Molecular phylogenetics of the hummingbird genus Coeligena

Advances in the understanding of biological radiations along tropical mountains depend on the knowledge of phylogenetic relationships among species. Here we present a species-level molecular phylogeny based on a multilocus dataset for the Andean hummingbird genus Coeligena. We compare this phylogeny to previous hypotheses of evolutionary relationships and use it as a framework to understand patterns in the evolution of sexual dichromatism and in the biogeography of speciation within the Andes. Previous phylogenetic hypotheses based mostly on similarities in coloration conflicted with our molecular phylogeny, emphasizing the unreliability of color characters for phylogenetic inference. Two major clades, one monochromatic and the other dichromatic, were found in Coeligena. Closely related species were either allopatric or parapatric on opposite mountain slopes. No sister lineages replaced each other along an elevational gradient. Our results indicate the importance of geographic isolation for speciation in this group and the potential interaction between isolation and sexual selection to promote diversification.     

Molecular phylogenetic analyses of nuclear and plastid DNA sequences support dysploid and polyploid chromosome number changes and reticulate evolution in the diversification of Melampodium (Millerieae, Asteraceae)

Chromosome evolution (including polyploidy, dysploidy, and structural changes) as well as hybridization and introgression are recognized as important aspects in plant speciation. A suitable group for investigating the evolutionary role of chromosome number changes and reticulation is the medium-sized genus Melampodium (Millerieae, Asteraceae), which contains several chromosome base numbers (x = 9, 10, 11, 12, 14) and a number of polyploid species, including putative allopolyploids. A molecular phylogenetic analysis employing both nuclear (ITS) and plastid (matK) DNA sequences, and including all species of the genus, suggests that chromosome base numbers are predictive of evolutionary lineages within Melampodium. Dysploidy, therefore, has clearly been important during evolution of the group. Reticulate evolution is evident with allopolyploids, which prevail over autopolyploids and several of which are confirmed here for the first time, and also (but less often) on the diploid level. Within sect. Melampodium, the complex pattern of bifurcating phylogenetic structure among diploid taxa overlain by reticulate relationships from allopolyploids has non-trivial implications for intrasectional classification.     

Multi-locus phylogeny clarifies the systematics of the Australo-Papuan robins (Family Petroicidae, Passeriformes)

The Australo-Papuan family Petroicidae (Aves: Passeriformes) has been the focus of much systematic debate about its relationships with other passerine families, as well as relationships within the family. Mostly conservative morphology within the group limits the effectiveness of traditional taxonomic analyses and has contributed to ongoing systematic debate. To assess relationships within the family, we sampled 47 individuals from 26 species, representing the majority of genera and species, for four loci: 528 base pairs (bp) of C-myc, 501 bp of BA20454 and 336 bp of BA23989 from nuclear DNA and 1005 bp of the mitochondrial ND2 gene. There was consensus between individual loci and overall support for major lineages was strong. Partitioned Bayesian analyses of all four loci produced a fully resolved and very well-supported phylogeny that addresses many of the previous systematic debates in this group. The Eopsaltriinae as construed is monophyletic with the exception of Eopsaltria flaviventris, which is nested within Microeca as an unremarkable member of that genus. This relationship is corroborated by morphology and egg color and pattern. Petroicinae as currently construed was not monophyletic and comprised two lineages that are paraphyletic with respect to each other. The third subfamily, Drymodinae, remains incertae sedis. The mangrove robin, Peneonanthe pulverulenta, of tropical Australia and New Guinea is nested within a clade that also contained the sampled species of Peneothello and Melanodryas, a novel relationship. Preliminary biogeographic and divergence time estimates from these results are discussed and a new subfamily arrangement proposed.     

