DNA barcoding a unique avifauna: an important tool for evolution, systematics and conservation.

BACKGROUND: DNA barcoding utilises a standardised region of the cytochrome c oxidase I (COI) gene to identify specimens to the species level. It has proven to be an effective tool for identification of avian samples. The unique island avifauna of New Zealand is taxonomically and evolutionarily distinct. We analysed COI sequence data in order to determine if DNA barcoding could accurately identify New Zealand birds. RESULTS: We sequenced 928 specimens from 180 species. Additional Genbank sequences expanded the dataset to 1416 sequences from 211 of the estimated 236 New Zealand species. Furthermore, to improve the assessment of genetic variation in non-endemic species, and to assess the overall accuracy of our approach, sequences from 404 specimens collected outside of New Zealand were also included in our analyses. Of the 191 species represented by multiple sequences, 88.5% could be successfully identified by their DNA barcodes. This is likely a conservative estimate of the power of DNA barcoding in New Zealand, given our extensive geographic sampling. The majority of the 13 groups that could not be distinguished contain recently diverged taxa, indicating incomplete lineage sorting and in some cases hybridisation. In contrast, 16 species showed evidence of distinct intra-species lineages, some of these corresponding to recognised subspecies. For species identification purposes a character-based method was more successful than distance and phylogenetic tree-based methods. CONCLUSIONS: DNA barcodes accurately identify most New Zealand bird species. However, low levels of COI sequence divergence in some recently diverged taxa limit the identification power of DNA barcoding. A small number of currently recognised species would benefit from further systematic investigations. The reference database and analysis presented will provide valuable insights into the evolution, systematics and conservation of New Zealand birds.

Bioacoustic and multi-locus DNA data of Ninox owls support high incidence of extinction and recolonisation on small, low-lying islands across Wallacea.

Known for their rich biodiversity and high level of endemism, the islands of Wallacea serve as natural laboratories for the study of spatio-temporal evolution and patterns of species diversification. Our study focuses on the owl genus Ninox, particularly the Southern Boobook (N. novaeseelandiae) and Moluccan Boobook (N. squamipila) complexes, which are widely distributed across Australasia. We conducted bioacoustic and multi-locus DNA analyses of 24 Ninox owl taxa to evaluate relationships and levels of divergence within the two complexes and ultimately assess the relationship between patterns of taxonomic differentiation and bioclimatic factors. We found that taxa that are vocally and/or genetically distinct from populations on the Australian mainland are found on islands that are significantly larger and higher in altitude than taxa that are vocally and/or genetically indistinct from populations on the Australian mainland. This pattern suggests that taxa occurring on small, low-lying Wallacean islands are likely to be recent colonisers that have dispersed from Australia. Overall, our observations demonstrate that the genus Ninox is likely to have colonised the Wallacean region multiple times as small, low-lying islands undergo frequent extinction, whereas populations on large and high-altitude islands are more resilient.

The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.

Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase/prokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A(2), sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., vampire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.

Early evolution of the venom system in lizards and snakes.

Among extant reptiles only two lineages are known to have evolved venom delivery systems, the advanced snakes and helodermatid lizards (Gila Monster and Beaded Lizard). Evolution of the venom system is thought to underlie the impressive radiation of the advanced snakes (2,500 of 3,000 snake species). In contrast, the lizard venom system is thought to be restricted to just two species and to have evolved independently from the snake venom system. Here we report the presence of venom toxins in two additional lizard lineages (Monitor Lizards and Iguania) and show that all lineages possessing toxin-secreting oral glands form a clade, demonstrating a single early origin of the venom system in lizards and snakes. Construction of gland complementary-DNA libraries and phylogenetic analysis of transcripts revealed that nine toxin types are shared between lizards and snakes. Toxinological analyses of venom components from the Lace Monitor Varanus varius showed potent effects on blood pressure and clotting ability, bioactivities associated with a rapid loss of consciousness and extensive bleeding in prey. The iguanian lizard Pogona barbata retains characteristics of the ancestral venom system, namely serial, lobular non-compound venom-secreting glands on both the upper and lower jaws, whereas the advanced snakes and anguimorph lizards (including Monitor Lizards, Gila Monster and Beaded Lizard) have more derived venom systems characterized by the loss of the mandibular (lower) or maxillary (upper) glands. Demonstration that the snakes, iguanians and anguimorphs form a single clade provides overwhelming support for a single, early origin of the venom system in lizards and snakes. These results provide new insights into the evolution of the venom system in squamate reptiles and open new avenues for biomedical research and drug design using hitherto unexplored venom proteins.

