Carmen M. Valiejo-Roman

Towards a molecular phylogeny of Apiaceae subfamily Apioideae: additional information from nuclear ribosomal DNA ITS sequences

Evolutionary relationships among 116 representatives (80 genera) ofApiaceae (Umbelliferae) subfam.Apioideae were investigated by comparative sequencing of the two internal transcribed spacers of the 18S–26S nuclear ribosomal DNA repeat. The resultant phylogenies, inferred using maximum parsimony and neighbor-joining methods, clarified the relationships of several genera whose phylogenetic placements have heretofore been problematic. Comparisons between the phylogenies inferred and the distribution of several phytochemical (coumarins, flavonoids, and phenylpropenes) and morphological (stomates, pollen, and cotyledonary shape) characters were also made, revealing that many of these characters (like those morphological and anatomical characters of the fruit) are highly homoplastic. It is not surprising then that systems of classification ofApioideae based on these characters, particularly with regard to tribal and subtribal designations and relationships, are unsatisfactory. The results of recent serological investigations of the subfamily support several relationships proposed herein using molecular data.

Relationships among genera in Saniculoideae and selected Apioideae (Umbelliferae) inferred from nrITS sequences

The internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were newly sequenced for eight species of Umbelliferae (six species from subfamily Saniculoideae: Actinolema macrolema, Astrantia minor, Eryngium giganteum, E. coeruleum, Hacquetia epipactis, and Lagoecia cum inoides, two species from subfamily Hydrocotyloideae: Dickinsia hydrocotyloides and Azorella trifurcata), as well as Hohenackeria exscapa, a species of uncertain position in the family. Phylogenetic analyses of new data, plus previously reported sequences of 52 other species using neighbor-joining, maximum parsimony, and maxim um likelihood methods yielded similar results: (1) Actinolema is sister to Astrantia corresponding to Drude’s treatments; (2) in Astrantia, molecular divergence is revealed between sects. Astrantiella (A. minor) and Astrantia (A. major, A. maxima); (3) Eryngium appears to be paraphyletic; (4) Hacquetia might be treated as a part of Sanicula; and (5) Lagoecia is very distant from all other Saniculoideae and close to some genera of Apioideae. Our results correspond to matK data previously published: (1) Hohenackeria forms a clade with Bupleurum, in a position near the base of the Apioideae tree; (2) Azorella is sister to a large cluster uniting all Saniculoideae and Apioideae, being slightly closer to them than to the Hydrocotyle-Araliaceae clade; (3) Dickinsia is very distant from phenetically similar Hydrocotyle, falling within a large cluster of Apioideae, but also including Lagoecia and Naufraga.

nrDNA ITS sequences and affinities of Sino-Himalayan Apioideae (Umbelliferae)

The internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were sequenced for 13 species of Apioideae from the Sino-Himalayan flora [Apium ventricosum (= Sium frigidum), Arcuatopterus thalictrioideus, Cyclorhiza peucedanifolia, Meeboldia achilleifolia, Notopterygium forbesii, N. weberbauerianum, Pternopetalum delavayi, P. vulgare, Pterocyclus rivulorum, Sinocarum cruciatum, Sinodielsia delavayi, Tongoloa elata, and Trachydium simplicifolium], relationships of which are controversial or obscure and unresolved on the basis of morphological data. Phylogenetic trees inferred by neighbor-joining, parsimony analysis, and Bayesian inference are topologically congruent, but not identical. The newly obtained data permit testing of several hypotheses regarding these taxa. Some of the early hypotheses treat local species under well-known European genera (e.g., Apium, Carum, Pimpinella, etc.), but these are not supported by phylogenetic analysis of nuclear rDNA spacer sequences. In particular, Sinodielsia, Meeboldia, Tongoloa, and Pternopetalum are distant from Pimpinella; Pternopetalum, Sinocarum and Tongoloa are remote from Carum; Arcuatopterus is separate from Peucedanum and Angelica; Pternopetalum is distinct from Cryptotaenia; and Notopterygium and Pterocyclus are separate from Pleurospermum. Chinese Notopterygium is shown to be closely related to Siberian and Mongolian Hansenia; this, plus similarity in fruit structure, suggests that they are congeneric. Apium ventricosum, more commonly known as Sium frigidum, appears a close relative of Sinocarum. Affinities of most Sino-Himalayan Apioideae, therefore, are found among taxa from the same and adjacent territories. Wide disjunction between presumptively related taxa has been revealed only in Cyclorhiza and Komarovia. Sinodielsia and Meeboldia are also revealed to be clearly distinct genera.

