Phylogeny of the saffron-crocus species group, Crocus series Crocus (Iridaceae).

Phylogenetic relationships among the taxa of Crocus series Crocus are still unclear, preventing the understanding of species diversity and the evolution of the important spice saffron (Crocus sativus). Therefore, we analyzed sequences of two chloroplast (trnL-trnF, matK-trnK) and three nuclear (TOPO6, ribosomal DNA ETS and ITS) marker regions to infer phylogenetic relationships among all species belonging to series Crocus. Our phylogenetic analyses resolved the relationships among all taxa of the series. Crocus hadriaticus and the former C. pallasii subspecies appeared polyphyletic. The latter deserve elevating the subspecies to species rank, while for C. hadriaticus a detailed study of species boundaries is necessary. Multi-locus and also genome-wide single nucleotide polymorphism data obtained through genotyping-by-sequencing placed C. sativus within C. cartwrightianus with no indication that other Crocus species contributed to the evolution of the triploid. Our analyses thus made an autotriploid origin of C. sativus from C. cartwrightianus very likely.

Causes of plant diversification in the Cape biodiversity hotspot of South Africa.

The Cape region of South Africa is one of the most remarkable hotspots of biodiversity with a flora comprising more than 9000 plant species, almost 70% of which are endemic, within an area of only ± 90,000 km2. Much of the diversity is due to an exceptionally large contribution of just a few clades that radiated substantially within this region, but little is known about the causes of these radiations. Here, we present a comprehensive analysis of plant diversification, using near complete species-level phylogenies of four major Cape clades (more than 470 species): the genus Protea, a tribe of legumes (Podalyrieae) and two speciose genera within the iris family (Babiana and Moraea), representing three of the seven largest plant families in this biodiversity hotspot. Combining these molecular phylogenetic data with ecological and biogeographical information, we tested key hypotheses that have been proposed to explain the radiation of the Cape flora. Our results show that the radiations started throughout the Oligocene and Miocene and that net diversification rates have remained constant through time at globally moderate rates. Furthermore, using sister-species comparisons to assess the impact of different factors on speciation, we identified soil type shifts as the most important cause of speciation in Babiana, Moraea, and Protea, whereas shifts in fire-survival strategy is the most important factor for Podalyrieae. Contrary to previous findings in other groups, such as orchids, pollination syndromes show a high degree of phylogenetic conservatism, including groups with a large number of specialized pollination syndromes like Moraea. We conclude that the combination of complex environmental conditions together with relative climatic stability promoted high speciation and/or low extinction rates as the most likely scenario leading to present-day patterns of hyperdiversity in the Cape.

New insights into Trimezieae (Iridaceae) phylogeny: what do molecular data tell us?

BACKGROUND AND AIMS: The Neotropical tribe Trimezieae are taxonomically difficult. They are generally characterized by the absence of the features used to delimit their sister group Tigridieae. Delimiting the four genera that make up Trimezieae is also problematic. Previous family-level phylogenetic analyses have not examined the monophyly of the tribe or relationships within it. Reconstructing the phylogeny of Trimezieae will allow us to evaluate the status of the tribe and genera and to examine the suitability of characters traditionally used in their taxonomy. METHODS: Maximum parsimony and Bayesian phylogenetic analyses are presented for 37 species representing all four genera of Trimezieae. Analyses were based on nrITS sequences and a combined plastid dataset. Ancestral character state reconstructions were used to investigate the evolution of ten morphological characters previously considered taxonomically useful. KEY RESULTS: Analyses of nrITS and plastid datasets strongly support the monophyly of Trimezieae and recover four principal clades with varying levels of support; these clades do not correspond to the currently recognized genera. Relationships within the four clades are not consistently resolved, although the conflicting resolutions are not strongly supported in individual analyses. Ancestral character state reconstructions suggest considerable homoplasy, especially in the floral characters used to delimit Pseudotrimezia. CONCLUSIONS: The results strongly support recognition of Trimezieae as a tribe but suggest that both generic- and species-level taxonomy need revision. Further molecular analyses, with increased sampling of taxa and markers, are needed to support any revision. Such analyses will help determine the causes of discordance between the plastid and nuclear data and provide a framework for identifying potential morphological synapomorphies for infra-tribal groups. The results also suggest Trimezieae provide a promising model for evolutionary research.

Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera.

BACKGROUND AND AIMS: Oil-producing flowers related to oil-bee pollination are a major innovation in Neotropical and Mexican Iridaceae. In this study, phylogenetic relationships were investigated among a wide array of New World genera of the tribes Sisyrinchieae, Trimezieae and Tigridieae (Iridaceae: Iridoideae) and the evolution of floral glandular structures, which are predominantly trichomal elaiophores, was examined in relation to the diversification of New World Iridaceae. METHODS: Phylogenetic analyses based on seven molecular markers obtained from 97 species were conducted to produce the first extensive phylogeny of the New World tribes of subfamily Iridoideae. The resulting phylogenetic hypothesis was used to trace the evolutionary history of glandular structures present in the flowers of numerous species in each tribe. Hypotheses of differential diversification rates among lineages were also investigated using both topological and Binary-State Speciation and Extinction methods. KEY RESULTS AND CONCLUSIONS: Floral glandular structures and especially trichomal elaiophores evolved multiple times independently in the American tribes of Iridoideae. The distribution pattern of species displaying glandular trichomes across the phylogeny reveals lability in the pollination system and suggests that these structures may have played a significant role in the diversification of the Iridoideae on the American continent.

The effect of mammalian herbivory on inflorescence architecture in ornithophilous Babiana (Iridaceae): implications for the evolution of a bird perch.

PREMISE OF THE STUDY: The showiness of floral displays is usually explained as an adaptation to attract pollinators. However, selection for less attractive displays imposed by non-pollinating agents, particularly herbivores, may balance pollinator-driven selection for highly visible inflorescences. We investigated whether inflorescence architecture, particularly the unusual ground-level flowering associated with a specialized bird perch in Babiana ringens may have originated, in part, as an adaptive response to mammalian herbivory. METHODS: We measured levels of herbivory by antelope in populations of B. hirsuta, the putative sister species of B. ringens, which possesses the likely ancestral form of inflorescence architecture. To test for position-dependent effects of herbivory on flowers, we compared the herbivory rates and seed production of manipulated inflorescences in a field experiment. We predicted that flowers at the base of inflorescences would suffer less herbivory than those in apical positions. RESULTS: We found herbivore damage to flowers in 50% of naturally occurring B. hirsuta plants. Manipulated inflorescences with only basal flowers, and consequently similar inflorescence architecture to B. ringens, experienced significantly lower herbivory and higher seed set than inflorescences manipulated to have only apical flowers. CONCLUSIONS: Our results are consistent with the hypothesis that position-dependent herbivory on inflorescences could have played a role in the evolution of inflorescence design. More specifically, position-dependent herbivory may have selected for the loss of apical flowers. Position-dependent herbivory may have contributed toward the evolution of a naked inflorescence axis, a structure that characterizes B. ringens and functions as a bird perch facilitating cross-pollination by sunbirds.

Evolution of oil-producing trichomes in Sisyrinchium (Iridaceae): insights from the first comprehensive phylogenetic analysis of the genus.

BACKGROUND AND AIMS: Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined. METHODS: Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species. KEY RESULTS AND CONCLUSIONS: Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.

Genetic melting pot and importance of long-distance dispersal indicated in the Gladiolus imbricatus L. populations in the Polish Carpathians.

The genetic diversity in 11 populations of Gladiolus imbricatus in five mountain ranges, including the Tatra, Pieniny, Gorce, Beskid Niski (Western Carpathians) and Bieszczady Mts (Eastern Carpathians), was studied with inter-simple sequence repeat (ISSR) markers. The species is a perennial plant occurring in open and semi-open sites of anthropogenic origin (meadows and forest margins). We checked a hypothesis on the microrefugial character of the plant populations in the Pieniny Mts, a small calcareous Carpathian range of complicated relief that has never been glaciated. Plant populations in the Tatra and Pieniny Mts had the highest genetic diversity indices, pointing to their long-term persistence. The refugial vs. the non-refugial mountain ranges accounted for a relatively high value of total genetic variation [analysis of molecular variance (AMOVA), 14.12%, p = 0.003]. One of the Pieniny populations was of hybridogenous origin and shared genetic stock with the Tatra population, indicating there is a local genetic melting pot. A weak genetic structuring of populations among particular regions was found (AMOVA, 4.5%, p > 0.05). This could be an effect of the frequent short-distance and sporadic long-distance gene flow. The dispersal of diaspores between the remote populations in the Western Carpathians and Eastern Carpathians could be affected by the historical transportation of flocks of sheep from the Tatra to Bieszczady Mts.

