Kerryn E Slack

Frédéric Delsuc

Trish Mclenachan

Ulfur Arnason

David Penny

Resolving the root of the avian mitogenomic tree by breaking up long branches

Keywords: avian evolution, long branches, galloanseres, Passeriformes, Neoaves, Cracrafti

Incomplete taxon sampling has been a major problem in resolving the early divergences in birds. Five new mitochondrial genomes are reported here and three break up long branches that tended to attract the distant reptilian outgroup. These long branches were to galliforms, and to oscine and suboscine passeriformes.

Breaking these long branches leaves the root between paleognaths and neognaths as inferred by maximum likelihood and Bayesian phylogenetic analyses. This means that morphological, nuclear and mitochondrial data are now in agreement on the position of the root of the avian tree and we can, for the moment, move on to other questions. An overview is then given of the deepest divisions in the mitogenomic tree inferred from complete mitochondrial genomes. The strict monophyly of both the galloanseres and the passerines is strongly supported, leaving the deep six-way split within Neoaves as the next major question for which resolution is still lacking. Incomplete taxon-sampling was a major problem earlier and although some resolution is now available there are still problems related to the fact that current phylogenetic methods still fail to account for real features of DNA sequence evolution.

Introduction

After a period of relative quiescence following the publication of Sibley and Ahlquist's DNA/DNA hybridisation tapestry (1990) the ever-decreasing cost of DNA sequences has led to a rebirth of deep-level avian systematics. As a part of this effort to acquire new data aiming at resolving the phylogeny of avian orders, complete mitochondrial genomes had been sequenced for an increasing number of taxa (for example, [START_REF] Mindell | Interordinal relationships of birds and other reptiles based on whole mitochondrial genomes[END_REF][START_REF] Paton | Complete mitochondrial DNA genome sequences show that modern birds are not descended from transitional shorebirds[END_REF][START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF][START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]. The amount of sequence data accumulated so far permits the evaluation of a range of prior hypotheses primarily proposed on morphological, paleontological and biogeographical grounds (for example, [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF][START_REF] Cracraft | Phylogenetic relationships among modern birds (Neornithes)[END_REF]). In the case of placental mammals, as expected on theoretical grounds, increasing the number of taxa led to an excellent agreement between nuclear and mitochondrial datasets [START_REF] Lin | Four new mitochondrial genomes, and the stability of evolutionary trees of mammals[END_REF][START_REF] Reyes | Congruent mammalian trees from mitochondrial and nuclear genes using Bayesian methods[END_REF]). Such agreement between different datasets is essential for corroboration [START_REF] Penny | Testing the theory of evolution by comparing phylogenetic trees constructed from five different protein sequences[END_REF] because model misspecification [START_REF] Buckley | Model misspecification and probabilistic tests of topology: Evidence from empirical data sets[END_REF]) can be hard to detect, Systematic biases can lead to 100% bootstrap support for conflicting trees [START_REF] Phillips | Genome-scale phylogeny: sampling and systematic errors are both important[END_REF], and even maximum likelihood methods can become inconsistent when the model is not specified accurately [START_REF] Chang | Inconsistency of evolutionary tree topology reconstruction methods when substitution rates vary across characters[END_REF]).

An early problem was that mitogenomic-based studies of avian phylogenetic relationships appeared to place the reptilian outgroup within Passeriformes (Härlid and Arnason 1999;[START_REF] Mindell | Interordinal relationships of birds and other reptiles based on whole mitochondrial genomes[END_REF]). Such a rooting was at odds with data from morphology [START_REF] Sibley | Phylogeny and classification of birds[END_REF][START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF][START_REF] Livezey | Higher-order phylogenetics of modern Aves based on comparative anatomy[END_REF][START_REF] Mayr | The deep divergences of neornithine birds: a phylogenetic analysis of morphological characters[END_REF], nuclear DNA (Garcia-Moreno and [START_REF] Garcia-Moreno | Rooting a phylogeny with homologous genes on opposite sex chromosomes (gametologs): a case study using avian CHD[END_REF] and genomic DNA strings (Edwards et al. 2002). These all supported a major division between paleognaths (tinamous and ratites) and neognaths (all other birds including the species-rich Passeriformes). The passerine rooting of the mitochondrial tree has been suggested to be the result of a possible long-branch attraction effect between the distant reptilian outgroup and the fast-evolving passerines [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF]. Indeed, in the unrooted avian tree the passerines were grouped together on the tree, but could become paraphyletic, or even diphyletic when the outgroup was added [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF]. Such disruption of the ingroup by a distant outgroup is known from simulation studies [START_REF] Holland | Outgroup misplacement and phylogenetic inaccuracy under a molecular clock: A simulation study[END_REF]) and here the ingroup tree by itself was more likely to be correct. A similar ingroup disruption was also found with mammals [START_REF] Lin | Four new mitochondrial genomes, and the stability of evolutionary trees of mammals[END_REF].

In general, distant outgroups are hard to place correctly into the ingroup tree [START_REF] Holland | Outgroup misplacement and phylogenetic inaccuracy under a molecular clock: A simulation study[END_REF]. Taxon sampling from Passeriformes has been sparse with only one suboscine (broadbill) and two oscines (rook and indigobird) included at that time [START_REF] Braun | Examining basal avian divergences with mitochondrial sequences: Model complexity, taxon sampling, and sequence length[END_REF]. Base composition bias might also have played a role since phylogenetic reconstructions under a non-homogeneous model dealing with base composition heterogeneity [START_REF] Paton | Complete mitochondrial DNA genome sequences show that modern birds are not descended from transitional shorebirds[END_REF] and RY-coding analyses [START_REF] Braun | Examining basal avian divergences with mitochondrial sequences: Model complexity, taxon sampling, and sequence length[END_REF] supported the classical rooting of the avian tree between paleognaths and neognaths. However, the position of the root of the avian tree is still difficult to resolve clearly with complete mitochondrial data and must be 28/8/08, page 4 of 28 tested by the inclusion of more basal neognaths (Galloanserae) as well as Passeriformes that appear especially fast evolving [START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]). Ideally, it is preferable for a result to be found by all good methods of phylogenetic analysis, not just by one specialised analysis. In difficult cases, it might thus be necessary for both theoretical and practical reasons to first build an unrooted tree, and then test the placement of the root secondarily.

The phylogeny of paleognaths, with a particular emphasis on both living and extinct ratites, has been well studied from the mitogenomic viewpoint (Härlid and Arnason 1999;Härlid et al. 1999;[START_REF] Cooper | Complete mitochondrial genome sequences of two extinct moas clarify ratite evolution[END_REF][START_REF] Haddrath | Complete mitochondrial DNA genome sequences of extinct birds: ratite phylogenetics and the vicariance biogeography hypothesis[END_REF]. Their origins and biogeographic evolution in relation to the tectonic fragmentation of Gondwana are now relatively well understood [START_REF] Cooper | Complete mitochondrial genome sequences of two extinct moas clarify ratite evolution[END_REF][START_REF] Haddrath | Complete mitochondrial DNA genome sequences of extinct birds: ratite phylogenetics and the vicariance biogeography hypothesis[END_REF], although some relationships within ratites and between deep ratites and tinamous are still uncertain.

Turning to neognaths, they are usually divided into Galloanserae (chicken, ducks and allies) and Neoaves (all other neognath birds). This subdivision is now well supported from molecular data by both mitochondrial [START_REF] Paton | Complete mitochondrial DNA genome sequences show that modern birds are not descended from transitional shorebirds[END_REF][START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]) and nuclear sequences (van Tuinen et al. 2000;[START_REF] Garcia-Moreno | Congruent avian phylogenies inferred from mitochondrial and nuclear DNA sequences[END_REF][START_REF] Chubb | New nuclear evidence for the oldest divergence among neognath birds: the phylogenetic utility of ZENK (i)[END_REF]). We have recently added the magpie goose (Anseranas semipalmata) a basal member of the water fowl lineage (Anseriformes) to the complete mitochondrial dataset, establishing an important calibration point for avian evolution [START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]). In the present study, we include a basal member (Ericson et al. 2002b) of the chicken-related lineage (Galliformes) by sequencing the complete mitochondrial genome of the Australian brush-turkey (Alectura lathami, Megapodiidae). This is expected to break the relatively long ancestral lineage leading to chicken and quail, and to test the relationships within Galloanserae further.

Phylogenetic relationships among the six or more major groups of Neoaves that contain the vast majority of extant bird species remain elusive and are usually represented as a multifurcation [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF][START_REF] Cracraft | Phylogenetic relationships among modern birds (Neornithes)[END_REF]. Within this unresolved phylogeny, Passeriformes are by far the most speciose group. The current taxonomy of oscines, based mainly on the results of DNA/DNA hybridizations [START_REF] Sibley | Phylogeny and classification of birds[END_REF], has recently been challenged by analyses of both nuclear and mitochondrial genes [START_REF] Irestedt | Phylogeny of major lineages of suboscines (Passeriformes) analysed by nuclear DNA sequence data[END_REF]Ericson et al. 2002a,b;[START_REF] Ericson | Phylogeny of Passerida (Aves: Passeriformes) based on nuclear and mitochondrial sequence data[END_REF][START_REF] Barker | Phylogeny and diversification of the largest avian radiation[END_REF]). Based on these new results a new taxonomy of major passerine groups has been proposed (Ericson et al. 2002b). Three major lineages have been distinguished: Acanthisittia (New Zealand wrens), suboscines (Eurylaimides or Old World suboscines and Tyrannides or New World suboscines) and oscines (Menurae and Euoscines).

Oscines and Suboscines are grouped together into Eupasseres to the exclusion of New Zealand wrens, the latter representing the most basal lineage of Passeriformes. The early emergence of New Zealand wrens 28/8/08, page 5 of 28 within Passeriformes is consistent with a Gondwanan origin for the whole passerine group [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF][START_REF] Barker | A phylogenetic hypothesis for passerine birds: taxonomic and biogeographic implications of an analysis of nuclear DNA sequence data[END_REF][START_REF] Edwards | Out of Gondwana: the origin of passerine birds[END_REF]Ericson et al. 2002a).

