Genetic diversity and differentiation of the Critically Endangered Hispaniolan palm Coccothrinax jimenezii M.M. Mejía & R.G. García based on novel SSR markers
Coccothrinax jimenezii is an endangered palm species from Haiti facing conservation challenges at the moment. The authors of this paper conducted experiments with hope of understanding the levels of genetic diversity of this species from the two populations of c.jimenezii in order to aid in the conservation of the palm species. This experiment demonstrated that the species investigated had more genetic variability than was previously thought indicating how the work the authors performed served a purpose in establishing improved conservation efforts for this species.
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Coccothrinax jimenezii M.M. Mejía & R.G. García is a Critically Endangered palm species restricted to Haiti (one population near the city of Gonaïves with 43 individuals) and the Dominican Republic (one population on the shores of Lago Enriquillo with 18 individuals). The species faces two major conservation challenges: (1) water level rise in the hypersaline Lago Enriquillo and (2) overexploitation of leaves for making brooms in Haiti. Six SSR microsatellite loci were used to access levels of genetic variation and the genetic structure of these two populations. Only the Gonaïves site had loci that deviated from Hardy–Weinberg Equilibrium (2 loci). Both populations exhibited a relatively large number of private alleles (13 in Lago Enriquillo and 14 in Gonaïves) and did not show evidence of genetic bottlenecks. Inbreeding coefficients were much larger in Gonaïves (Fis = 0.232) than in Lago Enriquillo (Fis = 0.093). We detected high genetic differentiation among these sites (Fst = 0.497) suggesting that additional taxonomic studies are needed to determine if individuals from these two sites should be recognized as belonging to two different taxa. Because of taxonomic uncertainties we recommend not to translocate individuals between sites in future conservation activities involving this species.
The Caribbean Island Biodiversity Hotspot has a high priority for conservation as the region has a high number of endemic species, a high deforestation rate, and environmental biology challenges pertinent to invasive species; unsustainable exploitation of natural resources; and rapid urban and rural development (Smith et al., 2004; Maunder et al., 2008, 2011). Within this conservation biology perspective, molecular tools can be useful to address issues pertinent to taxonomy and systematics of threatened species. They also can reveal patterns of genetic diversity of species with very narrow distribution that in many cases are in the verge of extinction (Allendorf and Luikart, 2007; Oleas et al., 2013). Caribbean Island palms have been the focus of conservation initiatives that include red-listing [sensu IUCN (2014) by Zona et al. (2007) and Peguero et al. (2015b)]; species inventories (Moya López and Leiva Sánchez, 2000; Leiva Sánchez, 2006); conservation genetic studies (Rodríguez-Peña et al., 2014a, 2014b); field surveys and conservation assessments (Henderson et al., 1990; Leiva Sánchez, 2008; Peguero et al., 2011, 2015a); molecular phylogenetics (Roncal et al., 2008); and outreach and education (Leiva Sánchez, 1999; Martínez Betancourt and Miranda, 2009–2010).
Coccothrinax is the most species-rich palm genus in the Caribbean Islands, where it has over 49 endemic species (Acevedo-Rodríguez and Strong, 2012; Mejía and García, 2013). Seven of these species (C. argentea, C. boschiana, C. gracilis, C. jimenezii, C. montana, C. scoparia, and C. spissa) are restricted to Hispaniola whilst four of them (C. barbadensis, C. ekmanii, C. fragrans, C. miraguama) occur both on Hispaniola and at least another Caribbean island (Peguero et al., 2015b).
Coccothrinax jimenezii M.M. Mejía & R.G. García is confined to two small populations (Fig. 1). One of them is found near the shoreline Lago Enriquillo, Dominican Republic (with 16 adults, 2 immature individuals, and several seedlings). The second population occurs near the city of Gonaïves, Haiti (with 43 immature individuals – all less than 2 m tall with no reproductive structures; neither adults nor seedlings have been located) (Peguero et al., 2015a).
Coccothrinax jimenezii is the most recently described species of the genus (Mejía and García, 2013) and no species of Coccothrinax is found with C. jimenezii, indeed no other species of the genus occurs within kilometers of the known two sites of C. jimenezii. As a Critically Endangered palm, C. jimenezii has been central to our conservation biology activities during the last 2 years as it is one of the most threatened species of this genus (Francisco-Ortega et al., in preparation). Extensive field work that included demographic studies and conservation assessments was conducted both in Haiti and the Dominican Republic. Distribution patterns and discussion of conservation concerns were reported by Peguero et al. (2015a). Our conservation initiatives included the production of printed educational material presented during the VI Simposio Flora de La Española in June 2015 at Santo Domingo, Dominican Republic. Here we present an additional conservation contribution based on the population genetic analyses of microsatellite data (SSRs).
