The age and biogeography of Citrus and the orange subfamily (Rutaceae: Aurantioideae) in Australasia and New Caledonia

The geological history of Australasia, New Caledonia, and Southeast Asia, has been complex, resulting in competing biogeographic hypotheses for taxa found here. Alternative hypotheses-Gondwanan vicariance, rafting terranes, long-distance dispersal-may be distinguished by different predicted divergence times between disjunct sister taxa. Taxa within Rutaceae subfamily Aurantioideae are ideal for testing these hypotheses because of their distributions. Therefore, the ages of Rutaceae and Aurantioideae were estimated using molecular dating. One data set comprised 51 sequences of rbcL and atpB with sampling across rosids and three fossil calibrations: crown Fabales+Fagales+Rosales (>94 Ma), Fabaceae (>51 Ma) and stem Ailanthus, Simaroubaceae (>52 Ma). Another data set comprised 81 Aurantioideae using >8 kb of chloroplast sequence and secondary calibration. Confidence in estimated divergence times was explored by varying the root age, dating method (strict, local, and relaxed clocks), and inclusion of internal calibrations. We conclude that the Rutaceae crown diverged in the Eocene (36.4-56.8 Ma, mean 47.6), whereas the Aurantioideae crown originated in the early Miocene (12.1-28.2 Ma, mean 19.8). This young age suggests that Gondwanan vicariance does not explain the distributions of extant Aurantioideae. Taxa found in New Caledonia may have arrived by separate transoceanic dispersal events.     

Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq

The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian “true citrus fruit trees” (genera Citrus, Poncirus, and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.     

Molecular phylogeny of Vincetoxicum (Apocynaceae-Asclepiadoideae) based on the nucleotide sequences of cpDNA and nrDNA

Molecular phylogenetic analyses of Vincetoxicum and Tylophora (Apocynaceae-Asclepiadoideae) were conducted based on the nucleotide sequences of cpDNA (two intergenic spacers of trnL (UAA)-trnF (GAA) and psbA-trnH and three introns, i.e., atpF, trnG (UCC) and trnL (UAA)), and nrDNA (ITS and ETS regions). Our phylogenetic analysis revealed two monophyletic groups; one consisted of seven taxa of Tylophora and Vincetoxicum inamoenum, Vincetoxicum magnificum and Vincetoxicum macrophyllum (Clade I) and the other consisted of 17 accessions of Vincetoxicum (Clade II). The monophyly of the genus Vincetoxicum was not supported. Although many nucleotide substitutions were observed in Clade I, the genetic differentiation within Clade II was small. Low genetic diversification but considerable morphological divergence suggests that the species in Clade II had undergone rapid diversification. Although most species in Clade I have tiny flowers, those in Clade II have larger and more nectariferous ones. Thus, we hypothesized that the rapid morphological radiation in Clade II may have been due to the gaining of floral characters such as large flowers and large amounts of nectar corresponding to diverse pollinators.     

A combined molecular approach to phylogeny of the jumping spider subfamily dendryphantinae (araneae: salticidae)

Four gene regions were sequenced for 30 species of jumping spiders, most from the subfamily Dendryphantinae, to investigate their molecular phylogeny and evolution. These are three regions from the mitochondria (ca. 560 bp of 16S plus adjacent tRNA, 1047 bp of cytochrome oxidase 1 (CO1), and 414 bp of NADH1 (ND1) and one region from the nuclear genome (ca. 750 bp of 28S). Parsimony and likelihood analyses of these gene regions separately and together support the monophyly of the dendryphantines as delimited previously by morphological characters. A group of elongate-bodied genera are placed as basal among the dendryphantines, and previously proposed relationships of Poultonella, Paraphidippus, and Sassacus vitis are confirmed. Comparison of overall rates of molecular evolution indicates striking differences across the gene regions, with highest divergence in ND1, CO1, 16S, and 28S in decreasing order. All four regions are characterized by both within- and among-site rate variation. Phylogenetic results from CO1 conflict conspicuously with phylogenetic results from the other genes and morphological data. Attempts to account for potential sources of this conflict (e.g., accommodating biased base composition, high homoplasy, within- and among-site rate variation, etc.) are largely unsuccessful.     

