Pollen morphology of the tribes Naucleeae and Hymenodictyeae (Rubiaceae – Cinchonoideae) and its phylogenetic significance

The tribe Naucleeae has recently been recircumscribed on the basis of both morphological and molecular [ rbcL , trnT-F , internal transcribed spacer (ITS)] evidence, and has been found to be the sister group of the tribe Hymenodictyeae Razafim. & B. Bremer. In order to find pollen morphological support for this new classification, the pollen and orbicules of 65 species, representing 23 Naucleeae and the two Hymenodictyeae genera, were investigated by scanning electron and light microscopy. Naucleeae pollen is very small (< 20 µm) to small (20–30 µm) and its shape in equatorial view is suboblate to spheroidal or, more rarely, subprolate. Three compound apertures are present, each comprising a long and narrow ectocolpus, a circular to slightly lolongate mesoporus, and an often H-shaped endoaperture. The sexine ornamentation is perforate, rugulate, or (micro)reticulate, and supratectal elements are always absent. Apart from the variation in sexine ornamentation, the tribe is rather stenopalynous. The pollen of Hymenodictyeae is very similar to that of Naucleeae. The H-shaped endoapertures often observed probably form a synapomorphy for the clade comprising Naucleeae and Hymenodictyeae. Our pollen morphological observations are not in conflict with the widened delimitation of Naucleeae. Unambiguous pollen support for the recent subtribal or generic concepts of Naucleeae could not be found because of a lack of variation of pollen characters within the tribe. Orbicules are invariably present in the ten Naucleeae taxa investigated. They are spheroidal and smooth or irregularly folded.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 329–341.     

Re-assessment of monophyly, evolution of myrmecophytism, and rapid radiation in Neonauclea s.s. (Rubiaceae)

The biologically interesting ant-plant association, myrmecophytism, occurs in ca. 140 of the 11,000 species and 22 of the 630 genera of the coffee family (Rubiaceae). These myrmecophytic Rubiaceae species are predominantly distributed in Southeast Asia, especially the Malesian region, with comparatively few species in mainland Africa and the Neotropics. The mostly Southeast Asian genus Neonauclea s.s is one of the three Rubiaceae genera with extensive radiation of myrmecophytes and also the most speciose genus of the tribe Naucleeae s.l. We perform parsimony phylogenetic analyses of Neonauclea s.s., previously resolved as paraphyletic, and its allied genera using both ETS and ITS sequencing data to test: (1) the paraphyly of Neonauclea s.s.; (2) the phylogenetic relationships within the Ludekia-Myrmeconauclea-Neonauclea complex; and (3) the evolution of myrmecophytism within the complex. The earlier proposed paraphyly of Neonauclea s.s. appears to be the result of the combined effects of parallel substitutions in Metadina trichotoma and the sampled ITS putative pseudogenes of Neonauclea longipedunculata and losses of some synapomorphies of Neonauclea s.s. in the latter. The analyses present strong support for the monophyly of Myrmeconauclea and Neonauclea s.s. and their sister-group relationships. Our findings additionally favor the hypothesis of multiple origins of myrmecophytism in the Bornean Neonauclea, which have independently been exploited by at least three Cladomyrma ant species. Furthermore, we interpret the low levels of variation in both the ETS and ITS sequences as indication of a recent and rapid radiation for Neonauclea s.s. (with 65 species) and a recent and slow radiation for Myrmeconauclea (with three species). We argue that the rapid diversification of Neonauclea s.s. is partly associated with the nature of its fruits and its ability to colonize a wide range of habitats. We postulate that both ecological and geographical events may have been responsible for the radiation of the non-myrmecophytic Neonauclea species. Finally, we argue that the acquisition of the pseudo-multiple fruits and long-tailed seeds has allowed Myrmeconauclea to specialize on rheophytic habitats but its narrow ecological tolerance may have hindered its speciation.     

