Piper species (Piperaceae) of Sri Lanka and the diversity of the economically important Piper nigrum (black pepper): An overview of what has been done and what is yet to be achieved

: Applying restriction site-associated DNA sequencing (RADseq) and target capture for Piper species from species-rich South America and India, Southeast Asia, and Africa will highlight the origin and evolution of Sri Lankan endemics, P. zeylanicum , P. walkerii , and P. trineuron . Looking into the genetic diversity of cultivated P. nigrum from different agroclimatic regions and available germplasm in Sri Lanka using RADseq will give an overview of the existing genetic diversity of black pepper, which is economically important and needs genetic improvement. Variation in flower composition (male, female or bisexual) across the spikes and their shape is of major interest to evolutionary and pollination biologists and plant systematists. The 3D shape models of flowers obtained by computed tomography of the wild species of Piper from Sri Lanka and cultivated P. nigrum will play an important role in revising the taxonomy and understanding the pollination biology of the genus.


INTRODUCTION
Piper L. is one of the most diverse genera among the basal clades of angiosperms and is widespread in tropical wet forests around the world (Dyer and Palmer, 2004), with about 2,000 species (Quijano-Abril et al., 2006). Many members of Piper are climbers, although few are shrubs and herbs, and they are abundant in the understory of the tropical wet forests. The monophyly of this large genus has been confirmed by molecular phylogenetic analyses of Piperales (Jaramillo et al., 2004;Wanke et al., 2007;Smith et al., 2008). Species of Piper are easily recognized by their swollen nodes, entire, distichously arranged leaves and terminal spikes that become leaf-opposed. The spikes vary in how they are held (erect vs. pendulous) and length, thickness and colour, but they always consist of minute perianth-less flowers subtended by bracts. The small size of the flowers makes it difficult to observe the number and position of the stamens, the main characters of Candolle´s system (1869,1923), and has thus caused instability in the classification. Indeed, the number of stamens is largely uninformative in Paleotropical Piper. In terms of stem anatomy, P. nigrum L. has a pattern of vascular bundle arrangement that is similar to that of monocots, whereas the vascular bundle arrangement of P. colubrinum Link is similar to that of eudicots (Empari and Sim, 1985). Trimen (1895) recorded nine Sri Lankan species. A survey done by the Department of Export Agriculture of Sri Lanka revealed the presence of eleven species, namely Piper nigrum L., P. betle L., P. longum Blume, P. thwaitesii C.DC., P. subpeltatum Willd., P. argyrophyllum Miq., P. sylvestre Lam., P. zeylanicum Miq., P. trineuron Miq., P. attenuatum Buch.-Ham. ex Miq., and P. chuvya (Miq.) C.DC., the last earlier considered a variety of P. betle (Samuel, 1981) (Figure 1). The Revised Handbook to the Flora of Ceylon (Huber, 1987) has recognized ten species. Of these, P. zeylanicum, P. walkerii Miq., and P. trineuron are endemics, and P. nigrum (black pepper), P. longum (thippili), and P. betle (betel leaves) are of economic importance in Sri Lanka ( Figure 2).
The greatest diversity of Piper is concentrated in the Neotropics (ca. 1300 species: 70%), and the Neotropical clade has been estimated to be 65 mya or younger, which indicates that diversification of the Neotropical clades occurred throughout the Cenozoic (Martínez et al., 2015). In India, Piper is represented by more than 100 species, most of which (65 species) are confined to the northeastern region. Evergreen forests of the southern Western Ghats also form a significant centre of diversity for Piper, with 18 taxa (17 species and two subspecies) reported from the Kerala region (Sasidharan, 2013). Among these taxa, ten are local endemics with one species categorized as critically endangered (MOE, 2012). In Thailand, 38 species are recorded, out of which eight are popularly used by Thai people as vegetables, spices, decoration and medicines and in traditional ceremonies: P. betle, P. longum, P. nigrum, P. pendulispicum C.DC., P. retrofractum Vahl, P. sarmentosum Roxb., P. wallichii (Miq.) Hand.-Maz., and an undescribed Piper species (Chaveerach et al., 2006). African Piper is estimated to comprise about 15 species found mostly in the tropical regions of East, Central, and West Africa. The most common of these species are P. guineense Schumach. & Thonn. and P. capense L. f., which are widespread across many African countries. Piper guineense, otherwise known as West African black pepper, originates from West Africa and compares favourably with black pepper (P. nigrum) as a flavouring for food (Oyemitan, 2017).
A new fossil Piper from the late Cretaceous of Colombia has been identified and described as P. margaritae (Martínez et al., 2015). Previous analyses for Piperales have shown contrasting results for the age of Piper, ranging from the Cretaceous (ca. 92 mya, Smith et al., 2008) to the Paleocene (ca. 57 mya, Symmank et al., 2011;appr. 31 mya, Naumann et al., 2013). Molecular phylogenetic analyses have indicated that Piperales are a relatively early divergent clade (Soltis et al., 2000, Jaramillo andKramer, 2007), with the age of Piperaceae being 91.2 mya, and the crown age of Piper being 71.75 mya. Piper has been

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hypothesized to have dispersed first from the Neotropics to Africa and then to the Pacific and Asia with subsequent dispersal from Asia to Australia and back to Africa .

