Phylogeny of the Dipsacales s.l. based on chloroplast trnL-F and ndhF sequences

Sequences of the chloroplast trnL-F region and 3(') end ndhF gene were used to elucidate phylogenetic relationships and the delimitation of families within Dipsacales s.l. Parsimony analyses of individual and combined data were conducted using maximum parsimony method. The most parsimonious tree based on combined trnL-F and 3(') end ndhF data set recognizes seven major clades of Dipsacales s.l. with the following relationships: Apiales (Adoxaceae ((Diervillaceae, Caprifoliaceae s.str.) (Linnaeaceae (Morinaceae (Dipsacaceae, Valerianaceae))))). Both Sambucus and Viburnum have close relationships with Adoxaceae, supporting their inclusion in this family. Caprifoliaceae s.l. (excluding Sambucus and Viburnum) is polyphyletic, and comprises three clades or families, i.e., Linnaeaceae (Abelia, Dipelta, Kolkwitzia, and Linnaea), Diervillaceae (Weigela and Diervilla) and Caprifoliaceae s.str. (Heptacodium, Leycesteria, Lonicera, Symphoricarpos, and Triosteum). This study focuses on the systematic position of Heptacodium, Triplostegia, and Morinaceae; and suggests that Heptacodium is closely related to the other Caprifoliaceae s.str.; Triplostegia is a sister to Dipsacaceae; Morinaceae, which has an affinity with Dipsacaceae, is possibly a sister group with Dipsacaceae-Valerianaceae clade. Our results are highly congruent with those of and.     

Revisiting of Carex sect. Confertiflorae s.l. (Cyperaceae): New data from molecular and morphological evidence and first insights on Carex biogeography in East Asia

Carex sect. Confertiflorae s.l. is a medium-sized species group (ca. 40 species) with its center of diversity in E Asia (China and Japan). According to morphological traits, the section has been proposed to split into two sections (sects. Confertiflorae sensu Ohwi and Molliculae Ohwi) up to five different ones (sects. Confertiflorae s.s., Molliculae, Dispalatae Ohwi, Ischnostachyae Ohwi, and Alliiformes Akiyama). Recent phylogenetic reconstructions showed Confertiflorae s.l. not to be monophyletic, as species traditionally considered part of it were found to belong to other clades, whereas species traditionally ascribed to other sections were nested within it. In this study, we investigated the phylogenetic structure, morphological affinities, and biogeographic history of sect. Confertiflorae s.l. We employed a taxon-based approach to explore the morphological affinities of the species considered in sect. Confertiflorae and compared the micromorphology of the nutlets of almost all the taxa using SEM. We included 40 samples representing 31 species/subspecies of sect. Confertiflorae s.l. and used two nuclear (ETS and ITS) and three plastid (trnL-F, matK, and rpl32-trnL ^UAG) molecular markers to reconstruct the phylogeny of the group. The phylogenetic analyses confirmed the polyphyly of sect. Confertiflorae s.l., whose representatives were found within five distinct clades. From these, two clades, sect. Confertiflorae and sect. Molliculae, were found to be closely related and contained the majority of the species. The composition of the two clades agreed with the morphological structure of the group, and we confirmed an exclusive combination of features (namely color of basal sheaths, presence of bract sheath, peduncle of lowest spike, inflorescence sex distribution, shape of pistillate glume apex, and color and veins of utricle, among others) characterizing each of the two clades. The origin of the two clades was found to be in the early Pliocene; however, the majority of the diversification events within each clade took place during the Pleistocene. This illustrates that although Asia has been regarded as having little potential ecological space for Carex to diversify due to its climate stability, groups of sedges sub-endemic from that area may have a fairly recent origin related to glaciations. We proposed the rearrangement of sect. Confertiflorae as previously conceived as three independent sections: the monotypic Alliiformes, sect. Molliculae, and sect. Paludosae.     