ITS phylogeny of West Asian Heracleum species and related taxa of Umbelliferae–Tordylieae W.D.J.Koch, with notes on evolution of their psbA-trnH sequences

SummaryHeracleum is a large and taxonomically complex genus of the Umbelliferae–Tordylieae. The phylogenetic relationships of West Asian Heracleum species and related taxa were explored using data from sequences of the internal transcribed spacer (ITS1 and ITS2) region of nuclear ribosomal DNA. The data set consists of 56 species, of which 47 were analyzed for the first time; it represents all subdivisions of the genus Heracleum, as well as some representatives of Pastinaca complex. Heracleum was shown to be a polyphyletic genus, as its species fall into two different clades, one of which comprises also Symphyoloma and Mandenovia. Section Pubescentia was confirmed, in contrast to the sections Villosa and Heracleum being polyphyletic. A separate position of the section Wendia was supported. H. marashicum was shown to be a member of a clade comprising Pastinaca and related genera. The sequences of chloroplast psbA-trnH intergenic spacer, the region recently proposed for DNA barcoding in plants, were also analyzed for 33 species, representing all principal clades within Heracleum and its relatives. They have been proven to be very similar and not suitable for DNA barcoding in this group. However, some sequence variation was revealed. This variation could be explained by the combination of such evolutionary events as inversion and duplication. It was shown that these events are rather common in Tordylieae and can occur independently in different lineages. The evolution patterns of psbA-trnH spacer are hypothesized.

Inflorescence and Early Flower Development in Loteae (Leguminosae) in a Phylogenetic and Taxonomic Context

Molecular phylogeny shows that the temperate legume tribe Loteae is close to the mostly tropical Robinieae and monogeneric Sesbanieae, but comparative morphological studies of these groups are limited. Unusual patterns of inflorescence symmetry and calyx development have been described in some Loteae, but taxon sampling was low. We studied these features with scanning electron microscopy in 25 species of Loteae plus in three Robinia species. Phylogenetic trees of Loteae based on nuclear ribosomal ITS sequences and 77 morphological characters are constructed. Our data show that whorled flower arrangement is a synapomorphy of Loteae; joint initiation of the two adaxial sepals is a synapomorphy of a clade containing Hippocrepis, Scorpiurus, and Coronilla; floral buds bent backward early in development are a synapomorphy of Coronilla; bilateral umbel symmetry and the presence of a single whorl of flowers are probably primitive within Loteae. Inflorescences of Robinia show no special similarities with those of Loteae. Developmental data support homologies between sterile bracts in all Loteae. Even if the sterile bract is situated at the top of the peduncle, it is morphologically the first leaf on the peduncle. Monosymmetric umbels of Loteae (including the model legume Lotus japonicus) could be useful for investigating genetic control of symmetry in structures of hierarchic levels higher than flowers.

A comparison of nrDNA ITS and ETS loci for phylogenetic inference in the Umbelliferae: An example from tribe Tordylieae

The Umbelliferae is a large and taxonomically complex family of flowering plants whose phylogenetic relationships, particularly at low taxonomic levels, are generally obscure based on current and widely used molecular markers. Thus, information on the phylogenetic utility of additional molecular markers at these levels is highly favorable. We investigate the utility of nuclear ribosomal DNA (nrDNA) external transcribed spacer (ETS) sequences for phylogenetic inference in Umbelliferae tribe Tordylieae, a group whose relationships have been previously difficult to resolve owing to low sequence variability, and compare the results to those obtained from the nrDNA internal transcribed spacer (ITS) region. We report that the ETS region evolves at a slightly faster rate and has a higher percentage of parsimony informative characters than that of ITS and all chloroplast DNA loci examined to date. The ETS region is a valuable phylogenetic marker in Umbelliferae for low level analysis, especially when used in combination with ITS.

An attempt to clarify taxonomic relationships in ‘‘Verwandtschaftskreis der Gattung Ligusticum’’ (Umbelliferae-Apioideae) by molecular analysis

Relationships among the taxa of Umbelliferae, presumably close to Ligusticum and Selinum were investigated by two independent molecular taxonomic methods. 134 ITS 1-2 sequences were analyzed (29 new and 2 reinvestigated species) and immunochemical comparison of storage seed proteins for 38 species of Apioideae of Ligusticum affinity was performed, eight reference systems (antisera) were used. Both approaches yield similar results, showing the extremely polyphyletic nature of this group and some large genera (Ligusticum s.l., Selinum s.l., Pachypleurum) in the Umbelliferae. The independent status of the genera Magadania, Sphaenolobium, Arafoe, Lomatocarpa, Dimorphosciadium and some other segregates of Ligusticum, Cnidium and Selinum have been confirmed, but Cnidium proved to be unnatural even as currently circumscribed. In the group of East Asian taxa the genera Oreocome, Ligusticopsis, Cortia and Cortiella appeared to be closely related. Haplosphaera was shown to be a genus of Hansenia-Notopterygium group.

