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Home , Asperula, Galium, Galium album, Galium glaucum, Galium humifusum, Galium lucidum

Biologia 69/3: 300—310, 2014 Section Botany DOI: 10.2478/s11756-013-0314-z

Genetic diversity and relationships among Egyptian () and related using ISSR and RAPD markers

Kadry Abdel Khalik1,2*, Magdy Abd El-Twab3 &RashaGalal3

1Botany Department, Faculty of Science, Sohag University, Sohag 82524, Egypt; e-mail: [email protected] 2Biology Department, Faculty of Science, Umm-Al-Qura University, 3Botany and Microbiology Department, Faculty of Science, Minia University, Egypt

Abstract: Genetic diversity and phylogenetic analyses of 24 species, representing nine sections of the Galium (Rubi- aceae), have been made using the Inter Simple Sequence Repeats (ISSR), Randomly Amplified Polymorphic DNA (RAPD), and combined ISSR and RAPD markers. Four ISSR primers and three RAPD primers generated 250 polymorphic ampli- fied fragments. The results of this study showed that the level of genetic variation in Galium is relatively high. RAPD markers revealed a higher level of polymorphism (158 bands) than ISSR (92 bands). Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrograms were compared. Six clades can be recognized within Galium, which mostly corroborate, but also partly contradict, traditional groupings. UPGMA-based dendrogram showed a close relationship between members of section Leiogalium with G. verum and G. humifusum (sect. Galium), and G. angusti- folium (sect. Lophogalium). Principal coordinated analysis, however, showed some minor differences with UPGMA-based dendrograms. The more apomorphic groups of Galium form the section Leiogalium clade including the perennial sections Galium, Lophogalium, Jubogalium, Hylaea and Leptogalium as well as the annual section Kolgyda. The remaining taxa of Galium are monophyletic. Key words: Galium; genetic diversity; ISSR; RAPD; Rubiaceae

Introduction distributed in temperate and tropical regions of the world (Willis 1985; Mabberley 1987). Galium itself is Rubiaceae is the fourth-largest angiosperm , com- problematic taxonomically, because taxa from different prising approximately 660 genera and 11,500 species sections exhibit similar habit, many species are widely and classified into 42 tribes (Robbrecht & Manen 2006; distributed and polymorphic, and species groups often Soza & Olmstead 2010a). Most of the family is trop- are poorly differentiated both morphologically and geo- ical and woody. is the only tribe centered in graphically (Schischkin 2000). This genus was described temperate regions, but has cosmopolitan distribution. by Linnaeus (1753) who established the occurrence of Most of its members are herbaceous and adapted to 26 species. He divided them into two groups according xeric habitats (Robbrecht 1988; Jansen et al. 2000). to fruit type (glabrous and hispid). Boissier (1881) con- Rubieae is a monophyletic group, sharing both mor- sidered 90 species and divided them into three sections phological and molecular synapomorphies (Manen et al. (Eugalium, Aparine and ) and 11 subsections. 1994; Natali et al. 1995, 1996; Bremer 1996; Andersson Ehrendorfer et al. (1976) recognized 145 species for Eu- & Rova 1999; Bremer & Manen 2000; Nie et al. 2005; ropean flora, classifying into 10 sections. Ehrendorfer & Backlund et al. 2007; Bremer & Eriksson 2009). How- Sch¨onbeck-Temesy (1982) listed for the flora of ever, classification and identification within Rubieae 101 species divided into 10 sections. have been problematic, especially for the larger genera In Egypt, Tackholm (1974) named 12 species of and Galium. A number of taxa within Aspe- Galium, Boulos (1995, 2000) recognized only 10 species. rula appear morphologically similar to Galium, differing Abdel Khalik et al. (2007; 2008a, b, c) studied 13 Egyp- only in corolla tube length, and these have been trans- tian taxa of Galium by different means such as mor- ferred from Asperula to Galium (Ehrendorfer 1958; Na- phological characters, including vegetative parts, flow- tali et al. 1995; Ehrendorfer et al. 2005; Abdel Khalik ers, fruits, seeds, pollen grains, anatomical structure. & Bakker 2007). Numerical analysis was conducted, and they classified Galium L. is one of the largest genera of Ru- these species into groups. bieae with more than 400 species included into 16 sec- Molecular markers are useful in identifying the tions containing annual and perennial herb that are maximally diverse parental genotypes through an eval-

* Corresponding author

c 2013 Institute of Botany, Slovak Academy of Sciences Genetic diversity and relationships among Galium 301

Table 1. List of the studied species of Galium sited according to traditional Boissier (1881), more recent traditional (Ehrendorfer & Sch¨onbeck-Temesy 1982; Ehrendorfer et al. 2005) and a recent phylogenetic classification based on molecular data (Soza & Olmstead 2010b).

