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Fig 1.

Geographic distribution of the two subspecies for each montane stag beetle.

Lucanus ogakii and L. kanoi (A) and Prismognathus davidis (B). The solid line shows the distribution region and the dashed line represents the likely distribution area. The mountain range shown in light gray represents an altitude between 1,000 and 2,000 m and dark gray represents an altitude of >2,000 m.

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Table 1.

Primers and their amplification size of each amplicon in this study.

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Fig 2.

Genetic variations (p-distance) of COI (A), 16S rDNA (B), and 28S rDNA (C) in Lucanidae.

Nucleotide divergence of pairwise comparisons for individuals within species (upper), for species within genera (middle), and among genera of Lucanidae (bottom) are shown. Relevant data excluding those of taxonomically debated species are shown in the upper right panel.

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Fig 3.

Divergence time estimation and pairwise divergences based on COI and 16S rDNA for taxonomically debated stag beetles.

Calibration dating based on COI+16S rDNA for (A) Lucanus ogakii, L. kanoi, and L. maculifemoratus taiwanus, (B) Prismognathus davidis nigerrimus and Pri. d. cheni, and (C) Pri. piluensis and Pri. formosanus. Interspecific pairwise comparison using p-distance for five Lucanus taxa (D) and four Prismognathus taxa (E) are shown. COI divergences: lower-left, and 16S rDNA divergences: upper-right (D, E).

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Fig 4.

Phylogenetic inferences based on COI+16S rDNA+28S rDNA using maximum likelihood (ML).

Both bootstrapping values of ML (left) and posterior probabilities of Bayesian inference (BI) (right) >50% are shown at nodes.

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Fig 5.

Multilocus species delimitation and hybridization test of the five Lucanus taxa.

The species delimitations suggested by *BEAST and PTP are shown at the right side of the phylogram. STRUCTURE analysis assumed the optimal Bayesian clustering (K = 2) of the addressed five taxa. Each bar stands for a single individual. Morphological taxa are represented by different colors. The two groups found in L. k. piceus by PTP are represented by a dashed line.

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Fig 6.

Multilocus species delimitation and hybridization test of the four Prismognathus taxa.

The species delimitations recommended by *BEAST and PTP are shown at the right side of the phylogram. STRUCTURE analysis assumed the optimal Bayesian clustering (K = 2) of the four taxa. Each bar stands for one individual. Morphological taxa are represented by different colors.

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Fig 7.

Multilocus species delimitation and hybridization test of the four Neolucanus taxa.

The species delimitations recommended by *BEAST and PTP are shown at the right side of the phylogram. STRUCTURE analysis assumed the optimal Bayesian clustering (K = 2) of the addressed taxa. Each bar stands for one individual. Morphological taxa are represented by different colors.

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Fig 8.

Haplotype networks of taxonomically debated Lucanus and Prismognathus stag beetles based on mitochondrial COI (A, C) and 16S rDNA (B, D).

Taxa are represented by different colors. With the smallest circle standing for one individual, the number of individuals for each haplotype is shown in the circle.

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