Exploring the Palynology of Coleus Lour. (Lamiaceae) from an Evolutionary Perspective

doi:10.21203/rs.3.rs-5304707/v1

Coleus Lour. is known for its medicinal properties. However, this genus faces taxonomic challenges due to the aggregation of species formerly classified under Plectranthus L'Hér., resulting in numerous synonyms. To support the current taxonomic circumscription, this research focused on the pollen evaluation of Coleus species from both palynotaxonomic and evolutionary perspectives. Seventeen species were examined, including three for which novel data are presented in this study: Coleus amboinicus Lour., C. barbatus (Andrews) Benth. ex G.Don, and C. neochilus (Schltr.) Codd. The pollen grains were acetolyzed and examined using scanning electron microscopy. A similarity dendrogram was constructed based on pollen morphometric data. The phylogeny was developed using Bayesian inference with the aim of mapping the evolution of pollen size and morphology. The similarity dendrogram revealed six distinct pollen groups, differing in ornamentation (rugulate, reticulate, and bireticulate, with perforations and/or granules) and shape (prolate-spheroidal, prolate, subprolate, and oblate). Ancestral state reconstruction indicates that the most recent common ancestor of Coleus likely had subprolate pollen approximately 35 µm in size (polar axis), with an initial rapid diversification in grain size followed by a period of stabilization. Despite the current scarcity of pollen data, Coleus demonstrates significant potential for further palynological studies.

evolutionary analysis

exine ornamentation

medicinal plants

palynotaxonomy

Plectranthus

Lamiaceae is a cosmopolitan family composed of around 230 genera and 7,300 species (Gbif 2023; Antar et al. 2024; Powo 2024). In Brazil, 600 species of 71 genera are spread across nearly the entire territorial area (Antar et al. 2024; Powo 2024). Among its subtribes, Plectranthinae has faced significant taxonomic revisions, particularly regarding the distinction between Coleus Lour. and Plectranthus L'Hér. (Paton et al. 2019). Previously, these genera belonged to a single grouping: the genus Ocimum L., along with Burnatastrum Briq., Englerastrum Briq., Neomuellera Briq., and Isodictyophorus Briq. (Morton 1962). Posteriorly, the separation of Coleus and Plectranthus was based on palynology: the main difference would be the densely perforated exine in Plectranthus, while in Coleus, this characteristic is less pronounced (Morton 1962; Halbritter and Buchner 2016). However, this criterion was considered insufficient, leading to a proliferation of synonyms within these genera (Morton 1962; Antar et al. 2024; Powo 2024).

Recent phylogenetic studies have identified Coleus and Plectranthus as being phylogenetically distant (Paton et al. 2018). Furthermore, there are morphological characters that support this division: Coleus is differentiated from Plectranthus primarily by the calyx, where the pedicel is asymmetrically attached to the base of the calyx tube (Paton et al. 2019). In contrast, Plectranthus exhibits a calyx throat with oblique lateral lobes, positioned closer to the lowermost pair of lobes than to the upper lobe (Paton et al. 2019). Further analysis still is needed to fully understand the intergeneric relationships within Plectranthinae, as well as among Coleus species (Paton et al. 2018, 2019).

Comprising approximately 300 species, Coleus originates from the Paleotropics and has been introduced in Brazil (Paton et al. 2019; Powo 2024). The genus consists of annual or perennial herbs or shrubs, characterized by opposite leaves (Paton et al. 2019). The inflorescence varies from compact to loose, with sessile or pedicellate flowers arranged in spirals or cymes (Paton et al. 2019; Powo 2024). The four stamens may have fused or free filaments, inserted into the anterior lip or exerted (Paton et al. 2019). The stigma is bifid, and the fruits are ovoid, often flattened dorso-ventrally (Paton et al. 2019). Moreover, Coleus presents a diverse array of both medicinal and ornamental uses (Kavitha et al. 2010; Machado et al. 2021; Reddymalla et al. 2021; Joshi 2022). Among its species, Coleus amboinicus Lour., C. barbatus (Andrews) Benth. ex G.Don, and C. neochilus (Schltr.) Codd stand out due to their ethnobotanical importance as herbal medicines, especially in Brazil (Cerqueira et al. 2020; Fernandes et al. 2021).

