Ancient mitochondrial genomes unveil the origins and evolutionary history of New Zealand's enigmatic takahe and moho.

Many avian species endemic to Aotearoa New Zealand were driven to extinction or reduced to relict populations following successive waves of human arrival, due to hunting, habitat destruction and the introduction of mammalian predators. Among the affected species were the large flightless South Island takahe (Porphyrio hochstetteri) and the moho (North Island takahe; P. mantelli), with the latter rendered extinct and the former reduced to a single relictual population. Little is known about the evolutionary history of these species prior to their decline and/or extinction. Here we sequenced mitochondrial genomes from takahe and moho subfossils (12 takahe and 4 moho) and retrieved comparable sequence data from takahe museum skins (n = 5) and contemporary individuals (n = 17) to examine the phylogeny and recent evolutionary history of these species. Our analyses suggest that prehistoric takahe populations lacked deep phylogeographic structure, in contrast to moho, which exhibited significant spatial genetic structure, albeit based on limited sample sizes (n = 4). Temporal genetic comparisons show that takahe have lost much of their mitochondrial genetic diversity, likely due to a sudden demographic decline soon after human arrival (~750 years ago). Time-calibrated phylogenetic analyses strongly support a sister species relationship between takahe and moho, suggesting these flightless taxa diverged around 1.5 million years ago, following a single colonisation of New Zealand by a flighted Porphyrio ancestor approximately 4 million years ago. This study highlights the utility of palaeogenetic approaches for informing the conservation and systematic understanding of endangered species whose ranges have been severely restricted by anthropogenic impacts.

Recent evolution of extreme sexual dimorphism in the huia (Heteralocha acutirostris; Callaeidae).

The New Zealand wattlebirds (Callaeidae) are an endemic New Zealand passerine family whose species show extreme variation in bill morphology. In particular, the extinct huia (Heteralocha acutirostris) has attracted considerable attention because it exhibited extreme sexual dimorphism in bill morphology. However, the phylogenetic relationships within the Callaeidae, crucial for understanding bill evolution in the family, have not been resolved to date. Here we present phylogenies based on complete mitochondrial genome sequences and nuclear ultraconserved elements. Kokako (Callaeas spp.) is strongly supported as sister taxon to saddleback/tieke (Philesturnus spp.) and huia, diverging around 6.8 Ma. Saddleback and huia are estimated to have split from each other 5 Ma, indicating that the extreme sexual bill dimorphism in huia has evolved within this time frame. Our estimates for the divergences within the Callaetidae are similar to, or younger than, those of most other endemic New Zealand avian families, therefore the observed bill variation is not a consequence of a longer divergence time. Instead, the expansion of the huia into the wood-foraging niche, combined with the sexual dimorphism it evolved in order to optimise feeding on this resource, has been the main contributor to the large variation of bill morphologies within this family.

Resolving the phylogenetic position of Darwin's extinct ground sloth (Mylodon darwinii) using mitogenomic and nuclear exon data.

Mylodon darwinii is the extinct giant ground sloth named after Charles Darwin, who first collected its remains in South America. We have successfully obtained a high-quality mitochondrial genome at 99-fold coverage using an Illumina shotgun sequencing of a 12 880-year-old bone fragment from Mylodon Cave in Chile. Low level of DNA damage showed that this sample was exceptionally well preserved for an ancient subfossil, probably the result of the dry and cold conditions prevailing within the cave. Accordingly, taxonomic assessment of our shotgun metagenomic data showed a very high percentage of endogenous DNA with 22% of the assembled metagenomic contigs assigned to Xenarthra. Additionally, we enriched over 15 kb of sequence data from seven nuclear exons, using target sequence capture designed against a wide xenarthran dataset. Phylogenetic and dating analyses of the mitogenomic dataset including all extant species of xenarthrans and the assembled nuclear supermatrix unambiguously place Mylodon darwinii as the sister-group of modern two-fingered sloths, from which it diverged around 22 million years ago. These congruent results from both the mitochondrial and nuclear data support the diphyly of the two modern sloth lineages, implying the convergent evolution of their unique suspensory behaviour as an adaption to arboreality. Our results offer promising perspectives for whole-genome sequencing of this emblematic extinct taxon.

