaSc NaSc ‘‘Lower Loaseae’’ . ur Biologie, Systematische Botanik undPflanzengeographie, Freie Universit triphylla Bremer et al. (2002 (Killip) Weigend‘‘Lower Loaseae’’ WeigendKlaptrothieae Ruprecht Hydrangeaceae Florence Klaprothieae D.Don — sequence from Griseb. Mentzeliodieae (Urb. & Gilg) Weigend Juss.) Weigendsubsp. (Steyerm.) WeigendNaVe (Urb. & Gilg) WeigendNaGr (Urb. & Gilg) WeigendNaCa (Urb. & Gilg) WeigendNaGr (Urb. & Gilg) WeigendNaSc (Urb. & Gilg) WeigendNaCa (Weigend) Weigend NaGr (Urb. & Gilg) WeigendNaSc Kunth Mentzelioideae Phil. LoDe (Urb. & Gilg) WeigendNaGr Weigend) Weigend NaAl (Urb. & Gilg) WeigendNaSc (Urb. & Gilg) WeigendNaGr (Standley & Barkley) Weigend NaSc (Weigend) Weigend NaSc Dostert & Weigend (Juss.) Weigendsubsp. WeigendNaAl (Kunth) WeigendNaAl (Urb. & Gilg) WeigendNaAl (J.F.Macbr.) WeigendNaSc Juss. LoMc WeigendNaGr (Urb. & Gilg) WeigendNaAl (Killip) WeigendNaAl triphylla (Weigend) Weigend NaGr (Jacq.) WeigendNaSc (Urb. & Gilg) WeigendNaAl (Killip) WeigendNaSc ) obliqua subsp. Loasa triphylla Nasa solata Nasa raimondii Nasa ramirezii Nasa trianae Pteridophylla Nasa pteridophylla Nasa magnifica Nasa rubrastra ( Nasa triphylla (= Nasa poissoniana Nasa macrothyrsa Nasa jungiaefolia Nasa laxa Nasa lenta Nasa loxensis Nasa macrantha Nasa urens Nasa hornii Nasa insignis Nasa humboldtiana subsp. Nasa vargasii Nasa venezuelensis Nasa driesslei Nasa ferruginea Nasa herzogii Nasa weberbaueri Philadelphus pekinensis Presliophytum arequipense Xylopodia klaprothioides Presliophytum heucheraefolium Weigend Plakothira parviflora Nasa carunculata Nasa cymbopetala Scyphanthus elegans Nasa aeqatoriana Mentzelia scabra Mentzelia albescens Loasa urmenetae Loasa triloba Species Classification continued Nasa triphylla . 1235 1244 1353 1380 1237 1333 1373 — 1243 1329 1377 1232 1239 1240 1330 1246 1378 1374 1365 1231 1366 1238 1242 1351 1337 1291 1288 1287 1369 1290 1236 1335 1467 1375 1286 1285 1465 Table 2 ( DNA numbers follow an internal numbering code of the Institut f 1 1388 DNA No. ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 79

Ph. pekinensis 1291 100 D. rubens 1292 96 D. discolor 1293 96 M. albescens 1285 96 M. scabra 1286 Ch. grandis 1472 90 X. klaprothioides 1287 99 Kl. fasciculata 1348 Klaprothieae Kl. mentzelioides 1349 67 Pl. parviflora 1290 Ki. capensis 1364 H. chilensis 1325 H. fruticosa 1327 "L." malesherbioides 1328 "Lower Loaseae" 84 "L." longiseta 1367 85 Pr. arequipense 1288 55 Pr. heucheraefolium 1369 A. plumierii 1387 A. rostrata 1376 A. rupestris 1289 63 N. carunculata 1236 "Carunculatae" 99 N. urens 1246 76 N. vargasii 1231 78 N. ferruginea 1242 N. poissoniana 1243 N. raimondii 1244 N. aequatoriana 1375 "Saccatae" 88 99 N. triphylla* 91 N. pteridophylla 1237 N. humboldtiana 1365 N. laxa 1232 N. ramirezii 1353 N. macrothyrsa 1329 "Carunculatae" 63 N. driesslei 1238 99 53 N. herzogii 1351 N. lenta 1239 N. solata 1235 Alatae N. trianae 1380 65 N. loxensis 1240 N. rubrastra 1373 N. venezuelensis 1366 N. insignis 1374 73 N. hornii 1378 61 N. jungiaefolia 1377 N. weberbaueri 1337 Grandiflorae 65 N. magnifica 1333 N. macrantha 1330 89 N. cymbopetala 1335 62 B. espigneira 590* 82 B. prietea 591* Angulatae B. sylvestris 1385 B. insignis 592* Blumenbachia B. latifolia 1383 B. exalata 1384 Gripidea L. triloba 1388 99 L. nitida 1339 94 63 L. acerifolia 1340 63 62 L. insons 1394 Macrospermae 67 L. intricata 1392 96 L. heterophylla 1323 80 L. cf. insons 1391 L. pallida 1386 72 65 82 L. floribunda 1393 Floribundae 76 75 L. illapelina 1390 83 L. elongata 1464 Deserticolae 86 L. urmenetae 1465 "L." micrantha 1466 "Volubiles" 86 "L." gayana 598* 99 "L." lateritia 1350 Acaules "L." nana 597* South Andean Loasas "L." bergii 596* Pinnatae "L." filicifolia 1354 "L." asterias 1355 S. elegans 1467 61 Ca. cf. pterosperma 1356 70 99 Ca. cirsiifolia 1345 79 Ca. chuquitensis 1341 59 Ca. andina 1342 89 Ca. madrequisa 1357 Ca. nivalis 1389 Fig. 1. Strict consensus tree of 13,284 most parsimonious trees (L ¼ 293; CI=0.78, RI=0.91) of Loasoideae (with Mentzelioideae andHydrangeaceaeas outgroups), with bootstrap support values (above branches) andPUZZLE support values (below branches). Areas shaded in grey indicate the ornithophilous taxa (all other taxa in Loasoideae are melittophilous and/or autogamous). ARTICLE IN PRESS 80 M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90

