Material and methods

This case study was carried out in the Research and Teaching Laboratory of Applied Botany under the auspices of the Department of Biology of the University of Joensuu, Finland (N 62° 36' E 29° 40') from 1999 to 2003 as a part of the larger project, "Quinolizidine alkaloids in arctic and sub-arctic flora". This large project explores the broader problem of QAs occurring in plants growing in northern ecosystems. This case study presents and discusses the research results from the first part of this project.

It has been qualitatively checked wild (X) and semi-wild (B) plants and some crops (?) of 40 legume species growing wild in Finland for alkaloids. The species studied were botanically determined with the use of a stereo light microscope at the beginning of the project, and for the experimental trials, the naturally growing vegetation places were pre-marked. In this way, the established trial area was 1 m2 for each species in each habitat. In the case of a genus with a high and broad habit of growth, an additional trial area was provided (Table 27). For each year, there were at least 20 plants within the pre-marked areas.

For each year, the botanical purity and identity of the studied species were monitored using a stereo microscope. During these microscope investigations, no significant overwintering or disease damage were observed. The populations studied had wild or semi-wild distributions. They were growing under wild conditions in naturally competitive habitats. These habitats were (a) roadside, (b) forest border stand, (c) former field and (d) in a garden area. The legume populations in the trial areas were observed and tested for QAs each year for 5 years. Leaves of plants were taken from a total of 4000 legume individuals, belonging to 13 different legume genera, of which 40 species and sub-species were studied (Table 27). The natural distribution of legume species in northern conditions served as the basis for the size of the genus studied. Some species studied are rare and others are very common in Finland (Table 27).

Table 27 Systematic division and habitat characteristics of the studied legume species

Genus

Number of Species

Number of Individuals

Habitat characteristics

Astragalus

500

5

(1) dBv a r+

Coronilla

100

1

(l) aXv r

Cytisus

100

1

(2) bXBv r

Lathyrus

600

6

(l), 3bcXBvr+

Lotus

100

1

(l) aXv+

Lupinus

100

1

(1) aBv Va+

Medicago

300

3

(l), 2acB?Vr+

Meliotus

100

1

(l) acXBv r

Ononis

100

1

(2) cXv r

Ornithopus

100

1

(l) d?Vr

Oxytropis

100

1

(l) bXv r

Trifolium

900

9

(l),(2) ac?BXvv r+

Vicia

900

9

(l),(2) abcdBX?VV r+

Total 13

4000

40

Notes: (1) = eastern Finland, (2) = southern Finland, (3) = western Finland; a = roadside, b = forest border stand, c = former field, d=garden area; X = wild plant, B = semi-wild plant, ? = crop; V = vegetative growth advanced, VV = vegetative growth advanced, the first phase of flowering; a = additional trial area; r = rare species; r+ = both rare and common species; + = common species.

Notes: (1) = eastern Finland, (2) = southern Finland, (3) = western Finland; a = roadside, b = forest border stand, c = former field, d=garden area; X = wild plant, B = semi-wild plant, ? = crop; V = vegetative growth advanced, VV = vegetative growth advanced, the first phase of flowering; a = additional trial area; r = rare species; r+ = both rare and common species; + = common species.

The number of species studied was the highest for the genera Trifolium and Vicia, Lathyrus, Astragalus and Medicago. Eight of the studied genera were represented by only one species (Table 27). The analysed samples were taken from plants in the stages of expanded vegetation, pre-flowering or at the beginning of flowering. The time of sampling fell between 20 June and 30 June of each year, which is known in sub-arctic flora ecosystems as a period of very active photosynthesis during the extended daylight conditions of the "white nights". During this time of year, light and temperature conditions were generally very similar at each experimental site. Each year, leaf samples were randomly selected from the same habitat and the same populations.