No. Taxon Voucher Boissier (1881) Ehrendorfer & Soza & Olm- Present study Sch¨onbeck-Temesy stead (2010b) RAPDs + (1982); Ehrendor- ISSRs fer et al. (2005)

1 L. Egypt, Gebel Elba, Gebel Sect. Aparine Sect. Kolgyda Clade III, sub- Group 6 Ekwal, Abdel Khalik et Subsect. Leucaprinea clade A al., s.n. (SHG) 2 Mill. XX-0-ULM-2004-F-13: –Sect.Leiogalium Clade III, sub- Group 1 subsp. pycnotrychum No. 2012/451 (ULM) clade D (Braun) Krendl. 3 Galium album Mill. DE-0-B-2040707: No. 2160 –Sect.Leiogalium Clade III, sub- Group 4 A subsp. album (B) clade D 4 Galium angustifolium Kew Garden, Millennium –Sect.Lophogalium Clade VII Group 4 B subsp. angustifolium seed bank, serial number: 0377463(K) 5 Galium asparagifolium GR-0-B-2713189: No. 2162 –Sect.Leiogalium –Group4B Boiss. and Heldr. (B) 6 Galium canum Req. Palestine, Wadi Sawaanit, Sect. Eugalium Sect. Jubogalium –Group4B in rocks, P.H. Davis 5038 Subsect. Chromogalia (K) 7 Galium circae Krendl GR-0-B-2647080: No. 2163 –Sect.Leiogalium -Group4B (B) 8 L. DE-0-B-1961900:No. 2167 –Sect.Leiogalium –Group4B (B) 9 Galium grande Mc RSABG, EX: 21746, No. –Sect.Baccogalium –Group3 Clatchie 21747 (RSABG) 10 M. Kew Garden, Millennium –Sect.Galium –Group4B Bieb seed bank, serial number: 0243746(K) 11 All. IT -0-B-2612099: No. 2169 –Sect.Leiogalium Clade III, sub- Group 1 (B) clade D 12 (L) All. Netherlands, Gelderland, Sect. Eugalium Sect. Leiogalium Clade III, sub- Group 1 NW of Wolfheze.Open low Subsect. Leiogalia clade D vegetation on sandy soil, C.C.H. Jongkind 5226 (WAG) 13 L. Egypt, in cultivated land, Sect. Aparine Sect. Kolgyda Clade III, sub- Group 4 B near Maruit,Letuneux 197 Subsect. Apera clade B (K) 14 Galium obliquum Vill. Kew Garden, Millennium –Sect.Leptogalium –Group4A seed bank, serial number: 0058584 (K) 15 (L.) DE-0-B-1870409: No. 2172 –Sect.Hylaea Clade III, sub- Group 4 A Scop. (B) clade A 16 Galium parisiense L. ,40kmS.of Sect. Aparine Sect. Kolgyda Clade III, sub- Group 4 A Tiznit on the road to Subsect. Xanthapari- clade B Bou-Izakarm, alt. 1000 nea m, W.J. de Wild and J. Dorgelo 1948 (WAG). 17 Galium scabrifolium GR-0-B-2312983: No. 2176 -Sect.Leiogalium –Group1 (Boiss.) Hausskn (B) 18 Galium schultesii Vest. RO-0-B-0751486: No. 2178 –Sect.Leiogalium –Group4B (B) 19 Galium setaceum Lam. Egypt, Gebel Elba, Wadi Sect. Aparine Sect. Jubogalium –Group5 subsp. setaceum. Yahameeb, N: 22◦ 12 Subsect. Xanthapari- 28,E:36◦ 20 12,Alt. nea 600m, Abdelkhalik 2033 (SHG). 20 Galium sinaicum Egypt, Wadi El Arbaeein, Sect. Eugalium Sect. Jubogalium –Group2 (Delileex Decne) Boiss. Wadi Gragena, St. Ka- Subsect. Chromogalia trein 19.6.2005 Abdel Khalik et al. sn. (SHG) 21 L. Egypt, Sohag city, in gar- Sect. Aparine Sect. Kolgyda –Group4B subsp. spurium. den near Akhmim bridge, Subsect. Leucaprinea Elkordy 1 (SHG). 302 K. Abdel Khalik et al.

