Li-Yun Jiang

The Effectiveness of Three Regions in Mitochondrial Genome for Aphid DNA Barcoding: A Case in Lachininae

Background The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding. Methodology/Principal Findings Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in “best match” and 90.8% in “best close match”) and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of “tag barcodes” is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the “barcoding overlap” can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the “best close match” technique. Conclusions/Significance A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of “tag barcodes” can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.

Use of a mitochondrial COI sequence to identify species of the subtribe Aphidina (Hemiptera, Aphididae)

Abstract Aphids of the subtribe Aphidina are found mainly in the North Temperate Zone. The relative lack of diagnostic morphological characteristics has hindered the identification of species in this group. However, DNA-based taxonomic methods can clarify species relationships within this group. Sequence variation in a partial segment of the mitochondrial COI gene was highly effective for identifying species within Aphidina. Thirty-six species of Aphidina were identified in a neighbor-joining tree. Mean intraspecific sequence divergence in Aphidina was 0.52%, with a range of 0.00% to 2.95%, and the divergences of most species were less than 1%. Mean interspecific divergence within previously recognized genera or morphologically similar species groups was 6.80%, with a range of 0.68% to 11.40%, with variation mainly in the range of 3.50% to 8.00%. Possible reasons for anomalous levels of mean nucleotide divergence within or between some taxa are discussed.

Widespread infection and diverse infection patterns of Wolbachia in Chinese aphids

Wolbachia are intracellular symbionts that infect a wide range of arthropods and filarial nematodes. Aphids are engaged in diverse and complex relationships with their endosymbionts. Four supergroups (A, B, M and N) of Wolbachia were previously detected in aphids and supergroups M and N were only found in aphids. In this study, we detected and described Wolbachia infections in natural populations of aphids in China. Three supergroups (A, B and M) were found in the examined aphid species. Supergroup M was preponderant, whereas supergroups A and B were only detected in certain species. Supergroup N was not found in this study. There were four infection patterns of Wolbachia in aphids, namely, infection with supergroup M alone, co‐infection with supergroups A and M, co‐infection with supergroups B and M, and co‐infection with supergroups A, B and M. The pattern of infection only with supergroup M was universal and was found in all evaluated subfamilies. Only two subfamilies, Aphidinae and Lachninae, manifested to present all four infection patterns. Three patterns were observed in Calaphidinae (M, A&M, B&M) and Eriosomatinae (M, B&M, A&B&M). Two patterns were observed in the Anoeciinae (M, A&M) and Greenideinae (M, B&M), and only one pattern (M) was observed in the remaining families and/or subfamilies of Aphidoidea. These results indicated that Wolbachia infections in Chinese aphids are widespread. Phylogenetic analyses suggest that Wolbachia supergroup M spread rapidly and recently among all host species of aphids in China. Reasons for this spread and its mechanisms are discussed along with the possible effects of Wolbachia on their aphid hosts.

Phylogenetic congruence between Mollitrichosiphum (Aphididae: Greenideinae) and Buchnera indicates insect-bacteria parallel evolution

We wanted to test whether Mollitrichosiphum, an aphid genus with life cycles on subtropical woody host plants, and Buchnera, the primary endosymbiont of aphids, evolve in parallel. We used three aphid genes (mitochondrial COI, cytochrome oxidase subunit I and Cytb, cytochrome b; nuclear EF1α, translation elongation factor 1 alpha) and two Buchnera genes (16S rDNA; gnd, gluconate‐6‐phosphate dehydrogenase) to reconstruct phylogenies. The congruence between the phylogenetic trees of aphids and Buchnera was then measured. The results present phylogenetic evidence for the parallel evolution of Mollitrichosiphum and Buchnera at the intraspecific as well as the interspecific levels. Our results support the possibility of using endosymbiont genes to study host evolutionary history and biogeographical patterns. We also investigated the usability of the Buchnera gnd gene as a barcoding marker for aphid identification.

Hemipteran mitochondrial genomes: features, structures and implications for phylogeny.

The study of Hemipteran mitochondrial genomes (mitogenomes) began with the Chagas disease vector, Triatoma dimidiata, in 2001. At present, 90 complete Hemipteran mitogenomes have been sequenced and annotated. This review examines the history of Hemipteran mitogenomes research and summarizes the main features of them including genome organization, nucleotide composition, protein-coding genes, tRNAs and rRNAs, and non-coding regions. Special attention is given to the comparative analysis of repeat regions. Gene rearrangements are an additional data type for a few families, and most mitogenomes are arranged in the same order to the proposed ancestral insect. We also discuss and provide insights on the phylogenetic analyses of a variety of taxonomic levels. This review is expected to further expand our understanding of research in this field and serve as a valuable reference resource.

An aphid lineage maintains a bark‐feeding niche while switching to and diversifying on conifers

Lachnine aphids are unusual among phytophagous insects because they feed on both leafy and woody parts of both angiosperm and conifer hosts. Despite being piercing‐sucking phloem‐feeders, these aphids are most speciose on woody parts of coniferous hosts. To evaluate the significance of this unusual biology on their evolution, we reconstructed the ancestral host and feeding site of the lachnine aphids and estimated important host shifts during their evolution. We sampled 78 species representing 14 of the 18 genera of Lachninae from Asia and North America. We performed parsimony, Bayesian and likelihood phylogenetic analyses of combined mitochondrial Cox1, Cox2, CytB and nuclear EF1a1 DNA sequences. We dated the resulting phylograms important nodes using Bayesian methods and multiple fossil and secondary calibrations. Finally, we used parsimony and Bayesian ancestral state reconstruction to evaluate ancestral feeding ecology. Our results suggest the lachnine common ancestor fed on a woody part of an angiosperm host in the mid‐Cretaceous. A shift to conifer hosts in the Late Cretaceous is correlated with a subsequent increased diversification in the Palaeogene, but a switch to leafy host tissues did not engender a similar burst of diversification. Extant lachnine lineages exhibit the full range of historical association with their hosts: some appeared before, some concomitant with and some after the appearance of their hosts. We conclude our study by placing all the lachnine genera in five tribes.