The species faces a main conservation concern in the Dominican Republic. Lago Enriquillo is a hypersaline lake located at 45 m below sea level and at only 9 km from Haiti. This is the largest lake in the Caribbean and its waters, coastal areas, and three islands are officially protected inside the Parque Nacional Lago Enriquillo e Isla Cabritos. The lake is part of a major geographical depression that splits the island of Hispaniola into two main geological units (Fig. 1). In the last ten years Lago Enriquillo has undergone unexpected rise in water level (Romero Luna, 2011). The environmental factors that have triggered these hydrological changes in this lake are unclear; however, it appears that they are related to unusual rain fluctuations (Romero Luna, 2011). Because the Dominican Republic population of C. jimenezii is located near the shoreline of this lake, there is concern that the soils where the species occur can be negatively affected by saline intrusion.
During our field research we did not find any use for this palm in the Dominican Republic. In contrast, in the Haitian population the leaves of the vast majority of its individuals had been harvested to make brooms. This ethnobotanical practice has a clear detrimental effect on the long term viability of this population and could be reason why in this site we did not find any plant that has more than 2 m in height (Peguero et al., 2015a). None of the plants had fruits or flowers and in the Haitian site we did not find either inflorescences or fructifications from previous seasons.
Molecular tools have not been used to address conservation genetics issues within any species of Coccothrinax. Micosatellite SSR data have proven useful to reveal patterns of genetic diversity and erosion in threatened Hispaniolan species of the palm genus Pseudophoenix (Rodríguez-Peña et al., 2014a). They have also helped to address biogeographical and conservation questions with other native/endemic species from this biodiversity hotspot [e.g., Zamia lucayana Britton (Zamiaceae) in the Bahamas (Calonje et al., 2013), Zamia in Puerto Rico (Meerow et al., 2012), and Ipomoea microdactyla Griseb. (Convolvulaceae) in Cuba and the Bahamas (Geiger et al., 2014)].
The aim of our study was to determine (1) levels of genetic diversity of the two known populations of C. jimenezii, (2) degree of genetic differentiation between these populations, and (3) to provide conservation recommendations based on these results. Our initial hypothesis was that these two small-size populations exhibit low levels of genetic variation and that they are co-specific within C. jimenezii. To address these hypotheses and objectives we used SSR markers originally developed for the Florida–Bahamas endemic Coccothrinax argentata (Zona et al., in preparation).
MATERIALS AND MATHODS
Sampling methods, primer development, and DNA extraction
The two known populations of this species were included in our study, and we sampled 18–30 individuals per population (Table 1). All of the individuals (18) from the Dominican Republic population were included in the analyses. Leaf material was fast dried in the field using Drierite (W. A. Hammond Drierite Co. Ltd., Xenia, Ohio, USA), subsequently this material was used for DNA isolations using the DNeasy Mini Plant kit (QIAGEN, Venlo Limburg, The Netherlands).
Total genomic DNA of fresh leaves of a single individual of C. argentata was also isolated with the DNeasy Mini Plant kit. This DNA sample was subsequently used to obtain microsatellite loci that were developed by the Georgia Genomics Facility at the University of Georgia (Athens, Georgia, USA). A next generation sequencing approach with an Illumina HiSeq 2000 (Illumina, San Diego, California, USA) was used to identify these microsatellite markers. The resulting 100 bp Illumina sequences were screened using the PERL script program PAL_FINDER_v0.02.03 to identify potential 4 bp microsatellite repeat elements among the reads (Castoe et al., 2012; Slashdot Media, San Jose, California, USA). Among these SSR loci we selected six that cross-amplified with samples of C. jimenezii and that were polymorphic with different samples of this species. Primer-pairs used to amplified these six SSR loci as well as details pertinent to their allele motif, annealing temperature, and allele size are shown in Table 2.
Demographics & genetic diversity statistics
This table includes demographic features and descriptive genetic diversity statistics for the two populations of Coccothrinax jimenezii.The six loci were polymorphic in the Haitian population but only five of them from the Dominican Republic exhibited variation in their alleles. The genetic diversity statistics looks at data based on the Hardy-Weinberg formula which can help determine the frequency of the heterozygous state.