The passerines have been represented in the mitogenomic database by only three taxa: the gray-headed broadbill (Smithornis sharpei; Eurylemidae) belonging to the Old World suboscines, and two Euoscines: the village indigobird (Vidua chalybeata; Estrildidae) and the rook (Corvus frugilegus; Corvidae). Under the new classification (Ericson et al. 2002b) rook and indigo bird are both in the Euoscines, leaving the Menurae unrepresented. A representative of New Zealand wrens, the rifleman (Acanthisitta chloris; Acanthisittidae) has been recently added to the complete mitochondrial dataset, but its position was still locally unstable within passerines [START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]. By incorporating the new complete mitochondrial genomes of the superb lyrebird (Menura novaehollandiae; Menuridae) representing the second major group of oscines, and the fuscous flycatcher (Cnemotriccus fuscatus; Tyrannidae) a member of the New World suboscines, we now have a much more representative taxon sampling of Passeriformes. We expect from theory [START_REF] Hendy | A framework for the quantitative study of evolutionary trees[END_REF]) and from simulations [START_REF] Holland | Outgroup misplacement and phylogenetic inaccuracy under a molecular clock: A simulation study[END_REF]) that these key taxa will stabilise the relationships within Passeriformes by subdividing the two long branches leading to oscines and suboscines, respectively. This is especially important in order to tackle issues such as the position of the rifleman within passerines, and especially the central question of the position of the root of the avian tree.

Another interesting aspect of the current mitochondrial tree is the difficulty [START_REF] Haring | The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae) indicates an early split in the phylogeny of raptors[END_REF][START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF][START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]) in recovering the expected monophyly of birds of prey (Falconiformes)currently represented by the buzzard (Buteo buteo; Accipitridae) and the falcon (Falco peregrinus; Falconidae). Falconiformes are part of a large group of Neoaves including seabirds, shorebirds, doves, cranes, rails, flamingos, penguins, loons and grebes [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF], a major grouping we informally call the Conglomerati (or Cracrafti). The buzzard and falcon share the same alternative mitochondrial gene order, but are fairly divergent from each other and seem to represent an early split in the raptor lineage [START_REF] Haring | The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae) indicates an early split in the phylogeny of raptors[END_REF]). Thus we again have sparse taxon sampling. Previously, only partitioned-likelihood phylogenetic analyses (with RY-coding third codon positions of the mitochondrial proteins plus nucleotides from RNAs) appearing to support their monophyletic origin [START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]).

As a first step we have sequenced the complete mitochondrial genome of the turkey vulture (Cathartes aura; Cathartinae). This provides an opportunity to test the Sibley and Ahlquist hypothesis (1990) that New World vultures (Cathartinae) are closer to storks (Ciconiinae) than to other birds of prey. It has long being realised that New World and Old World vultures may not share a most recent common ancestor, but may instead represent an example of morphological convergence resulting from adaptation to a scavenging way 28/8/08, page 6 of 28 of life (see [START_REF] Sibley | Phylogeny and classification of birds[END_REF] for an historical review). However, the question of whether New World vultures are more closely related to storks, as suggested by behavioural resemblances [START_REF] Rea | Cathartid affinities: a brief overview[END_REF] and DNA/DNA hybridisations [START_REF] Sibley | Phylogeny and classification of birds[END_REF], or to birds of prey (including Old World vultures) is still ambiguous [START_REF] Seibold | Evolutionary history of New and Old World vultures inferred from nucleotide sequences of the mitochondrial cytochrome b gene[END_REF]. Resolving this question certainly requires more molecular data [START_REF] Helbig | Are storks and New World vultures paraphyletic?[END_REF].

The order Charadriiformes represents a very large and diversified group of shorebirds which also belongs to the Conglomerati/Cracrafti [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF]. Shorebirds are usually divided in three major clades:

Charadrii (oystercatchers, thick-knees, sheathbills, plovers, and allies), Scolopaci (turnstones, sandpipers and jacanas), and Lari (gulls, coursers, pratincoles, terns, skimmers, and skuas) (see Van Tuinen et al. 2004). DNA/DNA hybridisation suggested an early emergence of Scolopaci with a sister-group relationship between Charadrii and Lari [START_REF] Sibley | Phylogeny and classification of birds[END_REF]. The first two shorebird mitochondrial genomes to be sequenced were the blackish oystercatcher (Haematopus ater; Haematopodidae) and the ruddy turnstone (Arenaria interpres; Scolopacidae) [START_REF] Paton | Complete mitochondrial DNA genome sequences show that modern birds are not descended from transitional shorebirds[END_REF]. Since then, data from nuclear genes have explored the relationships among shorebirds families (Ericson et al. 2003;[START_REF] Paton | RAG-1 sequences resolve phylogenetic relationships within Charadriiform birds[END_REF]Thomas et al. 2004). These have challenged the DNA/DNA hybridisation results by finding a closer relationship between Scolopaci and Lari with an early divergence of Charadrii. The sequencing of the southern blackbacked gull (Larus dominicanus; Laridae) mitochondrial genome offers the opportunity to test the new nuclear-based hypothesis by adding a representative of the previously unsampled third major group of Charadriiformes (Lari, gulls).

Here we report mitochondrial genomes for five birds chosen to help clarify the deepest divisions and test specific phylogenetic hypotheses in the avian tree. The sequences have been determined for the brush turkey (Alectura lathami, Megapodiidae), two passerines with a flycatcher (Cnemotriccus fuscatus; Tyrannidae) and the superb lyrebird (Menura novaehollandiae; Menuridae), the turkey vulture (Cathartes aura; Cathartinae) and a gull (Larus dominicanus; Laridae). The phylogenetic analyses using maximum likelihood and Bayesian methods support a growing consensus from nuclear, mitochondrial and morphological data for the position of the root of the avian tree and for its first main divisions. The turkey vulture is not positioned with the stork and the gull joins with the turnstone.

Materials and Methods

Tissue samples

The southern black-backed gull (Larus dominicanus [Lichtenstein, 1823]) came from Waikanae, New Zealand and was supplied by the New Zealand Department of Conservation (Kapiti Area branch). The 28/8/08, page 7 of 28

DNA extraction and sequencing

Genomic DNA was extracted from 25-50 mg of sample tissue using the High Pure TM PCR Template Preparation Kit (Protocol Vb; Boehringer Mannheim). To minimize the possibility of obtaining nuclear copies of mitochondrial (mt) genes (numts), the five mitochondrial genomes were amplified in two (turkey vulture, flycatcher) or three (gull, brush-turkey, lyrebird) overlapping fragments using long range PCR. The long range products (see below) were recovered from agarose gels using an appropriate kit (e.g. Concert TM

Rapid Gel Extraction System kit [GIBCO BRL/Life Technologies], AccuPrep™ Gel Purification Kit

[Bioneer]). They were then used as templates in a second round of PCR amplification (overlapping fragments of 0.3 -3.1 kb in length). PCR reactions were set up using the long range and reamplification conditions described in [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF]. Annealing temperatures for the reamplifications ranged from 48 to 60°C and extension times from 1 to 3 minutes. The conserved primers used were from [START_REF] Kocher | Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers[END_REF], [START_REF] Sorenson | Primers for a PCR-based approach to mitochondrial genome sequencing in birds and other vertebrates[END_REF], [START_REF] Cooper | Complete mitochondrial genome sequences of two extinct moas clarify ratite evolution[END_REF] and Cooper et al. (unpublished), with some being modified from the original, or were designed from alignments of bird mitochondrial genes. Where necessary (e.g. control region), specific primers were designed for individual birds using the Oligo® 4.03 program (National Biosciences, Inc.).

The positions and sizes of the long-range PCR products and the annealing temperatures, extension times and primers used to generate them are given below. Primer nomenclature: Av (avian) position gene direction (F: forward; R: reverse). Positions and genes indicate where the 3' end of the primer binds in the chicken mitochondrial genome. To avoid confusion, 'gene' and 'direction' are inverted for rRNA primers. The reamplification products were purified by treatment with 2 units of shrimp alkaline phosphatase (SAP) and 10 units of Exonuclease I (incubated at 37ºC for 30 min then 80ºC for 15 min) or were recovered from agarose gels as above. Two regions of the lyrebird mitochondrial genome were cloned: a 0.9 kb fragment spanning the end of tRNA-Phe plus the start of 12S rRNA and a 1.4 kb fragment covering part of the control region, tRNA-Pro and part of NADH6. The PCR products were ligated into the pGem®-T Easy vector (Promega) and transformed into MAX Efficiency® DH5α™ competent cells (Invitrogen) following manufacturer's instructions. Plasmid DNA was isolated from transformants containing inserts using the GenElute™ Plasmid Miniprep Kit (Sigma). Two clones were sequenced for the first region (plus direct sequence from a PCR product generated using a specific control region primer) and three clones for the second. Sequencing reactions were run on ABI 377 or ABI 3730 sequencers and were set up according to 28/8/08, page 9 of 28 manufacturer's instructions. Sequences were manually checked/corrected (including the removal of any primer sequence) and assembled using Sequencher TM 4.1 (Gene Codes Corp.). Overlaps between sequences were sufficient to ensure synonymy (usually ≥100 nt between individual sequences; a total of from 1 -4 kb between the different long range products). Sequence identity was confirmed through Fasta searches of the EMBL database (http://www.ebi.ac.uk/fasta33/nucleotide.html).

