First evidence of deviation from Mendelian proportions in a conservation programme.

Classic Mendelian inheritance is the bedrock of population genetics and underpins pedigree-based management of animal populations. However, assumptions of Mendelian inheritance might not be upheld in conservation breeding programmes if early viability selection occurs, even when efforts are made to equalise genetic contributions of breeders. To test this possibility, we investigated deviations from Mendelian proportions in a captive metapopulation of the endangered Tasmanian devil. This marsupial population is ideal for addressing evolutionary questions in conservation due to its large size, range of enclosure types (varying in environmental conditions), good genomic resources (which aid interpretation), and the species' biology. Devil mothers give birth to more offspring than they can nurse in the pouch, providing the potential for intense viability selection amongst embryos. We used data from 140 known sire-dam-offspring triads to isolate within-family selection from population-level mechanisms (such as mate choice or inbreeding), and compared observed offspring genotypes at 123 targeted SNPs to neutral (i.e., Mendelian) expectations. We found lower offspring heterozygosity than expected, and subtle patterns that varied across a gradient of management intensity from zoo-like enclosures to semi-wild environments for some loci. Meiotic drive or maternal-foetal incompatibilities are consistent with our results, although we cannot statistically confirm these mechanisms. We found some evidence that maternal genotype affects annual litter size, suggesting that family-level patterns are driven by differential offspring mortality before birth or during early development. Our results show that deviations from Mendelian inheritance can occur in conservation programmes, despite best-practice management to prevent selection.

Going under down under? Lineage ages argue for extensive survival of the Oligocene marine transgression on Zealandia.

Twenty-five years ago, it was suggested that current-day New Zealand, part of the largely sunken continent of Zealandia, could have been completely inundated during the Oligocene marine transgression (OMT) some 25-23 million years ago. Such an event would, of necessity, imply that all terrestrial, freshwater, and maybe coastal marine species must have dispersed there since. This idea has generated heated debate, on which geological, palaeontological and molecular data are being brought to bear. Here, we review the phylogeographic literature in the form of molecular estimates of divergence times between New Zealand lineages and their closest overseas sister groups. Using an event-based approach, we show that these divergence times follow approximately a smooth exponential over the last 50 Ma or more. Approximately 74 of these 248 lineages appear to have survived the OMT in situ; some of these major lineages comprise multiple additional lineages as a result of autochthonous speciation prior to the OMT. Non-volant terrestrial animals, freshwater animals and trees are particularly well represented in surviving lineages, whereas marine animals, herbs and shrubs tend to show more recent arrival times. There is no evidence for a deficit of pre-Oligocene lineages, nor an excess of ones arriving just afterwards. The pattern is one of geometric increase in new lineages with more recent time, reflecting a balance between immigration and extinction. Consequently, this large body of molecular data provides no evidence for complete inundation of New Zealand during the Oligocene. In conjunction with new geological and palaeontological findings, these data suggest that it is time to put the idea to rest.

Interspecific hybridization causes long-term phylogenetic discordance between nuclear and mitochondrial genomes in freshwater fishes.

Classification, phylogeography and the testing of evolutionary hypotheses rely on correct estimation of species phylogeny. Early molecular phylogenies often relied on mtDNA alone, which acts as a single linkage group with one history. Over the last decade, the use of multiple nuclear sequences has often revealed conflict among gene trees. This observation can be attributed to hybridization, lineage sorting, paralogy or selection. Here, we use 54 groups of fishes from 48 studies to estimate the degree of concordance between mitochondrial and nuclear gene trees in two ecological grades of fishes: marine and freshwater. We test the hypothesis that freshwater fish phylogenies should, on average, show more discordance because of their higher propensity for hybridization in the past. In keeping with this idea, concordance between mitochondrial and nuclear gene trees (as measured by proportion of components shared) is on average 50% higher in marine fishes. We discuss why this difference almost certainly results from introgression caused by greater historical hybridization among lineages in freshwater groups, and further emphasize the need to use multiple nuclear genes, and identify conflict among them, in estimation of species phylogeny.

