Phylogenetic Relationships of Dianthus (Caryophyllaceae) Species Found in South Africa

Abstract

This study addresses the phylogenetic relationships within the genus Dianthus in South Africa, employing a molecular analysis approach and the distribution pattern of the representatives of the genus. A robust phylogenetic tree comprising three plastids (trnH-psbA, trnK-matK, and trnK-psbA) and a nuclear region was assembled based on 94 taxa of Dianthus and Petrorhagia species using Mr Bayes analysis. Furthermore, distribution maps for each Dianthus species were generated using QGIS. Our results have revealed Dianthus as monophyletic when Petrorhagia is used as an outgroup. Four strongly supported clades were identified (Africa, Eurasian, Verruculosi and Armerium), indicating geographical distinctions. We identified potential discrepancies, notably in the placement of D. mooiensis and D. namaensis. An unexpected relationship between D. thunbergii and D. bolusii was also highlighted, which challenges current morphological classifications. Defined clades were further observed within African Dianthus (Ethiopian and Northern African species; BS/100, and Western and Eastern regions of South Africa; BS/100), while two subclades (BS/100 and BS/72) were observed within the South African clade. Additionally, ITS generated unsupported and unresolved trees independently, as with the three plastid markers (trnH-psbA, trnK-matK, and trnK-psbA). The identified clades and unresolved species placements warrant further investigation, possibly through additional molecular gene markers and broader taxon sampling.

1. Introduction

Dianthus L., commonly known as wild pink dianthus or carnation, is a large genus of ca. 384 accepted species, 150 subspecies, 12 heterotypic varieties and two forms distributed worldwide [1]. The genus Dianthus belongs to the family Caryophyllaceae, also known as the Carnation family [2]. It is native to Europe, Asia, and Africa and has been introduced to Australia, North and South America, and Australia, as illustrated in Figure 1 [2,3]. However, Constantinidis [4] and Valente et al. [3] noted the Mediterranean region as a center of diversity for the whole genus.

South Africa is home to 15 species, nine subspecies, and six nominal subspecies [1]. Most of these species are concentrated in the eastern regions of the country [5]. These species share similar physical features [6]. They are herbaceous and mostly have woody stems [6,7]. The leaves are opposite, simple, and linear in shape with smooth margins [2]. Dianthus flowers vary in color (Figure 2), ranging from pink, purple, lilac, violet, red, and white, with five serrated petals at the edges [6,7].

Each South African Dianthus species is adapted to a specific ecological niche and is recognized for its distinctive floral characteristics. Dianthus albens Aiton is commonly found in the Western Cape and grows in fynbos and renosterveld habitats. It is characterized by white flowers and the ability to thrive in nutrient-poor, acidic soils. Dianthus basuticus Burtt Davy is found in the high-altitude regions of the Drakensberg and Lesotho. It is well-suited to montane grasslands. It typically has narrow linear leaves and produces small pink to purple flowers. Dianthus basuticus consists of three lower ranks: subsp. basuticus, subsp. fourcadei S.S. Hooper and subsp. grandiflorus (E.Mey. ex Sond.) S.S. Hooper. Dianthus bolusii Burt Davy is known for its delicate, fringed pink flowers and is typically found in fynbos regions of the Western Cape. It grows in nutrient-poor, acidic soils, often in rocky and sandy environments. Dianthus burchellii Ser. is found in grasslands and rocky outcrops across South Africa. It has narrow leaves and pink flowers with fringed edges [2,8].

Dianthus caespitosus Thunb. has two subspecies, subsp. caespitosus Thunb. and subsp. pectinatus (E.Mey. ex Sond.) S.S. Hooper. It is found in rocky areas and grasslands, forms dense clumps, and produces small pink flowers. It is adapted to well-drained sandy soils. Dianthus crenatus Thunb. is widespread in South Africa. It grows in various habitats, including grasslands and rocky outcrops. It features erect stems and white-to-pale-pink flowers with fringed petals [2,8]. Dianthus holopetalus Turcz. is notable for its entire (non-fringed) petals, which are uncommon in this genus. It is typically found in grasslands and rocky areas at higher altitudes in South Africa [2,8].

Dianthus kamisbergensis Sond. is endemic to the Kamiesberg Mountains in the Northern Cape; this species grows in rocky outcrops and produces bright pink flowers with deeply fringed petals. Dianthus laingsburgensis S.S. Hooper is endemic to the Laingsburg area of the Western Cape. This species is adapted to the dry, rocky conditions of the Karoo and produces pink to white flowers. Dianthus micropetalus Ser. is a species with very small flowers. It is adapted to arid conditions and is often found in sandy or rocky soils in the Northern Cape. Dianthus mooiensis F.N. Williams is native to the Mpumalanga province, this species thrives in rocky, well-drained soils, and has three subspecies, subsp. dentatus F.N. Williams, subsp. kirkii S.S. Hooper and subsp. mooiensis. This species is characterized by tufted growth and pinkish-white flowers with fringed petals [2,8].