Gradual speciation in a global hotspot of plant diversity.

The speciation process that underlies recent, rapid radiations of plants is controversial, and suggested mechanisms range from pollinator or ecological niche differentiation to allopatry and nonadaptive divergence. Phylogenetic approaches to locating the most appropriate speciation models have been constrained by the low levels of molecular divergence between recently diverged species, which are typical of recent, rapid radiations. In this issue of Molecular Ecology, Rymer et al. (2010) used coalescence analyses of sequence data and genome scans of Amplified Fragment Length Polymorphism (AFLP) loci to demonstrate that in a species complex in the irid genus Gladiolus, a member of the hyper diverse Cape flora of southern Africa, speciation is a gradual process. Older divergences are genetically more differentiated, and show a greater difference in flowering time and floral morphology, than taxa that diverged more recently. There is no evidence of any abrupt events. Gene flow is limited by shifts in flowering time and floral morphology; thus, by pre-zygotic rather than by post-zygotic mechanisms, these evolved together with the occupation of somewhat different habitats. This research gives the first critical insight into how the remarkable diversity in a diversity hotspot could have arisen. More importantly, it demonstrates that the speciation process in recent, rapid radiations is tractable and can be investigated with suitable genetic tools.

Evidence of recent and continuous speciation in a biodiversity hotspot: a population genetic approach in southern African gladioli (Gladiolus; Iridaceae).

There has been much debate over the origin of species diversity in biodiversity hotspots, particularly the rate of speciation over extinction and the geographic mode of speciation. Here, we looked at speciation with varying degrees of sympatry in a biodiversity hotspot, focusing on a distinct morphological clade in the Cape Floristic Region in southern Africa, the Gladiolus carinatus species complex (Iridaceae). We investigate the mechanisms involved in population and species differentiation through a combination of ecological and genomic approaches. We estimated spatial and phenological overlap, differences in floral morphology, genetic isolation and genomic selection. A genetic coalescent analysis estimated that the time of divergence between lineages followed the establishment of available habitat in the Cape littoral plain where these species currently overlap geographically. Marked shifts in flowering time and morphology, which act as barriers to gene flow, have developed to varying degrees over the last 0.3-1.4 million years. An amplified fragment length polymorphism genome scan revealed signatures of divergent and balancing selection, although half of the loci consistently behaved neutrally. Divergent species outliers (1%) and floral morph outliers (3%) represent a small proportion of the genome, but these loci produced clear genetic clusters of species and significant associations with floral traits. These results indicate that the G. carinatus complex represents a continuum of recent speciation. We provide further evidence for ecological adaptation in the face of gene flow.

Foliar epidermal anatomical characteristics of taxa of Iris subg. Scorpiris Spach (Iridaceae) from Turkey.

Iris L. is one of the important genus of family Iridaceae, consist of 56 taxa naturally occurred in Turkey. The similarities and variations in the subgenus overlapping the taxonomic positions of the species in the subgenera and needs anatomical assessment especially by microscopic techniques. In this study, the taxonomic significance of leaf anatomical characters of 10 Iris subgenus Scorpiris taxa were studied in detail and the relationship among these taxa were evaluated using microscopy techniques. Fresh leaf samples of species were fixed in 70% alcohol solution for anatomical observation under microscope. Eleven different micromorphological features were statistically analyzed to delimit the species in subgenus. Based on morphological and anatomical similarities, we studied relationships among; (1) ssp. turcica, ssp. caucasica, I. nezahatiae and I. pseudocaucasica; (2) correlation between ssp. turcica and ssp. caucasica; (3) association of I. galatica, I. persica, ssp. margaretiae and ssp. stenophylla with each other; (4) relationship between ssp. stenophylla and ssp. margaretiae; and (5) relevance between I. aucheri and I. peshmeniana. Moreover, the taxonomy of subgenus Scorpiris has been discussed in detail with novel and diagnostic features based on micromorphological physiognomies. We found that four species in this study are endemic to Turkey, while seven are critically endangered geophytes in the country. The leaf anatomical characteristics of 10 taxa were divided into three groups. Main aim of this research was to study the taxonomy of the complex subgenus Scorpiris through microscopic techniques.

