n , THE RONDELETIA COMPLEX Johan E. Rova 2 Piero DelpreteP and G. ti. , (RUBIACEAE): AN ATTEMPT TO Birsitta Bremer4 AND USE TRNL-F RPS16, ITS, SEQUENCE DATA TO DELIMIT GUETTARDEAE, RONDELETIEAE, AND SECTIONS WITHIN RONDELETIA 1 Abstract In the present study, a molecular phytogeny of the Rondeietia L. complex (Rubiaceae, Rondeletieae) was constructed with the following main objectives: to evaluate the sections of Rondeietia proposed by Fernandez Zequeira; to test if Stevemia (1) (2) Poit. belongs to the Rondeletieae s. str.; (3) to check if ITS data from Rondeletieae support previous phylogenetic results from trnL-F data regarding circumscription of Rondeletieae; and to verify Hodgkimonia F. Muell. belongs to Guettardeae or (4) if elsewhere. Two analyses were performed, one with ITS sequences from 46 taxa in the Rondeletieae—Guettardeae complex, and the other mo th combined ITS, rps!6 and trnL-F sequences of 21 taxa. Representatives of nine of the 10 sections of Rondeietia , recognized by Fernandez Zequeira were included in the ITS analysis. Five of her sections could be tested for monophyly. Support was only found for Rondeietia, sect. Leoninae M. Fernandez Zeq., while representatives from section Chamaebuxifoliae M. Fernandez Zeq., section Hypoleucae Standi., and section Nipemes M. Fernandez Zeq. together form a well -supported clade that could be distinguished also based on morphology. The latter clade is sister to Stevemia which is thus placed within , Rondeietia s. str. In addition, ITS sequence data confirm the separation of Rovaeanthus Borhidi from Rogiera Planch. Support & is low for inclusion of Blepharidium Standi., Mazaea Krug L'rb., Phyllomelia Griseb., Rachicallis DC., part of Rogiera and , & Suberanthus Borhidi M. Fernandez Zeq. in Rondeletieae. Rachicallis Mazaea and Phyllomelia form a clade with strong , , support. The tribe Rondeletieae s. str. W'as found to be monophyletic in all trees, although with low support; however, a re- delimitation of the tribe is proposed here based on this study and previous phylogenetic analyses. The monophyly for the tribe Guettardeae is weakly supported, with the inclusion of Arachnoihryx Planch, (including Cuatrecasasiodendron Steyerm.), & Gonzalagunia Ruiz Pav., Hodgkinsonia and Timonius DC. Although was recently the subject of a molecular phylogenetic it , study, the tribe Guettardeae is still in need of a wide-ranging survey in order to confirm its monophyly and delimit its taxonomic boundaries. Because Cuatrecasasiodendro was found within the Arachnothryx clade, the twro genera are here synonvmized as Arachnothryx, and in turn positioned within the tribe Guettardeae. In addition, based on herbarium and field & studies, the tw7o species described under Cuatrecasasiodendron C speciabile Steyerm. and C. colombianum Standi. . ( & Steyerm.) are treated as synonyms to the new7 combination Arachnothryx spectabilis (Steyerm.) Rova, Delprete B. Bremer, which proposed here. is Key words: Arachnoihryx Cuatrecasasiodendron Guettardeae, Hodgkinsonia ITS, phylogeny, Rogiera Rondeietia , , , , , Rondeletieae, rpsl6 Rubiaceae, Stevemia trnL-F , , We 1 are grateful to Attila Borhidi (Janos Pannonius University, Pecs) for help with identification of specimens of Rondeietia and related taxa and to Nahid Heidari and the staff at the Evolutionary Biology Centre (Uppsala University) tor technical assistance. The National Tropical Botanical Garden, Lawai, Kauai, Hawaii, provided material for Rondeietia inermis and Rovaeanthus sirigosus This work was supported by a grant from the Bergius Foundation. Part of this research was realized . during a Visiting Scientist fellowship to Piero Delprete (PD) from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) at the Universidade Federal de Goias, G< Tania, Goias, Brazil. PD’s fieldwork in Cuba was supported by a grant from the MaeAr thur Foundation through the American Council of Learned Societies/Social Science Research Council W orking Group on Cuba. W e also thank the Cuban government for giving permission to PD to perform fieldwork, and Ramona Oviedo, Mayra Fernandez, Jaqueline Salazar, and Lazara Sotolongo Academia de Ciencias, Institute de Ecologia y ( HAC) PD Sistematica, La Habana, Cuba; for accompanying during fieldwork in 2002. • I Tagal >erg Baskarp, SE-566 92 Habo, Sweden. Author for correspondence: jorova@telia.com. 3 CNPq Visiting Scientist, Institute of Biological Sciences—ICBrl, Department of General Biology/Botany Universidade , Federal de Goias, Campus II, 74001-970 Goiania, Goias, Brazil. Current address: Ins titut de Recherche pour le Developpement, Botanique et Bioinformatique de FArchitecture des Plantes (AMAP), TA-A51/PS2, Blvd. de la Lironde, 34398 Montpellier Cedex France, pdelprete@hotmail.com. 5, 4 Bergius Foundation at the Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-106 91 Stockholm, Sweden. 