Systematic Rearrangement of Korean <Emphasis Type="Italic">Scirpus</Emphasis> L. s.l. (Cyperaceae) as Inferred from Nuclear ITS and Chloroplast <Emphasis Type="Italic">rbcL</Emphasis> Sequences

The genus Scirpus L. s.l. (Cyperaceae) has been accepted as a polyphyletic taxon by most plant taxonomists. This genus was separated into different genera by several different authors: Scirpus s. str., Trichophorum Pers., Bolboschoenus (Asch.) Palla, Schoenoplectus (Rchb.) Palla, and Schoenoplectiella Lye. The heterogeneity of Korean Scirpus s.l. has not yet been studied. We examined 17 taxa of Korean Scirpus s.l. by morphological characters and phylogenetic analyses based on nuclear ITS and chloroplast rbcL sequences. Phylogenetic analyses using maximum parsimony, maximum likelihood, and Bayesian method provided sufficient resolution. The phylogeny revealed the polyphyly of Korean Scirpus s.l. with five distinct clades. These clades correspond to Bolboschoenus, Schoenoplectus s.str., and Schoenoplectiella, Scirpus s.str., and Trichophorum, respectively. These five genera were delimited in terms of the morphology of tuber, bract, and inflorescence. By virtue of our findings, we suggest that the 17 taxa of Korean Scirpus s.l. should be placed into five genera as follows: Bolboschoenus (two species), Schoenoplectus (three species), Schoenoplectiella (six species), Scirpus s.str. (five species), and Trichophorum (one species).     

Phylogenetic reassessment of the Teloschistaceae (lichen-forming Ascomycota, Lecanoromycetes)

The Teloschistaceae is a widespread family with considerable morphological and ecological heterogeneity across genera and species groups. In order to provide a comprehensive molecular phylogeny for this family, phylogenetic analyses were carried out on sequences from the nuclear ribosomal ITS region obtained from 114 individuals that represent virtually all main lineages of Teloschistaceae. Our study confirmed the polyphyly of Caloplaca, Fulgensia and Xanthoria, and revealed that Teloschistes is probably non-monophyletic. We also confirm here that species traditionally included in Caloplaca subgenus Gasparrinia do not form a monophyletic entity. Caloplaca aurantia, C. carphinea and C. saxicola s. str. groups were recovered as monophyletic. The subgenera Caloplaca and Pyrenodesmia were also polyphyletic. In the subgenus Caloplaca, the traditionally recognized C. cerina group was recovered as monophyletic. Because this study is based solely on ITS, to maximize taxon sampling, the inclusion of phylogenetic signal from ambiguously aligned regions in MP (recoded INAASE and arc characters) resulted in the most highly supported phylogenetic reconstruction, compared with Bayesian inference restricted to alignable sites.     

Targeted sequencing supports morphology and embryo features in resolving the classification of Cyperaceae tribe Fuireneae s.l.

Molecular phylogenetic studies based on Sanger sequences have shown that Cyperaceae tribe Fuireneae s.l. is paraphyletic. However, taxonomic sampling in these studies has been poor, topologies have been inconsistent, and support for the backbone of trees has been weak. Moreover, uncertainty still surrounds the morphological limits of Schoenoplectiella, a genus of mainly small, amphicarpic annuals that was recently segregated from Schoenoplectus. Consequently, despite ample evidence from molecular analyses that Fuireneae s.l. might consist of two to four tribal lineages, no taxonomic changes have yet been made. Here, we use the Angiosperms353 enrichment panel for targeted sequencing to (i) clarify the relationships of Fuireneae s.l. with the related tribes Abildgaardieae, Eleocharideae, and Cypereae; (ii) define the limits of Fuireneae s.s., and (iii) test the monophyly of Fuireneae s.l. genera with emphasis on Schoenoplectus and Schoenoplectiella. Using more than a third of Fuireneae s.l. diversity, our phylogenomic analyses strongly support six genera and four major Fuireneae s.l. clades that we recognize as tribes: Bolboschoeneae stat.nov., Fuireneae s.s., Schoenoplecteae, and Pseudoschoeneae tr. nov. These results are consistent with morphological, micromorphological (nutlet epidermal cell shape), and embryo differences detected for each tribe. At the generic level, most sub-Saharan African perennials currently treated in Schoenoplectus are transferred to Schoenoplectiella. Our targeted sequencing results show that these species are nested in Schoenoplectiella, and their treatment here is consistent with micromorphological and embryo characters shared by all Schoenoplectiella species. Keys to recognized tribes and genera are provided.     