Dianthus namaensis Schinz is found in the Namaqualand region and is adapted to the arid conditions of the succulent Karoo biome. It has three subspecies: subsp. dinteri (Schinz) S.S. Hooper, subsp. junceus (Burtt Davy) S.S. Hooper and subsp. namaensis. It has small white-to-pale-pink flowers. Dianthus thunbergii S.S. Hooper, consists of two subspecies: subsp. maritimus S.S. Hooper and subsp. thunbergii. This species is widespread in various habitats in South Africa. It features pink to purple flowers and is often found in rocky and grassy areas. Dianthus transvaalensis Burtt Davy is primarily found in the Transvaal region (now part of Gauteng, Limpopo, and Mpumalanga). It grows in grasslands and has pink to white flowers with fringed petals. Dianthus zeyheri Sond. has two subspecies, subsp. natalensis S.S. Hooper and subsp. zeyheri. It is distributed mainly in the Eastern Cape and KwaZulu-Natal, but extends to the northern provinces of South Africa. This species is noted for its white or pale pink flowers with a slightly aromatic scent [2,8,9].

Furthermore, D. zeyheri has been found to be of taxonomic importance, because of its distribution that extends from the Eastern Cape, throughout the coastal region of KwaZulu-Natal to the northern border of Zululand and into Mozambique [6]. This is in contrast to other species, which are only distributed in one or two regions of the country. D. zeyheri grows in grasslands and forest margins, in well-drained, loamy soil, particularly on sandstone [2]. D. zeyheri is characterized by palmate petals with fimbriate margins and a very long 5-lobed green calyx which is fused at the base [7]. Even though Raimondo et al. [10] reported the conservation status (IUCN) of D. zeyheri as of least concern, population decline in the wild is a concern.

Fassou et al. [1] reported that Dianthus is a genus with many taxonomically complex species groups, and they believed that this may be the reason why there is no complete treatment of the whole genus. In addition, Burtt Davy [11] noticed that there is a specific limit among the South African Dianthus, which are often ill-defined. The characters used for specific delimitation are less amenable to precise definition compared to those of other genera. In addition, this misconception of the species’ characteristics has led botanists to assign more than one name to similar species due to incorrect descriptions of morphological characters [11].

Hooper [7] indicated that the species that are occurring in South Africa possess certain characteristics that coexist across all native species. These characters are perennial hemicryptophytic habits with a woody taproot and a well-developed base (7). They have setaceous to narrow-elliptic leaves and consist of solitary pedicellate flowers with outer-lower pairs of calyx-bracts smaller than the upper pairs, and the calyx is uniformly striate-nervose with hairy and globose petal laminae [7]. However, Burtt Davy [11] argued that the description employed by Sonder [12] to categorize South African species based on flowering stems is unreliable. This is because most Dianthus species in this region exhibit both simple and branched flowering stems within a single plant [11].

Some of the species that are of taxonomic concern, and which also show blurring of morphological boundaries between species and their subspecies, are D. zeyheri, which has been considered either as one coherent species [2,11,12] or consisting of two subsp.: zeyheri and natalensis [5,7]. These two subspecies are distinguished only by their petal structures [7]. Hooper [7] recognized D. zeyheri subsp. zeyheri having 8–24 mm long petals, fringed with deep incisions, and petals of subsp. natalensis with 8–17 mm long petals with fine incisions. D. zeyheri also shares similar characters with D. crenatus [7]. In addition, D. mooiensis and D. basuticus also have a resemblance, sharing diagnostic characters such as linear basal leaves and apical fimbriate petals [2,11].

This study aimed to address two key questions: Do South African Dianthus species form a monophyletic clade within this genus? Can these species be distinguished based on the sequences used? These questions were explored by (1) investigating the phylogenetic relationships of South African Dianthus species using DNA sequences from plastid (trnK-matK, trnK-psbA, and trnH-psbA) and nuclear (ITS) datasets; (2) investigating and clarifying the evolutionary relationships among species within the Dianthus genus in South Africa using phylogenetic analysis; and (3) analyzing the geographical distribution patterns of these species to clarify their evolutionary relationships.

It is thus hypothesized that molecular analysis of the Dianthus genus in South Africa will clarify the phylogenetic relationships among its species, resulting in a more precise classification and identification within the group. It is also hypothesized that Dianthus may not form a monophyletic group.

