Plant Syst. Evol. 231: 77±89 32002) Phylogenetic relationships in Rosaceae inferred from chloroplast matK and trnL-trnF nucleotide sequence data D. Potter1, F. Gao1, P. Esteban Bortiri1, S.-H. Oh1, and S. Baggett2 1Department of Pomology, University of California, Davis, USA 2Ph.D. Program Biology, Lehman College, City University of New York, New York, USA Received February 27, 2001 Accepted October 11, 2001 Abstract. Phylogenetic relationships in Rosaceae economically important fruits of temperate were studied using parsimony analysis of nucleo- regions is produced by members of this family, tide sequence data from two regions of the including species of Malus 3apples), Pyrus chloroplast genome, the matK gene and the trnL- 3pears), Prunus 3plums, peaches, cherries, trnF region. As in a previously published phylog- almonds, and apricots), Rubus 3raspberries), eny of Rosaceae based upon rbcL sequences, and Fragaria 3strawberries). The family also monophyletic groups were resolved that corre- includes many ornamentals, e.g., species of spond, with some modi®cations, to subfamilies Maloideae and Rosoideae, but Spiraeoideae were Rosa 3roses), Potentilla 3cinquefoil), and polyphyletic. Three main lineages appear to have Sorbus 3mountain ash). A variety of growth diverged early in the evolution of the family: 1) habits, fruit types, and chromosome numbers Rosoideae sensu stricto, including taxa with a base is found within the family 3Robertson 1974), chromosome number of 7 3occasionally 8); 2) which is traditionally divided into four sub- actinorhizal Rosaceae, a group of taxa that engage families on the basis of fruit type 3e.g., Schulze- in symbiotic nitrogen ®xation; and 3) the rest of the Menz 1964). Spiraeoideae are characterized by family. The spiraeoid genus Gillenia, not included follicles or capsules, Rosoideae by achenes, in the rbcL study, was strongly supported as the Amygdaloideae 3Prunoideae) by drupes, and sister taxon to Maloideae sensu lato. A New World Maloideae by pomes. In the traditionally origin of Maloideae is suggested. The position of circumscribed Rosoideae, the base chromo- the economically important genus Prunus and the some number is x 7, 8, or 9; in Amygdaloi- status of subfamily Amygdaloideae remain unre- solved. deae, x 8; in Spiraeoideae, x 9 315, 17); and in Maloideae, x 17. The division of the Key words: Rosaceae, Gillenia, Maloideae, family into four subfamilies has not been Amygdaloideae, Spiraeoideae, phylogeny, matK, followed universally. For example, Hutchinson trnL-trnF. 31964) recognized 20 tribes and did not group these into subfamilies. The large and economically important angio- Because of their economic importance and sperm family Rosaceae has a worldwide dis- diversity, the Rosaceae have been the subject tribution and includes over 3000 species in 122 of numerous taxonomic and evolutionary genera 3Heywood 1993). The vast majority of studies. The family is generally considered to 78 D. Potter et al.: Chloroplast DNA Phylogeny of Rosaceae form a natural group united by ¯oral charac- Prinsepia, Oemleria, and Exochorda. The teristics 3Robertson 1974, Dickson et al. 1992). results suggested that, since some fruit types Kalkman 31988) suggested that the presence of have evolved several times within the family, a hypanthium may be the only morphological they are not as reliable as indicators of synapomorphy for the group, but the uncer- relationship as chromosome numbers. tainty of the relationship of Rosaceae to other Takhtajan 31997) incorporated some results families casts some doubt on this conclusion. from recent phylogenetic studies, such as that of This, as well as the relationships among Morgan et al. 31994), into his classi®cation of subfamilies, genera, and species, are subjects the family, in which he recognized twelve of considerable discussion and investigation subfamilies. Exochorda, previously classi®ed in 3summarized in Morgan et al. 1994, Phipps Spiraeoideae, was included in Amygdaloideae, et al. 1991). Recent phylogenetic analyses have Maloideae were expanded to include the tradi- employed a variety of data, including vegeta- tionally spiraeoid genera Kageneckia, Vauqueli- tive, ¯oral, and fruit morphology 3Kalkman nia, and Lindleya, and both Rosoideae and 1988; Phipps et al. 1991; Rohrer et al. 1991, Spiraeoideae were subdivided. 