Efficient search for new resistant genotypes to the citrus tristeza closterovirus in the orange subfamily Aurantioideae

 Virulent isolates of the citrus tristeza virus (CTV) are continuously arising and their spread threatens the world citrus industry. Methods for effective utilization of material conserved in germplasm banks are needed in plant improvement. Two objectives are pursued in the present paper: a search for new CTV-resistant genotypes and tests of two strategies for this search. One of these tests is based on a study of genetic relationships among genera and species of the orange subfamily and the other on scores of molecular markers known to be linked to the CTV-resistant locus. Sampled plants were graft-inoculated with a mild CTV isolate (T-346) and two virulent ones (T-388 and T-305). Susceptible plants were those where CTV multiplication was detected beyond 4 months after inoculation. All cultivars of Poncirus trifoliata tested, as well as Severinia buxifolia and Atalantia ceylanica, were resistant to the three CTV isolates; Fortunella crassifolia (Meiwa kumquat) resists two of them. The finding of CTV resistance in this species, closely related to cultivated Citrus species, opens a new arena for CTV-resistance improvement of oranges and mandarines by sexual hybridization. The searching strategy based on phylogenetic data has been successful, whereas the other one may be worthwhile only when the search is restricted to the species where linkage analysis is available. A good documentation system that allows quick sampling of accessions to build up core collections and where the location of new and useful genes could be easily worked out, is suggested to enhance germplasm utilization. Received: 27 April 1997 / Accepted: 5 June 1997     

Genetic diversity in the orange subfamily Aurantioideae. I. Intraspecies and intragenus genetic variability

Despite the great economic importance of citrus, its phylogeny and taxonomy remain a matter of controversy. Moreover pathogens of increased virulence and dramatic environmental changes are currently spreading or emerging. The objectives of the present paper, measuring genetic variability and studying its pattern of distribution, are crucial steps to optimize sampling strategies in the search of genotypes that tolerate or resist these threatening factors within the huge array of Citrus and Citrus related species. Their intraspecific and intrageneric variability was studied comparatively by means of ten enzymatic systems using eight different measures. The analysis of ten enzymatic systems allowed us to distinguish all the species and all but one artificial hybrid. The species with the lowest genotypic variability are C. myrtifolia, C. deliciosa (willow leaf mandarin), C. paradisi (grapefruit), C. limon (lemon) and C. sinensis (sweet orange), while Severinia buxifolia shows the highest value. A broad spectrum of heterozygosity values was found in the collection. Lemons (C. limon, C. meyeri, C. karna, C. volkameriana), limes (C. aurantifolia, C. limettioides, C. lattifolia) and C. bergamia show a very high percentage of heterozygosity which indicates an origin through interspecific hybridization. Two main factors limit genetic intraspecific variability: apomictic reproduction, where nucellar embryos are much more vigorous than the zygotic ones, and nurserymen selecting against variability in the seedling stage of the rootstocks or in propagating the scion cultivars vegetatively. Additionally, self-pollination appears in some species mainly used as rootstocks which would explain their low heterozygosity values. Genetic differences between species and genera are in general high, which suggests that adaptation might have played an important role during the evolution of the orange subfamily.     

