C
4
I
d i
1
,.,„,
Figure 2-2. Representative specimens of Dryopteris caudipinna.
a. Ibaraki Prefecture, Tsukuba-shi, Tsukuba Shrine (1987-08-23, S. Matsumoto s.n., living collection of the Tsukuba Botanical Garden 77398, TNS-VS-9508257)
b. Aichi Prefecture, Atsumi-gun, Atsumi-cho, Yamada (1980-11-23, K. Inukai 4201,
TNS-VS-550286)
N
W
S
•
•
„int
l
• 0
•
y
0 400 km
Figure 2-3. Distribution map of Dryopteris caudipinna.
Open triangles, previously known localities; Closed circles, study.
new localities reported in this
N
W E
S
Kozu Isl.
Mikura Isl.
Hachij o Isl.
Aogashima Isl.
Oshima Isl.
Toshima Isl.
Ni] ima Isl.
Miyake Isl.
0 50 km
Figure 2-4. Pie charts showing the approximate ratio of sexual Dryopteris caudipinna (white) to apogamous Dryopteris erythrosora (black).
The counts are based on Nakaike and Yamamoto (1984) for Oshima Island, on Yamamoto
and Nakaike (1983) for Hachijojima Island and on spore observations made in this study
for the remaining islands.
Chapter 3. Cytological and genetic analyses in mixed populations of apogamously reproducing Dryopteris erythrosora and their sexual relative D. caudipinna, on Izu-Oshima Island
3.1. Introduction
In Chapter 2, the geographical distribution of Dryopteris caudipinna, a sexually reproducing close relative of the apogamously reproducing Dryopteris erythrosora, was clarified. According to the results obtained in the main islands of Japan, apogamous D.
erythrosora is widely distributed and extremely common, whereas sexual D. caudipinna is rare and its distribution is confined to coastal areas. In contrast, in the Izu Islands, Tokyo Prefecture, Japan, sexual D. caudipinna is dominant and common, whereas apogamous D. erythrosora is relatively rare there. In general, sexual species appear to have difficulty in growing together with closely related apogamous species because the fertility and propagation of sexual species has disadvantages compared with their apogamous relatives, which can reproduce without costly fertilization. This should be one of the major reasons for the dominant distribution of either apogamous D.
erythrosora or sexual D. caudipinna in a particular locality in Japan. However, these two fern species with different reproductive modes are observed growing together with competitive frequencies on Izu -Oshima Island, which lies in the northern most part of the Izu Islands and is closest to Honshu, the largest main island of Japan. Therefore,
Izu-Oshima Island may provide a rare opportunity to examine mixed populations of closely related sexual and apogamous fern species.
Moreover, putative hybrids between D. erythrosora and D. caudipinna have been reported on the Izu-Oshima Island (Nakaike and Yamoto 1984). As already mentioned in the General Introduction (Chapter 1), Walker (1962) documented the hybridization between related apogamous and sexual fern species in artificial crossing experiments using Pteris cretica. He attempted to hybridize sperms developed on the gametophytes of its triploid apogamous type and eggs developed on those of the diploid sexual type and succeeded in producing a tetraploid hybrid with the ability of apogamous reproduction. Thus, it appeared likely that similar hybridization events may take place in natural populations of the Dryopteris species growing on Izu-Oshima Island.
I selected the Izu-Oshima Island as my study site to examine mixed populations of apogamous D. erythrosora and sexual D. caudipinna. I considered the need to clarify the geographical distribution of the apogamous and the sexual species of Dryopteris and to determine exact localities of mixed populations on Izu-Oshima Island. Such a detailed distribution within the island was not elucidated by the investigation of Chapter 2 based only on herbarium specimens because the specimens were sporadically collected and did not cover the entire distribution within the island. Therefore, I should examine the possibility of hybridization between the apogamous and the sexual species in their natural mixed populations.
For this purpose, first, fresh leaf samples were collected from approximately 30 populations of the D. erythrosora complex (from either D. erythrosora or D. caudipinna, where the species could not be distinguished in the field based on their morphology), covering the entire distribution of the D. erythrosora complex on Izu-Oshima Island.
