Genetic variation of S. variegatus populations
We obtained 70 haplotypes of the COI gene and 67 haplotypes of the Cytb gene from 15 populations. The S. variegates COI alignment (652 bp) and Cytb alignment (421 bp) had 45 (6.9%) and 40 (9.5%) variable sites, respectively (Table 1), and of which 28 of COI and 23 of Cytb are parsimony informative. The base composition of the two genes was adenine (A) and thymine (T) (67.5% and 73.3%) biased, respectively, which is common for an insect mitochondrial genes. Haplotype diversity (Hd) ranged from 0.424 to 0.913 (mean = 0.865) and nucleotide diversity (π) ranged from 0.00072 to 0.00462 (mean = 0.00427) for the COI gene (Table 1). Similarly, Hd ranged from 0.464 to 0.833 (mean = 0.834) and π ranged from 0.00119 to 0.00539 (mean = 0.00479) for the Cytb gene (Table 1).
Table 1
Genetic diversity indices and neutrality test for mitochondrial COI and Cytb markers in all analyzed Strongyllodes variegates populations
Marker | Population code | S | Hn | Hd | π | k | Tajima's D | P | Fu's Fs | P |
COI | GDQH | 4 | 5 | 0.702 | 0.00166 | 1.082 | 0.263 | NS | -0.286 | NS |
| HZGS | 8 | 9 | 0.702 | 0.00192 | 1.255 | -1.059 | NS | -3.893 | ** |
| ZYGS | 5 | 6 | 0.649 | 0.00121 | 0.790 | -1.188 | NS | -2.707 | * |
| GYSC | 14 | 14 | 0.855 | 0.00378 | 2.467 | -1.008 | NS | -6.799 | *** |
| HZSX | 14 | 15 | 0.913 | 0.00462 | 3.018 | -0.487 | NS | -6.672 | *** |
| AKSX | 13 | 13 | 0.852 | 0.00431 | 2.810 | -0.351 | NS | -3.961 | * |
| FJCQ | 11 | 14 | 0.857 | 0.00328 | 2.138 | -0.740 | NS | -7.898 | *** |
| ESHB | 6 | 6 | 0.893 | 0.00257 | 1.679 | -1.280 | NS | -3.114 | ** |
| LCHB | 9 | 11 | 0.764 | 0.00320 | 2.085 | -0.206 | NS | -3.819 | * |
| AQAH | 13 | 11 | 0.580 | 0.00146 | 0.949 | -2.201 | *** | -8.187 | *** |
| LAAH | 4 | 5 | 0.424 | 0.00072 | 0.467 | -1.654 | * | -3.127 | *** |
| HFAH | 6 | 6 | 0.574 | 0.00119 | 0.775 | -1.306 | NS | -2.271 | NS |
| CHAH | 5 | 6 | 0.520 | 0.00092 | 0.597 | -1.543 | * | -3.524 | *** |
| NJJS | 5 | 4 | 0.458 | 0.00095 | 0.619 | -1.367 | NS | -0.697 | NS |
| ZJJS | 5 | 6 | 0.628 | 0.00118 | 0.770 | -1.041 | NS | -2.417 | * |
| All | 45 | 70 | 0.865 | 0.00427 | 2.786 | -1.628 | * | -25.887 | *** |
Cytb | GDQH | 6 | 6 | 0.708 | 0.00417 | 1.758 | 0.547 | NS | 0.186 | NS |
| HZGS | 4 | 4 | 0.469 | 0.00285 | 1.198 | 0.558 | NS | 1.002 | NS |
| ZYGS | 3 | 4 | 0.583 | 0.00161 | 0.678 | -0.394 | NS | -0.714 | NS |
| GYSC | 14 | 14 | 0.833 | 0.00539 | 2.271 | -1.192 | NS | -7.424 | *** |
| HZSX | 11 | 11 | 0.832 | 0.00472 | 1.986 | -0.916 | NS | -4.300 | * |
| AKSX | 13 | 13 | 0.810 | 0.00455 | 1.916 | -1.258 | NS | -6.437 | ** |
| FJCQ | 9 | 10 | 0.791 | 0.00300 | 1.262 | -1.381 | NS | -5.530 | *** |
| ESHB | 2 | 3 | 0.464 | 0.00119 | 0.500 | -1.310 | NS | -0.999 | NS |
| LCHB | 18 | 15 | 0.752 | 0.00359 | 1.51 | -2.252 | *** | -12.320 | *** |
| AQAH | 8 | 9 | 0.718 | 0.00255 | 1.075 | -1.273 | NS | -4.442 | ** |
| LAAH | 3 | 4 | 0.71 | 0.00215 | 0.905 | 0.223 | NS | -0.187 | NS |
| HFAH | 9 | 9 | 0.784 | 0.00251 | 1.239 | -1.322 | NS | -3.954 | ** |