sta ll sc

A B C

aa aa aa df

D E F

aa df aa

df df

GH I

Fig. 2. Morphological characters in Loasoideae. (A) Flowers of Xylopodia klaprothioides (Weigendet al., 97/450C) and Plakothira parviflora (Weigends.n.); note the apically free staminodes(sta) in Plakothira andthe distinctfloral scales (sc) in Xylopodia. Both species have longitudinal lamellae (ll) on their petals. (B) Erect flower of Presliophytum incanum (Weigendet al. 2000 /695). (C) Pendulous flower of Loasa insons (LoMc, Weigendet al., 5913). (D) Floral scale of Loasa acerifolia (LoMc, Weigendet al., 6848) with double arch (aa) and flag-shaped dorsal filaments (df). (E) Floral scale of Loasa nitida (LoMc, Weigendet al., s.n.) with double arch (aa) and flag-shaped dorsal filaments (df). (F) Floral scale of ‘‘Loasa’’ filicifolia (LoPi, Weigendet al., 5880) with doublearch (aa) and distally widened dorsal filaments (df). (G) Floral scale of Caiophora pterosperma (CaPt, Weigend& Dostert, 97/27) with double arch (aa) and distally widened dorsal filaments (df). (H) Young floral scale of Caiophora canarinoides (CaLa, Ackermann 402) with double arch (aa) and distally widened dorsal filaments (df). (I) Floral scale of ‘‘Loasa’’ gayana (LoVo, Weigendet al., 7057) with double arch (aa) and distally widened dorsal filaments (df). ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 81

spm

A B C

spm av

D E F

appap

G H I appap

Fig. 3. Morphological characters in Loasoideae. (A) Shortly petiolate, deeply pinnatifid leaf of Caiophora cirsiifolia (CaCi, Weigend & Dostert, 97/194). (B) Shortly petiolate, deeply pinnatifid leaves of Loasa nana (LoPi, Weigendet al., 7080). (C) Serrate petal margin (spm) in the bee-pollinatedflower of Caiophora pterosperma (CaPt, Weigend& Dostert, 97/27). (D) Serrate petal margin (spm) in ‘‘Loasa’’ filicifolia (LoPi, Weigendet al., 5880). (E) Capsule dehiscencewith apical valves (av) in Aosa rupestris (Weigend, 7138). (F) Capsules twistedantidromouslyin Caiophora carduifolia (CaCa, Weigend et al., 5470). (G) Longitudinal dehiscence in capsules of Caiophora scarlatina (CaCh, M. & K. Weigend, 2000/108). (H) Capsules twisted anticlockwise in Blumenbachia hieronymi (BlBl, Weigend s.n.). (I) Hood-shaped, abruptly apiculate petals (appapp) in Blumenbachia catarinensis (BlBl, Foto C. Schlindwein). ARTICLE IN PRESS 82 M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90

aw aw

dc df ns ns

A B C

D E F

G H I

Fig. 4. Morphological characters in Loasoideae. (A) Floral scale of Blumenbachia hieronymi (BlBl, Weigend, s.n.) with basally inserted dorsal filaments (df). (B) Floral scale of Nasa lenta (NaAl, Weigendet al., 5446) with nectar sacs (ns) andapical wings (aw), dorsal filaments absent. (C) Floral scale of Nasa urens (NaSc, Weigend& Skrabal, 5889) with nectar sacs (ns), apical wings (aw), and dorsal calli (dc), dorsal filaments absent. (D) Pendulous flower of bee-pollinated Nasa carunculata (NaCa, Weigendet al., 5035). (E) Pinnate leaf of Nasa aequatoriana (N. triphylla group, NaSc, Weigend3997). (F) Campanulate, hummingbird-pollinatedflower of Caiophora buraeavii (CaLa, Kraus s.n., cultivatedat Munich). (G) Campanulate, hummingbird-pollinatedflower of Caiophora rosulata (CaCh, M. & K. Weigend, 2000/23). (H) Star-shaped, hummingbird-pollinated flower of Nasa trianae (NaAl, Weigend, 3610). (I) Campanulate, hummingbird-pollinated flower of Nasa weberbaueri (NaGr, Weigend& Dostert, 98/261). ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 83