Plastid and nuclear phylogenomic incongruences and biogeographic implications of Magnolia s.l. (Magnoliaceae)

Magnoliaceae, an assemblage of early diverged angiosperms, comprises two subfamilies, speciose Magnolioideae with approximately 300 species in varying numbers of genera and monogeneric Liriodendroideae with two species in Liriodendron L. This family occupies a pivotal phylogenetic position with important insights into the diversification of early angiosperms, and shows intercontinentally disjunct distribution patterns between eastern Asia and the Americas. Widespread morphological homogeneity and slow substitution rates in Magnolia L. s.l. resulted in poorly supported phylogenetic relationships based on morphology or molecular evidence, which hampers our understanding of the genus’ temporal and spacial evolution. Here, based on the newly generated genome skimming data for 48 Magnolia s.l. species, we produced robust Magnolia phylogenies using genome-wide markers from both plastid genomes and single nucleotide polymorphism data. Contrasting the plastid and nuclear phylogenies revealed extensive cytonuclear conflicts in both shallow and deep relationships. ABBA-BABA and PhyloNet analyses suggested hybridization occurred within sect. Yulania, and sect. Magnolia, which is in concordance with the ploidy level of the species in these two sections. Divergence time estimates and biogeographic reconstruction indicated that the timing of the three tropical Magnolia disjunctions coincided with the mid-Eocene cooling climate and/or late Eocene climate deterioration, and two temperate disjunctions occurred much later, possibly during the warm periods of the Miocene, hence supporting the boreotropical flora concept of Magnolia s.l.     

Phylogenetic relationships of Thlaspi s.l. (subtribe Thlaspidinae, Lepidieae) and allied genera based on chloroplast DNA restriction-site variation

Chloroplast DNA restriction-site variation was analyzed in 30 accessions representing 20 species from the major lineages in Thlaspi s.l. (previously described as genera by Meyer 1973, 1979) and allied genera from the subtribe Thlaspidinae (Peltaria, Teesdalia, Cochlearia, Ionopsidium, Aethionema). A total of 161 variable restriction sites were detected. Phylogenetic analyses indicated a division of Thlaspi s.l. into three groups consistent with Meyer's genera Thlaspi s. str., Microthlaspi and Noccaea/Raparia. The genus Thlaspi s.l. as currently described proved to be paraphyletic because one of its major lineages, i.e. Thlaspi s. str., appeared to be more closely related to other genera (Peltaria, Teesdalia) than to the remaining lineages of Thlaspi s.l., i.e. Noccaea/Raparia and Microthlaspi. Sequence divergence values (100 x p) between the Thlaspi s.l. lineages were similar to values between these groups and related genera (Teesdalia, Peltaria), respectively. Chloroplast DNA variation was also used to assess subtribal classification of the genera studied. The cpDNA data were inconsistent with the controversial taxonomic classifications based on morphology. The molecular data would suggest that (1) the subtribe Thlaspidinae, as traditionally described, is not monophyletic; (2) the Thlaspidinae should be reduced to a group consisting of Thlaspi s. str., Peltaria, Teesdalia, Microthlaspi, Noccaea/Raparia, and that Aethionema should be excluded from the Thlaspidinae; and (3) Cochlearia and Ionopsidium represent the subtribe Cochleariinae.     

Phylogeny and classification of the Grimmiaceae/Ptychomitriaceae complex (Bryophyta) inferred from cpDNA