A time-calibrated phylogeny of southern hemisphere stoneflies: Testing for Gondwanan origins.

For more than two centuries biogeographers have attempted to explain why terrestrial or freshwater lineages have geographic distributions broken by oceans, with these disjunct distributions either attributed to vicariance associated with Gondwanan fragmentation or trans-oceanic dispersal. Stoneflies (order: Plecoptera) are a widespread order of freshwater insects whose poor dispersal ability and intolerance for salt water make them ideal candidates for Gondwanan relicts - taxa whose distribution can be explained by vicariant isolation driven by the breakup of Gondwana. Here we reconstruct the phylogenetic relationships among southern hemisphere stoneflies (5 families; 86 genera) using 2864bp of mitochondrial (COI) and nuclear (18S, H3) DNA, with a calibrated relaxed molecular clock used to estimate the chronology of diversification. Our analysis suggests that largely antitropical stonefly sub-orders, Arctoperlaria (northern hemisphere) and Antarctoperlaria (southern hemisphere), were formed approximately 121Ma (95% prior probability distribution 107-143Ma), which may reflect the vicariant rifting of the supercontinent Pangaea. Subsequently, we infer that a single Arctoperlaria lineage has dispersed into southern hemisphere 76Ma (95% range 65-98Ma). The majority of divergences between South American and Australian stonefly lineages appear to coincide with the opening of Drake Passage around 40Ma, suggesting vicariant isolation of these landmasses may be responsible for these biogeographic disjunctions. In contrast, divergences between New Zealand lineages and their sister taxa appear to post-date vicariant timeframes, implying more recent dispersal events.

Behavioural evolution in penguins does not reflect phylogeny.

Over the past two decades, behavioural biologists and ecologists have made effective use of the comparative method, but have often stopped short of adopting an explicitly phylogenetic approach. We examined 68 behaviour and life history (BLH) traits of 15 penguin species to: (i) infer penguin phylogeny, (ii) assess homology of behavioural characters, and (iii) evaluate hypotheses about character evolution and ancestral states. Parsimony analysis of the BLH dataset found either two shortest trees (characters coded as unordered) or a single shortest tree (characters coded as a combination of unordered and Dollo). The BLH data had significant structure. Kishino-Hasegawa tests indicated that BLH trees were significantly different from most previous estimates of penguin phylogeny. The BLH phylogeny generated from Dollo characters appeared to be less accurate than the tree derived from the completely unordered dataset. Dividing BLH data into display and non-display traits resulted in no significant differences in level of homoplasy and no difference in the accuracy of phylogeny. Tests for homology of BLH traits were performed by mapping the characters onto a molecular tree. Assuming that independent gains are less likely than losses of character states, 65 of the 68 characters were likely to be homologous across taxa, and at least several characters appeared to have been stable since the origin of modern penguins around 30 Myr. Finally, the likely BLH traits of the most recent common ancestor of extant penguins were reconstructed from character states along the internal branch leading to the penguins. This analysis suggested that the "proto-penguin" probably had a similar life history to current temperate penguins but few ritualized behaviours. A southern, cool-temperate origin of penguins is suggested.

Taxonomic validity and phylogenetic relationships of a newly-described tooth-carp, Aphanius mesopotamicus Coad, 2009 (Teleostei: Cyprinodontidae).

Variation among complete cytb sequences (1140 bp) of Aphanius mesopotamicus Coad, 2009 was compared with closely related species, to investigate the validity of this taxon as a newly-described tooth-carp based on morphological characteristics. Maximum likelihood and Bayesian likelihood trees supported the monophyly of A. mesopotamicus and its sister group relationship to A. sophiae. Some 10-16 differences were found when compared to four different population samples of A. sophiae, whereas, intraspecific differences were only up to 6 bp. These distances suggest divergence from a common ancestor with A. sophiae at roughly 1 million years ago. These results are congruent with morphology-based hypotheses, indicating a recent speciation event.

Genomics reveals repeated landlocking of diadromous fish on an isolated island.