1994), ¯oral ontogeny 3Evans and Dickinson A long-standing question concerns the or- 1999a, b), wood anatomy 3Zhang 1992), pollen igin of Maloideae. Because of a base chromo- morphology 3Hebda and Chinnappa 1994), some number of 17, it is generally accepted that chloroplast DNA sequences 3Morgan et al. the subfamily originated either via polyploidi- 1994), nuclear gene sequences 3Potter 1997, zation of a spiraeoid ancestor with x 9 3e.g. Evans et al. 2000), and combined data from Gladkova 1972) or by hybridization between multiple sources 3Evans and Dickinson 1997, two lineages, most likely a spiraeoid with x 9 1999c), to address questions about the place- and an amygdaloid with x 8, followed by ment of problematic genera and the relation- polyploidization 3e.g. Sax 1933). The exact ships of the subfamilies. Sequences of the identity of the putative parental lineage3s) internal transcribed spacer 3ITS) regions of remains controversial, however. The rbcL nuclear ribosomal DNA have been used in analysis by Morgan et al. 31994) indicated that phylogenetic studies of two of the subfamilies members of the spiraeoid tribe Sorbarieae 3plus 3Maloideae, Campbell et al. 1995, Rosoideae, the traditionally rosoid genus Adenostoma) are Eriksson et al. 1998). These studies have done sister to Maloideae sensu lato, a relationship much to improve our understanding of the consistent with data from carpel anatomy anities of particular taxa and the evolution 3Sterling 1966), but this relationship was weak- of speci®c characters in Rosaceae, but a variety ly supported. Morgan et al. 31994) pointed out of questions remain, and all of the cited papers that, since rbcL is a chloroplast-encoded gene, point out areas in which further resolution is phylogenetic hypotheses based upon it repre- needed. sent phylogenies of maternal lineages only. In a phylogenetic study of rbcL gene Thus, the results are not inconsistent with an sequence variation across the family 3Morgan ancestral member of Amygdaloideae with x 8 et al. 1994), monophyletic groups of genera having been one of the parents involved in an were identi®ed that corresponded, with some ancient hybridization that led to the origin of modi®cations, to all of the subfamilies except Maloideae 3Sax 1933, Phipps et al. 1991). Spiraeoideae, which was shown to be grossly Recent phylogenetic studies of the nuclear gene polyphyletic. The data strongly supported waxy by Evans et al. 32000), however, have not recognition of Rosoideae sensu stricto, exclud- provided any evidence for an amygdaloid ing several taxa with x 9, and of Maloideae ancestor having been involved in the origin of sensu lato, including several spiraeoid taxa Maloideae; nor have phylogenetic studies of with x 15 or 17. Weak support was found for several nuclear genes in our laboratory 3e.g. Amygdaloideae sensu lato, including Prunus, Potter et al. 1999). D. Potter et al.: Chloroplast DNA Phylogeny of Rosaceae 79 Recent molecular phylogenetic analyses exist 3Taberlet et al. 1991) and which has been 3Chase et al. 1993, Morgan et al. 1994, Soltis useful in phylogenetic studies of angiosperm et al. 1995, KaÈ llersjoÈ et al. 1998, Soltis et al. groups at various taxonomic levels 3e.g. Gielly 2000) place Rosaceae in a clade with Mora- and Taberlet 1996, Karol et al. 2000, Potter ceae, Rhamnaceae, Urticaceae, and several et al. 2000, Scott et al. 2000, Bortiri et al. in others, which together have been designated press). as order Rosales 3Angiosperm Phylogeny Group 1998). Data from several genes further support a sister relationship of Rosaceae to the Materials and methods rest of the families in the order 3Evans and The species sampled for this study are listed in Campbell 2000, Soltis et al. 2000). Table 1. Voucher specimens are deposited in the The goal of this study was to assess U. C. Davis Herbarium 3DAV). Total DNA was phylogenetic relationships across Rosaceae extracted from fresh leaf material using the CTAB using new chloroplast DNA data, with a method 3Doyle and Doyle 1987) or modi®cations primary emphasis on the mostly woody taxa thereof. In the case of two species 3Fragaria vesca not included in Rosoideae sensu stricto, i.e., and Oemleria cerasiformis), the matK and trnL- genera traditionally classi®ed in Spiraeoideae, trnF sequences were determined from dierent Maloideae, and Amygdaloideae, and taxa of accessions. Primers for PCR and sequencing were pur- Rosoideae with x 9. We wanted to test the chased from Genosys Biotechnologies, Inc. PCR relationships identi®ed by Morgan et al. 31994) ampli®cations were carried out using the Perkin- and we were especially interested in seeing Elmer GeneAmp II kit. For matK, a 1.5±1.9 kb whether or not we could obtain better resolu- fragment was ampli®ed using trnK685F and tion than was provided by the rbcL data in the trnK2R as primers as described by Hu et al. following areas: 1) the deep branches within 32000). The forward PCR primer, trnK685F, is the Rosaceae phylogenetic tree; 2) the anities located at 685 bp upstream of the trnK50 exon of Prunus and other taxa traditionally classi- relative to the Pisum sequence 3Boyer and Mullet ®ed in Amygdaoideae; and 3) the relationships 1988). The reverse PCR primer, trnK2R, occurs at 0 of Maloideae to spiraeoid and/or amygdaloid site 2475 on the trnK3 exon. The trnL-trnF region taxa. In addition, we included Gillenia of tribe was ampli®ed using primers c and f 3Taberlet et al. Gillenieae 3Spiraeoideae; Schulze-Menz 1964), 1991). For both regions, PCR conditions were as follows: 1 minute at 95 °C; 40 cycles of 30 seconds a group not represented in Morgan et al.'s at 95 °C, 1 minute at 55 °C, and 2 minutes at 31994) rbcL study. Several members of other 72 °C; 7 minutes at 72 °C.