Phylogenetics of Cranichideae with emphasis on Spiranthinae (Orchidaceae, Orchidoideae): evidence from plastid and nuclear DNA sequences

DNA sequences from plastid rbcL and matK genes and the trnL-F region, as well as the nuclear ribosomal ITS region, were used to evaluate monophyly and subtribal delimitation of Cranichideae and generic relationships in Spiranthinae. Cranichideae are moderately supported as monophyletic, with Chloraeinae and Pterostylis-Megastylis indicated as their collective sisters. Within Cranichideae, Pachyplectroninae and Goodyerinae form a well-supported monophyletic group sister to a "core spiranthid" clade that includes, according to their branching order, Galeottiellinae, Manniellinae, and a Prescottiinae-Cranichidinae-Spiranthinae subclade. Inclusion of Galeottiella in Spiranthinae, as in previous classifications, renders the latter paraphyletic to all other spiranthid subtribes. Cranichidinae and Spiranthinae (minus Galeottiella) are monophyletic and strongly supported, but Prescottiinae form a grade that includes a strongly supported prescottioid Andean clade and a weakly supported Prescottia-Cranichidinae clade sister to Spiranthinae. Well-supported major clades in Spiranthinae identified in this study do not correspond to previous alliances or the narrowly defined subtribes in which they have been divided recently. Morphological characters, especially those that have been used for taxonomic delimitation in Cranichideae, are discussed against the framework of the molecular trees, emphasizing putative synapomorphies and problems derived from lack of information or inadequate interpretation of the characters.     

Recent origin and phylogenetic utility of divergent ITS putative pseudogenes: a case study from Naucleeae (Rubiaceae)

The internal transcribed spacer (ITS) of nuclear ribosomal DNA has been widely used by systematists for reconstructing phylogenies of closely related taxa. Although the occurrence of ITS putative pseudogenes is well documented for many groups of animals and plants, the potential utility of these pseudogenes in phylogenetic analyses has often been underestimated or even ignored in part because of deletions that make unambiguous alignment difficult. In addition, long branches often can lead to spurious relationships, particularly in parsimony analyses. We have discovered unusually high levels of ITS polymorphism (up to 30%, 40%, and 14%, respectively) in three tropical tree species of the coffee family (Rubiaceae), Adinauclea fagifolia, Haldina cordifolia, and Mitragyna rubrostipulata. Both secondary structure stability and patterns of nucleotide substitutions in a highly conserved region (5.8S gene) were used for distinguishing presumed functional sequences from putative pseudogenes. The combination of both criteria was the most powerful approach. The sequences from A. fagifolia appear to be a mix of functional genes and highly distinct putative pseudogenes, whereas those from H. cordifolia and M. rubrostipulata were identified as putative pseudogenes. We explored the potential utility of the identified putative pseudogenes in the phylogenetic analyses of Naucleeae sensu lato. Both Bayesian and parsimony trees identified the same monophyletic groups and indicated that the polymorphisms do not transcend species boundaries, implying that they do not predate the divergence of these three species. The resulting trees are similar to those produced by previous analyses of chloroplast genes. In contrast to results of previous studies therefore, divergent putative pseudogenes can be useful for phylogenetic analyses, especially when no sequences of their functional counterparts are available. Our studies clearly show that ITS polymorphism may not necessarily mislead phylogenetic inference. Despite using many different PCR conditions (different primers, higher denaturing temperatures, and absence or presence of DMSO and BSA-TMACl), we recovered only a few functional ITS copies from A. fagifolia and none from H. cordifolia and M. rubrostipulata, which suggests that PCR selection is occurring and/or the presumed functional alleles are located at minor loci (with few ribosomal DNA copies).     

Phylogenetic relationships of Eurasian pines (Pinus, Pinaceae) based on chloroplast rbcL, MATK, RPL20-RPS18 spacer, and TRNV intron sequences

The sequence divergence of chloroplast rbcL, matK, trnV intron, and rpl20-rps18 spacer regions was analyzed among 32 Pinus species and representatives of six other genera in Pinaceae. The total aligned sequence length is 3570 bp. Of the four sequences examined, matK evolved much faster than rbcL in Pinus and in other Pinaceae genera. The two noncoding regions did not show more divergence than the two coding regions, especially within each Pinus subgenus. Phylogenetic analyses based on these four sequences gave consistent results and strongly supported the monophyly hypothesis for the genus Pinus and its two recognized subgenera. Pinus krempfii, the two-flat-needle pine endemic to Vietnam, was placed in subgen. Strobus and showed closer affinity to subsect. Gerardianae. The ancient character of sect. Parrya is further confirmed. However, monophyly of the sect. Parrya is not supported by our data. Among the Eurasian pines of subgen. Pinus, Mediterranean pines formed one clade and the Asian members of subsect. Sylvestres formed another. The Himalayan P. roxburghii showed considerable divergence from all the other hard pines from both regions. Pinus merkusii was distinctly separated from all the Asian members of subsect. Sylvestres. The implications of our results for Pinus classification are discussed.     

