Application of noninvasive sampling technique in mitochondrial genome intraspecific phylogeny of the endangered butterfly, Teinopalpus aureus (Lepidoptera: Papilionidae)

Abstract

The butterfly genus of Teinopalpus, endemic to Asia, embodies a distinct species of mountain-dwelling butterflies with specific habitat requirements. These species are rare in the wild and hold high conservation and research value. Similar to other protected species, the genetic analysis of the rare Teinopalpus aureus poses challenges due to the complexity of sampling. In this study, we successfully extracted DNA and amplified mitochondrial genomic DNA from various noninvasive sources such as larval feces, larval exuviae, larval head capsules, pupal exuviaes, and filamentous gland secretions, all integral parts of butterfly metamorphosis. This was conducted as part of a research initiative focused on the artificial conservation of T. aureus population in Jinggang Shan Nature Reserve. Our findings illustrated the successful extraction of DNA from multiple noninvasive sources, achieved through modified DNA extraction methodologies. Although the DNA concentration obtained from noninvasive samples was lower than that from muscle tissues of newly dead larvae during rearing, all samples met the requirements for PCR amplification and sequencing, yielding complete circular sequences. These sequences are pivotal for both interspecific and intraspecific genetic relationship analysis. Our methods can be extended to other insects, especially scarce species.

Keywords: Teinopalpus aureus; DNA extraction; intraspecific phylogeny; mitochondrial genome; noninvasive sampling.

Fig. 1.

Comparison of genomic DNA concentration and purity among invasive (LvH: larval head) and noninvasive (LvE: larval exuviae; PpE: pupal exuviae; LvSS: larval silk gland secretions; LvHC: larval head capsule) samples of the butterfly T. aureus, under different preservations (SGDP: silica gel dried-preserved; EtP: ethanol-preserved; FrP: freeze-preserved; DrP: dried-preserved) and preserved duration, including ST, short-term preserved (1–48 h); MT, middle-term preserved (within 1 month), and LT, long-term preserved (≧18 months).

Fig. 2.

Comparison of genomic DNA concentration and purity between the long-term preserved (≧18 months) noninvasive samples of larval exuviae (LvE) and pupal exuviae (PpE) of the butterfly T. aureus, under different preservations (SGDP: silica gel dried-preserved; EtP: ethanol-preserved).

Fig. 3.

Comparison of genomic DNA concentration and purity of larves feces, under different preservations (EtP: ethanol-preserved; FrP: freeze-preserved; SGDP: silica gel dried-preserved; DrP: dried-preserved) and preserved duration, including ST, short-term preserved (1–48 h); MT, middle-term preserved (within 1 month) and LT, long-term preserved (≧18 months).

Fig. 4.

Electropherogram of PCR amplification products of the 2 mitochondrial DNA fragments (TA1 and TA14, see Table 2) in noninvasive (LvE: larval exuviae; PpE: pupal exuviae; LvHC: larval head capsule; LvSS: larval silk gland secretions; LvF: larval feces) and invasive samples (LvH: larval head), M: DL2000 DNA Marker.

Fig. 5.

Three circular mitochondrial DNA from noninvasive (LvF: larval feces; PpE: pupal exuviae) and invasive (LvH: larval head) sample of T. aureus in Jinggangshan, South China.

Fig. 6.

Fragment length variation of mitochondrial genes from noninvasive (LvF: larval feces; PpE: pupal exuviae) and invasive (LvH: larval head) samples of T. aureus in Jinggangshan, South China.

Fig. 7.

Maximum likelihood (ML) and Bayesian (BI) phylogenetic tree constructed with 13 protein-coding gene sequences of mitochondrial genes of T. aureus, by using T. imperialis and Meandrusa sciron as an outgroup, showing ML 1,000 cycles of bootstrap test and BI posterior probability values, as well as the invasive (LvH: larval head) and noninvasive (LvF: larval feces; PpE: pupal exuviae) samples from Jinggangshan, South China.