Diversity and population genetic structure of the wax palm Ceroxylon quindiuense in the Colombian Coffee Region

The wax palm from Quindío (Ceroxylon quindiuense) is an icon of the cultural identity of the coffee growing eco-region and of all Colombia. Processes of urbanization, expansion of the agricultural and livestock area, among others, have increased its level of threat. Protecting this palm from extinction is important at an ecological level, given its function as a key species in Andean ecosystems. This work evaluated the diversity and population genetic structure of the wax palm from Quindío in five populations of the Colombian coffee region eco-region (Andean zone) by using ten microsatellite molecular markers. Two groups were identified at genetic diversity level, along with a heterozygote deficiency in all the populations possibly due to cryptic population structure effects mediated by loss of habitat. The five sampling units considered presented a total significant genetic structure, revealing a high degree of reproductive isolation. The results presented here add to the Conservation Plan for this species existing in Colombia.


Introduction 28
Loss of habitat is the processes with the highest impact on tropical ecosystems [1]. This process has obvious 29 effects, like modification of the landscape and local elimination of some species; but can also have effects on the long-

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The wax palm from Quindío (Ceroxylon quindiuense) is the Colombian national tree [5]; it is distributed in the 35 country's three mountain ranges of the Andes [6], often being a dominant plant from the montane rain forest canopy 36 and considered a key species within the ecosystems it inhabits [7]. It is a fundamental part of the structure and diversity 37 that makes up the Colombian coffee cultural landscape, playing a crucial role as source of food for wildlife, like birds 38 and small mammals [7,8].

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This species is currently in danger of extinction (EN), according to the red list of palm species [9]. Some of the threats 40 affecting C. quindiuense and, generally, palms of the genus include their commercial use through extraction of leaves 41 for the Catholic Holy Week, an activity that has been managed and reduced considerably [9]. Other threats include 42 paddocking processes which involves the loss of habitat and low regeneration because of the introduction of other uses 43 of the soil they occupy (mainly crops and livestock). This considerable reduction of its habitat has permitted estimating 44 that their population has diminished by over 50% in the last three generations (210 years) [10].

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Bearing in mind the latent threats this species has for its survival, it becomes relevant to know the effect caused by the 46 loss of habitat on the persistence, genetic diversity, and future viability of umbrella species, like C. quindiuense, which 47 currently has no defined conservation areas in Colombia. Thus, knowledge and understanding of the diversity and 48 genetic structure of a species plays an important role for the conservation and analysis of ecological and social aspects; 49 permitting among some points, to determine the response capacity of the populations and species against natural 50 environmental changes or those provoked by conscious or unconscious human activities [11]. Likewise, the study of 51 the genetic constitution of the populations and of the mechanisms of evolutionary change acting on them are the pillars 52 that constitute the tools to confront the loss of biological diversity [12]. 3

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For this reason, it is important to diagnose the current situation and conservation of the wax palm from Quindío, 54 focusing on the genetic level of distribution in the studied areas. This diagnosis will permit undertaking actions to 55 protect this palm from extinction, adding that the main problem identified, the loss of biological diversity of the species, 56 is the impact that can be generated in the continuity of the landscape and other species that depend on it. Likewise, C 57 quindiuense is the flagship species of the eco-region of the Colombian coffee region, a zone declared world heritage 58 by UNESCO in 2011, which generates a big opportunity to continue with conservation efforts of the Colombian 59 national tree.

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In genetic structure studies on Ceroxylon, Gaitán [13] investigated the genetic diversity and population structure in

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alpinum developed by Gaitán [13] demonstrated that the populations of this species in the central mountain range have 76 a high genetic diversity that permits establishing potentially successful repopulation programs. This also exhibits 77 significant genetic structuring, suggesting that we should seek the conservation of each unit that represents an 78 independent gene pool.

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Considering the studies mentioned, it is expected that populations of Ceroxylon quindiuense in the Colombian coffee 80 eco-region will be also highly structured with limited gene flow among them. This would generate patterns of low 4 81 genetic diversity and, with such, a high degree of inbreeding as a consequence of the loss of habitat present for this 82 species. Due to all the aforementioned, this study evaluated the diversity and population genetic structure of the wax 83 palm from Quindío (Ceroxylon quindiuense: Ceroxyloideae) in five populations of the Colombian coffee eco-region 84 by using microsatellite molecular markers.

Study area 87
The area of the Colombian coffee region represents a set of ecosystems distributed between the central and western

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The DNA was extracted by taking 1g of previously macerated tissue from the leaf collected and using the Qiagen

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Evaluation and amplification of loci microsatellites 122 Ten loci microsatellites (SSR) were evaluated, which were developed by Gaitán [13] for the species of the genus 123 Ceroxylon alpinum and C. sasaime (Table 1).

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Structure and diversity analysis

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In general, the allele frequencies for the 10 microsatellites presented a similar distribution with frequencies between 207 10 and 30%; however, frequencies above 50% were identified in four locations (Anaime, Pijao, San Félix, and 208 Tenerife) and frequencies below 42% in the population of Cocora. Allele 142 in loci Ca30 and Ca20 presented allele      (Table 3). Analysis of seedlings and juveniles for all the populations presented no significant 243 differences within each population (p > 0.05). found from different populations, except for a group that presents in its majority individuals from the population of 249 Tenerife (Fig 6). For other hand, Bayesian analysis using STRUCTURE identified two cluster (K=2), reflecting a small 250 mix of individuals between clusters (Fig 7).

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Plan for said species, that a considerable portion of the individuals that exist survive in paddocks. A large proportion 282 of individuals is found in relatively small forest fragments, whose long-term permanence is not guaranteed.

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The impact of loss of habitat due to human activities is reflected on the structure and on the amount of genetic variation

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They masticate while remaining in the inflorescence; part of the pollen is adhered to their bodies and they then transfer 295 it to another inflorescence.

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The relationship between the wax palm from Quindío and said beetles, as well as the effect of fragmentation in this 297 process, are aspects still not studied. However, it has been recorded that many species of pollinators, like the

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C. quindiuense has a strong genetic structure with all the populations differentiated, yielding significant structuring 308 values (Table 3)

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Even so, it is evident that the current state of conservation of its habitat is influenced considerably in said structuring,

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where landscape characteristics may modify gene flow between population pairs, directly when affecting the dispersion 316 rates between them, or indirectly upon affecting the spatial disposition of the dispersion rates and among the 317 subpopulations intervened [50]. For C. quindiuense, the isolation by resistance (IBR) model can be applied [50]. It is

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It was identified that the population of Tenerife had the highest number of private alleles, the lowest levels of genetic 338 diversity (Table 2), and significant structuring with respect to the other populations (Table 3); even the majority of 339 individuals formed one of the groupings identified in the DAPC ( Figure 6) and the STRUCTURE analysis (Figure 7).

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This population has not been studied previously; however, its low genetic diversity and population genetic structure

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The genetic structure of a population is determined by its evolutionary history and expresses the amount of genetic

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The genetic criteria obtained from the study of molecular markers could be useful to select wildlife populations as 356 candidates with priority for their conservation [53]. As the case of the populations of wax palm from Tolima and

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Quindío for which Sanín [14] states that the huge genetic diversity available in these populations, in spite of the current

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This study is, to date, the first work at genetic level developed for the populations of San Félix, Anaime, Tenerife, and

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Pijao that contributes to one of the first lines of action of the Conservation Plan for this Andean palm in Colombia,

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broadening the knowledge base in this species. Thus, we recommend considering the genetic diversity identified for