Genetic differentiation,polyploidization and hybridization in northern EuropeanDactylorhiza (Orchidaceae): Evidence from allozyme markers

Taxa endemic to North-western Europe are rare, but the orchid genusDactylorhiza contains several species restricted to this area. Evidence from morphological and cytological studies have indicated that some species may have arisen recently and may be of hybrid origin. In the present report, I use allozymes to characterize the genomes in various species ofDactylorhiza and evaluate the possibilities for rapid evolutionary change in the genus. Allotetraploid species have evolved repeatedly from two principal diploid ancestral lineages. These lineages include extant diploid and autotetraploid species, from which allotetraploid derivatives may still arise. It is suggested that allotetraploidization dominates over introgression as speciation mechanism in the genus. The more common and widespread allotetraploid species could be characterized by their allozyme characters over considerable distances, indicating that each of them may have a unique origin and that they have spread from their ancestral populations to the present distribution areas. However, it is also possible that some allotetraploid species contain local populations that have been independently derived from the ancestral lineages.     

Examining the phylogeny of the Australasian Lymnaeidae (Heterobranchia: Pulmonata: Gastropoda) using mitochondrial, nuclear and morphological markers

We examined the species groups relationships of the freshwater snail genus Austropeplea using mitochondrial, nuclear and morphological markers in addition to traditional methods of shell shape analysis. Based primarily on the results of a combined molecular and morphological analysis, samples of the nominal species A. tomentosa form distinct lineages. The New Zealand populations of A. tomentosa are a very distinct lineage from any of the Australian populations attributed to A. tomentosa. Furthermore, within the Australian group, three lineages, south Australia, Tasmania and eastern Australia, appear to have undergone recent and/or rapid speciation events. Samples assigned to A. lessoni were resolved as two distinct lineages, representing the eastern and northern Australian populations. Kutikina hispida was resolved within the Australian A. tomentosa clade. Molecular results for A. viridis suggests that it is also composed of at least two distinct lineages that could be treated as species. Incongruence observed between the single mitochondrial, nuclear and morphological topologies highlight the importance of using a number of different datasets in the delimitation of species-group taxa.     

Chloroplast DNA restriction site variation in theVernonieae (Asteraceae), an initial appraisal of the relationship of New and Old World taxa and the monophyly ofVernonia

Chloroplast DNA restriction site variation was examined for 35 taxa in theVernonieae and four outgroup tribes, using 17 restriction enzymes mapped for ca. 900 restriction sites per species; 139 mutations were found to be phylogenetically informative. Phylogenetic trees were constructed using Wagner and weighted parsimony, and evaluated by bootstrap and decay analyses. Relationships of Old and New World taxa indicate complex geographical relationships; there was no clear geographic separation by hemisphere. The relationships between Old and New World Vernonias found here support prior morphological analyses. The sister group to all New and most Old World taxa was composed of a small group of Old World species including yellow-flowered, trinervate-leaved species previously postulated to be basal in the tribe. The majority of both New and Old World taxa are derived from a lineage beginning with the monotypic genusStokesia, an endemic of the southeastern United States. The genusVernonia was also found to be paraphyletic within both the New and Old World. Available data do not support either the separation ofVernonia or the tribeVernonieae into geographically distinct lineages. The pattern of relationships within theVernonieae for taxa from North America, Asia, Africa, Central and South America is most similar to that of several other groups of both plants and animals with a boreotropical origin, rather than an origin in Gondwanaland. Such a pattern of distribution suggests more ancient vicariant events than are routinely postulated for theAsteraceae.     

Molecular phylogeny ofIva (Asteraceae,Heliantheae) based on chloroplast DNA restriction site variation