Phylogenetic relationships within the Grimmiaceae/Ptychomitriaceae were studied using a plastid tRNA cluster, including four tRNAs (trnS, trnT, trnL, trnF), a fast evolving gene (rps4), four spacers separating the coding regions, as well as one group I intron. Secondary structure analyses of the spacers as well as the trnL intron P8 domain identified several homoplastic inversions. Tracing the structural evolution of P8 we were able to identify lineage specific modifications that are mainly explained by inversions often in combination with large indel events. Phylogenetic analyses using maximum parsimony, maximum likelihood, and Bayesian methods indicate that Jaffueliobryum and Indusiella are closely related to Ptychomitrium and form the Ptychomitriaceae s. str. As Campylostelium is neither resolved within Ptychomitriaceae s. str. nor Grimmiaceae s. str., we prefer to treat it in its own family, Campylosteliaceae De Not. The systematic position of Glyphomitrium, as also found by other authors, should be considered in a broader analysis of haplolepidous mosses as our analyses indicate that it is not part of Campylosteliaceae, Grimmiaceae, or Ptychomitriaceae. Within Grimmiaceae s. str., Racomitrium is recognized as a monophyletic group sister to a clade including Dryptodon, Grimmia, and Schistidium. Coscinodon species appear disperse in Grimmia s. str. next to species sharing the same gametophyte morphology, and thus the genus is synonymized with Grimmia. Finally, Schistidium is resolved monophyletic with high statistical support, and seems to represent a rapidly evolving group of species. Our results are not fully congruent with recently published treatments splitting Grimmiaceae in a fairly high number of genera, neither with a comprehensive Grimmia including Dryptodon and Grimmia s. str.     

Phylogeny of Orthotrichum s.l. and Ulota s.l. (Orthotrichaceae,Bryophyta): Insights into stomatal evolution

Orthotrichum Hedw. s.l. and Ulota Mohr s.l. are two of the most speciose genera of the xerophytic moss family Orthotrichaceae. We reconstructed the phylogeny of these two genera using three data matrices: (i) organellar genomes and 33 taxa; (ii) six loci from three genomes and 144 taxa; and (iii) two plastid loci and 163 taxa. The present phylogeny, based on the maximum sampling of genes or taxa to date, generally confirms the new classification of Orthotrichum and Ulota, and indicated that all Ulota species, except Ulota phyllantha Brid., form a clade and three lineages comprise the cryptoporous Orthotrichum clade. We provided new morphological characters that support the present division of the two genera. Ancestral state reconstruction of stoma indicates that superficial stomata in Orthotrichum represent a plesiomorphic character and semi-immersed stomata were derived from immersed stomata. The results also suggest that immersed stomata independently arose once in Orthotrichum, whereas semi-immersed stomata probably arose more than once. Molecular dating analysis reveals that the occurrence of immersed stomata is probably related to arid environments during the early Oligocene to late Miocene, whereas the appearance of semi-immersed stomata might be associated with the mesic–xeric or semiarid environments during the middle Miocene to Pliocene. Ancestral state reconstruction of habitat indicates that the saxicolous habitat is apomorphic and independently evolved multiple times in Orthotrichum and Ulota, which supports the former hypothesis. Considering morphological statistics, the development of the cryptopore in Orthotrichum could provide increased resilience to dry habitats, and might promote their habitat shift during evolution.     

Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

Phylogeny and taxonomy of Afrocayratia,a new genus of Vitaceae from continental Africa and Madagascar

The genus Cayratia Juss. in the traditional sense (i.e., Cayratia s.l.) of the grape family has been shown to be non‐monophyletic. Previous studies supported the splitting of Cayratia s.l. into three genera, that is, Cayratia s.s., Causonis Raf., and a new genus representing the African Cayratia clade. However, the morphology of the African Cayratia clade has not been studied carefully and its phylogenetic position within Vitaceae remains unclear. Our study integrates molecular, distributional, and morphological data and supports the recognition of the new genus Afrocayratia from continental Africa and Madagascar. Phylogenetic analyses strongly support the monophyly of Afrocayratia and resolve it as a sister of Cayratia s.s. based on the chloroplast data, but it is placed sister to Cyphostemma based on the internal transcribed spacer dataset. Molecular dating suggests that Afrocayratia split with Cayratia s.s. during the Paleocene, but that the extant species of Afrocayratia did not diversify until the early Miocene. Afrocayratia differs from its allied genera in having short stigmas and seeds with subcircular ventral infold cavities in cross‐section. Three clades are detected within Afrocayratia, with A. debilis (Baker) J.Wen & L.M.Lu as the first diverged lineage. The second diverged lineage includes A. delicatula (Willems) J.Wen & Z.D.Chen and A. gracilis (Guill. & Perr.) J.Wen & Z.D.Chen. The third diverged lineage includes A. imerinensis (Baker) J.Wen & L.M.Lu, A. longiflora (Desc.) J.Wen & Rabarijaona, and A. triternata (Baker) J.Wen & Rabarijaona from Madagascar, which form a monophyletic group that diverged from the second lineage in the middle Miocene. Combining the morphological and molecular evidence, we formally describe the new genus Afrocayratia, make seven new combinations, and provide a key to species of the genus.     