4.2.1. Method of QAsi+) indications

A DRG indicator has been used in the test for QAs in all the species studied. This quick and accurate qualitative method was developed and used to detect alkaloids and heterocyclic nitrogen compounds, especially in plant drug analysis755. A DRG indicator has been successfully used to obtaining qualitative indications of total QAs in previous studies7'740'746'748'756. This method was re-evaluated before being implemented in this study. Pure alkaloids and alkaloid-rich extracts were placed separately on TLC plates and, together with a TLC plate without an alkaloid (the control TLC), they were precipitated by the DRG. On all of the TLC plates, with the exception of the control TLC, a colour change occurred. This establishes that the DRG method functions well. In the sample-testing process, indications were obtained from the juice of the leaves on the TLC plates precipitated by the DRG. A colour change from yellow to orange signalled a positive indication (+), whereas no colour change served as a negative indication (—). At this juncture, some critical remarks on this method should be mentioned. The DRG represents a means of indicating the total amount of QAs as a specific class of alkaloids as chemical compounds but not in terms of specific alkaloids. The DRG indication reflects only the presence or absence of this group of compounds. In reality, there can be different alkaloids or their derivatives in different plant species. A DRG positive indication was studied and qualified this in comparison with GC and GC MS+ methods. The (+) indication mechanism is based on a reaction between N+ constructed in the QA ring as a result of a DRG final reaction with H+

The orange colour change on the TLC plates was caused by I2 after a reaction with N+ in the alkaloid ring with H+ from HI. All QAs and their derivatives have a heterocyclic structure and N+ in the ring. The intra-molecular hydrogen bond N—H has been discussed in the literature757, and the alkaloid indicator method used in this research worked very well. Although differences in colour change intensity were observed, this did not lead to any methodological problems. The concentration of QA skeletons in the plants and species varied. Only indications of the presence or absence of total compounds has been studied in this work. All test indications were clearly readable for colour change or absence of change. One hundred tests per species were conducted during the research period (i.e. 20 individual plants from each species per year).

4.2.2. Mutational trajectories

Populations follow mutational trajectories that move them upwards within the genotypic fitness landscape758 759. Changes in plants first result in short-term evolutionary (micro-evolutionary) dynamics and subsequently, in a much more extended time-scale, the mutagenesis process. A succession of novel, beneficial mutations may appear and become fixed during the later macro-evolutionary process. Many authors consider both processes important in understanding natural selection758'759'760'761. For these categories changes in both individual alkaloid^-1 and individual alkaloid(—) plants within the populations were found. This question has not been previously studied. On the basis of changes and trends in the species' populations, the micro-evolution coefficient is expressed according to following established formula:

where MEC = micro-evolution coefficient; 2 = sum; Fy = average trait frequency of a plant with indication per year; Yn = number of years; 1 = constant hypothetical coefficient, which means that the trait of an individual plant is the same from year to year (i.e. no trends, no mutations, no trajectories).

The MECs for a species are coefficients for real trends of plant traits during the period studied. Numerically, it can have a value from 0.00 to 1.00 and can also be interpreted, if needed, as a percentage. The lower the MEC, the weaker the rate of micro-evolution for the period of time studied. A maximum value (1.00) for the MEC signifies that a micro-evolution (trait change) has occurred in all the plants studied and, therefore, macro-evolution has occurred in the population. In this case, the new hypothetical constant coefficient is valid (no trends, no mutations, no trajectories). The MECs can be positive or negative. Positive MECs show that the traits are developing in a micro-evolutionary process, and negative MECs indicate the reverse. The MECs represent very useful parameters for measurement of micro-evolution in plant species.

All statistics and graphs were generated using SPSS and SigmaPlot 2002 for Windows Version 8.02 (SPSS Inc.). ANOVA was used to analyze the experimental data on alkaloid(+- frequencies. The average frequency and standard deviation for each species were calculated, and five groups of species with different frequencies were established. The relative frequencies for each species with annual standard deviations for the populations of the species were found. The statistical significance of the variations in relative frequencies and the MECs for each species were calculated. Moreover, using ANOVA, the statistical significance of these coefficients and correlations with r between species were checked. To determine whether the MECs are similar or different, the BSR with R2 as the best criterion, and the significance of possible different variables with the model were checked. Using the best criterion, it was possible to study the position of different MECs in relation to MECs for other species and evaluate their significance.

4.3. Results

4.3.1. QAs(+) occurrence and frequency

The QAs occurred in all the legume genera, species and sub-species studied. There were both QAs(+} and QAs(-) plants (Table 28). The frequency of QA occurrence mostly varied (0.1-0.8) between species (P< 0.001). The smallest frequency was found in only two species, Trifolium arvense and Trifolium pratense, in which positive test results were not found more frequently than every tenth individual (Table 28).

