Phenetic and cladistic analyses of Boraginaceae Juss.

Abstract

Background

The systematics of family Boraginaceae draw attention of many botanists for many years. The current study's primary goals are to clarify phenetic and phylogenetic relationships within Boraginaceae according to morphology and molecular characteristics and to evaluate the morphological characters that can be applied in systematics of Boraginaceae.

Results

The macromorphological characters of 39 species, 2 subspecies and 5 varieties of wild boraginaceous plants were extracted and subjected to phenetic and principal component analysis that was performed for detecting the most important characters differentiating the studied taxa. The generated dendrogram is divided into five clear groups; Arnebia decumbens var. macrocalyx and Heliotropium curassavicum are the most distantly related species, while Echium angustifolium subsp. angustifolium and E. angustifolium subsp. sericeum are the most closely related species. The phylogenetic relations among the examined taxa were determined using DNA barcoding of the rbcl gene. The phylogenetic analysis generated a cladogram showing that among the studied taxa of Boraginaceae there is a bolster for three clear lineages with resolved relationships.

Conclusions

It is concluded that the chosen morphological characters were important in species delimitation, where more than half of the total morphological variations (67.94%) were explained by the first two principal components, indicating that the morphological characters showed high variability, which is useful for discrimination, and these characters, in addition to molecular characters, shared in drawing the phenetic and phylogenetic relationships within Boraginaceae that were considered not monophyletic groups. Boraginaceae contained some monophyletic genera such as Heliotropium and Alkanna, while the other studied taxa expressed a non-monophyletic relationships.

1 Background

Boraginaceae Juss. includes around 1600 to 1700 species in 90 genera [1] and is widely widespread in tropical (Northern and Central South America), subtropical, and temperate (Irano-Turanian and Mediterranean) regions of the world [2, 3]. This family is represented in the wild Egyptian flora by 15 genera [4] viz. Adelocaryum, Alkanna, Anchusa, Arnebia, Cordia, Echiochilon, Echium, Heliotropium, Lappula (= Echinosperma), Lithospermum, (= Moltiopsis), Myosotis, Nonnea, Onosma (= Podonosma), Paracaryum and Trichodesma. These genera include 44 species and 5 varieties. The largest genus is Heliotropium (11 species), Echium (7 species), and Anchusa (6 species). On the other hand, the genera Adelocaryum, Echiochilon, Myosotis, Nonnea and Podonosma are represented in Egypt by one species. [5] transferred Cordia species and Coldenia to the family Ehretiaceae and combined four additional genera: Asperugo, Eritrichium, Gastrocotyle and Hormuzakia, which include 52 species and 9 varieties.

Many botanists were interested in the systematic categorization of the family for many years, viz. [6,7,8,9,10].

The infrafamilial classification of Boraginaceae was traditionally divided into five subfamilies: Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae and Wellstedioideae. [11,12,13,14,15,16,17,18,19] accepted this subfamilial treatment although other scientists not. [20,21,22] moved Cordioideae, Heliotropioideae and Ehretioideae to Heliotropiaceae based on embryological criteria, while [17, 23,24,25] treated Wellstedioideae at familial level as Wellstediaceae. Conversely, Hoplestigmataceae, Hydrophyllaceae and Lennoaceae were widely recognized as different families. [3] recognize eight subfamilies, viz. Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae, Hydrophylloideae, Lennooideae, Namoideae and Wellstedioideae.

Boraginaceae comprises about 13 tribes divided into eight subtribes [26]. [27] recognizes six tribes Boragineae, Cynoglosseae, Eritrichieae, Lithospermeae, Myosotideae and Trigonotideae but molecular criteria of [10] nest Eritrichieae, Myosotideae and Trigonotideae within Cynoglosseae s. l. so support four tribes based on both molecular characteristics and morphology [28, 29] including Boragineae, Cynoglosseae, Echiochileae and Lithospermeae. Cynoglosseae s. l. is the largest and morphologically complex tribe that contains more than half of the family's species.

Boraginaceae is regarded as monophyletic due to morphological, molecular and phytochemical traits [30,31,32,33,34]. Other phylogenetic studies demonstrate that Boraginaceae traditionally is paraphyletic with regard to Hoplestigmataceae, Hydrophyllaceae and Lennoaceae [9, 10, 30, 32, 35, 36].