PC1/ PC1

B1/ B1 PC2/ PC2

/ B2 B2

PC1 PC1/

A B1/ B1

PC2 / PC2 PC3

B2 / B2 iAPC

B2 B PC1

/ PC1 PC1 B1

PC2

PC2 B2 PC3

B2 / C D Fig. 5. Inflorescence architecture of Loasoideae (bracts and primary flower in distal dichasium black, next-lower bracts or pair of bracts grey; free arrows indicate metatopia). (A–B) Typical frondose inflorescences of Loasa s.str., Caiophora and Scyphanthus with non-recaulescent bracts (B, B/) and asymmetrically dichasial (A) to monochasial (B) paraclades. (C) Inflorescence of Aosa (A. plumerii, AoPa), both the ebracteose (w) distal dichasium and one bract (B2) are metatopically displaced onto paraclade 2 (PC2), distal paraclades strictly monochasial. (D) Frondose inflorescence of Nasa (N. picta, NaSc), one distal paraclade absent (w), the other strictly monochasial with one metatopic bract per flower. ARTICLE IN PRESS 84 M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90

PC1

B1 PC1/ iAPC B1/

B2 B1 PC1 PC1/ A

B1/ B2 iAPC

sAPC B B1

PC1 B1/ B1 / PC1/ PC1 PC1

B1/ / B2 B2 B2

C D Fig. 6. Inflorescence architecture of Loasoideae (bracts and primary flower in distal dichasium black, next-lower bracts or pair of bracts grey; free arrows indicate metatopia). (A) Inflorescence of ‘‘Loasa’’ ser. Malesherbioideae (‘‘L.’’ longiseta, LoMa), primary flower andone bract (B1) metatopic on one primary paraclade(PC1), PC1 movedinto one line with primary axis. (B) Inflorescence of Huidobria (H. chilensis), as 6A, but with inferior accessory paraclades (iAPC). (C) Inflorescence of Presliophytum (P. incanum), as 6B, but with inferior andsuperior accessory paraclades(iAPC, sAPC). (D) Bracteose inflorescence of Klaprothia mentzelioides, distal paraclades once dichasial, second-order paraclades strictly monochasial, all bracts metatopic with their axillary products (if present). ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 85

Klaprothieae "Lower Loaseae" South Andean Loasas

. ae " (incl rbioideae)

caules Maleshe eserticolae . ãVolubiles Pinnatae . Loasa Macrosperm D Floribundae . A ser enbachia . . . ser " ser ser. " ser ser ser ser. L. " " issenia hichicaste PresliophytumL. asa " L. L. Klaprothia Plakothira Xylopodia K Huidobria C " Aosa N Blum L. L. L. L. " " Scyphanthus Caiophora

29 28

27 16 22 23 26 25 7 15 24

14 19

5 11 12 13 18 21 4

2 10 Legend

9 Apomorphy 8 High bootstrap support values

Fig. 7. Annotated cladogram of Loasoideae (for symbols see legend). (1) Staminodes united into antesepalous complexes (vs. absent or free). (2) Fruit wall strongly lignified (vs. not or weakly sclerified). (3) Inflorescences bracteose (vs. frondose or ebracteose). (4) Petals with longitudinal lamellae (Fig. 2A; vs. longitudinal lamellae absent). (5) Flowers tetramerous (Fig. 2A; vs. pentamerous). (6) Leaves opposite throughout (vs. alternate above). (7) Staminodes of floral scale free in distal part, apically lobed (vs. united to top, andentire). (8) Floral scales always of 3 unitedstaminodes(vs. number variable or greater than 3). (9) Stamens thigmonastic (vs. with autonomous movement). (10) Flower scales strongly contrasting in colour (vs. white, yellow or greenish andmore or less the same colour as petals). (11) Inflorescence with pronouncedcon- andrecaulescence ( Fig. 6; vs. metatopia absent or leading to different structure). (12) Inflorescences ebracteose (vs. bracteose or frondose). (13) Nectar scales with dorsal sacs (Fig. 4B andC ; vs. sacs absent). (14) Nectar scales with well-developed apical wings (Fig. 4B andC ; vs. wings absent or very short). (15) Each flower on paraclades with individual bract (Fig. 5D; vs. ebracteose or with 2 bracts). (16) 2n ¼ 28 (vs. 2n ¼ 12; 24, 26). (17) Metatopia in the inflorescence absent (or marginal recaulescence of bracts—Fig. 5A, B; vs. recaulescence with bracts movedto next flower). (18) Fruits twisted anticlockwise and with longitudinal dehiscence (Fig. 3H; vs. straight or twistedantidromously).(19) Petals apiculate (Fig. 3I; vs. petals acuminate). (20) Nectar scales with double arch on back (Figs. 2D–I; vs. double arch absent). (21) Complex hetero-oligomeric iridoids present (tricoloriside type, Weigendet al. 2000 ; vs. absent). (22) Dorsal filaments flag-shaped( Figs. 2D andE ; vs. filiform or dorsoventrally flattened). (23) Thyrsoids basitonic, with alternate paraclades (vs. acrotonic and/or with opposite paraclades). (24) Leaves pinnatifid (to pinnate, bipinnate or bipinnatisect—Figs. 3A andB ). (25) Leaves shortly petiolate (petiole less than 1/2 as long as lamina—Figs. 3A andB ; vs. petiole equal to or longer than lamina). (26) Petal margins serrate (Figs. 3C andD ; vs. margin entire). (27) Fruits with longitudinal sutures (Fig. 3G; vs. capsule opening with apical valves only). (28) 10- hydroxy-oleoside dimethyl ester present (vs. absent, Weigendet al. 2000 ). (29) Reduction of chromosome number (2n ¼ 14; 16 vs. 2n ¼ 24; 26). the secondgroup, four more or less well-supported (only low bootstrap support: 53 BS); (2d) N. ser. groups can be distinguished, of which the precise Grandiflorae (73 BS, 61 P). relationships are unresolved: (2a) N. venezuelensis The South Andean Loasas clade contains Blumenba- (Steyerm.) Weigend, the only species of the N. venezue- chia,‘‘Loasa’’ (excl. ser. Malesherbioideae), Scy- lensis group analyzed; (2b) the N. triphylla group (99 phanthus,andCaiophora. Blumenbachia is weakly BS); (2c) N. ser. Alatae anda part of N. ser. ‘‘Saccatae’’ supportedas monophyletic in the PUZZLE analysis ARTICLE IN PRESS 86 M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90