Table 1. (continued)

No. Taxon Voucher Boissier (1881) Ehrendorfer & Soza & Olm- Present study Sch¨onbeck-Temesy stead (2010b) RAPDs + (1982); Ehrendor- ISSRs fer et al. (2005)

22 Galium tricornutum XX-0-BONN-23771 Sect. Aparine Sect. Kolgyda Clade III, sub- Group 3 Dandy (BONN) Subsect. Camptopoda clade A 23 Galium uniflorum Kew Garden, Millennium –Sect.Bataparine –Group4B Michx. seed bank, serial number: 0532334(K). 24 Galium verum L. DE-0-BONN-14328 Sect. Eugalium Sect. Galium Clade III, sub- Group 1 (BONN) Subsect. Chromogalia clade D

Table 2. Characteristics of RAPD and ISSR primers sequence and amplification products generated by the studied taxa.

Methods Primer name Sequence 5-3 Number of polymorphic Size of DNA fragments (bp) fragments of DNA

Primer A GGTGCGGGAA 40 300–1000 RAPD Primer B GTTTCGCTCC 57 200–1200 Primer C GTAGACCCGT 61 200–1500

ISSR 13 GAGGAGGAGGC 15 500–1000 ISSR 15 GTGGTGGTGGC 22 300–1000 ISSR ISSR 16 AGAGAGAGAGAGAGT 50 100–1500 ISSR 17 ATATATATATATATATAG 5 200

Total number of polymorphic bands 250 –

uation of genetic diversity which is useful in culti- found by using RAPD and ISSR as the combined results var identification, seed purity analysis and breeding. would be more credible to analyze the genetic structure Among the various molecular markers, Random Am- of Galium species of Egypt and related species. plified Polymorphic DNA (RAPD) and Inter-Simple Se- quence Repeat (ISSR) are simple and quick techniques Material and methods and have become popular as their application does not need any prior information about the target sequences materials in the genome, high-efficiency and sharp sensibility, and The samples of Gallium seeds were taken from wild popu- these techniques have now been widely used for line lations and some herbarium specimens. Voucher specimens identification and genetic diversity. These markers have of the populations studied are deposited in the herbarium of the Department of Botany of Sohag University (SHG) been used for DNA fingerprinting, conservation biol- (Table 1). ogy (Martin & Sanchez-Yelamo 2000; Li et al. 2005), to identify and determine relationships at the species, Plant genomic DNA extraction population and cultivar levels in many (Pezh- Total genomic DNA was extracted from germinated seeds. manmehr et al. 2009; Manica-Cattani et al. 2009; Nan These were first ground into a fine powder in liquid nitro- et al. 2003; Fracaro et al. 2005; Mattioni et al. 2002; gen using a pestle and mortar following the steps of CTAB Zhang & Dai 2010), genetic diversity studies (Qian et al. protocol (Doyle & Doyle 1990; Doyle 1991). 2001; Pradeep et al. 2005; Josiah et al. 2008; Parmaksız ¨ & Ozcan 2011; Xavier et al. 2011; Shen et al. 2012; Random Amplified Polymorphic DNA (RAPD) analysis Aghaabasi & Baghizadeh 2012; Buldewo et al. 2012; RAPD was performed as described by Huang et al. (2000) Abdel Khalik et al. 2012), which utilize the advantages and Abd El-Twab & Zahran (2008). PCR reactions were of the two molecular marker techniques, reduce poten- carried out with several primers using 25 µLvolumePCR tial errors connected with each method and hence im- mixture containing 2.5 µL of buffer (Taq DNA polymerase prove the reliability of results. complete high specificity reaction buffer), 2.5 µLdNTPs Literature review also revealed that the phyloge- (from 10 mM stock, Bioron International, Germany), 12 ng netic (molecular markers, ISSR and RAPD) investiga- primers (Operon Nippon EGT Co. Ltd.), 1 U DFS-Taq DNA polymerase (Bioron International, Germany) and 100 ng of tion of the Galium species have not been documented DNA (Table 2). The thermal cycler (Thermo Hybaid) was until now. We focused on Egyptian species and related operated as follows: 1 cycle at 95 ◦C for 5 min followed by species of this genus to compare and align species with 40 cycles at 95 ◦C, 36 ◦Cand72◦C for 40 sec, 1 min and genetic markers. 2 min respectively; and a final amplification was carried out The present study describes the genetic variability at 72 ◦C for 10 min. Genetic diversity and relationships among Galium 303

Fig. 1. A representative agarose gel where PCR products of RAPDs (A: amplified by RAPD primer) and ISSRs (B: amplified by ISSR 16 primer) markers, respectively.

