Phylogeny problems of the genus Vaccinium L. and ways to solve them

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BACKGROUND

Vaccinium L. is an economically important genus belonging to the family Ericaceae Juss., tribe Vaccinieae Rchb. [1]. The tribe Vaccinieae is a taxon including a large number (~1000) of woody plant species of the family Ericaceae, different in morphological traits, and common in temperate and tropical zones of all continents except Australia and Antarctica [2]. Most of the species occur in the tropics, mainly in the mountain rain forests.

This tribe includes the genera Agapetes G.Don, Anthopteropsis A.C. Sm., Anthopterus Hook., Calopteryx Ruiz & Pav., Cavendishia Lindl., Ceratostema Juss., Costera J.J. Sm., Demosthenesia A.C. Sm., Didonica Luteyn & Wilbur, Dimorphanthera (Drude) F. Muell., Diogenesia Sleumer, Disterigma (Klotzsch) Nied., Gaylussacia Kunth, Gonocalyx Planch. & Linden, Laterospora A.C. Smith, Macleania Hook., Mycerinus A.C. Sm., Notopora Hook.f., Oreanthes Benth., Orthaea Klotzsch, Paphia Schltr., Pellegrinia Sleumer, Periclesia A.C. Sm., Plutarchia A.C. Sm., Polyclita A.C. Sm., Psammisia Klotzsch, Rusbya Britton, Satyria Klotzsch, Semiramisia Klotzsch, Siphonandra Klotzsch, Sphyrospermum Poepp. & Endl., Symphysia (Vahl) Wilbur & Luteyn, Themistoclesia Klotzsch, Thibaudia Ruiz & Pav., and Utleya Wilbur & Luteyn [3].

The genus Vaccinium includes approximately 500 species growing on all continents except Australia and Antarctica [4]. Most species occur in the tropics on open mountain slopes and the rest are distributed in subtropical, temperate, and boreal regions of the northern hemisphere [5]. Slightly less than two-thirds of the species are occur in south-east Asia, more than 50 species in America, and the rest are dispersed throughout the world [6].

Many species in the genus Vaccinium have colorful leaves, flowers, and fruits, making them valuable ornamental plants [7]. Some species have edible fruits, which are used as medicine in various communities [8]. Cranberries, blueberries, and lingonberries are among the most studied of these species, having been domesticated in the XIX and XX centuries [9, 10]. Currently, 42,746 hectares of land are used for growing cranberries, 126,144 hectares for blueberries, and 29 hectares for lingonberries [11].

Blueberries and a number of other species in the genus Vaccinium also have a great potential as new crops. The most economically important Vaccinium species belong to the sections Cyanococcus A. Gray, Oxycoccus (Hill) Koch, Vitis-Idaea (Moench) Koch, Myrtillus Dumortier, and Vaccinium L. [12].

The berries of these crops contain an average content of vitamin C, fiber and basic microelements, four organic acids, namely cinchona, citric, hydroxysuccinic, and benzoic acids [7]. A high content of flavonoids, mainly anthocyanins, imparts the fruits with a bright color [13]. The presence of these compounds accounts for the antioxidant, antimutagenic, and antitumor activity of berries in the geneus Vaccinium. As anthocyanins are the main consumed antioxidants in the Western diet, their content is one of the key indicators of the quality of berries and an important selection trait [14, 15].

Currently, there is a growing increase in the demand for berries of domesticated Vaccinium species, which necessitates the breeding of new crops [12]. When breeding for new crops, it is important to determine the phylogenetic relationships between representatives of the genus and their closest relatives; however, such studies on the genus Vaccinium are retarded by challenges arising from the peculiarities of speciation within this genus.

