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Genetic Diversity and Population Structure of Capirona (Calycophyllum Spruceanum Benth.) from the Peruvian Amazon Revealed by RAPD Markers

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Genetic Diversity and Population Structure of Capirona (Calycophyllum Spruceanum Benth.) from the Peruvian Amazon Revealed by RAPD Markers Article Genetic Diversity and Population Structure of Capirona (Calycophyllum spruceanum Benth.) from the Peruvian Amazon Revealed by RAPD Markers Carla L. Saldaña 1 , Johan D. Cancan 1, Wilbert Cruz 1 , Mirian Y. Correa 2 , Miriam Ramos 3, Eloy Cuellar 3 and Carlos I. Arbizu 1,* 1 Dirección de Desarrollo Tecnológico Agrario, Instituto Nacional de Innovación Agraria (INIA), Av. La Molina 1981, Lima 15024, Peru; [email protected] (C.L.S.); [email protected] (J.D.C.); [email protected] (W.C.) 2 Facultad de Ciencias Biológicas, Universidad Nacional Pedro Ruíz Gallo, Juan XXIII No 391, Lambayeque 14013, Peru; [email protected] 3 Facultad de Ciencias Forestales, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 15024, Peru; [email protected] (M.R.); [email protected] (E.C.) * Correspondence: [email protected] Abstract: Capirona (Calycophyllum spruceanum Benth.) is a tree species of commercial importance widely distributed in South American forests that is traditionally used for its medicinal properties and wood quality. Studies on this tree species have been focused mainly on wood properties, propagation, and growth. However, genetic studies on capirona have been very limited to date. Currently, it is possible to explore genetic diversity and population structure in a fast and reliable Citation: Saldaña, C.L.; Cancan, J.D.; Cruz, W.; Correa, M.Y.; Ramos, M.; manner by using molecular markers. We here used 10 random amplified polymorphic DNA (RAPD) Cuellar, E.; Arbizu, C.I. Genetic markers to analyze the genetic diversity and population structure of 59 samples of capirona that were Diversity and Population Structure of sampled from four provinces located in the eastern region of the Peruvian amazon. A total of 186 Capirona (Calycophyllum spruceanum bands were manually scored, generating a 59 × 186 presence/absence matrix. A dendrogram was Benth.) from the Peruvian Amazon generated using the UPGMA clustering algorithm, and, similar to the principal coordinate analysis Revealed by RAPD Markers. Forests (PCoA), it showed four groups that correspond to the geographic origin of the capirona samples 2021, 12, 1125. https://doi.org/ (LBS, Irazola, Masisea, Iñapari). Similarly, a discriminant analysis of principal components (DAPC) 10.3390/f12081125 and STRUCTURE analysis confirmed that capirona is grouped into four clusters. However, we also noticed that a few samples were intermingled. Genetic diversity estimation was conducted Academic Editors: Carol A. Loopstra considering the four groups (populations) identified by STRUCTURE software. AMOVA revealed the and Timothy A. Martin greatest variation within populations (71.56%) and indicated that variability among populations is Received: 14 July 2021 28.44%. Population divergence (Fst) between clusters 1 and 4 revealed the highest genetic difference Accepted: 20 August 2021 (0.269), and the lowest Fst was observed between clusters 3 and 4 (0.123). RAPD markers were Published: 22 August 2021 successful and effective. However, more studies are needed, employing other molecular tools. To the best of our knowledge, this is the first investigation employing molecular markers in capirona Publisher’s Note: MDPI stays neutral in Peru considering its natural distribution, and as such it is hoped that this helps to pave the way with regard to jurisdictional claims in towards its genetic improvement and the urgent sustainable management of forests in Peru. published maps and institutional affil- iations. Keywords: amazon forest; capirona; genetic diversity; molecular markers; population structure Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. Calycophyllum spruceanum (Benth.) (Rubiaceae) is a fast-growing forest species [1] with This article is an open access article a natural origin in the Amazon basin, covering the territories of Bolivia, Peru, Colombia, distributed under the terms and and Brazil [2]. Capirona is a valuable timber forest species in the Peruvian Amazon that conditions of the Creative Commons is widely used by local communities and is exported around the world. It has a high- Attribution (CC BY) license (https:// density, durable wood; therefore, it is used for the construction of economically valuable creativecommons.org/licenses/by/ products [3]. Capirona has high potential for cultivation in agroforestry systems, which 4.0/). Forests 2021, 12, 1125. https://doi.org/10.3390/f12081125 https://www.mdpi.com/journal/forests Forests 2021, 12, 1125 2 of 12 counters unsustainable land use due to activities such as slash-and-burn agriculture, and this species represents