Chung Ping Lin

How do insect nuclear and mitochondrial gene substitution patterns differ? Insights from Bayesian analyses of combined datasets

We analyzed 12 combined mitochondrial and nuclear gene datasets in seven orders of insects using both equal weights parsimony (to evaluate phylogenetic utility) and Bayesian methods (to investigate substitution patterns). For the Bayesian analyses we used relatively complex models (e.g., general time reversible models with rate variation) that allowed us to quantitatively compare relative rates among genes and codon positions, patterns of rate variation among genes, and substitution patterns within genes. Our analyses indicate that nuclear and mitochondrial genes differ in a number of important ways, some of which are correlated with phylogenetic utility. First and most obviously, nuclear genes generally evolve more slowly than mitochondrial genes (except in one case), making them better markers for deep divergences. Second, nuclear genes showed universally high values of CI and (generally) contribute more to overall tree resolution than mitochondrial genes (as measured by partitioned Bremer support). Third, nuclear genes show more homogeneous patterns of among-site rate variation (higher values of alpha than mitochondrial genes). Finally, nuclear genes show more symmetrical transformation rate matrices than mitochondrial genes. The combination of low values of alpha and highly asymmetrical transformation rate matrices may explain the overall poor performance of mitochondrial genes when compared to nuclear genes in the same analysis. Our analyses indicate that some parameters are highly correlated. For example, A/T bias was positively and significantly associated with relative rate and CI was positively and significantly associated with alpha (the shape of the gamma distribution). These results provide important insights into the substitution patterns that might characterized high quality genes for phylogenetic analysis: high values of alpha, unbiased base composition, and symmetrical transformation rate matrices. We argue that insect molecular systematists should increasingly focus on nuclear rather than mitochondrial gene datasets because nuclear genes do not suffer from the same substitutional biases that characterize mitochondrial genes.

How do insect nuclear ribosomal genes compare to protein-coding genes in phylogenetic utility and nucleotide substitution patterns?

Abstract.  The expanding data set on insect molecular systematics allows examination of phylogenetic performance and molecular evolution of different types of gene. Studies combining more than one gene in the same analysis allow examination of the relative contribution and performance of each gene partition and can help inform gene choice for resolving deep and/or problematic divergences. We compared results obtained from analyses of twelve insect data sets in which authors combined one or more nuclear ribosomal genes (28S and/or 18S) with one or more protein‐coding genes [elongation factor‐1α (EF‐1α), histone H3, carbamoylphosphate synthetase domain (CPS domain of CAD, or rudimentary), long‐wavelength rhodopsin (LW opsin), glucose‐6‐phosphate dehydrogenase (G6pd), phosphoenolpyruvate carboxykinase (PEPCK), arginine kinase, and white]. Data sets examined spanned eight orders of insects (Odonata, Ephemeroptera, Hemiptera, Coleoptera, Trichoptera, Lepidoptera, Diptera and Hymenoptera), providing a broad range of divergence times and taxonomic levels. We estimated the phylogenetic utility of the individual genes (using parsimony methods) and characterized the nucleotide substitution patterns (using Bayesian methods) to ask which type of data is preferable for phylogenetic analysis in insects. Nuclear ribosomal and protein coding genes differed little in our measures of phylogenetic performance and patterns of nucleotide substitution. We recommend combining nuclear ribosomal gene data with nuclear protein‐coding gene data because each data set has distinct advantages. We do not recommend using mitochondrial genes for higher‐level studies of insect phylogeny because reviewed studies demonstrate substitution patterns that lead to high levels of homoplasy.