Systematics and origin of moths in the subfamily Arctiinae (Lepidoptera,Erebidae) in the Neotropical region

The availability of standard protocols to obtain DNA sequences has allowed the inference of phylogenetic Hypotheses for many taxa, including moths. We here have inferred a phylogeny using maximum‐Likelihood and Bayesian approaches for a species‐rich group of moths (Erebidae, Arctiinae), with strong emphasis on Neotropical genera collected in different field campaigns in the Atlantic Forest of Brazil, eastern Amazon and southern Ecuador. A total of 277 species belonging to 246 genera were included in the analysis. Our main objectives were to shed light on the relationships between suprageneric groups, especially subtribes, and hypothesize colonization events in and out of the Neotropics. The monophyly of Arctiinae and its four tribes (Lithosiini, Amerilini, Syntomini and Arctiini) was recovered in the ML and Bayesian trees. Three Lithosiini subtribes previously found and two additional species groups were recovered monophyletic in both phylogenetic estimation methods. In Arctiini, the monophyly of Spilosomina and Arctiina was highly supported in the ML and Bayesian trees, but the monophyly of Ctenuchina and Echromiina was weakly supported in the ML tree and absent in the Bayesian tree; the remaining subtribes were paraphyletic and, in the case of Phageopterina, formed several species groups. The mapping of species occurrence in our ML tree suggests that Arctiinae have an Old World origin and that the Neotropical region was colonized at least six times independently. Our analysis also suggests that a number of species that occur in Neotropical and other zoogeographic regions may have originated in the Neotropics, although further taxon sampling is required to support this hypothesis. To our knowledge, this is the first time that a highly speciose group of tropical moths is well covered in a phylogeny, and it seems plausible that the results reported here may be extendable to other species‐rich tropical undersampled moth taxa.     

Molecular phylogenetic analysis of Passifloraceae sensu lato (Malpighiales) based on plastid and nuclear DNA sequences

A phylogenetic analysis of Passifloraceae sensu lato was performed using rbcL, atpB, matK, and 18S rDNA sequences from 25 genera and 42 species. Parsimony analyses of combined data sets resulted in a single most parsimonious tree, which was very similar to the 50% majority consensus tree from the Bayesian analysis. All nodes except three were supported by more than 50% bootstrap. The monophyly of Passifloraceae s.l. as well as the former families, Malesherbiaceae, Passifloraceae sensu stricto, and Turneraceae were strongly supported. Passifloraceae s.s. and the Turneraceae are sisters, and form a strongly supported clade. Within Passifloraceae s.s., the tribes Passifloreae and Paropsieae are both monophyletic. The intergeneric relationships within Passifloraceae s.s. and Turneraceae are roughly correlated with previous classification systems. The morphological character of an androgynophore/gynophore is better used for characterizing genera grouping within Passifloraceae s.s. Other morphological characters such as the corona and aril are discussed.     

Higher level phylogeny of Satyrinae butterflies (Lepidoptera: Nymphalidae) based on DNA sequence data

We have inferred the first empirically supported hypothesis of relationships for the cosmopolitan butterfly subfamily Satyrinae. We used 3090 base pairs of DNA from the mitochondrial gene COI and the nuclear genes EF-1alpha and wingless for 165 Satyrinae taxa representing 4 tribes and 15 subtribes, and 26 outgroups, in order to test the monophyly of the subfamily and elucidate phylogenetic relationships of its major lineages. In a combined analysis, the three gene regions supported an almost fully resolved topology, which recovered Satyrinae as polyphyletic, and revealed that the current classification of suprageneric taxa within the subfamily is comprised almost completely of unnatural assemblages. The most noteworthy findings are that Manataria is closely related to Melanitini; Palaeonympha belongs to Euptychiina; Oressinoma, Orsotriaena and Coenonympha group with the Hypocystina; Miller's (1968). Parargina is polyphyletic and its components group with multiple distantly related lineages; and the subtribes Elymniina and Zetherina fall outside the Satyrinae. The three gene regions used in a combined analysis prove to be very effective in resolving relationships of Satyrinae at the subtribal and tribal levels. Further sampling of the taxa closely related to Satyrinae, as well as more extensive sampling of genera within the tribes and subtribes for this group will be critical to test the monophyly of the subfamily and establish a stronger basis for future biogeographical and evolutionary studies.     