Dataset assembly

In addition to the five new birds from this paper, 25 other complete avian mitochondrial genomes were included in the analyses (18 neognaths and seven paleognaths). The 18 neognath taxa are: chicken (Gallus gallus; GenBank accession number AP003317), Japanese quail (Coturnix japonica; AP003195), magpie goose (Anseranas semipalmata; AY309455), redhead (duck, Aythya americana; AF090337), greater whitefronted goose (Anser albifrons; AF363031), rifleman (NZ wren, Acanthisitta chloris; AY325307), grayheaded broadbill (Smithornis sharpei; AF090340), village indigobird (Vidua chalybeata; AF090341), rook (Corvus frugilegus; Y18522), peregrine falcon (Falco peregrinus; AF090338), Eurasian buzzard (Buteo buteo; AF380305), blackish oystercatcher (Haematopus ater; AY074886), ruddy turnstone (Arenaria interpres; AY074885), Oriental stork (Ciconia boyciana; AB026193), red-throated loon (Gavia stellata; AY293618), little blue penguin (Eudyptula minor; AF362763), black-browed albatross (Diomedea melanophris; AY158677) and Kerguelen petrel (Pterodroma brevirostris; AY158678). The seven paleognath taxa are: emu (Dromaius novaehollandiae; AF338711), southern cassowary (Casuarius casuarius; AF338713), great spotted kiwi (Apteryx haastii; AF338708), greater rhea (Rhea americana; Y16884), ostrich (Struthio camelus; Y12025), great tinamou (Tinamus major; AF338707) and elegant crested tinamou (Eudromia elegans; AF338710). The most up-to-date version of each GenBank file was used and the original chicken mitochondrial genome (X52392) used in previous analyses (e.g. [START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF][START_REF] Slack | Early penguin fossils, plus mitochondrial genomes, calibrate avian evolution[END_REF]) has been replaced by a more recent sequence.

Six reptiles were used as outgroups: American alligator (Alligator mississippiensis; Y13113), spectacled caiman (Caiman crocodylus; AJ404872), eastern painted turtle (Chrysemys picta; AF069423), green turtle (Chelonia mydas; AB012104), blue-tailed mole skink (Eumeces egregius; AB016606) and common iguana (Iguana iguana; AJ278511). This is the same set of outgroups used previously (for example, [START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF][START_REF] Slack | Early penguin fossils, plus mitochondrial genomes, calibrate avian evolution[END_REF].

Two neoavian taxa (an owl and a parrot; [START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]) and three paleognaths (extinct NZ moas; Cooper et al. 2001, Haddrath and[START_REF] Haddrath | Complete mitochondrial DNA genome sequences of extinct birds: ratite phylogenetics and the vicariance biogeography hypothesis[END_REF] were omitted from the full analyses until paired taxa are available. In the meantime, the owl and parrot do not affect the position of the root of the avian tree that is 28/8/08, page 10 of 28 the focus of this study. We are in the process of sequencing a barn owl (Tyto), an African parrot (lovebird, Agapornis) and a forest falcon (Micrastur) (www.awcmee.massey.ac.nz/mt_genomes). Based on previous experience of improved taxon sampling [START_REF] Lin | Four new mitochondrial genomes, and the stability of evolutionary trees of mammals[END_REF][START_REF] Delsuc | Comment on "Hexapod origins: monophyletic or paraphyletic?[END_REF][START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]) the addition of these three mitochondrial genomes will help stabilize the position of both owls and parrots within Neoaves for future analyses. Similarly, there is some instability within paleognaths when moas are included [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF], and the issue of fine-tuning paleognath interrelationships will be readdressed once additional kiwi sequences become available (G.C. Gibb, in preparation).

Phylogenetic analysis

Sequences were aligned in SeAl v1 [START_REF] Rambaut | Se-Al: Sequence Alignment Editor[END_REF], at the amino acid level for protein-coding genes, and based on secondary structure for RNA genes. Each dataset has 12 protein-coding genes, two rRNAs and 21 tRNAs (lacking tRNA-Phe because it is still not available for the rifleman). Gaps, ambiguous sites adjacent to gaps, the NADH6 (light-strand encoded), and stop codons (often incomplete in the DNA sequence), were excluded from the alignment.

We made two alignments; with and without the 6 outgroup taxa. The 'birds-only' dataset was used first to study relationships within the ingroup (birds) in order to test whether there were any changes to the tree when the outgroup was added. In practice, the tree from the birds-only dataset was compared with the tree using only the birds from the full alignment (aligned including the outgroup). This allowed the separation of any effects of adding the outgroup, from any changes from the alignment. The full dataset had 11,737 unambiguously aligned nucleotide sites, the birds-only dataset has 13,440 nucleotides (15% longer).

In previous work, RY-coding of the most variable partitions of the nucleotide data (especially the 3 rd codon position) has been shown to be advantageous [START_REF] Delsuc | Comment on "Hexapod origins: monophyletic or paraphyletic?[END_REF][START_REF] Phillips | The root of the mammalian tree inferred from whole mitochondrial genomes[END_REF][START_REF] Phillips | Combined nuclear and mitochondrial protein-coding DNA sequences resolve the interrelations of the major Australasian marsupial radiations[END_REF]. The recoding increases the proportion of changes on internal branches of the tree (treeness) and alleviates the differences in nucleotide composition (Relative Compositional Variability, RCV). It also increases concordance between mitochondrial and nuclear datasets. RY-coding does increase the ML scores, but because RY-coding is not strictly nested within nucleotide coding (M.A. Steel, pers. comm.) it is not valid to compare their ML scores directly. However, because of the better fit of the data to the model (higher treeness, and less variability in nucleotide composition (lower RCV), this is our preferred method of analysis of vertebrate mitochondrial data. Thus the trees reported here were inferred with the third codon positions recoded as R and Y. Analysis is by standard programs including ModelTest [START_REF] Posada | Modeltest: testing the model of DNA substitution[END_REF], PAUP* [START_REF] Swofford | PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods)[END_REF] and MrBayes [START_REF] Ronquist | MrBayes3: Bayesian phylogenetic inference under mixed models[END_REF]. We ran 500 unconstrained ML bootstrap replicates with PAUP* on the Helix computing cluster 28/8/08, page 11 of 28 (www.helix.massey.ac.nz), plus a partitioned Bayesian analysis using four MCMCMC chains of 10 7 generations. The full data sets and command blocks for both PAUP* and MrBayes are available in nexus format at www.awcmee.massey.ac.nz/downloads. These files illustrate the procedures used both in finding optimal estimates for gamma and invariant sites for the different data partitions, as well running subsets of the data as RY coded.

Results

The five new avian complete mitochondrial genomes are deposited in GenBank under the following accession numbers: Australian brush-turkey (Alectura lathami, AY346091); superb lyrebird (Menura novaehollandiae, AY542313); fuscous flycatcher (Cnemotriccus fuscatus, AY596278); southern blackbacked gull (Larus dominicanus, AY293619); and turkey vulture (Cathartes aura, AY463690). The gull, brush-turkey and turkey vulture have the standard avian gene order first identified in the chicken [START_REF] Desjardins | Sequence and gene organization of the chicken mitochondrial genome -a novel gene order in higher vertebrates[END_REF]. This is expected in that the other galliforms in the dataset (chicken and quail) and the two other shorebirds (oystercatcher and turnstone) are already known to have the standard gene order. However, the turkey vulture also appears to have the standard avian gene order, and thereby differs from the two falconiform mitochondrial genomes (falcon and buzzard) already available. By itself, this is certainly of interest, but not sufficient yet to exclude it from the falconiforms, even though for characters with an extremely high number of states parsimony is an ML estimator [START_REF] Steel | Two further links between MP and ML under the Poisson model[END_REF]).

The lyrebird and flycatcher genomes have the alternative avian gene order first recognized by [START_REF] Mindell | Multiple independent origins of mitochondrial gene order in birds[END_REF]. The flycatcher result is as expected; [START_REF] Mindell | Multiple independent origins of mitochondrial gene order in birds[END_REF] examined nine major suboscine lineages (including a tyrannid flycatcher) and found that all had the alternative gene order. On the other hand, [START_REF] Mindell | Multiple independent origins of mitochondrial gene order in birds[END_REF] found that a set of 106 oscine species had the standard avian gene order. However, that set was mostly Passeridae (90 taxa) and Fringillidae (11 taxa) and the remaining five families (Corvidae, Sturnidae, Cisticolidae, Certhiidae, Estrildidae) were each represented by a single taxon.

Although [START_REF] Mindell | Multiple independent origins of mitochondrial gene order in birds[END_REF] concluded "that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines" [START_REF] Bensch | Mitochondrial genomic rearrangements in songbirds[END_REF] subsequently identified the alternative gene order in an oscine group (Phylloscopus warblers, Sylviidae).

The lyrebird (Menuridae) now provides the second example of the alternative gene order in oscine passerines. The duplicated control region associated with the alternative gene order has been reduced to a short non-coding region in both the lyrebird and the Phylloscopus warblers. However, if this non-coding region is still similar to the control region then the sequence fragment (most of NADH6, tRNA-Glu, part of the control region) that [START_REF] Mindell | Multiple independent origins of mitochondrial gene order in birds[END_REF] used to examine most of their oscine species (102 of 106) might not detect the alternative gene order when it is present. This could happen, for example, where the 28/8/08, page 12 of 28 rearrangement is recent, or if a form of concerted gene evolution homogenized the duplicates (G.C. Gibb et al. in prep.).