Landlocking of diadromous fish in freshwater systems can have significant genomic consequences. For instance, the loss of the migratory life stage can dramatically reduce gene flow across populations, leading to increased genetic structuring and stronger effects of local adaptation. These genomic consequences have been well-studied in some mainland systems, but the evolutionary impacts of landlocking in island ecosystems are largely unknown. In this study, we used a genotyping-by-sequencing (GBS) approach to examine the evolutionary history of landlocking in common smelt (Retropinna retropinna) on Chatham Island, a small isolated oceanic island 800 kilometres east of mainland New Zealand. We examined the relationship between Chatham Island and mainland smelt and used coalescent analyses to test the number and timing of landlocking events on Chatham Island. Our genomic analysis, based on 21,135 SNPs across 169 individuals, revealed that the Chatham Island smelt was genomically distinct from the mainland New Zealand fish, consistent with a single ancestral colonisation event of Chatham Island in the Pleistocene. Significant genetic structure was also evident within the Chatham Island smelt, with a diadromous Chatham Island smelt group, along with three geographically structured landlocked groups. Coalescent demographic analysis supported three independent landlocking events, with this loss of diadromy significantly pre-dating human colonisation. Our results illustrate how landlocking of diadromous fish can occur repeatedly across a narrow spatial scale, and highlight a unique system to study the genomic basis of repeated adaptation.

Genetic drift outweighs natural selection at toll-like receptor (TLR) immunity loci in a re-introduced population of a threatened species.

During population establishment, genetic drift can be the key driver of changes in genetic diversity, particularly while the population is small. However, natural selection can also play a role in shaping diversity at functionally important loci. We used a well-studied, re-introduced population of the threatened Stewart Island robin (N = 722 pedigreed individuals) to determine whether selection shaped genetic diversity at innate immunity toll-like receptor (TLR) genes, over a 9-year period of population growth following establishment with 12 genetic founders. We found no evidence for selection operating with respect to TLR diversity on first-year overwinter survival for the majority of loci, genotypes and alleles studied. However, survival of individuals with TLR4BE genotype was significantly improved: these birds were less than half as likely to die prior to maturity compared with all other TLR4 genotypes. Furthermore, the population frequency of this genotype, at a two-fold excess over Hardy-Weinberg expectation, was increased by nonrandom mating. Near-complete sampling and full pedigree and reproductive data enabled us to eliminate other potential causes of these patterns including inbreeding, year effects, density dependence, selection on animals at earlier life history stages or genome-level association of the TLR4E allele with 'good genes'. However, comparison of observed levels of gene diversity to predictions under simulated genetic drift revealed results consistent with neutral expectations for all loci, including TLR4. Although selection favoured TLR4BE heterozygotes in this population, these effects were insufficient to outweigh genetic drift. This is the first empirical study to show that genetic drift can overwhelm natural selection in a wild population immediately following establishment.

Extreme positive selection on a new highly-expressed larval glycoprotein (LGP) gene in Galaxias fishes (Osmeriformes: Galaxiidae).

We describe the intron-exon structure and DNA/protein sequences of a new larval glycoprotein (LGP) gene from nine species of galaxiid fish. The gene has a distant similarity to Danio THP (Tamm-Horsfall urinary glycoprotein; uromodulin) and cichlid SPP120 (seminal plasma glycoprotein) due to conserved features of its zona pellucida (ZP) domain, including eight highly conserved cysteines and a consensus furin cleavage site. Using a combination of 454 sequencing of cDNA and exon-primed intron-spanning sequencing of genomic DNA, we obtained full sequences of the coding region (996 bp) and its intervening sequences (1,459 bp). LGP shows an exceptionally strong signal of positive selection over the entire coding region, as evidenced by d(N)/d(S) values >1. Across nine species of Galaxias, 87/332 (26%) amino acid residues are variable, compared with 9/386 (2%) for mitochondrial cytochrome b (cytb) in the same group of species. Across 36 interspecific pairwise comparisons, genetic distances are in all cases larger for coding region than for introns, by a factor of 2.4-fold on average. Reading frame, gene structure, splice sites, and many ZP motifs are conserved across all species. Together with the fact that the gene is expressed in all species, these results argue clearly against the possibility of a pseudogene. We show by 454 sequencing and quantitative polymerase chain reaction that the transcript is abundant (ca. 0.5%) in newly hatched larvae and appears to be almost absent from a range of adult tissues. We postulate that the strong Darwinian evolution exhibited by this protein may reflect some type of immunoprotection at this vulnerable larval stage.

