Many described species in section Petota are very similar to each other and are able to cross, suggesting that this section is overclassified. We have tested this for the large group of South American species of the section Petota, using a population genetic approach that would allow us to identify any structure among this material, if present. The results obtained from the analysis of 566 South-American Solanum section Petota accessions with STRUCTURE showed an optimal overall subdivision of these accessions in 16 clusters. By maximizing the partitioning of genetic variation among groups (Fst) we obtained support for additional groups within these clusters, up to a total of 44 units (or 48 units including the unknown species accessions) (Table 2). This does not automatically mean that 44 is the correct number of species as genetic differentiation would be expected among separate species but it can also be found among populations within a species (see below). Nevertheless, the Fst values of the various species arrangements in Table 1 offer a clear indication of overclassification: Fst increases from 0.145 (the 566 accessions) to 0.273 (10 clusters) and to 0.312 (16 clusters). The highest value is obtained after the nested analysis, when 44 groups explain 35% of the genetic variation (the remainder being present within species). The Fst value of the 89 species arrangement (0.2953) is even lower than that of the 16 clusters (0.312), indicating that the current species arrangement is 'over the top' but still does explain a considerable part of the genetic variation within the dataset.
Misclassification and overclassification
If not all accessions of a species are in one cluster but one or a few are present in different clusters, this may indicate misclassification. Occurrence of different species labels intermingled within one cluster points at overclassification. From both situations we see examples in our dataset and these may have consequences for the (sub) species status of the present taxa.
Status of subspecies
In nearly all cases there was no support for maintaining taxa at the subspecies level. This is the case for the subspecies within the species S. microdontum, S. vernei, S. boliviense and S. megistacrolobum. Only one of the recognized subspecies was supported in our analysis: S. commersonii subsp. malmeanum could be differentiated genetically from S. commersonii subsp. commersonii (Table 3).
Some of these (sub) species have been extensively studied previously, using morphology. The subspecies S. microdontum subsp. gigantophyllum was already considered to be a synonym of S. microdontum [32] and should not be recognized, as this is a clear case of overclassification. Giannattasio and Spooner studied the boundaries between S. megistacrolobum subsp. megistacrolobum and S. megistacrolobum subsp. toralapanum using morphological data [33] and with molecular markers [34]. Based on their analysis they suggested to preserve S. megistacrolobum subsp. toralapanum as a distinct subspecies while our analysis does not find support for this. Spooner et al. [35] studied the relationships of S. boliviense and S. astleyi using RAPDs and concluded that S. astleyi should be reduced to a subspecies of S. boliviense. Our data do not provide support for a subspecies level in S. boliviense.
Some species are supported
The following species are supported as genetically distinct units: S. raphanifolium, S. verrucosum (with S. macropilosum as synonym), S. microdontum, S. commersonii, S. okadae (only the seven accessions in cluster 15), S. huancabambense, and S. sogarandinum. The seven S. okadae accessions that appear in cluster 3 together with S. venturii accessions turned out to be mislabeled and have been corrected as being S. venturii accessions (personal communication R. Hoekstra, CGN). The accessions labeled S. microdontum, S. huancabambense and S. sogarandinum share their cluster with accessions from other species, but the optimal partitioning of genetic variation within the cluster shows that they represent distinct genetic units. This is consistent with the results from Jacobs et al. [9] and most of these species were also recognized in one or more other studies [2, 6, 32, 36, 37].
Support for combinations of species, pointing at overclassification
Some species are assigned to one STRUCTURE cluster, but their accessions do not form distinct genetic units within the cluster on their own, but combined with accessions from another species they do (Table 2). These are probably cases of overclassification. Examples are the combination of S. verrucosum and S. macropilosum in cluster 2, of S. kurtzianum and S. maglia in cluster 3, of S. venturii and S. okadae in cluster 3, of S. sandemanii, S. weberbauerii, and S. medians in cluster 5. Some of these combinations have already been recognized in the literature, e.g. S. macropilosum is considered a synonym of S. verrucosum [6].
Spooner and Salas [2] recognized S. medians and S. sandemanii, but not S. weberbauerii, which name they apparently considered as a synonym (unfortunately, information about this was not provided). Spooner et al. [38] synonymized both S. sandemanii and S. weberbaueri under S. medians.
Accessions scattered across clusters, pointing at mislabelling
The analysis showed that accessions from some species were scattered across two or even three clusters. This was the case for the accessions with the following species labels: S. maglia, S. doddsii, S. chacoense, S. gourlayi, S. virgultorum, S. hoopesii, S. augustii, S. tarijense S. vernei, S. infundibuliforme, S. alandiae, S. neorosii, S. sucrense, S. pachytrichum, and S. violaceimarmoratum. A major cause for this situation is probably mislabeling of accessions, although some of these species may be the product of hybridization events that occurred a long time ago. For instance, Solanum doddsii from Bolivia has been hypothesized to be a hybrid between S. alandiae and S. chacoense [39].
Misclassifications do occur since identification is often problematic due to ambiguous species characteristics. Problems with the identification of species were already addressed by Spooner and Salas [2] and Spooner and van den Berg [40], who noted that many of the taxa are extremely similar in morphology and many species are distinguished only by minor characters with often overlapping character states.