2. Materials and Methods

2.1. Taxon Sampling and Collection of Plant Material

Between 100 and 105 specimens per species of South African Dianthus were examined, sourced from the Bews Herbarium (NU), KZN Herbarium (NH), and the National Herbarium (PRE) [13]. However, DNA extraction samples were specifically obtained from three specimens, each representing one of the three species (D. basuticus, D. kamisbergensis, and D. transvaalensis), and these samples were carefully prepared for further analysis. The reason for extracting DNA from only three species and downloading sequences for the others was the degradation of DNA in the preserved materials. Over time, DNA in herbarium specimens can deteriorate, making it challenging to obtain high-quality amplifiable DNA, particularly from older specimens. As a result, we downloaded publicly available genetic sequences from an online database (GenBank), one sequence for each species with existing sequences. The three species, however, had not been sequenced previously, nor did they have sequences available in online databases; therefore, DNA extraction was necessary from herbarium specimens. We were unable to find fresh material of these species in the field. Details of all the sequences of the three species and sequences from GenBank can be found in Table S1. DNA extraction and Polymerase Chain Reaction (PCR) procedures were conducted at the University of South Africa, Eureka Building, while sequencing reactions were performed at the Inqaba Laboratory Biotechnology, Pretoria, South Africa.

Moreover, among the 15 South African Dianthus species, D. burchellii, D. crenatus, and D. holopetalus were excluded from the DNA extraction and molecular analyses due to a lack of plant material from the field and herbarium collections. These species were not used in the study simply because the necessary plant specimens were not accessible at the time, which made it impossible to perform the required molecular analyses. To supplement the dataset, we obtained an additional 10 South African sequences and 85 non-South African sequences from GenBank/NCBI based on the studies of Valente et al. [3] and Fassou et al. [1]. This aggregation resulted in a total of 98 taxa, consisting of 94 Dianthus and 4 Petrorhagia as outgroups. Detailed voucher information and GenBank accession numbers are listed in Table S1.

2.2. Choosing Outgroups

Outgroups were chosen based on the molecular studies of Valente et al. [3] and Fassou et al. [1]. Previous studies have indicated that Velezia and some species of Petrorhagia are more closely related to Dianthus [14]. Therefore, Petrorhagia sp.; P. prolifera (L.) P.W. Ball & Heywood; Petrorhagia sp. and P. thessala (Boiss.) P.W. Ball & Heywood were chosen as outgroups, following the methods of Fassou et al. [1] and Valente et al. [3].

2.3. DNA Extraction, Amplification and Sequence Alignment

Genomic DNA was isolated from 0.01–0.3 g of herbarium-dried leaf material using a DNA extraction kit (i.e., Zymo Research, Murphy Avenue, Irvine USA). DNA extraction was performed according to the manufacturer’s protocol. Purification of samples was carried out using a Zymo Research kit and all the DNA extraction and PCR amplification reactions were completed at the University of South Africa, Eureka Building, Johannesburg.

All PCRs were performed using ReadyMix Master (Advanced Biotechnologies, Epson, Surrey, KT199AP, UK). The primers used for the PCR reactions are listed in

Table 1. Details of the primers used for PCR amplification.

Locus	Primer	Reference
ITS
ITS5	GGA AGT AAA AGT CGTAAC AAG G	[16]
ITS4	TCCTCCGCT TAT TGATAT GC	[16]
trnH-psbA
trnH	TGA TCC ACT TGG CTA CCG CC	[17]
psbA	GCT AAC CTT GGT ATG GAA GT	[17]
trnK-matK
trnK-F	GGG TTG CTA ACT CAA TGGTAG AG –	[18]
CARYmatK1440R	AKC GTA AAT GAG AGG ATT G	[19]
trnK-psbA
trnK	GGG TTG CTA ACT CAA TGGTAG AG –	[17]
psbA	GCT AAC CTT GGT ATG GAA GT	[17]

. Bovine serum albumin (3.2% BSA) was added to both the plastid reaction mixtures. This additive serves as a stabilizer for enzymes, reduces problems with secondary structures, and improves annealing [15]. PCR amplification was performed using a Fast Thermal Cycler machine.

