SYSTEMATICS AND MORPHOMETRICS OF THE SUBFAMILY BYTTNERIOIDEAE BURNETT IN BANGLADESH

Taxonomic revision and morphometrics are two crucial facets for the proper identification, updating of taxa, and strengthening phenetic relationships of angiosperms. This study focused on the systematics and morphometrics of the subfamily Byttnerioideae Burnett (Family Malvaceae) for the first time in Bangladesh, revealing eight taxa under seven genera, namely Abroma Jacq., Ayenia L., Guazuma Mill., Kleinhovia L., Melochia L., Theobroma L. and Waltheria L . These seven genera fall into three tribes: Byttnerieae, Theobromateae, and Hermannieae. Dichotomous bracketed keys to genera and species were generated. Detailed nomenclature, diagnostic characteristics, illustrations, representative specimens, notes on distribution and uses for each species were provided. Morphometrics study was conducted employing cluster and principal components analysis (PCA). Cluster analysis revealed close relationships between Theobromateae and Hermannieae tribes compared to Byttnerieae tribe in the form of UPGMA dendrogram. Similarity matrix revealed the highest affinity between Ayenia elegans Ridl. and Ayenia grandifolia (DC.) Christenh. & Byng. In the PCA analysis, the first two principal components explained approximately 82.26% cumulative variance. PCA-derived scatter and projection plots supported the topology and inter-relationships in the cluster analysis. These findings will enhance future conservation strategies, especially for the threatened and medicinally important taxa of Byttnerioideae in Bangladesh, and support molecular phylogenetic studies of the subfamily Byttnerioideae


Introduction
Byttnerioideae is one of the nine subfamilies of the angiosperm family Malvaceae (APG IV, 2016), consisting of 650 species belonging to 26 genera worldwide.Prior to the Angiosperm Phylogeny Group (APG) classification, members of Byttnerioideae were placed under the family Sterculiaceae (Cronquist, 1981).The 26 genera of Byttnerioideae have been incorporated under four tribes: Byttnerieae, Hermannieae, Lasiopetaleae and Theobromateae.The member taxa of Byttnerioideae inhabit predominantly in the tropical and subtropical regions (Lima et al., 2019).In Bangladesh, this subfamily is consisted of 8 taxa under 7 genera and 3 tribes (Ahmed and Rahman, 2022).Byttnerioideae is distinct by its simple, lobed or compound leaves, with five petals and numerous stamens, frequently connate at the base.Fruits typically manifest as capsules or schizocarps and seeds often adorned with hairs or other appendages (Colli-Silva et al., 2024).The importance of these species underscores the need for a taxonomic revision of Byttnerioideae.Such a revision is crucial in plant taxonomy, as it entails a thorough re-examination of a particular group to amend or enhance its description or diagnosis.Given that taxa often exhibit phenotypic plasticity, a taxonomic revision serves to update and refine the classification system (Baur et al., 2014).In Bangladesh, no efforts have been made so far for taxonomic revision of Byttnerioideae and thereby, this process is particularly necessary to ensure accurate identification and conservation of economically and medicinally important species within this subfamily.
Morphometrics, also known as numerical taxonomy or taximetrics, is a multidisciplinary approach that integrates mathematical principles with taxonomy to analyze character states through covariances between morphological variation and other associated or causal variables (Sneath and Sokal, 1973;Bookstein, 1991).Morphological data derived from taxonomic revision provides the pedestal of morphometric analysis because the diversity in morphological characters can be classified by the numeric gaps between taxa, which reflects their evolutionary relationships as revealed by morphological changes (Otte and Endler, 1989).This integrated approach allows for a more precise and quantifiable understanding of species differences and similarities, facilitating deeper insights into the classification and interrelationships of Byttnerioideae taxa in Bangladesh.There have been limited efforts to elucidate the morphometric relationships of angiosperm taxa in Bangladesh, and to date, no studies have focused specifically on the Byttnerioideae subfamily (Rahman and Rahman, 2013;Rahman et al., 2013).
An integrated approach combining taxonomic revision and morphometric analysis is critical for a detailed understanding of Byttnerioideae.Taxonomic revision ensures up-to-date classifications, while morphometrics provides detailed analysis of phenotypic variations and interrelationships.With these integrated approaches, we aimed to enhance species identification, update classifications, and clarify morphometric relationships, thereby providing a comprehensive understanding of systematic position of Byttnerioideae in Bangladesh and supporting conservation efforts for the threatened and medicinally important species.