Conversely, the highest frequency [F(A)] was found in 14 species, in which at least every second individual tested positive for QAs (Table 29). Seven species exhibited a frequency range of 31-50% [F(B)] and 12 species and sub-species 21-30% [F(C)]. Moreover, 5 species had a group frequency F(D) range of 11-20% (Table 29). The largest relative frequency (RF) changes occurred in Vicia spp. and Ononis repens, and the smallest changes occurred in L. polyphyllus and L. corniculatus (Figures 101-106). Statistically significant frequency fluctuation (RF) was also observed during different years and in different species. The number of positive and negative individuals fluctuated according to species (P < 0.001). The average number of frequency fluctuation (RF) was also observed during different years and in different species. The number of positive and negative individuals fluctuated according to species (P< 0.001).

The average number of positive individuals clearly doubled for Vicia hirsuta during 1999-2001, after which the increase became smaller (Figure 106).

4.3.2. Tendencies of QAs(+) plants to evolve and their MECs

Thirty-three species had a tendency to decrease the number of individuals with QAs(+) (P < 0.001) but in the case of four species there was a tendency to increase the number of individuals with QAs(+) (P < 0.001). Three species

Table 28 Quinolizidine alkaloid frequencies in plant populations of Fabaceae in the boreal zone during 1999-2003

Species Average Frequency in Populations QAs(+)

Table 28 Quinolizidine alkaloid frequencies in plant populations of Fabaceae in the boreal zone during 1999-2003

Astragalus alpinus

o.s

0.03)

Astragalus arenarius

0.9

0.04)

Astragalus cicer

0.9

0.05)

Astragalus frigidus

0.9

0.0ó)

Astragalus glycyphyllos

o.s

0.04)

Coronilla varia

0.4

0.03)

Cytisus scoparius

0.4

(0.02)

Lathyrus japonicus

0.?

(0.05)

Lathyrus linifolius

0.?

(0.04)

Lathyrus palustris

0.?

(0.04)

Lathyrus pratensis

0.?

(0.04)

Lathyrus sylvestris

o.s

(0.06)

Lathyrus vernus

0.?

(0.04)

Lotus corniculatus

0.6

(0.04)

Lupinus polyphyllus

0.9

(0.0?)

Medicago sativa ssp. sativa

0.2

(0.02)

Medicago sativa ssp. falcate

0.3

(0.02)

Medicago lupulina

0.3

(0.03)

Meliotus officinalis

0.3

(0.03)

Ononis repens

0.3

(0.04)

Ornithopus perpusillus

0.4

o.oi)

Oxytropis campestris

0.6

(0.05)

Trifolium arvense

0.1

(0.02)

Trifolium aureum

0.3

(0.04)

Trifolium hybridum

0.3

(0.02)

Trifolium fragiferum

0.3

(0.03)

Trifolium medium

0.4

(0.05)

Trifolium montanum

0.4

(0.04)

Trifolium pratense

0.1

(0.0i)

Trifolium spadiceum

0.4

(0.03)

Trifolium repens

0.4

(0.04)

Vicia cassubica

0.3

(0.03)

Vicia cracca

0.2

(0.03)

Vicia hirsuta

0.2

(0.03)

Vicia lathyroides

0.3

(0.03)

Vicia sativa

0.2

(0.03)

Vicia sepium

0.2

(0.02)

Vicia villosa

0.3

(0.03)

Vicia sylvatica

0.3

(0.04)

Vicia tetrasperma

0.3

(0.05)

Abbreviations: X - number of individuals with QAs'+'; Xn - total number of individuals; In parenthesis standard deviation of mean frequency; P < 0.001*".

Abbreviations: X - number of individuals with QAs'+'; Xn - total number of individuals; In parenthesis standard deviation of mean frequency; P < 0.001*".

Table 29 Frequencies of QAs^+ in the legume species studied

Species with F(A)

Species with

Species with

Species with

Species with

F (B)

F (C)

F (D)

F(E)