The reproductive mode of each sample was subsequently examined to clarify the detailed distribution of each of these species on the island.
Second, I determined genotypes of all samples using allozyme variations as genetic markers. Allozyme variations have been documented as powerful codominant genetic markers for population genetic analyses of wild plants, including ferns (Gastony and Gottlieb 1982, Soltis et al. 1983, Soltis and Soltis 1990, Werth and Windham 1991).
I compared the genetic variation of apogamous D. erythrosora in mixed populations with that in pure apogamous populations. If the amount of observed genetic variation within the apogamous plants in mixed populations was significantly higher than that in pure apogamous populations, it would strongly suggest that the apogamous ferns acquired genetic variation from the sexual ferns in mixed populations.
This study attempted to address the following three questions:
(1) How are apogamous D. erythrosora and sexual D. caudipinna distributed on Izu-Oshima Island?
(2) Do apogamous D. erythrosora and sexual D. caudipinna share their allozyme alleles in mixed populations?
(3) Is there evidence suggesting that apogamous fern variation from their sexual relatives when they grow together?
species acquire genetic
3. 2. Materials and Methods
3.2.1. Collection of plant materials and recording of location information.
In this study, a total of 874 individuals of the Dryopteris erythrosora complex, consisting of apogamous D. erythrosora and sexual D. caudipinna, were collected from 30 populations on Izu-Oshima Island, Tokyo Prefecture, Japan. These populations are at least 0.5 km apart and equally scattered across the island, particularly in the growing area of D. erythrosora complex. The accurate location of each examined population was recorded using a GPS recorder, GPSmap 60CSx (Garmin), and the locations obtained are shown in Table 3-1. One whole leaf with spores was collected from each matured sporophyte of the D. erythrosora complex. Whole leaf samples were collected from approximately 20 (11-26) sporophytes in each population. A small amount of fresh leaf material (one-two upper pinnae, approximately 10 mg) was subsequently collected from each whole leaf sample, packed in a small plastic bag, and stored at 4°C for enzyme electrophoresis. The remaining part of each leaf was placed between old newspapers and dried as a voucher specimen. All voucher specimens have been deposited in the Makino Herbarium of Tokyo Metropolitan University (MAK).
3.2.2. Estimation of reproductive mode.
As already noted in Chapter 2, reproductive modes in most homosporous ferns
can be determined simply by counting the number of spores in a sporangium.. Sexually
reproducing homosporous ferns usually have 64 spores in a sporangium, whereas apogamous ferns usually have 32 spores (Walker 1962). Also, in this chapter, the reproductive mode for all plant materials was estimated by counting spore numbers per sporangium for each voucher specimen (64 spores: sexual; 32 spores: apogamous).
3.2.3. Enzyme electrophoresis.
Fresh leaf material was used for enzyme electrophoresis. Approximately 200 mg of fresh leaf material was ground in 0.8 mL of extraction buffer [0.1 M Tris-HC1 (pH 7.5), 5% Sigma PVP-40T, 0.5% sodium metabisulfite, 0.5% sodium ascorbate, and 0.2% 2-mercaptoethanol]. The resulting slurry was centrifuged at 10,000 rpm for 10 min and the supernatant was used for electrophoresis. Electrophoresis was performed on a polyacrylamide gel in two layers. The concentration gel consisted of 3.75%
polyacrylamide [10% acrylamide, 2.5% methylene-bis-acrylamide, 0.25 M Tris-HC1 (pH6.7)] and the separation gel consisted of 7.5% polyacrylamide [30% acrylamide, 0.8% methylene-bis-acrylamide, 1.5 M Tris-HC1 (pH 8.9)]. Electrophoresis was performed for 3 h at 4°C and 100 mA. In the present study, phosphoglucoisomerase (PGI) was analyzed. Enzyme staining was performed according to the methods described by Wendel and Weeden (1989). Because more than one locus was observed for Pgi, the loci were numbered sequentially, with the most anodally migrating locus named 1..