The programs used for PCR amplification were as follows: (a) for ITS, the protocol used consisted of pre-melt at 94 °C for 60 s, denaturation at 94 °C for 60 s, annealing at 48 °C for 60 s, extension at 72 °C for 3 min (for 28 cycles), followed by a final extension at 72 °C for 7 min; (b) for matK, the protocol used consisted of pre-melt at 94 °C for 3 min, denaturation at 94 °C for 60 s, annealing at 52 °C for 60 s, extension at 72 °C for 2 min (for 30 cycles), final extension was at 72 °C for 7 min; (b) for trnH-psbA, the protocol used consisted of a pre-melt at 94 °C for 3 min, denaturation at 94 °C for 60 s, annealing at 48 °C for 68 s, extension at 72 °C for 1 min (for 28 cycles), final extension at 72 °C for 7 min; (c) for trnK-psbA, the protocol used consisted of a pre-melt at 96 °C for 1 m 30 s, denaturation 95 °C for 30 s, annealing 50 °C for 1 min, extension 72 °C for 1 min 30 s (for 35 cycles) and final extension at 72 °C for 20 min; (d) for trnK-matK, the protocol is similar to trnK-psbA except that the extension step was only 1 min. The PCR products were verified by electrophoresis on 1% agarose gels stained with ethidium bromide. The PCR products were sent to Inqaba Laboratory Biotechnology, Pretoria, South Africa, for cycle sequencing. Cycle sequencing reactions were carried out at Inqaba Laboratory Biotechnology.

2.4. Data Analyses

Complementary strands were assembled and edited using Sequencer 3.1 (Gene Codes, Ann Arbor, MI, USA). ITS, trnH-psbA; trnK-matK and trnK-psbA were aligned manually in PAUP version 4.0b.10 [20]. All sequences were aligned using the MUSCLE program, and any necessary alignment adjustments or exclusions of ambiguously aligned sequences were also performed using MUSCLE.

2.5. Molecular Phylogenetic Analyses

A maximum parsimony (MP) tree was constructed based on plastid trnK-matK, trnK-psbA, trnH-psbA, and nuclear (ITS) sequences. Tree searches were analyzed using a heuristic search with 1000 replicates of random taxon addition, holding 10 trees at each step during stepwise addition with the tree bisection reconnection (TBR) branch swapping algorithm and saving multiple equal parsimonious trees (MulTrees). All analyses were conducted using the PAUP version.4.0b10 program [20] and all character transformation were treated as Fitch parsimony [21]. Branch lengths were calculated using DELTRAN (Delayed transformation) character optimization instead of ACCTRAN (accelerated transformation) due to report errors with PAUP version.4.0b10.

Bootstrap analysis was used to estimate the support for each clade [22] using TBR swapping with Fitch weights and retaining 10 trees per replicate. Bootstrap support was categorized as high (85–100), moderate (75–84), or low (50–74).

Bayesian Inference (BI) was conducted with MrBayes v.3.2.7 [23], using four parallel Markov Chain Monte Carlo (MCMC) running for a total of 10 million generations and MODELTEST version.3.06 [24]. A model test was performed to determine the most appropriate model for each gene based on the Akaike information criterion (AIC) [25]. All tree file outputs were processed using tree annotator v. 2.7.4 and the trees were visualized using FigTree v1.4.4, a graphical viewer of phylogenetic trees.

2.6. Distribution Maps Analyses

Occurrence data, including latitude and longitude coordinates for 14 Dianthus species, each represented by more than 100 specimens, were extracted from the SANBI online BRAHMS database [26] and from herbarium specimens examined in the study. These coordinates were converted from degrees, minutes, and seconds (DMS) to decimal degrees (DD). Subsequently, these points were incorporated and visualized in the Quantum Geographic Information System (QGIS version 3.16) software as a shapefile. To ensure accuracy, outliers were scrutinized against the South Africa_Province border shapefile and removed when necessary. In the layout manager, distribution maps were generated for each species and a combined distribution map of the 14 species. A map of Dianthus burchellii could not be generated due to insufficient locality data.

3. Results

3.1. Statistics of Molecular Data

A robust phylogenetic tree of three plastids (trnH-psbA, trnK-matK, and trnK-psbA) and the ITS nuclear region was assembled using 98 taxa of Dianthus and Petrorhagia species. Among the gene regions, trnH-psbA sequences were the shortest, and trnK-matK sequences were the longest. A summary of the DNA matrix and maximum parsimony statistics for individual gene regions is presented in

Table 2. Summary of DNA matrix and maximum parsimony statistics for the aligned, analyzed, and number of informative individual gene regions. The best JMODELTEST analysis results are also presented.

Gene Regions	No. of Characters	No. of Taxa	Characters Constant	Parsimony Uninformative	Parsimony Informative	Missing	jMODELTEST AIC	Number of Trees per Run
ITS	659	95	181	361	117	>5%	F81+I+G
trnH-psbA	258	93	183	39	36	>5%	TN93+I+G
trnK-matK	1376	95	1252	72	52	>5%	GTR+I+G
trnK-psbA	772	91	668	83	21	>5%	HKY85+I+G
Combined regions	3065	98	2284	555	226	>5%	-	20,001

, along with the best JMODELTEST analysis results.