10.3417/2006179 doi: - Ann. Missouri Bot. Card. 96 182 193 Published on 23 April 2009 : . . n Volume Number Rova 183 96, 1 et al. 2009 The Complex Rondeletia The tribe Rondeletieae (Rubiaceae, Cinchonoi- are closely related, and this study also aimed to deae) includes predominantly shrubs and trees and investigate ITS data would suggest a similar if New is mostly distributed in the World tropics circumscription of Rondeletieae as previous studies (Robbrecht, 1988; Delprete, 1999a), with the main had. Finally, the study was aimed to investigate ITS if A center of diversity in the Greater Antilles. thorough sequence data would support Bremer’s (1992) inclu- description of the taxonomic and systematic history of sion of the Australian genus Hodgkinsonia F. Muell. the tribe found in Delprete (1999a). in the Guettardeae or Delprete (1996) transfer of the is ’s The largest genus of the tribe, Rondeletia L., is genus from the Guettardeae to the Chiococceae. mainly Antillean and comprises approximately 120 species. Standley (1918) divided Rondeletia into 15 M M ATERIA AND ETHOD LS S sections based on morphological and distributional data. Since then, several morphological and molecular For the ITS analyses, material was sampled from as studies in the Rondeletieae have argued about the many Rondeletia species and subspecies as possible. An made statu s of Stand ley's sections and the circumscrip - effort was to include representatives tion of the genus Rondeletia One opinion is that from all genera in Rondeletieae sensu Rova et al. . Rondeletia should be regarded as a narrowly circum- (2002). The outgroup consisted of Luculia Sweet scribed genus, separated from morphologically similar (basal in Rubiaceae, e.g., Bremer et ah, 1995), genera such as Arachnothryx Planch., javorhaea Cateshoea fueitesu Urb., Chiococca alba (L.) Hitchc. & Borhidi Jarai-KomL, Rogiera Planch., Roigella (Chiococceae and 12 accessions representing 11 s.l.), & Borhidi M. Fernandez Zeq., Rovaeanthus Borhidi, species in the following six genera of the tribe and Suberanthws Borhidi <& M. Fernandez Zeq. Guettardeae (based on available material and the & A (Stevermark, 1967; Borhidi Fernandez Zequeira, results from Rova et ah, 2002): racknothryx , & 1981a, b; Borhidi, 1982, 1989, 1994; Borhidi Jarai- Cuatrecasasiodendro Steyerm., Gonzalagunia Ruiz & Kornlodi, 1983; Fernandez Zequeira, 1994; Delprete, Pav., Guettarda L., Rogiera and Tirnonius DC. , 1999a, as Rondeletia complex sensu Delprete; Rova, Authors of species names are given in fable or 1, 1999; Rova et ah, 2002; Borhidi et ah, 2004; Rova, otherwise when first mentioned in the text. DNA On unpublished). the other hand, Lorenee (1991) Fresh or silica gel—dried leaves were used for recognized Rondeletia as a widely circumscribed extraction when available, but often herbarium DNA germs, treating the names applied to Mexican and material had to be used. was extracted using CTAB & Central American taxa of the complex as synonyms. the method (Doyle Doyle, 1987) and cleaned Based on morphological data, Fernandez Zequeira with the QIAquick PC R Purification Kit (QIAGEN (1994) made an attempt to classify the Greater GmbH, Hi! den, Germany). The cocktail for polymer- Antillean (especially the Cuban) Rondeletia species ase chain reaction (PCR) amplification w as mixed as 7 X into 10 sections. Her classification comprised 104 follows (to ca. 25 pi): 2.5 pi 10 buffer, 2.5 pi DNA species, most of them endemic to Cuba. This means 25 niM MgCl 2 pi dNTP, 0.125 pi Taq poly- 2 , pM that a majority of the species of Rondeletia were merase, 0.625 pi 10 forward primer, 0.625 pi s. str. M pM included in her study. According to Fernandez 10 reverse