Unrooted tree

We know from both simulations [START_REF] Holland | Outgroup misplacement and phylogenetic inaccuracy under a molecular clock: A simulation study[END_REF] and empirical studies on mammals [START_REF] Lin | Four new mitochondrial genomes, and the stability of evolutionary trees of mammals[END_REF] and birds [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF]) that an outgroup can disrupt a previously established ingroup tree (see Introduction). Therefore we first report an unrooted ML tree for the 30 bird sequences, together with the results of 500 full ML bootstrap runs. The data is the combined protein coding and RNA genes for both datasets -aligned with or without the 6 outgroup taxa, giving a combined length of 11,737 and 13,440 nucleotides respectively. Again, we find that re-coding the 3 rd position (cdp) as R/Y characters markedly reduces the relative compositional variability (RCV), and increases the signal on the internal branches compared with the external branches (the treeness value is increased). As before, the models were optimized separately for each of the four data partitions using ModelTest [START_REF] Posada | Modeltest: testing the model of DNA substitution[END_REF]. The four partitions were: codons 1&2; codon 3; RNA stems; and RNA loops. The unrooted maximum likelihood tree for the birds-only dataset is shown in Figure 1.

The position of the megapode (brush turkey) is, as expected, a deep lineage in Galliformes, and the three galliforms (chicken, quail and brush-turkey) then group with the three anseriforms (duck, goose, and magpie goose [Anseranas]), forming Galloanserae. The tyrant flycatcher, as expected, joins with the other suboscine (broadbill) and their union is quite deep. Similarly, the lyrebird is deeper in the oscines than the indigo bird/rook divergence. It is worth noting that with the new oscine and suboscine included that the NZ wren (rifleman) now comes basal to the passerines. Overall, the results strongly support the revised classification of oscines (Ericson et al. 2002b) into Menurae (which includes lyrebird) and Euoscines (which includes rook and indigo bird). These three new taxa (brush turkey, tyrant flycatcher and lyrebird) were selected from prior knowledge in anticipation that they would break up long branches that could be attracting the outgroup (a long-branch attraction problem, Hendy and Penny 1989) when rooting the avian tree. This will be re-examined later when examining the position of the root.

The gull joins strongly with the turnstone, rather than with the other shorebird (oyster catcher). More formally, the Lari (gulls) are closer to the Scolopaci (turnstones) than to the Charadrii (oyster catcher). The result is in agreement with the results from nuclear data of Ericson et al. (2003) and [START_REF] Paton | RAG-1 sequences resolve phylogenetic relationships within Charadriiform birds[END_REF]. It is not in agreement with [START_REF] Sibley | Phylogeny and classification of birds[END_REF], but it is only a single interchange (nearest neighbor interchange) on the tree. We say that a tree is 'locally stable' if taxa shift no more than one branch on the 28/8/08, page 13 of 28 tree [START_REF] Cooper | Mass survival of birds across the Cretaceous-Tertiary boundary: Molecular evidence[END_REF]. Again, the agreement of nuclear and mitochondrial data is pleasing, but expected.

The fifth new species is the turkey vulture -a New World vulture. The two issues here are whether the falconiforms (in a broad sense) are monophyletic, and if not, whether the turkey vulture goes with the stork.

On the present dataset, turkey vulture does not come with the two other falconiforms, but nor is it particularly close to the stork. Thus there is some support for the independent origin of core falconiforms and New World vultures, but we need a wider selection of taxa, such as flamingos, grebes, herons, rails, etc. before firmly identifying the closest relative of New World vultures. However, it is also desirable to have additional core falconiforms, because the falcon still has a tendency to go deeper in the tree, disrupting the falcon/buzzard group. Similarly, [START_REF] Fain | Parallel radiations in the primary clades of birds[END_REF] do not recover the falcon/buzzard clade, emphasizing the need for additional taxa. On the present data set the falconiforms (in the broad sense, including New World vultures) are not monophyletic, but we cannot distinguish yet between their being polyphyletic (diphyletic in this case) or paraphyletic. This latter case is potentially interesting from the ecological/life histories viewpoint in that many of the sea-and shorebirds may have diversified from carnivores (raptors) into a related niche in a marine environment. At present, the position of both the peregrine falcon and the stork are still locally unstable and improved taxon sampling is thus required.

Rooted tree

The incorporation of the brush turkey, the tyrant flycatcher and the lyrebird breaks up long unbranched edges situated deep in the avian tree. Therefore the next step is to identify what (if any) effect this has for locating the position of the root in the avian tree. Figure 2 is the rooted tree, using the six taxa outgroup constituted of two crocodilians, two turtles and two lizards. This reptilian outgroup joins with strong support between paleognaths and neognaths (Figure 2), and is thus consistent with most recent work on both nuclear genes (van Tuinen et al. 2000;[START_REF] Chubb | New nuclear evidence for the oldest divergence among neognath birds: the phylogenetic utility of ZENK (i)[END_REF][START_REF] Garcia-Moreno | Congruent avian phylogenies inferred from mitochondrial and nuclear DNA sequences[END_REF]) and mitochondrial genomes [START_REF] Paton | Complete mitochondrial DNA genome sequences show that modern birds are not descended from transitional shorebirds[END_REF][START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF]). Of the outgroup taxa, crocodilians are again closest to birds.

An important observation is that the addition of the outgroup does not make major disruptions to the ingroup tree as was previously the case with fewer taxa [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF]. As mentioned earlier, simulations [START_REF] Holland | Outgroup misplacement and phylogenetic inaccuracy under a molecular clock: A simulation study[END_REF] have shown that the addition of the outgroup can lead to disruption of the previously established relationships within the ingroup as it has been observed in placental mammal mitogenomic trees for example [START_REF] Lin | Four new mitochondrial genomes, and the stability of evolutionary trees of mammals[END_REF]. It is therefore pleasing that the basic ingroup tree (from the unrooted tree of Figure 1) is not altered when the outgroup is included, giving us more confidence in 28/8/08, page 14 of 28 the present rooting. As a result, the paleognath/neognath division is supported as well as the strict monophyly of passerines. As before, there is increased support for the chicken/duck grouping (Galloanseriforms), again in agreement with morphological [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF]), nuclear (van Tuinen et al. 2000;[START_REF] Chubb | New nuclear evidence for the oldest divergence among neognath birds: the phylogenetic utility of ZENK (i)[END_REF][START_REF] Garcia-Moreno | Congruent avian phylogenies inferred from mitochondrial and nuclear DNA sequences[END_REF] and mitochondrial [START_REF] Paton | Complete mitochondrial DNA genome sequences show that modern birds are not descended from transitional shorebirds[END_REF][START_REF] Harrison | Four new avian mitochondrial genomes help get to basic evolutionary questions in the Late Cretaceous[END_REF] datasets. Within the Neoaves, there is strong support for Passeriformes occupying a basal position, and the Conglomerati (Cracrafti) still holds up, though significant groups such as rails and pigeons are not yet represented and there are differences in predictions between [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF] and [START_REF] Cracraft | Phylogenetic relationships among modern birds (Neornithes)[END_REF].

There is one interesting difference between the birds-only results (Figure 2), and the tree derived from the alignment that includes the 6 outgroup taxa (Figure 1). This is that the first divergence is between shorebirds and raptors/ seabirds on the birds-only dataset, but the falconiforms (buzzard and falcon in particular) are deeper on the tree from the alignment containing the 6-taxon outgroup. This difference, especially the deeper divergence of shorebirds is worth following up, given the comment of [START_REF] Feduccia | Mesozoic aviary takes form[END_REF] that shorebirds appear to be a late Cretaceous lineage, and from the results of [START_REF] Slack | Early penguin fossils, plus mitochondrial genomes, calibrate avian evolution[END_REF] that discusses the agreement here between estimates of times of divergence from molecular data and existence of early fossil footprints of birds. However, some relationships within the Conglomerati remain unresolved with low bootstrap values except for the monophyly of Charadriiformes and for the putative grouping of Procellariformes with Gaviiformes, Sphenisciformes and Ciconiiformes (Figures 2 and3). The lack of conflicting signal for this latter grouping (see Figure 3) is interesting in that it argues against there being any strong systematic bias [START_REF] Phillips | Genome-scale phylogeny: sampling and systematic errors are both important[END_REF]).

Increased stability from breaking up long branches (edges).

A primary interest here is testing whether the improved taxon sampling (breaking up long branches within the avian tree) leads to improved stability. Qualitatively it appears so since the addition of brush turkey, tyrant flycatcher and lyrebird, has significantly increased the strength of the rooting between paleognaths and neognaths. To make this conclusion quantitative we deleted each of the 30 avian taxa in turn, and ran 100 bootstrap samples on each of the reduced 30 data sets, using ML. Thus each dataset had 29 birds and the 6 outgroup taxa. This is essentially a jackknife approach [START_REF] Lanyon | Detecting internal inconsistencies in distance data[END_REF], deleting taxa sequentially and measuring the effect [START_REF] Penny | The use of tree comparison metrics[END_REF]. For the 30 bootstrap runs, the outgroup was constrained so that the six outgroup taxa always grouped together, as well as the two turtles, the two crocodilians and the two lizards. There were no constraints on the avian tree. For each of the 30 datasets the percentage of trees from the 100 bootstrap samples were summed under the categories of paleo/neognath rooting, rooting within paleognaths, galloanseriform rooting, passerine rooting, or within the Neoaves generally (Table 1).

For both the galloanseriform and passerine cases the results include the rooting within, for example, galloanseriforms or between galloanseriforms and all other birds. 28/8/08, page 15 of 28 With phylogenetic trees (including our own studies) it is traditional to give the results first and subsequently think up explanations! Here we reverse the process and give our predictions first (Table 1, column 2) and then ran the bootstrap samples before comparing the predictions and the results. Because our theme has been that breaking up long branches improves the stability of the tree, we focus our predictions on how we expect the additional sequences to affect the bootstrap values for different rootings. The second column in Table 1 indicates our predictions on how the bootstrap values might change for each jackknife sample, relative to the bootstrap values with the full 30 bird dataset. The predictions are whether we expect that removal of a particular avian taxon will increase the bootstrap values for a competing placement of the root. For example, we expect that removing the brush turkey will lead to the root appearing significantly more often on the galloanseriform lineage, indicated as '>Ga'. Concomitably, there would have to be a reduction in cases where the root comes into the expected position between paleognaths and neognaths.