| CHAH | 7 | 9 | 0.726 | 0.00272 | 1.145 | -1.151 | NS | -5.076 | *** |
| NJJS | 9 | 9 | 0.776 | 0.00347 | 1.462 | -1.082 | NS | -3.413 | * |
| ZJJS | 8 | 9 | 0.697 | 0.00215 | 0.903 | -1.655 | * | -5.812 | ** |
| All | 40 | 67 | 0.834 | 0.00479 | 2.015 | -1.819 | ** | -26.759 | *** |
For each population, the number of variable sites (S), number of haplotypes (Hn), haplotype diversity (Hd), nucleotide diversity (π), average number of nucleotide differences (k) and Tajima's D and Fu's Fs test statistics for selective neutrality are given. |
Values are significant at * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS, not significant |
Haplotype Analyses Of The Coi And Cytb Genes
Among 70 identified COI haplotypes (H1-H70), and the haplotypes sequence were deposited in GenBank (Accession number: MN935027–MF935096), 34 (48.6%) were unique (haplotypes represented by one sample) (Table S2). The four most frequent haplotypes (H1-H4) were found in 132 (30.2%), 59 (13.5%), 29 (6.6%), and 60 (13.7%) samples (Table S2; Fig. 2a). Haplotype 1 was found in almost all except for GDQH, FJCQ and ESHB populations, whereas Haplotype 2 was only discovered in GYSC, HZSX, AKSX, FJCQ, ESHB and LCHB populations (Table S2). Among 67 identified Cytb haplotypes (H1-H67), and the haplotypes sequence were deposited in GenBank (Accession number: MN935097–MF935163), 35 (52.2%) were unique and 32 were observed in more than one sample (Table S2). Three most frequent haplotypes (H1-H3) were found in 158 (36.2%), 61(14.0%) and 48 (10.9%) samples (Table S2; Fig. 2b). Haplotype 1 was found in all populations except for ESHB population, whereas Haplotype 3 was only discovered in AQAH, LAAH, HFAH, CHAH, NJJS and ZJJS populations (Table S2). The haplotype analyses and haplotype network analyses (see below) of both COI and Cytb genes revealed that S. variegates populations could be divided into three major geographical distribution regions or haplogroups: SP haplogroup (GDQH, HZGS and ZYGS populations), UY haplogroup (GYSC, HZSX, AKSX, FJCQ, ESHB and LCHB populations) and LY haplogroup (AQAH, LAAH, HFAH, CHAH, NJJS and ZJJS populations) (Fig. 1, Table S1).
For the haplotype network of the COI gene, there was no common haplotype between SP/UY and LY haplogroups except for Haplotype 1 (H1), which was observed in three haplogroups. Haplotype 2 (H2), the most abundant haplotype, was only detected in UY haplogroup. Haplotype 3 (H3) was only discovered in LY haplogroup. There were six common haplotypes (H4-H9) between SP and UY haplogroups. A total of five missing haplotypes were observed in all populations (Fig. 2a). Similarly, for the haplotype network of Cytb gene, there were two common haplotypes (H1, H4) in three haplogroups. Haplotype 2 (H2), the most abundant, was only detected in UY haplogroup. Haplotype 3 (H3) was only discovered in LY haplogroup. Haplotype 5–6, 7, 8–9 (H5-H6, H7, H8-H9) were common in SP and UY haplogroup, SP and LY haplogroup, UY and LY haplogroup, respectively. A total of four missing haplotypes were observed in UY haplogroup (Fig. 2b).