Functional and structural diversification of the Anguimorpha lizard venom system.

Venom has only been recently discovered to be a basal trait of the Anguimorpha lizards. Consequently, very little is known about the timings of toxin recruitment events, venom protein molecular evolution, or even the relative physical diversifications of the venom system itself. A multidisciplinary approach was used to examine the evolution across the full taxonomical range of this ∼130 million-year-old clade. Analysis of cDNA libraries revealed complex venom transcriptomes. Most notably, three new cardioactive peptide toxin types were discovered (celestoxin, cholecystokinin, and YY peptides). The latter two represent additional examples of convergent use of genes in toxic arsenals, both having previously been documented as components of frog skin defensive chemical secretions. Two other novel venom gland-overexpressed modified versions of other protein frameworks were also recovered from the libraries (epididymal secretory protein and ribonuclease). Lectin, hyaluronidase, and veficolin toxin types were sequenced for the first time from lizard venoms and shown to be homologous to the snake venom forms. In contrast, phylogenetic analyses demonstrated that the lizard natriuretic peptide toxins were recruited independently of the form in snake venoms. The de novo evolution of helokinestatin peptide toxin encoding domains within the lizard venom natriuretic gene was revealed to be exclusive to the helodermatid/anguid subclade. New isoforms were sequenced for cysteine-rich secretory protein, kallikrein, and phospholipase A(2) toxins. Venom gland morphological analysis revealed extensive evolutionary tinkering. Anguid glands are characterized by thin capsules and mixed glands, serous at the bottom of the lobule and mucous toward the apex. Twice, independently this arrangement was segregated into specialized serous protein-secreting glands with thick capsules with the mucous lobules now distinct (Heloderma and the Lanthanotus/Varanus clade). The results obtained highlight the importance of utilizing evolution-based search strategies for biodiscovery and emphasize the largely untapped drug design and development potential of lizard venoms.

A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus.

The predatory ecology of Varanus komodoensis (Komodo Dragon) has been a subject of long-standing interest and considerable conjecture. Here, we investigate the roles and potential interplay between cranial mechanics, toxic bacteria, and venom. Our analyses point to the presence of a sophisticated combined-arsenal killing apparatus. We find that the lightweight skull is relatively poorly adapted to generate high bite forces but better adapted to resist high pulling loads. We reject the popular notion regarding toxic bacteria utilization. Instead, we demonstrate that the effects of deep wounds inflicted are potentiated through venom with toxic activities including anticoagulation and shock induction. Anatomical comparisons of V. komodoensis with V. (Megalania) priscus fossils suggest that the closely related extinct giant was the largest venomous animal to have ever lived.

Novel venom proteins produced by differential domain-expression strategies in beaded lizards and gila monsters (genus Heloderma).

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.

Multilocus analysis of a taxonomically densely sampled dataset reveal extensive non-monophyly in the avian family Locustellidae.

The phylogeny of most of the species in the avian passerine family Locustellidae is inferred using a Bayesian species tree approach (Bayesian Estimation of Species Trees, BEST), as well as a traditional Bayesian gene tree method (MrBayes), based on a dataset comprising one mitochondrial and four nuclear loci. The trees inferred by the different methods agree fairly well in topology, although in a few cases there are marked differences. Some of these discrepancies might be due to convergence problems for BEST (despite up to 1×10(9) iterations). The phylogeny strongly disagrees with the current taxonomy at the generic level, and we propose a revised classification that recognizes four instead of seven genera. These results emphasize the well known but still often neglected problem of basing classifications on non-cladistic evaluations of morphological characters. An analysis of an extended mitochondrial dataset with multiple individuals from most species, including many subspecies, suggest that several taxa presently treated as subspecies or as monotypic species as well as a few taxa recognized as separate species are in need of further taxonomic work.

A multigene phylogeny examining evolutionary and ecological relationships in the Australo-papuan wrens of the subfamily Malurinae (Aves).