Astragalus alpinus

Coronilla varia

Medicago

Medicago

Trifolium

sativa spp

sativa ssp

arvense

falcata

sativa

Astragalus arenarius

Cytisus

Medicago

Vicia cracca

Trifolium

scoparius

lupulina

pratense

Astragalus cicer

Ornithopus

Meliotus

Vicia hirsuta

perpusillis

officinalis

Astragalus frigidus

Trifolium

Ononis repens

Vicia sativa

medium

Astragalus glycyphyllos

Trifolium

Trifolium

Vicia sepium

montanum

aureum

Lathyrus japonicus

Trifolium

Trifolium

spadiceum

hybridum

Lathyrus linifolius

Trifolium

Trifolium

repens

fragiferum

Lathyrus palustris

Vicia cassubica

Lathyrus pratensis

Vicia

lathyroides

Lathyrus sylvestris

Vicia villosa

Lathyrus vernus

Vicia sylvatica

Lotus corniculatus

Vicia

tetrasperma

Lupinus polyphyllus Oxytropis campestris

Lupinus polyphyllus Oxytropis campestris

Abbreviations: F(A) = frequency >50%; F(B) = frequency 31-50%; F(C) = frequency 21-30%; F(D) = frequency 11-20%; F(E) = frequency 1-10%.

had no tendency to change (P < 0.001) the number of individuals with QAs(+) (Figures 107-112). Only four species (Astragalus arenarius [R2 = 0.65], Astragalus cicer [R2 = 0.552] Astragalus frigidus [R2 = 0.64] and L. polyphyllus [R2 = 0.706]) had positive MECs (P < 0.001), which indicates that these species, more clearly than other species, are increasing and will consequently increase the number of individuals with QAs(+) (Figures 107-108). Astragalus alpinus (R2 = 0.551), Astragalus glycyphyllos (R2 = 0.065) and Lathyrus sylvestris (r2 = 0.91) (Figures 107 and 109) had the MEC value of 0.0, indicating that these species have no tendency towards micro-evolution in the case of QAs(+) and QAs(-). All other species studied have a micro-evolutionary tendency to reduce their QAs(+) frequencies (Figures 107 and 112). This trend was highest in T. arvense (R2 = 0.87) and T. pratense (R2 = 0.94) (in both cases 0.7) and Vicia cracca, V. hirsuta, Vicia sativa and Vicia sepium (R2 = 0.079) (-0.6 in each case) (Figures 111 and 112). Statistical analysis strongly confirmed the validity of these results.

g 16

1999

2000

2001 Years

2002

- Coronilla varia •■'.— Cytisus scoparius ■■■ - Lotus corniculatus Lupinus polyphyllus -■-- Meliotus officinalis —--■■ Ononis repens -«— Ornithopus perpusillus —'.■.— Oxytropis campestris P < 0.001**

1999

2000

2002

2003

2003

Figure 101. RF of QAs« individuals in Coronilla varia, Cytisus scoparius, Lotus corniculatus, Lupinus polyphyllus, Meliotus officinalis, Ononis repens, Ornithopus perpusillus, Oxytropis campestris during 1999-2003.

Years

1999

2000

2001 Years

Lathyrus japonicus Lathyrus linifolius Lathyrus palustris Lathyrus pratensis Lathyrus sylvestris Lathyrus vernus P<0.001***

1999

2000

2001 Years

2002

2003

Figure 103. RF of QAs(+) individuals in Lathyrus spp. during 1999-2003.

2002

2003

Figure 103. RF of QAs(+) individuals in Lathyrus spp. during 1999-2003.

Years

1999

2000

2002

2003

Trifolium arvense Trifolium aureum Trifolium hybridum Trifolium fragiferum Trifolium medium Trifolium montanum Trifolium pratense Trifolium spadiceum Trifolium repens P< 0.001***

1999

2000

2001 Years

2002

2003

Figure 105. RF of QAs« individuals in Trifolium spp. during 1999-2003.

Vicia cracca —f— Vicia hirsuta

Vicia lathyroides Vicia sativa Vicia sepium Vicia vilosa Vicia sylvatica Vicia tetrasperma P< 0.001***

1999

2000

2001 Years

2002

2003

0.20

0.15

0.05

• Astragalus alpinus (1) O Astragalus arenarius (2) ▼ Astragalus deer (3) V Astragalus frigidus (4) ■ Astragalus glycyphyllos (5) P<0.001"'

Astragalus species

Species r

2

3

4

5

1

-0.094

-0.037

0.33***

-0.11

2

0.495***

0.42***

-0.28**

3

0.49***

-0.32**

4

-0.29**

Species model ABCDEF

R2

A. cicer*** A

0.24***

A. cicer*** - A. glycyphyllos*** B

0.46***

A. alpinus*** - A. cicer*** - A. glycyphyllos*** C

0.551***

A. alpinus*** - A. arenarius - A. cicer*** - A. glycyphyllos*** D

0.552***

A. arenarius*** - A. frigidus*** - A. glycyphyllos*** E

0.64***

A. alpinusns - A. arenarius*** - A. frigidus***- A. glycyphyllos*** F

0.65***

Figure 107. MEC of Astragalus species.

