PLAN DE INVESTIGACIÓN

(Anexo a la Solicitud)

1. Nombre del Proyecto o Estudio.

Sistemática, Evolución, y biogeografía del clado Viburnum (Adoxaceae)

2. Área que cubre el Proyecto o Estudio:

The study area will be focused on individuals of Viburnum located in the regions of
Amazonas, Cajamarca, Cusco, Junín, Pasco and Piura (Table 1), outside of protected
natural areas and buffer zones. Access to most of these areas will be by carreteras y
caminos utilizados por los pobladores locales.

Table 1. Regions, Provinces, and Districts where sampling will occur.

Region Province District
Amazonas Bongará Florida
Amazonas Bongará Yambrasbamba
Amazonas Chachapoyas Chachapoyas
Amazonas Chachapoyas Chuquibamba
Amazonas Chachapoyas Leimebamba
Amazonas Chachapoyas Molinopampa
Cajamarca Celendin Celendin
Cajamarca San Ignacio Huarango
Cajamarca San Ignacio San Jose de Lourdes
Cajamarca San Ignacio Tabaconas
Cusco La Convención Huayopata
Cusco La Convención Maranura
Cusco La Convención Santa Ana
Cusco La Convención Santa Teresa
Cusco Quispicanchi Marcapata
Cusco Quispicanchi Camanti
Cusco Urubamba Ollantaytambo
Junín Chanchamayo San Ramon
Junín Chanchamayo Vitoc
Junín Tarma Palca
Junín Tarma Tarma
Pasco Oxapampa Chontabamba
Pasco Oxapampa Huancabamba
Pasco Oxapampa Oxapampa
Piura Ayabaca Ayabaca

3. Antecedentes del Proyecto o Estudio.

This proposal is for obtaining permission to collect samples for the systematic study of
the major Neotropical clade (the Orienotinus clade) of Viburnum, which occurs in
mountanous areas from Mexico to southern Boliva and northern Argentina.
The woody angiosperm clade Viburnum (Adoxaceae) has an almost global distribution
and is constituted by approximately 165 species of shrubs and small trees. In a
continuous progression of phylogenetic analyzes over many years, we have concluded
that Viburnum originated in rainforests of the tropical mountains of Southeast Asia and
that at least 10 lineages adapted to temperate climates of the northern hemisphere
(Donoghue et al., 2004; Winkworth & Donoghue, 2004, 2005; Donoghue & Clement,
2011, 2012; Clement et al., 2014; Spriggs et al., 2015). From temperate environments
of the Asian continent, four lineages of Viburnum spread to Europe, while five other
lineages entered the new world, probably using the Beringia Strait. Of the five lineages
present in the new world, the oldest and most widespread lineage is Porphyrotinus,
which is characterized by purple fruits. The Porphyrotinus clade is made up of several
subclades, one of these subclades includes the species complex V. dentatum present in
North America and its sister clade, Oreinotinus, which contains ca. 36 species
distributed in rain forests in the Caribbean, the mountains of Mexico and Central
America, and the Andes to southern Bolivia and northern Argentina. The Oreinotinus
species are the focus of this study within Peru. In Peru, 11 species of have been
described within this clade (Killip & Smith, 1930; Killip, 1937; Diels, 1942; Dudley,
1969).

In Neotropical Viburnum, we have discovered evidence that indicates the existence of

several pairs of sister species in mountainous regions. These species differ from one
another by a remarkable set of foliar characters. A group of species presents large
leaves, pubescent (with hairs) and serrated margins. A second group of species
presents small leaves, glabrous (without hair) and continuous margins. In the recent
past, the presence of these two groups of species was interpreted as evidence that the
ecological niche of these species did not change, that is, these two types of plants did
not evolve to colonize other environments (niche conservation).

However, new evidence suggests that the two morphological groups have differentiated
one from the other repeatedly, evidence that these plants adapt to new environments
when this is possible. If this is the case, the Viburnum species would represent a case
in which the same adaptation process has occurred repeatedly and thus contributed to
the speciation of the Viburnum clade. In this sense, the objective of our research is to
study the evolution of the Viburnum clade as a model group for the study of ecological
speciation in plants.