Shotgun Mitogenomics Provides a Reference Phylogenetic Framework and Timescale for Living Xenarthrans.

Xenarthra (armadillos, sloths, and anteaters) constitutes one of the four major clades of placental mammals. Despite their phylogenetic distinctiveness in mammals, a reference phylogeny is still lacking for the 31 described species. Here we used Illumina shotgun sequencing to assemble 33 new complete mitochondrial genomes, establishing Xenarthra as the first major placental clade to be fully sequenced at the species level for mitogenomes. The resulting data set allowed the reconstruction of a robust phylogenetic framework and timescale that are consistent with previous studies conducted at the genus level using nuclear genes. Incorporating the full species diversity of extant xenarthrans points to a number of inconsistencies in xenarthran systematics and species definition. We propose to split armadillos into two distinct families Dasypodidae (dasypodines) and Chlamyphoridae (euphractines, chlamyphorines, and tolypeutines) to better reflect their ancient divergence, estimated around 42 Ma. Species delimitation within long-nosed armadillos (genus Dasypus) appeared more complex than anticipated, with the discovery of a divergent lineage in French Guiana. Diversification analyses showed Xenarthra to be an ancient clade with a constant diversification rate through time with a species turnover driven by high but constant extinction. We also detected a significant negative correlation between speciation rate and past temperature fluctuations with an increase in speciation rate corresponding to the general cooling observed during the last 15 My. Biogeographic reconstructions identified the tropical rainforest biome of Amazonia and the Guiana Shield as the cradle of xenarthran evolutionary history with subsequent dispersions into more open and dry habitats.

Closing the gap: Avian lineage splits at a young, narrow seaway imply a protracted history of mixed population response.

The evolutionary significance of spatial habitat gaps has been well recognized since Alfred Russel Wallace compared the faunas of Bali and Lombok. Gaps between islands influence population structuring of some species, and flightless birds are expected to show strong partitioning even where habitat gaps are narrow. We examined the population structure of the most numerous living flightless land bird in New Zealand, Weka (Gallirallus australis). We surveyed Weka and their feather lice in native and introduced populations using genetic data gathered from DNA sequences of mitochondrial genes and nuclear ß-fibrinogen and five microsatellite loci. We found low genetic diversity among extant Weka population samples. Two genetic clusters were evident in the mtDNA from Weka and their lice, but partitioning at nuclear loci was less abrupt. Many formerly recognized subspecies/species were not supported; instead, we infer one subspecies for each of the two main New Zealand islands. Although currently range restricted, North Island Weka have higher mtDNA diversity than the more wide-ranging southern Weka. Mismatch and neutrality statistics indicate North Island Weka experienced rapid and recent population reduction, while South Island Weka display the signature of recent expansion. Similar haplotype data from a widespread flying relative of Weka and other New Zealand birds revealed instances of North Island-South Island partitioning associated with a narrow habitat gap (Cook Strait). However, contrasting patterns indicate priority effects and other ecological factors have a strong influence on spatial exchange at this scale.

Molecular phylogeny, biogeography, and habitat preference evolution of marsupials.

Marsupials exhibit great diversity in ecology and morphology. However, compared with their sister group, the placental mammals, our understanding of many aspects of marsupial evolution remains limited. We use 101 mitochondrial genomes and data from 26 nuclear loci to reconstruct a dated phylogeny including 97% of extant genera and 58% of modern marsupial species. This tree allows us to analyze the evolution of habitat preference and geographic distributions of marsupial species through time. We found a pattern of mesic-adapted lineages evolving to use more arid and open habitats, which is broadly consistent with regional climate and environmental change. However, contrary to the general trend, several lineages subsequently appear to have reverted from drier to more mesic habitats. Biogeographic reconstructions suggest that current views on the connectivity between Australia and New Guinea/Wallacea during the Miocene and Pliocene need to be revised. The antiquity of several endemic New Guinean clades strongly suggests a substantially older period of connection stretching back to the Middle Miocene and implies that New Guinea was colonized by multiple clades almost immediately after its principal formation.

Deep global evolutionary radiation in birds: diversification and trait evolution in the cosmopolitan bird family Rallidae.

