Molecular diversity and genetic relationships among Geranium pusillum and G. pyrenaicum with inter simple sequence repeat (ISSR) regions

Figures & data

Table 1. Location and herbarium accession numbers of the studied populations of G. pyrenaicum Burm and G. pusillum L. collected by Esfandani in Iran.

Species	Population	Locality	Latitude	Longitude	Altitude (m)	Voucher no.
G. pyrenaicum	1	Mazandaran, SangDeh Forest	36°01ʹ11ʺ	53°13ʹ26ʺ	1720	HSBU 201,666
G. pyrenaicum	2	Mazandaran, Veresk Bridge	35°54ʹ12ʺ	52°59ʹ27ʺ	1579	HSBU 201,667
G. pyrenaicum	3	East Azerbaijan Kaleybar	38°52ʹ39ʺ	47°23ʹ92ʺ	1144	HSBU 201,668
G. pyrenaicum	4	East Azerbaijan Kaleybar cheshme Ali-Akbar	38°52ʹ39ʺ	47°25ʹ92 ʺ	1133	HSBU 201,669
G. pyrenaicum	5	East Azerbaijan Kaleybar, Shojabad	38°52ʹ39ʺ	47°25ʹ92ʺ	1137	HSBU 201,670
G. pyrenaicum	6	East Azerbaijan Kaleybar, road side	38°52ʹ39ʺ	47°25ʹ92 ʺ	1122	HSBU 201,671
G. pyrenaicum	7	East Azerbaijan, Babak fort	38°51ʹ51ʺ	47°02ʹ28ʺ	1155	HSBU 201,672
G. pusillum	1	East Azerbaijan Kaleybar	38°52ʹ39ʺ	47°23ʹ92ʺ	1144	HSBU 201,673
G. pusillum	2	Mazandaran, SangDeh Forest	36°01ʹ11ʺ	53°13ʹ26ʺ	1720	HSBU 201,674
G. pusillum	3	East Azerbaijan Kaleybar cheshme Ali-Akbar	38°52ʹ39ʺ	47°25ʹ92ʺ	1133	HSBU 201,675
G. pusillum	4	East Azerbaijan Kaleybar, Shojabad	38°52ʹ39ʺ	47°25ʹ92 ʺ	1137	HSBU 201,676
G. pusillum	5	East Azerbaijan Kaleybar, road side	38°52ʹ39ʺ	47°25ʹ92ʺ	1122	HSBU 201,677

Figure 1. Distribution map of the populations studied.

Table 2. Evaluated morphological characters.

NO	Characters	Numerical code	No.	Characters	Numerical code
1	Plant height	mm	20	Mericarp length	mm
2	Length of stem leaves petiole	mm	21	Mericarp width	mm
3	Length of stem leaves	mm	22	Mericarp length/mericarp width	mm
4	Width of stem leaves	mm	23	Seed length	mm
5	Length of stem leaves/width of stem leaves	mm	24	Seed width	mm
6	Width of stem leaves/length of stem leaves	mm	25	Seed length/seed width	mm
7	Number of segment stem leaves	mm	26	Stipules length	mm
8	Length of basal leaves petiole	mm	27	Stipules width	mm
9	Length of basal leaves	mm	28	Stipules length/stipules width	mm
10	Width of basal leaves	mm	29	Bract length	mm
11	Length of basal leaves/width of basal leaves	mm	30	Bract width	mm
12	Width of basal leaves/length of basal leaves	mm	31	Bract length/bract width	mm
13	Number of segment basal leaves	-	32	Pedicel length	mm
14	Calyx length	mm	33	Peduncle length	mm
15	Calyx width	mm	34	Rostrum length	mm
16	Calyx length/calyx width	mm	35	Style length	mm
17	Petal length	mm	36	Stamen filament length	mm
18	Petal width	mm	37	Fruit length	mm
19	Petal length/petal width	mm	38	Number of flowers per inflorescence	-
39	Type root		60	Bract shape
40	Vegetation-forms		61	Stipules shape
41	State of stem strength		62	Bract and stipules hair density
42	State of stem branches		63	Bract and stipules hair
43	Leaf shape		64	Shape of segments cauline leaves
44	Phylotaxy		65	Shape of calyx
45	Leaf tips		66	Calyx apex
46	Shape of segments basal leaves		67	Petal shape
47	Stamen filament color		68	State of petal ligule
48	Stigma hair		69	Shape of petal lobes
49	Mericarp shape		70	State of petal ligule hair
50	Mericarp surface		71	Stamen filament hair
51	Mericarp hair		72	Mericarp hair density
52	Mericarp rostrum hair		73	Mericarp color
53	Sepal hair		74	Seed color
54	Sepal hair density		75	Seed shape
55	Peduncle and pedicel hair		76	Seed surface ornamentation
56	Anther color		77	Peduncle and pedicel hair density
57	Stem hair		78	Petioles hair
58	Stem hair density		79	Petioles hair density
59	Leaf hair		80	Leaf hair density

Table 3. The main differences between G. pusillum and G. pyrenaicum.

