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Supplemental Information

Estimates of evolutionary divergence over sequence pairs (means ± standard errors of p-distance), between and within groups

a Q represents samples genetically classified as F. quadaranus. b T represents samples genetically classified as F. taihangnica. c reference F. quadaranus, F. kangxianensis, or F. taihangnica

DOI: 10.7287/peerj.preprints.2058v2/supp-1

Raw data of morphometric measurements

It is also available at https://figshare.com/s/be10cb912d81f0c419aa

DOI: 10.7287/peerj.preprints.2058v2/supp-2

The mean ± standard deviation and the difference in the means (p-values) of 34 morphometric indices for 110 specimens were grouped in five ways

"16S" indicates being grouped by the 16S rRNA gene (Q and T); "morp3" indicates being grouped by the three morphotypes (VV, vw&wv, and ww); "sex" indicates being grouped by the sexes (female and male); "F_morp3" indicates the females being grouped together and "M_morp3" indicates the males being grouped together. P-values were calculated by Bartlett’s test, ANOVA, Kruskar–Wallis test, and the t-test. The significance level for each index in each group is estimated using the p-value of t-test as the first priority; if there is no t-test result, then the ANOVA is used, unless the p-value of Bartlett’s test is below 0.05, as marked in red, meaning that the variance of the means is significant different, and thus, the ANOVA is not applicable. In this situation, the p-value of the Kruskar–Wallis test is used instead. Red highlight and postfixed ***: p-value < 1.0E-09; red highlight and postfixed **: 1.0E-09 ≤ p-value < 0.01; yellow highlight and postfixed *: 0.01 ≤ p-value < 0.1; green highlight and postfixed `: 0.1 ≤ p-value < 0.3; and blue highlight and no postfix: p-value > 0.5. It is also available at https://figshare.com/s/be10cb912d81f0c419aa

DOI: 10.7287/peerj.preprints.2058v2/supp-3

The p-values that estimate the significance level in Table S3 for the five groups

"16S" column and "sex" column calculated by t-tests, refer to the difference in means between Q and T, and between sexes, respectively; "morp3" column, "F_morp3" column and "M_morp3" column calculated by ANOVA or Kruskar–Wallis tests, refer to the differences in means among the three morphotypes (VV, vw&wv, and ww) in all 110 specimens, in 52 females, and in 58 males, respectively. Red highlight and red text: p-value < 1.0E-09; red highlight and dark red text: 1.0E-09 ≤ p-value < 0.01; yellow highlight: 0.01 ≤ p-value < 0.1; green highlight: 0.1 ≤ p-value < 0.3; and blue highlight: p-value > 0.3. See plot version in Fig. S1.

DOI: 10.7287/peerj.preprints.2058v2/supp-4

Means and standard deviations (mean ± s.d.) of morphometric indices for the three morphotypes in both female and male sets

a sample sizes are shown in parentheses. b Dark, medium, and light pink represent the highest, middle, and lowest values, respectively, for each index. c ***: p-value < 1.0E-09; **: 1.0E-09 ≤ p-value < 0.01; *: 0.01≤ p-value < 0.1; `: 0.1 ≤ p-value < 0.3; and none: p-value > 0.3. The p-values correspond to those in the last two columns of Table S4 (for details see Table S3). d Measured characteristics: SVL: snout-vent length, used to eliminate body size effects; HL: head length, from posterior end of mandible to tip of snout; HW: head width, measured at corners of the mouth; SL: snout length, distance between anterior edge of orbit and tip of snout; NED: nostril-to-eye distance, distance between centre of nostril and anterior edge of orbit; IND: internarial distance, distance between inner ends of nostrils; IOD: interorbital distance, shortest distance between inner edges of upper eyelids; IAE: distance between anterior corners of eyes; IPE: distance between posterior corners of eyes; LHL: length of lower arm and hand, from elbow to tip of third finger; HAL: hand length, from base of outer palmar tubercle to tip of third finger; TEL: femur length, from vent to knee; TL: tibia length; TFL: tibiofibula length (length of tarsus and foot), from base of tarsus to tip of fourth toe; FL: foot length, from proximal end of inner metatarsal tubercle to tip of fourth toe; T5FFL: length of free flap of the fifth toe, length of cutaneous fringe along the outer margin of the fifth toe; F1L: first finger length, from proximal end of thenar tubercle to tip of first finger. F3L: partial 3rd finger length, distance between basal border of third finger to tip of third finger; F4L: fourth finger length, from proximal end of thenar tubercle to tip of fourth finger. e Extent indices (scored between 0, indicating the trait was not seen, and 1, indicating the maximum extent): BBE: extent of big bumps above the anal region; SBE: extent of small bumps above the anal region; VBE: extent of bumps in the shape of inverted "V" between shoulder blades; VSE: extent of patch in the shape of inverted "V" between shoulder blades; LBE: extent of line-shaped bumps on the back; BSE: the extent of spot pattern on the back; LSE: extent of strip or or spot pattern on legs (scored between 0, indicating no obvious pattern, 1, indicating pure strip pattern, and 2, indicating pure spot pattern).