Sufficient breadth of taxon sampling in major organisms groups is important to identify more realistic biological diversification processes that reveal the degree of historical biogeographic signal and net diversification retained in the current lineage distribution. We examine the mechanisms driving diversity in one of the major avian clades with an exceptional large-scale radiation, the family Rallidae, using the most complete species-level (∼70%) time calibrated hypothesis of evolutionary relationships produced to date. We find that Rallidae exhibit a pattern of diversification involving episodes of range expansion and regional speciation that results in most clades represented in all habitable continents. Our results suggest that several features may have played an important role on the diversification rates in Rallidae. Lineage accumulation is nearly constant and morphology (frontal shield and body size), innovate (flightlessness), habitat (forest) and distribution (insular) traits are possibly associated with increasing diversification rates along with spatial and ecological processes during the Miocene and Pliocene. Diversification and the global retention of lineage diversity have occurred in multiple lineages in Rallidae due to their dispersal ability and exploitation of ecological opportunities.

De-novo assembly of four rail (Aves: Rallidae) genomes: A resource for comparative genomics.

Rails are a phenotypically diverse family of birds that includes 130 species and displays a wide distribution around the world. Here we present annotated genome assemblies for four rails from Aotearoa New Zealand: two native volant species, pukeko Porphyrio melanotus and mioweka Gallirallus philippensis, and two endemic flightless species takahe Porphyrio hochstetteri and weka Gallirallus australis. Using the sequence read data, heterozygosity was found to be lowest in the endemic flightless species and this probably reflects their relatively small populations. The quality checks and comparison with other rallid genomes showed that the new assemblies were of good quality. This study significantly increases the number of available rallid genomes and will enable future genomic studies on the evolution of this family.

Beyond phylogeny: pelecaniform and ciconiiform birds, and long-term niche stability.

Phylogenetic trees are a starting point for the study of further evolutionary and ecological questions. We show that for avian evolutionary relationships, improved taxon sampling, longer sequences and additional data sets are giving stability to the prediction of the grouping of pelecaniforms and ciconiiforms, thus allowing inferences to be made about long-term niche occupancy. Here we report the phylogeny of the pelecaniform birds and their water-carnivore allies using complete mitochondrial genomes, and show that the basic groupings agree with nuclear sequence phylogenies, even though many short branches are not yet fully resolved. In detail, we show that the Pelecaniformes (minus the tropicbird) and the Ciconiiformes (storks, herons and ibises) form a natural group within a seabird water-carnivore clade. We find pelicans are the closest relatives of the shoebill (in a clade with the hammerkop), and we confirm that tropicbirds are not pelecaniforms. In general, the group appears to be an adaptive radiation into an 'aquatic carnivore' niche that it has occupied for 60-70 million years. From an ecological and life history perspective, the combined pelecaniform-ciconiform group is more informative than focusing on differences in morphology. These findings allow a start to integrating molecular evolution and macroecology.

Tinamous and moa flock together: mitochondrial genome sequence analysis reveals independent losses of flight among ratites.

Ratites are large, flightless birds and include the ostrich, rheas, kiwi, emu, and cassowaries, along with extinct members, such as moa and elephant birds. Previous phylogenetic analyses of complete mitochondrial genome sequences have reinforced the traditional belief that ratites are monophyletic and tinamous are their sister group. However, in these studies ratite monophyly was enforced in the analyses that modeled rate heterogeneity among variable sites. Relaxing this topological constraint results in strong support for the tinamous (which fly) nesting within ratites. Furthermore, upon reducing base compositional bias and partitioning models of sequence evolution among protein codon positions and RNA structures, the tinamou-moa clade grouped with kiwi, emu, and cassowaries to the exclusion of the successively more divergent rheas and ostrich. These relationships are consistent with recent results from a large nuclear data set, whereas our strongly supported finding of a tinamou-moa grouping further resolves palaeognath phylogeny. We infer flight to have been lost among ratites multiple times in temporally close association with the Cretaceous-Tertiary extinction event. This circumvents requirements for transient microcontinents and island chains to explain discordance between ratite phylogeny and patterns of continental breakup. Ostriches may have dispersed to Africa from Eurasia, putting in question the status of ratites as an iconic Gondwanan relict taxon.

Mutation and evolutionary rates in adélie penguins from the antarctic.

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (micro) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29-0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent.