Hybrid accessions
Many authors [1, 2, 4, 41, 42] have suggested that certain recognized species in Solanum sect. Petota are the results of hybridization. Recent hybridizations can readily be recognized from the STRUCTURE analysis by the probability with which they are assigned to a particular cluster. While most accessions have a very high probability (usually around 0.9) to belong to one cluster, hybrid individuals tend to have a much lower probability (< 0.5) and have a, often only slightly lower, probability to belong to another cluster. Schulte et al. [43] also argue that a posterior probability lower than 0.5 provides strong evidence for a recent hybrid origin of individuals.
To practically present our results, we have assigned all accessions to the cluster to which it had the highest probability, but Additional file 1 lists all probabilities for all accessions. Hybrid accessions thus identified include amongst others accessions of the species S. spegazzinii and S. gourlayi, which co-occur in northern Argentina. The S. spegazzinii accession SPG386 was assigned to cluster 3 with a probability of 0.459 and with 0.262 to cluster 16. Another example of recent hybridization is NRS737 which shows probabilities of 0.435 and 0.434 with the clusters 13 and 15, respectively. However, in all cases the actual parents are unknown.
Non-supported species
Some species do appear in one cluster in the STRUCTURE analysis, but their accessions do not form a separate group in the Fst analysis, not even as part of a fixed combination with another species label. This concerns the following species: S. mochiquense, S. immite, S. chancayense in cluster 7, S. canasense, S. bukasovii, S. candolleanum, S. coelestipetalum, S. pampasense, S. ambosinum, S. marinasense, S. multidissectum, S. velardei in cluster 10, S. arnezii, S. yungasense, in cluster 12, S. incamayoense in cluster 13, S. tarijense, S. berthaultii in cluster 14, S. arac-pappa, S. leptophyes, S. ugentii, S. oplocense, S. sparsipilum, and S. brevicaule in cluster 16.
Many species mentioned in this category are part of what is termed the 'brevicaule complex' [7, 8, 44]: S. canasense S. bukasovii, S. candolleanum, S. coelestipetalum, S. pampasense, S. ambosinum, S. marinasense, S. velardei, S. incamayoense, S. leptophyes, S. ugentii and S. sparsipilum. Ugent [45] already proposed in 1970 that these should be reduced to one species. The division of the species according to our analysis in two clusters (10 and 16) reflects the presence of the northern and southern subgroups of the brevicaule-complex (see below). Solanum oplocense was shown to be a well-defined species using morphological data [7] and molecular data [8], but it was not distinct in an AFLP study [46] nor in ours. Previous results from a morphological study [47] and a more recent molecular study [48] had already suggested that the species S. berthaultii and S. tarijense should be combined. The species in cluster 7 were studied morphologically by Ames and collaborators [49], who placed Solanum immite and S. chancayense among the 6 distinctive species in a group of 29 species, the remainder of which were 'difficult to distinguish'.
Clusters correspond to the geographical origin of the accessions
Many accessions within a cluster come from the same geographical region (Additional file 1). For the largest and most complicated clusters (7, 10, 12, 14, 16) the information on the geographic origin of the accessions allows to draw some tentative conclusions. Cluster 16 contains mostly accessions from Argentina and Bolivia from the southern brevicaule complex and cluster 10 consist mostly of accessions from Peru (and northern Bolivia) that can be considered as belonging to the northern brevicaule complex. This separation of the brevicaule complex in a northern and southern part was already noted by Kardolus [50], was confirmed by Spooner and Salas [2] and is accepted in the treatment of this group on the Solanaceae Source website (http://www.solanaceaesource.org), where Spooner and his collaborators maintain two species, S. candolleanum for the northern representatives, and S. brevicaule for the southern representatives. Cluster 7 contains almost exclusively Peruvian accessions, and some species labels in cluster 7 (S. albornozii, S. augustii, S. chancayense, S. dolichocremastrum, S. immite) are associated with series Piurana [1, 2], but Jacobs et al. [9] could not find support for these species to be included in one of the recognized Piurana species groups. Ames and collaborators [49, 51] studied putative members of series Piurana with, respectively, morphological data and COSII markers, and concluded that based on morphology only three out of a total of 33 species could be recognized. The molecular data supported more species, some of them lacking morphological support, and the authors announced that decisions on species boundaries will be formalized in a forthcoming taxonomic monograph.
Cluster 14 contains all S. berthaultii accessions and almost all S. tarijense accessions, plus a few accessions with other species labels, which mostly come from Bolivia and Argentina. Cluster 12 contains accessions from various geographical origins, most of them from Bolivia and Argentina but some are from Peru and Paraguay. This group may represent accessions that relatively easily exchanged genetic material. The geographical distribution of accessions within clusters is consistent with the notion that our approach produces a meaningful arrangement of the accessions into groups that may (have) exchange(d) genetic material. For exchange of genetic material at least the accessions with the different species labels should have overlapping or adjacent geographical areas, at present or in the recent past.
Indeed, information on the distribution areas of the species of sect. Petota given in Hijmans et al. [52] confirms overlapping areas for many species within the recognized clusters, e.g. the species S. augustii, S. immite and S. dolichocremastrum in cluster 7, and S. berthaultii and S. tarijense in cluster 14.