Gene trees versus species trees: reassessing life-history evolution in a freshwater fish radiation.

Mechanisms of speciation are best understood in the context of phylogenetic relationships and as such have often been inferred from single gene trees, typically those derived from mitochondrial DNA (mtDNA) markers. Recent studies, however, have noted the potential for phylogenetic discordance between gene trees and underlying species trees (e.g., due to stochastic lineage sorting, introgression, or selection). Here, we employ a variety of nuclear DNA loci to reassess evolutionary relationships within a recent freshwater fish radiation to reappraise modes of speciation. New Zealand's freshwater-limited Galaxias vulgaris complex is thought to have evolved from G. brevipinnis, a widespread migratory species that retains a plesiomorphic marine juvenile phase. A well-resolved tree, based on four mtDNA regions, previously suggested that marine migratory ability has been lost on 3 independent occasions in the evolution of this species flock (assuming that loss of diadromy is irreversible). Here, we use pseudogene (galaxiid Numt: 1801 bp), intron (S: 903 bp), and exon (RAG-1: 1427 bp) markers, together with mtDNA, to reevaluate this hypothesis of parallel evolution. Interestingly, partitioned Bayesian analysis of concatenated nuclear sequences (3141 bp) and concatenated nuclear and mtDNA (4770 bp) both recover phylogenies implying a single loss of diadromy, not three parallel losses as previously inferred from mtDNA alone. This phylogenetic result is reinforced by a multilocus analysis performed using Bayesian estimation of species trees (BEST) software that estimates the posterior distribution of species trees under a coalescent model. We discuss factors that might explain the apparently misleading phylogenetic inferences generated by mtDNA.

New Zealand phylogeography: evolution on a small continent.

New Zealand has long been a conundrum to biogeographers, possessing as it does geophysical and biotic features characteristic of both an island and a continent. This schism is reflected in provocative debate among dispersalist, vicariance biogeographic and panbiogeographic schools. A strong history in biogeography has spawned many hypotheses, which have begun to be addressed by a flood of molecular analyses. The time is now ripe to synthesize these findings on a background of geological and ecological knowledge. It has become increasingly apparent that most of the biota of New Zealand has links with other southern lands (particularly Australia) that are much more recent than the breakup of Gondwana. A compilation of molecular phylogenetic analyses of ca 100 plant and animal groups reveals that only 10% of these are even plausibly of archaic origin dating to the vicariant splitting of Zealandia from Gondwana. Effects of lineage extinction and lack of good calibrations in many cases strongly suggest that the actual proportion is even lower, in keeping with extensive Oligocene inundation of Zealandia. A wide compilation of papers covering phylogeographic structuring of terrestrial, freshwater and marine species shows some patterns emerging. These include: east-west splits across the Southern Alps, east-west splits across North Island, north-south splits across South Island, star phylogenies of southern mountain isolates, spread from northern, central and southern areas of high endemism, and recent recolonization (postvolcanic and anthropogenic). Excepting the last of these, most of these patterns seem to date to late Pliocene, coinciding with the rapid uplift of the Southern Alps. The diversity of New Zealand geological processes (sinking, uplift, tilting, sea level change, erosion, volcanism, glaciation) has produced numerous patterns, making generalizations difficult. Many species maintain pre-Pleistocene lineages, with phylogeographic structuring more similar to the Mediterranean region than northern Europe. This structure reflects the fact that glaciation was far from ubiquitous, despite the topography. Intriguingly, then, origins of the flora and fauna are island-like, whereas phylogeographic structure often reflects continental geological processes.

Do insects lose flight before they lose their wings? Population genetic structure in subalpine stoneflies.