Inter Simple Sequence Repeats (ISSR) analysis or dissimilarity (DNA electrophoretic patterns contain vis- ISSR procedure was achieved as described by Dogan et ible bands assigned to specific positions in an individual al. (2007). PCR amplification was carried out with sev- lane). Pairwise similarity of the genotypes or genetic phe- eral primers using 100 ng of genomic DNA (Table 2). The notypes represented in the different lanes can be quantified 25 µL PCR mixture contained 2.5 µL of buffer (Taq DNA using indexes or coefficients of similarity. These estimators polymerase complete high specificity reaction buffer (10X) define genetic distances that portray DNA divergence be- containing 500 mM KCl, 100 mM TrisHCl pH 8, 0.1% tween organisms in phenetic and cladistic analyses (Huang 20 and 15 mM MgC12, Bioron International, Germany), et al. 2000). For each primer, the consistent amplified prod- 2.5 µL dNTPs (from 10 mM stock, Bioron International, ucts were recorded. The polymorphic fragments (RAPD and Germany), 12 ng primers (Operon Nippon EGT CO. LTD.) ISSR) were named by the primer code followed by the size 1 U DFS-Taq DNA polymerase (Bioron International, Ger- of the amplified fragment in base pairs. For phylogenetic many), and 100 ng of DNA. The thermal cycler was operated analysis, each amplified band was treated as a unit charac- as follows: 1 cycle at 94 ◦C for 1.5 min; 35 cycles at 9, 40 and ter regardless of its intensity and scored in terms of a binary 72 ◦C for 40, 45 sec and 1.5 min respectively; 1 cycle at 94 ◦C code, based on presence (1) and absence (0) of bands. Only for 45 sec; 1 cycle at 44 ◦C for 45 s and a final amplification clear and reproducible bands were considered for scoring. at 72 ◦Cfor5min. For phylogenetic analysis, all the members of Galium were included. To analyse data obtained from the binary ma- Gel-electrophoretic analysis trices, the NTSYS-pc version 2.1 statistical package (Rohlf Gel electrophoresis following Abd El-Twab & Zahran (2008) 2000) was used. Three datasets were used, viz. RAPD, ISSR, was used to determine the presence/absence of the total ge- and combined datasets of RAPD and ISSR. The statisti- nomic DNA and size of the DNA fragments after RAPD cal method took into account the presence or absence of and ISSR loaded using loading buffer in 1.5% Agarose Gel, each band as differential features. The binary qualitative which carries DNA from negative to positive side. DNA was data matrices were then used to construct similarity matri- stained in gel by ethidium bromide (0.5 µgmL−1), that com- ces based on Jaccard similarity coefficients (Jaccard 1908). bines with DNA fragments and gives violet light under UV The similarity matrices were then used to construct dendro- light, at that time; photographs were taken using a digital grams using unweighted pair group method with arithmetic system (Past software) (Fig. 1). average (UPGMA). To compare RAPD- and ISSR-based dendrograms, Data analysis cophenetic matrices were derived from the dendrograms us- RAPD and ISSR markers produce DNA amplification sig- ing the COPH (cophenetic values) program, and the good- nals that can be converted into measurements of similarity ness of fit of the clustering to the 2 data matrices was 304 K. Abdel Khalik et al.