Convergent evolution of cucurbitacin feeding in spatially isolated rootworm taxa (Coleoptera: Chrysomelidae; Galerucinae,Luperini)

Historically, chemical ecologists assumed that cucurbitacin feeding and sequestration in rootworm leaf beetles is a remnant of an ancient association between the Luperini (Coleoptera: Chrysomelidae; Galerucinae) and Cucurbitaceae (ancestral host hypothesis). Under this premise, rootworms that do not develop on cucurbits but undergo pharmacophagous forays for cucurbitacins are thought to do so to supplement novel host diets that lack these bitter compounds. The ancestral host hypothesis is supported from studies of pyrrolizidine alkaloid pharmacophagy in Lepidoptera but has not been subjected to phylogenetic analysis within the Luperini. New evidence that this feeding behavior is better correlated with an adult affinity for pollen than with larval host offers the possibility that Old and New World rootworm species with an affinity for cucurbitacins converged on this behavior through apomorphic taste receptor modifications (loose receptor hypothesis). Here we test the monophyly of cucurbitacin feeding within the Luperini by using nuclear and mitochondrial sequence data to infer phylogenetic relationships among 49 taxa representing tribes of the Galerucinae and subtribes of the Luperini. The resulting phylogenetic hypothesis is mostly concordant with existing tribal and subtribal delineations within the Subfamily Galerucinae sensu stricto (Galerucinae not including the flea beetles). The establishment of ancestry among the subtribes of the Luperini refutes the monophyly of cucurbitacin feeding and cucurbit specialization, with the New World Diabroticina being paraphyletic to the Old World Aulacophorina and cosmopolitan Luperina. These data unambiguously support the convergent evolution of cucurbitacin feeding in rootworms and are inconsistent with the ancestral host hypothesis.     

Discordance of chloroplast and nuclear ribosomal DNA data in Osmorhiza (Apiaceae)

Phylogenetic studies were conducted to evaluate interspecific relationships in Osmorhiza (Apiaceae: Apioideae) using sequences of the ITS regions of nuclear ribosomal DNA, the chloroplast ndhF gene, and two noncoding regions (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer). All data sets suggest the monophyly of the New World taxa and showed that Osmorhiza aristata from Asia is relatively divergent from other members of the genus, even though it is morphologically similar to the eastern North American O. claytonii and O. longistylis. The ITS and chloroplast DNA trees differ in the relationships among the New World taxa, especially the phylogenetic position of O. occidentalis, O. glabrata, and O. depauperata. The lack of congruence between the two data sets may be a result of hybridization or introgression. Although there is high discordance between nrITS and two chloroplast DNA data sets, the latter two show similar topologies.     

Subtribal and generic relationships of Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: combined molecular evidence

The monophyly of and phylogenetic relationships within the orchid tribe Maxillarieae Pfitzer were evaluated using parsimony analyses of combined nuclear ribosomal and plastid DNA sequence data of ITS 1 and 2, matK, and the trnL intron and the trnL-F intergene spacer. Each of the separate analyses produced highly congruent but weakly supported patterns (by the bootstrap), so these were combined in a single analysis. Analysis of 90 ingroup taxa (representing ～35% of currently recognized genera) and four outgroup taxa produced resolved and highly supported cladograms. Based on the cladograms, we recognize six subtribes: Eriopsidinae, Oncidiinae (including Pachyphyllinae, Ornithocephalinae, and Telipogoninae), Stanhopeinae, Coeliopsidinae, Maxillariinae (including Lycastinae and Bifrenariinae), and Zygopetalinae (including Cryptarrheninae, Dichaeinae, Huntleyinae, and Warreinae). Stanhopeinae were sampled most intensively; their generic relationships were highly resolved in the analysis and largely agree with currently accepted generic concepts based on morphology. Coeliopsidinae (Coeliopsis, Lycomormium, Peristeria) are sister to Stanhopeinae. Correlations are drawn among phylogeny, pollination mechanisms, and life history traits.     