Toward resolving deep neoaves phylogeny: data, signal enhancement, and priors.

We report three developments toward resolving the challenge of the apparent basal polytomy of neoavian birds. First, we describe improved conditional down-weighting techniques to reduce noise relative to signal for deeper divergences and find increased agreement between data sets. Second, we present formulae for calculating the probabilities of finding predefined groupings in the optimal tree. Finally, we report a significant increase in data: nine new mitochondrial (mt) genomes (the dollarbird, New Zealand kingfisher, great potoo, Australian owlet-nightjar, white-tailed trogon, barn owl, a roadrunner [a ground cuckoo], New Zealand long-tailed cuckoo, and the peach-faced lovebird) and together they provide data for each of the six main groups of Neoaves proposed by Cracraft J (2001). We use his six main groups of modern birds as priors for evaluation of results. These include passerines, cuckoos, parrots, and three other groups termed "WoodKing" (woodpeckers/rollers/kingfishers), "SCA" (owls/potoos/owlet-nightjars/hummingbirds/swifts), and "Conglomerati." In general, the support is highly significant with just two exceptions, the owls move from the "SCA" group to the raptors, particularly accipitrids (buzzards/eagles) and the osprey, and the shorebirds may be an independent group from the rest of the "Conglomerati". Molecular dating mt genomes support a major diversification of at least 12 neoavian lineages in the Late Cretaceous. Our results form a basis for further testing with both nuclear-coding sequences and rare genomic changes.

Two aspects along the continuum of pigeon evolution: A South-Pacific radiation and the relationship of pigeons within Neoaves.

Phylogenetics explores the continuum of shallower to deeper genetic divergences between taxa. Along this continuum increasing lengths of DNA sequence can be used to answer deeper and deeper questions about biological relationships. We use shorter, and then longer mitochondrial DNA sequences to address two aspects of pigeon evolution. Firstly, we examine the phylogenetic relationships of the eight genera within the South Pacific Ducula-Ptilinopus radiation, and examine how this radiation fits into pigeons generally. Within Ducula, taxa are closely related, whereas Ptilinopus is very diverse, and paraphyletic. One third of all pigeon species are within the Ducula-Ptilinopus radiation, however all are very similar ecologically. Secondly, we study the deeper phylogenetic question regarding the relationship of pigeons to other birds. To this end, we report the complete mitochondrial genome of Hemiphaganovaeseelandiae, a member of the Ducula-Ptilinopus radiation. We use this mitochondrial genome, along with additional sandgrouse (Pterocles namaqua) mitochondrial genes to assess various candidates for the closest relative of pigeons. Of parrots, shorebirds, and sandgrouse, we find highest support for the sandgrouse-pigeon grouping. Furthermore in these analyses the pigeon and sandgrouse group closer to the falcons than any other included taxon. The finding that pigeons and sandgrouse may be more closely related to falcons than to previous candidates such as shorebirds or parrots invites further investigation.

Convergent morphological responses to loss of flight in rails (Aves: Rallidae).

The physiological demands of flight exert strong selection pressure on avian morphology and so it is to be expected that the evolutionary loss of flight capacity would involve profound changes in traits. Here, we investigate morphological consequences of flightlessness in a bird family where the condition has evolved repeatedly. The Rallidae include more than 130 recognized species of which over 30 are flightless. Morphological and molecular phylogenetic data were used here to compare species with and without the ability to fly in order to determine major phenotypic effects of the transition from flighted to flightless. We find statistical support for similar morphological response among unrelated flightless lineages, characterized by a shift in energy allocation from the forelimbs to the hindlimbs. Indeed, flightless birds exhibit smaller sterna and wings than flighted taxa in the same family along with wider pelves and more robust femora. Phylogenetic signal tests demonstrate that those differences are independent of phylogeny and instead demonstrate convergent morphological adaptation associated with a walking ecology. We found too that morphological variation was greater among flightless rails than flighted ones, suggesting that relaxation of physiological demands during the transition to flightlessness frees morphological traits to evolve in response to more varied ecological opportunities.

Ancient Mitogenomes Reveal the Evolutionary History and Biogeography of Sloths.

