Caught in the act: Incipient speciation at the southern limit of Viburnum in the Central Andes

A fundamental objective of evolutionary biology is to understand the origin of independently evolving species. Phylogenetic studies of species radiations rarely are able to document ongoing speciation; instead, modes of speciation, entailing geographic separation and/or ecological differentiation, are posited retrospectively. The Oreinotinus clade of Viburnum has radiated recently from north to south through the cloud forests of Mexico and Central America to the Central Andes. Our analyses support a hypothesis of incipient speciation in Oreinotinus at the southern edge of its geographic range, from central Peru to northern Argentina. Although several species and infraspecific taxa of have been recognized in this area, multiple lines of evidence and analytical approaches (including analyses of phylogenetic relationships, genetic structure, leaf morphology, and climatic envelopes) favor the recognition of just a single species, V. seemenii. We show that what has previously been recognized as V. seemenii f. minor has recently occupied the drier Tucuman-Bolivian forest region from Samaipata in Bolivia to Salta in northern Argentina. Plants in these populations form a well-supported clade with a distinctive genetic signature and they have evolved smaller, narrower leaves. We interpret this as the beginning of a within-species divergence process that has elsewhere in the neotropics resulted repeatedly in Viburnum species with a particular set of leaf ecomorphs. Specifically, the southern populations are in the process of evolving the small, glabrous, and entire leaf ecomorph that has evolved in four other montane areas of endemism. As predicted based on our studies of leaf ecomorphs in Chiapas, Mexico, these southern populations experience generally drier conditions, with large diurnal temperature fluctuations. In a central portion of the range of V. seemenii, characterized by wetter climatic conditions, we also document what may be the initial differentiation of the leaf ecomorph with larger, pubescent, and toothy leaves. The emergence of these ecomorphs thus appears to be driven by adaptation to subtly different climatic conditions in separate geographic regions, as opposed to parapatric differentiation along elevational gradients as suggested by Viburnum species distributions in other parts of the neotropics.

Methods
We collected leaf tissues from herbarium samples or from our own recent collections of these plants from central Peru to southern Bolivia. We included five samples previously classified as V. incarum, 29 as V. seemenii, and 13 as “New Name 2”. Whenever possible multiple individuals per population were included, and all collections were deposited in the Yale Herbarium (YU) except for three specimens located in NY and MO (Appendix 1). Total DNA was extracted from leaf tissues using DNeasy plant extraction kits (Qiagen Inc., Hilden, Germany).

RAD-seq data were generated by Floragenex Inc. (Eugene, Oregon) by digesting genomic DNA with the PstI restriction enzyme, followed by sonication and size selection at 400 bp. Samples were ligated with 10bp barcodes for multiplexing. Then samples were pooled and sequenced on Illumina HiSeq 2500 or 4000 to produce 100bp SE reads.

Samples were demultiplexed and assembled into orthologous loci with ipyrad v.0.9.85 (Eaton and Overcast 2020) using a reference-guided method. For this purpose, we used the draft reference genome of V. lautum (Donoghue et al. 2022). We discarded paralog reads that mapped to multiple locations in the reference.