HISTORY OF THE TAXONOMY OF THE GENUS VACCINIUM

The genus Vaccinium was first described by Carl Linnaeus in 1753. It included the species V. frondosum L., V. album L., V. stamineum L., V. uliginosum L., V. vitisidaea L., V. oxycoccos L., V. myrtillus L., V. corymbosum L., V. arctostaphylos L., V. hispidulum L., V. ligustrinum L., and V. mucronatum L. [16]. No major systematic analysis of the described new species was performed until the beginning of the XX century when active breeding work for cranberries and blueberries began in the USA [10, 17]. Therefore, the first work in their taxonomy was of an applied nature and mainly concerned species distributed in North America. The main difficulties in such studies became apparent, namely the absence of fertility barriers in morphologically different organisms, which leads to the formation of several hybrids, and the occurrence of both autopolyploidy and allopolyploidy throughout the genus [17]. For example, V. myrsinites Lam. is an allopolyploid species resulting from the hybridization of V. tenellum Ait. and V. darrow Camp species [18].

Camp was among the first to present work on the genus Vaccinium in 1945 [18]. He classified the genus according to morphological features. However, this work was confined to species growing in the northern hemisphere, with special attention to North American blueberries. Therefore, the genus was divided into several sections, with 9 diploid, 12 tetraploid, and 3 hexaploid species included in the North American blueberry section Cyanococcus [9]. During World War II, field work was limited. The incompleteness of field data, resulted in errors in classification, leading to some organisms being designated as separate species, which later turned out to be hybrids or polyploids [19]. To resolve this problem, several species were combined into one with different levels of ploidy, and in the new classification, Kloet retained the previous division into sections [6], and the Cyanococcus section included 6 diploid, 5 tetraploid, and 1 hexaploid blueberry species [9]. This reduction in the number of species was due to the inclusion of all crown-forming species of North American blueberries into one species, V. corymbosum, with three levels of ploidy.

Thus, the differentiation of Vaccinium species is complicated by polyploidy, similar morphology, and introgression during hybridization [9]. Therefore, the use of morphological characteristics in phylogenetic studies of this genus does not always enable us to assess unambiguously the evolutionary relationships between the studied species, which necessitates use of modern methods of phylogeny.

MODERN PHYLOGENETIC METHODS

The development of molecular genetics and biochemical studies has brought new approaches to phylogenetic studies. In the mid-to-late XX century, molecular labeling began to develop rapidly, and at the turn of the century, the Barcode of Life project was launched [20]. The main thrust of the Barcode of Life was to combine the efforts of classical and molecular biologists to improve taxonomy and phylogeny. It involves selecting taxonomically significant DNA sequences and entering them into databases together with taxonomic information, which enables creation of a system of molecular identification of organisms, including plants.

Despite progress in the approach, markers for DNA barcoding have certain disadvantages. Based on Matveeva et al. [20], we compiled the characteristics of the main markers used in DNA barcoding of plants (Table 1).

Table 1. Characterization of the main markers for DNA barcoding of plants

Таблица 1. Характеристика основных маркеров для ДНК-штрихкодирования растений

Table 1 shows that traditional markers used for DNA barcoding in plants can be conventionally divided into nuclear and cytoplasmic ones. Cytoplasmic markers are usually maternally inherited. Nuclear markers, such as ITS, are multicopy, but in species of hybrid origin, the proportion of sequences of the ribosomal gene cluster inherited from one of the parents can be reduced to such an extent that ITSs of this parent are not detected using traditional DNA barcoding protocols [21]. The features of these markers can lead to ambiguities in the reconstruction of phylogenetic relationships, which results in trees of the same species with slightly different topologies. To solve this problem, multilocus analysis methods are used, which use information on several marker sequences.

Since the development of these algorithms, sequencing methods have developed significantly. Thus, the Next-Generation Sequencing algorithms developed in the 2000s enabled simplification and reduction of the cost of whole-genome projects, resulting in the generation of unprecedented amounts of data on the sequences of both model and non-model organisms. Open access to these data gave rise to a new approach, phylogenomics, which determines the phylogenetic relationships of species based on the analysis of their genomes [22]. Phylogenomic methods are based on whole-genome sequences or on the traits of the entire genome [23].

Methods based on nucleotide sequence information from genomes require alignment of orthologous genes from which a phylogenetic tree is derived using two alternative approaches, namely the supermatrix approach, which is based on combining all genomic regions into a single matrix that includes all taxa, then such a combined dataset is subjected to phylogenetic inference using the desired phylogenetic method (distance, maximum likelihood, maximum parsimony, Bayesian approach) [24], and the supertrees approach (or a tree of trees), which involves combining trees obtained based on the analysis of individual genes [23].