Living sloths represent two distinct lineages of small-sized mammals that independently evolved arboreality from terrestrial ancestors. The six extant species are the survivors of an evolutionary radiation marked by the extinction of large terrestrial forms at the end of the Quaternary. Until now, sloth evolutionary history has mainly been reconstructed from phylogenetic analyses of morphological characters. Here, we used ancient DNA methods to successfully sequence 10 extinct sloth mitogenomes encompassing all major lineages. This includes the iconic continental ground sloths Megatherium, Megalonyx, Mylodon, and Nothrotheriops and the smaller endemic Caribbean sloths Parocnus and Acratocnus. Phylogenetic analyses identify eight distinct lineages grouped in three well-supported clades, whose interrelationships are markedly incongruent with the currently accepted morphological topology. We show that recently extinct Caribbean sloths have a single origin but comprise two highly divergent lineages that are not directly related to living two-fingered sloths, which instead group with Mylodon. Moreover, living three-fingered sloths do not represent the sister group to all other sloths but are nested within a clade of extinct ground sloths including Megatherium, Megalonyx, and Nothrotheriops. Molecular dating also reveals that the eight newly recognized sloth families all originated between 36 and 28 million years ago (mya). The early divergence of recently extinct Caribbean sloths around 35 mya is consistent with the debated GAARlandia hypothesis postulating the existence at that time of a biogeographic connection between northern South America and the Greater Antilles. This new molecular phylogeny has major implications for reinterpreting sloth morphological evolution, biogeography, and diversification history.

The complete mitochondrial genome of the eastern grey kangaroo (Macropus giganteus).

We present the complete mitochondrial genome (accession number: LK995454) of an iconic Australian species, the eastern grey kangaroo (Macropus giganteus). The mitogenomic organization is consistent with other marsupials, encoding 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, an origin of light strand replication and a control region or D-loop. No repetitive sequences were detected in the control region. The M. giganteus mitogenome exemplifies a combination of tRNA gene order and structural peculiarities that appear to be unique to marsupials. We present a maximum likelihood phylogeny based on complete mitochondrial protein and RNA coding sequences that confirms the phylogenetic position of the grey kangaroo among macropodids.

The phylogenetic affinities of the extinct glyptodonts.

Among the fossils of hitherto unknown mammals that Darwin collected in South America between 1832 and 1833 during the Beagle expedition were examples of the large, heavily armored herbivores later known as glyptodonts. Ever since, glyptodonts have fascinated evolutionary biologists because of their remarkable skeletal adaptations and seemingly isolated phylogenetic position even within their natural group, the cingulate xenarthrans (armadillos and their allies). In possessing a carapace comprised of fused osteoderms, the glyptodonts were clearly related to other cingulates, but their precise phylogenetic position as suggested by morphology remains unresolved. To provide a molecular perspective on this issue, we designed sequence-capture baits using in silico reconstructed ancestral sequences and successfully assembled the complete mitochondrial genome of Doedicurus sp., one of the largest glyptodonts. Our phylogenetic reconstructions establish that glyptodonts are in fact deeply nested within the armadillo crown-group, representing a distinct subfamily (Glyptodontinae) within family Chlamyphoridae. Molecular dating suggests that glyptodonts diverged no earlier than around 35 million years ago, in good agreement with their fossil record. Our results highlight the derived nature of the glyptodont morphotype, one aspect of which is a spectacular increase in body size until their extinction at the end of the last ice age.

New Zealand Passerines Help Clarify the Diversification of Major Songbird Lineages during the Oligocene.

Passerines are the largest avian order, and the 6,000 species comprise more than half of all extant bird species. This successful radiation probably had its origin in the Australasian region, but dating this origin has been difficult due to a scarce fossil record and poor biogeographic assumptions. Many of New Zealand's endemic passerines fall within the deeper branches of the passerine radiation, and a well resolved phylogeny for the modern New Zealand element in the deeper branches of the oscine lineage will help us understand both oscine and passerine biogeography. To this end we present complete mitochondrial genomes representing all families of New Zealand passerines in a phylogenetic framework of over 100 passerine species. Dating analyses of this robust phylogeny suggest Passeriformes originated in the early Paleocene, with the major lineages of oscines "escaping" from Australasia about 30 Ma, and radiating throughout the world during the Oligocene. This independently derived conclusion is consistent with the passerine fossil record.