Multiple phylogenetic analyses on Boraginaceae are centered on connections inside a genus or among genera that are closely related [37, 38], although other studies carried on tribal level [28, 29, 35, 39] that resolve the interrelationship among tribes but the relationships inside each tribe still largely unsettled [37].

The main objectives of the present study were to clarify phenetic and phylogenetic relationships within Boraginaceae according to morphology and molecular characters and to evaluate the morphological characters that can be used in systematics of Boraginaceae.

2 Methods

2.1 Plant material

The current study was carried out on 46 taxa (39 species, 2 subspecies, 5 varieties) belonging to 14 genera (Table 1) representing more than 93% of the Boraginaceae in the flora of Egypt according to [4].

Table 1 Voucher specimens of Boraginaceae, their numbers that were kept in the public herbarium of Ain Shams University and their accession numbers in GenBank (46 taxa, 14 genera)

2.2 Macromorphological characters investigation

The easily observable character states of (42) morphological characters are summarized in Table 2. These characters were investigated from herbarium specimens deposited at the Herbaria of Ain Shams University, Faculty of Science (CAIA), Cairo University, Faculty of Science (CAI), Flora and Phytotaxonomy Research Department (CAIM) and Orman Botanical Garden, Giza. Published descriptions also were consulted [40]. The identification and nomenclature were authenticated using [5, 41] and International Plant Name Index [42].

Table 2 The extracted morphological characters (42), their states (107) and codes of the studied taxa

2.3 Extraction of DNA and amplification of rbcL primers

In an Eppendorf tube, liquid nitrogen was used to crush 100 mg of leaves into a powder, and then, DNA was extracted with the aid of CTAB (cetyltrimethylammonium bromide) protocol of Doyle and Doyle (1987). The rbcL region of the purified DNA was amplified with the aid of PCR with the following universal primers:

Forward primer: 5′-ATG TCA ACA CAA ACA GAG ACT AAA GC-3′;

Reverse primer: 5′-GAA ACG GTC TAT CCA ACG CAT-3′.

The reactions of the amplification were performed in 25 μL as follows: 5 × GoTaq® Flexi buffer 5 μL, MgCl2 (25 mM) 2.5 μL, dNTPs (10 mM each) 0.5 μL, forward primer (10 μM) 1.2 μL, reverse primer (10 μM) 1.2 μL, Go Taq™ (5 U/μL) 5 μL, DNA stock 2 μL, H₂O 7.6 μL up to make 25 μL total volume. The following were the reaction conditions: initial denaturation at 95 °C for 5 min, 40 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s and 72 °C for 10 min. The purification kit of the PCR product (Thermo PCR Purification Kit, USA) was used to separate all positive PCR amplicons from other unwanted materials such as dimers, RNA, free nucleotides, and unamplified DNA fragments. It is a necessary step prior to the automated DNA sequencing. Macrogen Korea, 6F, 172, Dolma-ro, Bundang-gu, Seongnam-si, Gyeonggi-do (Jeongja-dong, Seoul National University Bundang Hospital Healthcare Innovation Park) received the purified DNA for sequencing.

2.4 Phenetic analysis

Character states (107) by taxon (46) matrix (Additional file 1: Appendix A) were subjected to phenetic analysis by use of PAleontological STatistics version 3.23 [43]. PCA (principal component analysis) ordination and similarity matrix were created using the same software, based on the investigated morphological characters of the studied taxa.

2.5 Phylogenetic analysis

Phylogenetic analyses that are based on maximum parsimony were performed on the produced data matrix using MrBayes 3.2 [44] with Markov chain Monte Carlo simulation. The sample and print frequency is 500, the diagnostic frequency is 5000 and the run length is 1,000,000. Vahlia digyna (Vahliaceae) was used as an outgroup for rooting the cladogram.

3 Results

The phenetic analysis of the coded data matrix of the investigated morphological character states generated a dendrogram (Fig. 1) that is divided into five clear groups: The first one consists of Alkanna strigosa, Arnebia tinctoria, Echiochilon fruticosum, Echium horridum, Lappula spinocarpos, Moltkiopsis ciliata and Nonea vivianii at 0.6 taxonomic distance. The second group comprises Alkanna orientalis, A. tinctoria, Anchusa aegyptiaca, A. hispida, A. humilis, A. milleri, A. undulata, Arnebia decumbens var. decumbens, A. decumbens var. macrocalyx, A. hispidissima, A. linearifolia, Asperugo procumbens, Buglossoides incrassata, B. tenuiflora, Paracaryum intermedium and P. rugulosum at about 0.675 similarity index. The third group includes Echium angustifolium subsp. Anugstifolium, E. angustifolium subsp. sericeum, E. rauwolfii, E. rubrum and E. sabulicolum at taxonomic distance (0.675). All the studied taxa of genus Heliotropium are nested in the fourth group at about 0.45 taxonomic distance. The last group comprises Trichodesma africanum and T. ehrenbergii at about 0.375 similarity index. Coldenia procumbens and Lappula sinaica are separated as distinct identities at taxonomic distances 0.375 and 0.525, respectively.

