Genetic diversity and neutrality tests calculated for
Abstract
This chapter provides a case of genus Polytremis Mabille, 1904 (Lepidoptera: Hesperiidae), to explain the molecular phylogeny and taxonomy of Lepidoptera and the influence of Wolbachia infection. Earlier studies of Lepidoptera were focused mainly on the morphological classification, population distribution, and identification of new species. As the supplementary to morphological research, analysis of DNA has been widely used in the phylogenetic studies of Lepidoptera. The study provides a conservative estimate that the Wolbachia infection rate in Polytremis nascens Leech (1893) is 31%, and no significant difference in the prevalence is found between the sexes. The Wolbachia infection mainly prevails in populations of P. nascens in southern China, which influence the diversity of mtDNA in P. nascens by a Wolbachia-induced sweep. The Wolbachia infection rate in Polytremis fukia Evans (1940) is 47% and shows a weak association existed between mitochondrial DNA haplotypes and wFuk1 infection status.
Keywords
- Lepidoptera
- microsatellite
- mitochondrial genome
- molecular phylogeny
- taxonomy
- Wolbachia
1. Introduction to molecular phylogeny and taxonomy of Lepidoptera
Butterflies and moths (
2. The phylogeny of the butterfly genus Polytremis
The family Hesperiidae includes more than 4000 species, commonly known as “skippers,” of which Hesperiinae is the largest subfamily.
2.1. The method of constructing and analyzing phylogenetic tree
The specimens from 15 of the estimated 18 species in the genus
2.2. Genetic divergence, phylogenetics, and network of genus Polytremis
Figure 1A reveals five main clades and shows the ML tree based on the data set of COI. Clade I contained eight species:
The level of DNA sequence divergence reflected the taxonomic hierarchy of the original species. The lowest intraspecific COI genetic distance was observed between
The average interspecific rDNA genetic distance (K2P distance 1.0%) was far less than that of COI (K2P distance 7.9%). Except for
In
2.3. Conclusion: combined morphological and molecular analysis
A total of 20 morphological characters yield a two-cluster solution with hierarchical cluster analysis. The first cluster includes 12 species of
3. Taxonomic status of two sibling species of Polytremis (Lepidoptera : Hesperiidae)
The skipper
3.1. Genetic divergences and haplotype networks
All 46 samples yielded high quality of DNA. A total of 19 haplotypes were identified in all 46 samples, and the haplotype network was constructed and presented in Figure 2. There was no shared haplotype among the four taxa. Haplotypes of the same taxon differed from each other by no more than five mutation distance. The five mutation distances existed between the haplotype Ptt I and Ptt III of
The data set of nuclear
Overall,
Ns | Nh | Hd | Nv | π | SD (π) | D | F | |
---|---|---|---|---|---|---|---|---|
All | 33 | 12 | 0.875 | 38 | 0.0207 | 0.0019 | 0.281 | 2.368 |
8 | 3 | 0.750 | 6 | 0.0064 | 0.0048 | 1.598 | 2.631 | |
25 | 9 | 0.803 | 13 | 0.0050 | 0.0070 | −1.014 | −1.886* | |
All | 33 | 5 | 0.773 | 9 | 0.0078 | 0.0057 | 1.122 | 1.509 |
8 | 2 | 0.571 | 1 | 0.0015 | 0.0010 | 1.444 | 1.100 | |
25 | 3 | 0.640 | 3 | 0.0030 | 0.0020 | 1.080 | 1.159 |
3.2. Population structure and phylogenetic analysis
The analysis of molecular variance (AMOVA) for the COI sequences of
Source of variation | Sum of squares | Variance components | Percentage variation | Φ statistic | |
---|---|---|---|---|---|
Among populations | 1 | 169.650 | 9.98266 Va | 88.53 | – |
Within populations | 11 | 45.269 | 1.29341 Vb | 11.47 | 0.896 ( |
Total | 12 | 214.919 | 11.27607 | ||
Fixation index | 0.8853 |
Mitochondrial haplotypes sampled from
3.3. Demographic inference and estimation of divergence times
Demographic history changes were analyzed for