Figure 107. MEC of Astragalus species.

O Cytisus scoparius (2) ▼ Lotus corniculatus (3) V Lupinus polyphyllus (4) ■ Meliotus officinalis (5) O Ononis repens (6)

♦ Ornithopus perpusillus (7) O Oxytropis campestris (8)

}

i 5

í í

1

2

S

4 S Species

6

l

B

Species r

2

S

4

S

6

l

B

1

0.104

-0.109

0.164

0.06S

0.49S***

0.066

0.20l

2

0.326***

-0.16l

j -0.304**

0.411***

0.319***

0.39S***

S

-0.099

-0.0BB

-0.312**

-0.009

-0.0B6

4

-0.04S

0.0S

-0.032

-0.031

S

-0.S12***

0.101

0.396***

6

i

0.286**

0.389***

l

i

1 0.030

Species model ABCDEFG

R2

Oxytropis campestris*"' A

0.SSS***

Oxytropis campestris*** - Coronilla varia *** B

0.60S***

Oxytropis campestris*** - Coronilla varia *** - Lotus corniculatus* C

0.623***

Ononis repens - Oxytropis campestris *** - Coronilla varia'" - Lotus corniculatus" D

0.63l***

Ononis repens**- Ornithopus perpusillus'" - Oxytropis campestris***- Coronilla varia*" - Lotus corniculatus*** E

0.6l2***

Ononis repens*** - Ornithopus perpusillus'" - Oxytropis campestris***- Coronilla varia*** - Cytisus scoparius**- Lotus corniculatus*** F

0.696***

Ononis repens*** - Ornitbhopus perpusillus'" - Oxytropis campestris***- Coronilla varia* **- Cytisus scoparius* - Lotus corniculatus*** - Lupinus polyphyllus G

0.l06***

Figure 108. MEC of Coronilla, Cytisus, Lotus, Lupinus, Meliotus, Ononis, Ornithopus, Oxytropis spp.

0.10

0.05

0.00

Lathyrus species o Lathyrus japonicus (1) ■ > Lathyrus linifolius (2) v Lathyrus palustris (3) Lathyrus pratensis (4) =3 Lathyrus sylvestris (5) 11 Lathyrus vernus (6)

Species r

2

3

4

5

6

1

0.06

0.061

0.015

-0.06

-0.06

2

0.053

0.047

0.07

0.052

3

0.58***

0.163

-0.47

4

-0.36***

0.08

5

0.18

Model ABCDE

R2

Lathyrus japonicus - Lathyrus palustris*** A

0.95***

Lathyrus japonicus - Lathyrus palustris*** - Lathyrus sylvestris*** B

0.97***

Lathyrus japonicus - Lathyrus palustris*** - Lathyrus pratensis - Lathyrus sylvestris*** C

0.97***

Lathyrus japonicus - Lathyrus palustris*** - Lathyrus pratensis - Lathyrus sylvestris*** -Lathyrus vernus D

0.97***

Lathyrus japonicus - Lathyrus linifolius - Lathyrus palustris*** - Lathyrus pratensis -Lathyrus sylvestris*** - Lathyrus vernus E

0.91***

Figure 109. MEC of Lathyrus species.

Figure 109. MEC of Lathyrus species.

4.3.3. Tendencies and dependencies of species

The genus Astragalus (Figure 107) clearly had different tendencies in evolutionary trait variations (P < 0.001). Astragalus arenarius, A. cicer and A. frigidus had similar evolutionary tendencies (MEC = 0.10) but their SDs differed, the

Medicago species and sub-species

Medicago species and sub-species

Medicago sativa ssp. sativa (1) Medicago sativa ssp. falcata (2) Medicago lupulina (3)

Species and sub-species r

2

3

1

-0.15

-0.05

2

0.49 ***

Model AB

R2

Medicago sativa ssp. sativa *** - Medicago sativa ssp. falcata A

0.022

Medicago sativa ssp. sativa *** - Medicago sativa ssp. falcata - Medicago lupulina B

0.023

Figure 110. MEC of Medicago sativa and Medicago lupulina.