Alphabetical letters were given to alleles of allozymes at individual loci, with the fastest
migrating allozyme named a, the second fastest named b, and so on. During genotype estimation in this study, sexual D. caudipinna and apogamous D. erythrosora were supposed to be diploid and triploid, and therefore, to have two and three alleles, respectively, though the ploidy of each sample was not examined in this chapter. As will be noted in the next chapter, these suppositions are mostly correct and do not appear to cause problems in the analyses of Chapter 3.
3.2.4. Quantification of genetic variation.
To quantitatively describe the clonal diversity, Simpson's diversity index (D) was calculated for every population as well as for the entire species: D = 1—EPi2, where Pi isfreaquency of each clone i.
3.3. Results
3.3.1. Estimation of reproductive mode.
The spore number per sporangium was counted for 617 samples from the 30 populations on Izu-Oshima Island. Among them, 53 samples (5.9%) had 64 spores per sporangium and were thus recorded as sexual D. caudipinna, whereas the remaining 564 samples (94.1%) had 32 spores per sporangium and were recorded as apogamous D.
erythrosora (Table 3-1). These results indicated that the apogamous species is more common than the sexual species on Izu-Oshima Island.
Distribution of individuals of the sexual and apogamous species in each of the 30 populations is shown in Figure 3-1. Figure 3-1 also shows the relative frequencies of the sexual (red) and apogamous (blue) individuals of the D. erythrosora complex in each population. Seven mixed populations containing both apogamous D. erythrosora and sexual D. caudipinna and 23 pure apogamous populations were identified. The former contained a total of 100 individuals of the apogamous species and 53 individuals of the sexual species and the latter contained a total of 464 individuals of the apogamous species. In this study, pure sexual populations consisting of only sexual D.
caudipinna were not observed on Izu-Oshima Island. In other words, the sexual species was found only in mixed populations, together with its closely related apogamous species.
On Izu-Oshima Island, sexual D. caudipinna (within mixed populations) was observed mostly in the northeastern part of the island (Figure 3-1). These places were the areas where recent lava flow had been recorded (The Geospatial Information Authority of Japan 2006, Kawabe 1998) (Figure 3-2).
3.3.2. Allozyme analysis.
For 786 individuals of the D. erythrosora complex sampled from the 36 populations on Izu-Oshima Island, genotypes of the Pgi-2 locus were estimated on the basis of enzyme electrophoresis results (Table 3-1). Four alleles were recognized, either in D. erythrosora or D. caudipinna. Thus, these two related species share the same alleles. These four alleles seemed to correspond to a, b, d, and f alleles reported by Ishikawa (1997). In this study, the four alleles observed in the D. erythrosora complex are called a, b, d, and f, from the most anodal alleles to the cathodal ones (Table 3-2, 3-3, and 3-4). Allele frequencies in sexual D. caudipinna were as follows: a, 0.10; b, 0.23; d, 0.12; f,, 0.55. Those in apogamous D. erythrosora, when each individual of the apogamous species is supposed to have three independent alleles, were as follows: a, 0.06; b, 0.32; d, 0.47;f, 0.15 in mixed populations, and in pure apogamous populations, a, 0.06; b, 0.35; d, 0.54;f, 0.05. The frequency of allele f was significantly higher in the
sexual species than in the apogamous species (Table 3-4, Figure 3-3) (t-test, P - 0.007), among which 16 different genotypes (aab, aad, abd, add, bbb, bbd, bdd, ddd, fff, aaf,
adf, bbf, bdf, bff, ddf, and dff) were observed on Izu-Oshima Island (Table 3-2, Figure 3-4). Among these genotypes, the following three were common: bdd (35%), bbd (26%),
and ddd (13%). Several genotypes, particularly those containing several f alleles (e.g., fff, dff) were observed only in mixed populations. Moreover, frequencies of allele f were higher in the apogamous individuals of mixed populations (average, 0.19; N = 7) than in those of pure apogamous populations (average, 0.05; N = 23) (t-test, P =7
3.3.3. Comparison of genetic diversity among populations.
To compare the amount of clonal diversity in the apogamous species among populations, Simpson's diversity index D (D = 1—Epi2) was calculated using the frequency of each genotype in each population (pi) (Table 3-3). The average D values among the populations of the apogamous species was 0.74 (N = 7) in mixed populations (growing with sexual D. caudipinna), whereas that in pure apogamous populations was 0.66 (N = 23). Thus, D values for mixed populations were significantly higher than those for pure apogamous populations (t-test, P - 0.018).