Fig. 2. UPGMA phenogram showing genetic diversity of 24 taxa of Galium based on RAPDs characters. calculated by comparing the original similarity matrices nutum, G. aparine and G. lucidum with about 0.63 with the cophenetic value matrices using the Mantel ma- similarity. (4) A cluster with Galium album, G. mol- trix correspondence test (Mantel 1967) in the MXCOMP lugo and G. verum with 0.66 genetic similarities. (5) program. Principal co-ordinate analysis (PCOORDA) was A cluster comprises Galium spurium, G. obliquum and performed based on the similarity coefficients. Lastly, a com- G. scabrifolium showing about 0.73 genetic similarity. bined dataset was prepared using both RAPD and ISSR data and used to calculate the combined similarity matrix, (6) A cluster which is divided into two subgroups. (a) which was ultimately used to construct the phylogenetic tree A subgroup includes cluster of G. murale and G. odor- and PCOORDA. The combined phylogenetic tree was com- atum with about 0.83 similarity. (b) A subgroup which pared with RAPD- and ISSR-based trees using the Mantel can divided into two subgroups. (I) A subgroup con- matrix correspondence test. tains Galium grande, G. sinaicum and G. angustifolium subsp. angustifolium with about 0.80 similarity. (II) A Results subgroup comprises Galium parisiense, G. uniflorum, G. humifusum, G. canum,G. asparagifolium, G. circae RAPD analysis and G. glaucum with about 0.78 genetic similarity. Twenty six primers were used for the RAPD analysis to investigate the pattern of genetic variation among ISSR analysis 24 species of the genus Galium growing wild in Egypt In total 18 primers for the ISSR were used to investi- and related species. Among the primers tested only gate the pattern of genetic variation among 24 species three revealed a polymorphism (Table 2). Each of these of the genus Galium growing wild in Egypt and related primers was tested on all samples studied and were se- species. Among the primers tested only four (Table 2) lected for genotype analysis because their patterns were produced clear bands and had reproducibility, so they reproducible and stable. Polymorphic bands were se- were selected for further analysis. A total number of lected for identifying the genetic similarity for the group 92 reproducible polymorphic bands were resulted after of species. One hundred and fifty eight reproducible 4 ISSR primers; those bands were used for studying polymorphic bands were produced after 3 RAPD-PCR the genetic similarity among the species. The average primers. The average similarity coefficient ranged from similarity coefficient was ranged from 0.59 to 0.95. The 0.49 to 1.00. The highest number of polymorphic am- results showed that primer ISSR 17 was monomorphic plification DNA fragments obtained per primer C was and the rest of the primers polymorphic. The highest 61 bands with size ranged from 200 to 1500 bp (Ta- number of polymorphic amplification DNA fragments ble 2). Relations between the studied taxa are pre- obtained per primer ISSR 16 was 50 bands with size sented in a dendrogram built on the basis of similar- ranged from 100 to 1500 bp, while the lowest number of ity coefficients. For ease of comparison, the 158 bands 5 bands were generated with primer ISSR 17 (Table 2). were taken together and the number of bands from The results of the consensus tree from ISSR data indi- each size of DNA fragments (bp) was scored for ev- cated that tree was divided into 7 main branches and ery species. Six main branches and clusters with about clusters with 0. 77 similarity (Fig. 3). (1) A branch in- 0.61 similarity were obtained (Fig. 2). (1) A branch in- cludes Galium aparine. (2) A branch comprises Galium cludes Galium setaceum. (2) A branch comprises Gal- setaceum. (3) A branch contains Galium schultesii.(4) ium schultesii. (3) A cluster contains Galium tricor- AbranchwithGalium spurium. (5) A branch includes Genetic diversity and relationships among Galium 305

Fig. 3. UPGMA phenogram showing genetic diversity of 24 taxa of Galium based on ISSRs characters.

Fig. 4. UPGMA phenogram showing genetic diversity of 24 taxa of Galium based on combination of RAPDs and ISSRs characters.

Galium parisiense. (6) A major cluster includes Gal- Combined RAPD and ISSR analysis ium tricornutum, Galium album subsp. pycnotrychum, The UPGMA dendrogram obtained from the cluster G. grande, G. uniflorum, G. cannum, G. humifusum analysis of RAPD and ISSR combined data gave near and G. murale with about 0.80 similarity. (7) A major similar clustering pattern, with Jaccard’s similarity co- cluster divided into 2 sub-clusters with about 0.82 sim- efficient ranging from 0.58 to 0.95. The results indicated ilarity: first sub-cluster with Galium sinaicum, G. an- that the consensus tree was divided into 6 major clus- gustifolium subsp. angustifolium, G. odoratum, G. as- ters and branches with 0.66 similarity and confirmed paragifolium, G. circae and G. glaucum with about 0.86 by PCOA (Figs 4, 5). The groups corresponding with genetic similarity; second sub-cluster includes Galium species were clearly defined by the first and second prin- mollugo, G. verum, G. obliquum, G. lucidum, G. scabri- cipal coordinates which represented 16% and 11% of folium and G. album subsp. album with 0.83 similar- total variation, respectively. (1) A branch includes Gal- ity. ium aparine. (2) A branch comprises Galium setaceum. 306 K. Abdel Khalik et al.

Fig. 5. Principal Coordinates Analysis of RAPDs and ISSRs combined constructed using 250 variable DNA bands from 24 species of Galium.