Taxonomic revision
Twenty-five field visits were undertaken in different areas of Bangladesh to collect plant samples.These specimens were meticulously studied and identified, with confirmations made through consultation of standard literatures (Ahmed et al., 2009;Ashrafuzzaman and Sarwar, 2021).Additional voucher specimens of Byttnerioideae deposited at the DUSH, DACB, HCU and BFRIH were studied.To facilitate identification, taxonomic keys were generated for genera and species.Nomenclature was updated using recent standard floras and trustworthy web-based data sources (www.powo.science.kew.org; www.ipni.org).Each species is accompanied by technical descriptions, up-to-date nomenclature, phenology, representative specimens, habitat, global distribution, illustrations and uses.Voucher specimens were deposited at DUSH.

Morphometric analysis
Eight species of the Byttnerioideae used for morphometric analysis are presented in Table 1.Thirty-one characters, both qualitative and quantitative, were investigated and analyzed for morphometrics endeavor (Table 2).Both vegetative and reproductive characters were considered and coded as binary-states using NTedit v.1.2module of NTSYSpc v.2.10e software package (Rohlf, 2012).The Similarity module was employed to construct similarity matrix using the Simple Matching coefficient.Subsequently, cluster analysis was performed using the UPGMA method through the SAHN module.Principal components analysis (PCA) was conducted employing the Ordination module and Eigen package to construct the two-dimensional scattered diagram.The Eigen package was used to generate Eigenvector and Eigenvalues.Afterwards, the Mod3D tool from the Graphics module was implemented to generate three-dimensional diagram of PCA.All analyses were conducted using the NTSYSpc v.2.10e software package (Rohlf, 2012).
Habitat: Near water course, in the hilly forest areas.Distribution: Bhutan, Cambodia, China, Hong Kong, India, Lao PDR, Nepal, Thailand and Viet Nam.
Uses: A. grandifolia leaves possess anticancer property (Lalawmpuii et al., 2017).Ethnic ladies of Chittagong Hill Tracts wash their hair with macerated young parts and barks (Uddin and Hassan, 2018).
Uses: Guazuma ulmifolia is administered for treatment of diarrhea, hemorrhages, inflammatory disorders, and as a uterine contraction stimulant.Aerial portions of the plant have been demonstrated to protect the stomach from the harmful effects of NSAIDs (non-steroidal antiinflammatory medicines), mostly through anti-inflammatory and radical-scavenging processes.Flavanocoumarins isolated from G. ulmifolia showed promising anti-cancer effects in-vitro against human monocytic leukemia cell line TPH-1 (Maldini et al. 2013) (Fig. 3A).Vernacular names: Guest Tree (English); Bola, Bholla (Bangla).A small tree, up to 12 m tall.Leaves broadly ovate, 5.5-18 x 5.5-18 cm, abaxially puberulent when young, adaxially glabrous, acuminate or acute at the apex, cordate or subcordate at the base, margin entire or sparsely dentate, axils of venation and midrib sometimes with minute simple hairs, lateral veins 4-6 pairs; petiole 3.0-5.5 cm long, glabrous or with dense, minute simple hairs.Inflorescence a thyrse, up to 45 cm long.Sepals c. 6 mm long, pink; petals pink, yellow at apex; ovary globose, hairy, ovule 1 in each locule; style glabrous; stigma protruding 1-2 mm long.Capsule pyriform to globose, 5-angled, greenish pink at maturity.Seeds globose, dark brown or black.Flowering and fruiting: February-April.
Habitat: The plant thrives in the understory of tropical rainforests that are evergreen.It grows in bunches along river banks, wherein roots are frequently soaked throughout the year.Planted in gardens as well.
Uses: Cocoa is economically significant as used in various industries, including confectionery, food and beverage, and, more recently, pharmaceuticals and cosmetics.It is used for the treatment of cardiovascular problems (Rusconi and Conti, 2010).
Habitat: Occurs mostly in waste places as a weed, and sometimes is grown as ornamentals.Distribution: Argentina, Brazil, Cambodia, Haiti, Honduras, India, Mexico, Pakistan, Sri Lanka, Taiwan, Tanzania, Tonga, Thailand, Uganda, USA, Viet Nam, Yemen, Zambia and Zimbabwe.
Uses: The species is valued for its medicinal properties, and administered for sore throat, cough inflammation and asthma.The species is also ethnomedicinally important, and used for rheumatism, diarrhea and infertility (Zongo et al., 2013;Chitra et al., 2022).