an alternative to increase rural incomes and development [4]. Thus, capirona is currently the object of research in the Peruvian Amazon basin in order to estab- lish participatory research with local communities, optimizing genetic and silvicultural management strategies including community conservation methods [1] since it is known that the indiscriminate use of forest species is generating deforestation in various regions of the planet [5–7]. Genetic diversity studies are indispensable for conducting conservation programs and sustainable management. Moreover, these studies based on molecular mark- ers provide important information on the genetic makeup of the population because they are independent of environmental factors [8]. A demographic study would not provide this kind of information, influencing decision making. In recent years, molecular markers such as DNA-based markers like random-amplified polymorphic DNA (RAPD), microsatellite or simple sequence repeat (SSR) and inter simple sequence repeat (ISSR) have been used in studies for the analysis of phylogeny, inter-species relationships and genetic diversity for forest species like Pinus leucodermis, Cedrela odorata Eucalyptus globulus, Swietenia macrophylla, Bertholletia excelsa and Populus deltoides [9–14]. Previous research determined the genetic variation of capirona by using amplified fragment length polymorphisms (AFLP) among nine populations from the Peruvian Ama- zon [1]. They demonstrated that variation among individuals within populations is pre- dominant. However, they did not employ samples from the Madre de Dios department, which is considered a place where capirona exists naturally [15]. On the other hand, Tauchen et al. [3] identified the genetic variability of capirona at the level of DNA polymor- phism evaluated by non-specific ITS primers of plant materials from Pucallpa, department of Ucayali, in the Peruvian Amazon. Their results showed that morphological diversity is greater than genetic diversity of capirona. They also found that environmental factors had a greater impact on the phenotype in those accessions. Finally, their results are in line with the claims of previous studies on C. spruceanum, suggesting greater variation within provenances than among provenances. Capirona is an orphan forest species, and no further studies of genetic diversity with molecular markers have been reported. RAPD is an especially useful DNA-based method for initial research of genetic diver- sity in plant species. These studies are crucial for the initiation of conservation programs, and the rapidity of working with RAPD markers is an advantage because they can deliver crucial information in a short time [16–18]. They have been used to differentiate cultivars and also on genetic mapping [19–21]. Although some researchers have shown reservations regarding the usefulness of RAPD, basically due its poor reproducibility [22,23], other studies mentioned that RAPD can be employed for genetic analyses [24–28]. Additionally, other studies demonstrated a high level of reproducibility [29]. A great potential for the analysis of polymorphism and genetic diversity has also been demonstrated by RAPD markers as they are easy to test and require low concentrations of DNA [17]. The objective of this study was to determine the genetic diversity and population structure of capirona from primary forests located in the departments of Ucayali, San Martín and Madre de Dios located in the Peruvian Amazon to help pave the way towards its modern breeding program, the sustainable conservation of this tree species in the near future. 2. Materials and Methods 2.1. Plant Material We collected 59 samples of capirona from Ucayali, San Martín and Madre de Dios departments in the Peruvian Amazon considering their natural range of distribution. Leaf samples were collected in paper envelopes, stored in airtight containers with silicone gel, and transported to the National Institute of Agricultural Innovation (INIA) for genomic DNA extraction. Further details of the samples examined in this study are available in Table1. Forests 2021, 12, 1125 3 of 12 Table 1. Codes and geographic information of the 59 samples of capirona from the Peruvian Amazon characterized in this study. Code Region Department Latitude Longitude Code Region Department Latitude Longitude cap018 Irazola Ucayali 8◦510 S 75◦70 W cap057 LBS San Martín 6◦280 S 76◦200 W cap019 Irazola Ucayali 8◦510 S 75◦70 W cap058 LBS San Martín 6◦280 S 76◦200 W cap020 Irazola Ucayali 8◦510 S 75◦70 W cap059 LBS San Martín 6◦280 S 76◦200 W cap021 Irazola Ucayali 8◦510 S 75◦70 W cap060 LBS San Martín 6◦280 S 76◦200 W cap022 Irazola Ucayali 8◦510 S 75◦70 W cap061 LBS San Martín 6◦280 S 76◦200 W cap023 Irazola Ucayali 8◦510 S 75◦70 W cap062 LBS San Martín 6◦280 S 76◦200 W cap024 Irazola Ucayali 8◦510 S 75◦70 W cap063 LBS San Martín 6◦280 S 76◦200 W cap030 Masisea
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