(50 P), but not in the bootstrap analysis, except for two distally free staminodes (Fig. 2A). Since all other of the taxa in Blumenbachia sect. Angulatae (63 BS, Loasoideae have floral scales (and in all other sub- 84 P). In the parsimony tree, all species of ‘‘Loasa’’ families of Loasaceae staminodes are either free or ser. Pinnatae (‘‘L.’’ bergii,‘‘L.’’ filicifolia,‘‘L.’’ nana, absent) an apomorphic reversion, andnot a retained ‘‘L.’’ asterias) andone species of ‘‘ L.’’ ser. ‘‘Volubiles’’ ancestral character, is most parsimonious for Klaprothia (‘‘L.’’ micrantha) are foundas a basal polytomy, with and Plakothira in this respect. Huffordet al. (2003) ‘‘L.’’ gayana (‘‘L.’’ ser. ‘‘Volubiles’’) and‘‘ L.’’ lateritia retrieve the African genus Kissenia as sistergroup to (‘‘L.’’ ser. Acaules) retrievedas sister taxa (86 BS, 99 P). Klaprothieae (not resolvedin our analysis), which However, all taxa sampledof ‘‘ L.’’ ser. Pinnatae, renders further support for this hypothesis, since ‘‘L.’’ ser. Acaules,‘‘L.’’ ser. ‘‘Volubiles’’, Scyphanthus, Kissenia also has staminodes united into a distinct and Caiophora constitute a monophyletic group in the nectar scale. The close relationship between Neotropical PUZZLE analysis (67 P). Scyphanthus and Caiophora Klaprothia, including K. (Sclerothrix) fasciculata are invariably retrievedas sister taxa (61 BS, 70 P). (C.Presl) Poston as suggestedby Poston andNowicke There was little sequence variation in Caiophora. Loasa (1990), andthe Marquesas Islandsendemic Plakothira is ser. Macrospermae, which was broadly sampled, is reflectedin the phylogeny, and Plakothira may indeed retrievedas a well-supportedmonophyletic group (99 have arisen from epizoochorous ancestors (Klaprothia BS), andthe two closely alliedseries Floribundae mentzelioides has tardily dehiscent, burr-like capsules), (L. illapelina, L. floribunda, L. pallida; 53 P) and with subsequent modifications due to the island Deserticolae (L. urmenetae, L. elongata; 83 BS, 86 P) environment (loss of dispersal mechanism, island are identified as another monophylum (65 BS, 76 P). woodiness). Although its position remains unresolved in the present analysis, Kissenia probably is the sistergroup of Klaprothieae (Huffordet al. 2003 ), since morphological characters such as strongly lignifiedfruits Discussion andbracteose inflorescences ( Fig. 6D) can be regarded as synapomorphic (Fig. 7: characters 2 and3). Systematics and plausibility of the molecular Another aspect that is entirely congruent between the analyses data presentedhere andthose publishedby Hufford et al. (2003) is that the two species of Huidobria are not The trnL data confirm the conclusions of Weigend retrievedas monophyletic. They are essentially held (1997) in showing the polyphyly of ‘‘Loasa’’ sensu together by the fact that the number of staminodes in Urban andGilg (1900) andin confirming the mono- the antesepalous groups is not fixed, but the same is true phyly of the segregate genera Nasa and Presliophytum, for Klaprothieae (whereas all other Loaseae have the of the re-defined genus Caiophora with the exclusion of apomorphic, fixednumber of three staminodes; Fig. 7: two sections of Blumenbachia (B. sect. Angulatae, B. character 8), indicating the plesiomorphic condition of sect. Gripidea), andof tribe Klaprothieae. ‘‘ Loasa’’ this character state. Furthermore, both Huidobria remains paraphyletic (also indicated in Weigend1997 ), species have a peculiar inflorescence morphology (Fig. even after the exclusion of ‘‘L.’’ ser. Malesherbioideae. 