Fig. 1

UPGMA clustering of the studied boraginaceous taxa based on (42) morphological characters

PCA ordination and matrix of similarity that based on the investigated morphological characteristics of the studied taxa are presented in Fig. 2 and Additional file 1: Appendix B. Among the investigated taxa, the most distant and the closest species are determined. Arnebia decumbens var. macrocalyx and Heliotropium curassavicum are the species that are the most distantly linked (percentage dissimilarity: 13.49074), while Echium angustifolium subsp. angustifolium and E. angustifolium subsp. sericeum are the species that are most closely linked (percentage dissimilarity: 1.7320508). The outline of the analysis indicated that contributions for the first two principal components to total variation of 42 characters were (42.79%) and (25.15%) eigenvalues, respectively. The biological meaning of the components was analogized by the correlation between the component and character (Table 3). The first component is positively correlated with inflorescence type (0.29), petal color (0.23), stigma length (0.24) and ovary texture (0.23), and negatively with upper leaves petioles (−0.29), bracteoles (−0.29), style origin (−0.28) and stigma shape (−0.32). The second component is positively correlated with petal texture (0.29), petal fusion (0.26), style texture (0.24), style position (0.36) and nectar disk (0.23) and negatively with anthers level (−0.36), filament texture (−0.24) and style shape (−0.28).

Fig. 2

Principal component analysis of the studied boraginaceous taxa based on (42) morphological characters

Table 3 PCA variable loadings of a two-dimensions, eigenvalues, contributions and scores of the components for (42) morphological characters of the studied taxa of Boraginaceae

The sequences were submitted to GenBank and assigned accession numbers from OP933830 to OP933875. The phylogenetic analysis generated a cladogram (Fig. 3) showing that among the studied taxa of Boraginaceae there is a bolster for three clear lineages with resolved relationships viz. Heliotropium lineage that included all studied taxa of genus Heliotropium, lineage II (Anchusa undulata, Arnebia hispidissima, A. tinctoria, Echium angustifolium subsp. angustifolium, E. angustifolium subsp. sericeum, E. rubrum) and lineage III (Alkanna orientalis, A. strigosa, A. tinctoria, Anchusa aegyptiaca, A. hispida, A. humilis, A. milleri, Asperugo procumbens, Buglossoides tenuiflora, Lappula sinaica, Paracaryum intermedium, P. rugulosum, Trichodesma africanum and T. ehrenbergii).

Fig. 3

Neighbor joining phylogenetic tree of the studied boraginaceous taxa based on chloroplast rbcL sequence

4 Discussion

From phenetic point of view as revealed in the produced phenogram, the studied taxa Alkanna strigosa, Arnebia tinctoria, Echiochilon fruticosum, Echium horridum, Lappula spinocarpos, Moltkiopsis ciliata and Nonea vivianii are grouped together in a single phenetic group. This is in accord with [28] where Alkanna, Arnebia, Echiochilon and Echium are included in the same tribe Lithospermeae. [45] agree with this but exclude Echiochilon in tribe Echiochileae and [15, 26, 38, 46] treated Moltkiopsis in tribe Lithospermeae along with Alkanna, Arnebia and Echium, while [27, 28] put it under tribe Trigonotideae. On the other hand, [10] put Nonea and Lappula in two distinct tribes: Boragineae and Cynoglosseae, respectively.

Alkanna orientalis, A. tinctoria, Anchusa aegyptiaca, A. hispida, A. humilis, A. milleri, A. undulata, Arnebia decumbens var. decumbens, A. decumbens var. macrocalyx, A. hispidissima, A. linearifolia, Asperugo procumbens, Buglossoides incrassata, B. tenuiflora, Paracaryum intermedium and P. rugulosum are grouped together in an exclusive group. This is consistent with the positioning of Alkanna, Arnebia and Buglossoides in the same tribe Lithospermeae [10, 27, 28, 45]. Taxonomic systems, viz. [10, 15, 27, 28, 46, 47], distribute Asperugo, Anchusa and Paracaryum in tribes Eritrichieae, Boragineae and Cynoglosseae, respectively.