Iva s.str. (comprising ten species) was examined by cpDNA restriction site variation to determine phyletic relationships within the group. The results were compared with relationships proposed from other data. A total of 86 restriction site mutations was detected, 47 of which proved phylogenetically informative. A single most parsimonious tree was obtained using both Wagner and Dollo parsimony. The tree revealed three main lineages that are congruent with the three chromosome lineages (base numbers of x = 16, 17, 18). The monophyly of the x = 16 and 18 groups was supported strongly by molecular data, while the monophyly of x = 17 lineage was only supported moderately. Relationships among the three lineages indicate that the sect.Iva is paraphyletic because sect.Linearbractea is nested within it. Both morphological data and the secondary chemical data are in agreement with the proposed cpDNA phylogeny. Because of this agreement, sect.Iva is revised such that,I. axillaris was excluded and positioned within the newly proposed sect.Rhizoma. Patterns and rates of cpDNA evolution were also examined. The results indicated an uneven evolution in the chloroplast genome with different rates of cpDNA evolution in at least a few species ofIva. However, the evolutionary clock hypothesis can not be rejected within most of the lineages inIva.     

Phylogeographic patterns in Leccinum sect. Scabra and the status of the arctic-alpine species L. rotundifoliae

We investigated inter- and intraspecific phylogenetic relationships in the ectomycorrhizal fungal genus Leccinum section Scabra. Species of this section are exclusively associated with Betula and occur throughout the Northern Hemisphere. We compared the phylogenetic relationships of arctic, alpine, boreal and temperate accessions of section Scabra based on DNA sequences of the single-copy nuclear gene Gapdh and the multiple-copy nuclear region 5.8S-ITS2. Exclusively arctic lineages were not detected in species that occur both in arctic-alpine or boreal regions, except in L. rotundifoliae that was restricted to cold climates. L. scabrum and L. holopus showed an intercontinental phylogeographic pattern, and L. variicolor showed a pattern unrelated to geographical distribution. Molecular clock estimates indicated that L. rotundifoliae is as old as other species in section Scabra. Individual gene trees suggest that interspecific hybridisation occurred several times in the evolution of section Scabra.     

Molecular phylogeny of the speciose vole genus Microtus (Arvicolinae, Rodentia) inferred from mitochondrial DNA sequences

Voles of the genus Microtus represent one of the most speciose mammalian genera in the Holarctic. We established a molecular phylogeny for Microtus to resolve contentious issues of systematic relationships and evolutionary history in this genus. A total of 81 specimens representing ten Microtus species endemic to Europe as well as eight Eurasian, six Asian and one Holarctic species were sequenced for the entire cytochrome b gene (1140 bp). A further 25 sequences were retrieved from GenBank, providing data on an additional 23, mainly Nearctic, Microtus species. Phylogenetic analysis of these 48 species generated four well-supported monophyletic lineages. The genus Chionomys, snow voles, formed a distinct and well-supported lineage separate from the genus Microtus. The subgenus Microtus formed the strongest supported lineage with two sublineages displaying a close relationship between the arvalis species group (common voles) and the socialis species group (social voles). Monophyly of the Palearctic pitymyid voles, subgenus Terricola, was supported, and this subgenus was also subdivided into two monophyletic species groups. Together, these groupings clarify long-standing taxonomic uncertainties in Microtus. In addition, the "Asian" and the Nearctic lineages reported previously were identified although the latter group was not supported. However, relationships among the main Microtus branches were not resolved, suggesting a rapid and potentially simultaneous radiation of a widespread ancestor early in the history of the genus. This and subsequent radiations discernible in the cytochrome b phylogeny, show the considerable potential of Microtus for analysis of historical and ecological determinants of speciation in small mammals. It is evident that speciation is an ongoing process in the genus and that the molecular data provides a vital insight into current species limits as well as cladogenic events of the past.     