Genetic diversity in the orange subfamily Aurantioideae. II. Genetic relationships among genera and species

Genetic relationships were studied by means of ten isoenzymatic systems, at the genus and species level, using two distances and four methods of aggregation in a germplasm collection of 198 cultivars and accessions of 54 species belonging to Citrus and 13 related genera. The most consistent results were obtained by the chord distance and the neighbor-joining clustering method. Citrus species were distributed in two main groups: the orange-mandarin group and the lime lemon-citron-pummelo group. The species C. halimii and C. tachibana are not included in these groups. Mandarin species fall into three main subgroups: one includes C. sinensis; the second, C. aurantium, the third, small-fruit species. The citron, the pummelo and the ancient lemon subgroups form a cluster to which the species belonging to subgenus Papeda and the cultivated limes, lemons and bergamots are related. Microcitrus spp, to which Severinia buxifolia and Atalantia ceylanica seem to be related, cluster with the lime lemon-citron-pummelo group while Fortunella is close to the orange-mandarin group. Poncirus trifoliata, the most important species for citrus rootstock improvement is located far from Citrus but connected to it through Fortunella spp. A broad distribution of species has been found that should be taken into account to sample new genotypes in the search of desired characters in order to fully and efficiently use genetic resources for citrus improvement.     

A molecular framework for the phylogeny of the ant subfamily dolichoderinae

Partial sequences are reported for the mitochondrial genes for cytochrome oxidase subunits 2 and 3 and for cytochrome b, and the entire sequence of the gene for tRNA(Leu)(UUR) for species from 14 genera of dolichoderine ants and from three outgroup genera. Considerable variation was observed between tRNA genes in the size of the TPsiC arm and the DHU and anticodon loops and whether or not the TPsiC stem possesses a GC pair. The outgroup taxa showed complete TAA CO1 stop codons, but dolichoderines have either TA or T. The outgroup taxa showed a noncoding gap between the CO1 and the tRNA(Leu)(UUR) genes. A phylogeny-independent compatibility test using the amino acid sequences showed differences between the genes consistent with variation in evolutionary rates, according with other studies. Base compositions proved heterogeneous between species, hence phylogenetic analysis was restricted to the protein sequences using maximum likelihood and the mtREV24 replacement matrix. A maximum-likelihood consensus tree has similarities to those from morphological studies with some exceptions such Leptomyrmex falling within the dolichoderine genera rather than basally, and the accretion of genera formerly included under Iridomyrmex. Features of the tRNA genes and the CO1 termination codons agree quite well with the molecular phylogeny.     

Molecular phylogeny of the subfamily Tephritinae (Diptera: Tephritidae) based on mitochondrial 16S rDNA sequences

The phylogeny of the subfamily Tephritinae (Diptera: Tephritidae) was reconstructed from mitochondrial 16S ribosomal RNA gene sequences using 53 species representing 11 currently recognized tribes of the Tephritinae and 10 outgroup species. The minimum evolution and Bayesian trees suggested the following phylogenetic relationships: (1) monophyly of the Tephritinae was strongly supported; (2) a sister group relationship between the Tephritinae and Plioreocepta was supported by the Bayesian tree; (3) the tribes Tephrellini, Myopitini, and Terelliini (excluding Neaspilota) were supported as monophyletic groups; (4) the non-monophyletic nature of the tribes Dithrycini, Eutretini, Noeetini, Tephritini, Cecidocharini, and Xyphosiini; and (5) recognition of 10 putative tribal groups, most of which were supported strongly by the statistical tests of the interior branches. Our results, therefore, convincingly suggest that an extensive rearrangement of the tribal classification of the Tephritinae is necessary. Since our sampling of taxa heavily relied on the current accepted classification, some lineages identified by the present study were severely under-sampled and other possible major lineages of the Tephritinae were probably not even represented in our dataset. We believe that our results provide baseline information for a more rigorous sampling of additional taxa representing all possible major lineages of the subfamily, which is essential for a comprehensive revision of the tephritine tribal classification.     