3.4. Discussion
The geographical distribution of the sexually reproducing fern species, Dryopteris caudipinna on Izu-Oshima Island was described in this study for the first time . Its distribution was confined to the northeastern part of Izu Oshima Island, where lava flow had occurred relatively recently (approximately 200-400 years ago; Kawabe 1998).
Thus, sporophytes of the sexual species were observed in the localities where strong disturbances had recently occurred. Therefore, it appears that the sexual species are better adapted to disturbed habitats than its related apogamous species. At first glance , this appears anomalous. As discussed in the General Introduction of this thesis, the apogamous species have advantages in colonizing new habitats because fertilization between two independent gametophytes derived from two different spores of the same species is not necessary. Moreover, it is apparent that apogamous fern species were derived from sexual species. It is natural to consider that sexual species tend to survive in stable habitats. However, in evolutionary biology, the long-and short-term advantages of sexual reproduction over those of asexual reproduction have been demonstrated not in stable but in constantly changing environments (Crow and Kimur 1965, Maynard Smith 1978, Hamilton et al. 1990). This is because sexually reproducing species may adapt to newly changed environments more easily and/or quickly than asexually reproducing ones. Therefore, it may be reasonable to consider that sexually reproducing D. caudipinna tend to be distributed in areas recently disturbed by lava, where both the
environment and the biome would have changed even annually. I consider that it may be interesting to continue such an observation of mixed populations for several decades, if possible, so as to record the frequency change of the two closely related fern species with different reproductive modes in each population.
On Izu-Oshima Island, the apogamous fern species D. erythrosora, and its closely related sexual species shared a, b, d, and f alleles of Pgi-2. However, differences in the frequencies of these four alleles were observed between the two specie. In particular, the f allele frequency was higher in sexual species than in its apogamous relative. The f allele frequency was higher in individuals of the apogamous species within the mixed populations than those within the pure apogamous population. In addition, apogamous individuals in mixed populations were genetically more variable than those in pure apogamous populations. In other words, the clonal diversity observed in apogamous species was higher in mixed populations than in pure apogamous populations. Therefore, it was suggested that apogamous fern species acquired genetic variation from their closely related sexual species, possibly through hybridization.
However, on Izu-Oshima Island, individuals of the apogamous D. erythrosora from pure apogamous populations showed high genetic variation. Therefore, I
considered the possibility that the sexual D. caudipinna may be distributed even in the pure apogamous populations in the recent past and that the apogamous species may have obtained genetic variation from their sexual relatives at that time. As shown in the
results of Chapter 2, . sexual D. caudipinna prevails in most of the Izu Islands. It would not be so surprising if sexual species had also occurred in higher frequencies on Izu-Oshima Island in the past. In addition, apogamous species may obtain high clonal diversity in all of their populations through frequent genetic segregation by homoeologous chromosome pairing.
In this chapter, I concluded as follows:
(1) The apogamous species Dryopteris erythrosora was widely distributed on Izu-Oshim aIsland, while its closely related sexual species D. caudipinna was confined to the northeastern part of the islands, where the environment had recently been disturbed by lava flows.
(2) The apogamous D. erythrosora and the sexual D. caudipinna shared all four of the Pgi-2 alleles, though their frequencies differed from each other to some extent.
(3) Individuals of the apogamous species in mixed populations tended to be more similar to their sexual relatives in allele frequencies and showed higher clonal diversity than those in pure apogamous populations. This constitutes positive evidence that apogamous fern species incorporate genetic variation from sexual species when they
grow together.
