Preliminary phylogenetic analysis of the Andean clade and the placement of new Colombian blueberries (Ericaceae, Vaccinieae)

Abstract The blueberry tribe Vaccinieae (Ericaceae) is particularly diverse in South America and underwent extensive radiation in Colombia where many endemics occur. Recent fieldwork in Colombia has resulted in valuable additions to the phylogeny and as well in the discovery of morphologically noteworthy new species that need to be phylogenetically placed before being named. This is particularly important, as the monophyly of many of the studied genera have not been confirmed. In order to advance our understanding of the relationships within neotropical Vaccinieae and advice the taxonomy of the new blueberry relatives, here we present the most comprehensive phylogenetic analysis for the Andean clade. Anthopterus, Demosthenesia, and Pellegrinia are among the putative Andean genera recovered as monophyletic, while other eight Andean genera were not. The analyses also showed that genera that have been traditionally widely defined are non-monophyletic and could be further split into more discrete groups. Four newly discovered Colombian Vaccinieae are placed in the monophyletic Satyria s.s. and the Psammisia I clade. Although these new species are endemic to the Colombian Western Cordillera and Chocó biogeographic region and three are not known outside of Las Orquídeas National Park, they do not form sister pairs.


Introduction
In the neotropical regions, the most extensive radiation of the plant family Ericaceae took place in Colombia where there are 24 genera and 278 described species. Notably, about 55% of the Colombian Ericaceae are endemic to the country (Pedraza-Peñalosa unpubl.). Within Colombia, the greatest documented diversity of Ericaceae is found in the Western Cordillera and adjacent Chocó region, which belong to the Tropical Andes and Chocó biodiversity hotspots (Mittermeier et al. 1998;Myers et al. 2000), respectively. Th ese hotspots have the highest angiosperm diversity in N South America (Morawetz and Raedig 2007), but despite their importance for the understanding of the genesis of the neotropical fl ora, entire lineages particularly rich in NW Colombia are missing in modern monographic and phylogenetic research, and not only in Ericaceae.
Over the past 40 years, major U.S. herbaria have observed a sharp decline (ca. > 85%; calculated between 1970-2009) in the number of Colombian specimens received and databased. Th is decline refl ects the decrease in fi eld-based projects and exchange of herbarium specimens due to a combination of safety concerns, complicated Colombian permitting legislation, and lack of funding.
It is only recently that Colombian Ericaceae are being included in molecular analyses (see Disterigma (Klotzsch) Nied. in Pedraza-Peñalosa 2009, 2010a, 2010b. Th e ongoing inventory of the vascular plants of Las Orquídeas National Park (LONP), strategically located in the confl uence of the Colombian Tropical Andes and Chocó regions, has made available interesting new material of Ericaceae. LONP is a poorly known and isolated protected area in NW Antioquia within the general region with the greatest documented diversity and endemism of Ericaceae in the Neotropics (Luteyn 2002). Four new members of the tribe Vaccinieae stand out among the many other new plant species discovered in LONP. All of them are endemic to the Colombian Western Cordillera and Chocó biogeographic region, and three of them are endemic to LONP. Vaccinieae include the edible North American blueberries and South American mortiños and make up the bulk of Ericaceae in Colombia.
Th ere are about 600 species of Vaccinieae in the Neotropics, currently placed in 30 genera, 28 of them endemic to the region (Luteyn 2002). Large-scale phylogenetic analyses of Vaccinieae are few. In a preliminary analysis of the entire tribe, Kron et al. (2002) found that the great majority of the neotropical taxa are resolved within an Andean clade. Th is clade has about 500 species (Pedraza-Peñalosa unpubl.) and includes species growing as far south as Bolivia. Th e only study of the N Andean blueberries is one that included 55 species and 14 Andean genera (Powell and Kron 2003). However, because Colombian taxa have been largely unavailable, Colombian species were largely absent from these studies. Kron et al. (2002) and Powell and Kron (2003) are studies with diff erent scopes, but both found a striking disparity between phylogenetic relationships and the current classifi cation system, with 60-80% of the genera sampled resolved as not monophyletic. However, because tropical Ericaceae are very diverse, hard to collect and sometimes hard to sequence, phylogenetic analyses comprehensive enough to readdress generic limits are not available yet. In the absence of a phylogenetic based classifi cation for Vaccinieae, the generic placement of novel species remains challenging, at best.
Morphology is most frequently the only tool available to determine the taxonomic identity of a new Vaccinieae and the morphology of the four new species from LONP place them in Satyria Klotzsch and Psammisia Klotzsch, both broadly-circumscribed groups that have been shown to be non-monophyletic. Satyria and Psammisia are both placed by nuclear and chloroplast molecular data within the Andean clade (Kron et al. 2002, Powell andKron 2003). Psammisia, the second largest neotropical Ericaceae genus, is paraphyletic with respect to Macleania Hook., while the small-sized Satyria is polyphyletic. While the phylogenetic relationships of Psammisia and Macleania have not been addressed in more detail, molecular analyses revealed that the species of Satyria are placed in two clades that are not closely related to each other. Satyria from Central America and N South America are congeneric and form Satyria s.s. (including the type species, S. warszewiczii Klotzsch), whereas species of Satyria from S Peru and Bolivia cluster with representatives of Th ibaudia Ruiz & Pav. ex J. St.-Hil. from the same geographic region, forming the Th ibaudia clade, which is placed in a distant part of the phylogenetic tree (Powell and Kron 2003). All the previously mentioned groups, except for Th ibaudia, are most diverse in N South America.
Th e objectives of this study are twofold, fi rst, to provide the most comprehensive phylogenetic analysis yet for the entire Andean clade, second, to discover the evolutionary affi nities of the novel taxa from LONP. Th ese phylogenetic results will be used to guide their future naming. Although special emphasis has been placed in the representation of the neglected Colombian taxa, making of this dataset the largest published for neotropical Vaccinieae, more work is still necessary to elucidate intergeneric relationships within the Andean clade. Consequently, our results are still only preliminary, but they lay the groundwork for future detailed studies within and across Andean Vaccinieae. Lastly, although no attempt to reconstruct the evolution of morphological characters is made here, the morphology associated with the best-supported clades is briefl y discussed when relevant.