A molecular phylogeny of the grass subfamily Panicoideae (Poaceae) shows multiple origins of C4 photosynthesis

DNA sequence data from the chloroplast gene ndhF were analyzed to estimate the phylogeny of the subfamily Panicoideae, with emphasis on the tribe Paniceae. Our data suggest that the subfamily is divided into three strongly supported clades, corresponding to groups with largely identical base chromosome numbers. Relationships among the three clades are unclear. In unweighted parsimony analyses, the two major clades with x = 10 (Andropogoneae and x = 10 Paniceae) are weakly supported as sister taxa. The third large clade corresponds to x = 9 Paniceae. In analyses under implied weight, the two clades of Paniceae are sisters, making the tribe monophyletic. Neither resolution is strongly supported.Our molecular phylogenies are not congruent with previous classifications of tribes or subtribes. Based on this sample of species, we infer that C(4) photosynthesis has evolved independently several times, although a single origin with multiple reversals and several reacquisitions is only slightly less parsimonious. The phosphoenol pyruvate carboxykinase (PCK) subtype of C(4) photosynthesis has evolved only once, as has the NAD-malic enzyme (ME) subtype; all other origins are NADP-ME. Inflorescence bristles are apparently homologous in the genera Setaria and Pennisetum, contrary to opinions of most previous authors. Some genera, such as Digitaria, Echinochloa, and Homolepis are supported as monophyletic. The large genus Paspalum is shown to be paraphyletic, with Thrasya derived from within it. As expected, Panicum is polyphyletic, with lineages derived from multiple ancestors across the tree. Panicum subg. Panicum is monophyletic. Panicum subg. Dichanthelium, subg. Agrostoides, and subg. Phanopyrum are unrelated to each other, and none is monophyletic. Only Panicum subg. Dichanthelium sect. Dichanthelium, represented by P. sabulorum and P. koolauense, is monophyletic. Panicum subg. Megathyrsus, a monotypic subgenus including only the species P. maximum, is better placed in Urochloa, as suggested by other authors.     

A molecular phylogeny of celtidaceae and ulmaceae (Urticales) based onrbcL Nucleotide sequences

On the basis of 1,290 bp sequences of the chloroplast generbcL, a molecular phylogeny of seven of nine genera of the Celtidaceae and four of six genera of the Ulmaceae was produced. These data were analyzed together with some other urticalean genera using three methods (i.e., maximum parsimony, maximum likelihood, and neighbor joining methods). Maximum likelihood topology among 18 trees obtained indicated that the Urticales are monophyletic with its common clade splitting basally into two: one leading to a line comprisingAmpelocera (traditionally placed in Celtidaceae) and Ulmaceae, and the other leading to a line comprising the remaining genera of Celtidaceae, Moraceae, and other Urticales. Ulmaceae, to whichAmpelocera is a sister group, are monophyletic, as supported by many lines of morphological evidence. In contrast to Ulmaceae, the monophyly of Celtidaceae (excludingAmpelocera) was not supported, and resolution of relationships of Celtidaceae with other Urticales, as well as of those within the family, is left for future study.     

A molecular phylogeny of the groupers of the subfamily Epinephelinae (Serranidae) with a revised classification of the Epinephelini

The phylogenetic relationships among the fishes in the perciform tribe Epinephelini (Serranidae) have long been poorly understood, in large part because of the numerous taxa that must be considered and the large, circumtropical distribution of the group. In this study, genetic data from two nuclear (Tmo-4C4 and histone H3) and two mitochondrial (16S and 12S) genes were gathered from 155 serranid and acanthomorph species as a means of developing a phylogenetic hypothesis using both maximum-likelihood and -parsimony criteria. The maximum-parsimony analysis recovered 675 most parsimonious trees of length 5703 steps (CI = 0.2523, HI = 0.7477, RI = 0.6582), and the maximum-likelihood analysis recovered 1 tree at −lnLikelihood = 28279.58341. These phylogenetic hypotheses are discussed in light of previous morphological evidence to evaluate the evolutionary history of the group and their implications for the currently recognized taxonomy. Our results question the monophyly of the Serranidae, as well as the genera Cephalopholis, Epinephelus, and Mycteroperca as currently defined. The Serranidae is monophyletic only with the exclusion of the genera Acanthistius and Niphon. We propose a revised classification of the tribe Epinephelini that reflects the hypothesized shared ancestry of the group and recognizes 11 genera: Alphestes, Cephalopholis, Dermatolepis, Epinephelus, Gonioplectrus, Hyporthodus (which is resurrected for 11 species of deep-bodied groupers), Mycteroperca (including 7 species heretofore allocated to Epinephelus), Plectropomus, Saloptia, Triso, and Variola.     