Molecular Phylogeny of the North American Enchenopa binotata (Homoptera: Membracidae) Species Complex

Abstract The North American Enchenopa binotata (Say) species complex is a model of sympatric speciation in which phytophagous insects are hypothesized to diverge through host-plant specialization resulting from changes in host plant usage that alter life history timing. A robust phylogeny is needed to evaluate the historical relevance of the prediction that sister taxa differ in critical life-history traits. Phylogenetic analysis using parsimony and likelihood criteria of 2305 nucleotides in sequences from mitochondrial COI, COII, tRNA-Leucine, and 12S genes revealed two pairs of sister taxa. Both pairs of sister taxa differ from each other in the timing of egg hatch in the spring that is mediated by differences in host-plant phenology. Host plant mediated timing of egg hatch results in asynchronous life histories among sister taxa facilitating reproductive isolation. Sister taxa of Enchenopa from Celastrus and from Viburnum differ in their diurnal and temporal spans during which mating occurs. Mating of Enchenopa from Liriodendron takes place after that of its sister species on Cercis. These results support the hypothesis that speciation could have been initiated through a shift to a host plant that alters life-history timing.

Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists: Leishmania-Specific Informative Sites in Hydrophilic Loops of Trans- Endoplasmic Reticulum N-Acetylglucosamine-1-Phosphate Transferase

ABSTRACT A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.

Molecular Phylogenetics and Evolution of Maternal Care in Membracine Treehoppers

The treehopper subfamily Membracinae (Insecta: Hemiptera: Membracidae) comprises the majority of genera and species diversity in the New World tropics. These treehoppers exhibit a wide range of social behaviors, making them an excellent group for studying patterns of social evolution in insects. However, to date the tribal and generic relationships have remained unclear. We reconstructed the phylogeny of the Membracinae using a combined mitochondrial (COI, COII, tRNA-Leu, and 12S) and nuclear (Wg) gene data set. A total of 2608 aligned nucleotide sites were obtained for 112 species, representing 25 of 38 currently recognized genera and all four tribes. A strict consensus of five equally parsimonious trees recovered the subfamily and three of its four tribes. The majority rule consensus tree derived from the Bayesian analyses based on the GTR+I+G and mixed-models recovered many clades shared with the parsimony trees and is identical to the single best tree inferred from maximum likelihood analysis, aside from the rearrangement of one node. A comparison of mitochondrial and nuclear genes indicated that Wg provided higher consistency index (CI), data decisiveness (DD), partitioned Bremer support (PBS) than any of the mitochondrial genes analyzed. The combined mitochondrial and nuclear DNA provide strong support for the monophyly of the subfamily and three of its four tribes (Aconophorini, Hoplophorionini, and Hypsoprorini). Membracini is paraphyletic with respect to Hoplophorionini and contains two lineages, the Membracini sensu strictu and the newly resurrected tribe Bolbonotini. Our analyses show that there is a strong phylogenetic component to the evolution of maternal care. Given the widespread occurrence of maternal care within the subfamily, this trait is estimated to have < or = 3 origins, two reversals, and one loss. Our results suggest that the evolution of maternal care in insects may not be as evolutionarily labile as previously thought.

The complete mitochondrial genome and phylogenomics of a damselfly, Euphaea formosa support a basal Odonata within the Pterygota

This study determined the first complete mitochondrial genome of a damselfly, Euphaea formosa (Insecta: Odonata: Zygoptera), and reconstructed a phylogeny based on thirteen protein-coding genes of mitochondrial genomes in twenty-five representative hexapods to examine the relationships among the basal Pterygota. The damselflys mitochondrial genome is a circular molecule of 15,700bp long, and contains the entire set of thirty-seven genes typically found in insects. The gene arrangement, nucleotide composition, and codon usage pattern of the mitochondrial genome are similar across the three odonate species, suggesting a conserved genome evolution within the Odonata. The presence of the intergenic spacer s5 likely represents a synapomorphy for the dragonflies (Anisoptera). Maximum parsimony, maximum likelihood, and Bayesian analyses of both nucleotide and amino acid sequences cannot support the three existing phylogenetic hypotheses of the basal Pterygota (Palaeoptera, Metapterygota, and Chiastomyaria). In contrast, the phylogenetic results indicate an alternative hypothesis of a strongly supported basal Odonata and a sister relationship of the Ephemeroptera and Plecoptera. The unexpected sister Ephemeroptera+Plecoptera clade, which contradicts with the widely accepted hypothesis of a monophyletic Neoptera, requires further analyses with additional mitochondrial genome sampling at the base of the Neoptera.