The gnd gene of Buchnera as a new, effective DNA barcode for aphid identification

DNA barcoding uses a standard DNA sequence to facilitate species identification. Although the COI gene has been adopted as the standard, COI alone is imperfect due to several shortcomings. The primary endosymbiont of aphids, Buchnera, has higher evolutionary rates and interspecies divergence than its co‐diverging aphid hosts, making it a potential tool for resolving the ambiguities in aphid taxonomy. We compared the effectiveness of employing two different DNA regions, gnd and COI, for the discrimination of over 100 species of aphids. The mean interspecific divergence of the gnd region was significantly higher than the mean intraspecific variation; there were nearly nonoverlapping distributions between the intra‐ and interspecific samples. In contrast, COI showed a lower interspecific divergence, which led to difficulties in identifying closely related species. Our results show that gnd can identify species in the Aphididae, which suggests that the gnd region of Buchnera is a potentially effective barcode for aphid species identification. We also recommend the 2‐locus combination of gnd + COI as the aphid barcode. This will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of aphids.

A total-evidence phylogenetic analysis of Hormaphidinae (Hemiptera: Aphididae), with comments on the evolution of galls

A phylogenetic analysis of Hormaphidinae is presented based on a total‐evidence approach. Four genes (two mitochondrial, COI and CytB, and two nuclear, EF‐1α and LWO) are combined with 65 morphological and seven biological characters. Sixty‐three hormaphidine species representing three tribes and 36 genera as well as nine outgroups are included. Parsimony and model‐based approaches are used, and several support values and implied weighting schemes are explored to assess clade stability. The monophyly of Hormaphidinae and Nipponaphidini is supported, but Cerataphidini and Hormaphidini are not recovered as monophyletic. Based on the parsimony hypothesis from the total‐evidence analysis, the phylogenetic relationships within Hormaphidinae are discussed. Cerataphidini is re‐delimited to exclude Doraphis and Tsugaphis, and Hormaphidini is redefined to include Doraphis. Ceratocallis Qiao & Zhang is established as a junior synonym of Ceratoglyphina van der Goot, syn. nov. Lithoaphis quercisucta Qiao, Guo & Zhang is transferred to the genus Neohormaphis Noordam as Neohormaphis quercisucta (Qiao, Guo & Zhang) comb. nov. Galls have evolved independently within three tribes of Hormaphidinae. In Cerataphidini, pseudogalls are ancestral, both single‐cavity and multiple‐cavity galls have evolved once, and galls appear to have evolved towards greater complexity. Galling on secondary hosts has evolved twice in hormaphidines.

DNA barcoding of Greenideinae (Hemiptera:Aphididae) with resolving taxonomy problems

Abstract. Species of the Greenideinae are distributed mainly throughout South-east Asia and include some important agricultural and horticultural pests. Rapid and accurate species circumscription and identification in this subfamily are very difficult because similar morphological traits are shared among congeneric species. Here, we test the efficiency of DNA barcoding in the Greenideinae by analysing 214 samples covering 42 species belonging to nine genera using two mitochondrial gene fragments (COI barcode fragment and Cytb gene fragment). The results show that DNA barcoding is a useful species identification method in this subfamily. Both genes can correctly identify most species using neighbour-joining tree analyses and distance-based analyses. Based on the molecular and morphological evidence, we question the validity of two species, Mollitrichosiphum rhusae Ghosh, 1917 and Schoutedenia emblica (Patel & Kulkarni, 1953). Further analysis of the COI barcode fragment shows that Greenidea psidii van der Goot, 1917, an invasive species in Hawaii, is possibly from China. This is a preliminary DNA barcoding study in Greenideinae, and comprehensive sampling is needed to rigorously test the usefulness of DNA barcoding in this subfamily.

Species Differentiation of Chinese Mollitrichosiphum (Aphididae: Greenideinae) Driven by Geographical Isolation and Host Plant Acquirement

The impact of both the uplift of the Qinghai-Tibetan Plateau (QTP) and the separation of the Taiwan and Hainan Islands on the evolution of the fauna and flora in adjacent regions has been a topic of considerable interest. Mollitrichosiphum is a polyphagous insect group with a wide range of host plants (14 families) and distributions restricted to Southeast Asia. Based on the mitochondrial Cytochrome C Oxidase Subunit I (COI) and Cytochrome b (Cytb) genes, the nuclear elongation factor-1α (EF-1α) gene, and the detailed distribution and host plant data, we investigated the species differentiation modes of the Chinese Mollitrichosiphum species. Phylogenetic analyses supported the monophyly of Mollitrichosiphum. The divergence time of Mollitrichosiphum tenuicorpus (c. 11.0 mya (million years ago)), Mollitrichosiphum nandii and Mollitrichosiphum montanum (c. 10.6 mya) was within the time frame of the uplift of the QTP. Additionally, basal species mainly fed on Fagaceae, while species that fed on multiple plants diverged considerably later. Ancestral state reconstruction suggests that Fagaceae may be the first acquired host, and the acquisition of new hosts and the expansion of host range may have promoted species differentiation within this genus. Overall, it can be concluded that geographical isolation and the expansion of the host plant range may be the main factors driving species differentiation of Mollitrichosiphum.