Methods based on whole-genome traits involve the reconstruction of phylogenetic relationships not by nucleotide sequences, but by the presence of genes (gene repertoire) or by the order of genes [23]. In the first case, the presence, absence, or duplication of genes constitutes the phylogenomic data, whereas in the second case, large-scale karyotypic changes in the genome constitute the phylogenomic data [24]. These methods are associated with rare genomic changes that are less prone to homoplasia and therefore more informative than methods based on the analysis of nucleotide sequences.

These methods include the obligatory stage of assembly and annotation of the genome, as well as the search for orthologous sequences, which is complicated when dealing with non-model organisms. To simplify this analysis, the assembly and alignment-free (AAF) method was developed [25]. This method allows phylogeny to be determined directly from unassembled genome sequence reads, making phylogenomic analysis available when dealing with species without an appropriate reference genome or large sequence coverage.

Apart from DNA marking, chemosystematics, an interdisciplinary field that uses information about the chemical composition of plants to determine interspecific and intraspecific phylogenetic relationships, has begun to develop in recent years [26]. The occurrence of chemical compounds and their structures are often taxonomically specific, so they can be used as markers for distinguishing between taxa [27]. As such, both primary and secondary metabolites can be used in plants. However, the same compounds are often formed during completely different biosynthetic pathways in unrelated plants; therefore, such methods can be useful in determining the boundaries of lower taxonomic ranks [28].

To date, various methods have been developed that allow reconstructing phylogenetic relationships without using morphological traits. Phylogenetic markers have advantages such as presence in a wide range of plant species, low search, and sequencing costs, and disadvantages such as different rates of divergence and ambiguity of interpretation in taxa of hybrid origin. Despite their disadvantages, phylogenetic markers enable determination of phylogenetic relationships between different taxa when used together.

APPLICATION OF MODERN METHODS IN PHYLOGENETIC STUDIES OF THE GENUS VACCINIUM

The methods described above, such as DNA barcoding, fingerprinting, phylogenomics, and chemosystematics, began to be used in the study of the genus Vaccinium, allowing revision of its taxonomy proposed by Kloet. In the first such work, the matK and ITS markers were used to determine the phylogenetic relationships of various representatives of the entire tribe Vaccinieae [2]. Based on the data of K. Kron et al. [2] supplemented with new sequences from NCBI (www.ncbi.nlm.nih.gov), we reconstructed the phylogeny of the genus (Fig. 1). The resulting dendrograms and those of Kron et al. [2] did not confirm the traditional genus boundaries, but several well-supported clades were found on the tree, namely Andean; Mesoamerican/Caribbea, East Malaysian; Agapetes, consisting of some Asiatic Vaccinium and Agapetes; Bracteata-Oarianthe, including representatives of the respective sections; Orthaea/Notopora, which includes the genera Orthaea and Notopora; Myrtillus and Vaccinium, including some Vaccinium. Moreover, most of the recovered clades in their composition-united representatives of various genera, whereas the clades Vaccinium and Myrtillus included species of the genus Vaccinium, which had been assigned to different sections in the previous classifications. Based on these results, Kron et al. concluded that it is necessary to reassess the taxonomy of the genus Vaccinium as the genus is not monophyletic. Although the work on this reassessment started in 2003 [29], many researchers have reported radical differences from the theoretical genus structure, which could be due to the difficult interpretation of the results of phylogenetic analysis obtained based on the classical markers, such as ITS and matK, in species in which evolution, the processes of hybridization and polyploidization played a significant role [30].

Fig. 1. The phylogenetic tree obtained from the analysis of the matK and ITS sequences of various species of the Ericaceae family based on the data of K. Kron et al. [2], supplemented by us

Рис. 1. Филогенетическое дерево, полученное при анализе последовательностей matK и ITS различных видов семейства Ericaceae на основе данных K. Kron и соавт. [2], дополненных нами. Эволюционная история была выведена с использованием метода максимального правдоподобия и модели General Time Reversible [33]. Эволюционные анализы проводили в MEGA X [34]

To date, due to the lack of a well-resolved molecular phylogeny of the entire genus, the well-founded phenotypic classification of the genus proposed by Kloet [31] is a priori accepted in the studies. Thus, the modern classification assumes the division of the genus Vaccinium into two subgenera and includes 33 sections. Using GRIN data, we described the species mentioned in this review, taking into account their taxonomic position and geographical distribution [3] (Table 2).