3.4. Conclusion
There is a small region of overlap in west Sichuan province in the distribution of
Wing | Genital | |||||
---|---|---|---|---|---|---|
Color of cilia of wings | Color of underside ground | Number of spots in space Cu2 of the forewing | Color of scales scattered in costa and subapical area of forewing | Color of scales scattered in discal area and dorsum of hind wing | Ductus bursae | |
Brown | Yellowish brown | 0 | Greenish ochreous | Greenish ochreous | Thin | |
Grayish white | Greenish ochreous | 1 or 2 | Grayish white | Grayish white | Thick |
4. The application of mitochondrial genome and microsatellite DNA in Polytremis
With the development of the research, the single molecular fragment cannot meet the research requirements. New molecular markers need to be explored. Recently, the mitochondrial genome has become one of the important molecular markers to explore different categories of
4.1. Complete mitochondrial DNA genome of P. nascens and Polytremis jigongji
The complete mtDNA genome of
The complete mtDNA genome of
4.2. Isolation and characterization of microsatellite loci in P. nascens and P. fukia
Microsatellites are highly polymorphic and codominant molecular markers based on simple repeated and frequent sequences common in the all-living organisms, which have proven to be a powerful tool available in population genetic and evolutionary studies [34]. The ideal molecular marker has become very prevalent in studies of insects over the last 10 years [35]. Variability of the 12 polymorphic microsatellites was surveyed in 53 individuals of
Baishanzu | Hailuogou | Lianglu | Shengtangshan | Maoershan | Kuankuoshui | |
---|---|---|---|---|---|---|
Baishanzu | ||||||
Hailuogou | 0.044 | |||||
Lianglu | 0.050 | 0.003 | ||||
Shengtangshan | 0.029 | 0.025 | 0.022 | |||
Maoershan | 0.023 | 0.021 | 0.020 | 0.013 | ||
Kuankuoshui | 0.026 | 0.012 | 0.014 | 0.022 | 0.017 |
The variability of the 11 polymorphic microsatellites was surveyed in 21 individuals of
5. Wolbachia infection and influence in Polytremis species
5.1. Wolbachia infection status and genetic structure in natural populations of P. nascens
We surveyed
Many explanations have been proposed that deviation from neutral evolution in a species and the absence of diversity in mtDNA can be associated with either a genome-wide bottleneck effect or a selective sweep on mtDNA [43]. In our study, the uninfected butterflies show higher mtDNA polymorphism than butterflies infected with
Although there seems to be a strong association existed between mtDNA haplotypes and
The network analysis reflects genealogical relationships of the mtDNA haplotypes [31]. The single mutation steps separate adjacent haplotypes in the network and older haplotypes are placed at internal branching points, whereas younger ones occur toward the tip positions [50]. The haplotype network of
5.2. A prevalence survey of Wolbachia in P. fukia
Of the butterflies examined by diagnostic PCR for 16S rRNA, 47% (15/32) were
Figure 7A shows the ML tree based on the data set of the concatenated sequences and supports the monophyly of
Number of haplotypes | Hd | SD (Hd) | Number of variable sites (S) | π | SD (π) | D | F | ||
---|---|---|---|---|---|---|---|---|---|
All sequences | 32 | 23 | 0.980 | 0.012 | 50 | 0.008 | 0.001 | 0.568 | −3.913 |
Infection with | 8 | 4 | 0.821 | 0.101 | 5 | 0.001 | 0.000 | 0.840 | 0.428 |
Infection with | 7 | 7 | 1.000 | 0.076 | 32 | 0.008 | 0.001 | 0.077 | −1.085 |
Free from infection | 17 | 14 | 0.978 | 0.027 | 36 | 0.007 | 0.002 | 0.522 | −2.510 |
Acknowledgments
This study was financially supported by grants from National Natural Science Foundation of China (no. 31401997) and Young Teacher Training Scheme for Colleges and Universities in Shanghai (no. ZZssd15068).
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