Population Genetic Differentiation
Strong genetic divergence was observed across populations (FST = 0.425, P < 0.0001, Table 2). Between the spring oilseed rape regions and winter oilseed rape regions, the FCT value was not significant (FCT = 0.071, P = 0.153, Table 2). However, the FCT value among three haplogroups was highly significant (FCT = 0.470, P < 0.0001, Table 2), suggesting that geographical features constitute a strong natural barrier to gene flow. The results further demonstrate that S. variegates populations in China is divided into three haplogroups, SP, UY and LY, in three geographical regions. Significant genetic differentiation was observed among populations within haplogroups (FSC = 0.072, P < 0.0001; Table 2), and within populations (FST = 0.508, P < 0.0001, Table 2) based on the combined COI and Cytb genes.
Table 2
Hierarchical analysis of molecular variance (AMOVA) in collected Strongyllodes variegates from 15 populations
Source of variation | df | Sum of squares | % of variation | Fixation indices |
(a) | | | | |
Among populations | 14 | 446.669 | 42.50 | |
Within populations | 422 | 599.926 | 57.50 | FST = 0.425*** |
(b) | | | | |
Among regions | 1 | 58.936 | 7.11 | FCT = 0.071ns |
Among populations within regions | 13 | 387.734 | 38.01 | FSC = 0.409*** |
Within populations | 422 | 599.926 | 54.88 | FST = 0.451*** |
(c) | | | | |
Among regions | 2 | 391.765 | 47.01 | FCT = 0.470*** |
Among populations within regions | 12 | 54.904 | 3.80 | FSC = 0.072*** |
Within populations | 422 | 599.926 | 49.18 | FST = 0.508*** |
SP vs. UY | | | | |
Among regions | 1 | 124.847 | 33.89 | FCT = 0.339** |
Among populations within regions | 7 | 46.483 | 5.95 | FSC = 0.090*** |
Within populations | 248 | 438.452 | 60.16 | FST = 0.398*** |
SP vs. LY | | | | |
Among regions | 1 | 89.300 | 38.88 | FCT = 0.389** |
Among populations within regions | 7 | 26.418 | 5.11 | FSC = 0.084*** |
Within populations | 263 | 265.672 | 56.00 | FST = 0.440*** |
UY vs. LY | | | | |
Among regions | 1 | 332.830 | 54.95 | FCT = 0.550*** |
Among populations within regions | 10 | 36.907 | 2.23 | FSC = 0.050*** |
Within populations | 333 | 495.727 | 42.82 | FST = 0.572*** |
AMOVA partitioned among (a) all populations. (b) two regions: spring oilseed rape regions (GDQH, HZGS, ZYGS) and winter oilseed rape regions (GYSC, HZSX, AKSX, FJCQ, ESHB, LCHB, AQAH, LAAH, HFAH, CHAH, NJJS, ZJJS). (c) three regions: SP regions (GDQH, HZGS, ZYGS), UY regions (GYSC, HZSX, AKSX, FJCQ, ESHB, LCHB) and LY regions (AQAH, LAAH, HFAH, CHAH, NJJS, ZJJS). |
**P ≤ 0.001, *** P ≤ 0.0001 after 1,023 permutations; ns, not significant |
The percentages of genetic variation within populations (60.16% between SP and UY haplogroups, and 56.00% between SP and LY haplogroups) were significantly higher than those of the comparisons between haplogroups (33.89% between SP and UY regions, 33.88% between SP and LY regions) (Table 2). However, in the comparison between UY and LY haplogroups, the percentage of genetic variations among haplogroups (54.95%) was higher than that of 42.82% within populations (Table 2), an indicator that there is limited gene flow between UY and LY haplogroups. Pairwise FST values of combined COI and Cytb genes were significant among geographical regions (FST > 0.25, Table 3), and gene flow among haplogroups was estimated extremely low (Nm < 1; Table 3).