The magnitude of the effect is hard to predict, but based on prior experience (for example, [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF] we would expect bootstrap values to decrease by 10-30%, and possibly even more. Where it seemed that a smaller effect could occur we have added question marks, for example '>Ga??'. Note that to estimate the increased stability from adding a taxon, we are measuring the decrease in bootstrap values from removing the taxon. More than one value can occur in a column, for example in column 3, the first value is the paleognaths occurring as a monophyletic group; the second for the neognaths. Similarly, within the galloanseriforms, the root could come on the branch basal to the group, or within the chicken or the duck group. One aberrant bootstrap sample could affect several internal branches on the tree. For example, a '1+1+1' will usually be one bootstrap sample that changes three internal branches. Having given our predictions, the actual results are given in Table 1. Surprisingly, our predictions were relatively poor; there was virtually no loss of stability by removing any individual taxon. That is, the results were considerably more robust than we expected. Overall this implies that there is some cumulative effect from adding a range of taxa that break up long branches, leading to the increased stability. Nevertheless there are interesting individual effects especially evidenced when the rows are summed to give the 'total' effect of removing each taxon. Removal of the New Zealand wren (rifleman) had the largest effect, by which we infer that adding this taxon into the dataset contributed the most to stabilizing the tree. As expected, removing a deeper branching taxon had more effect than removing one member of a pair of more closely related taxa. For example, removing the brush turkey had more effect than deleting either the chicken or quail. Overall, removal of a passerine had a relatively large effect, a result that might be explained by their higher evolutionary rates. However, we were surprised that the removal of the penguin has also a significant effect, despite its relatively short branch and its internal 28/8/08, page 16 of 28 position in the avian tree. We had assumed that the problem was that the stork was relatively unstable on the tree, but we must consider penguin as a potentially difficult taxon.

In an additional analysis, we computed a Z-closure super-network [START_REF] Huson | Phylogenetic super-networks from partial trees[END_REF]), using SplitsTree 4.1 [START_REF] Huson | Application of phylogenetic networks in evolutionary studies[END_REF], from the 30 jackknife ML trees where each avian species was omitted in turn from the rooted tree (Figure 3). This method offers a graphical summary of the topologies of the 30 jackknifed trees and allows identification of the parts of the phylogeny which were in agreement for all the 30 trees with 29 avian taxa. Areas of conflict are represented as rectangles in the super-network, reflecting any instability caused by removing individual taxa. The results in Figure 3 are striking in that there is no incongruence detected with respect to the position of the root of the avian part of the tree. This again shows the lack of effect of removing any single taxon and illustrates that a robust rooting of the avian tree can be obtained by using an expanded number of mitochondrial genomes. In contrast, uncertainty is detected in three places within the avian tree corresponding to areas of instability identified previously (see Figure 2). The first is between the tinamou and the deeper ratites (ostrich and rhea) where the tree is not completely stable and therefore removing taxa has an effect. Similarly, there is a tendency for the rifleman to come onto the basal suboscine branch as represented by a cycle in Figure 3. However, the major effect was inside the Conglomerati (Cracrafti) with a series of boxes involving the raptors (including turkey vulture) and the sea birds and shore birds. Either the core raptors (falcon and buzzard) or the shore birds can come out basal in this Conglomerati group. This relatively unstable part of the avian tree is being studied further by sequencing additional mitochondrial genomes, including osprey, a forest falcon, and additional potential relatives of stork and penguin. However, although the variability within the Conglomerati (Cracrafti) is very interesting, it is not relevant to the question at hand of demonstrating the stability of the root of the avian tree.

Discussion

The long-term goal of this project is to use nuclear and mitochondrial sequences, together with fossil data, to test modes of macroevolution in the Late Cretaceous [START_REF] Penny | The rise of birds and mammals: Are microevolutionary processes sufficient for macroevolution[END_REF]. The primary aim of this study is to determine whether the breaking up of some long branches on the avian tree leads to agreement between nuclear and mitochondrial data on the position of the root. In this respect, the position of the root now appears in agreement between morphological, nuclear and mitochondrial data, and thus it is time to move on to other questions. Data from complete mitochondrial genomes obviously takes longer to obtain for each taxon than sequences from a single nuclear gene. This has lead to some false starts concerning the position of the avian root due in part to incomplete taxon sampling (Härlid and Arnason 1999;Mindell et al. 1999), though earlier analyses by [START_REF] Braun | Examining basal avian divergences with mitochondrial sequences: Model complexity, taxon sampling, and sequence length[END_REF] and [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF] indicated that the 28/8/08, page 17 of 28 paleognath/neognath division could not be rejected. There appears to have been an 'urban myth' that mitochondrial genomes could not recover the same avian root as morphological and nuclear data. In fact, the main problem was incomplete taxon sampling, a problem that is better solved by more data collection than by polemics. Indeed, it is hard to imagine theoretical reasons that would lead to different roots from different datasets. It is unlikely that the trees would be fundamentally different and therefore additional sequences and better methods of analysis are a more likely solution to the problem. We should all aim at improving data sets to test whether they lead to consensus.

With the major early divisions having been resolved, perhaps the next step on the avian tree is to provide resolution within Neoaves and especially within the Conglomerati (Cracrafti). For this specific question, breaking up long branches does appear to be an effective strategy, but obviously depends on appropriate taxa being available. When there was only a single suboscine (broadbill) and a single Falconiform (falcon) in the dataset, there was a strong tendency for them to come together (see discussion in [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF]).

However, with additional passerine being incorporated, we now have strong support for the respective monophyly of both Passeriformes and Conglomerati. The falcon and the stork have both been difficult to place in the mitogenomic tree in that both still appear to be uncertain about their final position. In early datasets, the falcon could even occur at the base of the passerines [START_REF] Slack | Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features[END_REF]. The addition of the buzzard has reduced the wandering of the falcon, but the falcon/buzzard grouping is relatively weak, and the falcon can go deeper within the neoaves. We are currently sequencing a forest falcon (Micrastur) to determine its effect because in [START_REF] Sibley | Phylogeny and classification of birds[END_REF] this was the deepest divergence among relatives of the falcon. The present data set has no species from within the proposed Metaves group of [START_REF] Fain | Parallel radiations in the primary clades of birds[END_REF], and thus we cannot yet comment on that hypothesis. Mitochondrial genomes from members of that group are being completed and then we will be able to test the predicted distinction of Neoaves into Metaves and Coronaves.

The problem of the early divisions of Neoaves is going to be difficult. [START_REF] Cracraft | Avian evolution, Gondwana biogeography and the Cretaceous-Tertiary mass extinction event[END_REF] proposed six unresolved groups, and this increased to nine in [START_REF] Cracraft | Phylogenetic relationships among modern birds (Neornithes)[END_REF]. It has been suggested that the early divergence of neoavian birds was an 'explosive radiation' (as just one example, see [START_REF] Poe | Birds in a bush: Five genes indicate explosive evolution of avian orders[END_REF].

However, for a radiation to be 'explosive' it requires both a rapid series of lineage divergences, combined with simultaneous morphological and/or ecological adaptations. It would not be sufficient just for divergences of lineages to be close together -that could occur very easily by a rapidly dispersing taxon even though the subsequent ecological and morphological divergences occurred many millions of years later. Such a delayed adaptation would scarcely be an 'explosive' radiation. The first aspect (rate of diversification of lineages) is best studied from molecular data (as is done here). The second, the timing of 28/8/08, page 18 of 28 adaptations to new environments/niches is perhaps better studied from the fossil record. As yet, in the present data we do not see any evidence whatsoever for an 'explosive radiation' of neoavian birds, even though the early divergences may have been relatively rapid in Neoaves. We are more cautious, and want to see data on the speed of morphological and ecological changes before coming to such dramatic conclusion about an explosive radiation. We prefer at present to consider the early diversification of Neoaves as an adaptive radiation, indicating that it is a relatively fast radiation but strictly according to known microevolutionary principles. Indeed, as mentioned earlier, one of our main goals is to determine the extent to which the processes of microevolution are sufficient to explain macroevolution. This is the theme behind our models of avian and mammal diversification in the Late Cretaceous [START_REF] Penny | The rise of birds and mammals: Are microevolutionary processes sufficient for macroevolution[END_REF], and whether (by comparing the human and chimpanzee genomes) there is any aspect of the human genome that is not a normal microevolutionary (genetic) process [START_REF] Penny | Our relative genetics[END_REF]). We think that the 'explosive radiation' should be to restrict to possible cases where normal microevolutionary process are clearly insufficient to account for macroevolution. To test such hypotheses we need a tree sufficiently stable in order that good timing estimates are possible, preferably on combined nuclear and mitochondrial data. All progress in this direction is welcomed.

28/8/08, page 23 of 28 Thomas, G.H., Wills, M.A. Székely, T., 2004. Phylogeny of shorebirds, gulls, and alcids (Aves: Charadri) from the cytochrome c gene: parsimony, Bayesian inference, minimum evolution and quartet puzzling. Mol. Phyl. Evol. 30,[516][517][518][519][520][521][522][523][524][525][526] Van Tuinen, M., Sibley, C. Hedges, S. B., 2000. The early history of modern birds inferred from DNA sequences of nuclear and mitochondrial ribosomal genes. Mol. Biol. Evol. 17, 451-457. Van Tuinen, M., Waterhouse, D., Dyke, G.J., 2004. Avian molecular systematics on the rebound: a fresh look at modern shorebird phylogenetic relationships. J. Avian Biol. 35,[191][192][193][194] 28/8/08, page 24 of 28

  Australian brush-turkey (Alectura lathami[Gray, 1831]) and superb lyrebird (Menura novaehollandiae [Latham, 1802]) samples came from Australia. Darryl Jones (Australian School of Environmental Studies, Griffith University, Brisbane) and Ian Owens (Queensland University) provided the brush-turkey, and Cathy Nock (Centre for Animal Conservation Genetics, Southern Cross University [Lismore campus], New South Wales) the lyrebird. The turkey vulture (Cathartes aura [Linnaeus, 1758]) came from Texas, USA and the fuscous flycatcher (Cnemotriccus fuscatus [Wied-Neuwied, 1831]) from Peru. Both samples were supplied by the Louisiana State University Museum of Natural Science Collection of Genetic Resources (sample numbers: turkey vulture LSUMNS B-17242, flycatcher LSUMNS B-7284).