Social behaviour and life history of membracine treehoppers

Social behaviour in the form of parental care is widespread among insects but the evolutionary histories of these traits are poorly known due to the lack of detailed life history data and reliable phylogenies. Treehoppers (Hemiptera: Membracidae) provide some of the best studied examples of parental care in insects in which maternal care involving egg guarding occurs frequently. The Membracinae exhibit the entire range of social behaviour found in the treehoppers, ranging from asocial solitary individuals, nymphal or adult aggregations, to highly developed maternal care with parent–offspring communication. Within the subfamily, subsocial behaviour occurs in at least four of the five tribes. The Aconophorini and Hoplophorionini are uniformly subsocial, but the Membracini is a mixture of subsocial and gregarious species. The Hypsoprorini contains both solitary and gregarious species. Accessory secretions are used by many treehoppers to cover egg masses inserted into plant tissue while oviposition on plant surfaces is restricted to a few species. Presumed aposematic colouration of nymphs and teneral adults appears to be restricted to gregarious and subsocial taxa. Ant mutualism is widespread among membracine treehoppers and may play an important role in the evolutionary development of subsocial behaviour. The life history information provides a basis for comparative analyses of maternal care evolution and its correlation with ant mutualism in membracine treehoppers. The results show that there is a strong phylogenetic component to the evolution of maternal care in membracine treehoppers, and provide the first quantitative evidence of correlated evolution of maternal care and ant mutualism in treehoppers. Further research on natural history, particularly of the tropical fauna, will be necessary to better understand the evolution of social behaviour and life history in treehoppers.

Ultrastructure of dragonfly wing veins: composite structure of fibrous material supplemented by resilin

Dragonflies count among the most skilful of the flying insects. Their exceptional aerodynamic performance has been the subject of various studies. Morphological and kinematic investigations have showed that dragonfly wings, though being rather stiff, are able to undergo passive deformation during flight, thereby improving the aerodynamic performance. Resilin, a rubber‐like protein, has been suggested to be a key component in insect wing flexibility and deformation in response to aerodynamic loads, and has been reported in various arthropod locomotor systems. It has already been found in wing vein joints, connecting longitudinal veins to cross veins, and was shown to endow the dragonfly wing with chordwise flexibility, thereby most likely influencing the dragonflys flight performance. The present study revealed that resilin is not only present in wing vein joints, but also in the internal cuticle layers of veins in wings of Sympetrum vulgatum (SV) and Matrona basilaris basilaris (MBB). Combined with other structural features of wing veins, such as number and thickness of cuticle layers, material composition, and cross‐sectional shape, resilin most probably has an effect on the vein′s material properties and the degree of elastic deformations. In order to elucidate the wing vein ultrastructure and the exact localisation of resilin in the internal layers of the vein cuticle, the approaches of bright‐field light microscopy, wide‐field fluorescence microscopy, confocal laser‐scanning microscopy, scanning electron microscopy and transmission electron microscopy were combined. Wing veins were shown to consist of up to six different cuticle layers and a single row of underlying epidermal cells. In wing veins of MBB, the latter are densely packed with light‐scattering spheres, previously shown to produce structural colours in the form of quasiordered arrays. Longitudinal and cross veins differ significantly in relative thickness of exo‐ and endocuticle, with cross veins showing a much thicker exocuticle. The presence of resilin in the unsclerotised endocuticle suggests its contribution to an increased energy storage and material flexibility, thus to the prevention of vein damage. This is especially important in the highly stressed longitudinal veins, which have much lower possibility to yield to applied loads with the aid of vein joints, as the cross veins do. These results may be relevant not only for biologists, but may also contribute to optimise the design of micro‐air vehicles.

