The delimitation of species boundaries has been a constant challenge to the fields of systematics, natural history, and conservation biology. Subtle and minor morphological differences in a widespread species complex make delimiting species boundaries particularly difficult. High throughput targeted sequencing of hundreds of loci has allowed researchers to obtain improved insights into evolutionary processes and resolved previously ambiguous phylogenetic relationships. Piper amalago (subgenus Gonistum) is morphologically similar to, and geographically overlaps with, many other Neotropical Piper species that have narrow restricted distributions, or occur as narrow endemics. Taxonomists have debated whether morphological differences between P. amalago and other similar species merit distinct species status or if these taxa represent a single widespread species exhibiting extensive intraspecific variation. Recent molecular analyses demonstrated that P. amalago is paraphyletic with morphologically similar species, notably P. martensianum, but these findings lack phylogenetic support. This study investigates the phylogenetic relationships of P. amalago and several closely related species using multiple DNA sequences, and seeks to determine whether species can be delimited on the basis of phylogenetic, geographical, and morphological information. The Angiosperms353 bait set was used to retrieve nuclear genes to build maximum likelihood and multispecies coalescent phylogenetic hypotheses. Available DNA sequence data are consistent with the presence of a single, monophyletic, widespread species complex, with considerable morphological variation and some geographic structure. While the drivers of the morphological variation within this complex are not yet fully understood, we have a better understanding of evolutionary relationships and species boundaries within Piper, a giant genus.

Piper (Piperaceae) is a species-rich pantropical genus (∼2000 species) with distinctive morphological characteristics such as swollen nodes, lack of a perianth, and one-seeded berry fruits. Sub-generic lineages within Piper are often associated with morphological traits and are well supported with molecular data, but sampling in previous studies was incomplete and conflict exists between morphological data and molecular inference. In this paper, Piper section Enckea is evaluated for monophyly by pairing DNA, primarily from herbarium material, with the Angiosperms353 bait set, high throughput sequencing, recently developed bioinformatic techniques, and two methods of phylogenetic reconstruction, maximum likelihood and multi-species coalescence. The results suggest continued support for recognition of Piper sect. Enckea as a monophyletic section and contribute to a better understanding of relationships among species within the section. However, they also raise questions about the morphological features used to define P. sect. Enckea and delimit species within the section.