Molecular systematics of the fern genus Hymenophyllum s.l. (Hymenophyllaceae) based on chloroplastic coding and noncoding regions

We investigated the phylogenetic relationships of the filmy fern genus Hymenophyllum s.l. using the rbcL and rps4 genes and the intergenic spacer rps4-trnS. Because of variation in length of the noncoding marker, we tested and compared three methods for integrating indels. They proved to be useful for estimating a phylogeny of the genus. The rps4-trnS marker, with coded indels integrated, produced better resolution than analysis of either rps4 or rbcL, and combining the three data sets allowed us to obtain a well resolved and strongly supported topology. We interpret our data as showing support for the classical bigeneric system for the family, and call into question several classifications proposed in the past century. The segregate genera Cardiomanes, Hymenoglossum, Serpyllopsis, and Rosenstockia are embedded within Hymenophyllum s.l. Although the deepest relationships within the genus remain uncertain, two subgenera described by Morton do have some support: (1) Sphaerocionium, in which the problematic section Microtrichomanes is embedded; and (2) a diverse Hymenophyllum, including species that were placed originally in Serpyllopsis, Rosenstockia, Hemicyatheon, and Craspedophyllum by Copeland. Subgenus Mecodium appears to be polyphyletic; nevertheless, a subgroup within Mecodium is strongly supported. Several unexpected associations gain support from cytological data and certain morphological characters not previously used to distinguish species groups within Hymenophyllum s.l.     

A phylogenetic analysis of tribe Areceae (Arecaceae) using two low-copy nuclear genes

 A phylogenetic study of the largest tribe of palms, the Areceae, was conducted using sequences of two low-copy nuclear genes. Previous morphological and plastid DNA studies have not supported the monophyly of the tribe, but have placed its members in a large clade that includes the monophyletic tribes Geonomeae, Cocoeae, Podococceae, and Hyophorbeae. We analyzed this large clade to test the monophyly of tribe Areceae with nuclear data, to explore relationships among its subtribes, and to identify other monophyletic groups. For 54 palm species, including members of all 17 subtribes of tribe Areceae, we sequenced regions of the malate synthase (MS) and phosphoribulokinase (PRK) genes. Simultaneous analysis of these regions revealed 52 shortest trees, all of which resolved tribe Areceae as polyphyletic. Subtribes Iguanurinae, Dypsidinae, Oncospermatinae, and Arecinae were also resolved as polyphyletic. A clade of Indo-Pacific taxa was resolved with strong support, and would be a suitable target for more focused study. Received February 7, 2001; accepted April 9, 2002 Published online: December 3, 2002     

Phylogeny and morphological evolution of tribe Menispermeae (Menispermaceae) inferred from chloroplast and nuclear sequences

The Menispermaceae family contains ca. 72 genera with 450 species that are almost entirely tropical. Its phylogeny at the tribal level has never been examined using molecular data. Here we used DNA sequences of the chloroplast matK gene and trnL-F regions, and the nuclear ITS region to study the delimitation and position of the tribe Menispermeae within the family and its subtribal monophyletic groups. Family-wide phylogenetic analyses of the chloroplast data produced two strongly supported clades. The first clade contains two subclades: Coscinieae including Arcangelisia and Anamirta, and Tinosporeae sensu lato including Fibraureae, supported by morphological characters, such as traits of the cotyledon, stylar scar and embryo. The second clade consists of the tribes Menispermeae sensu DC. and Tiliacoreae Miers. All our analyses surprisingly recognized that tribe Menispermeae is not monophyletic unless tribe Tiliacoreae is included, suggesting that characters of cotyledon and stylar scar are very important for the infrafamilial classification, and that endosperm presence vs. absence was over-emphasized in traditionally tribal division of the family. Our topologies indicate a secondary loss of endosperm. The monophyly of two subtribes of the tribe Menispermeae, Stephaniinae and Cissampelinae, is supported by the cpDNA and ITS data, as well as by morphological characters, including aperture types and shapes, and colpal membrane features of pollen grains, and sepal number of male flowers. The Cocculinae was recognized as a paraphyletic group containing the remaining genera of the tribe Menispermeae.