Morphology of Piper
Although Piper is easy to recognize by a combination of vegetative and reproductive characters, the apparent uniformity of its diminutive flowers presents a significant challenge for developing an infrageneric classification. The earliest classifications of Piperaceae emphasizing Piper recognized 7-15 genera (Kunth, 1839;Miquel, 1843Miquel, -1844. The infrageneric taxonomy of Piper is currently unresolved, and most local treatments have described species without subgeneric affiliation (Yuncker, 1953(Yuncker, , 1972(Yuncker, , 1973Chew, 1972;Yongqian, Nianhe, and Gilbert, 1999). Given their minute size, changes in floral structure may be evolutionarily less important than variation in inflorescence type. It is likely that pollination and dispersal biology have been influenced more by whole inflorescence structure than by individual flower structure (Thies, Kalko, and Schintzler, 1998). In this perspective, modern approaches of imaging hold great promise for finding characters capable of differentiating between species with similar phenotypes (Sedeek et al., 2014) because the most complete way to describe the morphological phenotype is to build a three-dimensional (3D) model (Staedler et al., 2013). The immense amount of data contained in an accurate 3D model can then be mined not only to uncover traits that allow discrimination between closely related taxa but also to explore correlation with other types of data, e.g. pollinators ( Van der Niet et al., 2010) or crop yield (Jiang et al., 2012).

Molecular phylogenetics of Piper
Molecular phylogenetic studies using nuclear ribosomal (nr) ITS DNA and plastid intron psbJ-petA, has been useful in examining the monophyly of Piper (Jaramillo and Manos, 2001;Jaramillo and Callejas, 2004a, b;Tepe et al., 2004;Jaramillo et al., 2008). So far, no study has examined the phylogenetic relationships of the Sri Lankan species of Piper, except for nrITS barcoding work (Jayarathna et al., 2016a). The origin and evolution of Piper in Peninsular India were investigated using nrITS and plastid intron psbJ-petA (Sen et al., 2019). The plastid markers ndhF and trnL intron showed a close relationship of the two Malagasy species with the African P. guineense, but the sequencing of the nuclear G3pdh gene showed some hybridization and introgression with Asian and African species (Palchetti et al., 2020). In addition to DNA sequencing, molecular markers such as RAPD (randomly amplified polymorphic DNA) have been used to characterize genetic diversity among six species of Piper (Shivashankar, 2014).
The 4C content of four New World and five Old World Piper species ranged overall from 2.73 pg in the tetraploid P. longum to 9.62 pg in the octoploid P. nigrum (Samuel et al., 1986). Genome size reports are also available for a few more species from India (Chikkaswamy et al., 2007).

Phytochemical survey of Piper
Piper is one of the most important genera of medicinal plants (Mgbeahuruike et al., 2017;Salehi et al., 2019;Kumar et al., 2020). Species have been reported to possess various pharmacological activities such as insecticidal, antibacterial, anti-inflammatory, antithyroid, antitumor, and hepato properties. Piper nigrum, which is known as the king of spices, contains the major pungent alkaloid piperine that also possesses medicinal properties (Srivastava and Sing, 2017).
The chemical composition and morphology of the Sri Lankan Piper species were investigated for utilizing their valuable traits for crop improvement. Piper nigrum, P. betle, P. longum, P. chuvya, P. siriboa L., P. sylvestre, P. zeylanicum, and P. walkeri were examined in morphological and phytochemical studies (Jayarathna et al. 2016b) using the specimens collected from natural habitats and cultivation. Thirteen morphological and anatomical characters were subjected to cluster analysis to infer species relationships. According to the phenogram in Jayarathna et al. (2016b), the taxa were divided into two main clusters: P. longum, P. zeylanicum, P. sylvestre, P. nigrum, and P. walkeri formed one cluster, whereas P. chuvya, P. betle, and P. siriboa formed a second cluster. This study has played an important role in the crop improvement of P. betle. Leaves of P. betle are considered to be rich in phenolic compounds with antiproliferative, antimutagenic, antibacterial, antidiabetic, and antioxidant properties. Studies on P. betle have reported that it contains important chemical constituents such as chavibetol, chavibetol acetate, and caryophyllene (Arambewela et al., 2011;Shah et al., 2016). Piper longum is used as an important ingredient in traditional medicine in Asia and the Pacific islands and is widely cultivated in Sri Lanka for medicinal purposes. The dried spikes and roots are used in a decoction for acute and chronic bronchitis, fever, and cough (Kumari and Yakandawala, 2008).