The use of diverse approaches for accurate botanical identification is crucial for both ensuring the safe use of herbal medicines (Ekor 2014; Rivera et al. 2014; Bussmann 2015) and conducting evolutionary studies (Kress 2017). However, diagnosing Coleus using morphological characteristics is challenging due to the continuous nature of these traits and the lack of clear apomorphies within the genus (Paton et al. 2019). Palynological analyses have the potential to resolve such taxonomic issues, as pollen grains often display consistent structures at the genus level, and sometimes even at the species level (Cantino et al. 1992; Gonçalves-Esteves et al. 2022). In this context, Lamiaceae serves as a compelling example of the application of palynotaxonomy across different genera (Gul et al. 2021; Özaltan and Koçyi̇ği̇t 2022). Recognizing the importance of palynology in taxonomic delimitation, this study aims to provide new descriptions for Coleus, including C. amboinicus, C. barbatus, and C. neochilus. Additionally, we compiled the currently available palynological data for species of the genus, developed a similarity dendrogram, and examined the evolutionary implications based on phylogenetic information.

Plant Material and Pollen Characterization

Inflorescences of C. amboinicus, C. barbatus and C. neochilus were collected at the Medicinal Plant Garden of the Federal University of Lavras - Brazil. The material was fixed and maintained in ethyl alcohol:propionic acid (3:1) and stored at -20°C. Voucher specimens were deposited at the Research Center for Chemistry, Biology, and Agriculture (CPQBA) and the State University of Campinas Herbarium (UEC) in São Paulo, Brazil, under the following records: C. amboinicus (CPQBA 364), C. barbatus (UEC 121403), and C. neochilus (CPQBA 1388).

Slides were prepared according to the acetolysis method of Erdtman (1952). A total of fifty pollen grains were examined using an optical microscope (Leica DMLS) equipped with a Nikon Digital Sight DS-Fi1 micro-camera. The variables evaluated were: polar diameter (P); equatorial diameter (E); colpus diameter (D); apocolpium side (d); colpus width (CW); and colpus length (CL) and thickness of nexin and sexin. The pollen shape was defined by the P/E ratio. The measurements were carried out with the aid of Image Tool v. 3.0 software.

In scanning electron microscopy analyses, anthers were subjected to three consecutive ten-minute washes in cacodylate buffer, followed by postfixation in 1% osmium tetroxide for a minimum of one hour at room temperature. Subsequently, the material underwent three rinses in distilled water, followed by dehydration in a series of acetone concentrations (25%, 50%, 75%, 90%, and 100%), and was then transferred to the critical point apparatus (CDP 030). The pollen grains were extracted from the anthers on stubs, which were submitted to the sputter coater (SCD 050), and finally analysed on a LEO EVO 40 scanning electron microscope.

Cluster Analysis

A palynological survey was conducted, covering 14 species of Coleus available in the literature (Halbritter 2016; Khalik 2016; Smitha et al. 2018) and three from this study (Coleus amboinicus, C. barbatus and C. neochilus). A dendrogram was constructed based on morphometric data (see Table 2). The analysis utilised the Euclidean distance method implemented in R software (R Core Team 2023), along with the factoextra (Kassambara and Mundt 2020), dendextend (Galili 2015), and igraph (Csardi and Nepusz 2006) packages.

Phylogenetic Inference

Phylogenetic reconstruction was performed using the ITS, matK, rbcL, rps16 and trnL-F markers. Sequences were obtained from the GenBank platform (Table S1). Of the 17 Coleus species previously sampled in the dendrogram, only C. bishopianus (Gamble) Smitha & A.J. Paton, C. intraterraneus (S.T. Blake) P.I. Forst. & T.C. Wilson, C. neochilus (Schltr.) Codd, and C. fruticosus Wight ex Benth. lacked available sequences and could not be included in the phylogeny. The outgroup was represented by Alvesia clerodendroides (T.C.E.Fr.) B.Mathew, Alvesia rosmarinifolia Welw., Tetradenia fruticosa Benth., and Tetradenia riparia (Hochst.) Codd.