6D) which is, however, very similar to that foundin These results are congruent with the data of Hufford Presliophytum. Grau (1997) discusses the profound et al. (2003), which also underscore the naturalness of differences between the two species of Huidobria Caiophora, Presliophytum, and Nasa, andthe justifica- regarding seed, leaf, and flower morphology. On tion for the segregation of Huidobria and Aosa from balance, the available data suggest that the two species ‘‘Loasa’’. While the recent morphological classification may indeed represent two only distantly related lineages. of Weigend(1997) is thus largely vindicated, the The term ‘‘Lower Loaseae’’ was informally intro- infrageneric groups (i.e., sections andseries) of the duced for Chichicaste, Huidobria, Kissenia, Presliophy- much older Urban andGilg (1900) classification are also tum and‘‘ Loasa’’ ser. Malesherbioideae (without largely retrievedin Loasa s.str. (L. ser. Deserticolae, L. indication of any close relationship), as a working ser. Macrospermae, L. ser. Floribundae). concept to name the taxa lacking both thigmonastic Klaprothieae (with Xylopodia as sister to Klaprothia stamens andcoloredfloral scales ( Weigend1997 ). and Plakothira) is identified as monophyletic by However, green-house experiments by the present apomorphic characters such as longitudinal lamellae authors have recently shown that Presliophytum on the petals, tetramerous flowers (Figs. 2A and7 : (P. heucheraefolium, P. incanum) does indeed show characters 4, 5, 6), andstrictly opposite, usually entire thigmonastic stamens, andat least one species leaves (Fig. 7: character 7). The position of Xylopodia is (L. malesherbioides) has colorednectar scales. By crucial to understanding the evolution of Loasoideae definition, Presliophytum andser. Malesherbioideae flowers: It has antesepalous staminodes united into wouldthus have to be placedinto Higher Loaseae, nectar scales, whereas the other Klaprothieae have although molecular resolution is satisfactory neither in ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 87 this analysis nor in Huffordet al. (2003) . An expansion Grandiflorae, a mostly High Andean lineage with erect of Presliophytum s.l. to include ‘‘L.’’ ser. Malesherbioi- wings on the floral scales andsubcircular to peltate deae, as was suggestedin Huffordet al. (2003) ,is foliage leaves (WeigendandRodriguez 2002 ). probably the sensible taxonomical consequence. Pre- The South Andean Loasas sensu Weigend(1997) are sliophytum s.l. is then heldtogether by its peculiar retrievedwith moderatesupport. They includenearly all inflorescence morphology with extreme metatopia (Figs. the pre-1997 genera of Loaseae, namely Blumenbachia, 6A, C and7 : character 11), leaf andseedcharacters. Caiophora, ‘‘Loasa’’,andScyphanthus. The group is The remaining taxa of Loasoideae (Aosa, Blumenba- morphologically readily circumscribed on the basis of chia, Caiophora,‘‘Loasa’’ in a narrow sense, Nasa, and the absence of metatopia in its inflorescences (Figs. 5A, Scyphanthus) were unitedinto Higher Loaseae, which B and7 : character 17), whereas at least the bracts are are likely monophyletic basedon apomorphies such as metatopic in all other Loasaceae (indicating the thigmonastic stamens andcoloredfloral scales ( Weigend plesiomorphic condition). All South Andean Loasas 1997; Fig. 7: characters 9 and10). The molecular results except Blumenbachia have a distinct double arch on the do not contradict this view if we include Presliophytum back of each nectar scale (Figs. 2D–I), andthis in this clade. The small Brazilian-Hispaniolan genus uniqueness suggests the monophyly of the correspond- Aosa (6 spp.) is not retrievedas a clade,but shows ing group (Fig. 7: character 20). Blumenbachia has been various morphological characters (e.g., characteristic redefined by removing B. sect. Angulatae and B. sect. ebracteose inflorescences: Figs. 5C and7 : character 12) Gripidea from Caiophora (Weigend1997 ), andthe which render its monophyly likely. distinctness from Caiophora of these two groups is Nasa is resolvedas a well-supportedmonophyletic clearly confirmedby molecular data from the present group in the present analysis as well as in Huffordet al. study. Like Caiophora, Blumenbachia has fruits with (2003, albeit with a smaller taxon sampling), andthis is longitudinal dehiscence (Fig. 3H), but this character congruent with various unique morphological characters appears to be convergent, since the two groups differ (presence of dorsal sacs and apical wings on the nectar profoundly in other aspects of morphology (e.g. scale scales: Figs. 4B andC ; inflorescence morphology: Fig. morphology: Fig. 4A, petal morphology: Fig. 3I). 