Nucleotide sequences from four mitochondrial genes and three nuclear introns were used to examine phylogenetic relationships within the Australo-papuan fairy-wrens (Passeriformes: Maluridae: Malurinae). A well-resolved and well-supported phylogenetic hypothesis of all species in the subfamily was generated. The tree contained three clades corresponding to groups with similar plumages previously identified in earlier studies: the "bi-color," "blue," and "chestnut-shouldered" groups. The genus Malurus was not monophyletic -Malurusgrayi formed a clade with two New Guinean genera Sipodotus and Clytomyias. We recommend M. grayi be reclassified into the genus Chenorhamphus Oustalet 1898. One other taxonomic change is recommended based on the large genetic distance between the two subspecies of Chenorhamphus grayi - the elevation of C. g.campbelli to specific status (= C. campbelli). Although the family Maluridae appears to have had its origins in Australia, the DNA data supports a New Guinean origin for the Malurini (Sipodotus, Clytomyias, Chenorhamphus, Malurus).

Mitochondrial and nuclear DNA phylogenies reveal a complex evolutionary history in the Australasian robins (Passeriformes: Petroicidae).

The Australasian robins (Petroicidae) comprise a relatively homogeneous group of small to medium-sized insectivorous birds. Their center of diversity is Australia and New Guinea (40 species) but seven species have managed to colonize geographically distant islands such as Tanimbar, New Britain, New Zealand, New Caledonia, Norfolk Island, Vanuatu, Solomon Islands, Fiji and Samoa. To resolve the evolutionary relationships within the Petroicidae, we here present the results of a phylogenetic analysis of sequence data from two mitochondrial genes (ND2, CO1) and one nuclear intron (ß-Fibrinogen intron 5) for all 14 genera and 40 of the 46 currently recognized species. All phylogenetic analyses identified six primary lineages, treated here as subfamilies, within the Petroicidae: (1) Eopsaltriinae comprising Eopsaltria (excluding E. flaviventris), Tregellasia, Peneothello, Melanodryas, Poecilodryas and Heteromyias; (2) Drymodinae comprising Drymodes; (3) Microecinae comprising Microeca, Monachella and Eopsaltria flaviventris; (4) Petroicinae comprising Petroica and Eugerygone; (5) Pachycephalopsinae comprising Pachycephalopsis; and (6) Amalocichlinae comprising Amalocichla. The genera Eopsaltria, Microeca, Peneothello and Poecilodryas were found to be paraphyletic. Based on assessments of phylogenetic branching patterns and/or DNA divergence it also was apparent that Eopsaltriaaustralis, Tregellasialeucops, Melanodryascucullata, Heteromyiasalbispecularis, Drymodessupercilious and Microecaflavigaster may each comprise more than one species. The Petroicidae display a complex biogeographical history involving repeated radiations both within, and across Australia and New Guinea. It appears that dispersal into smaller islands such as New Britain, Tanimbar and the South Pacific has only been undertaken by species with a "flycatcher" body form.

Trophic niche shifts and phenotypic trait evolution are largely decoupled in Australasian parrots.

BACKGROUND: Trophic shifts from one dietary niche to another have played major roles in reshaping the evolutionary trajectories of a wide range of vertebrate groups, yet their consequences for morphological disparity and species diversity differ among groups. METHODS: Here, we use phylogenetic comparative methods to examine whether the evolution of nectarivory and other trophic shifts have driven predictable evolutionary pathways in Australasian psittaculid parrots in terms of ecological traits such as body size, beak shape, and dispersal capacity. RESULTS: We found no evidence for an 'early-burst' scenario of lineage or morphological diversification. The best-fitting models indicate that trait evolution in this group is characterized by abrupt phenotypic shifts (evolutionary jumps), with no sign of multiple phenotypic optima correlating with different trophic strategies. Thus, our results point to the existence of weak directional selection and suggest that lineages may be evolving randomly or slowly toward adaptive peaks they have not yet reached. CONCLUSIONS: This study adds to a growing body of evidence indicating that the relationship between avian morphology and feeding ecology may be more complex than usually assumed and highlights the importance of adding more flexible models to the macroevolutionary toolbox.

Plumage patterns are good indicators of taxonomic diversity, but not of phylogenetic affinities, in Australian grasswrens Amytornis (Aves: Maluridae).