A great deal of statistical software was used to generate the values reported in Table 2.
Some examples include:
• Micro-Checker used to evaluate the presence of null alleles (nonfunctional copy of a gene caused by a genetic mutation) and allelic dropouts (Loss of one allele during polymerase chain reaction (PCR) amplification of DNA
• GenePop used to calculate heterozygote excess or deficiency
• BOTTLENECK used to test for recent genetic bottlenecks (sharp reduction in the size of a population due to environmental events or human activities) in the populations The Gonaïves locality showed clear evidence of inbreeding with a large Fis positive value of 0.232 (Table 2). The Dominican Republic population had a very low Fis value of 0.092 showing only a small degree of inbreeding.
Population genetic analysis
The program Micro-Checker v. 2.2.3 (Van Oosterhout et al., 2004) was used to evaluate the presence of null alleles and allelic dropouts, employing 3000 randomizations. Descriptive statistics (Table 2), analysis of molecular variance (AMOVA) with 10,000 permutations, estimates of genetic differentiation (Fst), and mean number of migrants (Nm) were obtained with GenAlEx v. 6.5 (Peakall and Smouse, 2006, 2012). Inbreeding coefficients (Fis) were computed with GDA (Weir, 1996). Tests for Hardy–Weinberg Equilibrium (HWE) and the U test (Rousset and Raymond, 1995) for heterozygote excess or deficiency were run with GenePop v. 4.2 (Raymond and Rousset, 1995, 2008) using 10,000 Monte Carlo Markov chain iterations (Guo and Thompson, 1992). Linkage disequilibrium (LD) was tested for each population with ARLEQUIN v. 188.8.131.52 (Excoffier and Lischer, 2010) using a likelihood ratio test (Slatkin and Excoffier, 1996). A Monte Carlo Markov chain method was applied with 100,000 iterations, a burn-in of 10,000 and the significance level set at P < 0.001. Principal coordinate analysis (PCO) among all the individuals included in our study was computed with GenAlEx based on the algorithm developed by Orloci (1978) after conversion of the individual-by-individual genetic distance matrix, as defined by Smouse and Peakall (1999), to a covariance matrix and data standardization. This ordination technique was used to detect the genetic structure of populations projected in a continuous space (Abbott et al., 1985). The aim of this multivariate analysis was to produce a scatter diagram that summarized the original multidimensional data set and revealed the presence of groups. The Bayesian clustering program STRUCTURE v.2.3.4 (Pritchard et al., 2000) was used to estimate the underlying genetic structure among populations. K values of 1–10 were simulated across 20 replicate runs of 1,000,000 iterations after a burn-in of 100,000. The Δk method of Evanno et al. (2005) as implemented in STRUCTURE HARVESTER (Earl and vonHoldt, 2012) was used to determine the ‘true’ value of K across samples. After the likely level of K was estimated, a consensus Q-matrix from the 20 runs was constructed using CLUMPP (Jakobsson and Rosenberg, 2007) for visualization with DISTRUCT (Rosenberg, 2004) through the program ClumPAK (Kopelman et al., 2015). BOTTLENECK 1.2.02 (Cornuet and Luikart, 1996) was used to test for recent genetic bottlenecks in the populations under the infinite allele model (Kimura and Crow, 1964).
The output from Micro-Checker did not provide evidence for allele dropouts and yielded a general homozygote excess for two of the loci in the Haitian population suggesting the presence of null alleles. However, the high proportion of homozygotes detected in these two loci could also be the result of stochastic processes associated to genetic drift and inbreeding.
Each of the individuals had a unique genetic profile and therefore we did not identify repeated multi-locus genotypes (Table 2). The six loci were polymorphic in the Haitian population but only five of them exhibited allelic variation in the site from the Dominican Republic. The mean number of alleles per locus ranged between 3.0 (Dominican Republic) and 3.1 (Haiti), but the two populations had high number of private alleles, with 14 for Gonaïves and 13 for Lago Henriquillo. The population from Haiti had two loci that were not under HWE, and the global test showed that this population has heterozygote deficiency (P < 0.001). In contrast the exact test found that there was no significant departure from HWE for any of the loci of the Lago Henriquillo site (Table 2). Linkage disequilibrium values were of 20% for the population from Haiti and 6% for that from the Dominican Republic. The Gonaïves locality showed clear evidence of inbreeding with a large Fis positive value of 0.232 (Table 2). The Dominican Republic population had a very low Fis value of 0.092 showing only a small degree of inbreeding. The Fst value was 0.497 and Nm between the two sites was 0.253. Over 50% of the molecular variation (AMOVA test) was between populations.