Taxon sampling
Sequence data from 94 terminals (91 species), belonging to 20 putatively neotropical Vaccinieae genera, were analyzed. Th e sampling strategy followed that of Pedraza-Peñalosa et al. (2013), but with emphasis on taxa of Andean origin. Eighteen species endemic to Colombia were newly sequenced and for fi ve other species that also grow outside Colombia, a population from Colombia was chosen. Th e sampled taxa exemplify diff erent aspects of reproductive and vegetative morphology. Th ey also represent the major clades recovered within Neotropical Vaccinieae in previous phylogenetic analyses, and also include species from the Caribbean/Mesoamerican clade. Emphasis was placed on sampling Satyria, Macleania, Psammisia, Th ibaudia and Cavendishia Lindl. For polymorphic species hard to identify or those whose variation is insufficiently known, more than one specimen was sequenced. For the plastic Satyria grandifolia Hoerold, two specimens recently collected in LONP (NW Colombia), each representing a separate morphospecies, were sequenced. Th ese were analyzed along with the sequence already available in GenBank and originally collected in SW Colombia. Two specimens were also sequenced of the very rare and morphologically insuffi ciently understood Psammisia mediobullata Luteyn & Sylva, endemic to a small region of NW Antioquia (Colombia). Trees were rooted with Gaylussacia baccata (Wangenh.) K.Koch, a species from a genus of extra-neotropical origin that is basal with respect to all New World taxa (Kron et al. 2002).

Analytical methods
A multiple sequence alignment was produced using MUSCLE (Edgar 2004) and the model of sequence evolution was estimated with jModelTest 2 (Guindon andGascuel 2003, Darriba et al. 2012). Maximum Likelihood (ML) estimation of the phylogenetic relationships was conducted using RAxML (Stamatakis 2006), employing one thousand replicates with stepwise random taxon addition, starting MP trees, the GTRGAMMA model of evolution, and 1000 bootstraps (BS) inferences. Th e best ML tree was visualized with FigTree 1.3.1 (http://tree.bio.ed.ac.uk/). Th e ML analyses were performed for each individual loci, partition (nuclear vs. plastid), and lastly, for the entire dataset.