Organization of chloroplast psbA-trnH intergenic spacer in dicotyledonous angiosperms of the family umbelliferae

Chloroplast intergenic psbA-trnH spacer has recently become a popular tool in plant molecular phylogenetic studies at low taxonomic level and as suitable for DNA barcoding studies. In present work, we studied the organization of psbA-trnH in the large family Umbelliferae and its potential as a DNA barcode and phylogenetic marker in this family. Organization of the spacer in Umbelliferae is consistent with a general pattern evident for angiosperms. The 5′-region of the spacer situated directly after the psbA gene is more conserved in length compared to the 3′-region, which has greater sequence variation. This pattern can be attributed to the maintenance of the secondary structural elements in the 5′-region of the spacer needed for posttranscriptional regulation of psbA gene expression. In Umbelliferae only, the conserved region contains a duplication of the fragment corresponding to the loop of the stem-loop structure and an independent appearance of identical sequence complementarities (traits) necessary to stabilize the stem-loop structure in different lineages. The 3′-region of the spacer nearest to trnH ranges greatly in size, mainly due to deletions, and the decrease in spacer length is a general trend in the evolution psbA-trnH in Umbelliferae. The features revealed in spacer organization allow us to use it as phylogenetic marker, and indels seem to be more informative for analyses than nucleotide substitutions. However, high conservation among closely related taxa and occurrence of homoplastic inversions in the stem-loop structure limit its application as DNA barcode.

Phylogenetics of Anthyllis (Leguminosae: Papilionoideae: Loteae): partial incongruence between nuclear and plastid markers, a long branch problem and implications for morphological evolution

Phylogenetic relationships in the genus Anthyllis (Leguminosae: Papilionoideae: Loteae) were investigated using data from the nuclear ribosomal internal transcribed spacer regions (ITS) and three plastid regions (psbA-trnH intergenic spacer, petB-petD region and rps16 intron). Bayesian and maximum parsimony (MP) analysis of a concatenated plastid dataset recovered well-resolved trees that are topologically similar, with many clades supported by unique indels. MP and Bayesian analyses of the ITS sequence data recovered trees that have several well-supported topological differences, both among analyses, and to trees inferred from the plastid data. The most substantial of these concerns A. vulneraria and A. lemanniana, whose placement in the parsimony analysis of the ITS data appears to be due to a strong long-branch effect. Analysis of the secondary structure of the ITS1 spacer showed a strong bias towards transitions in A. vulneraria and A. lemanniana, many of which were also characteristic of certain outgroup taxa. This may contribute to the conflicting placement of this clade in the MP tree for the ITS data. Additional conflicts between the plastid and ITS trees were more taxonomically focused. These differences may reflect the occurrence of reticulate evolution between closely related species, including a possible hybrid origin for A. hystrix. The patterns of incongruence between the plastid and the ITS data seem to correlate with taxon ranks. All of our phylogenetic analyses supported the monophyly of Anthyllis (incl. Hymenocarpos). Although they are often taxonomically associated with Anthyllis, the genera Dorycnopsis and Tripodion are shown here to be more closely related to other genera of Loteae. We infer up to six major clades in Anthyllis that are morphologically well-characterized, and which could be recognized as sections. Four of these agree with various morphology-based classifications, while the other two are novel. We reconstruct the evolution of several morphological characteristics found only in Anthyllis or tribe Loteae. Some of these characters support major clades, while others show evidence of homoplasy within Anthyllis.

Pollination of Vietnamese Aspidistra xuansonensis (Asparagaceae) by female Cecidomyiidi flies: Larvae of pollinator feed on fertile pollen in anthers of anthetic bisexual flowers

UNLABELLED • PREMISE OF THE STUDY Aspidistra is a species-rich, herbaceous monocot genus of tropical Southeast Asia. Most species are recently discovered and apparently endangered, though virtually nothing is known about their biology. Species of the genus are primarily distinguished using flower morphology, which is enormously diverse. However, the pollination process has not been directly observed in the center of diversity of the genus (N Vietnam and S China). Indirect and partly direct data on the only widely cultivated species of the genus (A. elatior) placed it among angiosperms with the most unusual pollination biology, though these data are highly controversial, suggesting pollen transfer by mollusks, crustaceans, flies, or possibly tiny soil invertebrates such as collembolans.• METHODS Pollination of Aspidistra xuansonensis in the center of diversity of the genus was studied using visual observations and videos and light and scanning electron microscopy investigation of flowers and their pollinators. Pollinators and their larvae were molecularly barcoded.• KEY RESULTS Aspidistra xuansonensis is pollinated by female cecidomyiid flies (gall midges). They oviposit on anthers, and larvae develop among the pollen mass. Molecular barcoding proved taxonomic identity of the larvae and the flies. The larvae neither damage floral parts nor cause gall formation, but feed on pollen grains by sucking out their content. The larvae move out of the flowers before decomposition starts. Carebara ants steal developing larvae from flowers but do not contribute to pollination.• CONCLUSIONS More than one kind of myiophily is present in Aspidistra. Brood site pollination was documented for the first time in Aspidistra. The pollination system of A. xuansonensis differs from other kinds of brood site pollination in the exit of the larvae prior to the decomposition of floral parts.