Table 2. Brief description of some Vaccinium species

Таблица 2. Краткое описание некоторых видов Vaccinium

As the use of DNA barcoding does not allow unambiguous reconstruction of the phylogeny of the genus Vaccinium, more time-consuming and expensive methods of molecular phylogenetics are used in genetic studies of economically significant species. In particular, the genomic and transcriptome resources of the American cranberry (V. macrocarpon) were used in the development of nuclear [31], chloroplast, and mitochondrial SSR markers [32] for the analysis of genetic diversity and genetic mapping within a species. The dendrogram plotted on their basis distributed species into genera and sections within Vaccinium in a manner similar to the morphological classification. Figure 2 presents the phylogenetic relationships determined by Schlautman et al. [32] based on cytoplasmic SSR markers between species, which were previously investigated using ITS and matK markers. In fact, the analysis performed became a kind of approximation to multilocus analysis since the markers used were relatively evenly distributed throughout the genome and plasmon.

Fig. 2. Phylogenetic tree of economically important species of the genus Vaccinium, built on the basis of the SSR loci of mitochondria and chloroplasts [32]. I — Species belonging in the Oxycoccus section, II — Vitis-idaea, III — Batodendron, IV — Cyanococcus

Рис. 2. Филогенетическое дерево экономически важных видов рода Vaccinium, построенное на основе локусов SSR митохондрий и хлоропластов [32]. I — Вид, входящий в секцию Oxycoccus, II — Vitis-idaea, III — Batodendron, IV — Cyanococcus

Phylogenetic analysis using SSR markers identified the genus Vaccinium as monophyletic [32], and also showed the monophyly of the sections Cyanococcus, Oxycoccus, Vitis-idaea, whereas data based on DNA barcoding revealed polyphyly of the genus, but defined the tribe Vaccinieae as monophyletic [2], with members of different genera clustered according to their geographical origin. For example, the Andean clade includes representatives of 17 genera, which diversity is concentrated in the region of the northern Andes, or the Mesoamerican/Caribbean clade includes 6 genera, which representatives are common in Central America and the Caribbean islands. Perhaps such discrepancies are related to the different breadth of species coverage, as information on microsatellite repeats was obtained only for economically important species. Therefore, further more extended analysis will help resolve the remaining taxonomic issues within Ericaceae and its many genera. Additionally, it may be practical to use other chloroplast markers presented in Table 1.

SSR markers also allow assessment of genetic diversity in populations of wild relatives of cultivated species. This knowledge helps develop effective conservation strategies and facilitates their use for agricultural purposes.

The natural habitats of V. macrocarpon and V. oxycoccos overlap in many areas. Thus, in order to understand better the relationship between the two cranberry species, Rodriguez-Bonilla et al. [35] estimated the genetic distance on microsatellite sequences of organisms from wild populations. Consequently, the populations were divided into two main clusters, one of which contained all accessions of V. oxycoccos, and the other included all accessions of V. macrocarpon. This was also confirmed by principal component analysis, which also revealed geographic clustering within species.

Genetic evaluations of both species showed very high levels of heterozygosity. These results are consistent with the biology of cranberries, which is characterized by cross-pollination and reduced fertility in experimentally obtained inbred lines [36]. These features contribute to the maintenance of a high level of genetic diversity in wild cranberry populations [35].

NGS DATA FOR STUDYING THE SYSTEMATICS AND PHYLOGENY OF THE GENUS VACCINIUM

Recent assemblies of the genomes of V. macrocarpon, V. microcarpum, V. oxycoccos, and V. corymbosum facilitated their comparative genomics [15, 37]. Molecular age determination revealed that V. macrocarpon diverged from V. oxycoccos approximately 2 million years ago, and 4.5 million years ago from V. microcarpum the results indicate that divergence of V. macrocarpon and V. corymbosum occurred between 5 and 10.4 million years ago. Additionally, the analysis showed that the divergence of Vaccinium from the more distant relatives, Rhododendron williamsianum Rehder & E.H. Wilson (order Ericales, family Ericaceae) and Actinidia Lindl. (order Ericales, family Actinidiaceae) occurred 22 and 52.1 million years ago, respectively.