Results
Th e resulting topologies of the individual, partitioned (nuclear vs. plastid), and combined best ML trees are in general agreement (trees not shown) and no signifi cant confl icts were detected. Th us, the results and discussion will focus on the best ML tree obtained from the combined analysis ( Fig. 1). Only the more robust groups recovered by this analysis and those relevant to the placement of the new species from LONP will be discussed in detail. Th e general topology of the most likely reconstruction (lnL= -13984.363979) is congruent with previous phylogenetic analyses of neotropical Vaccinieae. Here, some species from the Guyana Shield and the South American páramos are of extra-neotropical origin (98% bootstrap) and sister to a large neotropical clade. Th e neotropical clade (97% bootstrap) comprises a small Mesoamerican/Caribbean clade (100% bootstrap) sister to a large Andean clade (100% bootstrap), where the vast majority of the species are found. Th e Andean clade is divided into two major groups, named here A and B (Fig. 1).

Discussion
Th e two main groupings within the Andean clade, clades A and B (Fig. 1), also appear in previous studies with large generic coverage (see Kron et al. 2002, Pedraza-Peñalosa et al. 2013. However, these studies diff er in their generic sampling and the groups in which they disagree are either poorly supported or sampled, making detailed comparisons diffi cult. Th e following are the most signifi cant fi ndings.

HtAP clade
Th is strongly supported group (99% bootstrap; Fig. 1) is among the largest Andean Vaccinieae subclades with an estimated 170 species, most of them Cavendishia. Heteroandrous taxa are very distinctive within Vaccinieae and are characterized by having strongly unequal stamens in which the fi laments and/or anthers of adjacent stamens (each corresponding to one of the two staminal whorls) alternate in length. When there are diff erences in anther size, dimorphism can be also expressed in anther shape (Satyria s.s.) and orientation of the tubule aperture (Satyria s.s., Cavendishia). However, diff erences in this latter feature are poorly documented in species descriptions and are at times slight, and neither its extent nor its consistency is clear.
A close evolutionary relationship between Satyria s.s. and Cavendishia was fi rst proposed by Smith (1932) who suggested that even though the genera are distinct, they form a separate group within Vaccinieae having staminal dimorphism in common. However, the nature of the dimorphism is quite diff erent in each group, as pointed out below, and it should be further examined. Smith included the also dimorphic Orthaea within this group, but Andean species of Orthaea were not included in this analysis as it is currently being studied in detail (revision in preparation by N. R. Salinas).
Lastly, the HtAP species are characterized by having the pedicel articulate with the calyx (seen as a constriction at the point of attachment), a homoplasic feature that is traditionally used as part of the key characters useful to tell genera apart.