Phylogenetically independent associations between autonomous self-fertilization and plant invasiveness.

Many plant species have been introduced from their native ranges to new continents, but few have become naturalized or, ultimately, invasive. It has been predicted that species that do not require the presence of compatible mates and the services of pollinators for reproduction will be favored in establishment after long-distance dispersal. We tested whether this hypothesis, generally referred to as Baker's law, holds for South African species of Iridaceae (iris family) that have been introduced in other regions for horticultural purposes. Fruit and seed production of flowers from which pollinators had been experimentally excluded was assessed for 10 pairs of species from nine different genera or subgenera. Each species pair comprised one naturalized and one nonnaturalized species, all of which are used in international horticulture. On average, species of Iridaceae that have become naturalized outside their native ranges showed a higher capacity for autonomous fruit and seed production than congeneric species that have not become naturalized. This was especially true for the naturalized species that are considered to be invasive weeds. These results provide strong evidence for the role of autonomous seed production in increasing potential invasiveness in plants.

Nucleotide diversity and phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae.

The plastid genome regions of two intergenic spacers, psbA-trnH and trnL-trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA-trnH region was higher than trnL-trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA-trnH and trnL-trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative's lineage. Present study revealed that psbA-trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL-trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.

The complete chloroplast genome sequences of Iris sanguinea donn ex Hornem.

The complete chloroplast genome of Iris sanguinea was sequenced newly in this study. The total chloroplast genome size of I. sanguinea was 152 408 bp, its structure and gene contents were well conserved as typical chloroplast characteristics. Large single copy (LSC) and small single copy (SSC) of 82 340 bp and 18 016 bp, respectively, were separated from a pair of inverted repeats (IRA and IRB) of 26 026 bp. A total of 112 genes, i.e. 78 protein-coding genes, 30 tRNA genes, and four rRNA genes, were encoded in the chloroplast genome of I. sanguinea. Overall GC content of I. sanguinea was 36.83%. Phylogenetic analysis with the reported chloroplast genomes revealed that I. sanguinea is most closely related to I. gatesii.

[RbcL sequence analysis of Belamcanda chinensis and related medicinal plants of Iris].

AIM: To identify "Shegan" [Belamcanda chinensis (L.) DC.] and relative medicinal plants of Iris including Iris tectorum Maxim., I. dichotoma Pall., I. germanica L. and I. japonica Thunb. by ribulose 1,5-bisphosphate carboxylase Large Gene (rbcL) sequence analysis. METHODS: General DNA was isolated from the fresh leaves of Belamcanda chinensis and 4 Iris spp. by CTAB. A pair of primers was designed to amplify the rbcL gene and PCR Preps DNA kit was used to purify the PCR products. The rbcL sequences were determined by ABI (Applied Biosystems Inco.) Prism 310 Genetic Analyzer. RESULTS: A fragment of about 750 bp of rbcL gene from Belamcanda chinensis and 4 Iris spp. were amplified and sequenced. The rbcL sequences of Iris tectorum, I. dichotoma Pall. and I. japonica were reported for the first time. The rbcL sequences of 5 species of Iridaceae were aligned and analyzed using Clustal (Version 8.0) and MEGA (Version 2.0.) programs. The nucleotide number of difference is from 1.000 to 20.000. The tranversions is from 0.000 to 9.000 and the transitions is from 0.000 to 14.000. Phylogenetic tree based on rbcL partial sequence data indicated that the eleven samples of 5 species clustered separately. CONCLUSION: The sequence variation of rbcL can be used to identify Belamcanda chinensis and 4 species of relative medicinal plants of Iris. The molecular phylogenetic tree accords with the classical taxonomy.