Pachyphragma andGagria (Brassicaceae) Revisited: Molecular data indicate close relationship toThlaspi S.Str.

The systematic position ofPachyphragma macrophyllum (Brassicaceae) is unsettled. This species was either treated as a monotypic section ofThlaspi s.l. or was treated as a distinct genus.Gagria lobata apparently closely related toPachyphragma, was also given generic rank. To unravel the phylogenetic relationships ofPachyphragma andGagria, ITS sequence studies were conducted using as a reference representatives of the main lineages ofThlaspi s.l. The molecular data indicate thatGagria lobata andPachyphragma macrophyllum are closely related to members ofThlaspi s.str., an assemblage of species well separated from the otherThlaspi s.l. lineages.     

Phylogenomic conflict analyses in the apple genus Malus s.l. reveal widespread hybridization and allopolyploidy driving diversification,with insights into the complex biogeographic history in the Northern Hemisphere

Phylogenomic evidence from an increasing number of studies has demonstrated that different data sets and analytical approaches often reconstruct strongly supported but conflicting relationships. In this study, 785 single-copy nuclear genes and 75 complete plastomes were used to infer the phylogenetic relationships and estimate the historical biogeography of the apple genus Malus sensu lato, an economically important lineage disjunctly distributed in the Northern Hemisphere and involved in known and suspected hybridization and allopolyploidy events. The nuclear phylogeny recovered the monophyly of Malus s.l. (including Docynia); however, the genus was supported to be biphyletic in the plastid phylogeny. An ancient chloroplast capture event in the Eocene in western North America best explains the cytonuclear discordance. Our conflict analysis demonstrated that ILS, hybridization, and allopolyploidy could explain the widespread nuclear gene tree discordance. One deep hybridization event (Malus doumeri) and one recent event (Malus coronaria) were detected in Malus s.l. Furthermore, our historical biogeographic analysis integrating living and fossil data supported a widespread East Asian-western North American origin of Malus s.l. in the Eocene, followed by several extinction and dispersal events in the Northern Hemisphere. We also propose a general workflow for assessing phylogenomic discordance and biogeographic analysis using deep genome skimming data sets.     

A molecular phylogenetic study of Cucujidae s.l. (Coleoptera: Cucujoidea)

Of all the superfamilies within the megadiverse order Coleoptera (Insecta), Cucujoidea (Cucujiformia) is arguably the most problematic taxonomically. The families comprising Cucujidae s.l. (Silvanidae, Laemophloeidae, Passandridae and Cucujidae s.s. represent a large portion of cucujoid diversity. Herein we present the results of a rigorous molecular phylogenetic analysis of Cucujidae s.l. using maximum‐likelihood and Bayesian analyses of seven genes. Representatives of over half of the families of Cucujoidea (excluding the cerylonid series), as well as a broad sampling of Silvanidae and Laemophloeidae, were analysed. The monophyly of Cucujidae s.l. is rejected but a subgrouping of taxa that may form the core of a natural cucujoid lineage is recovered. This clade consists of two large monophyletic groups including several families each. Relationships among these smaller cucujoid groups are discussed, including several novel phylogenetic hypotheses, whereas morphological characters considered significant for classification in Cucujidae s.l. are evaluated in light of these phylogenetic hypotheses. Silvaninae, Telephanini, Brontini and Brontinae are recovered as monophyletic in the Bayesian analysis, but the former two are recovered as paraphyletic in the maximum‐likelihood analysis. Our results support the placement of Psammoecus Latreille within Telephanini and also recover a paraphyletic Telephanus Erichson. Silvaninae is divided into three lineages, each representing a potential tribal lineage. Laemophloeidae is rendered paraphyletic in all analyses by Propalticidae and the latter is herein formally transferred to Laemophloeidae stat.n . Several suprageneric laemophloeid clades are recovered and discussed as potential higher‐level groups. Laemophloeus Dejean is not recovered as monophyletic.     