Origins of Horseshoe Bats (Rhinolophus, Rhinolophidae) in Southern Africa: Evidence from Allozyme Variability

Morphological analyses indicate that horsehose bats in the genus Rhinolophus constitute a monophyletic group which most likely originated in southeastern Asia but which presently inhabits Oriental, Australian, Palaearctic, and Ethiopian zoogeographical provinces. Ten species occur in southern Africa, but it is uncertain which species represent dispersals from Eurasia through North Africa and which have resulted from speciation in Africa. Analyses of 34 allozyme encoding loci in these 10 species and in 2 southern African species of leafnose bats in the sister genus Hipposideros reveal the presence of at least three lineages of Rhinolophus in southern Africa. One lineage includes R. clivosus, R. darlingi, R. fumigatus, and R. hildebrandtii, all of which, except R. clivosus, are endemic to sub-Saharan Africa. Rhinolophus blasii is genetically allied with, but distinct from this group, and appears to be a recent migrant from another lineage centered on the Mediterranean. A third lineage, including at least R. capensis, R. denti, R. simulator, and R. swinnyi, is endemic to sub-Saharan Africa. The phylogenetic position of R. landeri is uncertain, most likely because of the small sample size used to estimate allelic frequencies for this species. The biochemical genetic definitions of these lineages largely agree with previous morphological analyses of Rhinolophus species. Divergences between species within two lineages (R. clivosus, R. darlingi, R. fumigatus, and R. hildebrandtii; and R. capensis, R. denti, R. simulator, and R. swinnyi) appear to reflect two bursts of speciation in the Plio-Pleistocene period within Africa.     

Phylogeography and species boundaries of the western North American Nightsnake (Hypsiglena torquata): revisiting the subspecies concept

The subspecies concept has received considerable debate throughout the past century. Subspecies were originally used to delineate potential incipient species, but were later employed to simply capture geographical variation. There is a recent trend to eliminate the trinomial in light of new evidence. Discrete, diagnosable lineages are elevated to specific status, while those that show clinal variation and/or appear to represent ecological pattern classes are placed in synonymy with the parent species and the subspecific epithets are disregarded. Here, I examine the species boundaries of nightsnakes (Hypsiglena torquata) using standard phylogeographic methods and mtDNA data from 178 individuals. Previously, seventeen subspecies of H. torquata were described. In this study, I recognize six species in what was previously considered H. torquata: one is novel, two were previously recognized subspecies, while the remaining three are wide-spread, polymorphic lineages, composed of multiple subspecies. I make the case to maintain the subspecific lineages in these wide-ranging species because they are geographically cohesive, morphologically discrete, and may represent incipient species within each complex, which have not yet achieved speciation. These subspecies are maintained, not only pending future investigations, but because they provide a useful identity for the taxonomy of this diverse lineage.     

Reconstructing the history of Campanulaceae with a Bayesian approach to molecular dating and dispersal–vicariance analyses

We reconstruct here the spatial and temporal evolution of the Campanula alliance in order to better understand its evolutionary history. To increase phylogenetic resolution among major groups (Wahlenbergieae–Campanuleae), new sequences from the rbcL region were added to the trnL-F dataset obtained in a previous study. These phylogenies were used to infer ancestral areas and divergence times in Campanula and related genera using a Bayesian approach to molecular dating and dispersal–vicariance analyses that takes into account phylogenetic uncertainty. The new phylogenetic analysis confirms Platycodoneae as the sister group of Wahlenbergieae–Campanuleae, the two last ones inter-graded into a well-supported clade. Biogeographic and dating analyses suggest that Western Asia and the Eastern Mediterranean have played a major role as centers of migration and diversification within the Campanula alliance, probably in relation to the intense orogenic activity that took place in this region during the Late Neogene, and that could have promoted isolation and allopatric speciation within lineages. Diversification rates within several Campanula lineages would have increased at the end of the Miocene, coinciding with the Messinian Stage. Strong selective pressures from climate changes and the expansion of mountainous regions during this period are suggested to explain the adaptation to drought, cold or disturbed environments observed in many Campanula species. Several independent long-distance dispersal events to North America are inferred within the Rapunculus clade, which seem to be related to high ploidy levels.     