Morphometric analysis
The morphometric analysis of eight taxa within the subfamily Byttnerioideae (Table 1) yielded insights into their inter-relationships.Thirty-one characters, both qualitative and quantitative, were investigated to infer inter-relationships among the taxa employed.The characters and character states of the taxa studied are presented in Table 2. To corroborate systematic position of the member taxa of Byttnerioideae, a combinatory approach was undertaken with cluster analysis (CA) and principal components analysis (PCA), revealing agreeable and congruent results among the member taxa.Cluster analysis revealed distinct groupings among the eight species within the Byttnerioideae, as shown in the UPGMA dendrogram.The studied species were grouped into two major clusters: major cluster 1 and major cluster 2 (Fig. 5).The major cluster 1 included all the members of the tribe Byttnerieae, namely A. augustum, A. elegans, A. grandifolia, and Kleinhovia hospita.In major cluster 2, four species from two tribes, Theobromateae and Hermannieae were grouped together, forming two subclusters within the second major cluster.G. ulmifolia was accompanied by T. cacao in the first subcluster, while M. corchorifolia was grouped with W. indica in the second subcluster of the major cluster 2. The similarity matrix indicated the highest affinity (0.935) between A. elegans and A. grandifolia (Table 3).On the contrary, the lowest morphological affinity (0.516) was observed between M. corchorifolia and K. hospita (Fig. 5).The UPGMA-based cluster analysis was applied to Acacia senegal to understand its morphometric relationships in Uganda (Mulumba and Kakudidi, 2010).Similarly, this procedure was used to reveal morphological relationships of Salvia fruticosa in Greece (Bertsouklis et al., 2021).These studies validate our choice of using UPGMA for this morphometric investigation, reinforcing its effectiveness in analyzing and understanding the morphological similarities and variations within Byttnerioideae.2010).PCA, by reducing the dimensionality and noise of multivariate data, enables the adjustment of univariate trait models to specific principal components.It operates by analyzing a data table consisting of observations described by multiple inter-correlated dependent variables.This revolutionary method extracts essential information from the data table and represents it as a series of new orthogonal variables.Additionally, PCA illustrates the similarity patterns of the data and variables by presenting them as points on maps (Uyeda et al., 2015).CA can provide a hierarchical classification of entities using similarity or dissimilarity matrix.This efficacious technique helps to characterize and compare communities (assemblages) of organisms in varied habitats on a geographical and temporal scale.Some properties of clustering include high scalability, high dimensionality, algorithm usability with multiple data types and interpretability.Different methods are employed for clustering, such as partitioning, hierarchical, density-based, grid-based and constraint-based strategies (Kettenring, 2006).In a previous study, the UPGMA results were compared with PCA outcomes to understand the morphometrics of Tibouchina hatschbachii and T. marumbiensis, showing consistent findings between the UPGMA and PCA (Maia and Goldenberg, 2019).Another study combined these techniques to understand species delimitation in Argyreia using leaf anatomical characters, revealing a new species, Argyreia gyrobracteata Traiperm & Chitchak (Chitchak et al., 2018).In our study, the coherence of UPGMA and PCA, therefore, strengthens the inter-relationships of the three tribes of Byttnerioideae.The current morphometric findings, along with a comprehensive taxonomic revision of Byttnerioideae, provide essential insights for future conservation strategies and phylogenetic studies.By analyzing morphometric data and revising taxonomic classifications, the study clarifies species boundaries and inter-relationships, enhancing our understanding of species diversity and evolution.This combined approach guides conservationists in prioritizing species and habitats for protection based on unique morphological traits and environmental adaptations, particularly for the economically and medicinally important taxa.The coherence between UPGMA and PCA techniques ensures robust, reproducible results, offering a reliable foundation for future studies.These findings will serve as a crucial resource for constructing accurate phylogenetic trees and developing effective conservation strategies, ensuring the sustainable management of Byttnerioideae species in Bangladesh.