  12S or 16S rRNA to tRNA-Leu(CUN): Turkey vulture and flycatcher (11.3 kb): 60°C, 8 min Gull, brush-turkey and lyrebird (9.3 kb): 53°C, 9.5 min and 2 x 54°C, 10 min, respectively Turkey vulture and flycatcher: Av1753F12S (25 nt): 5'-AAACTGGGATTAGATACCCCACTAT-3' 28/8/08, page 8 of 28 Gull, brush-turkey and lyrebird: Av3725F16S-LR (32 nt): 5-turkey (5.5 kb): 54°C, 5.5 and 7 min respectively; lyrebird (7.6 kb): 52°C, 8 min Av10647COIIIF (23 nt): 5'-TTTGAAGCAGCAGCCTGATAYTG-3' Av16137tProR (23 nt): 5'-ARAATRCCAGCTTTGGGAGTTGG-3' Cytb to tRNA-Met: Gull (6.2 kb): 53°C, 8 min Av15656CytbF (20 nt): 5'-AACCTGTTAGGRGAYCCAGA-3' Av5201tMetR (20 nt): 5'-CCATCATTTTCGGGGTATGG-3' tRNA-Leu(CUN) to 16S rRNA: Brush-turkey (7.4 kb) and lyrebird (8.5 kb): 55°C and 54°C respectively, 10 min Both: Av13063tLeuF-LR (38 nt): 5'-TGGTCTTAGGARCCATCTATCTTGGTGCAAMTCCAAGT-3' Brush-turkey: Av3782R16S (22 nt): 5'-CGGTCTGAACTCAGATCACGTA-3' Lyrebird: Av3797R16S (22 nt): 5'-CGACCTGGATTTCTCCGGTCTG-3' COIII to 16S rRNA: Turkey vulture (9.9 kb) and flycatcher (10.3 kb): Av10647COIIIF and Av3797R16S (60°C, 8 min)

Figure Captions Figure 1 .

 Captions1 Figure Captions

Figure 2 .

 2 Figure 2. Rooted tree using six outgroup taxa. The same coding as for Figure 1. Bootstrap values are shown; * is 100%.

Figure 3 .

 3 Figure 3. Phylogenetic super-network obtained by applying the Z-closure method to the 30 jackknife ML trees where each individual bird taxon has been removed in turn from the complete dataset. This figure represents a graphical summary of multiple phylogenies reconstructed from the different sets of taxa. It shows on which parts of the phylogeny all partial trees are in agreement (dichotomous parts) and where there exist contradicting signals (networking parts).

Table 1 .

 1 Predicted and actual effects on the position of the root from taxon removal

	species omitted	predicted	paleo/	Paleognaths	Galloanseres	Passerines	Neoaves	Total
			neognaths ‡	Pg	Ga	Pa
	None, all 30 birds	n.a.
	emu	-	100/99	-	1	-	-	1
	cassowary	-	99/97	-	2+1	1+1+1	-	6
	kiwi	-	100/97	-	1+1	1	1	4
	rhea	>Pg??	100/95	-	1+1	3+4x1	4	13
	ostrich	>Pg??	100/93	-	3+1	3+1	4	12
	tinamou	>Pg	100/91	-	1	2+2+2	8	15
	gs_tinamou	>Pg	100/95	-	3+1	1+1+1	2	9
	magpie goose	>Ga	100/94	-	3	1+1	3	8
	goose	-	100/98	-	1+1	1+1+1	1	6
	duck	-	99/96	1	2	-	2	5
	brush turkey	>Ga	100/92	-	7+3	-	1	11
	chicken	-	100/99	-	1+1	-	-	2
	quail	-	100/97	-	2	1+1	1	5
	rifleman	>Pa?	100/91	-	2	5+5+3	7	22
	broadbill	>Pa	100/94	-	3+1	2	3	9
	flycatcher	>Pa	98/96	2	-	2+1+1	4	10
	lyrebird	>Pa	99/93	1	3+1	3+2+2	4	16
	rook	>Pa??	100/94	-	4+3x1	1+1	2	11
	indigobird	>Pa??	100/93	-	3+1	1+2x1*	4	11
	falcon	-	99/97	1	3+2	-	-	6
	buzzard	>Oth??	100/96	-	-	2+1+1	4	8
	turkey vulture	-	99/94	-	2+1	1	4	8
	oystercatcher	-	100/95	-	2+1	1	3	7
	turnstone	-	100/97	-	2+3x1	1	1	7
	gull	-	99/98	1	1+1+1	-	-	4
	stork	-	100/98	-	1	1	1	3
	penguin	-	100/91	-	4+1	3+1+1	5	15
	albatross	-	100/98	-	1	-	1	2
	petrel	-	100/95	-	2	-	3	5
	loon	-	100/95	-	3	1+1	2	7
	‡ Expected position of the root from nuclear and morphological data.
	**crested tinamou joins rhea, inside ratites

Acknowledgements

We especially thank Dick Gill, Darryl Jones, Ian Owens, Cathy Nock, Fred Sheldon and Donna Dittman for the samples. Tim White and Warwick Allen assisted with computer programs for running analyses on the Helix supercomputer and Gillian Gibb for considerable assistance in manuscript preparation. F.D. acknowledges the support of a Lavoisier Postdoctoral Grant from the French Ministry of Foreign Affairs.

Both the New Zealand Marsden Fund and the Centre for Research Excellence funds supported the work.

28/8/08, page 28 of 28


Weili Wang

Dominique Agustin
email: dominique.agustin@iut-tlse3.fr.

Rinaldo Poli

Influence of ligand substitution on molybdenum catalysts with tridentate Schiff base ligands for the organic solvent-free oxidation of limonene using aqueous TBHP as oxidant

Keywords: Epoxidation, Molybdenum, Limonene, Solvent-free process, Ligand effect

may come

Influence of ligand substitution on molybdenum catalysts with tridentate Schiff base ligands for the organic solvent-free oxidation of limonene using aqueous TBHP as oxidant

Introduction

The use of natural feedstock as chemical source is of constantly increasing interest. 1 Terpenes, due to their diverse structures, represent an interesting available ore for several chemical transformations. 2 As an example of terpene, limonene (Lim) is an inexpensive substrate obtained as waste from the orange juice industry. [START_REF] Burdock | Fenaroli's handbook of flavor ingredients[END_REF]4 The limonene epoxides (LimOs) and diols (LimDs), obtained by oxidation of Lim (Scheme 1), are added-value renewable-based compounds, [START_REF] Ciriminna | [END_REF]6 with applications in the synthesis of fragrances, flavors, food additives, or compounds with biological and therapeutic activity. 7,8 Both LimO isomers, cis-LimO and trans-LimO, find applications in medicine, 9 metal coatings, varnishes, printing inks, and in natural based polymers 10 but exist in Nature in insufficient quantities to satisfy the needs. 11 Both LimD isomers, eq-LimD and ax-LimD, generated from the LimOs by epoxide ring opening with water, 12 are also precursors of interest for the pharmaceutical industry. 13 Scheme 1. Oxidation of limonene and potential LimO and LimD products.

The Lim epoxidation may occur at the internal and/or terminal alkene moiety and the presence of water facilitates the epoxide ring opening. In most cases, the main products are cis-LimO and trans-LimO deriving from the regioselective epoxidation of the endocyclic double bond, which is more reactive because of its higher electron density. The terms cis and trans, for this family of products, refer to the relative position of the Me and iPr substituents relative to the cyclohexane ring. Although four different stereoisomers may result in principle from the ring opening of cis-LimO and trans-LimO, only two of them, the diequatorial cis isomer (eq-LimD) and the diaxial trans isomer (ax-LimD), have been obtained by this procedure as shown in Scheme 1. Traces of other compounds such as carvone and carveol, resulting from allylic oxidation, may also be observed.

For the non-catalyzed processes leading to endocyclic epoxidation, organic peracids typically yield a 1:1 cis/trans-LimO mixture, 14 whereas N-sulfonyloxaziridines show stereoselectivities in favor of cis-LimO. 15 In the extensive literature on metal catalyzed Lim epoxidation, the cis/trans stereoselectivity is rarely indicated. A variety of mononuclear molybdenum catalysts in combination with the TBHP oxidant were reported to yield equimolar amounts of the two isomers, 16 while a greater stereoselectivity in favor of the trans isomer has been recently reported for a dinuclear Mo catalyst. 16b A mononuclear iron catalyst yields predominantly the cis-LimO product. 17 Concerning the selectivity of the ring opening step leading from LimO to LimD, it has been reported that both cis-and trans-LimO lead to the same ax-LimD product under acidic conditions (pH 3), 18 which was attributed to a selective axial nucleophilic attack that can be rationalized by the Fürst-Plattner rule. 19 However, at an only slightly higher pH (4), only cis-LimO is opened stereospecifically to yield ax-LimD while trans-LimO remains unreacted. 18 Further studies have shown that the stereospecific opening of the cis-LimO isomer to ax-LimD also occurs in the presence of lanthanide 20 or Mo 21 catalysts. This ring opening selectivity has been exploited in some cases as a way to generate isomerically pure ax-LimD and/or to kinetically accumulate pure trans-LimO from the isomer mixture. The opposite preference for the trans-LimO ring opening has been accomplished by use of other conditions such as the use of nucleophilic amines, 22 yielding again a trans-diaxial addition product, or water in the presence of mercuric reagents at pH 7, which leads, after treatment of an alkoxymercury intermediate with NaBH4, to the stereospecific generation of the eq-LimD product. 18,23 The LimO ring opening has also been explored with the use of enzymes.