Additionally, this analysis revealed two polyploidization events in the evolution of the genus Vaccinium, namely an ancient γ-tripplication and a later whole-genome duplication (Vm-α) shared with other members of the Ericaeae, Theaceae D. Don, and Actinidiaceae Gilg & Werderm families approximately 58 million years ago. This age determination is consistent with the Dl-α duplication of the genome in Diospyros L. (order Ericales, family Ebenaceae Gürke) and the Ad-α duplication in Actinidia (Fig. 3).

Fig. 3. Whole genome duplications in cranberry evolution based on pooled data [15, 37]. I — represents a γ-triplication, II — represents a Vm-α duplication, both of which formed the modern Vaccinium genome; III — Dl-α duplication of the genome characteristic of the genus Diospyros; IV — Ad-α duplication of the Actinidia genome

Рис. 3. Полногеномные дупликации в эволюции клюквы на основе объединенных данных [15, 37]. I — γ-трипликация, II — Vm-α-дупликация, оба этих события сформировали современный геном Vaccinium; III — Dl-α-дупликация генома, характерная для рода Diospyros; IV — Ad-α-дупликация генома Actinidia

Blueberry breeding has a short history and began in the 20th century in the USA. To improve the basic qualities of the crop, breeders used interspecific hybridization of tetraploid and hexaploid blueberry species, which formed naturally through unreduced gametes. This is why cultivated blueberries have several levels of ploidy, namely, tetraploid lowbush V. angustifolium, tetraploid highbush V. corymbosum, and hexaploid Rabbit-Eye blueberry V. virgatum. Additionally, when breeding highbush varieties, species common in the northern states were used, which led to their resistance to the northern climate. These cultivars later came to be known as northern highbush blueberries, which were then crossed with southern blueberries to produce varieties adapted to cultivation in the southern states, southern highbush blueberries.

Since the history of blueberry breeding is well documented, Nishiyama et al. [38] used ddRAD sequencing to perform its genetic population analysis. This algorithm enabled the sequencing of the genome regions associated with restriction sites, which were selected on the basis of the published blueberry V. corymbosum genome. Thus, rapid and economical detection of SNPs and indels in the studied genomes was achieved.

An analysis of the population structure suggests that the blueberry cultivar Rabbit Eye and northern highbush blueberries are relatively homogeneous, but southern highbush blueberries contain a much more mixed genetic background. Taking into account the pedigree of blueberries, the most optimal was the division of the entire data set into nine hypothetical genomes, which correspond to the number of species actively used in the course of breeding (V. darrowii, V. elliottii, V. tenellum, V. angustifolium, V. corymbosum, V. constablaei, V. virgatum, V. myrtilloides, and V. pallidum). The trends identified are consistent with the history of blueberry breeding [38].

Thus, this method has shown its adequacy for the genus Vaccinium and can be further used for other taxa under study.

In phylogenetic studies, data on biochemical composition can also be useful, given that Vaccinium species are producers of important secondary metabolites. One of these metabolites is the iridoid glycoside monotropein, which was found in the fruits of cultivated species of cranberries, lingonberries, bilberries, and bog whortleberry V. uliginosum, but does not occur in close relatives, namely North American blueberries V. corymbosum, V. angustifolium, and V. virgatum. A more detailed analysis, including both cultivated blueberry varieties and wild species, revealed monotropein in five varieties (Bluehaven, Blue Ridge, Ornablue, Ozarkblue, and Summit) and in all 13 wild Vaccinium species analyzed (V. arboreum, V. calycinum, V. consanguineum, V. meridionale, V. cylindraceum, V. elliottii, V. floribundum, V. fuscatum, V. ovatum, V. padifolium, V. reticulatum Nene, V. reticulatum Red Button, and V. stamineum). Ecotypic and pedigree analysis showed that only the Bluehaven variety belonged to the northern highbush ecotype, i.e., blueberry species common in the northern states of the USA were used in its breeding, and the Ornablue variety is a hybrid of the cultivated Concord and wild V. pallidum. In the breeding of each of these five varieties, there was hybridization with wild monotropein-positive species. Therefore, we suggest that the presence of monotropein in these varieties is associated with the introgression of wild species into cultivated blueberries [39]. A similar approach can be further used to establish phylogenetic relationships between accessions.