The phylogeny and evolution of thePterygodium—Corycium complex (Coryciinae,Orchidaceae)

The leaf-anatomy, palynology, seed-morphology, vegetative morphology and especially the highly complicated floral morphology of theCoryciinae s. str. (Diseae: Orchidoideae: Orchidaceae) are described and illustrated in detail. On the basis of these characters the presumed phylogeny, based on a rigorous cladistic analysis, is presented. The cladistic biogeographical analysis of theCoryciinae s. str. shows that it is a member of the Afrotemperate Track, with a pattern of vicariance events typical of the members of this track. An analysis of the patterns of speciation shows that allopatric speciation appears to be rare, and that parapatric speciation across edaphic boundaries may be the most important factor. Proceeding from the information presented, a new classification of the group is proposed in which we recognize the four generaCeratandra, Evotella, Pterygodium andCorycium. The new monotypic genusEvotella comprises a species originally described asPterygodium rubiginosum. The three species of the genusAnochilus are transferred toCorycium andPterygodium. P. magnum, which was originally described asPterygodium but was transferred toCorycium lately, is placed in a monotypic section ofPterygodium.     

Nuclear and mitochondrial phylogeography of the Atlantic forest endemic Xiphorhynchus fuscus (Aves: Dendrocolaptidae): biogeography and systematics implications

We studied the intraspecific evolutionary history of the South American Atlantic forest endemic Xiphorhynchusfuscus (Aves: Dendrocolaptidae) to address questions such as: Was the diversification of this bird's populations associated to areas of avian endemism? Which models of speciation (i.e., refuges, river as barriers or geotectonism) explain the diversification within X. fuscus? Does the genetic data support subspecies as independent evolutionary units (species)? We used mitochondrial (n=34) and nuclear (n=68) DNA sequences of X. fuscus to study temporal and spatial relationships within and between populations. We described four main monophyletic lineages that diverged during the Pleistocene. The subspecies taxonomy did not match all the evolutionary lineages; subspecies atlanticus was the only one that represented a monophyletic and isolated lineage. The distribution of these lineages coincided with some areas of endemism for passerines, suggesting that those areas could be regions of biotic differentiation. The ancestor of X. fuscus diverged approximately 3 million years ago from Amazonian taxa and the phylogeographic pattern suggested that X. fuscus radiated from northeastern Brazil. Neither the riverine nor the geotectonic vicariance models are supported as the primary cause for diversification of geographic lineages, but rainforest contractions and expansions (ecological vicariance) can explain most of the spatial divergence observed in this species. Finally, analyses of gene flow and divergence time estimates suggest that the endangered subspecies atlanticus (from northeastern Brazil) can be considered a full species under the general lineage species concept.     

Evidence for Vertical Inheritance and Loss of the Leukotoxin Operon in Genus <Emphasis Type="Italic">Mannheimia</Emphasis>

The Mannheimia subclades belong to the same bacterial genus but have taken divergent paths toward their distinct lifestyles. M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that horizontal gene transfer of the leukotoxin operon has catalyzed pathogenic adaptation and speciation of M. haemolytica + M. glucosida, or other major subclades, by using a strategy that combines compositional and phylogenetic methods. We show that it has been vertically inherited from the last common ancestor of the diverging Mannheimia subclades, although several strains belonging to M. ruminalis have lost the operon. Our analyses support that divergence within M. ruminalis following colonization of the ovine rumen was very rapid and that functional decay of most of the leukotoxin operons occurred early when the adaptation to the rumen was fastest, suggesting that antagonistic pleiotropy was the main contributor to losses in the radiating lineages of M. ruminalis. To sum up, the scenario derived from these analyses reflects two aspects. On one hand, it opposes the hypothesis of horizontal gene transfer as a catalyst of pathogenic adaptation and speciation. On the other hand, it indicates that losses of the leukotoxin operons in the radiating lineages of M. ruminalis have catalyzed their adaptation to a commensal environment and reproductive isolation (speciation). [Reviewing Editor: Dr. David Guttman] Jesper Larsen and Anders G. Pedersen contributed equally to this work.     