primer, 2.5 pi 0.1 EM A Cl, 2 pi Zequeira (1994), the sections are distinguished by dimethyl sulfoxide (DMSO), 2 pi template, and 10 pi various combinations of (often multistate) morpholog- water. In some cases, the amount of primer or template ical characters such as position and shape of was doubled (replacing some of the water). Primers & inflorescence, flower merosity, calyx lobe shape, leaf P17 and 26S-82R (Popp Oxelman, 2001) were used indumentum, and phyllotaxy (leaves opposite for amplification. Sequencing reactions were realized vs. DYEnamic ET However, her focus on Cuban species using the terminator Cycle Sequencing verticil late). did little to resolve the problem in the larger kit (Amersham Biosciences, Buckinghamshire, Eng- Rondeletia complex. The aim of the present land) following the protocol of the manufacturer first (DMSO study was to test Fernandez Zequeira’s sections of was added in the same concentration as in if DNA PCR MegaBACE Rondeletia are supported by phylogenies obtained the mix) anti run on a 1000 from molecular sequences. The second aim was to test Analysis System (Amersham Biosciences). Eor se- PCR if Stevensia Poit. was closely related to Rondeletia or quencing, the same primers were used as in the not. Stevensia, has not been included previously in amplification. molecular phylogenies, but morphology suggests a For the ITS study, 50 new ITS sequences were close affinity between the genera. Earlier studies produced, and five additional sequences were down- & (Bremer et ah, 1995; Bremer Thu in, 1998; loaded from Gen Bank and included in the data matrix. 1 & Andersson Rova, 1999; Rova et al., 2002) have Taxon names, authors, vouchers, and Gen Bank shown that the tribes Guettardeae and Rondeletieae accession numbers are presented in Table 1. 6 6 s 184 Annals of the Garden Missouri Botanical Manual alignment and gapcoding of the ITS and resulted in high-quality sequences. was not It sequences were performed with the following criteria: possible to obtain sequences from section Lindenianae an effort was made to see if gaps/insertions could be M. Fernandez Zeq., although extractions were at- (1) interpreted as caused by repeats or inversions, and tempted from two different specimens. was also not if It PGR so, sequences were aligned to fit these possible events; possible to obtain products from Roigella & gaps (he., inferred insertion/deletion events) were eorreifolia (Griseb.) Borhidi M. Fernandez Zeq., (2) introduced into the sequences to keep the number of which trnL-F data showed to be closely related to substitutions in an aligned region to a minimum; (3) Rondeletia s. str. (Rova et ah, 2002). For four sections insertions/deletions and substitutions were considered Rondeletia sect. Odoratae Standi., section Pedicel- (j equally probable events; and gaps/insertions of lares Standi., section Rigidae M. Fernandez Zeq., and (4) equal length shared by two or more taxa were inferred section Chamaebuxifoliae was only possible to it ), We to be homologous and binary coded. Gaps of more than sequence one species from each section. were one position in length introduced due to multiplication unfortunately not able to sequence the type species of of single nucleotides, e.g., poly-A, were not coded. Rondeletia R. americana L. This species seems to be , Regions where alignment could not be unambiguously very rarely collected, and extractions made from the interpreted were removed from the analysis. After herbarium material that we found in the Swedish Museum alignment, two ITS matrices were produced, one of Natural History Herbarium did not amplify We including gap codings and the other without them. despite several attempts. were not able to Two parsimony analyses, conducted with PA UP* establish contact with anyone who could assist us version 4. Ob 10 (Swofford, 2000), were performed for with recently collected material from St. Vincent, each matrix. The ITS analysis was a heuristic where the species endemic, and was not possible first is it search (random addition sequence with 1000 repli- to do such fieldwork ourselves. Ten species that cates, tree hi section-reconnec tion [TBR] branch swap- yielded sequences were not listed under any section in ping, and MU LTREES option in effect), and the second the