Satyria s.s. clade
Th ibaudia costaricensis is sister to Satyria s.s., a clade that is in agreement with the molecular circumscription of Satyria by Powell and Kron (2003). Both taxa have connate fi laments (Fig. 1). Satyria s.s. species have in common markedly alternately unequal stamens with rigid dimorphic anthers, the longer of which have fl aring tubules that are often ornamented or recurved into hooks; their fi laments are equal in length (Powell and Kron 2003). Powell and Kron (2003) sampled 11 out of the 22 then recognized species and noted that the Colombian Satyria (10 species, 9 of them endemic) needed to be included in future studies to better elucidate evolutionary patterns. Seven Colombian species, including two new to science, are added to this analysis for a total of 18 Satyria s.l.
Fifteen morphospecies are placed within Satyria s.s., mostly South American (Fig.  1). However, despite the increased taxonomic coverage, support at the basal nodes is weak and the only well-supported major subclade is that of the Central American species (S. allenii-S. meiantha, 96% bootstrap). Th e derived position of these Central American species indicate they are the result of relatively recent dispersals from a South American ancestor. However, in the case of S. panurensis (Benth. ex Meisn.) Hook. f. ex Nied., a species found from Mexico to southern Bolivia (including the Guianese Shield to the east) and not included within the Central American clade of Satyria s.s., the direction of the migration of is not clear.
Although the new species are endemic to the same general region, have similar corolla colors and shape, and are the only Satyria s.s. known to have an ornamented calyx (winged and/or lobed), they are not sister species. Satyria sp. nov. 1, sister to a clade containing species from both Central and South America, is easily diff erentiated from all other Satyria s.s. because of its pseudoverticillate leaves. S. pilosa A.C.Sm., another newly sequenced species, present in Antioquia but also beyond, being endemic to the greater Chocó biogeographic region, is sister to S. sp. nov. 2 (87% bootstrap).
Some of the molecular-based relationships here obtained using a larger sampling of Satyria s.s. do not agree with some of the taxonomic rearrangements of a recent monographic study (Powell 2005). Although the proposed changes have not been formally published, the unpublished names and combinations have already appeared in taxonomic and record-based public databases, as well on annotations of herbarium specimens from several American and European herbaria. Hence the comments below.
Satyria warszewiczii is a species thought to be confi ned to Central America (southern Mexico to Panama), with a broad altitudinal gradient [(100-)300-2500 m] and consequently morphological variation (Luteyn and Wilbur 2005). Based on Principal Components Analyses of the morphological variation, Powell (2005) suggests that 11 species, mostly from South America, are indistinguishable from it and therefore should be synonymized.
Molecular data for Satyria latifolia A.C.Sm. and S. ventricosa, as well as two morphospecies of the variable S. grandifolia (Colombia-Peru) from NW Colombia , all putative synonyms of S. warszewiczii according to Powell (2005), were sampled. A third collection of S. grandifolia from SW Colombia, which was used in previous phylogenetic analyses (see Powell 2005, Powell andKron 2003), was also analyzed.
Th e Central American Satyria meiantha, S. warszewiczii, and S. ventricosa form a clade (95% bootstrap; Fig. 1), in agreement with Powell's taxonomic proposal, and they may as well be conspecifi c, although terminal branch lengths are very long. However, they are more closely related to S. allenii A.C.Sm., which was considered by Powell (2005) a distinct species, than to either S. latifolia or S. grandifolia.
On the other hand, the two newly sequenced specimens of Satyria grandifolia from NW Colombia form a well-supported clade sister to the Central American Satyria s.s., but with little support, while the S. grandifolia from SW Colombia is placed with species of extra-Andean distribution, although again with little support (Fig. 1). As for Satyria latifolia, this species is part of a poorly supported tetratomy and is not immediately related to either S. warszewiczii or any accession of S. grandifolia, as hypothesized. Moreover, there are no big diff erences between the samples of S. grandifolia from NW Colombia, whereas considerable changes have accumulated on the branches and terminals in their sister group, S. warszewiczii and relatives.
Altogether, the results suggest that the Satyria grandifolia from NW and SW Colombia are not conspecifi c. Th e herbarium vouchers of the S. grandifolia from NW Colombia were collected in the same biogeographic region (Chocó) where the type species was procured, about 200 km from the type locality. To rule out contamination of our sample, some molecular markers were independently re-sequenced and identical results were obtained. Th e herbarium voucher of the S. grandifolia from SW Colombia (Luteyn 15204) was collected much farther away from the type locality, but still within the Chocó biogeographic region. Unfortunately, because Luteyn 15204 does not have fl owers (only fruits), it is not possible to reassess its taxonomic identity.
In the same general area of Luteyn 15204 there are specimens very similar to those from NW Colombia, however, others have fl oral and vegetative characteristics that subtly diverge from them and which have not been observed in other studied S. grandifolia collections. Without doubt Powell was right at pointing out that species delimitation within Satyria s.s. is complicated and that more fi eldwork in western Colombia was advised.
It was also (Powell 2005) suggested that Satyria arborea A.C.Sm., endemic to Colombia, should be synonymized with S. allenii, endemic to Panama. However, in this analysis they are not sister species, with S. allenii placed at a more derived position within a well-supported clade that includes other Central American Satyria s.s. (Fig.  1). Th e newly sequenced S. arborea was collected relatively close to the type locality and it is easily diff erentiated from S. allenii by anther length (about twice as long in S. arborea). Anther length may be one of the best characters, beyond geographic distribution, to diff erentiate among these species, as other traditionally used features such as length of the petiole and pedicel and life form have been shown to be inadequate (Powell 2005).