Table 3-1. Genotype and estimated reproductive populations (A Z, a—d) on Izu-Oshima Island.
mode of each sample from the 30
Sample number
Genotype of Reproductive
Pgi-2 mode
Sample number
Genotype of Pgi-2
Reproductive mode Al
A2 A3 A4 A5 A6 A7 A8 A9 A10 All Al2 A13 Al4 A15 A16 A17 A18 A19 A20 A21 A22 A23
ddd bbd bbd bbd bbd bdd bbd bbd bbd bbd bbd bbd bbd bdd bdd bbd bdd bdd bdd bdd bbd bbd bbd
A A A A A A A A A A A A A A A A A A A A A A A
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 Dll D12
D13 D14 D15 D16 D17 D18 D19 D20 D21 D22 D23
bbd bbd dff bdd bbd ddd df bf bdd bdd bdd bdf ad aad bdd
bdf ddd ddd add add add adf add
A A A A A A S S A A A A S A A A A A A A A A A B1
B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18
ddd bdd ddd ddd ddd bbd dff bbd ddd ddd ddd ddd ddd bbd bbd bdd bdd bbd
A A A A A A A A A A A A A A A A A A Cl
C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12
bdd ddd bdf bdf bbd bdd bdf bdd bdd bbd bdd bdd
El E2 E3 E4 E5 E6 E7 E8 E9 El0 Ell
A A A A A A A 'A A A A A
adf adf adf bdd ddd bdf bbd ddd bbd adf adf
A A A A A A A A A A A F1
F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18
bbd ddd adf bbd ddf bbd ddd bbd bbd bbd add bbd ddd bdd bdf bbd bbd bdf
A A A A A A A A A A A A A A A A A A
Sample number
Genotype of Pgi-2
Reproductive mode G1
G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 G23 G24 G25
ff df ff bf bb ddd bbd bdd bbd bbd bbd bd ff bdd bdd bbd ff bdd bbd bdd bdd bdd bff ff bdd
A S S S S A A A A A A S A A A A A A A A A A A A A H1
H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H2O H21 H22 H23 H24
bdd bdf bbd bdd bdd bdd bdd aad add ddf bbd bdd bbd bdf bbd bdf bdd bdd bdd bbd bdd bbd bbd bbd
A A A A A A A A A A A A A A A A A A A A A A A A I1
12 13 14 15 I6 I7 I8 I9 110 Il l 112 113 114
bdf ad dff bdd ff df ff ff bbd bdd bdf bdf bbd bbd
A S A A S S A A A A A A A A
Sample number
Genotype of Reproductive Pgi-2mode
J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13
bff bf bdd
bf bf bb ff ff bdf ff bdd
bbf df
A S A S S S S S A S A A S
J14 bfS
K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 K13 K14 K15 K16 K17 K18 K19 K20 K21 K22 K23
as
bf aaf bf ff ff ddd
ff ff af aab
ff ff aab
af af ff if ff ff af af af
S S A S S S A S A S A S A A S S S S S S S S S Ll
L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24
bbd bdd bbb adf bbd bbd bdd bdd bdd bdd bdd ddd bdd bdd bdd bbd bbd bb add bbd bdd bbd bbd add
A A A
A A A A A A A A A A A A A A S A A A A A A
Sample number
Genotype of Reproductive
Pgi-2 mode
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17
bdd add add bdd bdd bbd add add bbd ddd ddd bbd ddd bbd bdd bbd bdd
A A A A A A A A A A A A A A A A A N1
N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 N22 N23 N24 N25
bf bf if if bb bdd bbd df bf ddf add add bf bbd ddd of ddd
if bdd bbd ddf
bd bbd
df bd
S S S S S A A S S A A A S A A S A S A A A S A S S 01
02 03 04 05 06 07 08 09 010 011 012 013 014 015 016 017 018 019
ddd bdd add bbd ddd ddd bdd bdd add bbd bdf bbd bdf ddd bdd bbd add
bbd bbd
A A A A A A A A A A A A A A A A A A A
Sample number
Genotype of Reproductive
Pgi-2 mode