A molecular phylogeny of the cotingas (Aves: Cotingidae)

The phylogenetic relationships of members of Cotingidae were investigated using >2100 bp of sequence data from two nuclear introns (myoglobin intron 2 and G3PDH intron 11) and one protein-coding mitochondrial gene (cytochrome b). Strong support was found for a monophyletic clade including 23 traditional cotingid genera, corresponding to the Cotingidae sensu [Remsen, J.V. Jr., Jaramillo, A., Nores, M., Pacheco, J.F., Robbins, M.B., Schulenberg, T.S., Stiles, F.G., da Silva, J.M.C., Stotz, D.F., Zimmer, K.J., 2005. Version 2005-11-15. A classification of the bird species of South America. American Ornithologists' Union. ]. Neither Oxyruncus nor any of the genera in Tityrinae sensu [Prum, R.O, Lanyon, W.E., 1989. Monophyly and phylogeny of the Schiffornis group (Tyrannoidea). Condor 91, 444-461.] are members of Cotingidae. Within Cotingidae a polytomy of four well-supported clades was recovered: (1) the fruiteaters Pipreola and Ampelioides; (2) the Ampelion group, including Phytotoma; (3) Rupicola and Phoenicircus; and (4) the 'core cotingas' consisting of the remainder of the Cotingas (e.g. fruitcrows, Cotinga, Procnias, Lipaugus, and Carpodectes), with Snowornis in a basal position. The separation of Snowornis from Lipaugus [Prum, R.O, Lanyon, W.E., 1989. Monophyly and phylogeny of the Schiffornis group (Tyrannoidea). Condor 91, 444-461.] was strongly supported, as were the close relationships between Gymnoderus and Conioptilon, and between Tijuca and Lipaugus. However, basal relationships among 'core cotinga' clades were not resolved.     

A molecular phylogeny of the Chalcidoidea (Hymenoptera)

Chalcidoidea (Hymenoptera) are extremely diverse with more than 23,000 species described and over 500,000 species estimated to exist. This is the first comprehensive phylogenetic analysis of the superfamily based on a molecular analysis of 18S and 28S ribosomal gene regions for 19 families, 72 subfamilies, 343 genera and 649 species. The 56 outgroups are comprised of Ceraphronoidea and most proctotrupomorph families, including Mymarommatidae. Data alignment and the impact of ambiguous regions are explored using a secondary structure analysis and automated (MAFFT) alignments of the core and pairing regions and regions of ambiguous alignment. Both likelihood and parsimony approaches are used to analyze the data. Overall there is no impact of alignment method, and few but substantial differences between likelihood and parsimony approaches. Monophyly of Chalcidoidea and a sister group relationship between Mymaridae and the remaining Chalcidoidea is strongly supported in all analyses. Either Mymarommatoidea or Diaprioidea are the sister group of Chalcidoidea depending on the analysis. Likelihood analyses place Rotoitidae as the sister group of the remaining Chalcidoidea after Mymaridae, whereas parsimony nests them within Chalcidoidea. Some traditional family groups are supported as monophyletic (Agaonidae, Eucharitidae, Encyrtidae, Eulophidae, Leucospidae, Mymaridae, Ormyridae, Signiphoridae, Tanaostigmatidae and Trichogrammatidae). Several other families are paraphyletic (Perilampidae) or polyphyletic (Aphelinidae, Chalcididae, Eupelmidae, Eurytomidae, Pteromalidae, Tetracampidae and Torymidae). Evolutionary scenarios discussed for Chalcidoidea include the evolution of phytophagy, egg parasitism, sternorrhynchan parasitism, hypermetamorphic development and heteronomy.     

