A two-step DNA barcoding approach for delimiting moth species: moths of Dongling Mountain (Beijing, China) as a case study

Abstract

DNA barcoding, based on a fragment of cytochrome c oxidase I (COI) mtDNA, is as an effective molecular tool for identification, discovery, and biodiversity assessment for most animals. However, multiple gene markers coupled with more sophisticated analytical approaches may be necessary to clarify species boundaries in cases of cryptic diversity or morphological plasticity. Using 339 moths collected from mountains surrounding Beijing, China, we tested a pipeline consisting of two steps: (1) rapid morphospecies sorting and screening of the investigated fauna with standard COI barcoding approaches; (2) additional analyses with multiple molecular markers for those specimens whose morphospecies and COI barcode grouping were incongruent. In step 1, 124 morphospecies were delimited into 116 barcode units, with 90% of the conflicts being associated with specimens identified to the genus Hypena. In step 2, 55 individuals representing all 12 Hypena morphospecies were analysed using COI, COII, 28S, EF-1a, Wgl sequences or their combinations with the BPP (Bayesian Phylogenetics and Phylogeography) multigene species delimitation method. The multigene analyses supported the delimitation of 5 species, consistent with the COI analysis. We conclude that a two-step barcoding analysis pipeline is able to rapidly characterize insect biodiversity and help to elucidate species boundaries for taxonomic complexes without jeopardizing overall project efficiency by substantially increasing analytical costs.

Figure 1

(a) The detailed topology of Hypena sequences. Different font colors indicates different morphospecies. Vertical line represents GMYC MOTU. (b) MOTU delimitation of Hypena under five approaches.

Figure 2

GMYC analysis of the 176 COI unique haplotypes for all 351 moth specimens. The red vertical lines on the ultrametric trees indicate the maximum likelihood transition point of the switch in branching rates, as estimated by a general mixed Yule-coalescent (GMYC) model. The GMYC analysis was performed using a single threshold. (b) Lineages-through-time plot based on the time calibrated tree obtained with all 176 COI unique haplotypes. The sharp increase in branching rate, corresponding to the transition from interspecies to intra-species branching events, is indicated by a vertical red line. 95% confidence intervals estimated ranges from 116 to 118.

Figure 3

Species delimitations based on distance threshold. (a) jMOTU analysis of COI variation in the number of MOTUs inferred at 1–100 cut-off values. Critical cutoff intervals are indicated with shaded sections. (b) Automatic partition of the COI gene from Automatic Barcode Gap Discovery (ABGD).

Figure 4

Species tree based upon combined analysis of five loci and posterior probabilities of each node for different datasets. (a) Species relationships among 12 Hypena species and 7 Notodontidae (outgroup) species. (b,c) Posterior probabilities of species delimitation with different datasets: 5 loci n + mtDNA (COI + COII + 28S rDNA + EF-1α + Wgl); 3loci nDNA (28S rDNA + EF-1α + Wgl) and 2 loci mtDNA (COI + COII); numbers in brackets beside node numbers indicate posterior probability support (red numbers are greater than or equal to 95, green numbers are less than 95, numbers less than 50 not shown).

Figure 5

Geometrical morphology analysis of 55 Hypena specimens with forewings and hind wings respectively. (a,g) Scatter plots of the scores for the first two PCs (PC1 and PC2). (b,h) Percentages of total variance accounted for by PCs. (c,k) Transformation grids for visualizing shape change of forewing and hind wing (for PC1 and PC2). (d,i) Location of the wing landmarks used in the morphometric analysis, 24 and 17 landmarks for forewing and hind wing respectively. (e,j) Procrustes superimposition of the forewings and hindwings. (f) Dorsal view of Hypena (Schrank, 1802).