The grasswrens (Maluridae: Amytornis) are elusive songbirds from the arid zones of Australia. Although some other Australian bird genera are also largely restricted to arid regions, none show the level of localized taxonomic diversity seen in Amytornis. Furthermore, their cryptic plumage patterns provide excellent camouflage but make it difficult to determine whether shared patterns reflect phylogenetic relationships or adaptations to similar terrain. To resolve the systematics and patterns of ecological diversification within Amytornis, we here present the results of a phylogenetic analysis of mitochondrial and nuclear multi-locus data for all recognized species and most subspecies, using traditional concatenation-based methods as well as a coalescent-based species-tree approach. Phylogenetic patterns retrieved by the species-tree approach were highly congruent with traditional methods, although branch support was generally higher in concatenation-based analysis, suggesting that species-tree methods may furnish more conservative results. In terms of identifying taxonomic diversity there was good concordance between plumage-based assessments and DNA distances. The same concordance was not found when comparing plumage-based and DNA-based predictions of phylogenetic relationships. Four primary lineages were identified: (a) barbatus; (b) merrotsyi; (c) the textilis complex, purnelli, ballarae, goyderi and housei; and (d) woodwardi, dorotheae, and the striatus complex. There was no robust resolution of relationships between lineages. It appears that in Amytornis, plumage differentiation between discrete populations is taxonomically significant, and not greatly influenced by ecophenotypic variation. However, at the deeper phylogenetic level, similar suites of plumage characters may be phylogenetically uninformative because of homoplasy. The study reveals higher levels of taxonomic diversity in Amytornis than previously recognized, with many taxa being highly localized. Such extensive short range endemism is mainly encountered in poorly-dispersing invertebrates and is unique in Australian birds. The identification here of the additional restricted range taxa has important conservation implications.

Molecular systematics and evolutionary origins of the genus Petaurus (Marsupialia: Petauridae) in Australia and New Guinea.

The glider genus Petaurus comprises a group of arboreal and nocturnal marsupial species from New Guinea and Australia. Molecular data were generated in order to examine phylogenetic relationships among species within the genus and explore the time-scale of diversification and biogeographic history of the genus in Australia and New Guinea. All known species and subspecies of Petaurus (with the exception of P. biacensis) were sequenced for two mitochondrial genes (ND2 and ND4) and one nuclear marker (omega-globin gene). Phylogenetic analyses confirmed the monophyly of the genus relative to other petaurids and showed a sister relationship of P. australis to the rest of Petaurus. The analyses revealed that currently recognised species of Petaurus formed distinct mitochondrial DNA (mtDNA) clades. Considerable mtDNA diversity and seven distinct clades were identified within the species P. breviceps, with the distribution of each clade showing no correspondence with the distributional limits of known subspecies. Molecular dating analyses using BEAST suggested an early to mid-Miocene origin (18-24 mya) for the genus. Ancestral area reconstructions, using BayesTraits, did not resolve the location for the centre of origin of Petaurus, but provided evidence for at least one dispersal event from New Guinea to Australia that led to the evolution of extant Australian populations of P. breviceps, P. norfolcensis and P. gracilis. The timing of this dispersal event appears to pre-date the Pleistocene, adding to the growing number of studies that suggest faunal connections occurred between Australia and New Guinea in the Late Miocene to Pliocene period.

Evolution of an arsenal: structural and functional diversification of the venom system in the advanced snakes (Caenophidia).

Venom is a key innovation underlying the evolution of advanced snakes (Caenophidia). Despite this, very little is known about venom system structural diversification, toxin recruitment event timings, or toxin molecular evolution. A multidisciplinary approach was used to examine the diversification of the venom system and associated toxins across the full range of the approximately 100 million-year-old advanced snake clade with a particular emphasis upon families that have not secondarily evolved a front-fanged venom system ( approximately 80% of the 2500 species). Analysis of cDNA libraries revealed complex venom transcriptomes containing multiple toxin types including three finger toxins, cobra venom factor, cysteine-rich secretory protein, hyaluronidase, kallikrein, kunitz, lectin, matrix metalloprotease, phospholipase A(2), snake venom metalloprotease/a disintegrin and metalloprotease, and waprin. High levels of sequence diversity were observed, including mutations in structural and functional residues, changes in cysteine spacing, and major deletions/truncations. Morphological analysis comprising gross dissection, histology, and magnetic resonance imaging also demonstrated extensive modification of the venom system architecture in non-front-fanged snakes in contrast to the conserved structure of the venom system within the independently evolved front-fanged elapid or viperid snakes. Further, a reduction in the size and complexity of the venom system was observed in species in which constriction has been secondarily evolved as the preferred method of prey capture or dietary preference has switched from live prey to eggs or to slugs/snails. Investigation of the timing of toxin recruitment events across the entire advanced snake radiation indicates that the evolution of advanced venom systems in three front-fanged lineages is associated with recruitment of new toxin types or explosive diversification of existing toxin types. These results support the role of venom as a key evolutionary innovation in the diversification of advanced snakes and identify a potential role for non-front-fanged venom toxins as a rich source for lead compounds for drug design and development.