3.2. Phylogenetic Analyses of All the Dianthus Species in This Study

The results of the separate analysis of plastid sequences did not support the independent use of plastid gene markers to assess phylogenetic relationships within the genus Dianthus. However, the combination of both nuclear (ITS) and plastid (trnH-psbA, trnK-matK, and trnK-psbA) datasets was well-supported with 0.92 PP (Figure 3) and 100/BS (Figure 4). In contrast, separate analyses of ITS sequences (Figure S1) revealed unsupported topological conflicts in resolving the two D. caespitosus as a sister group to the subclade of D. namaensis. According to the ITS phylogenetic tree, South African species are monophyletic, with the exception of one species, D. laingsburgensis. Dianthus laingsburgensis was nested within the Ethiopian species clade, forming a sister group/species to D. leptoloma.

Phylogenetic analyses of the combined datasets (Figure 3 and Figure 4) revealed that Dianthus was monophyletic when Petrorhagia was used as an outgroup. Four strongly supported clades were identified (Africa lineage, Eurasian radiation, Section Verruculosi, and Section Armerium), indicating geographical distinctions (Figure 3). The Africa lineage is primarily found in southern and eastern Africa, including countries like South Africa and Ethiopia. Eurasian radiation is widespread across Europe and Asia, particularly in regions extending from the Mediterranean to Central Asia. Section Verruculosi is concentrated in the eastern Mediterranean, with significant representation in countries like Greece and Turkey. Section Armerium is also mainly found in the Mediterranean region, particularly in southwestern Europe, including Spain and France. These distributions highlight the diverse habitats and evolutionary adaptations of the Dianthus genus across different continents [1].

To gain a deeper understanding of the relationships within the African clades, particularly focusing on the South African species, it is crucial to consider the results of the Bayesian analyses. In Figure 4, the outgroup Petrorhagia supports the monophyletic state of the Dianthus. P. thessala was resolved as sister to the African Dianthus species, while Petrorhagia sp. B was resolved (BS/100) possibly as P. prolifera, but a physical specimen was not checked to confirm this assumption due to the logistics of obtaining the material from Turkey. According to the Bayesian Analyses of the African lineage or species (Figure 4), two geographically distinct clades were identified: the Ethiopian and Northern African species (BS/100) and the Western and Eastern regions of South Africa (BS/100). Within the South African species, two more subclades with strong bootstrap support can be identified. One subclade consists of D. laingsburgensis, D. transvaalensis, D. basuticus, D. caespitosus, D. namaensis, D. mooiensis, D. micropetalus, and D. kamisbergensis, while the other subclade consists of D. thunbergii, D. bolusii, D. albens, and D. zeyheri.

3.3. Phylogenetic Analysis and Relationships Among South African Dianthus

In both Figure 3 and Figure 4, all African species were grouped within the African Lineage, with South African species forming one cluster and Ethiopian and Northern African species forming another. However, D. excelsus, D. serrulatus, and D. longiglumis exhibited unresolved phylogenetic positions. Notably, D. zeyheri was consistently identified as the closest species to D. albens (BS/88; Figure 3), with both species sharing fimbriated petal margins, a characteristic used for their identification [7].

The Bayesian Inference (BI) tree (Figure 4), based on combined gene analysis, revealed that South African Dianthus species are grouped into two distinct clades, each containing smaller subclades. D. zeyheri emerged as the closest species to D. albens, while D. thunbergii and D. bolusii, which come from different regions (eastern and western South Africa, respectively), were found to be closely related. Both species share similar floral characteristics, particularly in terms of petal shape and size, as well as fringed petals, a common trait among Dianthus species.

In the Western and Eastern South African clade (Figure 4), a well-supported group (100% bootstrap support) comprised three subclades. Specifically, D. caespitosus was the sister taxon to D. mooiensis, D. namaensis was the sister taxon to D. micropetalus, and both were sisters to D. kamisbergensis. The close relationship between D. namaensis and D. micropetalus was consistently supported by both phylogenetic trees (Figure 3 and Figure 4). Additionally, D. laingsburgensis was found to cluster with D. basuticus, both of which are closely related to D. transvaalensis. However, the placement of D. transvaalensis in Figure 3 is close to that of Northern African and South African species. This suggests that D. transvaalensis could represent a potential link between species diversity in both regions and that shares a common ancestor with species from both regions. Also, the connection between D. basuticus and D. transvaalensis may be due to their shared basal leaves, which are distinguishing features for their identification.