5D; karyology: 2n ¼ 28; Fig. 7: characters 13–16). The Moreover, apart from the opening mode, actual fruit internal phylogeny of Nasa is partly resolved, with a basal morphology is also quite different: In Caiophora, dichotomy of a core clade comprising taxa of all five capsules are straight, andtwistedantidromously if infrageneric groups anda smaller clade( N. urens group), twistedat all ( Fig. 3F), whereas those of Blumenbachia but none of these clades are held together by any obvious are always twisted, and twisted anticlockwise only (Figs. morphological character. The N. urens group comprises a 3H and7 : character 18). The monophyly of Blumenba- range of morphologically rather heterogenous species chia sensu Weigend(1997) is weakly supportedin the from Nasa ser. ‘‘Saccatae’’ (N. picta, N. chenopodiifolia, quartet puzzling analysis (but not with the parsimony N. urens, N. vargasii), the morphologically coherent N. criterion). Huffordet al. (2003) did not address this poissoniana group (N. ferruginea, N. poissoniana, N. problem, since they only included two species of sect. raimondii), and N. carunculata, the type species of N. ser. Blumenbachia (i.e., Blumenbachia sensu Urban andGilg ‘‘Carunculatae’’. Nasa carunculata has been considered as 1900) in their analysis. However, all species of Blumen- closely alliedto N. macrothyrsa, primarily because of its bachia share abruptly apiculate petals (Figs. 3I and7 : strikingly similar habit (strongly branchedshrubs; character 19), identical nectar scales (Fig. 4A), and Weigendet al. 2003 ), but N. macrothyrsa is nestedin capsules twistedanticlockwise ( Fig. 3H), andare there- the core clade, thus N. ser. ‘‘Carunculatae’’ appears to be fore likely to represent a monophyletic group in spite of polyphyletic. The paraphyly of Nasa ser. ‘‘Saccatae’’, the lack of molecular resolution. A sistergroup relation- previously postulatedby WeigendandRodriguez (2003) , ship between Blumenbachia andthe remainderof the is again evident, involving at least three lineages. Nasa South Andean Loasas is plausible. ser. ‘‘Saccatae’’ was establishedby Urban andGilg Within South Andean Loasas, two monophyletic (1900) on the basis of annual habit, andflowers with clades with annual species of Loasa are clearly retrieved spreading petals andcontrastingly colorednectar scales in the molecular tree: L. ser. Macrospermae,with (Fig. 4D), but these character states are clearly plesio- extremely large andsmooth seedsandvery conspicuous, morphic for Higher Loaseae, since they are foundin flag-shaped dorsal threads on their nectar scales (Figs. nearly all lineages (Loasa: Fig. 2C, Caiophora: Fig. 3C, 2D, E and7 : character 22), andanother clade Blumenbachia: Fig. 3I). Within the well-supportedcore comprising L. ser. Floribundae and L. ser. Deserticolae. clade in Nasa, two groups are strongly supportedas The latter two groups show apomorphies in floral monophyletic which are also well-circumscribedmorpho- morphology (Urban andGilg 1900 ) andhave identical logically: the N. triphylla group from N. ser. ‘‘Saccatae’’, inflorescences (basitonic thyrsoids with alternate para- which has deeply divided (trifoliolate to pinnate) leaves clades, unique in Loasoideae; Fig. 7: character 23). (Fig. 4E; Dostert andWeigend1999 ); and N.ser. Phytochemistry further indicates that all three series ARTICLE IN PRESS 88 M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90