Using the Rhodotorula glutinis hydrolytic enzyme, optically pure (1S,2R,4S)-cis-LimO was more rapidly and stereospecifically hydrolyzed to (1S,2R,4S)-ax-LimD, whereas optically pure (1S,2R,4R)-trans-LimO was opened more slowly to yield a mixture of (1R,2R,4R)-eq-LimD (65%) and (1S,2S,4R)-ax-LimD (35%). 24 By direct enzymatic oxidation of Lim using a chloroperoxidase enzyme from Caldariomyces fumago, the LimD products were directly generated without detection of the LimO intermediates. 25 Only ax-LimD was selectively obtained when the procedure was carried out in the absence of KCl and proposed to result from the stereospecific opening of cis-LimO, whereas addition of KCl yields a mixture of ax-LimD and eq-LimD, proposed to derive from a mixture of cis-LimO and trans-LimO. These enzymatic studies are apparently the only ones highlighting the direct formation of eq-LimD by LimO ring opening.

It is also pertinent to highlight that the production of LimO and LimD has typically been accomplished using non-green conditions (oxidants such as peracids 14 and/or organic solvents; 26 or use of mercuric reagents for separation as highlighted above). In order to develop a greener access to these compounds, safer oxidants and non-hazardous conditions are required. 27 Recently, we have reported the solvent-free epoxidation of cyclooctene and cyclohexene catalyzed by different molybdenum 28 and vanadium 29 complexes or by polyoxometalates. 30 The concept was then further applied to terpenic compounds. 31 A particular class of compounds that we have explored in some detail is the salicylidene amino(thio)phenolate family shown in Scheme 2. It was therefore of interest to apply these precatalysts to the Lim epoxidation under simpler and cleaner operating conditions, without organic solvents and with aqueous TBHP as oxidant. Preliminary results on the greener epoxidation of Lim using a few related Mo catalysts, yielding a mixture of cis-LimO, trans-Limo and ax-LimD, have been included in a recent publication. 31b Here, we report a fuller investigation demonstrating how the ligand nature, particularly the presence of the sulfur atom or an OH group in the R1 position, affects the selectivity and the kinetics of the various transformations.

Scheme 2. Pre-catalysts used in this study

Results and discussion

Catalyzed limonene oxidation

The results of initial catalytic tests, run with all the pre-catalysts shown in Scheme 2, are collected in Table 1. In the absence of catalyst (run 1), aqueous TBHP leads only to a 7.6% conversion at 80°C after 4 hours, with detected traces of LimOs and LimDs. Using a 0.5% catalyst loading, Lim conversions of 79% (after 4 hours with [MoO2(SAP)]2, run 2) and 93% (after 30 minutes with [MoO2(SATP)]2, run 3) demonstrated the catalytic activity of both complexes as well as the greater activity of the sulfur-containing complex. The conversion monitoring for runs 1, 2 and 3 is shown in Figure 1. The much higher activity for the catalyst with the ONS coordination sphere in Lim oxidation parallels that observed previously for the epoxidation of cyclooctene. 28b b Select = nproduct formed/nLim transformed obtained from GC analysis of the organic phase. c TOF is calculated using the time interval with maximum slope in the conversion plot. Monitoring the evolution of the various products in runs 2 and 3 revealed additional interesting features (see Figure 2). For the experiment with [MoO2(SAP)]2 (Figure 2, left), the two LimO isomers are initially produced in a ca. 1:1 ratio (13.8% and 15.3% after 60 min), but subsequently the amount of cis-LimO slowly decreased (2.6% after 4 h) while that of trans-LimO continued to increase (19.8% after 4 h). These data indicate that the rate of cis-LimO ring opening exceeds that of its generation at longer reaction times, while trans-LimO opens the ring more slowly. At the same time, the ax-LimD amount increased from 4.7% to 26.5% The initial observation of ax-LimD as the predominant diol in solution while only the cis-LimO intermediate is being consumed at significant rate suggests that the faster ring opening of cis-LimO leads selectively to this product. This observation is consistent with previous literature reports. On the other hand, the slower generation of eq-LimD suggests that this isomer results from the slower ring opening of trans-LimO. We are unaware of other contributions where the direct generation of eq-LimD from metal-catalyzed Lim epoxidation has been highlighted (see also the Introduction). Whether the ring opening of trans-LimO is selective or leads to both diol products cannot be established from these data (see Scheme 3), but will be detailed later. The mass balance (cis-LimO + trans-LimO + eq-LimD + ax-LimD + residual Lim) is low in both experiments: 80% after 2 h and 70% after 4 h for run 2; 56% after 0.5 h for run 3. This loss of material can be attributed to the further oxidative transformations of the LimD isomers to non-volatile products. Traces of carvone and carveol, plus other unidentified products, were also visible in the gas-chromatogram for both runs and were not quantified but could be estimated as < 5 %, hence could not account for the lack of mass balance. The final reaction mixture consisted of a single phase in each case, presumably because the organic compounds and the water originating from the TBHP reagent solution, which generates two phases at the beginning of the reaction, are progressively compatibilized by the LimD and tBuOH products.

The kinetic profiles for the same oxidation processes in the presence of the precatalysts [MoO2L a-c ]2 (runs 4-6) are shown in Figure 3 in comparison with those of runs 1 and 2. The conversion after 4 h was very good for each catalyst (see Table 1), in the order [MoO2L a ]2 complexes was similar (ca. 1:1, see Figure 4) to that observed for the experiment with [MoO2(SAP)]2 (Figure 2), whereas the most active [MoO2L a ]2 catalyst yields an initial cis/trans ratio of ca. 1:2 (Figure 4). Once produced, the LimO isomers are consumed to yield the two LimD products, mainly the axial isomer. The ring opening data are also in line with the behavior reported above for the parent [MoO2(SAP)]2 pre-catalyst: rapid for cis-LimO with selective formation of ax-LimD and slower for trans-LimO (see further details later). In addition, the LimD selectivity correlates with the Lim conversion, with the relative amount of the minor eq-LimD product increasing with the rate of Lim conversion. These data further confirm that the epoxide ring opening process is, like the epoxidation, metal-catalyzed and that the better epoxidation catalyst is also a better ring opening catalyst. The same trends for the epoxide/diol selectivity vs. the rate of conversion, although quantitatively less pronounced, have also been previously observed for the oxidation of cyclohexene catalyzed by the same ONO complexes but no conclusion concerning the epoxide ring opening catalysis was made. 28b The Green metrics for these catalyzed reactions have been analyzed

(90%) > [MoO2(SAP)]2 (79%) ~ [MoO2L c ]2 (77%) > [MoO2L b ]2 (68%).
in terms of the influence of catalyst and temperature on the LimO and LimD production (See SI). In order to better understand the catalytic epoxidation and epoxide ring opening for this substrate, additional experiments were carried out for the more active [MoO2(SATP)]2 systems at variable temperature and catalyst loading (Table 2 andFigures 5-7). A temperature increase without catalyst leads to greater Lim transformation although the conversion and selectivity remained poor (runs 9 and 12). With 0.5% molar ratio [MoO2(SATP)] and 2 eq TBHP per substrate in the 30-80°C range, the Lim conversion after 4 h increased dramatically, from 36% at 30°C after 5 h to 93% at 80°C after 30 min (runs 8, 11 and 15; Figure 5). At both 50°C (runs 6-11) and 80°C (runs 12-15) the conversion becomes faster as expected upon increasing the catalyst loading, see also Figure 6. The effect of the TBHP/substrate ratio has also been investigated, both at 50°C and at 80°C, with a 0.5% loading of the [MoO2(SATP)] catalyst (Table 3). Lowering the TBHP amount leads, as expected, to a slower conversion (cf. run 16 with run 11 of Table 2 and run 17 with run 15 of Table 2). It is of interest to note that this also results in a slight decrease of the eq-LimD selectivity and an increase of the ax-LimD selectivity. b Select = nproduct formed/nLim transformed obtained from GC analysis of the organic phase. c TOF is calculated using the time interval with maximum slope in the conversion plot.

Catalyzed limonene oxide ring opening

As shown in the previous section, the Lim epoxidation produced LimOs and/or LimDs in a catalyst-and temperature-dependent process and there is clear evidence that the more active epoxidation catalysts (e.g. the SATP complex) also have higher activity in the ring opening process. It was therefore of interest to carry out more detailed investigations on the catalyzed epoxide ring opening process. For this purpose, both a commercial LimO isomer mixture (cis/trans = 47:53) and the pure individual isomers have been investigated (Table 4). (cf. runs 19 and 20). The greater catalytic activity of the ONS ligand in ring opening is confirmed by the study of the isomerically pure cis- LimO (cf. run 22 and 23), which results in the stereospecific production of ax-LimD. Only the most active [MoO2(SATP)]2 catalyst was tested in the opening of pure trans-LimO (run 24): in spite of the more sluggish ring opening of this isomer, quantitative conversion could be achieved after 1.5 h. This run confirms that the eq-LimD isomer results from the opening of trans-LimO, but the product ratio is still in favor of the ax-LimD isomer. The mass balance suggests that either other reactions also occur, or that the LimD products are partially transferred in aqueous phase.

The separate investigations of pure cis-LimO and trans-LimO provide additional useful information on the stereochemistry of the ring opening process. None of the eq-LimD was generated by the ring opening of cis-LimO, either with or without catalyst (runs 21-23). Thus, both non-catalyzed and catalyzed cis-LimO ring openings lead stereospecifically to the ax-LimD isomer, in agreement with previous reports. 18,21,24 However, the ring opening of pure trans-LimO (run 24) provides a mixture of ax-LimD and eq-LimD in a ca. 4:3 ratio. To the best of our knowledge, this is the first report of the metal-catalyzed opening of trans-LimO.