The Evanno method of determining the true K identified K = 2 as optimal across the two C. jimenezii populations. These two clusters corresponded to the two known populations of the studied species (Fig. 2). The first two axes of the PCO accounted for 58.5% of the variance. Results from this analysis were in agreement with the Bayesian clustering results as the genotypes formed two groups across the first two axes (Fig. 2). Individuals from the Dominican Republic displayed positive values along the first coordinate, whereas those from Haiti had negative scores. There was no evidence of genetic bottleneck for any of the populations under the infinite allele model (P > 0.05).
Genetic diversity and conservation
It is expected that populations with few number of individuals to harbor low levels of genetic diversity and to have many loci that deviate from HWE. This is because of the combined effects that genetic drift and inbreeding have on populations exposed to population size reduction across several generations (Leimu et al., 2006; Höglund, 2009). The two known populations of C. jimenezii have a small number of individuals; however, it seems that stochastic events associated with drift and inbreeding have not been fully operating in these populations despite their very small size. Plants of Coccothrinax have hermaphroditic flowers, but it is unknown if they have self-incompatibility genetic systems to prevent autogamy and therefore to enhance heterozygosity. No evidence for bottlenecks was found in our study, and all of the loci of the Lago Enriquillo population and four of those from the Haitian locality were in HWE. In addition, values of observed heterozygosity (HO = 0.342 for the Dominican Republic and HO = 0.294 for Haiti) were within the higher range of those reported in other SSR studies that focused on threatened species of palms [0.019 for Livistona carinensis (Chiov.) J. Dransf. & N.W. Uhl from Yemen, Djibouiti, and Somalia (Shapcott et al., 2009); 0.143 for Lepidorrhachis mooreana (F.Muell.) O.F.Cook from Lord Howe Island (Shapcott et al., 2012); 0.143 for Pseudophoenix lediniana Read and 0.459 P. ekmanii Burret from Hispaniola (Rodríguez-Peña et al., 2014a); and 0.40 for Butia eriospatha (Mart. ex Drude) Becc. from Brazil (Nazareno and Dos Reis, 2014)]. Contrary to our expectations there is not a clear positive association between the small population sizes of C. jimenezii and the patterns of genetic variation detected in our study.
Lack of a positive correlation between observed heterozygosity and population size was also reported by Rodríguez-Peña et al. (2014b) in their study focusing on Caribbean Island species of Pseudophoenix. It was found that the small populations of P. sargentii H. Wendl. ex Sarg. from the Turks and Caicos, Isla Saona (Dominican Republic) and Pamilla (Parque Nacional del Este, Dominican Republic) have the largest HO values among the 18 sites included in their study. As an explanation for these results these authors suggested that a combination of the long generation time of palms and relatively recent habitat fragmentations might explain why populations of Caribbean palms lack strong positive associations between population size and levels of genetic diversity. Similar results have been reported in other plant groups occurring in areas that have undergone recent habitat fragmentation because anthropogenic activities (Wang et al., 2012). We have not been able to find data regarding the environmental history in these two populations since the 15th century. However, there is agreement among conservation biologists that since the arrival of the Europeans to this island there has been habitat fragmentation and deforestation linked to rapid expansion of urban and rural activities (Sambrook et al., 1999; Alscher, 2011; Foxx, 2012). Palms have long-life cycles; therefore, the detrimental consequences of genetic drift on genetic diversity can take a long period of time to manifest because of their long generation time and the presence of overlapping cohorts (Duminil et al., 2009). Despite the relatively high levels of genetic diversity found in the two populations of C. jimenezii there are challenges concerning their long term viability. The two known sites have fewer than 50 individuals and based on this small population size (Effective Population Size, Ne < 100) it is a matter of time for inbreeding and subsequent associated fitness loss to drive this species to extinction (Frankham et al., 2014).
Our results and those reported by Rodríguez-Peña et al. (2014b) for Pseudophoenix could have consequences for the remaining 13 Critically Endangered species of the genus [listed in Rankín Rodríguez and Areces Berazaín (2003), Berazaín Iturralde et al. (2005), González-Oliva et al. (2014, 2016), Peguero et al. (2015b)]. We argue that it cannot be assumed that these threatened species of Coccothrinax, despite their red-listing status and their relatively small population size, harbor low levels of genetic diversity as in many cases their current demographic patterns could be the result of recent and rapid changes in the original habitat associated with urban or rural developments.