(Deserticolae, Floribundae, Macrospermae) probably the timing of divergence events appears to be impossible share exclusive common ancestry, since they are the from the data available (see Table 1 for distribution only groups containing a particular type of complex areas). ‘‘Huidobria’’ (N Chile), Klaprothia (Bolivia to hetero-oligomeric iridoids (e.g., tricoloriside; Weigend Mexico), Xylopodia (N Peru: Amotape-Huancabamba et al. 2000; Fig. 7: character 21). Loasa ser. Loasa, the Zone; Weigend, 2000), Kissenia (Africa), Presliophytum type section of the genus with only two species (L. (NW Chile, W Peru), Plakothira (Marquesas Islands in acanthifolia Desr., L. sclareifolia Juss.), is morphologi- Polynesia), and Aosa (Brasil andHispaniola) are cally closely alliedto L. ser. Macrospermae andshares geographically widespread in the tropical regions pri- its two most striking characters (see above). Loasa marily of South America, but they are completely absent acanthifolia was sequenced, but the trnL sequence has a from higher elevations andthe temperate andmediter- very long delition (more than 250 bp in the alignment, ranean regions. They are foundwith a series of evident representing helix 8 of the trnL secondary structure; paleoendemics in the coastal deserts of western South Kuhsel et al. 1990). Because its inclusion thus eliminates America (Presliophytum and Huidobria), the rain forests many informative alignment positions, it hadto be of Central America (Chichicaste), in Africa (Kissenia), removedfrom the analysis. However, the close relation- andBrazil ( Aosa). Conversely, High Andean and south- ship between L. ser. Loasa and L. ser. Macrospermae is ern temperate habitats have only been colonizedby two, evident, thus the group comprising four series (Deserti- species-rich groups: the South Andean Loasas and Nasa. colae, Floribundae, Loasa, Macrospermae) likely repre- Nasa is restrictedto the American Cordillera, andthe sents Loasa s.str. in the very narrowest sense. limits of its distribution coincide rather precisely with The exact placement of ‘‘L.’’ ser. Pinnatae,‘‘L.’’ ser. the limits of the tropical region (southern limit: ‘‘Volubiles’’, and‘‘ L.’’ ser. Acaules remains unresolved Department Santa Cruz in Bolivia, northern limit: in the bootstrap analysis, but the quartet puzzling Province Chiapas in Mexico). Nasa ser. Grandiflorae is analysis indicates a sistergroup relationship with the the only genuinely High Andean group (2500–4500 m). (well-supported) Caiophora/Scyphanthus clade, which is This monophylum is apparently derived from plants congruent with two unique morphological characters: growing at lower elevations in the (paraphyletic) the vast majority of species in these groups have deeply remainder of Nasa, such as the only coastal species pinnatifid, very shortly petiolate leaves (Figs. 3A, B and (200–1000 m; N. urens (Jacq.) Weigend), various species 7: characters 24 and25). In addition,‘‘ L.’’ ser. Pinnatae, from moderate elevations in inner-Andean valleys Caiophora,andScyphanthus share the predominance of (o2500 m; N. poissoniana, N. vargasii), or cloudforest serrate petal margins (Figs. 3C, D and7 : character 26), taxa (2000–3000 m; N. triphylla group, N. laxa (Killip) the latter being entire in all other groups. Scyphanthus Weigend, N. ramirezii (Weigend) Weigend, N. ser. contains two annual herbs from the mediterranean Alatae). The Amotape-Huancabamba Zone in N Peru climate in Chile, whereas Caiophora is a High Andean and adjacent Ecuador seems to be the primary centre of taxon containing over 50 species andranging from diversification of Nasa, andascent into the higher Central Argentina into southern Ecuador, with a single elevations as well as ecological diversification may have annual species in Uruguay andSE Brazil. Caiophora and taken place largely in this region (Weigend2002 ). Scyphanthus were also retrievedas sister taxa in the Within the South Andean Loasas, it is also possible to analyses of Moody and Hufford (2000) and Hufford trace geographical patterns: all species of Loasa s.str. et al. (2003), andthey are evidently closely alliedsince (i.e. L. ser. Loasa, L. ser. Macrospermae, L. ser. they share apomorphic characters. The fruits of Deserticolae, L. ser. Floribundae) are endemic to the Scyphanthus and Caiophora open both with apical mediterranean region or coastal desert of Chile, with valves andwith longitudinalsutures (synapomorphic; only few species extending into similar habitats in Fig. 7: character 27; in the derived taxa of Caiophora the Argentina or Peru; Blumenbachia is nearly exclusively capsule apex remains coherent: Fig. 3G), whereas other south-east South American andis foundin a widearch Loasoideae typically have fruits opening with apical from Brazil to Chile, with the morphologically most valves only (Fig. 3E). The monophyly of Caiophora primitive group (B. sect. Angulatae) endemic to the sensu Weigend(1997) is basedon karyology (2 n ¼ 14; southern Andes. The other groups of ‘‘Loasa’’ (‘‘L.’’ ser. 16) and phytochemistry (10-hydroxyoleoside-dimethyl Acaules,‘‘L.’’ ser. ‘‘Volubiles’’, ‘‘L.’’ ser. Pinnatae) are ester; Weigendet al. 2000 ; Fig. 7: character 28), andis largely Patagonian. Scyphanthus is restrictedto the confirmedby the present analysis. mediterranean region of Chile. Caiophora is widespread at elevations above 3500 m in the Andes (Argentinian Andes to Ecuador), but has its morphologically most Historical biogeography primitive taxa (i.e., those most similar to L. ser. Pinnatae and Scyphanthus) in SW Brazil andUruguay ( C. Some aspects of historical biogeography can be arechavaletae), the Argentinian Andes (C. pulchella, addressed on the basis of the data presented here, but C. nivalis), andat moderateelevations of the Peruvian ARTICLE IN PRESS M. Weigendet al. / Organisms, Diversity & Evolution 4 (2004) 73–90 89