The Bayesian analysis of the plastid dataset (Figure S2) placed D. albens weakly supported by D. leptoloma (Ethiopian species). The southern African taxa showed a scattered distribution and lacked a clear definition, with low posterior probability values in their clades. The monophyly of the ITS BI tree (Figure S1) offered better resolution than that of the plastid BI tree (Figure S2). However, topological incongruence and insufficient support between the ITS and plastid datasets remain, especially in the placement of D. zeyheri, which is closely related to D. albens in the ITS tree (Figure S1) but remains unresolved in the plastid tree (Figure S2).

Dianthus thunbergii and D. bolusii were nested together in the fourth subclade (Figure 4), despite their distinct regional occurrences. These species share morphological features such as fimbriated petals, 2–6 bract pairs, and blue-gray leaves, with D. bolusii exhibiting densely clumped leaves close to the ground. Additionally, our findings identify D. thunbergii as being closely related to D. albens, with both species located within the fourth subclade, which is sister to both D. thunbergii and D. bolusii (Figure 4). Distinctions between D. thunbergii and D. albens can be made based on morphological traits, such as the narrower-ovate lobes at the tip of the calyx and lanceolate-elliptic bracts in D. thunbergii, compared to the ovate-elliptic bracts in D. albens.

In the first subclade (Figure 4), D. laingsburgensis, D. transvaalensis, and D. basuticus emerge as the species closest to each other in this subclade based on the phylogenetic tree from the combined datasets. A close relationship between D. laingsburgensis, D. transvaalensis, and D. basuticus could be inferred based on the characteristics of the basal leaves, calyx length, and bract pairs. The close grouping of D. basuticus, D. laingsburgensis, and D. transvaalensis in the phylogenetic tree suggests that these species share a common ancestor and have diverged relatively recently in evolutionary terms.

The second subclade is sister to the third subclade and contains species that are more closely related to those in the third subclade than to any other in the combined dataset (Figure 4). Th molecular grouping likely reflects shared morphological features, such as fimbriate and exserted petals. Our results have indicated a strong molecular support with a close relationship between D. caespitosus and D. mooiensis.

Within the third subclade, D. namaensis and D. micropetalus are closely related, supported by a moderate bootstrap (BS/63; Figure 4). This relationship is further confirmed by their adjacency in Figure 3, although with weak support (BS/51). Additionally, D. kamisbergensis is strongly supported (BS/100; Figure 4) as the sister species to D. namaensis and D. micropetalus.

Within the clade consisting of species from the Western and Eastern regions of South Africa, the Bayesian Inference tree highlighted four subclades among South African Dianthus species (Figure 4). The first subclade consists of D. laingsburgensis-D. basuticus, the second subclade consists of D. caespitosus–D. mooiensis, the third subclade consists of D. namaensis–D. kamisbergensis, and the fourth subclade consists of D. thunbergii-D. albens. In the third subclade, D. namaensis is resolved as the closest sister to D. micropetalus (Figure 4).

3.4. Dianthus Distribution in South Africa

Locality data collected from herbarium specimens and the SANBI online BRAHMS database indicated that Dianthus species are distributed across all nine provinces of South Africa (Figure 5; species distribution map). Individual species distribution maps are presented in Figures S3–S16. Some species are exclusively found in coastal areas along the Western Cape coastline (e.g., D. albens, D. bolusii, D. holopetalus, and D. caespitosus, extending to the Eastern Cape province). D. basuticus is distributed across seven provinces, spanning Western Cape, Eastern Cape, Free State, KwaZulu-Natal, Mpumalanga, certain areas of Gauteng, and part of Northern Cape. Notably, D. crenatus is confined to the eastern region of South Africa.

4. Discussion

4.1. Phylogenetic Incongruence and Topological Conflicts

Although the combination of the nuclear marker ITS and plastid markers trnH-psbA, trnK-matK, and trnK-psbA was statistically supported for homogeneity, the individual trees (Figures S1 and S2) displayed poorly supported topologies. While statistical tests indicated congruence between ITS and plastid markers, the resulting tree topologies differed in some respects. According to Wendel and Doyle [27], such conflicts and poorly supported trees may arise due to factors including inappropriate model choice, homoplasy, insufficient signal strength, data paucity, horizontal gene transfer, and incomplete lineage sorting.

Our study contests the claims made by Álvarez and Wendel [28], Hershkovitz and Zimmer [29], and Soltis et al. [30], which suggest that well-supported taxa remain unaffected by adjusting alignments or discarding ambiguously aligned sequences. In the current study, similar alignment adjustments were performed; however, the ITS did not provide reliable resolution at the species level.

Among the Petrorhagia species used as the outgroup in this study, P. thessala was the closest relative to the Dianthus species, whereas the remaining Petrorhagia species were more distantly related. This suggests that the genus Petrorhagia may be paraphyletic, raising classification concerns. These findings align with those of Madhani et al. [31], who also linked the sections Pseudotunica and Pseudogypsophila with Dianthus.