Current issues in species identification for forensic science and the validity of using the cytochrome oxidase I (COI) gene.

Species identification techniques commonly utilized in Australian Forensic Science laboratories are gel immunodifussion antigen antibody reactions and hair comparison analysis. Both of these techniques have significant limitations and should be considered indicative opinion based tests. The Barcode of Life Initiative aims to sequence a section of DNA (~648 base pairs) for the Cytochrome Oxidase I mitochondrial gene (COI) in all living species on Earth, with the data generated being uploaded to the Barcode of Life Database (BOLD) which can then be used for species identification. The COI gene therefore offers forensics scientists an opportunity to use the marker to analyze unknown samples and compare sequences generated in BOLD. Once sequences from enough species are on the database, it is anticipated that routine identification of an unknown species may be possible. However, most forensic laboratories are not yet suited to this type of analysis and do not have the expertise to fully interpret the implications of matches and non matches involving a poorly sampled taxa (for example where there are cryptic species) and in providing the required opinion evidence. Currently, the use of BOLD is limited by the number of relevant species held in the database and the quality assurance and regulation of sequences that are there. In this paper, the COI methodology and BOLD are tested on a selection of introduced and Australian mammals in a forensic environment as the first step necessary in the implementation of this approach in the Australian context. Our data indicates that the COI methodology performs well on distinct species but needs further exploration when identifying more closely related species. It is evident from our study that changes will be required to implement DNA based wildlife forensics using the BOLD approach for forensic applications and recommendations are made for the future adoption of this technology into forensic laboratories.

The timing of neotropical speciation dynamics: a reconstruction of Myiopagis flycatcher diversification using phylogenetic and paleogeographic data.

Neotropical forests have brought forth a large proportion of the world's terrestrial biodiversity, but the underlying evolutionary mechanisms and their timing require further elucidation. Despite insights gained from phylogenetic studies, uncertainties about molecular clock rates have hindered efforts to determine the timing of diversification processes. Moreover, most molecular research has been detached from the extensive body of data on Neotropical geology and paleogeography. We here examine phylogenetic relationships and the timing of speciation events in a Neotropical flycatcher genus (Myiopagis) by using calibrations from modern geologic data in conjunction with a number of recently developed DNA sequence dating algorithms and by comparing these estimates with those based on a range of previously proposed molecular clock rates. We present a well-supported hypothesis of systematic relationships within the genus. Our age estimates of Myiopagis speciation events based on paleogeographic data are in close agreement with nodal ages derived from a "traditional" avian mitochondrial 2%/My clock, while contradicting other clock rates. Our comparative approach corroborates the consistency of the traditional avian mitochondrial clock rate of 2%/My for tyrant-flycatchers. Nevertheless, our results argue against the indiscriminate use of molecular clock rates in evolutionary research and advocate the verification of the appropriateness of the traditional clock rate by means of independent calibrations in individual studies.

A multi-gene phylogeny reveals novel relationships for aberrant genera of Australo-Papuan core Corvoidea and polyphyly of the Pachycephalidae and Psophodidae (Aves: Passeriformes).

The core Corvoidea is the largest and most diverse oscine assemblage within the Australo-Papuan region. Although central to an understanding of the evolutionary history and biogeography of the group the composition and intergeneric relationships of the Australo-Papuan radiation remain poorly understood. Here we analysed DNA sequence data from two nuclear gene regions and the mitochondrial cytochrome b gene, for 40 species of core Corvoidea to test the systematic affinities of key Australo-Papuan lineages. The families Pachycephalidae (whistlers, shrike-thrushes and allies) and Psophodidae (whipbirds, quail-thrush and allies) were both recovered as polyphyletic assemblages. The core pachycephaline assemblage comprised Pachycephala, Colluricincla, parts of Pitohui, and Falcunculus with the remaining genera resolving as four divergent lineages with no clearly defined affinities. Ptilorrhoa and Cinclosoma (Cinclosomatidae) formed a clade separate from Psophodes (Psophodidae) but neither clade showed clear affinities to any other taxa. Novel relationships were also identified for three aberrant New Guinean genera; ditypic Machaerirhynchus and monotypic Rhagologus were both nested within an assemblage that included the Artamidae and African malaconotoids (bush-shrikes and allies) while the enigmatic Ifrita was found to be part of an assemblage that included the Monarchidae and Paradisaeidae.