A molecular phylogeny of Heterodonta (Bivalvia) based on small ribosomal subunit RNA sequences

Within Heterodonta, phylogenesis has so far been studied almost exclusively on the basis of morphological data. Results have often been discordant, and an exhaustive molecular approach has not yet been attempted. The present study was undertaken to clarify the phylogenetic relationships obtaining among Heterodonta families through the analysis of 18S rRNA gene. To do this, the whole sequence of this gene was analyzed in 29 species of eight superfamilies of the order of Veneroida (Arcticoidea, Cardioidea, Galeommatoidea, Mactroidea, Solenoidea, Tellinoidea, Tridacnoidea, and Veneroidea) and in two superfamilies of Myoida (Pholaloidea and Myoidea). The study was extended by constructing phylogenetic trees using partial sequences. This strategy made it possible to include 11 additional species by introducing three further superfamilies: Chamoidea, Corbiculoidea, and Hiatellinoidea. At variance with the conclusions reached on the basis of morphological features, the molecular data clearly show that the Myoida species included in this study belong to Veneroida, thus undermining the legitimacy of the division of Heterodonta into two orders, and that considerable differences in the phylogenetic relationships obtain among superfamilies.     

A molecular phylogeny of the genus Gammarus (Crustacea: Amphipoda) based on mitochondrial and nuclear gene sequences

A phylogeny of the genus Gammarus Fabricius, 1775 was constructed using DNA sequence data from the mitochondrial genes COI and 16S, and the nuclear genes 18S and 28S. Both parsimony and Bayesian analyses were conducted on separate and combined data partitions. The Bayesian phylogeny from the combined analysis was selected as the preferred phylogenetic hypothesis. The hypothesis supports monophyly of the genus Gammarus, paraphyly of the European-North American Gammarus, and monophyly of the Asian Gammarus. The Asian clade was further split into a southeastern group and a northwestern group. The dramatic climate change following the uplift of the Tibetan Plateau was probably the most important factor in triggering the diversification of southeastern and northwestern groups. The genus Sinogammarus is invalid and should be part of the genus Gammarus.     

Parsimony analysis of cpDNA restriction site variation in subfamily Nepetoideae (Labiatae)

Parsimony analysis of cpDNA restriction site variation supports monophyly of subfamily Nepetoideae. However, a close relationship among Nepetoideae and other gynobasic-styled Labiatae is not supported, indicating that a gynobasic style has evolved independently in at least two clades of Labiatae. The inferred relationships are congruent with the classification of Cantino, Harley, and Wagstaff (1992, Advances in labiate sciences, 27–37, Royal Botanic Gardens, Kew) but conflict to varying degrees with traditional classifications. Monophyly of four tribes of Nepetoideae also is supported.     

A molecular phylogeny of New Zealand's Petroica (Aves: Petroicidae) species based on mitochondrial DNA sequences

The New Zealand robin (Petroica australis), tomtit (P. macrocephala), and Chatham Island black robin (P. traversi) are members of the Petroicidae family of Australo-Papuan robins, found throughout Australasia and the western Pacific. In the nearly 200 years since the New Zealand members of Petroicidae were first described, the division of species, subspecies, and even genera has undergone many changes. In this study, we investigate whether molecular phylogenies based on mitochondrial DNA sequences support current taxonomic classifications based on morphology. Petroica traversi, P. australis, and P. macrocephala form distinct clades in phylogenetic trees constructed from Cytochrome b and control region sequences, however the position of the black robin is at odds with the morphological and behavioral data. The black robin does not appear to be a derivative of the New Zealand robin, instead it groups strongly with the tomtit, indicating that lineage sorting and/or introgressive hybridization may have occurred. There is some evidence to support the hypothesis that two invasions of Petroica from Australia have occurred, however additional data from Australian Petroica taxa are required to confirm this. Control region sequences confirm a deep split between the North and South Island P. australis lineages, but suggest a recent radiation of P. macrocephala.