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23
bdd bdd ddd bdd bbd bdd bbd bbd bdf bbd bdf bdd bbd bbd bdd bdf bdd bbd bbd bbd bbd bdd bdd
A A A A A A A A A A A A A A A A A A A A A A A
Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18 Q19 Q20 Q21
bbd bdd abd add bbd bdd aad aad bbd add add aad bdd bdd bdd bdf bbd bdd bbd bbd bdf
A A A A A A A A A A A A A A A A~
A A A A A R1
R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18
bbd bbd bdd ddd bbd bbd bbd bdd bdd bdd bdd bbd bbd bbd bbd bbd bdf bdd
A A A A A A A A A A A A A A A A A A
Sample number
Genotype of Reproductive Pgi-2mode
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26
bdf bdd bbd bdf bbd bdd add
dff add add bdd add
add add add add add bdd bbd bbd bdd aad bdd add bbd bbd
A A A A A A A A A A A A A A A A A A A A A A A A A A T1
T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18
add bdd bdd ddd bbd bbd bdf bbd bdf bbd bdf add bdd bdd bbd bdd dff ddd
A A A A A A A A A A A A A A A A A A U1
U2 U3 U4 U5 U6 U7 U8 U9 U10 U11 U12 U13 U14 U15 U16 U17 U18 U19 U20
ddd bbd ddd bbd ddd bbd bdf bbd bdd bdd bdd bbd bdf ddd bdd bbd bbd bbd bbd bbd
A A A A A A A A A A A A A A A A A A A A
Sample number
Genotype of Reproductive
Pgi-2 mode
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16 V17 V18 V19 V20
dff add add add ddd ddd ddd ddd ddd ddd ddd bbd ddd bdd bbd ddd ddd ddd bbd bbd
A A A A A A A A A A A A A A A A A A A A W1
W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20
ddd bbd bbd bbd bbd bbd bdd bbd bdd bbd bbd bbd bdd bdf bdd fff bbd bbd add bbd
A A A A A A A A A A A A A A A A A A A A X1
X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21 X22
bbd bbd bbd bbd bbd bdd bbd bdd bbd bbd bdf bbd bdd ddd ddd ddd bdd ddd ddd abd bbd bbd
A A A A A A A A A A A A A A A A A A A A A A
Sample number
Genotype of Reproductive
Pgi-2 mode
Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10 Yl1
Y12 Y13 Y14 Y15 Y16 Y17 Y18 Y19 Y20 Y21 Y22 Y23
bdd bbd bbd ddd bbd bbd bbd bbd bbd ddd ddd ddd bdd ddd ddd ddd ddd bdd ddd ddd ddd ddd ddd
A A A A A A A A A A A A A A A A A A A A A A A Z1
Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Z11 Z12 Z13 Z14
bdd bdd bdd bdd bdd bdd bdd bdd bdd bbd bbd bdd bdd dff
A A A A A A A A A A A A A A al
a2 a3 a4 a5 a6 a7 a8 a9 al 0 all al2 a13 al4 a15 a16 al 7 al 8 a19
aad aad add aad add adf aad bbd ddd bdd bdd bbd bbd bbd bbd add bdd bdf adf
A A A A A A A A A A A A A A A A A A A a20bdfA
Sample number
Genotype of Pgi-2
Reproductive mode b
b2 b3 b4 b5 b6 b7 b8 b9 b10 bll b12 b13 b14 b15 b16 b17 b18 b19 b20 b21 b22 b23 b24 b25 b26
abd bbd bbd add bbd bbd add
bdd bdd bbd bbd ddd bbd bbd bdd bdd bbd bbd add bbd adf bbd adf bbd bbd bdd
A A A A A A A A A A A A A A A A A A A A A A A A A A cl
c2 c3 c4 c5 c6 c7 c8 c9 c10 cl1 c12 c13 c14 c15 c16 c17 c18 c19 c20 c21 c22 c23 c24 c25
adf adf ddd bdd bbd bbd bbd adf bdf bdf add add ddd add adf adf bbd adf bbd add bdd bdd bdd add bbd
A A A A A A A A A A A A A A A A A A A A A A A A A dl
d2 d3 d4 d5 d6 d7 d8 d9 dl 0 dl1
bdd bdd bbd aad bbd bdd bdd bdd bbd ddd bdd
A A A A A A A A A A A
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