Habitat shifts in the evolutionary history of a Neotropical flycatcher lineage from forest and open landscapes.

BACKGROUND: Little is known about the role ecological shifts play in the evolution of Neotropical radiations that have colonized a variety of environments. We here examine habitat shifts in the evolutionary history of Elaenia flycatchers, a Neotropical bird lineage that lives in a range of forest and open habitats. We evaluate phylogenetic relationships within the genus based on mitochondrial and nuclear DNA sequence data, and then employ parsimony-based and Bayesian methods to reconstruct preferences for a number of habitat types and migratory behaviour throughout the evolutionary history of the genus. Using a molecular clock approach, we date the most important habitat shifts. RESULTS: Our analyses resolve phylogenetic relationships among Elaenia species and confirm several species associations predicted by morphology while furnishing support for other taxon placements that are in conflict with traditional classification, such as the elevation of various Elaenia taxa to species level. While savannah specialism is restricted to one basal clade within the genus, montane forest was invaded from open habitat only on a limited number of occasions. Riparian growth may have been favoured early on in the evolution of the main Elaenia clade and subsequently been deserted on several occasions. Austral long-distance migratory behaviour evolved on several occasions. CONCLUSION: Ancestral reconstructions of habitat preferences reveal pronounced differences not only in the timing of the emergence of certain habitat preferences, but also in the frequency of habitat shifts. The early origin of savannah specialism in Elaenia highlights the importance of this habitat in Neotropical Pliocene and late Miocene biogeography. While forest in old mountain ranges such as the Tepuis and the Brazilian Shield was colonized early on, the most important colonization event of montane forest was in conjunction with Pliocene Andean uplift. Riparian habitats may have played an important role in facilitating habitat shifts by birds expanding up the mountains along streams and adapting to newly emerging montane forest habitat.

DNA evidence shows vocalizations to be a better indicator of taxonomic limits than plumage patterns in Zimmerius tyrant-flycatchers.

We investigated mitochondrial and nuclear DNA in the small tyrant-flycatcher genus Zimmerius (Tyrannidae) and show that molecular data are in strong disagreement with morphology-based taxonomy, but in good concordance with vocal characters. Our molecular data identified two independent cases of well-supported polyphyletic species arrangements within this genus that indicate the following taxonomic changes: elevation of Z. acer and Z. albigularis to species level, separation of northern populations of Z. chrysops as a species and inclusion of southern populations of Z. chrysops into Z. viridiflavus. Although polyphyly has rarely been encountered in bird systematics it has previously been shown for two other tyrannid genera and suggests that tyrannid taxonomy may be poorly resolved, presumably as a consequence of the conserved plumage patterns observed in many tyrannid genera. Our study suggests that vocalizations can be a better indicator of taxonomic limits than plumage pattern in tyrannids.

Speciation in Indo-Pacific swiftlets (Aves: Apodidae): integrating molecular and phenotypic data for a new provisional taxonomy of the Collocalia esculenta complex.

White-bellied swiftlets of the Collocalia esculenta complex constitute a radiation of colony-breeding swifts distributed throughout the tropical Indo-Pacific region. Resolution of their taxonomy is challenging due to their morphological uniformity. To analyze the evolutionary history of this complex, we combine new biometric measurements and results from plumage assessment of museum specimens with novel as well as previously published molecular data. Together, this body of information constitutes the largest systematic dataset for white-bellied swiftlets yet compiled, drawn from 809 individuals belonging to 32 taxa for which new molecular, biometric, and/or plumage data are presented. We propose changing the classification of white-bellied swiftlets, for which two species are currently recognized, to elevate eight regional forms to species level, and we also describe two new subspecies. The ten taxa we recommend recognizing at the species level are: Collocalia linchi (Java to Lombok, Sumatran hills), C. dodgei (montane Borneo), C. natalis (Christmas Island), C. affinis (Greater Sundas, including the Thai-Malay Peninsula and Andaman-Nicobar Islands), C. marginata (Philippines), C. isonota (Philippines), C. sumbawae (west Lesser Sundas), C. neglecta (east Lesser Sundas), C. esculenta (Sulawesi, Moluccas, New Guinea, Bismarck Archipelago, Solomon Islands), and C. uropygialis (Vanuatu, New Caledonia). Future molecular and morphological work is needed to resolve questions of speciation and population affinities in the Philippines, Christmas Island, Wallacea and central Melanesia, and to shed light on historic diversification and patterns of gene flow in the complex.