Wing reduction and flightlessness are common features of alpine and subalpine insects, and are typically interpreted as evolutionary adaptations to increase fecundity and promote local recruitment. Here, we assess the impact of wing reduction on dispersal in stoneflies (Plecoptera: Gripopterygidae: Zelandoperla) in southern New Zealand. Specifically, we present comparative phylogeographic analyses (COI; H3) of strong-flying Zelandoperla decorata (144 individuals, 63 localities) vs. the co-distributed but weak-flying Zelandoperla fenestrata species group (186 individuals, 81 localities). The latter group exhibits a variety of morphotypes, ranging from fully winged to completely wingless. Consistent with its capacity for strong flight-mediated dispersal, Z. decorata exhibited no substantial phylogeographic differentiation across its broad South Island range. Conversely the weak-flying fenestrata species group exhibited substantial genetic structure across both fine and broad geographic scales. Intriguingly, the variable degrees of wing development observed within the fenestrata species group had no apparent impact on levels of phylogeographic structure, which were high regardless of morphotype, suggesting that even fully winged specimens of this group do not fly. This finding implies that Zelandoperla flight loss occurs independently of wing loss, and might reflect underlying flight muscle reduction.

Transverse Alpine Speciation Driven by Glaciation.

The allopatric model of biological speciation involves fracturing of a pre-existing species distribution and subsequent genetic divergence in isolation. Accumulating global evidence from the Pyrénées, Andes, Himalaya, and the Southern Alps in New Zealand shows the Pleistocene to be associated with the generation of new alpine lineages. By synthesising a large number of genetic analyses and incorporating tectonic, climatic, and population-genetic models, we show here how glaciation is the likely driver of speciation transverse to the Southern Alps. New calibrations for rates of molecular evolution and tectonic uplift both suggest a ∼2 million-year (Ma) time frame. Although glaciation is often seen as destructive for biodiversity, here we demonstrate its creativity, and suggest a general model for speciation on temperate mountain systems worldwide.

A comparison of five hybrid zones of the weta Hemideina thoracica (Orthoptera: Anostostomatidae): degree of cytogenetic differentiation fails to predict zone width.

Tension zones are maintained by the interaction between selection against hybrids and dispersal of individuals. Investigating multiple hybrid zones within a single species provides the opportunity to examine differences in zone structure on a background of differences in extrinsic factors (e.g., age of the zone, ecology) or intrinsic factors (e.g., chromosomes). The New Zealand tree weta Hemideina thoracica comprises at least eight distinct chromosomal races with diploid numbers ranging from 2n = 11 (XO) to 2n = 23 (XO). Five independent hybrid zones were located that involve races differing from one another by a variety of chromosomal rearrangements. The predicted negative correlation between extent of karyotypic differentiation (measured in terms of both percent of genome and number of rearrangements) and zone width was not found. Conversely, the widest zones were those characterized by two chromosome rearrangements involving up to 35% of the genome. The narrowest zone occurred where the two races differ by a single chromosome rearrangement involving approximately 2% of the genome. The five estimates of chromosomal cline width ranged from 0.5 km to 47 km. A comparative investigation of cline width for both chromosomal and mitochondrial markers revealed a complex pattern of zone characteristics. Three of the five zones in this study showed cline concordance for the nuclear and cytoplasmic markers, and at two of the zones the clines were also coincident. Zones with the widest chromosomal clines had the widest mitochondrial DNA clines. It appears that, even within a single species, the extent of karyotypic differentiation between pairs of races is not a good predictor of the level of disadvantage suffered by hybrids.

Episodic positive selection in the evolution of avian toll-like receptor innate immunity genes.