Cavendishia clade
Filaments and anthers of adjacent stamens are of diff erent lengths in Cavendishia, a diagnostic character that has been invoked by most of the taxonomic classifi cations of the neotropical Vaccinieae of the last century (Luteyn 1983, Smith 1932, Sleumer 1941. However, the Cavendishia clade also includes Th ibaudia jahnii (but < 50% bootstrap) toward its base (Fig. 1), a taxon that lacks the characteristic heteroandrous morphology of the genus Cavendishia and of the entire HtAP clade. Besides sharing free stamens, there are no other apparent morphological characters that satisfactorily explain the placement of T. jahnii within this clade Cavendishia has approximately 130 species, most of which are native/endemic to Colombia. Th e 15 species of Cavendishia here analyzed include representatives from the two currently recognized subgenera of Cavendishia: Chalmydantha and Cavendishia, as well as of four of the fi ve sections of subgenus Cavendishia (Foreroa is missing), all but one of the series of section Cavendishia (Unifl orae is missing), and all but series Lactiviscidae of section Engleriana (see Table 1). Unfortunately, none of the subgenera, sections, or series of Cavendishia is resolved as monophyletic. However, it must be cautioned that the phylogenetic relationships recovered within Cavendishia are too poorly supported to draw strong conclusions and a more extended taxonomic/ molecular sampling of it is needed.

HmCP clade
Th e relationships recovered for this clade (Fig. 1) are consistent with those found by Pedraza-Peñalosa et al. (2013). Taxa in this clade all have equal stamens and their pedicels are continuous with the calyx, unlike the HtAP clade, the sister group. Th at being said, the sampled species of Demosthenesia A.C.Sm. have slightly unequal fi laments, but not as markedly as in the HtAP clade.
Th e HmCP clade unites groups with diverse morphologies. Th e only monophyletic genera within it are relatively small (up to 12 spp.) and have contrasting geographic patterns: Anthopterus is widely distributed in the neotropics, while Pellegrinia and Demosthenesia are both endemic to a small area of the Peruvian and Bolivian Andes (Fig. 1). However, many subclades within HmCP are well supported and the two largest stand out. Th e four species of the Anthopterus revolutus-Th ibaudia fl oribunda clade (77% bootstrap) have in common winged or angulate calyces and corollas, whereas the seven species of the Diogenesia alstoniana-Demosthenesia spectabilis clade (96% bootstrap) in contrast all have terete calyces and corollas.
Only one species of Th emistoclesia Klotzsch was included in this analysis. However, Th emistoclesia with articulate calyces have recently been described, but unfortunately, none of them was available for sequencing. Th ese taxa also have other characteristics not previously thought to occur in the genus and it has been hypothesized they may represent a geographically and morphologically distinct clade (Pedraza-Peñalosa and Luteyn 2010). Th us, future analyses should sample in more detail the morphological diversity of Th emistoclesia, as well as that of other members of the HmCP clade.

Thibaudia p.p. clade
All species in the Th ibaudia p.p. clade have pedicels articulated with the calyx; it is precisely the presence of such articulation that initially defi ned Th ibaudia section Eurygania, currently a synonym of Th ibaudia. However, the staminal characters that seem to be important in defi ning other larger clades are absent here. Th is analysis supports Powell and Kron's (2003) assessment that species of Satyria in this clade, all endemic to S Peru and N Bolivia, should be segregated from Satyria s.s. (Fig. 1). Th e anther tubules of these Satyria do not diverge much distally, their sides being more parallel, and their tips lack ornamentations, so diff ering from Satyria s.s. (Powell 2005). Th e Th ibaudia and Satyria species that cluster together all have equal or slightly unequal stamens and connate fi laments.
On the other hand, although the T. macrocalyx-T. pachyantha subclade (74% bootstrap) also includes species with equal stamens, their fi laments are free. Other characters that are alos shared by some of the members of the Th ibaudia p.p. are thick corolla and calyx limb, anthers with poor distinction between tubules and thecae, thecae papillose and dehiscence by ventral clefts.
Th e unifying staminal features of Psammisia s.l. include stout anthers, free tubules, and connectives, the region where fi laments adhere to the anthers, that are 2-spurred, alternately spurred (i.e. only one staminal cycle is spurred), or rarely unspurred. It is precisely because of the presence of spurs that Smith (1932) linked Psammisia to a group of Macleania whose connectives are faintly thickened distally. Th ese thickened connectives were interpreted as a step toward the spurred condition observed in some Psammisia s.l. A morphological connection between members of Psammisia and Macleania is suggested by these results, as Psammisia s.l. is resolved in two clades, one of them sister to Macleania. However, the morphological basis of this relationship it is not yet known. As for the second grouping of Psammisia s.l., it is part of a more basal tritomy. Unfortunately, because taxon sampling is still inadequate and the type species of the genus was not available for sequencing, it is uncertain which Psammisia clade will retain the name.