Echium angustifolium subsp. Anugstifolium, E. angustifolium subsp. sericeum, E. rauwolfii, E. rubrum and E. sabulicolum are clustered together. This is consistent with the positioning of Echium in tribe Echieae according to [15, 26, 48] and in tribe Lithospermeae according to [10, 27, 28, 47].

The clustering of all the studied taxa of genus Heliotropium in a single group is confirmed by [26] classification system in placing all the taxa of Heliotropium at the same tribe Heliotropieae also [46] in placing them at the same subfamily Heliotropioideae.

T. africanum and T. ehrenbergii are grouped together in a single phenetic group. This is in accord with [10, 15, 28, 47, 48] where the present genera were included in the same tribe Cynoglosseae. [26, 27] placed it under tribe Trichodesmeae but [49] put it in subtribe Rindereae under the tribe Boragineae. Coldenia procumbens is separated as a distinct identity, and this is in accord with [46] in placing it under subfamily Cordioideae.

PCA can be useful in providing information on character variability [50]. The cumulative variance values of the main components obtained reveal the investigated features in boraginaceous taxa, because of their large variance value that can be useful in explaining discrepancies among taxa. Furthermore, among the examined specimens, the morphological features were chosen for PCA to assess the qualities that are relevant in description change.

PC1 explained 42.79% of total morphological variation which was positively and negatively determined by some floral characters, while PC2 explained 25.15% of total morphological variability that related to floral characters as the same as PC1; accordingly, more than half of total information (67.94%) could be explained by the first two principal components. This indicates that the component was determined by flower variables. So, the results indicate that floral structure showed variability, which is useful for discrimination. In this regard, [51] indicates that the morphological variability in Boraginaceae is explained to greater degree by floral variables.

From phylogenetic point of view, the produced cladogram showed that Boraginaceae are not monophyletic group contrary to some previous studies based on the data of morphology, phytochemistry and molecular structure that indicate the monophyly of Boraginaceae within its specific boundaries [31, 33, 34, 52]. Heliotropium lineage included all exemplars of genus Heliotropium confirming that Heliotropium is a monophyletic group. Tribe Heliotropieae is now typically recognized as subfamily Heliotropioideae [2, 28, 52].

Some exemplars of Anchusa, Arneba and Echium were grouped together in lineage II, and this is in accordance with placement of Arneba and Echium in tribe Lithospermeae according to [10, 27, 28, 46, 47], while [53] placed Arneba and Echium in one tribe Boraginoideae. Some taxonomic systems, viz. [10, 15, 26,27,28, 46,47,48, 53], put Anchusa in tribe Boragineae. Previous phylogenetic studies found a sister relationship between Boragineae and Lithospermeae [10, 39].

Alkanna orientalis, A. strigosa, A. tinctoria, Anchusa aegyptiaca, A. hispida, A. humilis, A. milleri, Asperugo procumbens, Buglossoides tenuiflora, Lappula sinaica, Paracaryum intermedium, P. rugulosum, Trichodesma africanum and T. ehrenbergii were grouped together. This is in accord with placing Alkanna and Anchusa in the same tribe Boragineae [10, 15, 26,27,28, 46,47,48, 53] keep placing Anchusa in tribe Boragineae but place Alkanna in tribe Lithospermeae. Taxonomic systems viz. [10, 15, 27, 28, 46, 47] placed Asperugo in tribe Eritrichieae. [10, 48, 54] put it in tribe Cynoglosseae. [26] put it in tribe Asperugeae. Phylogenetic studies found that Cynoglosseae is closest relative to Boragineae and Lithospermeae [10, 39]. Lappula and Trichodesma were included in the same tribe Cynoglosseae. [15, 47] also placed Trichodesma and Paracaryum in the same tribe Cynoglosseae.

5 Conclusion

It is concluded that the chosen morphological characters were important in species delimitation, where more than half of total morphological variations (67.94%) were explained by the first two principal components, indicating that the morphological characters showed high variability, which is useful for discrimination, and these characters shared in drawing the phenetic relationships within Boraginaceae. In addition, the phylogenetic relationships clarified that Boraginaceae is not a monophyletic group, but it contained some monophyletic genera such as Heliotropium and Alkanna, while the other studied taxa expressed non-monophyletic relationships.