CONCLUSION

In summary, we conclude that currently, the taxonomy of the genus Vaccinium is not well established owing to many difficulties faced by researchers. First, the DNA barcoding data clearly showed the polyphyletic nature of the genus Vaccinium, as well as the joint clustering of species with similar geographical localization. Second, species that were previously considered to be more phylogenetically distant were included in the same clade, whereas species that were considered close were placed in different clades. One of the reasons for these ambiguous results may be hybridization and polyploidization during speciation. Due to the existence of these ambiguities, botanists still adhere to the traditional genus system based on morphological characteristics. According to this system, the genus includes 33 sections, but the species composition of the sections and the evolutionary relationships between them remain controversial. Constructions based on the analysis of NGS sequencing results often coincide better with those based on traditional taxonomy than those based on DNA barcoding methods. Although more recent NGS sequencing methods provide new data that expand our knowledge about the origin and evolution of the Vaccinium genus and its relatives, they remain more laborious and expensive than DNA barcoding methods. This is why researchers studying the taxonomy, evolution, and domestication of these organisms still use additional sets of molecular markers to conduct large-scale studies of representatives of the genus Vaccinium and the tribe Vaccinieae.

In some cases, unique DNA sequence fragments recently introduced into genomes as a result of horizontal gene transfer can be regarded as markers. This approach enables combining individual species into clusters and tracking the relationship of species within clusters [20]. Analysis of the published genomes of some representatives of the genus indicates that the genus Vaccinium has such sequences. Naturally transgenic organisms were found in the genus [40], containing the sequence of the rolB/C-like gene. The presence of this conserved sequence in several species and a common localization site may indicate the transformation of their common ancestor with the subsequent transfer of this DNA fragment to descendants and its gradual divergence. Thus, the rolB/C-like gene can be used in the future as a phylogenetic marker for the genus Vaccinium.

ADDITIONAL INFORMATION

Author contribution. Thereby, all authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work. Contribution of the authors: R.R. Zhidkin — drawing up a plan, literature review, writing the main part of the text; T.V. Matveeva — drawing up a plan, literature review, making final edits.

Competing interests. The authors declare that they have no competing interests.

Funding source. This work was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with the agreement No. 075-15-2022-322 dated 22.04.2022 on the grant in the form of a subsidy from the federal budget of the Russian Federation. The grant was provided for state support for the creation and development of a world-class Scientific Center “Agrotechnologies of the Future”.

About the authors

Roman R. Zhidkin

Author for correspondence.
Email: zhidkinr@gmail.com

Student

Tatyana V. Matveeva

Email: radishlet@gmail.com
ORCID iD: 0000-0001-8569-6665
SPIN-code: 3877-6598
Scopus Author ID: 7006494611

Dr. Sci. (Biol.), Professor

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2. Fig. 1. The phylogenetic tree obtained from the analysis of the matK and ITS sequences of various species of the Ericaceae family based on the data of K. Kron et al. [2], supplemented by us

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3. Fig. 2. Phylogenetic tree of economically important species of the genus Vaccinium, built on the basis of the SSR loci of mitochondria and chloroplasts [32]. I — Species belonging in the Oxycoccus section, II — Vitis-idaea, III — Batodendron, IV — Cyanococcus

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4. Fig. 3. Whole genome duplications in cranberry evolution based on pooled data [15, 37]. I — represents a γ-triplication, II — represents a Vm-α duplication, both of which formed the modern Vaccinium genome; III — Dl-α duplication of the genome characteristic of the genus Diospyros; IV — Ad-α duplication of the Actinidia genome

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