Based on our knowledge of Viburnum in other Neotropical countries (e.g., Mexico), we

hypothesized that Viburnum plants with the two types of leaves (large / serrated /
pubescent vs. small / continuous margin / glabrous) have evolved independently in Peru
and other South American countries. Additionally, we hypothesize that the two forms
have diverged from a common ancestor, and that from this common ancestor two types
of species have evolved in order to occupy different climatic niches (ecosystems). To
verify this hypothesis, we propose to collect specimens of Viburnum in Peru in multiple
localities in mountainous regions of Peru, in order to study the geographic distribution
patterns of plants with different leaf types.
To achieve this goal, we need to collect herbarium specimens (with their respective
leaves) and small tissue samples (leaves) from each of these locations, to incorporate
them into a phylogenetic analysis using molecular data. In this way, we can determine if
the Viburnum species present in the mountains of Peru have differentiated according to
the same pattern of ecological differentiation of other Neotropical species.

4. Justificación del Proyecto o Estudio. (Justification of the Project or Study)

Viburnum possesses a series of comparative advantages for the study of ecological
speciation, and its study will result in important discoveries in the fields of evolution and
ecology. Our work is funded by the National Science Foundation (NSF) of the United
States, and consists of characterizing leaf morphology and of using sequencing
associated with restriction sites (RADseq) to identify pairs of sister species in a
phylogenetic context. In this way, it is expected to elucidate the importance of the
morphological differences mentioned above.

Peruvian Viburnum exhibit the patterns of morphological variation that are observed in
other Viburnum occurring elsewhere in the Neotropics, and thus may be an additional
example of replicated speciation. Understanding the evolution of the Peruvian species is
crucial to establishing the extent to which replicated evolution has played a role in the
evolution of the of Viburnum. The results of this project will better document the diversity
Peruvian Viburnum and provide a better understanding of the evolutionary processes
that generated the incredible plant diversity of Peru and South America. Our work will
also allow for a better delimitation of species in Peru and South America. All of these
results will increase general scientific knowledge and will enhance conservation and
education efforts centered on the protection and preservation of the Peruvian flora.

5. Objetivos: General y Específicos.

This project is part of a broader set of studies aimed at understanding the patterns of
diversification and speciation in Neotropical Viburnum. The main objectives of this work
are to evaluate the diversification patterns of Peruvian Viburnum, focusing in particular
patterns of replicated evolution.

Concrete objectives:
• Study the evolutionary history and infer the possible mechanisms and processes
responsible for diversification and morphological variation in Neotropical Viburnum
using (a) morphological data and (b) DNA sequence data.
• Develop hypotheses of evolutionary history of Neotropical Viburnum throughout its
distribution, using molecular phylogenetic analysis.
• Verify the delimitation between species and characterize the morphological variation,
using morphometric analyses.

Objectives of the project in Peru:

• Collect specimens of Viburnum for morphological and molecular phylogenetic studies.

6. Métodos y Técnicas detallados (Detailed methods and techniques).

Collection of biological material and extraction of DNA
The materials and equipment are traditionally used by botanists to make collections of
samples of vascular plants for herbarium: wooden press, hand pruning shears, digital
camera, GPS unit, newspaper, 70% alcohol, permanent markers, bags plastic, field
book. Photographs will be taken to document the habitat and habit, leaves, flowers, and
fruits. The specimens will be preserved in alcohol for morphological analysis and in
silica gel for molecular analysis.

Samples will be taken from specimens of Viburnum covering the entire geographical
distribution of the Orienotinus clade from Mexico to Bolivia. Total DNA extraction of the
individuals will be made from dried leaves preserved in silica gel using DNeasy Plant
Mini Kit (QIAGEN). In addition, samples of the flowers, fruits, leaves, and twigs of these
individuals will be taken and preserved in 70% alcohol for morphological and anatomical
analysis. The herbarium material will be deposited in the San Marcos Herbarium (USM)
and Yale University Herbarium (YU).

Phylogenetic analyses
We will infer the phylogenetic history of Peruvian Viburnum along with other species in
Oreinotinus clade using restriction site associated DNA (RADseq) data. RAD
sequencing, data assembly, and tree inference will be conducted following
methodologies presented in Eaton et al. (2016) and Spriggs et al. (2019).

Morphometric analysis
Quantitative morphological characters will be measured with a calipers and
stereoscopic microscope. Multivariate analyses will be used to visualize and explore
morphological variation and detect coorilations among characters. The multivariate
analyses will be conducted usign the R software library (R Development Core Team,
2008) and relevant packages.