Andes (C. pterosperma group). This distribution pattern 2002. Phylogenetics of asterids based on 3 coding and 3 indicates a primary radiation of the South Andean non-coding chloroplast DNA markers and the utility of Loasas in the southern temperate andmediterranean non-coding DNA at higher taxonomic levels. Mol. Phylog. zones. Subsequent dispersal and diversification, essen- Evol. 24, 274–301. tially of Caiophora, ledto the current distribution Darlington, J., 1934. A monograph of Mentzelia. Ann. patterns. Within South Andean Loasas, only Caiophora Missouri Bot. Gard. 21, 103–226. Davis, W.S., Thompson, H.J., 1967. A revision of Petalonyx has High Andean groups, and it seems to have colonized (Loasaceae) with a consideration of affinities in subfamily the High Andean region from the south temperate zone. Gronovioideae. Madron˜ o 19, 1–18. In the colonization of tropical High Andean habitats Dostert, N., Weigend, M., 1999. A synopsis of the Nasa by Caiophora and Nasa, two independent, alternative triphylla complex (Loasaceae), including some new species routes have thus been explored: Caiophora took the andsubspecies. HarvardPap. Bot. 4, 439–467. southern track by adaptation to temperate climate, Gottschling, M., Hilger, H.H., 2001. Phylogenetic analysis and whereas Nasa took the direct route with progressive character evolution of Ehretia and Bourreria (Ehretiaceae, adaptation to colder climates in the central Andes. In Boraginales) basedon ITS1 sequences. Bot. Jahrb. Syst. both groups, the widely open, contrastingly colored 123, 249–268. flowers typical for Loasoideae with melittophily (Figs. 2C, Grau, J., 1997. Huidobria, eine isolierte Gattung der Loasaceae 3C, D, I and4D ) are ancestral, andthey independently aus Chile. Sendtnera 4, 77–93. developed into superficially similar, much larger, orange Hempel, A.L., Reeves, P.A., Olmstead, R.G., Jansen, R.K., or red, hummingbird-pollinated flowers (Figs. 1 and4F–I ) 1995. Implications of rbcL sequence data for higher order in their High Andean representatives only. relationships of the Loasaceae andthe anomalous aquatic plant Hydrostachys (Hydrostachyaceae). Plant Syst. Evol. 194, 25–37. Acknowledgements Hufford, L., McMahon, M.M., Sherwood, A.M., Reeves, G., Chase, M.W., 2003. The major clades of Loasaceae: phylogenetic analysis using the plastid matk and trnl-trnf We wouldlike to express our sincere gratitudeto the regions. Am. J. Bot. 90, 1215–1228. National Tropical Botanical Gardens Hawaii (NTBG, Kuhsel, M.G., Strickland, R., Palmer, J., 1990. An ancient Hawaii), Jon Ricketson (Missouri Botanical Gardens), group I intron sharedby Eubacteria andChloroplasts. St. Vogel (Institut fur. Botanik undBotanischer Science 250, 1570–1573. Garten der Universitat. Wien, Austria), J. Grau and Moody, M.L., Hufford, L., 2000. Floral ontogeny and Ch. Ehrhardt (Institut fur. Systematische Botanik morphology of Cevallia, Fuertesia, and Gronovia Munchen,. Germany), J. Grant (Neufchatel, Switzer- (Loasaceae subfamily Gronovioideae). Int. J. Plant Sci. land), and L. Struwe (New Brunswick, USA) for leaf 161, 869–883. samples, as well as to the herbaria B, BSB, LPB, andM Posada, D., Crandall, K.A., 1998. Modeltest: testing the model for access to andloans from their collections. We want of DNA substitution. Bioinformatics 14, 817–818. to thank T. Hofreiter, M. Binder, T. Franke and H. Poston, M.E., Nowicke, J.W., 1990. A reevaluation of Forther. (Munich, Germany), N. Dostert, T. Henning Klaprothia and Sclerothrix (Loasaceae, Klaprothieae). Syst. Bot. 15, 671–677. and C. Schneider (Berlin, Germany), E. Rodriguez R. Poston, M.E., Thompson, H.J., 1977. Cytotaxonomic obser- (Trujillo, Peru), A. Cano E. (Lima, Peru), G. Vobis, and vations in Loasaceae subfamily Loasoideae. Syst. Bot. 2, C. Ezcurra (Bariloche, Argentina) for help in the field. 28–35. . We wouldlike to thank Mrs. C. M uller andA. Biesek Rambaut, A., 2001. Se–Al. Sequence alignment program (Berlin) for the laboratory work. The funds kindly v2.0a72. Oxford. provided by the following institutions at various stages Sleumer, H., 1956. Die Loasaceen Argentiniens. Bot. Jahrb. of the project are here gratefully acknowledged: Syst. 76, 411–462. Studienstiftung des Deutschen Volkes (1992–1997), Swofford, D.L., 1998. PAUPÃ. Phylogenetic Analysis Using Deutscher Akademischer Austauschdienst (1999–2000), Parsimony Ãandother Methods.Version 4. Sinauer Deutsche Forschungsgemeinschaft (Grant No. WE Associates, Sunderland. 2330/1, 2001–2003), Lewis B. andDorothy Cullman Taberlet, P., Gielly, L., Patou, G., Bouvet, J., 1991. 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