Characters	G. pusillum	G. pyrenaicum
Length and width of corolla	2–3.5 mm× 1.1–1.7 mm	5–8 mm× 2–5 mm
Length and width of leaf	1–2 × 1.5–3.5 cm	1–3.5 × 2–6 cm
Length and width of mericarp	1.7–2 mm× 0.9–1 mm	2.5–3 mm× 1–1.3 mm
Seed length	1.5–1.7 mm	2–2.2 mm
Plant height	9–30 cm	30–60 cm
Vegetation-forms	Annual or biennial	Perennial
Hair of stem and petioles	Hair of glandular and non-glandular, short fine, coarse	Hair of glandular and non-glandular, short and tall fine, Coarse
Hair of sepal, peduncle and pedicel	Long hair and medium non-glandular and glandular	Hair of glandular and non-glandular, short fine, Coarse
Shape of petal lobes	Apex emarginate with a 0.2–0.5 mm notch	Apex emarginate with a 1–2.5 mm notch

Table 4. Evaluated characters of pollen grains in studied Geranium species.

characters	G. pusillum	G. pyrenaicum
Polar axis length (p)	39.65	64
Exine thickness	3.7	4.3
Equatorial axis length (E)	38.22	59
Apocolpium length	26–27	33
Mesocolpium length	24	30
Colpus length	15.66	23
Pore diameter	7.87	4
P/E ratio	1.02	1.08
Polar shape	Circular	Circular
Equatorial shape	Circular	Circular
Homogeneity of pollen sculpture	Present	Present
Pollen shape	Spheroidal	Prolate-spheroidal
Exine ornamentation type	Reticulate-cristatum-clavate	Reticulate-cristatum-clavate

Table 5. Fruit characters of the Geranium species.

Taxon	Mericarp shape	Mericarp surface	Mericarp hair	Mericarp color	Mericarp ornamentation	Mericarp length (mm)	Mericarp width (mm)	L/W Mericarp (mm)	Rostrum length (mm)	Fruit length (mm)
G. pyrenaicum	Suborbicular	Smooth	Short hairs of non-glandular type	Brown	Reticulate	2.4–3	1–1.3	2–2.7	10–12	15
G. pusillum	Suborbicular	Smooth	Short hairs of non-glandular type	Brown	Reticulate	1.7–2	0.9–1	1.7–2.1	5–8	8–12

Table 6. Seed characters of the Geranium species.

taxon	Seed color	Seed shape	Seed surface	Seed length (mm)	Seed width (mm)	L/W seed (mm)	Hilum	Cell shape	Anticlinal wall
G. pyrenaicum	Reddish brown	Narrow elliptical	Reticulate-foveate	2–2.2	0.8–1	2.2–2.4	Basal	Polygonal	Shallow, thick, undulate
G. pusillum	Reddish brown	Narrow elliptical	Reticulate-foveate	1.5–1.7	0.8–1	1.6–2.1	Basal	Polygonal	Shallow, thick, undulate

Figure 2. Multidimensional scaling plots of morphological characters, separating two Geranium species groups from each other.

Figure 3. Pollen micrographs of Geranium species: G1–G3: G. pyrenaicum; H1–H3: G. pusillum. G1, H1: equatorial view; G2, H2: polar view; G3, H3: exine sculpture.

Figure 4. Scanning electron micrographs (SEM) of seed and fruit coat in Geranium species. (A1–A2) (C1–C2) G. pusillum; (B1–B2), (D1–D2) G. pyrenaicum.

Figure 5. MDS plot of morphological characters in G. pyrenaicum populations studied. Different colors indicate the plant specimens (numbers 1–8) studied from each geographical population.

Table 7. Genetic diversity parameters in the studied populations. (N = number of samples, Ne = number of effective alleles, I = Shannon information index, He = gene diversity, UHe = unbiased gene diversity, P% = percentage of polymorphism, populations).

Pop	N	Na	Ne	I	He	UHe	%P
pop1	7.000	1.311	1.377	0.331	0.221	0.238	62.30%
pop2	5.000	1.066	1.268	0.233	0.155	0.172	45.90%
pop3	5.000	0.443	1.059	0.057	0.037	0.041	11.48%
pop4	8.000	0.885	1.236	0.209	0.139	0.148	39.34%
pop5	8.000	0.639	1.148	0.129	0.086	0.092	24.59%
pop6	11.000	0.984	1.202	0.194	0.125	0.131	42.62%
pop7	5.000	0.311	1.020	0.017	0.011	0.012	3.28%

Populations 1–7 are according to Table 1.

Figure 6. MDS plot of ISSR data in G. pyrenaicum populations studied.

Figure 7. STRUCTURE plot and neighbor-net network of G. pyrenaicum populations based on k = 6 of ISSR data.

Figure 8. MDS plot of morphological characters in G. pusillum populations studied. Different colors indicate the plant specimens (numbers 1–8) studied from each geographical population.

Table 8. Genetic diversity parameters in the studied populations. (N = number of samples, Ne = number of effective alleles, I = Shannon information index, He = gene diversity, UHe = unbiased gene diversity, P% = percentage of polymorphism, populations).

Pop	N	Na	Ne	I	He	UHe	%P
pop1	5.000	0.922	1.190	0.173	0.114	0.127	33.33%
pop2	6.000	0.843	1.168	0.156	0.102	0.112	31.37%
pop3	9.000	1.275	1.261	0.256	0.164	0.173	56.86%
pop4	9.000	1.137	1.292	0.267	0.176	0.186	52.94%
pop5	6.000	0.471	1.054	0.051	0.033	0.036	9.80%

Figure 9. MDS plot of ISSR in G. pusillum populations studied. Different colors indicate the plant specimens (numbers 1–8) studied from each geographical population.

Figure 10. (a) Reticulogram, (b) STRUCTURE plot, (c) neighbor-net network of G. pyrenaicum populations based on k = 3 of ISSR data.

Figure 11. Consensus tree of morphological and molecular data in G. pusillum populations.