Alignment was performed using the MEGA program v. 2 (Stecher et al. 2020). Evolutionary model selection was determined by the Akaike criterion using the MrModelTest v. 2.3 program (Nylander 2004). The concatenated matrix was generated using the Mesquite v. 3.61 program (Maddison and Maddison 2021). Phylogenetic reconstruction was conducted through Bayesian inference using the MrBayes v. 3.2.2 program (Huelsenbeck and Ronquist 2001) available on the CIPRES platform (www.phylo.org). The following settings were applied: nchains = 4; nruns = 2; ngen = 107 and burnin = 0.25; samplefreq = 1,000.

Ancestral state reconstruction for categorical data

We assessed pollen grain shape from the perspective of marginal reconstruction of ancestral states using the maximum likelihood method. We performed this analysis in the R software (R Core Team 2023) with the packages corHMM (Beaulieu et al. 2013), phytools (Revell 2022), geiger (Pennell et al. 2014), and evobiR (Heath and Adams 2015). For the selection of the evolutionary models, we used the MK model for discrete data (Pagel 1999a,b; Lewis 2001). The Mk model can include the ‘equal rates’ (ER), in which the transitions between states occurred at equal rates, ‘All-rates-different’ (ARD), in which the transition rates (total change of state per branch) are variables, and the Symmetric Model (SYM) which considers that the transition rates are symmetrical, being used specifically for multistate data (Harmon 2019). The selection of the model for each trait considered the corrected Akaike criterion (AICc) and Akaike weight (AICw).

Phylogenetic mapping of continuous data

For continuous data, evolutionary models may follow a process of “Brownian motion”, in which the character's value changes randomly, both in direction and magnitude, over any time interval; “Early-Burst”, in which character change tends to concentrate at the base of the tree; and “Ornstein-Uhlenbeck”, which exhibits a tendency toward a central value under stabilizing selection (Harmon 2019). The evolutionary model testing for pollen size (polar diameter) was conducted using the R software (R Core Team 2023) with the geiger (Revell et al. 2023) and phytools (Revell 2022) packages. The model with the lowest AICc and highest AICw was selected for analysis. For character state mapping in phylogenetics, we utilized the ape package (Paradis et al. 2023).

Pollen description

The pollen grains of Coleus amboinicus, C. barbatus and C. neochilus species are hexazonocolpate (Fig. 1). Among them, Coleus amboinicus is clearly distinguished from C. barbatus and C. neochilus, which are more similar to each other (Table 1). This differentiation is particularly evident in the ornamentation: rugulate in C. amboinicus and bireticulate in the other species (Fig. 2). Additionally, there was observed an intraspecific variation in pollen grain size (polar and equatorial diameters), with differences of up to 15 µm between the smallest and largest grains within the studied species (Table 1).

Coleus amboinicus Lour. (Fig. 1a-b; 2a-b)

Isopolar pollen, 6-zonocolpate, exhibiting radial symetry, medium size (P: 30.24 [39.06] 46.76 μm, E: 28.99 [35.64] 41.59 μm) and a prolate spheroidal shape. The outline is circular or elliptical. The exine is tectate and columellate. The ornamentation is rugulate, without granules or perforations.

Coleus barbatus (Andrews) Benth. ex G.Don (Fig. 1c-d; 2f-h)

Isopolar pollen, 6-zonocolpate, exhibiting radial symetry, medium size (P: 36.83 [40.99] 47.10 μm, E: 31.35 [36.46] 42.75 μm) and a subprolate shape. The outline is circular or elliptical. The exine is tectate and columellate. The ornamentation is bireticulate, with granules and absent of perforations.

Coleus neochilus (Schltr.) Codd (Fig. 1e-f; 2i-l)

Isopolar pollen, 6-zonocolpate, exhibiting radial symetry, medium size (P: 35.14 [40.30] 45.29 μm, E: 27.61 [33.74] 39.26 μm) and a subprolate shape. The outline is circular or elliptical. The exine is tectate and columellate. The ornamentation is bireticulate, with granules and absent of perforations.