Allopolyploid origin of highly invasive Centaurea stoebe s.l. (Asteraceae)

Spotted knapweed (Centaurea stoebe) occurs from Western Asia to Western Europe both as diploid and tetraploid cytotypes, predominantly in single-cytotype populations with higher frequency of diploid populations. Interestingly, only tetraploids have been recorded so far from its introduced range in North America where they became highly invasive. We performed phylogenetic and network analyses of more than 40 accessions of the C. stoebe and C. paniculata groups and other related taxa using cloned internal transcribed spacer (ITS) and sequences of the chloroplast trnT-trnL and atpBrbcL regions to (i) assess the evolutionary origin of tetraploid C. stoebe s.l., and (ii) uncover the phylogeny of the C. stoebe group. Both issues have not been studied so far and thus remained controversial. Cloned ITS sequences showed the presence of two slightly divergent ribotypes occurring in tetraploid cytotype, while only one major ribotype was present in diploid C. stoebe s.str. This pattern suggests an allopolyploid origin of tetraploids with contribution of the diploid C. stoebe s.str. genome. Although we were not able to detect the second parental taxon, we hypothesize that hybridization might have triggered important changes in morphology and life history traits, which in turn may explain the colonization success of the tetraploid taxon. Bayesian relaxed clock estimations indicate a relatively recent--Pleistocene origin of the tetraploid C. stoebe s.l. Furthermore, our analyses showed a deep split between the C. paniculata and C. stoebe groups, and a young diversification of the taxa within the C. stoebe group. In contrast to nrDNA analyses, the observed pattern based on two cpDNA regions was inconclusive with respect to the origin and phylogeny of the studied taxa, most likely due to shared ancient polymorphism and frequent homoplasies.     

Pleistocene range expansions might explain striking disjunctions between eastern Brazil,Andes and Mesoamerica in Leandra s.str. (Melastomataceae)

Leandra s.str. clade has around 200 species nearly restricted to eastern Brazil. Most species in this group are narrow endemics, but a few present striking disjunct distributions between eastern Brazil and Andes or Mesoamerica. Given the predominantly “montane” distribution observed in most Leandra s.str., we hypothesized that cyclical range expansions during colder Pleistocene periods, followed by local extinctions during warmer interglacial periods, could have shaped the distribution of the disjunct species in this clade. In order to gather support for this biogeographical scenario in a phylogenetic framework, the species that occur outside eastern Brazil were identified, ages of the dispersal events estimated, climatic niche models for the disjuncts were generated, and the climatic envelope of these species compared. Our results place all dispersal events from eastern Brazil to Andes or Mesoamerica during the Pleistocene. Climatic niche modeling indicates a potential range expansion during the Pleistocene colder times for the disjunct species. Although the surpassing of the “dry diagonal” could have been facilitated during glacial periods, this open corridor is an effective barrier for Leandra, given the reduced number of species that dispersed beyond an eastern Brazilian origin. Additionally, the disjunct species do not present significant differences in their climatic envelopes to the non‐disjunct species. Our results provide support to a short‐dispersion/stepping‐stone migration scenario to account for the observed disjunctions in this clade. Range expansions during Pleistocene colder periods followed by local extinctions during interglacial periods could have shaped the distribution of Leandra s.str.     

Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species

Legumes from the genus Pachyrhizus, commonly known as yam bean, are cultivated in several countries from the American continent and constitute an alternative source for sustainable starch, oil and protein production. The endosymbionts of these legumes have been poorly studied although it is known that this legume is nodulated by fast and slow growing rhizobia. In this study we have analyzed a collection of strains isolated in several countries using different phenotypic and molecular methods. The results obtained by SDS-PAGE analysis, LPS profiling and TP-RAPD fingerprinting showed the high diversity of the strains analyzed, although all of them presented slow growth in yeast mannitol agar (YMA) medium. These results were confirmed using 16S-23S internal transcribed spacer (ITS) region and complete sequencing of the 16S rRNA gene, showing that most strains analyzed belong to different species of genus Bradyrhizobium. Three strains were closely related to B. elkanii and the rest of the strains were related to the phylogenetic group constituted by B. japonicum, B. liaoningense, B. yuanmingense and B. betae. These results support that the study of rhizobia nodulating unexplored legumes in different geographical locations will allow the discovery of new species able to establish legume symbioses.