(3) A branch contains Galium sinaicum.(4)Acluster dendrograms generated on the basis of similarity matri- with Galium grande and G. tricornutum.(5)Acluster ces. A highly significant correlation between these two includes Galium mollugo, G. verum, G. album subsp. dendrograms suggested that both markers were equally pycnotrychum, G. lucidum and G. scabrifolium. (6) A efficient for assessing phylogenetic relationships among major cluster divided into 2 sub-clusters with about the investigated taxa. Moreover, the genotype distri- 0.70 similarity: first sub-cluster with Galium odoratum, bution on the consensus tree based on the combined G. album subsp. album, G. obliquum and G. parisiense banding patterns of RAPD and ISSR may significantly with about 0.75 genetic similarity; second sub-cluster differ because it is possible that each technique amplify includes Galium uniflorum, G. spurium, G. humifusum, different parts of the genome. The RAPD markers cover G. canum, G. murale, G. angustifolium subsp. angusti- the whole genome for amplification, ISSR amplify the folium, G. circae, G. schultesii, G. asparagifolium and region between two micro satellites. Hence, the poly- G. glaucum with 0.71 similarities. morphisms reflect the diversity of these regions of the genome. It is therefore better to use the combination Discussion of banding patterns of the two techniques in order to use more segments sites of the genome that will increase Morphological characters in plants may be affected by the validity of the consensus tree. In general, our results environmental conditions. Thus, the use of morpholog- obtained from the RAPD and ISSR analyses, suggested ical characters for classification may result in discrep- groups and partially confirmed the sectional classifica- ancies. Productivity of a molecular marker technique tion of Galium by the most recent traditional (Ehren- depends on the amount of polymorphism it can de- dorfer & Sch¨onbeck-Temesy 1982; Ehrendorfer et al. tect among the set of accessions under investigation. 2005) and a recent phylogenetic classification based on RAPD and ISSR markers have been used in many stud- molecular data (Soza & Olmstead 2010b). ies for DNA fingerprinting and phylogenetic analyses. Galvan et al. (2003) concluded that ISSR would be a Sections Leiogalium, Galium and Lophogalium (groups better tool than RAPD for phylogenetic studies. The 1 and 4 B) present study, however, has demonstrated that both According to the combined RAPD and ISSR tree RAPD and ISSR technique along with suitable statis- (group 1) there is a close relationship between four tical tools could be successfully applied to assess the species of section Leiogalium (G. mollugo, G. album genetic diversity and phylogenetic analysis in Galium. subsp. pycnotrychum, G. lucidum and G. scabrifolium) Although RAPD and ISSR markers showed consider- and one species (G. verum) of section Galium with 0.70 able differences in detecting polymorphism and discrim- genetic similarities. These species are morphologically inating capacity, they showed nearly similar topology in similar in the structure by having consistently Genetic diversity and relationships among Galium 307 whorled leaves with six or more organs. Kliphuis (1986) of the clade represented by perennial sections Galium, presented a cytological and morphological analysis of Leiogalium, Leptogalium, Hylaea and the annual Kol- 19 species of Galium from the Balkans, and he classi- gyda. Mitova et al. (2002) have found iridoid esters fied the species of sectionLeiogalium into four groups with p-hydroxyphenylpropoionic acid, loganin 13 and and counted Galium mollugo (2n = 22), G. album 6-acetylscandoside 8 which are found also in G. verum (2n = 44), G. lucidum (2n = 44) and G. scabrifolium and members of section Leiogalium. These results re- (2n = 22). Also, he concluded that G. mollugo and flect the close relationship between G. humifusum with G. album constituted a polyploid complex. Moreover, members of section Leiogalium. Since our results are in- Manen et al. (1994) investigated phylogenetic analy- herited data, and we suggest that (1) species of section sis of 25 species of the tribe Rubieae using sequence Leiogalium form a polyphyletic group, and (2) there data from the atpB-rbcL intergene region. They showed are close relationships between members of this section very little change in the intergene cpDNA region in the with G. verum and G. humifusum (sect. Galium)and perennials studied from section Galium (G. verum,2x, G. angustifolium (sect. Lophogalium). Therefore, these 4x, Eurasian) and section Leiogalium which is a poly- data agree with those of Manen et al. (1994), Natali et