Table 3
Pairwise FST values (below diagonal) and gene flow (above diagonal) between regions based on the combined COI and Cytb genes
Regionsa | SP | UY | LY |
SP | | 0.457b | 0.373 |
UY | 0.354 *** | | 0.202 |
LY | 0.401 *** | 0.553 *** | |
a Regions as defined in Fig. 1 and Table 2 |
b Gene flow (Nm) was calculated from Fst as: Nm = (1- Fst) / 4 Fst |
*** P < 0.001 |
The Mantel test results based on combined COI and Cytb genes revealed a significant correlation between genetic distance and geographical distances among all populations (r = 0.500, P < 0.0001, Fig. 3).
Demographic Analyses
Tajima’s D and Fu’s Fs values of all S. variegates populations in SP, UY and LY haplogroups were negative and highly significant (P < 0.05), expect that the values for SP haplogroup based on Cytb were not significant (P > 0.05, Table 4). Distributions of pairwise differences obtained with COI and Cytb data had a small difference in UY haplogroup (Fig. 4). As for COI, there was a nonsmooth and unimodal relationship of pairwise mismatch distribution (Fig. 4a) with non-significant SSD and Rag values (Table 4). For Cytb, a sudden expansion was supported by historically mismatched distributions, expressing a smooth and unimodal pattern (Fig. 4b), with non-significant SSD and Rag values based on Cytb (Table 4). For the other two haplogroups (SP and LY), the sudden expansion hypothesis was rejected based on Tajima's D and Fu's Fs test statistics and P values for SSD and Rag (Table 4; Fig. 4). The tau values (τ), a rough estimate of the population expansion, were approximately 3.842 and 2.016 mutation units for UY haplogroup in both COI and Cytb, respectively. For SP and LY haplogroups, τ was 1.344 and 0.766 in the COI gene, and 3.693 and 0.875 in the Cytb gene (Table 4).
Table 4
Genetic diversity indices, neutrality test and mismatch distribution parameter of Strongyllodes variegates for mitochondrial COI and Cytb genes
Gene | Regionsa | N | Hn | Hd | π | k | D (p) | Fs (p) | τ | PSSD | PR |
COI | SP | 92 | 15 | 0.713 | 0.00183 | 1.193 | -1.565 (0.043) | -9.255 (< 0.001) | 1.344 | 0.030 | 0.130 |
| UY | 165 | 43 | 0.856 | 0.00397 | 2.587 | -1.471 (0.032) | -26.732 (< 0.001) | 3.842 | 0.520 | 0.790 |
| LY | 180 | 20 | 0.544 | 0.00113 | 0.736 | -2.132 (0.001) | -21.274 (< 0.001) | 0.766 | 0.010 | 0.060 |
Cytb | SP | 92 | 9 | 0.638 | 0.00354 | 1.492 | -0.393 (0.413) | -1.395 (0.260) | 3.693 | 0.310 | 0.330 |
| UY | 165 | 43 | 0.826 | 0.00436 | 1.837 | -1.992 (0.003) | -27.537 (< 0.001) | 2.016 | 0.520 | 0.360 |
| LY | 180 | 24 | 0.741 | 0.00276 | 1.162 | -1.799 (0.012) | -21.480 (< 0.001) | 0.875 | 0.000 | 0.000 |