At least 23 genera instead of one: the case of Iris L. s.l. (Iridaceae).

BACKGROUND: Iris L. s.l. is one of the most diverse and well-known genera in the Asparagales, with approximately 250-300 circumscribed species and significant economic impact. The taxonomy of the genus has suffered dramatic changes in the last century, particularly in the last decades after the application of molecular techniques. As a result several contrasting systematic arrangements are currently available to taxonomists. Many genera that were split from Iris s.str. in the past, on the basis of morphology (e.g., Hermodactylus, Iridodictyum, Juno, Pardanthopsis, and Xiphion, among others), are now a priori re-included in a very widely circumscribed Iris s.l. (incl. Belamcanda). This resulted in a more heterogeneous genus that is more difficult to define on morphological grounds. Testing congruence between taxonomic treatments and the results of recent molecular studies of Iris has never been performed, mostly due to the lack of proper taxonomic context. RESULTS: We generated several conventional phylogenies for Iris & outgroups using extensive sampling of taxa (187) and characters (10 plastid loci). We demonstrate that the natural history of Iris, written either as conventional molecular phylogenies or, if viewing in the context of the comparative approach, as a nested most parsimonious hierarchy of patterns, appear to be fully congruent with the narrow taxonomical treatment of the genus, restricted to the rhizomatous "bearded" taxa. The resulting topologies place Belamcanda, Pardanthopsis, and Gattenhofia as sisters to Iris s.str. and genus Siphonostylis as sister to Iris s.l. CONCLUSION: The present study clearly justifies the splitting of Iris s.l. into at least 23 genera, 18 of which have already been accepted in the past by numerous authorities. These genera are characterized by unique combinations of partly overlapping morphological characters and biogeography. Moreover, nearly the same entities, which we here recognize at a generic rank, were for centuries frequently referred to by horticulturists as "working-name" groups.

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus.

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH-psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity-based methods and tree-based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH-psbA. The ITS provided better results with 30.61-38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree-based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.

Radiation in the Cape flora and the phylogeny of peacock irises Moraea (Iridaceae) based on four plastid DNA regions.

Phylogenetic analyses of four plastid DNA regions, the rbcL exon, trnL intron, trnL-trnF intergenic spacer, and rps16 intron from each of 73 species in the African genus Moraea (Iridaceae: Irideae) including accessions of all major species clusters in the genus, show Moraea to be paraphyletic when Barnardiella, Galaxia, Hexaglottis, Homeria (all southern African), and Gynandriris (Eurasian as well) were recognized as separate genera. There are several small, isolated species clusters at the basal nodes of the tree that are all restricted to the winter-rainfall zone of southern Africa (the Greater Cape floral kingdom) and a few, highly derived, large species groups that have radiated extensively within the winter-rainfall zone. Mapping of floral traits shows that an Iris-type flower is ancestral in Moraea. Floral changes are associated with shifts in pollination systems, either from passive pollen deposition on long-tongued bees foraging for nectar to active pollen collection by female bees foraging for pollen, fly, or hopliine scarab beetle pollination. Dating the nodes of the phylogenetic tree using non-parametric rate smoothing with a calibration point derived from broad dating of the angiosperms indicates that the divergence between Moraea and its sister genus Ferraria occurred about 25 mya in the early Miocene. The early radiation of Moraea took place against a background of aridification and the spread of open habitats, such as desert, shrubland, and fynbos.

Phylogeny of Iris based on chloroplast matK gene and trnK intron sequence data.

Phylogenetic analyses of 46 species of Iris, representing all subgenera and all sections except Regelia, Brevituba, and Monolepis, utilized matK gene and trnK intron sequence data. Sequence data show that Iris is paraphyletic because Belamcanda chinensis is resolved within the genus. The two largest subgenera, Iris and Limniris, are both resolved as polyphyletic. With the removal of section Hexapogon, subgenus Iris is weakly supported as monophyletic. Relationships within subgenus Limniris are more complex with the subgenus as currently circumscribed representing eight independent origins among the species included in this study. Several potential monophyletic groups are identified including subgenus Scorpiris, series Spuria (subgenus Limniris section Limniris), and a clade of section Limniris species from North America and Asia.