Can whole-drawer images measure up? A reply to Johnson et al. (2013)?

Johnson et al. (2013) found that morphometric measurements of dragonfly wings taken from actual specimens and measurements taken from whole-drawer images of those specimens were equally accurate. We do not believe that their conclusions are justified by their data and analysis. Our reasons are, first, that their study was constrained in ways that restrict the generalisability of their results, but second, and of far greater significance, their statistical approach was entirely unsuited to their data and their results misled them to erroneous conclusions. We offer an alternative analysis of their data as published. Our reanalysis demonstrates, contra Johnson et al., that measurements from scanned images are not a reliable substitute for direct measurement.

Relaxed phylogenetics and the palaeoptera problem: resolving deep ancestral splits in the insect phylogeny.

The order in which the 3 groups of winged insects (the Pterygota) diverged from their common ancestor has important implications for understanding the origin of insect flight. But despite this importance, the split between the Odonata (dragonflies and damselflies), Ephemeroptera (mayflies), and Neoptera (the other winged orders) remains very much unresolved. Indeed, previous studies have obtained strong apparent support for each of the 3 possible branching patterns. Here, we present a systematic reinvestigation of the basal pterygote split. Our results suggest that outgroup choice and limited taxon sampling have been major sources of systematic error, even for data sets with a large number of characters (e.g., in phylogenomic data sets). In particular, a data set of 113 taxa provides consistent support for the Palaeoptera hypothesis (the grouping of Odonata with Ephemeroptera), whereas results from data sets with fewer taxa give inconsistent results and are highly sensitive to minor changes in data and methods. We also focus on recent methods that exploit temporal information using fossil calibrations, combined with additional assumptions about the evolutionary process, and so reduce the influence of outgroup choice. These methods are shown to provide more consistent results, for example, supporting Palaeoptera, even for data sets that previously supported other hypotheses. Together, these results have implications for understanding insect origins and for resolving other problematic splits in the tree of life.

Molecular genetic analysis and ecological evidence reveals multiple cryptic species among thynnine wasp pollinators of sexually deceptive orchids.

Sexually deceptive Chiloglottis orchids lure their male thynnine wasp pollinators to the flower by emitting semiochemicals that mimic the specific sex pheromone of the wasp. Sexual deception is possible because chemical rather than visual cues play the key role in wasp mate search, suggesting that cryptic wasp species may be frequent. We investigated this prospect among Neozeleboria wasp pollinators of Chiloglottis orchids, drawing on evidence from molecular phylogenetic analysis at three genes (CO1, rhodopsin and wingless), population genetic and statistical parsimony analysis at CO1, orchid associations and their semiochemicals, and geographic ranges. We found a compelling relationship between genetically defined wasp groups, orchid associations, semiochemicals and geographic range, despite a frequent lack of detectable morphological differences. Our findings reveal multiple cryptic species among orchid pollinators and indicate that chemical changes are important for wasp reproductive isolation and speciation. The diversity of Neozeleboria may have enabled, rather than constrained, pollinator-driven speciation in these orchids.

Phylogeny and evolution of the Meliphagoidea, the largest radiation of Australasian songbirds.

The Meliphagoidea comprises the largest radiation of Australasian passerines. Here we present the first detailed molecular phylogenetic analysis of its families and genera, particularly the Acanthizidae, using sequences from nine gene regions including both mitochondrial and nuclear DNA. Our results support some suggested relationships but challenge other groupings, particularly in Meliphagidae and Acanthizidae. Maluridae is sister to all other members of the superfamily. With appropriate taxon sampling and multilocus data, we provide the first strong molecular evidence supporting earlier recognition of bristlebirds, Dasyornis, as a separate family, Dasyornithidae. We further clarify its position as sister to Acanthizidae+Pardalotidae+Meliphagidae. Pardalotidae is sister to Acanthizidae, and thus its retention as a separate family is arbitrary. The meliphagid genus Lichenostomus is polyphyletic. We find no support for the current subfamily structure within Acanthizidae but recognise a clade that includes members of the subfamily Sericornithinae excluding Oreoscopus and Acanthornis. Subfamily Acanthizinae is paraphyletic. Surprisingly, the Tasmanian island endemic Acanthornis magna of mesic habitats is sister to the Aphelocephala whitefaces of mainland Australian xeric zones. This is one of several unexpected alignments of taxa as sisters that probably reflects the age of the Meliphagoidea. We find no evidence for separate radiations of New Guinean and Australian members of the Meliphagoidea.

Conservation of essential design features in coiled coil silks.

Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended beta-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small ( approximately 30 kDa each) and nonrepetitive and adopt a coiled coil structure. We examined silks from the 3 superfamilies of the Aculeata (Hymenoptera: Apocrita) by infrared spectroscopy and found coiled coil structure in bees (Apoidea) and in ants (Vespoidea) but not in parasitic wasps of the Chrysidoidea. We subsequently identified and sequenced the silk genes of bumblebees, bulldog ants, and weaver ants and compared these with honeybee silk genes. Each species produced orthologues of the 4 small fibroin proteins identified in honeybee silk. Each fibroin contained a continuous predicted coiled coil region of around 210 residues, flanked by 23-160 residue length N- and C-termini. The cores of the coiled coils were unusually rich in alanine. There was extensive sequence divergence among the bee and ant silk genes (<50% similarity between the alignable regions of bee and ant sequences), consistent with constant and equivalent divergence since the bee/ant split (estimated to be 155 Myr). Despite a high background level of sequence diversity, we have identified conserved design elements that we propose are essential to the assembly and function of coiled coil silks.

An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in Thalassarche albatrosses.

Molecular ecologists, in search of suitable molecular markers, frequently PCR-amplify regions of mitochondrial DNA from total DNA extracts. This approach, although common, is prone to the co-amplification of nuclear copies of transposed DNA sequences (numts), which can then generate apparent mitochondrial sequence heteroplasmy. In this study we describe the discovery of apparent mitochondrial sequence heteroplasmy in Thalassarche albatrosses but eliminate the possibility of true sequence heteroplasmy and numts and instead reveal the source of the apparent heteroplasmy to be a duplicated control region. The two control regions align easily but are not identical in sequence or in length. Comparisons of functionally significant conserved sequence blocks do not provide evidence of degeneration in either duplicate. Phylogenetic analyses of domain I of both control region copies in five Thalassarche species indicate that they are largely evolving in concert; however, a short section within them is clearly evolving independently. To our knowledge this is the first time contrasting evolutionary patterns have been reported for duplicate control regions. Available evidence suggests that this duplication may be taxonomically widespread, so the results presented here should be considered in future evolutionary studies targeting the control region of all Procellariiformes and potentially other closely related avian groups.

PERMUTATION TESTS AND OUTGROUPS.

- Commonly used permutation tail probability (PTP) and topology dependent permutation tail probability (T-PTP) tests incorporate an inappropriate treatment of designated outgroup taxa, and for that reason are biased either for (PTP) or for or against (T-PTP) rejection of the null hypothesis. A modified test is proposed, in which this source of bias is eliminated.