Cluster analysis

The data for species with currently available pollen descriptions are presented in Table 2. The pollen similarity dendrogram revealed six groups (Fig. 3). Coleus amboinicus and C. fruticosus have pollen grains with variations in the common birreticulate ornamentation pattern, which can be rugulate or reticulate. The group consisting of C. comosus and C. caninus features pollen grains with birreticulate ornamentation with perforations. Coleus cylindraceus, C. neochilus, and C. bishopianus exhibit birreticulate ornamentation with granules. The group represented by C. scutellarioides, C. intraterraneus, and C. australis has prolato-spheroidal pollen grains with birreticulate ornamentation. The remaining groups have pollen grains that are prolate or subprolate, with the former having significantly larger grains.

Ancestral state reconstruction

The phylogenetic informative characters, such as size (P) and shape (P/E), were analyzed for ancestral state reconstruction. The most recent common ancestor had subprolate pollen grains with a polar diameter of approximately 35 µm. The earliest-diverging clade showed a tendency towards subprolate grains, while the most recently diverged clade revealed greater variability in shape. Pollen size in Coleus species evolved under an Early-Burst (EB) model, indicating rapid diversification early in the genus's evolutionary history, followed by a decrease in the transition rate over time.

Pollen grains in Coleus exhibit an evolutionary trend towards a subprolate shape, a pattern commonly observed in the family (Gupta and Sharma 1990; Harley et al. 1992; Arogundade and Adedeji 2009; Özler et al. 2011; Firdous et al. 2015; Atalay et al. 2016; Azzazy 2016; Ma et al. 2016; Doaigey et al. 2018). The Early-Burst evolutionary diversification model identified in the pollen size evolution of Coleus suggests that the genus underwent an initial phase of rapid diversification, followed by a stabilization of the rates of change over time (Martin and Richards 2019). This pattern may be closely related to historical events that favoured an initial adaptive diversification (Martin and Richards 2019). For example, significant past climate changes could have created new habitats or altered existing ones, providing opportunities for Coleus species to colonize and specialize in different ecological niches. Additionally, geographical expansion and variation in available pollinators might have encouraged the evolution of different pollen sizes as an adaptive response to these varied conditions. After this period of intense diversification, the observed stabilization in pollen size may indicate that the genus reached an adaptive equilibrium, with current species well-adjusted to their respective environments, resulting in a reduced need for significant morphological changes in pollen (Martin and Richards 2019).

Pollen size variation is often observed due to the accumulation of substances during the maturation process, such as vesicles, lipid droplets, and starch grains (Kuang and Musgrave 1996; Knight et al. 2010). This variation can influence the length of the polar axes of pollen grains, which, in turn, can provide insights into the ploidy level of the plants (Erdtman 1952; Knight et al. 2010; Chaves et al. 2022). The average pollen sizes observed for C. amboinicus, C. barbatus, and C. neochilus closely align with those previously reported for diploid Coleus species (Reddy 1952; Nani et al. 2015). In contrast, triploid and tetraploid genotypes displayed larger mean pollen sizes, measuring 43.07 ± 1.63 µm and 52.7 ± 1.53 µm, respectively (Reddy 1952). Additionally, selective forces driven by male competition and specific pollination system conditions influence pollen size and shape (Harder 1998; Hao et al. 2020).

Hexacolpate pollen grains, such as those described for Coleus, also occur in other genera of the Lamiaceae family, such as Plectranthus, Hymenocrater Fisch. & C.A.Mey. (syn. of Nepeta L.), and Lycopus L. (Moon and Hong 2003; Jafari and Jafarzadeh 2008; Montero et al. 2011; Khalik 2016; Doaigey et al. 2018). In contrast, pollen with three, four, or five colpi is less common (Gupta and Sharma 1990; Pozhidaev 1992; Celenk et al. 2008; Moon et al. 2008; Özler et al. 2011). The higher number of colpi is considered a derived characteristic in Lamiaceae, as older tribes like Prostantheroideae and Ajugoideae have tricolpate pollen grains (Atalay et al. 2016).The presence of hexacolpate pollen in the family does not appear to correlate with tricolpate types, suggesting these variations may have evolved independently during Lamiaceae diversification (Pozhidaev 1992; Yu et al. 2018).