morphic Mediterranean-European polyploidy complex al. (1995), Mitova et al. (2002) and Soza & Olmstead with [G. mollugo (2x), G. album (4x), G. lucidum (4x), (2010b). and G. corrudifolium (2x)]. Natali et al. (1995) pre- sented phylogenetic analysis of 39 species of the tribe Section Jubogalium (groups 2, 4Band5) Rubieae using sequence data from the atpB-rbcL in- Concerning the clades of G. canum, G. setaceum and tergene region. They showed that the section Leiogal- G. sinaicum, our results do not support the mono- ium including G. mollugo, G. album, G. corrudifolium phyly of the artificial section Jubogalium. This is due and G. lucidum was closely related to the section Gal- to the placement of Galium sinaicum, G. canum and ium (with G. verum) and exhibited practically no se- G. setacum within three separate clades with 0.84 ge- quence differences. Furthermore, Mitova et al. (2002) netic similarities. Boissier (1881) classified these species presented iridoid patterns in 19 species of Galium.They into two sections on the basis of annual or perennial treated G. mollugo and G. album as one group and in- habit, flowers hermaphrodite or polygamous, and pe- cluded secogalioside 18 that considered it as an impor- duncle erect or recurved: sect. Aparine and sect. Eugal- tant chemotaxonomic marker. Also, they showed the ium. He placed G. setaceum in the former, with annual affinity of G. verum to the groups of G. mollugo and habit, flower hermaphrodite or polygamous and pedun- G. album by presence of loganin 13 and 6 acetylscan- cle erect or recurved, while G. canum and G. sinaicum doside 8. Likewise, Soza & Olmstead (2010b) studied were placed in the second section on the basis of peren- phylogenetically 126 old and new world taxa of Ru- nial habit, flower hermaphrodite and erects peduncle. bieae using sequence data from three chloroplast re- Furthermore, Ehrendorfer et al. (1976) and Ehrendorfer gions. They indicated seven major clades and identi- &Sch¨onbeck-Temesy (1982) placed those three species fied one clade (Clade D) comprising members of section in a separate section (Jubogalium). Abdel Khalik et Galium (G. verum) and sect. Leiogalium (G. mollugo, al. (2007) studied the pollen morphology of Galium G. album, and G. lucidum). in Egypt and indicated that G. canum has 5–7 colpi, Within the subgroup 4B, species of section Leio- G. sinaicum 5–6 colpi and G. setaceum 6–7 colpi. Fur- galium (G. glaucum, G. asparagifolium, G. schultesii thermore, Abdel Khalik et al. (2008b) investigated the and G. circae) and one species of section Lophogalium fruit and seed morphology of 13 Egyptian species of (G. angustifolium) have been recognized as a distinct Galium and showed that mericarp surface is micropapil- sub-group with 0.84 genetic similarities. These species late inG. sinaicum, with hooked or depressed hairs can be clearly defined on the basis of various features: in G. setaceum and with long white straight hairs in perennials, forming rhizomes with sexual and vegeta- G. canum. Moreover, Abdel Khalik et al. (2008c) in- tive reproduction, polyploids, and with narrow leaves, vestigated 50 morphological characters, including veg- mesoxerophylous plants. Ančev & Krendl (2011) inves- etative parts, flowers, fruits, seeds, pollen grains, and tigated the 18 species of section Leiogalium in anatomical structure by means of numerical analysis, of based on morphology and chromosome numbers and 13 taxa belonging to genus Galium from Egypt. They considered G. glaucum (4x = 44), G. asparagifolium concluded that species of section Jubogalium are het- (4x = 44) and G. schultesii (6x = 66) as polyploids. erogeneous. Our results disagree with those of Boissier Within section Leiogalium reticulate relationships and (1881), Ehrendorfer et al. (1976) and Ehrendorfer & affinities linked to hybridization, polyploidy, and ac- Sch¨onbeck-Temesy (1982), and agree with Abdel Kha- tive recent evolutionary differentiation are obvious. On lik et al. (2007, 2008a, b, c). the other hand, G. angustifolium (sect. Lophogalium)is characterized as a perennial herb, bearing linear leaves Section Kolgyda (groups 3, 4,and6) with apically directed, short hairs along the margins Within Kolgyda group, three major clades were iden- and polyploid (Ehrendorfer 1956; Dempster 1993). tified (Fig. 4). The first clade includes Galium tri- Additionally, Galium humifusum (sect. Galium) cornutum; the second clade includes G. aparine and separated with above members with 0.77 genetic sim- the third clade contains G. parisiense, G. murale and ilarities. Natali et al. (1995) suggested the monophyly G. supurum with 0.70 genetic similarities. Boissier 308 K. Abdel Khalik et al.