7. Detalle y Justificación de la Colecta definitiva y/o Captura temporal:

Table 2. List of species to be collected and number of individuals to be sampled.

Species Number of Individuals to be
Sampled
Viburnum ayavacense Kunth. 5
Viburnum incarum Graebn. 5
Viburnum hallii (Oerst.) Killip & A.C.Sm. 10
Viburnum mathewsii (Oerst.) Killip & A.C. Sm. 5
Viburnum reticulatum Ruiz & Pav. 10
Viburnum seemenii Graebn. 5
Viburnum tridentatum Killip & A.C. Sm. 5
Viburnum triphyllum Benth. 10
Viburnum witteanum Graebn. 5
Viburnum wurdackii T.R. Dudley 5

8. Cronograma de trabajo detallado:

Table 3. Cronograma de trabajo detallado

2019 2020 2021

1° 2° 3° 4° 1° 2° 4° 1°
Atividades Tri Tri Tri Tri Tri Tri 3° Tri Tri Tri 2° Tri

Muestras de muestreo X

Extracción de ADN X X X

Obtención de los datos

morfológicos X X X X

Análisis de datos
morfológicos X X X

Construcción y
secuenciación de la
biblioteca RAD-seq X X X

Análisis de datos
SNPs obtenidos a
través de RAD-seq X X

Preparación de
trabajos científicos X

9. Referencias bibliográficas (Bibliographic references):

Clement, W. L., & Donoghue, M. J. (2011). Dissolution of Viburnum section Megalotinus

(Adoxaceae) of Southeast Asia and its implications for morphological evolution and
biogeography. International Journal of Plant Sciences, 172(4), 559-573.

Clement, W. L., & Donoghue, M. J. (2012). Barcoding success as a function of

phylogenetic relatedness in Viburnum, a clade of woody angiosperms. BMC
evolutionary biology, 12(1), 73.

Clement, W. L., Arakaki, M., Sweeney, P. W., Edwards, E. J., & Donoghue, M. J.
(2014). A chloroplast tree for Viburnum (Adoxaceae) and its implications for
phylogenetic classification and character evolution. American journal of botany, 101(6),
1029-1049.

Diels, L. (1942). Neue Arten aus Ecuador V. Notizblatt des Botanischen Gartens und
Museums zu Berlin-Dahlem. 15: 784-787.

Donoghue, M. J., Baldwin, B. G., Li, J., & Winkworth, R. C. (2004). Viburnum phylogeny
based on chloroplast trnK intron and nuclear ribosomal ITS DNA
sequences. Systematic Botany, 29(1), 188-198.

Dudley, T. R. (1969). Viburnum wurdackii, a new Peruvian species. Rhodora, 71(788),

554-559.

Eaton, D. A., Spriggs, E. L., Park, B., & Donoghue, M. J. (2017). Misconceptions on
missing data in RAD-seq phylogenetics with a deep-scale example from flowering
plants. Systematic Biology, 66(3), 399-412.

Killip, E. P., & Smith, A. C. (1930). The South American species of Viburnum. Bulletin of
the Torrey Botanical Club 56(5), 245-258.

Killip, E. P. (1937) Caprifoliaceae. In: Macbride, J. F. (ed), Flora of Peru. Field Museum
of Natural History, Botany Series 13, 282-287.

R Development Core Team (2008). R: A language and environment for statistical

computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-
0, URL http://www.R-project.org.

Spriggs, E. L., Clement, W. L., Sweeney, P. W., Madriñán, S., Edwards, E. J., &
Donoghue, M. J. (2015). Temperate radiations and dying embers of a tropical past: the
diversification of Viburnum. New Phytologist, 207(2), 340-354.

Spriggs, E. L., Eaton, D. A., Sweeney, P. W., Schlutius, C., Edwards, E. J., &
Donoghue, M. J. (2018). Restriction-Site-Associated DNA Sequencing Reveals a
Cryptic Viburnum Species on the North American Coastal Plain. Systematic
Biology, 68(2), 187-203.

Winkworth, R. C., & Donoghue, M. J. (2004). Viburnum phylogeny: evidence from the
duplicated nuclear gene GBSSI. Molecular Phylogenetics and Evolution, 33(1), 109-
126.

Winkworth, R. C., & Donoghue, M. J. (2005). Viburnum phylogeny based on combined

molecular data: implications for taxonomy and biogeography. American Journal of
Botany, 92(4), 653-666.