Additionally, Coleus displays a pattern of complex bireticulate ornamentation, which may be associated with efficient pollination strategies (Mander et al. 2021). The rugulate ornamentation observed in C. amboinicus diverges from the typical pattern found in the genus (Montero et al. 2011; Khalik 2016), highlighting the need for taxonomic revision of the species, especially considering the high number of synonyms attributed to this taxon (Powo 2024). Despite the limited sampling of pollen descriptions in Coleus, the significant variation among pollen grains of different species suggests that palynological characteristics can be useful for species delimitation, especially for those with a complex taxonomic history. However, some species, such as Coleus intraterraneus (S.T.Blake) P.I.Forst. & T.C.Wilson, Coleus neochilus (Schltr.) Codd, and Coleus fruticosus Wight ex Benth., currently lack available markers, which complicates a more detailed analysis. Future studies with a more comprehensive analysis, including more species and populations from diverse geographical regions, could deepen the understanding of intraspecific and interpopulation variations. This would open new perspectives on the evolutionary mechanisms that have shaped the diversification of the genus.

This study reveals that pollen morphometrics can effectively differentiate Coleus species, showing distinct patterns in grain shape and ornamentation, particularly concerning the presence of granules and perforations on the exine. The lack of pollen data within the genus highlights the importance of palynotaxonomic studies to fill taxonomic gaps. The evolutionary patterns identified provide a foundation for future research, which should integrate pollen data with morphological and genetic information for a more accurate taxonomic revision.

Equatorial diameter (E)

Polar Axis (P)

Colpus Length (CL)

Colpus Width (CW)

Apocolpium Side (d)

Equatorial Diameter (D)

Sexina (SE)

Nexina (NE)

Total Exine (TE)

Competing Interests

The authors declare that they all agree with this publication. There is no conflict of interest of any kind.

Author Contribution

T. F. Nani: Investigation, Conceptualization, Writing - Review & Editing; A. L. A. Chaves: Cluster and phylogenetic analysis, writing - review and editing; J. V. B. Calvelli: Investigation, Writing - review and editing; S. Barbosa: Writing - Review & Editing, Supervision; L. C. Davide: Supervision, Project administration. All authors read and approved the final manuscript.

Acknowledgments

We would like to thank the Medicinal Plant Garden of the Federal University of Lavras (UFLA), for providing the botanical material; and the Electron Microscopy Laboratory of UFLA, for support in the analyses. The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) Código de Financiamento 001, PDPG nº 1026/2022. Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), ao MEC/SESu/FNDE/PET e ao Programa de pós-graduação em Ciências Ambientais da Universidade Federal de Alfenas, UNIFAL-MG.

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Table 1 Pollen characteristics of C. amboinicus, C. barbatus and C. neochilus, showing mean values and standard deviations (maximum and minimum values are given in parentheses). (E, P, CC and LC) – measurements made in equatorial view. (D and d) – measurements made in polar view

Parameters	C. amboinicus	C. barbatus	C. neochilus
Equatorial diameter (E)	35.64±2.86 (28.99 - 41.59)	36.46±2.64 (31.35 - 42.75)	33.74±2.41 (27.61 - 39.26)
Polar diameter (P)	39.06±3.96 (30.24 - 46.76)	40.99±2.5 (36.83 - 47.10)	40.30±2.09 (35.14 - 45.29)
Colpus Length (CL)	29.53±3.46 (23.08 - 36.8)	26.38±2.17 (21.24 - 32.88)	27.41±2.41 (23.24 - 32.49)
Colpus Width (CW)	2.13±0.4 (1.34 - 3.44)	1.75±0.29 (0.86 - 2.56)	1.99±0.35 (1.36 - 2.92)
Apocolpium Side (d)	7.44±1.42 (4.6 - 10.9)	8.88±1.51 (5.38 - 11.99)	8.53±1.55 (5.21 - 13.05)
Colpus Diameter (D)	16.92±1.61 (14.03 - 20.35)	17±1.23 (14.18 - 19.71)	16.34±1.55 (12.29 - 20.51)
Sexine (SE)	1.26±0.21 (0.94 - 1.8)	1.02±0.14 (0.77 - 1.38)	1.33±0.22 (0.69 - 1.7)
Nexine (NE)	1.15±0.25 (0.06 - 1.73)	1.17±0.12 (0.45 - 1.92)	1.25±0.15 (0.86 - 1.55)
Total Exine (TE)	2.42±0.38 (1.2 - 3.13)	2.2±0.19 (1.89 - 2.57)	2.58±0.3 (1.74 - 3.02)
Ornamentation	rugulate	bireticulate	bireticulate
Shape (P/E)	prolate spheroidal	subprolate	subprolate
Herbarium - Voucher	CPQBA 364	UEC 121403	CPQBA 1388