Sample size (N), number of haplotypes (Hn), haplotype diversity (Hd), nucleotide diversity (π), average number of nucleotide differences (k), Tajima's D (D) and Fu's Fs (Fs) test statistics for selective neutrality and index of population expansion (τ) are given. Significance values (p) of the parameters were evaluated with 1,000 simulations; PSSD: P value for SSD (sum of squared deviations) PR: P value for Rag (Harpending’s raggedness index). |
a Regions as defined in Fig. 1 and Table 2. |
Additional Files |
Additional file 1: Table S1 Sample information of Strongyllodes variegatus (Fairmaire) specimens collected for the present study |
Province | Location | Abbreviation | Longitude | Latitude | Years | Sample size |
Qinghai | Guide | GDQH | 101.43 | 36.05 | 2012 | 34 |
Gansu | Hezheng | HZGS | 103.35 | 35.43 | 2013, 2015 | 34 |
| Zhenyuan | ZYGS | 107.32 | 35.53 | 2019 | 24 |
Sichuang | Guangyuan | GYSC | 105.79 | 32.59 | 2015 | 30 |
Shaanxi | Hanzhong | HZSX | 106.67 | 33.16 | 2015 | 30 |
| Ankang | AKSX | 108.25 | 32.05 | 2015, 2017 | 35 |
Chongqing | Fengjie | FJCQ | 109.44 | 31.01 | 2015 | 30 |
Hubei | Enshi | ESHB | 109.72 | 30.61 | 2015 | 8 |
| Lichuang | LCHB | 108.77 | 30.48 | 2019 | 32 |
Anhui | Anqing | AQAH | 116.58 | 30.63 | 2016, 2017 | 37 |
| Liu'an | LAAH | 116.70 | 31.79 | 2016, 2017 | 21 |
| Hefei | HFAH | 117.23 | 31.88 | 2015, 2016, 2017 | 34 |
| Caohu | CHAH | 117.89 | 31.62 | 2012, 2015 | 26 |
Jiangsu | Nanjing | NJJS | 118.47 | 32.05 | 2019 | 31 |
| Zhenjiang | ZJJS | 119.18 | 31.94 | 2015 | 31 |
Additional file 2: Table S2 Geographical distribution of (A) COI and (B) Cytb haplotypes of Strongyllodes variegates (Hap.= Haplotype; N = total number) |
(A) Hap. | GDQH | HZGS | ZYGS | GYSC | HZSX | AKSX | FJCQ | ESHB | LCHB | AQAH | LAAH | HFAH | CHAH | NJJS | ZJJS | N |
H1 | | 1 | 3 | 3 | 3 | 3 | | | 1 | 24 | 16 | 21 | 18 | 22 | 17 | 132 |
H2 | | | | 11 | 8 | 12 | 10 | 3 | 15 | | | | | | | 59 |
H3 | | | | | | | | | | 3 | | 8 | 2 | 7 | 9 | 29 |
H4 | 14 | 18 | 14 | | 3 | 6 | 2 | | 3 | | | | | | | 60 |
H5 | 7 | 2 | | | | | | | 1 | | | | | | | 10 |
H6 | 1 | | | | 1 | | | | | | | | | | | 2 |
H7 | | 2 | | | 2 | 2 | | | | | | | | | | 6 |
H8 | | | 1 | | | 2 | | | | | | | | | | 3 |
H9 | | | 2 | 1 | | 1 | | | | | | | | | | 4 |
H10 | 11 | 5 | | | | | | | | | | | | | | 16 |
H11 | 1 | | | | | | | | | | | | | | | 1 |
H12 | | 2 | | | | | | | | | | | | | | 2 |
H13 | | 1 | | | | | | | | | | | | | | 1 |
H14 | | 1 | | | | | | | | | | | | | | 1 |
H15 | | 2 | | | | | | | | | | | | | | 2 |
H16 | | | 2 | | | | | | | | | | | | | 2 |
H17 | | | 2 | | | | | | | | | | | | | 2 |
H18 | | | | 1 | | | 1 | | | | | | | | | 2 |