Diversification in subtropical mountains: Phylogeography, Pleistocene demographic expansion, and evolution of polyphenic mandibles in Taiwanese stag beetle, Lucanus formosanus

Pleistocene glacial oscillations have had profound impacts on the historical population dynamics of extant species. However, the genetic consequences of past climatic changes depend largely on the latitude and topography of the regions in question. This study investigates the effect of Pleistocene glacial periods and the Central Mountain Range on the phylogeography, historical demography, and phenotypic differentiation of a montane forest-dwelling stag beetle, Lucanus formosanus (Coleoptera: Lucanidae), which exhibits extensive mandible variations across mountain ranges in subtropical Taiwan. Analyses of mitochondrial (cox1) and nuclear (wg) loci reveal that L. formosanus originated nearly 1.6 million years ago (Mya) in the early Pleistocene period and consisted of geographically overlapping Alishan and Widespread clades. A drastic population expansion starting approximately 0.2 Mya in the Widespread clade likely resulted from altitudinal range shift of the temperate forests, which was closely tied to the arrival of the Riss glacial period in the late Middle Pleistocene. A ring-like pattern of historical gene flow among neighboring populations in the vicinity of the Central Mountain Range indicates that the mountains constitute a strong vicariant barrier to the east-west gene flow of L. formosanus populations. A geographic cline of decreasing mandible size from central to north and south, and onto southeast of Taiwan is inconsistent with the low overall phylogeographic structures. The degree of mandible variation does not correlate with the expected pattern of neutral evolution, indicating that the evolutionary diversification of this morphological weapon is most likely subject to sexual or natural selection. We hypothesize that the adaptive evolution of mandibles in L. formosanus is shaped largely by the habitat heterogeneity.

Lineage-specific late pleistocene expansion of an endemic subtropical gossamer-wing damselfly, Euphaea formosa, in Taiwan

BackgroundPleistocene glacial oscillations have significantly affected the historical population dynamics of temperate taxa. However, the general effects of recent climatic changes on the evolutionary history and genetic structure of extant subtropical species remain poorly understood. In the present study, phylogeographic and historical demographic analyses based on mitochondrial and nuclear DNA sequences were used. The aim was to investigate whether Pleistocene climatic cycles, paleo-drainages or mountain vicariance of Taiwan shaped the evolutionary diversification of a subtropical gossamer-wing damselfly, Euphaea formosa.ResultsE. formosa populations originated in the middle Pleistocene period (0.3 Mya) and consisted of two evolutionarily independent lineages. It is likely that they derived from the Pleistocene paleo-drainages of northern and southern Minjiang, or alternatively by divergence within Taiwan. The ancestral North-central lineage colonized northwestern Taiwan first and maintained a slowly growing population throughout much of the early to middle Pleistocene period. The ancestral widespread lineage reached central-southern Taiwan and experienced a spatial and demographic expansion into eastern Taiwan. This expansion began approximately 30,000 years ago in the Holocene interglacial period. The ancestral southern expansion into eastern Taiwan indicates that the central mountain range (CMR) formed a barrier to east-west expansion. However, E. formosa populations in the three major biogeographic regions (East, South, and North-Central) exhibit no significant genetic partitions, suggesting that river drainages and mountains did not form strong geographical barriers against gene flow among extant populations.ConclusionsThe present study implies that the antiquity of E. formosas colonization is associated with its high dispersal ability and larval tolerance to the late Pleistocene dry grasslands. The effect of late Pleistocene climatic changes on the subtropical damselflys historical demography is lineage-specific, depending predominantly on its colonization history and geography. It is proposed that the Riss and Würm glaciations in the late Pleistocene period had a greater impact on the evolutionary diversification of subtropical insular species than the last glacial maximum (LGM).