4.2. Phylogenetic Relationships Within Dianthus

The findings of our study align with recent molecular phylogenetic studies on Dianthus [1,3], which highlight the need for taxonomic revisions and support the inclusion of Petrorhagia thessala within Dianthus, as previously suggested [1,3,30].

Our results (Figure 3 and Figure 4) provide evidence for the monophyly of Dianthus, supported by synapomorphic characters shared among the species grouped within the phylogenetic trees; however, due to limited sampling, this conclusion cannot be fully confirmed. These findings align with those of Valente et al. [3] and Fassou et al. [1], who also observed monophyletic Dianthus when using the sister genera Petrorhagia and Velezia as outgroups. Furthermore, our study identified four well-supported clades (Figure 3), with species grouped according to their geographic regions, highlighting the importance of geography in Dianthus evolution.

Our findings align with the classification of Dianthus species based on geographic distribution, as observed in the study by Valente et al. [3], in which species were grouped into lineages, Eurasian, African, Verruculosi, and Armerium, according to their region of occurrence. Interestingly, D. transvaalensis is the only South African species nested within the Northern African species (Figure 3). One possible explanation for this could be historical biogeographic events, such as long-distance dispersal or past land connections between Africa and Eurasia, which may have facilitated its migration and establishment in South Africa. This suggests that D. transvaalensis may have a distinct evolutionary history compared to other South African Dianthus species. Alternatively, its placement may result from an ancestral lineage connection, sharing a more recent common ancestor with species outside South Africa, indicating that its lineage diverged before the diversification of the South African clade. In contrast, Fassou et al. [1] provide a different perspective from Valente et al. [3], particularly regarding the placement of South African species such as D. mooiensis, D. namaensis, and D. zeyheri, which were observed to be nested within the Eurasian radiation clade.

Our findings further reveal that the African lineage is not divided into two subclades in Figure 3, but into two distinct clades in Figure 4. The first subclade consists of species from Ethiopia and Northern Africa, while the second includes species from South Africa, suggesting a classification based on the geographic origin within Africa. However, in Figure 3, the placement of certain species, such as D. laingsburgensis, D. serrulatus B, D. crinitus A, D. crinitus B, D. transvaalensis, and D. excelsus, remains unresolved. This may be due to factors such as inappropriate model selection, homoplasy, insufficient phylogenetic signals, data limitations, horizontal gene transfer or incomplete lineage sorting. As noted by Wendel and Doyle [27], these challenges can result in poorly resolved or misclassified species in phylogenetic trees.

Our results (Figure 4) established P. thessala as the sister group to the African Dianthus clade. Additionally, Petrorhagia sp. B was strongly resolved (BS/100), likely corresponding to P. prolifera (Figure 4), suggesting that the specimen used could be accurately identified as P. thessala. This strong resolution suggests that Petrorhagia sp. B is a distinct species closely related to P. prolifera based on genetic data, although both specimens need to be physically examined for morphological similarities. The high support for this identification indicates well-defined traits that differentiate it from other species in the genus, reducing the likelihood of misclassification and reinforcing its phylogenetic placement. Within the African Dianthus lineage, geographically distinct clades were observed, including an Ethiopian and Northern Africa clade (BS/100) and a Western and Eastern South African clade (BS/100). Additionally, two subclades (BS/100 and BS/72) were identified within the South African lineage (Figure 4).

Our study found D. zeyheri to be consistently emerging as the closest species to D. albens (Figure 4), a relationship that was strongly supported in our analysis (BS/88; PP/0.87; Figure 3), contradicting a previous study [1], where the relationship between D. zeyheri and other South African species remained unresolved. The isolation of D. zeyheri and the placement of D. mooiensis as the sister species to D. namaensis in the previous study may be the result of the limited sampling of South African species by previous study. This was also evident in Valente et al. [3], where the inclusion of only a few representative South African species resulted in unclear relationships among species. Thus, the limited sampling in both studies may have contributed to the unresolved phylogenetic relationships observed in their results. It should be emphasized that the current findings deviate from those of Fassou et al. [1]. In their study, D. mooiensis was placed within the clade containing Euro-Asian species, specifically embedded within the D. namaensis species. Notably, Fassou et al. [1] did not provide evidence of similar morphological features that would support the emergence of these two species as the closest related species in their phylogenetic analysis.

The strong molecular support for the close relationship between D. caespitosus and D. mooiensis aligns with previous studies, which highlight similar morphological traits between these species [7,11,32]. However, they remain morphologically distinct, with D. caespitosus having longer basal leaves than cauline leaves, while D. mooiensis exhibits predominantly cauline leaves with shorter basal leaves. Additionally, D. caespitosus has fewer and linear cauline leaves, whereas D. mooiensis has broader and more abundant leaves [7,11].