Economically important Piper species
Cultivated Piper nigrum: the pepper of commerce comprises dried mature berries of Piper nigrum, the second most widely traded spice worldwide after cinnamon. Black pepper has been considered to have its origin in the Western Ghats of Kerala, India, and may be an allotetraploid (hybrid) due to normal pairing of chromosomes at meiosis (Mathew, 1998). Based on morphological and biosystematic studies, it was suggested that three species, namely P. wightii, P. galeatum (Miq.) C.DC, and P. trichostachyon (Miq.) C.DC, are among the putative parents of P. nigrum (Neema, 2008;Lekhak et al., 2014). In the past, forests were continuous, which might have led to overlapping species ranges and facilitated natural crossing, and such polyploid hybrids might have originated many times at different locations, gradually leading to the establishment of large populations (Abbott et al., 2013). The allotetraploids once formed are effectively genetically isolated from the parents by their different ploidies.
In Sri Lanka, although pepper cultivation is concentrated in the Kandy, Matale, Kegalle, Kurunegala, Badulla, Monaragala, and Ratnapura districts, and it is found in ten of the 25 districts. The area of pepper cultivation has been estimated to be around 39,284 hectares (statistics from Dept. of Export Agriculture 2018). Germplasm collections of Piper nigrum in Sri Lanka hold several local selections (MB12, GK49, DM7, IW5, KW27, and a few others selected based on their yield and quality) and two introduced varieties, Panniyur (from India) and Kuching (from Malaysia), introduced in 1970. Three artificial hybrids between the introduced variety Panniyur and local selections have high yields and high oleoresin and piperine content. Genetic diversity in the germplasm of black pepper has been investigated by various molecular markers such as EST-SSR (Wu et al., 2016) and nuclear microsatellites (Joy et al., 2011;Kumariet al., 2019). The quality of black pepper apart from appearance and diversity is judged by both odour and pungency (Kay, 1970). The degree of pungency and flavour is primarily determined by the intrinsic characteristics of the variety or cultivar, and quality attributes can be improved by the selection and propagation of suitable strains (Purseglove et al., 1981). The black pepper cultivars of Malabar, Lampong, and Sri Lanka have high volatile and non-volatile solvent extracts (Nambudiri et al., 1970). The piperine content of Sri Lankan pepper is higher (7-15%) when compared with the commercial Indian and Malaysian varieties (Jennings and Wrolstad, 1965).
Piper betle: in addition to the extensive and wellestablished domestic market, betel has gained a significant position in the export market since 1974 (Arambewela et al., 2011). Betel leaves are closely associated with many cultural events and rituals, where they are traditionally offered as a mark of respect and auspicious beginnings. Betel leaves have been used for chewing purposes along with other condiments. Six cultivars of P. betle, namely 'Galdalu,' 'Mahamaneru,' 'Kudamaneru,' 'Ratadalu,' 'Nagawalli', and 'Malabulath' are cultivated in Sri Lanka (Arambewela et al., 2011).
Piper longum: this is naturally distributed in the wet and intermediate zones and included in the ten largest important herbal materials in Sri Lanka. A recent study using morphological characters identified three distinct phenetic groups, which highlights the importance of the correct identification of the specimen for ayurvedic treatments (Kumari and Yakandawala, 2008).