H19 | | | | 1 | 1 | | | | | | | | | | | 2 |
H20 | | | | 1 | 2 | | | | | | | | | | | 3 |
H21 | | | | 2 | 2 | | 1 | | | | | | | | | 5 |
H22 | | | | 1 | | | | | | | | | | | | 1 |
H23 | | | | 3 | 1 | | | | 3 | | | | | | | 7 |
H24 | | | | 2 | | | | | | | | | | | | 2 |
H25 | | | | 1 | | | | | | | | | | | | 1 |
H26 | | | | 1 | | | | | 4 | | | | | | | 5 |
H27 | | | | 1 | | | | | | | | | | | | 1 |
H28 | | | | 1 | | | | | | | | | | | | 1 |
H29 | | | | | 1 | 1 | | | | | | | | | | 2 |
H30 | | | | | 2 | 2 | | 1 | | | | | | | | 5 |
H31 | | | | | 1 | | 1 | 1 | 1 | | | | | | | 4 |
H32 | | | | | 1 | | | | | | | | | | | 1 |
H33 | | | | | 1 | | | | | | | | | | | 1 |
H34 | | | | | 1 | | | | | | | | | | | 1 |
H35 | | | | | | 1 | | | | | | | | | | 1 |
H36 | | | | | | 1 | | | | | | | | | | 1 |
H37 | | | | | | 2 | 2 | | 1 | | | | | | | 5 |
H38 | | | | | | 1 | | | | | | | | | | 1 |
H39 | | | | | | 1 | | | | | | | | | | 1 |
H40 | | | | | | | 1 | | | | | | | | | 1 |
H41 | | | | | | | 6 | | | | | | | | | 6 |
H42 | | | | | | | 1 | | | | | | | | | 1 |
H43 | | | | | | | 1 | | | | | | | | | 1 |
H44 | | | | | | | 1 | | | | | | | | | 1 |
H45 | | | | | | | 1 | | | | | | | | | 1 |
H46 | | | | | | | 1 | | | | | | | | | 1 |
H47 | | | | | | | 1 | | 1 | | | | | | | 2 |
H48 | | | | | | | | 1 | | | | | | | | 1 |
H49 | | | | | | | | 1 | | | | | | | | 1 |
H50 | | | | | | | | 1 | | | | | | | | 1 |
H51 | | | | | | | | | 1 | | | | | | | 1 |
H52 | | | | | | | | | 1 | | | | | | | 1 |
H53 | | | | | | | | | | 1 | 1 | | 2 | 1 | 2 | 7 |
H54 | | | | | | | | | | 1 | 2 | 1 | 1 | | | 5 |
H55 | | | | | | | | | | 1 | 1 | | | | | 2 |
H56 | | | | | | | | | | 1 | | | | | | 1 |
H57 | | | | | | | | | | 1 | | | | | | 1 |
H58 | | | | | | | | | | 1 | | | | | | 1 |
H59 | | | | | | | | | | 1 | | | | | | 1 |
H60 | | | | | | | | | | 1 | | | | | | 1 |
H61 | | | | | | | | | | 2 | | | | | | 2 |
H62 | | | | | | | | | | | 1 | | | | | 1 |
H63 | | | | | | | | | | | | 2 | | | | 2 |
H64 | | | | | | | | | | | | 1 | | | | 1 |
H65 | | | | | | | | | | | | 1 | | | | 1 |
H66 | | | | | | | | | | | | | 1 | | 1 | 2 |
H67 | | | | | | | | | | | | | 2 | | | 2 |
H68 | | | | | | | | | | | | | | 1 | | 1 |
H69 | | | | | | | | | | | | | | | 1 | 1 |
H70 | | | | | | | | | | | | | | | 1 | 1 |
(B) Hap. | GDQH | HZGS | ZYGS | GYSC | HZSX | AKSX | FJCQ | ESHB | LCHB | AQAH | LAAH | HFAH | CHAH | NJJS | ZJJS | N |
H1 | 12 | 24 | 15 | 3 | 11 | 10 | 3 | | 3 | 18 | 6 | 13 | 13 | 12 | 15 | 158 |
H2 | | | | 9 | 5 | 12 | 13 | 6 | 16 | | | | | | | 61 |
H3 | | | | | | | | | | 8 | 9 | 9 | 5 | 8 | 9 | 48 |
H4 | | 1 | | | 2 | 2 | | | | 1 | | 3 | | | | 9 |
H5 | 1 | | | | | | | | | 1 | | | 1 | | | 3 |