Psammisia I clade
Most of the sampled Psammisia s.l. are found within the Psammisia I clade (P. grandifl ora-P. pedunculata, < 50% bootstrap), which is dominated by species from the N Andes (Fig. 1). Psammisia dolichopoda A.C.Sm. is the only species that is also present in Central America. However, ongoing studies suggest that it has considerable morphological variation and may represent more than one species (Pedraza-Peñalosa pers. obs.).
Two of the new species from LONP are placed here, within a clade (77% bootstrap) dominated by Colombian taxa; the only exception is the rare Psammisia oreogenes Sleumer, which was earlier thought to be exclusive to Ecuador but is now known to also occur in the Colombian portion of the Chocó biogeographic region (Fig. 1). Th e new species from LONP portray morphological characters that are either unusual in Psammisia s.l. (e.g., P. sp. nov. 1 has large leaves with pinnate venation) or previously unknown (i.e., P. sp. nov. 2 is the only known taxon with pseudoverticillate leaves). In general, the novel taxa are morphologically diff erent and molecular sequence data indicate they are not immediately related.
It is diffi cult to fi nd unifying morphological characters for Psammisia I. Moreover, the clade lacks support and has a tritomy at its base. Within it, only the P. sp. nov. 1-P. sp. nov. 2 subclade has moderate support (77% bootstrap). All its species share chartaceous to subcoriaceous leaves, pinnate venation, racemes with short rachises typically less than 1.6 cm long making the infl orescences look fasciculate, and medium sized corollas 8-22 mm long.

Psammisia II
Sequence data shows that Psammisia II taxa are more closely related to Macleania than to other congeneric species (Fig. 1). Unifying characteristics of the Psammisia II group comprise coriaceous leaves, plinerved venation, racemes with usually conspicuous rachises (1-26 cm long), and long corollas (17-40 mm). Th e inclusion of Th ibaudia infl ata within Psammisia II in a relatively derived position, deserves further scrutiny as its pedicel and calyx are continuous, whereas in most Macleania, and in all Psammisia s.l. it is articulated.
Furthermore, other morphological features seem to also help to diff erentiate between the Psammisia I and II clades. Th e leaves of Psammisia II have laminar glands at the base of the abaxial side, while the leaves of Psammisia I do not have basal glands, or when laminar glands are present, they are then spread through the entire abaxial surface (e.g., P. sodiroi, see Pedraza-Peñalosa et al. 2013). Anther morphology also seems to be helpful, with Psammisia II possessing well-developed tubules of a diameter similar to that of the theca, while in Psammisia I tubules are smaller and narrower, of a basal diameter only a fraction of the theca width.