work of Fernandez Zequeira, but four of them analysis was a jackknife search (Iasi step search option, could be assigned to sections based on the key 10,000 replicates, and Jac resample emulation). provided in her paper (Fernandez Zequeira, 1994): R. & For the combined analyses, the data matrix from the inermis (Sprung.) Krug Urb. and R. pilosa Sw r . ITS study was combined with the entire matrices from belonging to section Leoninae and R. hameliifolia , & the trnL-F study of Rova et ah (2002) and from an Dwyer M. V. Hayden and R. purdiei Hook. f. rpsld analysis (Rova, unpublished), keeping the ind belonging to section Calophyllae M. Fernandez Zeq. el codings from each matrix. Previous analyses of each Sectional affinities are indicated in Figure 1. separate data set resulted in similar trees, which The first ITS matrix, without indel coding, included implied that the data sets were congruent. Taxa not 699 characters, of which 174 were parsimony included in the combined analyses were then deleted informative. The second ITS matrix, where indel using the command DELETE in the PA U P block. The were coded, included 723 characters, of which 198 resulting set of sequences comprised 20 ingroup taxa. were parsimony informative. The combined ITS, This set included all taxa where all three sequences rpsl and trnL-F matrix included 2751 characters, , were available and all Rondeletia species where at 1451 of which were parsimony informative. least ITS and rps 1 sequences were available. The strict consensus tree obtained from the ITS Chiococca alba was used as outgroup, because, analyses is presented in Figure 1. Heuristic searches previously, it had been clearly shown not to be part of both data sets each resulted in 48 most parsimo- of the ingroup (Rova et ah, 2002). The data were nious trees. Tree lengths were 768 (consistency index = — analyzed by a heuristic search (random addition [CI] 0.56, retention index RE 0.76) in the TBR sequence with 1000 replicates, branch swap- heuristic search of the data set without indel coding MU ETHELS — — ping, and option in effect). and 805 (CI 0.65, RI 0.77) in the heuristic search where indels were coded. Strict consensus Results trees were identical for both data sets, jackknife support was not found for all clades in the strict DNA More than 50 extractions were obtained from consensus trees from the heuristic searches, and Rondeletia representatives, but only 27 of these jackknife support values for a clade could vary up to PGR (representing 23 species) were amplified by more than 10 units between the two data Tree sets. and yielded sequences. Extractions that did not topologies differed only marginally between the two produce usable sequences were mostly made from jackknife searches. The jackknife search without herbarium material more than 50 years old. Material indel codings found one clade that was not found in PGR collected in silica gel almost always worked for the other jackknife search (or in the heuristic , Volume Number Rova 185 96, 1 et al. 2009 The Complex Rondeletia searches), and the jackknife search with hide codings both belong to Rondeletia sect. Leoninae. In our I resulted in two c lades not found in the jackknife analysis, they form a clade with strong support. search without indeis coded (Fig. Support for these Rondeletia sect, leoninae would thus be the only one 1). c lades was low in all cases. of Fernandez Zequeira’s sections that is supported by The analysis of the combined matrix resulted in 12 our ITS sequence data. — equally parsimonious trees (length 2046, Cl 0,91 RI In all analyses, there is moderate support for a , = A 0.97). strict consensus of these trees is shown in clade with Rondeletia alaternoides R. odorata jacq., v. , & U Figure 2, and branches from the consensus tree that also and R. pachyphylla Krug rb (Fig. 1, clade F), . occur in the ITS analysis are marked in bold in Figure which represent sections Calophyilae Qdoratae and 1. , , Pedicellar respectively. Following the diagnostic es, table of sections in Fernandez Zequeira (1994), we Discussion were unable to find any morphological characters that support this group. SECTIONAL CLASSIFICATION OF RONDELETIA Rondeletia intermixta Britton and R. ochraeea Urb. The main aim of our study was to test