ID: 727

DNA barcoding for the identification of endangered plants with

commercial use: Gentianella spp. (Gentianaceae) from the
Central Andes of Peru
Susy J. Castillo1, 2 , Mónica Arakaki1 , Asunción Cano1 , José Roque1, Joaquina Albán1 & José Olivera3
1Universidad Nacional Mayor de San Marcos . Museo de Historia Natural. Lima – Perú. 2E-mail:susy_827@yahoo.com
3Instituto de Investigación de Bioquímica y Nutrición. Facultad de Medicina de la UNMSM. Lima – Perú.

Abstract
Gentianella (Gentianaceae), a genus with 74% of its species being endemic at the national level, includes at least four endangered species exploited by its medicinal properties,
all together commercialized under Gentianella alborosea "hercampuri". As part of a larger phylogenetic study, barcode markers were used to resolve relationships, and define
species boundaries, among Peruvian Gentianella. Even with low supports, Gentianella clades share affinities in their geographical distribution and growth form, with few
exceptions.

Introduction G1
Gentianella, a genus with more than 300 species, is distributed
worldwide, however most of the species diversity is found in the Andes
G2
of South America1, 2. Peru holds 100 species, being 74% of them
national endemisms, mostly distributed above 3800 m3,4,5,6,7,8,9.
There are at least four species (G. alborosea, G. nitida, G. tristicha, G. G3
thyrsoidea) known as "hercampuri“ (Figs.1B, C, G, H), with significant
demand for their medicinal properties in national and international
G4
markets10, 11, 12(Figs.1.B, C, G, H). Even when exploitation requires state
regulations, the mechanisms are not effective, mainly due the informal
extraction and incorrect taxonomic identification. G5
Our study is focused on resolving phylogenetic relationships among
Gentianella from South America. At the same time, cover a necessity
for enrichment of the DNA reference library for Gentianaceae. Intensive
field work and morphological characterization has allowed the
establishment of their real distribution, as well as to establish a
hypothesis of relationships that is being tested, in the present work,
with molecular markers traditionally used in barcoding (ITS, matK).

Figure 2. Preliminary phylogenetic analyses based on Maximum Likelihood

for ITS2+matK (A), and ITS2 (B, C), for species of Gentianella, plus outgroups, included in the
present study. Bootstrap values (10000 replicates) are above the branches.

Results and Discussion

In our analyzes Gentianella species form a monophyletic clade that has
Swertia (ITS) or Halenia (ITS2-matK) as the sister group (Fig.2).
Gentianella campestris, type species of the genus, is found at the base.
Preliminary results show low support for most of the branches, thus it
is not possible at this point to establish a hypothesis of relationships
among Peruvian Gentianella. However, in the ITS2-matK analysis
(Fig.2,A), several of the groupings contain species with similar growth
form and/or geographical distribution. Groups G1 and G2 are mostly
perennial, robust, monocarpic or polycarpic, distributed in central-
north Peru (Ancash and Cajamarca), being exceptions G. nitida, G.
persquarrosa and Gentianella sp.1. Groups G3, G4 and G5 correspond
Figure 1. A) Study area, B) Gentianella alborosea “hercampuri”, C) G. nitida “hercampuri”, D)
Yanasinga mountain range (Ticlio, Lima), E) Sampling work, F) G. incurva, G) G. tristicha to species from central Peru (Junín, Lima, Pasco). G3 contains delicate
“hercampuri”, H) G. thyrsoidea “japallanshacoc”, I) Imaging work in the laboratory. and robust small herbaceous, distributed very closely. G4 includes
Species were identified with the help of specialized literature and examination of Type subshrubs, the exception being G. nitida. G. nitida, together with G.
specimens from national (USM, MOL, HAO, HUT) and international (B, F, G, NY, P, US)
herbaria. ruizii, have non-vascularized fimbriae in the corolla, a character only
seen in these two species. G5 contains small caespitose herbs. The
Methods highest percentages of support correspond to specimens of the same
Study area & fieldwork. Samples from previous collections, plus newly species from different populations, for example G. thyrsoidea (99), and
collected specimens were used. Additionally, we added samples and G. ruizii (99).
results from previous Gentianella taxonomic treatments13 developed in
the Central Andes (Fig. 1,A ). Most of the collections for the molecular Conclusions
portion of the study were done during 2017. We obtained a total of
130 samples, belonging to 40 Gentianaceae species, between herbaria Preliminary results are concordant with what is known for the
vouchers and samples recently preserved in silica gel. A few sequences Gentianeae tribe. Until this work, little work has been done with
representing the type species of genera Gentianella, Gentiana and Gentianella of South America at the molecular level. Also, collections
Halenia, from other regions, were obtained from GenBank. The project and treatments were scarce or incomplete. Our results show the
counts with the corresponding permits from SERFOR, the national effectiveness of ITS2 in the differentiation of species and placement
regulatory authority (Fig.1, E, F, I). within recognized clades, and concordant with patterns of
DNA extraction, amplification and sequencing. DNA isolations were distribution and growth form. However, these results are not
performed with a CTAB protocol14. Standard PCRs were carried out with sufficient to establish a phylogenetic hypothesis. A better sampling in
primers ITS2F and ITS4 (for ITS), and KIM_3F and KIM_1R (for matK). specimens and molecular markers should improve our results. The
PCR products were sequenced in both directions by Macrogen Inc. species with commercial use showed marked differences with the
(Macrogen Inc., Seoul, Korea). Sequences were edited in Codon Code ones sold in markers, thus we see a good potential in the
Aligner and aligned automatically. Maximum Likelihood analyses were development of identification protocols based on the data obtained.
performed in IQ-tree15, with 10000 bootstrat replicates.
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