Macleania clade
Macleania is sister to Psammisia II and there is good support for this clade and this relationship; the former clade includes M. fl oridunda Hook., the type species of the genus (Fig. 1). Because of its morphological range, Smith (1932) considered Macleania a coherent genus from which other genera were in the process of being derived. However, the subgenera Aponema and Macleania, which were thought to be morphologically well delimited, are not monophyletic. Moreover, although most sampled Ceratostema form a clade (but unsupported) placed within the basal tritomy of the Psammisia-Ceratostema-Macleania clade, C. reginaldii is resolved within Macleania.
As for Ceratostema, although as currently circumscribed is morphologically recognizable, it is not monophyletic and the sampled species fail to form a well supported clade. Ceratostema was one of the fi rst genera to be erected, and because Ericaceae is a predominantly montane group with a high number of endemics, it is not diffi cult to imagine the challenges earlier taxonomists faced to procure suffi cient specimens for their studies. Limited collections and fi eld observations led to problematic generic classifi cations. Consequently, not only many species have been transferred out of Ceratostema, but also entire genera have been segregated from within it such as Demosthenesia and Pellegrinia. Both genera are here resolved as monophyletic and not closely related to any sampled Ceratostema. Unfortunately, both taxon (16 out of ca. 75 spp.) and molecular sampling are still insuffi cient to better discern the evolutionary relationships between Macleania and Ceratostema.

Conclusions
With the study of the undersampled Colombian taxa, a critical component of neotropical Vaccinieae, a more complete picture of the complexity of the phylogenetic relationships within the Andean Vaccinieae has emerged.
Th e molecular results suggest that the observed diversity of neotropical Vaccinieae is mostly due to the diversifi cation of several clades of Andean origin that do not necessarily correspond to the current taxonomic classifi cation. Several genera that have been traditionally broadly defi ned are resolved as non-monophyletic and it seems likely they could be further split into more discrete groups. All this points to the need for broadscale comparative anatomical and morphological studies to reevaluate homologies, synapomorphies and clade support in general.
Indeed, although a cladistic analysis of morphological characters is premature at this point, it was still possible to identify morphological characters that seem to dif-ferentiate among some major clades and subclades. Most notable are the HtAP and HmCP clades, both strongly supported molecularly and easily diagnosable morphologically -they refl ect the diversity of arrangements and morphologies of the stamens and fl owers of Vaccinieae. However, although morphological characters may diagnose clades in one part of the tree, they may vary within a clade in another part of the tree. Such is the case of the fusion of staminal fi laments, a character diagnostic for Satyria s.s., the HmCP, and Cavendishia clades, but which is variable within the well-supported Macleania clade.
Th is analysis unequivocally places the newly discovered Colombian Vaccinieae within Satyria s.s. and the Psammisia I clade, 2 species in each clade, but not closely related to each other within their respective clades.
Th e small Satyria s.s. is a complex genus with species limits that are hard to elucidate. It is now clear that many morphological characters previously thought to diff erentiate species, especially those based on the scant voucher specimens available to earlier workers, do not work in the light of today's better documented intraspecifi c variation. Molecular results suggest that continuous characters (i.e., size) include informative data and can be used to distinguish species. Fieldwork in LONP suggests that characters that are not reported in herbarium labels and that cannot be recognized in herbarium specimens may be useful to work out species hard to diff erentiate (i.e. tridimensional shape of calyx, corolla, fruits, seed and embryo color) and these characters need to be reported on a regular basis.
Taxon sampling was nearly doubled in this analysis when compared to previous studies dealing with Andean species and robust monophyletic groups such as, Anthopterus, Demosthenesia, Gonocalyx, Pellegrinia, and Satyria s.s., were identifi ed within the Andean clade, although admittedly the fi rst four genera included only two species each. However, Gonocalyx, present in both Central and South America, may be of Mesoamerican origin. Clearly, more exhaustive analyses are necessary to fully resolve intergeneric relationships, and even the monophyletic groups here discerned (with exception of Satyria s.s.) need better representation. Further phylogenetic work is obviously needed for the large and non-monophyletic Psammisia s.l., Macleania, Th ibaudia s.l. and Ceratostema, accompanied of fi eld and herbarium work.
Lastly, it is also important to increase the representation of Central American species in order to further explore diversifi cation and colonization of that region. In these results, there is evidence of multiple dispersals of Andean Vaccinieae to Central America and of at least one radiation within Central America (Central American Satyria s.s.) of a genus of Andean origin. fi eld expeditions. Lastly, we are also grateful to the Herbario Nacional Colombiano and Instituto de Ciencias Naturales for their support and help procuring herbarium voucher specimen of Ericaceae for their study.