Fernandez form a clade with strong support. While R. intermixta M Zequeira s (1994) classification with 10 sections of belongs to section Rondeletia Fernandez Zeq., FI. Rondeletia using molecular phylogenetic analyses. ochraeea has not been previously classified to any This goal was hard to reach satisfactorily; despite an section. It is thus possible to argue that R. ochraeea extensive search, it was difficult to find herbarium or should also belong to section Rondeletia The only . silica gel—dried material that would work for PC R and other known representative of section Rondeletia & sequencing. For five of the sections, only one included in our analysis, R. portoricensis Krug representative of each could be sequenced. Further- Urb., is placed in an unresolved relationship to the R. more, ITS data are obviously not variable enough to intermixta—R. ochraeea clade, although jackknife provide resolution among sections Hypoleucae and support for this is below 50. we J\lipenses Nevertheless, obtained several interest- Our study does not show any support for a . ing results with regard to the circumscription of separation of sections Hypoleucae and Nipenses in Rondeletia and some of Fernandez Zequeira's sec- sequenced Rondeletia. All representatives of these tions. sections, except R. berteroana DC., are found in a There is strong support for the Rondeletia s. str. strongly supported but unresolved clade (Fig. clade 1, clade (Fig. clade E). This clade consists of No morphological character combination seems 1, PI). to predominantly Antillean species. The only exceptions be unique for these two sections as one group, to this distribution are R. hameliifolia from Central according to the character list in Fernandez Zequeira America (Panama) and R. purdiei from South America (1994). Rondeletia berteroana differs from the other (Ecuador). Neither R. hameliifolia nor R. purdiei were sequenced species of section Hypoleucae (and section included in Fernandez Zequeira’s (1994) treatment, Nipenses) in being from Hispaniola. Phis species is H but according to her identification key, both species found as sister to clade but with very low support would belong to section Calophyilae In our study, the (Fig. . 1). A two species form a clade with strong support. third Rondeletia chamaebuxifolia Griseb., the only se- representative of this section is R. alaternoides A. quenced representative of section Chamaebuxifoliae , Rich, from Cuba, which found in clade F is (Fig. 1). is found closely related to the species from sections Thus, ITS sequence data do not support a monophy- Hypoleucae and Nipenses Following the diagnostic . letic section Calophyilae. characters provided in Fernandez Zequeira (1994) for Rondeletia deamii (Donn. Sm.) Standi, is found just sections Chamaebuxifoliae Hypoleucae and Nipenses, , , The outside the Rondeletia clade. generic s. str. this clade (Fig. clade G) could be distinguished 1, position of this Central American species has recently from other sections by having - to 3-flowered 1 been under debate. Lorence (1999) supported its inflorescences and retrorse -pilose flowers. position in Rondeletia but Borhidi (2001a) positioned in Arachnothryx. Our ITS sequence data suggest that it STEVEN A SI this species should be treated as a Rondeletia , although support for this hypothesis is less than 50. The second aim of our study was to investigate the Rondeletia inemiis and R. pilosa Sw. were not relationships between Stevensia and Rondeletia . included in Fernandez Zequeira’s (1994) treatment of Stevensia is here for the first time included in a Cuban Rondeletia as these species occur in Puerto molecular phylogenetic study. According to ITS data, , Rico and the U.S. Virgin Islands, respectively. there is strong support for an inclusion of at least S. However, according to her key to sections, they would minutifolia Alain in Rondeletia, s. str. 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The Rondeletia Complex (Rubiaceae): An Attempt to Use ITS, rps16 , and trnL-F Sequence Data to Delimit Guettardeae, Rondeletieae, and Sections within Rondeletia 1

Similar The Rondeletia Complex (Rubiaceae): An Attempt to Use ITS, rps16 , and trnL-F Sequence Data to Delimit Guettardeae, Rondeletieae, and Sections within Rondeletia 1