Table 2 Pollen information for Coleus species used in cluster analysis and ancestral state reconstruction

Species	P (µm)	E (µm)	Colpi	Shape (P/E)	Ornamentation	Granules	Perforations	Reference	Name in the Reference (Synonym)
Coleus amboinicus Lour.	30	29	6	prolate spheroidal	rugulate	absent	absent	This study	Coleus amboinicus Lour.
Coleus arabicus Benth.	26	22	6	subprolate	bireticulate	absent	absent	Khalik (2016)	Plectranthus asirensis J.R.I.Wood
Coleus australis (R.Br.) A.J.Paton	30	34	6	prolate spheroidal	bireticulate	absent	absent	Australasian Pollen and Spore Atlas	Plectranthus parviflorus Willd.
Coleus aegyptiacus (Forssk.) A.J.Paton	41	29	6	prolate	bireticulate	absent	absent	Khalik (2016)	Plectranthus tenuiflorus (Vatke) Agnew
Coleus barbatus (Andrews) Benth. ex G.Don	36	31	6	subprolate	bireticulate	absent	absent	This study	Coleus barbatus (Andrews) Benth. ex G.Don
Coleus bishopianus (Gamble) Smitha & A.J.Paton	16	23	6	oblate	bireticulate	present	absent	Smitha et al. (2018)	Plectranthus bishopianus Gamble
Coleus caninus (B.Heyne ex Roth) Vatke	43	52	6	oblate	bireticulate	absent	present	Halbritter (2016)	Plectranthus caninus B.Heyne ex Roth
Coleus comosus Hochst. ex Gürke	48	55	6	oblate	bireticulate	present	present	Halbritter (2016)	Plectranthus ornatus Codd
Coleus congestus (R.Br.) A.J.Paton	52	35	6	prolate	bireticulate	absent	absent	Australasian Pollen and Spore Atlas	Plectranthus congestus R.Br.
Coleus cylindraceus (Hochst. ex Benth.) A.J.Paton	23	18	6	subprolate	bireticulate	present	absent	Khalik (2016)	Plectranthus cylindraceus Hochst. ex Benth.
Coleus fruticosus Wight ex Benth.	24	26	6	spheroidal	reticulate	absent	absent	Smitha et al. (2018)	Plectranthus deccanicus Briq.
Coleus hijazensis (Abdel Khalik) A.J.Paton	23	15	6	prolate	bireticulate	absent	absent	Khalik (2016)	Plectranthus hijazensis Abdel Khalik
Coleus intraterraneus (S.T.Blake) P.I.Forst. & T.C.Wilson	32	34	6	prolate spheroidal	bireticulate	absent	absent	Australasian Pollen and Spore Atlas	Plectranthus intraterraneus S.T.Blake
Coleus neochilus (Schltr.) Codd	35	27	6	subprolate	bireticulate	present	absent	This study	Coleus neochilus (Schltr.) Codd
Coleus scutellarioides (L.) Benth.	33	31	6	prolate spheroidal	bireticulate	absent	absent	Australasian Pollen and Spore Atlas	Coleus scutellarioides (L.) Benth.
Coleus succulentus Pax	20	16	6	subprolate	bireticulate	absent	absent	Khalik (2016)	Plectranthus pseudomarrubioides R.H.Willemse
Coleus villosus (Forssk.) A.J.Paton	23	18	6	subprolate	bireticulate	absent	absent	Khalik (2016)	Plectranthus arabicus E.A.Bruce

No competing interests reported.

TableS1.docx
Table S1 Information on molecular markers from GenBank used in the phylogenetic inference of Coleus. Species with missing molecular data are represented with a '-'

Exploring the Palynology of Coleus Lour. (Lamiaceae) from an Evolutionary Perspective

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Abstract

Figures

1 Introduction

2 Materials and Methods

3 Results

4 Discussion

5 Conclusions

Abbreviations

Declarations

Competing Interests

Author Contribution

Acknowledgments

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1