Interestingly, the stereoselectivity of this Mo-catalyzed ring opening process is quite similar to that reported for the opening catalyzed by the hydrolytic enzyme Rhodotorula glutinis (65:35). 24 In terms of mechanistic interpretation, the generation of the eq-LimD product cannot result by an effect of the Mo complex identical to that proposed for Hg 2+ in the selective generation of this diol from trans-LimO, for two reasons: i) the Hg 2+ reagent accelerates the opening of trans-LimO relative to cis-LimO, whereas the Mo catalyst leads to faster opening of the cis isomer; ii) the Hg 2+ -promoted opening of the trans-LimO is stereospecific, leading solely to eq-LimD, whereas the Mo catalyst generates a mixture of both LimD isomers. Indeed, the action of Hg 2+ was rationalized though a conformational change induced by the interaction between the soft Hg 2+ ion and the isopropenyl substituent, which is not possible for the Lewis-acidic Mo VI center because this d 0 metal ion has no affinity toward C=C unsaturations. We can rather invoke a mechanistic scheme with formation of Mo VI

Conclusions

Lim epoxidation is catalyzed very efficiently by [MoO2L]2 with salicylidene amino(thio)phenolate ligands, particularly [MoO2(SATP)]2. These complexes promote not only the Lim regioselective endocylic epoxidation to yield both cis-and trans-LimO isomers in a kinetically controlled 1:1 ratio, but also the subsequent epoxide ring opening in the presence of water to obtain a mixture of ax-and eq-LimD. The high activity of the [MoO2(SATP)]2 precatalyst promotes the efficient ring opening of the less reactive trans-LimO isomer, which is shown to proceed unselectively to a mixture of ax-and eq-LimD by analogy with a previous report of hydrolytic enzymatic ring opening, whereas the more reactive cis-LimO is stereospecifically opened to ax-LimD.

Experimental part

Materials and methods

All manipulations were carried out in air. Water was deionized twice before use. Organic solvents (ethanol, methanol, diethylether: synthesis grade, Aldrich) were employed as received without any purification. All molybdenum complexes were synthesized as previously described. 28a-c Limonene (98% Aldrich), the LimO mixture (Aldrich cis/trans 47/53, 98%), ax-LimD (Aldrich, 98%)) and TBHP (70% in water, ACROS) were used as received. The pure cis-LimO, trans-LimO and eq-LimD were synthesized according to literature procedure. 18,22,23 The catalytic reactions were monitored by gas chromatography on an Agilent 6890A chromatograph equipped with an FID detector, a HP5-MS capillary column (0.30 m x 0.25 mm x 0.25 m) and an autosampler, or on a Fisons GC 8000 chromatograph equipped with an FID detector and with a SPB-5 capillary column (30 m x 0.32 mm x 0.25 m). Authentic samples of all reactants and products were used for calibration. The Lim conversion and the formation of LimOs and limDs were calculated from the calibration curves (r 2 = 0.999) and an internal standard.

Catalytic Procedure.

In a typical experiment, limonene (1 equiv) and catalyst (x equiv, see tables) were mixed together in air in a round bottom flask. Acetophenone was added as internal standard. The flask was then immersed in a thermostated bath set at the desired temperature (see Tables )   and magnetically stirred. After thermal equilibration, aqueous THBP (70% in water, y equiv see tables) was added to the mixture, starting the reaction. Samples were periodically withdrawn and quenched by the addition of MnO2, followed by the addition of diethylether and removal of the manganese oxide and residual water by filtration through silica before GC analysis.

Figure 1 .

 1 Figure 1. Kinetic profiles for limonene oxidation: without catalyst (), with [MoO2(SAP)]2 (♦), with [MoO2(SATP)]2 (■). [Mo]/limonene/TBHP = 0.5/100/200, T = 80°C.

  of eq-LimD were detected initially and significant amounts are only formed after 3h.

Figure 2 .

 2 Figure 2. Kinetic profile for the limonene oxidation catalyzed by [MoO2(SAP)]2 (left), [MoO2(SATP)]2 (right): [Mo]/limonene/TBHP = 0.5/100/200, T = 80°C. Mass balance (…), limonene (□),cis-LimO (▲),trans-LimO (),eq-LimD (•),ax-LimD ().

Scheme 3 .

 3 Scheme 3. Selectivities for the LimO ring opening

  The [MoO2L a ]2 complex (R1 = OH) exhibited a much higher activity than the other pre-catalysts. The initial cis/trans ratio for the LimO intermediates for the experiments with the [MoO2L b ]2 and [MoO2L c ]2

Figure 3 .Figure 4 .

 34 Figure 3. Kinetic profile for limonene oxidation: without catalyst (-), with [MoO2(SAP)]2 (■), with [MoO2L a ]2(▲), with [MoO2L b ]2 (•), with [MoO2L c ]2(♦). [Mo]/limonene/TBHP = 0.5/100/200, reaction time = 4 h, T = 80°C.

Figure 5 .Figure 6 .Figure 7 .

 567 Figure 5. Influence of temperature on the [MoO2(SATP)]2-catalyzed limonene oxidation: 30°C (), 50°C (), 60°C (□), 80°C (). Conditions: [Mo]/limonene/TBHP = 0.5/100/200.

  [Mo]/limonene/TBHP = 0.5/100/200; T = 80°C. b Select = nproduct formed/nLimO(cis+trans) transformed obtained from GC analysis of the organic phase. c TOF is calculated using the time interval with maximum slope in the conversion plot. d Commercially available sample (cis/trans = 47/53). The epoxidation of the trans/cis LimO mixture confirms that cis-LimO is opened faster than trans-LimO, whether in the presence or absence of catalyst. The rate of the transformation increases in the order: no catalyst (run 18) < [MoO2(SAP)]2 (run 19) < [MoO2(SATP)]2 (run 20). Both LimO isomers are opened faster by the more active [MoO2(SATP)]2 catalyst

  Scheme 4. Proposed mechanism for the Mo-catalyzed ring opening of the LimO isomers.

Table 1 .

 1 Oxidation of limonene in the presence of various Mo catalysts. a

	Run	Catalyst	Time (h)	Lim Conv (%)	Selectivity (%) b LimO LimD Cis trans eq ax	TON	Highest TOF (h -1 ) c
	1	-	4	7.6	5.3 14.5	0.4	6.6	-	-
	2	[MoO2(SAP)]2	4	79	14.6 25.1	2.3 33.5 158	130
	3	[MoO2(SATP)]2	0.5	93	0	0	12.4 40.6 185	371
	4	[MoO2L a ]2	4	90	2.1	4.2	4.1 44.3 180	308
	5	[MoO2L b ]2	4	68	19.0 29.0	1.3 30.3 136	77
	6	[MoO2L c ]2	4	77	10.9 20.8	3.6 35.7 154	110

a Reaction conditions: [Mo]/limonene/TBHP = 0.5/100/200, T = 80°C.

Table 2

 2

	. Influence of temperature and catalyst loading for limonene epoxidation catalyzed by
	[MoO2(SATP)]2. a
	Run	T (°C)	time (h)	X (%)	Lim Conv. (%)	Selectivity (%) b LimO LimD Cis trans eq	ax	TON	TOF (h -1 ) c
	7 8	30	5	0 0.5	3.2 36	9.4 3.7	9.4 32.2	1.6 1.7 35.3 5.6	-70	-26
	9			0	5.3	10.4	0.6	0.6	5.8
	10	50	5	0.1	62.5	0.3	20.3	4.6 42.2 627	312
	11			0.5	83	0.1	10.4	5.8	50	166	161
	12		4	0	7.7	5.2	14.3	0.4	6.5
	13 14	80	2 0.5 0.25 0.1	85 87	0 0	0 0	7.9 72.9 850 11.4 46.3 348	1042 1743
	15		0.5	0.5	93	0	0	12.4 40.6 185	371

a [Mo]/limonene/TBHP = x/100/200. b b Select = nproduct formed/nLim transformed obtained from GC analysis of the organic phase. c TOF is calculated using the time interval with maximum slope in the conversion plot.

Table 3 .

 3 Influence of the TBHP amount on the [MoO2(SATP)]2-catalyzed limonene epoxidation. a

	Run	T (°C)	Time (h)	y	Conversion (%)	Selectivity (%) b LimO LimD cis trans eq ax	TON	TOF c (h -1 )
	16	50	5	110	61	0	4.9	9.5 58.5 123 109
	17	80	0.5 150	89	0	0	12.2 47.4 178 357

a Conditions: [Mo]/limonene/TBHP = 0.5/100/y. Reaction time = 4 h. T = 50°C.

Table 4 -

 4 Ring opening of limonene oxides catalyzed by molybdenum complexes. a

	Run substrate	Time (h)	catalyst	Conv (%)	LimD selectivity b (%)	TON	TOF (h -1 ) c
					cis	trans	eq	ax
	18 LimO 19 mixture d 20	4 4 0.5 [MoO2(SATP)]2 No catalyst [MoO2(SAP)]2	60.7 93.3 100	12.0 11.7 58.7 -26.2 2.6 58.5 119 45 -100 12.1 45.0 200 400
	21		4	No catalyst	13.5		0	6.4 -	-
	22	cis-LimO	4	[MoO2(SAP)]2	26.9		0	28.4 52	27
	23		0.5 [MoO2(SATP)]2	100		0	25.3 200 400
	24 trans-LimO	1.5 [MoO2(SATP)]2		100	15.8 20.8 200 130
	a

Acknowledgements

We acknowledge the Centre National de la Recherche Scientifique (CNRS) and the Institut Universitaire de France (IUF) for financial support, the Université Paul Sabatier and its Institut Universitaire Technologique for the facilities and the Chinese Scholarship Council for the fellowship of W.W. No.201208370239