(1881) treated G. aparine, G. tricornutum, G. spurium, ilarities. Natali et al. (1995) recommended a close rela- G. murale and G. parisiense as members of section tionship between G. odoratum and G. corsicum (sect. Aparine, and classified them in different subsections. Leptogalium). These species can be defined on the ba- However, Ehrendorfer et al. (1976) and Ehrendorfer sis of perennial habit. Soza & Olmstead (2010b) iden- and Sch¨onbeck-Temesy (1982) considered these taxa as tified Clade III including G. odoratum (sect. Haylaea, good members in section Kolgyda (synonym of sect. sub-clade A) and species of both section Leptogalium Aparine). Ehrendorfer (1971) considered the autoga- (sub-clade B). Our results suggest close relationships mous G. aparine complex as probably originating by between species of both sections and agree with those allopolyploidy from three racial stocks of Southwest of Natali et al. (1995) and Soza & Olmstead (2010). Asian origin. The species seems to include tetraploid, hexaploid and octaploid cytotypes (2n = 42, 44, 48, Section Bataparine (group 4B) 62, 66 and 68). Hanf (1983) showed that variants of This section comprises only G. uniflorum. It has been G. spurium with setose fruits, besides the flower char- recognized as a distinct clade with 0.77 genetic similar- acters and the diploid chromosome number are often ities. This species can be clearly defined on the basis of not easy to distinguish from G. aparine. Also, Kli- various features: perennial, hermaphroditic, glabrous, phuis (1986) investigated the section Kolgyda and in- fleshy fruits to hooked-hairy. Soza & Olmstead (2010b) cluding Galium aparine (2n = 42, 44, 48, 64, 66) and settled that section Bataparine is a paraphyletic grade. G. parisiense (2n = 22, 44, 66), and concluded that Our results show that G. uniflorum belongs to the clade both species constituted a polyploid complex. Natali et includes species from different sections as sister to this al. (1995, 1996) exhibited that G. aparine, G. spurium clade. More data are necessary for deciding this assump- and G. tricornutum were joined together in the same tion that the section Bataparine is paraphyletic or not. clade. In conclusion, the present study demonstrated that With Egyptian material, Abdel Khalik et al. (2007) RAPD, ISSR and cost-effective markers such as RAPD indicated that G. aparine has 7–9 colpi, G. spurium 6–8 and ISSR together with good statistical tools can be colpi, G. murale 6–7 colpi, G. parisiense 8–10 colpi, and successfully applied to study phylogenetic relationships G. tricornutum 8–9 colpi. Moreover, Abdel Khalik et al. at the intraspecific level in Galium. Additionally, the (2008c) settled that results of both cluster and PCO- large number of polymorphic bands obtained in the ORDA analyses confirmed the group of G. aparine, present study signifies the power of RAPD and ISSR G. tricornutum, G. ceratopodum and G. spurium as markers in fingerprinting and diversity analyses. Six a well-distinguished group and showed that G. aparine clades can be recognized within Galium,whichmostly and G. tricornutum form a subgroup, and another sub- corroborate, but also partly contradict, traditional tax- group includes G. ceratopodium and G. spurium.Sim- onomic treatments. A remarkable result from this study ilarly, Soza & Olmstead (2010b) identified one clade was to identify a close relationship between members (Clade III) comprising members of section Kolgyda of section Leiogalium and with G. verum and G. hu- with other sections, but under this clade (III) distin- mifusum (sect. Galium)andG. angustifolium (sect. guished two subclades (A and B); subclade A included Lophogalium). Further support comes from the molec- G. aparine and G. tricornutum and subclade B com- ular data of RAPD and ISSR which indicate that the prised G. murale and G. parisiense; unfortunately, they more apomorphic groups of Galium form the section did not included G. spurium in their study. In general, Leiogalium clade including the perennial sections Gal- our results mainly do not support the taxonomic sys- ium, Lophogalium, Jubogalium, Hylaea and Leptogal- tem of the section Kolgyda proposed by Boissier (1881), ium as well as the annual section Kolgyda. However, we Ehrendorfer et al. (1976), Ehrendorfer & Sch¨onbeck- believe that molecular and morphological approaches Temesy (1982), Natali et al. (1995, 1996) and Abdel should be combined in order to arrive at a broadly ac- Khalik et al. (2008c), but partially agree with Soza & cepted phylogenetic reconstruction of the genus Gal- Olmstead (2010b) in the classification of the species of ium. Moreover, comprehensive study covering further this section into two sub-clades. species from different sections of Galium would be nec- essary to make a more thorough classification. Section Baccogalium (group 3) This section includes only G. grande. It has been rec- ognized as a distinct clade with 0.67 genetic similari- Acknowledgements ties and can be clearly defined on the basis of various features: perennial, fleshy-fruited, dioecious and polyg- We are grateful to the Director and Curator of Kew herbar- amous species. Soza and Olmstead (2010a) studied the ium (K), Berlin herbarium (B), Bonn botanical garden’s fruit morphology and evolution of Galium and they con- herbarium (BONN), botanical gardens of the University of cluded that section Baccogalium is a monophyletic. Our Ulm (ULM), Rancho Santa Ana Botanic Garden’s herbar- ium, USA (RSABG) and Wageningen University herbar- results agree with those authors. ium (WAG). We are indebted to Prof. Dr. Ana Ortega Olivencia, Area of Botany, Faculty of Science, University of Sections Hylaea and Leptogalium (group 4 A) Extremadura, Badajoz, for going through the man- This group includes G. odoratum (sect. Hylaea) and uscript and making valuable suggestions. Special thanks G. obliquum (sect. Leptogalium) with 0.78 genetic sim- to Dr. Valerie Soza, Department of Biology, University of Genetic diversity and relationships among Galium 309

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Received July 17, 2013 Accepted October 30, 2013