4.3. Geographical Distribution of Dianthus in South Africa

The phylogenetic analyses presented in Figure 3 and Figure 4 consistently group Dianthus species according to geographical location, supporting previous research by Valente et al. [3]; this finding contrasts with that of Fassou et al. [1], who observed South African species clustering within the Eurasian radiation, complicating their classification. These results provide partial support for Madhani et al.’s [31] theory that geography has played a critical role in the evolution of Dianthus in Eurasia and other regions. Madhani et al. [31] also noted that while Dianthus species show limited ecological differentiation, many are narrow endemics, suggesting that geographical factors have been a significant driver of speciation within the genus.

In Africa, Dianthus species are primarily found north of the Sahara and are more widespread in the southern regions (Figure 1 and Figure 5). The African Dianthus species are largely confined to Eastern Tropical Africa, as noted by Burtt Davy [11]. Burtt Davy [11] further suggested that South African Dianthus species likely reached their current range via high mountain corridors in Eastern Tropical Africa, reflecting an earlier phase of diversification and evolution.

Dianthus kamisbergensis and Dianthus laingsburgensis have historically been documented in the Northern Cape Province, particularly in the Namaqualand region [6,11]. However, recent distribution data show that these species now also occur in the Western Cape province of South Africa (Figure 5, Figures S9 and S10), primarily within the subtropical biome of the Cape provinces. Notably, in the phylogenetic tree (Figure 4), these species are grouped separately, likely due to specific morphological differences.

Distribution maps (Figure 5 and Figures S3–S16) reveal the diverse ecological niches occupied by various South African Dianthus species. Coastal species like D. albens and D. bolusii are restricted to the Western Cape, while D. basuticus has a broader distribution across multiple provinces. Additionally, D. albens is predominantly found in the far Western Cape but extends its range to the Eastern Cape, where it is sparsely distributed, and further through the high mountain grasslands of the Free State to the Northwest Province. Within these regions, D. albens co-occurs with related species such as D. thunbergii, D. caespitosus, and D. bolusii, whose distribution is limited to the Eastern Cape (Figure 5 and Figures S3–S16). The distinct eastern distribution of D. crenatus (Figure S7) highlights the significance of geographic factors in the evolution and distribution of Dianthus species in South Africa, although the underlying reasons for this remain unclear.

One major trend is apparent within the winter-rainfall season: most South African species, particularly those known to be endemic to the Western Cape province, are observed in the current results extending to the Eastern Cape, through the Free State to the Northwest Province, as well as the Northern Cape. These trends, however, are based on current locality distribution records, and they need to be analyzed more critically using biogeographical and phylogenetic methods.

A further trend is evident in the well-known species of Lesotho, D. basuticus. According to Hooper [6], this species was previously known to occur only in the high mountain areas of the eastern region grasslands of the Eastern Cape, Free State, KwaZulu-Natal, and Mpumalanga provinces. However, the current results indicate that this species is now distributed in the fynbos vegetation of the winter-rainfall region of the Western Cape of South Africa (Figure 5 and Figure S4).

The only two species that seem to be adapted solely to the eastern regions up to the northern regions of South Africa are D. mooiensis and D. zeyheri (Figure 5, Figures S12 and S16). No records of these species were found in the Northern and Western Cape provinces of South Africa (Figure 5, Figures S12 and S16). D. micropetalus is the only species of Dianthus that occurs in seven provinces of South Africa, excluding the KwaZulu-Natal and Mpumalanga provinces (Figure 5 and Figure S11). In the literature, its range is known to extend from Cradock, through Kalahari, to the dry areas of Great Namaqualand [11].

5. Conclusions

The identified clades and unresolved species placements warrant further investigation, possibly through the use of additional molecular markers or broader taxon sampling. This study lays the foundation for future research aimed at unraveling the intricate evolutionary history and ecology of Dianthus in South Africa. The presence of well-supported subclades within the South African clade indicates the potential for further exploration of regional evolutionary patterns.

Furthermore, the results of this study propose a comprehensive examination of the taxonomy of Dianthus and Petrorhagia to arrive at a definitive conclusion regarding the classification of these genera. This echoes the sentiment of previous studies [1,2,3], which recommended further investigation incorporating more material from Dianthus, Gypsophila, Petrorhagia, and Saponaria, as these genera were inadequately represented in previous analyses. Additionally, the current study unequivocally demonstrates the immense value of molecular phylogenies in understanding the evolutionary history of complex taxa, as exemplified by Dianthus.