Amplified fragment length polymorphism (AFLP) profiles generated from a subset of the investigated Fosterella specimens after selective amplification with a combination of unlabeled Mse+CAA and IRDye700-labeled Hind+AAG primers. PCR products were separated on a 6% denaturing polyacrylamide gel in an automated Li-COR sequencer. 

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... 0.25 U Taq DNA polymerase (Invitrogen). A touchdown protocol was followed for 47 cycles, each consisting of 94 8 C for 30 s, 65 8 to 56 8 C for 30 s, and 72 8 C for 60 s. Starting at 65 8 C, the annealing temperature was reduced by 0.8 8 C per cycle during the first 11 cycles, and then left con- stant at 56 8 C. Final extension was at 72 8 C for 7 min. Final products of the selective PCR were mixed with an equal volume of loading buffer, containing 98% ( v / v ) forma- mide, and denatured at 80 8 C for 5 min. Samples (0.5 m L) of each reaction were electrophoresed on denaturing 41 cm  0.2 mm polyacrylamide gels (6% Sequagel XR (National Diagnostics, Atlanta, Ga.) in 1æ TBE buffer (Sambrook and Russell 2001)), using an automated sequencer (LI-COR 4200 IR 2 ; LI-COR Inc., Lincoln, Nebr.). Fragment mobility was measured with real-time laser scanning. Gel images were stored electronically for further analysis. AFLP banding patterns were recorded as the presence (1) or absence (0) of a band at a particular position, with the help of GenImageIR version 4.02 software (Scanalytics Inc., Fairfax, Va.). The raw data were edited manually, after visual inspection. Faint or fuzzy bands were generally ignored. Scored fragment sizes ranged from 40 to 575 nucleotides. Standard lanes carrying identical samples were run on each gel, which facilitated the combination of datasets from 2 or more independent runs. For tree construction, the binary character matrix was converted into a distance matrix based on the Nei and Li (1979) ( = Dice) index of similarity, using NTSYSpc 2.10p (Rohlf 2000) or Treecon 1.3b software (Van de Peer and De Wachter 1994). Phenograms were generated either by unweighted pair group method with arithmetic mean (UPGMA) cluster analysis (Sneath and Sokal 1973) or by the neighbour-joining (NJ) algorithm (Saitou and Nei 1987), using the same program packages. An unweighted maximum parsimony analysis of the AFLP character matrix was con- ducted using PAUP* 4.0b10 (Swofford 2002). Character changes were interpreted under ACCTRAN optimization on, random addition, tree bisection-reconnection swapping options, and Maxtrees set to increase without limits. Statistical support for individual furcations in both the NJ and maximum parsimony trees was evaluated with bootstrapping (Felsenstein 1985). Bootstrap analyses used 1000 replicates. The complemented programs INTERVAL DATA, DCENTER, and EIGEN implemented in the NTSYSpc 2.10p software package (Rohlf 2000) were used to perform a principle coordinates analysis. Mature leaves were used fresh, or fixed in formalin/acetic acid/alcohol (FAA) and stored in 70% ethanol. Complete transverse-hand sections of the leaf blade (about halfway between the leaf tip and base) were prepared from unstained fresh material. Microtome sections were obtained from material either embedded in Paraplast and stained with safranine-astrablue or embedded in HEMA (Igersheim and Cichocki 1996 ) and stained with toluidine. The sections were investigated and documented with light microscopy (Leica Dialux 22), a digital camera system (Leica DC 300), and camera lucida drawings. For measuring and analyzing the sections, IM 1000 software (Leica) was applied. The assessment of morphologic, ecologic, and biogeographic characters was derived from a database comprising more than 360 specimens of all known Fosterella species. The database includes data from the literature (Smith and Downs 1974; Rauh 1979, 1987; Luther 1981, 1997; Smith and Read 1992; Ibisch et al. 1997, 1999, 2002; Kessler et al. 1999), information obtained from the study of cultivated plants (mainly in the Living Collection of the Fundaci ́n Amigos de la Naturaleza, Santa Cruz, Bolivia), and personal observations that were made during numerous field trips to all ecoregions of Bolivia (Pierre L. Ibisch, 1993–2003). The classification of the Bolivian ecoregions follows that described by Ibisch et al. (2004). In pilot experiments, 54 combinations of labeled Hin dIII- primer and unlabeled Mse I-primer with +3 selective bases each were screened with a small set of template DNAs. Well-resolved banding patterns were obtained with 13 of these combinations, and 8 primer pairs were eventually re- tained to analyse the entire set of 112 samples (Table 2). A total of 77 template DNA samples produced distinct AFLP profiles with all primer sets, and were included in the phylogenetic analysis (Table 1). Unfortunately, we were not able to generate consistent AFLP patterns from F. micrantha , which is the only Fosterella species present in Central America. The reproducibility of banding patterns was exemplarily tested with 2 primer combinations and 61 taxa. Numbers of unequivocally scorable band positions varied from 30 to 52, depending on the primer pair. Of 