Application of next-generation sequencing in Piper
For non-model plants such as black pepper, next-generation sequencing (NGS) technologies offer a great opportunity for rapid access to genetic information (Lister et al., 2009;Hawkins et al., 2010). High-throughput sequencing of the Piper nigrum (black pepper) root transcriptome has been reported (Gordo et al., 2012). Transcripts were functionally annotated and represent the first sequences derived from the transcriptomes of P. nigrum. The complete plastid genome of P. kadsura (Choisy) Ohwi was sequenced and includes 131 genes, including four unique rRNA plus 30 tRNA and 79 protein-coding genes (Lee et al., 2015). In addition, de novo assembly and characterization of fruit transcriptome in P. nigrum is also available (Hu et al., 2015). Recently, comparative transcriptome analysis of several plant tissues including leaves, roots, and spikes has been reported for P. longum L (Dantu et al., 2021). A reference genome of black pepper (P. nigrum) comprised 761.2 Mb sequences assembled into 26 pseudochromosomes provides insights into the molecular basis of species-specific piperine biosynthesis (Hu et al., 2019), the major alkaloid responsible for the pungency and flavour of black pepper. These genomic studies are important for further evolutionary investigations and biological and agronomic research on Piper.
The rapid expansion of NGS has yielded a powerful array of tools to address biological questions at a scale that was not possible until a few years ago. Restrictionsite-associated DNA sequencing (RADseq; Baird et al., 2008;Wang et al., 2012) and whole transcriptome shotgun sequencing (WTS; Wang et al., 2009) have quickly become the predominant genomic methods used in evolutionary studies. As a simple NGS derivative of a more traditional marker-based approaches, such as AFLP (amplified fragment-length polymorphisms; Miller et al., 2007), RADseq in particular has emerged as the gateway genomic approach for most non-model species. It is also referred to as "genotyping by sequencing" (Narum et al., 2013), a term that includes other genomic fragmentsequencing approaches. In addition, high-throughput target capture (hyb-seq) is a general class of methods that achieve genome partitioning through selective enrichment of specific subsets of the genome prior to NGS. Hybseq (sometimes referred to as exon-capture sequencing) consists of enriching genomic libraries for regions of interest (nuclear or organellar), such as highly conserved regions or more variable, low-copy orthologous (Weitemier et al., 2014) or functional loci (Moore et al., 2018). Sequencing 424 genes from over 130 herbarium specimens of Erythroxylum species demonstrated the efficacy of hybseq with highly degraded DNA (White et al., 2021). In land plants, the hyb-seq method has recently become a standard procedure for generating large amounts of sequence data for phylogenomics of non-model organisms (Gernandt et al., 2018;Medina et al., 2019).
High throughput DNA sequencing technologies and analysis algorithms provide an opportunity to study in detail genome-wide variability across groups of individuals, adding significant power to evolutionary investigations. In particular, RADseq (Baird et al., 2008) was established as a powerful tool for interspecific comparisons across a large number of loci in non-model organisms (Cariou et al., 2013). RADseq had been used in phylogenetics of tribe Shoreae, Dipterocarpaceae (Heckenhauer et al., 2018) and the European shrub willows (Salix L subgens. Chamaetia and Vetrix; Wagner et al., 2018). In Heliosperma pusillum, RADseq has been successfully used to examine parallel ecological divergence (Trucchi et al., 2017), whereas in radiating New Caledonia Diospyros this method could be used to evaluate substrate effects on speciation (Paun et al., 2016). In Nicotiana (Solanaceae), RADseq was employed to evaluate the possible interactions between chromosome number and genome size changes, which are both controlled by epigenetic mechanisms (Chase et al., 2022).
RADseq analyses of wild and cultivated species of Sri Lankan Piper will contribute to the taxonomic revision of this genus. Inclusion of selected species from South America, India, and Southeast Asia will help to detect the origin and timing of the endemic radiation of P. zeylanicum, P. trineuron, and P. walkerii. However, RADseq requires good quality DNA, in general, isolated from fresh or silicagel dried plant material, which becomes disadvantageous if herbarium specimens are expected to be used for phylogenomics. Investigations at the population level for P. nigrum using RADseq will give an overview of the genetic diversity of this cultivated, economically important species across the different agroclimatic regions, which will help selection for high-yielding varieties and thus the economy.

Computed tomography of inflorescence
In addition to the genetic diversity, it is important to investigate the floral structure and composition of bisexual flowers across the spikes of P. nigrum, which plays a decisive role in the yield of black pepper. Application of computed tomography to obtain the 3D structure of flowers in spikes of the wild and selected cultivated P. nigrum accessions (composition of male, female and bisexual flowers in the spikes) will provide an overview of pollination and fruit set in the spikes. A detailed study of inflorescences of the wild species will help to establish a phylogenetically based infrageneric classification of the genus Piper.

CONCLUSION
The NGS-based technique, RADseq, has been suggested as a powerful tool for intra-and interspecific comparisons across a large number of loci in non-model organisms, delivering clear phylogenetic information. This technique will enable to resolve phylogenetic relationships among the Piper species. It will also help to date the origin of the endemic taxa as well as to identify new species. In addition, the use of this technology at the population level in P. nigrum will give a clear overview of the genetic diversity of black pepper growing in different agroclimatic regions in Sri Lanka and thus be an aid for crop improvement. Further examination of looking into floral morphology of the wild species using 3D computed tomography may help in the taxomic revision of Piper. In addition computed tomography of the inflorescence (spikes) in cultivated P. nigrum will provide insights into pollination biology and fruit set to improve the yield of black pepper. Studies on chromosome and genome size can play an important role in black pepper breeding programs.