Figure 110. MEC of Medicago sativa and Medicago lupulina.

most stable being the case of A. arenarius. Changes in the MECs of this species correlated significantly with the MECs of A. cicer (r = 0.495) and A. frigidus (r = 0.42). However, A. glycyphyllos displayed a negative correlation (r = -0.28). Changes in A. cicer correlated positively with A. frigidus (r = 0.49) and negatively with A. glycyphyllos (r = -0.32). Moreover, A. alpinus also had a negative correlation with A. arenarius (r = -0.094) and A. cicer (r = -0.037).

Lupinus polyphyllus had negative correlations with other species (Figure 108). Within genus Lathyrus, there were similar tendencies with the exception of L. sylvestris (Figure 109). Medicago sativa ssp. sativa and Medicago sativa ssp. falcata had a slight negative correlation, and they both had negative MECs.

4 5 6 Trifolium species

• Trifolium arvense (1) O Trifolium aureum (2) ▼ Trifolium hybridum (3) v Trifolium fragiferum (4) ■ Trifolium medium (5)

O Trifolium montanum (6)

♦ Trifolium pratense (7) <> Trifolium spadiceum (8)

* Trifolium repens (9)

species r

0.08

-0.07 i 0.19 !-0.17 -0.005 j -0.111 MD. 15 i-0.48**1 0.22 ! 0.48**

0.07

0.15

0.01

-0.004 j 0.06 i -0.07 I _0.05 i 0 008 ' z005 * 0.18 I

0.13

0.13

ABCDEFGH

R2

T. arvense*** - T. pratense*** A

0.42***

T. arvense*** - T. aureum*** - T. pratense*** B

0.54***

T. arvense*** - T. aureum - T. montanum*** - T. pratense*** C

0.61***

T. arvense'" -T. aureum"'- T. médium'" - T. montanum'" - T. pratense'"

D

0.65***

T. arvense***- T. aureum***- T. médium***- T. montanum***-T. pratense*** - T. spadiceum*** E

0.68***

T. arvense'"-T. aureum'"- T. hybridum-T. fragiferum'"- T. montanum'"-T. pratense'"-T. spadiceum'"

F

0.87***

T. arvense'" - T. hybridum'"- T. fragiferum"* -T. médium*"- T. montanum*** -T. pratense*** - T. spadiceum*** - T. repens

G

0.94***

4se Vicia species

4se Vicia species

0 Vicia cassubica (1) Vicia cracca (2)

1 Vicia hirsuta (3) Vicia lathyroides (4)

a Vicia sativa (5)

Vicia sylvatica (8) i Vicia tetrasperma (9)

Species i

2

3

4

s

e

7

s

9

1

-0.01

o.os

-o.oe

o.o9

-o.o9

o.o7

0.14

o.o1

2

0.29**

o.o9

o.so***

o.os

o.o4

o.24*

o.4s***

3

0.23*

o.4o***

-o.39***

0.01

o.o9

o.33

4

o.7s

-o.27**

0.21*

0.1s

-o.os

s

o.o7

-o.2e**

o.so***

o.2e**

e

0.22*

-o.os

-o.os

7

-o.o7

-o.os

s

-0.14

Model

ABCDEFGH

R2

V. cassubica*** - V. syivatica A

o.o2

V. cassubica*** - V. viiiosa - V. syivatica B

o.o3

V. cassubica*** - V. iathyroides - V. viiiosa - V. syivatica* C

o.os

V. cassubica*** - V. cacca - V. iathyroides - V. sepium* - V. viiiosa D

o.oss

V. cassubica* - V. cacca - V. iathymides - V. sepium - V.viiiosa - V.syivatica E

o.oe

V. cassubica**- V. cacca - V. iathyoides - V. sepium - V.viiiosa - V.syivatica -V. tetiasepium F

0.076

V. cassubica**- V.cacca - V. birsuta - V. iathyroides - V. sepium* - V.viiiosa* -V .syivatica - V. tetrasepium G

0.07s

V. cassubica* - V. cracca - V. birsuta - V. iathyroides - V. sativa - V. sepium* -V. viiiosa - V. syivatica - V. tetrasepium H

0.079

The latter-mentioned Medicago sub-species exhibited a positive correlation (r = 0.49) with M. lupulina (Figure 110). Differences were also found within the genus Trifolium and the genus Vicia, and the evolutionary tendencies of both species were shown to be similar (Figures 111-112). The analyses of the correlations and the best R2 strongly confirmed the utilization of MECs as a method of evolutionary analysis.

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