310 band positions totally scored, 96% were variable (Table 2). AFLP fin- gerprints generated by the primer combination Mse +CAA and Hind +AAG from a subset of samples are exemplarily shown in Fig. 1. Pairwise Nei and Li (1979) similarity values ranged from 0.39 to 0.95 for all samples, and generally exceeded 0.66 at the species level. The single plant of F. spectabilis and 1 of the 2 investigated samples of F. graminea exhibited the largest distances from the other specimens and were arbitra- rily chosen to root the trees. Defining alternative outgroups or using the midpoint rooting option had no major influence on tree topology (not shown). Both the UPGMA and NJ tree received cophenetic correlation coefficients > 0.83, and exhibited almost identical branching patterns. Only the NJ tree is shown here (Fig. 2). It is subdivided into 12 clusters that receive various levels of bootstrap support (BS). The moderately well-supported cluster A (73% BS) comprises all specimens of F. elata . Clusters B and C each unite 2 con- specific accessions from closely adjacent localities (i.e., F. vasquezii (87% BS) and F. floridensis (100% BS), respectively). Cluster D (87% BS) harbours 2 accessions of F. heterophylla and 2 accessions of the unclassified F. spec. 2. The monophyly of these 2 species also receives good support. Cluster E is again moderately well-supported (69% BS) and contains all specimens of F. nowickii and F. weddelliana , with the clonotype specimen of F. cotacajensis in a basal position. The monospecific cluster F (58% BS) is formed by the 2 accessions of F. caulescens . F. rexiae , together with the unclassified F. spec. 3, makes up cluster G (73% BS). All but 1 of the investigated accessions of F. albicans , plus the unclassified F. spec. 4, constitute the unsupported cluster H. Species groups A–H are united in a large (but unsupported) supercluster, with F. graminea (RM216), F. gracilis , and F. albicans (RV4023) forming a basal grade. The weakly defined cluster I contains a second F. graminea (RM22) and the unclassified F. spec. 5 and F. spec. 6, whereas the well-supported cluster J (98% BS) comprises all accessions of F. villosula. Internal bootstrap values within cluster J are also relatively high, suggesting considerable sub- division within F. villosula . Cluster K (72% BS) comprises all specimens of F. penduliflora, F. latifolia , and F. chiquitana , plus the unclassified F . spec. 7. The samples from these 4 apparently closely related species are intermingled with each other. Cluster L (57% BS) harbours all sampled plants of F. weberbaueri , together with an unclassified species ( F. spec. 8), which was collected in Brazil. With few exceptions, relationships between the clusters outlined above remain obscure. Clusters D and E are sister groups, as are J and K, but each with very low support (BS 57% and 50%, respectively). The latter relationship was also observed in our preliminary RAPD study, where these 2 clusters were referred to as villosula and penduliflora groups, respectively (Ibisch et al. 2002). Finally, clusters I, J, and K are united in a larger cluster that receives 53% bootstrap support. The original binary character matrix was also subjected to a phylogenetic analysis using maximum parsimony as an op- timality criterion, as was also done in other AFLP studies (e.g., Kardolus et al. 1998; Koopman et al. 2001; Despr ́s et al. 2003). Defining F. spectabilis as an outgroup, PAUP* found 4 trees with 2491 steps, a consistency index of 0.12 and a retention index of 0.5164 (not shown). The low consistency index value indicates a high level of homoplasy, as would be expected with the application of AFLPs to a large dataset (see Despr ́s et al. 2003 for a discussion). The strict consensus tree displays a basal polytomy of various clades, the species compositions of which are generally congruent with the clusters defined above in the NJ tree. The following clades (clusters) receive bootstrap supports above 50% in the maximum parsimony tree: A (56%), B (81%), C (99%), D (57%), E (65%), J (63%), K (97%), and L (53%). Other clades, and relationships between clades, are not resolved. Relationships among Fosterella specimens investigated by AFLP are further illustrated in the results of a principal coordinates analysis (Fig. 3). The first 3 coordinates together account for 52% (i.e., 28.4%, 12.2%, and 11.4%) of the total variance. Essentially, the same groups are revealed as in the NJ tree, but resolution varies between the individual groups. Whereas clusters A, B, D, J, K, and L are each relatively well resolved in the 3-dimensional principle coordinates plot, other groups show considerable overlap. One group of 5 species ( F. villosula , F. micrantha , F. penduliflora , F. latifolia , and F. chiquitana ), referred to as leaf anatomy group 1, is characterized by a set of common leaf anatomical characters (exemplified in Figs. 4 a – 4 c ): cell walls of the adaxial epidermis are uniform and hardly thickened; sclerotic hypodermis is adaxially absent (Fig. 4 b ); chlorenchyma consists of isodiametric cells only, and is sharply demarcated from the adaxial water storage tissue; ...