H6 | | | 4 | | | | | | | 1 | | | | | | 5 |
H7 | | | 2 | 1 | | | | | | | | | | | | 3 |
H8 | | | | 1 | | | | | | | | 1 | 1 | | | 3 |
H9 | | | | 1 | 1 | | 4 | | | | | 2 | | | 1 | 9 |
H10 | 14 | 7 | | | | | | | | | | | | | | 21 |
H11 | 4 | 2 | | | | | | | | | | | | | | 6 |
H12 | 2 | | | | | | | | | | | | | | | 2 |
H13 | 1 | | 3 | | | | | | | | | | | | | 4 |
H14 | | | | 1 | | 1 | | | | | | | | | | 2 |
H15 | | | | 2 | 1 | | 1 | | | | | | | | | 4 |
H16 | | | | 5 | 4 | | | | | | | | | | | 9 |
H17 | | | | 2 | | | | | | | | | | | | 2 |
H18 | | | | 1 | | | | | | | | | | | | 1 |
H19 | | | | 1 | | | | | | | | | | | | 1 |
H20 | | | | 1 | | | | | 1 | | | | | | | 2 |
H21 | | | | 1 | | | | | | | | | | | | 1 |
H22 | | | | 1 | | | | | | | | | | | | 1 |
H23 | | | | | 1 | | 1 | | | | | | | | | 2 |
H24 | | | | | 1 | | | | | | | | | | | 1 |
H25 | | | | | 1 | | | | | | | | | | | 1 |
H26 | | | | | 2 | | | | | | | | | | | 2 |
H27 | | | | | 1 | | | | | | | | | | | 1 |
H28 | | | | | | 1 | | | | | | | | | | 1 |
H29 | | | | | | 1 | | | | | | | | | | 1 |
H30 | | | | | | 1 | 2 | | | | | | | | | 3 |
H31 | | | | | | 2 | 3 | | | | | | | | | 5 |
H32 | | | | | | 1 | | | | | | | | | | 1 |
H33 | | | | | | 1 | | | | | | | | | | 1 |
H34 | | | | | | 1 | 1 | | | | | | | | | 2 |
H35 | | | | | | 1 | | | | | | | | | | 1 |
H36 | | | | | | 1 | | | | | | | | | | 1 |
H37 | | | | | | | 1 | 1 | 1 | | | | | | | 3 |
H38 | | | | | | | 1 | | | | | | | | | 1 |
H39 | | | | | | | | 1 | | | | | | | | 1 |
H40 | | | | | | | | | 1 | | | | | | | 1 |
H41 | | | | | | | | | 1 | | | | | | | 1 |
H42 | | | | | | | | | 1 | | | | | | | 1 |
H43 | | | | | | | | | 1 | | | | | | | 1 |
H44 | | | | | | | | | 1 | | | | | | | 1 |
H45 | | | | | | | | | 1 | | | | | | | 1 |
H46 | | | | | | | | | 1 | | | | | | | 1 |
H47 | | | | | | | | | 1 | | | | | | | 1 |
H48 | | | | | | | | | 1 | | | | | | | 1 |
H49 | | | | | | | | | 1 | | | | | | | 1 |
H50 | | | | | | | | | 1 | | | | | | | 1 |
H51 | | | | | | | | | | 2 | | | 1 | 1 | 1 | 5 |
H52 | | | | | | | | | | 4 | 5 | 3 | 1 | | | 13 |
H53 | | | | | | | | | | 1 | | | | | | 1 |
H54 | | | | | | | | | | 1 | | | | | | 1 |
H55 | | | | | | | | | | | 1 | 1 | | | | 2 |
H56 | | | | | | | | | | | | 1 | | 1 | | 2 |
H57 | | | | | | | | | | | | 1 | | | | 1 |
H58 | | | | | | | | | | | | | 2 | 1 | | 3 |
H59 | | | | | | | | | | | | | 1 | | | 1 |
H60 | | | | | | | | | | | | | 1 | | | 1 |
H61 | | | | | | | | | | | | | | 5 | | 5 |
H62 | | | | | | | | | | | | | | 1 | 1 | 2 |
H63 | | | | | | | | | | | | | | 1 | 1 | 2 |
H64 | | | | | | | | | | | | | | 1 | | 1 |
H65 | | | | | | | | | | | | | | | 1 | 1 |
H66 | | | | | | | | | | | | | | | 1 | 1 |
H67 | | | | | | | | | | | | | | | 1 | 1 |