Toll-like receptors (TLRs) are a family of conserved pattern-recognition molecules responsible for initiating innate and acquired immune responses. Because they play a key role in host defence, these genes have received increasing interest in the evolutionary and population genetics literature, as their variation represents a potential target of adaptive evolution. However, the role of pathogen-mediated selection (i.e. episodic positive selection) in the evolution of these genes remains poorly known and has not been examined outside of mammals. A recent increase in the number of bird species for which TLR sequences are available has enabled us to examine the selective processes that have influenced evolution of the 10 known avian TLR genes. Specifically, we tested for episodic positive selection to identify codons that experience purifying selection for the majority of their evolution, interspersed with bursts of positive selection that may occur only in restricted lineages. We included up to 23 species per gene (mean = 16.0) and observed that, although purifying selection was evident, an average of 4.5% of codons experienced episodic positive selection across all loci. For four genes in which sequence coverage traversed both the extracellular leucine-rich repeat region (LRR) and transmembrane/intracellular domains of the proteins, increased positive selection was observed at the extracellular domain, consistent with theoretical predictions. Our results provide evidence that episodic positive selection has played an important role in the evolution of most avian TLRs, consistent with the role of these loci in pathogen recognition and a mechanism of host-pathogen coevolution.

Variation at innate immunity Toll-like receptor genes in a bottlenecked population of a New Zealand robin.

Toll-like receptors (TLRs) are an ancient family of genes encoding transmembrane proteins that bind pathogen-specific molecules and initiate both innate and adaptive aspects of the immune response. Our goal was to determine whether these genes show sufficient genetic diversity in a bottlenecked population to be a useful addition or alternative to the more commonly employed major histocompatibility complex (MHC) genotyping in a conservation genetics context. We amplified all known avian TLR genes in a severely bottlenecked population of New Zealand's Stewart Island robin (Petroica australis rakiura), for which reduced microsatellite diversity was previously observed. We genotyped 17-24 birds from a reintroduced island population (including the 12 founders) for nine genes, seven of which were polymorphic. We observed a total of 24 single-nucleotide polymorphisms overall, 15 of which were non-synonymous, representing up to five amino-acid variants at a locus. One locus (TLR1LB) showed evidence of past directional selection. Results also confirmed a passerine duplication of TLR7. The levels of TLR diversity that we observe are sufficient to justify their further use in addressing conservation genetic questions, even in bottlenecked populations.

The Invertebrate Life of New Zealand: A Phylogeographic Approach.

Phylogeography contributes to our knowledge of regional biotas by integrating spatial and genetic information. In New Zealand, comprising two main islands and hundreds of smaller ones, phylogeography has transformed the way we view our biology and allowed comparison with other parts of the world. Here we review studies on New Zealand terrestrial and freshwater invertebrates. We find little evidence of congruence among studies of different taxa; instead there are signatures of partitioning in many different regions and expansion in different directions. A number of studies have revealed unusually high genetic distances within putative species, and in those where other data confirm this taxonomy, the revealed phylogeographic structure contrasts with northern hemisphere continental systems. Some taxa show a signature indicative of Pliocene tectonic events encompassing land extension and mountain building, whereas others are consistent with range expansion following the last glacial maximum (LGM) of the Pleistocene. There is some indication that montane taxa are more partitioned than lowland ones, but this observation is obscured by a broad range of patterns within the sample of lowland/forest taxa. We note that several geophysical processes make similar phylogeographic predictions for the same landscape, rendering confirmation of the drivers of partitioning difficult. Future multi-gene analyses where applied to testable alternative hypotheses may help resolve further the rich evolutionary history of New Zealand's invertebrates.

Onset of glaciation drove simultaneous vicariant isolation of Alpine insects in New Zealand.

The origin of the New Zealand "beech gap," a low-diversity zone in the central South Island corresponding with a disjunction in the distribution of many taxa, has been the focus of biogeographical debate for many decades. Here, we use comparative phylogeographic analysis (COI; H3) of six alpine stonefly genera (116 individuals, 102 localities) to test a vicariant evolutionary hypothesis for the origin of this "biotic gap." We find strikingly similar phylogeographic patterns in all six genera, with the deepest genetic divergences always found between samples north and south of the beech gap. The magnitude of north-south genetic differentiation for COI is similar across all six genera (ranging from 0.074 to 0.091), with a test for simultaneous vicariance confirming that divergence is consistent with a single evolutionary event. The concordant cladogenesis detected across multiple taxa is consistent with vicariant isolation caused by the onset of glaciation in the late Pliocene. This study thus indicates an important cladogenetic role for glaciation, an abiotic evolutionary process that is more typically associated with loss of biodiversity.