DOI: 10.7287/peerj.preprints.2058v2/supp-5

Maximum likelihood phylogenetic tree based on 16S rRNA gene partial sequences

The bootstrap support values are shown below branches (The percentage of trees in which the associated taxa clustered together is shown below the branches). Scale bar indicates an evolutionary distance of 0.05 nucleotides per position in the sequence (The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.). Feirana specimens are named by a combination of voucher number and corresponding morphotype, the F. quadranus, F. taihangnica and F. kangxianensis references are named by a combination of GenBank number and species name, and the two R. rugosa outgroups are named by a combination of voucher number and species name. Pink and blue rectangles indicate four specimens with conflicting morphotypical classifications.

DOI: 10.7287/peerj.preprints.2058v2/supp-6

Heat-maps of weighted or non-weighted rotation matrices of the PCA (A–D), and coefficient matrices of the linear discriminant functions of the LDA (E–H)

In the weighted matrix, the corresponding proportion of explained variance for each PC or the corresponding proportion of explained discriminability for each LD is in parentheses. Red and blue palettes distinguish between weighted values (red) and non-weighted values (blue). (A) The first 10 PCs for females, corresponding to Fig. 5A; (B) The first 10 PCs for males, corresponding to Fig. 5E; (C) The first 10 PCs for the total specimens, corresponding to Fig. 5B; (D) The first two PCs of the extent indices for the total specimens, corresponding to Fig. 5H; (E) LD for females, grouped in three morphotypes, corresponding to Fig. 6A; (F) LD for males, grouped in three morphotypes, corresponding to Fig. 6E; (G) LD for the total specimens, grouped in three morphotypes, corresponding to Fig. 6B; (H) LD for the total specimens, grouped in five morphotypes, corresponding to Fig. 6F; (I) LD of the 27 ratio indices, grouped in three morphotypes, corresponding to Fig. 6D; (J) LD of the 7 extent indices, grouped in three morphotypes, corresponding to Fig. 6H.

DOI: 10.7287/peerj.preprints.2058v2/supp-7

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Yang Song conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables.

Xin Sui conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, prepared figures and/or tables, reviewed drafts of the paper.

Yuhong Bian contributed reagents/materials/analysis tools, he did important field observations.

Junfang Zhang performed the experiments, contributed reagents/materials/analysis tools, she helped raise frogs at home, did the artificial hybridization experiment (although it failed and didn't show the results in the paper) and collected morphometric analysis.

Junqiang Zheng contributed reagents/materials/analysis tools, reviewed drafts of the paper.

Pipeng Li conceived and designed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

Permission was granted by the College of Life Science, Shenyang Normal University (Approval No. SNY-LS-2009001).

Field Study Permissions

The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers):

With the permission for sampling frog specimens issued by the College of Life Science, Shenyang Normal University (Approval No. SNY-LS-2009001), the Forestry Department of Shaanxi Province, China, approved of the field work orally.

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

GenBank, accession numbers: KU865180-KU865298

Data Deposition

The following information was supplied regarding data availability:

figshare

https://figshare.com/s/a76953fe8b682d7d1220

https://figshare.com/s/be10cb912d81f0c419aa

https://figshare.com/s/7cbc902b516ac0863f86

https://figshare.com/s/72714eccf03e323ec38b

Funding

The authors received no funding for this work.


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