Normapolles plants: a prominent component of the Cretaceous rosid diversification

The Normapolles complex, characterised by its oblate and triaperturate pollen, constitutes an important and diverse element of many Late Cretaceous and Early Cainozoic floras of the Northern Hemisphere. Based on the dispersed pollen record alone it has been difficult to assess systematic affinities, but relationships with Fagales have been proposed. Over the past twenty years several exquisitely preserved Late Cretaceous reproductive structures with Normapolles type pollen in situ have been described. In this study we provide a summary and new information of these floral structures. Further, a new genus, Dahlgrenianthus, is described from the Late Cretaceous of southern Sweden. The genus includes the type species Dahlgrenianthus suecicus, a number of reproductive structures referred to Dahlgrenianthus sp., and Dahlgrenianthus trigonus (Knobloch et Mai) comb. nov. from the Maastrichtian flora of Walbeck, Germany. Dahlgrenianthus comprises small flowers with pentamerous perianth and androecium and a tricarpellate gynoecium. It is distinguished from all other Normapolles floral structures in its hypogynous floral organisation. All Normapolles floral structures described so far are thought to be related to various members of the core Fagales, but the group is obviously not monophyletic. The stratigraphic range of the Normapolles taxa and other fagalean fossils strongly suggests that all major fagalean lineages were present by the Cenomanian or earlier.     

Systematics and Evolution of the Dasyurid Marsupial Genus Sminthopsis: I. The Macroura Species Group

Genetic variation within the macroura species group, which includes Sminthopsis macroura, S. virginiae, S. douglasi, and S. bindi, was examined through analyses of complete mitochondrial 12S rRNA gene sequences, partial control-region DNA sequences, and allozymes. Divergent genetic lineages appear to be present within S. macroura and S. virginiae, and it is likely that this genetic divergence equates to currently unrecognized taxonomic diversity. Specimens of S. macroura (as currently recognized) belong to three genetically distinct lineages that are highly divergent from one another. Two of these lineages may be synonymous with two previously recognized dunnart species—S. froggatti and S. stalkeri. The third appears to represent "true" S. macroura and is itself genetically heterogeneous, with a number of subgroups present within it that may also represent currently unrecognized taxa. The mitochondrial DNA sequence divergences observed between S. virginiae nitela and the two other S. virginiaesubspecies are equivalent to, or greater than, those noted between other dunnart species. Allozyme divergences between these subspecies were however slightly lower, and determination on whether S. virginiae nitela should be returned to full species status (S. nitela) may require further evidence. Phylogenetic relationships between species in the macroura group appear to have been partially resolved, with individual 12S rRNA and combined mitochondrial DNA analyses recovering S. bindi as the earliest diverging taxon. Other relationships between species in the group were either not consistently recovered or lacked strong support.     

Development of neural lineages derived from the sine oculis positive eye field of Drosophila

The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the so-positive lineages as a tool to reconstruct the morphogenesis of the larval brain.     

Intralineage variation in the pattern ofrbcL nucleotide substitution

Variation in chloroplastrbcL sequences was studied in representative species of four different lineages: the tribeRubieae (Rubiaceae), and the generaDrosera (Droseraceae),Nothofagus (Nothofagaceae) andIlex (Aquifoliaceae). Each lineage has its particular non-overlapping set ofrbcL polymorphic sites, indicating that common unconstrainedrbcL sites are not shared. Large differences in the rate and pattern of nucleotide substitution are observed among the four lineages. The genusIlex has the lowest rate of substitution, the lowest transition/transversion ratio, the lowest synonymous/replacement ratio and the lowest number of substitutions at the third codon position. An apparent relationship of these measures to the age of the lineages is observed. The A + T content and codon use among the four lineages are very similar and, apparently, cannot account for the observed differences in patterns of nucleotide substitution. However, the A + T content of the two bases immediately flanking the polymorphic sites is higher inIlex than in the other lineages. This could be correlated with the transversion/transition bias observed inIlex. The particularly low synonymous/replacement ratio found inIlex could also be explained by the small population sizes of species in this genus.