Conservation and taxonomy
Both the STRUCTURE and PCO analyses supported two distinct clusters within C. jimeneziiand these groups correspond to the two known populations of this species. These clusters were also supported by the large Fst value (0.497) exhibited by these two populations. Among the studies on population genetics of palms that reported this coefficient (Trénel et al., 2008; Cibrián-Jaramillo et al., 2009; Shapcott et al., 2009, 2012; Namoff et al., 2011; Giovino et al., 2014; Nazareno and Dos Reis, 2014; Santos-Oliveira et al., 2014; Lanes et al., 2015; Zehdi-Azouzi et al., 2015), only Trénel et al. (2008) found Fst values higher than 0.4, for populations of Ceroxylon echinulatum in the Andean region. In an unpublished study focusing on the genetic structure of con-specific populations of Coccothrinax argentata from the Lower Florida Keys and the Florida mainland, Zona et al. (in preparation) found a much lower Fst value of 0.24.
The high levels of genetic differentiation detected within C. jimenezii raise questions whether these two populations can be treated as different varieties/subspecies within this taxon or if indeed they may represent two different species. The importance of taxonomy in conservation biology has been already highlighted by several studies (e.g., Mace, 2004; Costello et al., 2015; Halme et al., 2015), and there is agreement among conservationists that taxonomy and systematics provide valuable research tools in the current human-driven biodiversity crisis (Wen et al., 2015).
Coccothrinax has not been the subject of comprehensive taxonomic monographs. The only available phylogenetic analysis within this genus was produced by Nauman and Sanders (1991a) who suggested that it is composed of three major groups: the argentata group (with ten species, including the Critically Endangered C. crinita, C. cupularis, C. leonis, C. montana, C. victorinii, C. borhidiana), the argentea group (with six species, including the Critically Endangered C. spissa), and the pauciramosa group (with 31 species, including the Critically Endangered C. nipensis, C. pauciramosa and C. yuraguana). Nauman and Sander's (1991a) phylogenetic research was based on morphological traits. As part of their morphological conclusions they also indicated that homoplasy was common within Coccothrinax (Nauman and Sanders, 1991b). The molecular phylogeny presented by Roncal et al. (2008) based on nucleotide sequences of the nuclear genes PRK and RPB2 did not have enough resolution to resolve the main clades of the genus.
Based on the analysis of herbarium specimens there are no obvious morphological differences to distinguish among plants from the two populations of C. jimenezii. However, within this genus, distinctive three-dimensional features pertinent to how leaves and inflorescences are displayed are not well preserved on herbarium specimens (Zona, pers. comm.); therefore, morphological studies for future taxonomic revisions will need to include traits based on the examination of living material. In their original description Mejía and García (2013) indicated that C. jimenezii is morphologically similar to C. gracilis, but the former has leaves with whitish indumentum on both surfaces and a blade that is not fully circular in outline, and with a truncate hastula that is only a little visible on the underside; leaf sheaths thin and soft-textured; and fruits yellowish white at maturity. In contrast, C. gracilishas leaves with indumentum only on the underside and a blade that is fully circular in outline and with a triangular hastula; leaf sheaths have thicker fibers that are coarsely woven; and fruits purple at maturity (Henderson et al., 1995; Mejía and García, 2013). Based on our preliminary field observations in Hispaniola C. jimenezii appears also to be morphologically similar to C. ekmanii, as they both have leaf blades that are not fully circular in outline, but wedge-shaped (Henderson et al., 1995; Mejía and García, 2013). Regarding the type localities of these two putative relatives, C. gracilis was originally described from the north coast of Haiti; whereas, C. ekmanii was from southeastern Haiti (Burret, 1929).
The taxonomic uncertainties between the two populations of C. jimenezii have important implications for reintroduction programs. These two sites of C. jimenezii represent two clear management units for conservation, and the population genetic data suggest that we cannot rule out that they are two distinct taxa. Therefore we recommend not translocating material between these two populations for genetic conservation or ecological restoration programs until the taxonomy of this species within Coccothrinax is further studied.
We dedicate this paper to our colleague Walter Judd for his contributions to the flora of Haiti and his mentorship in plant systematics to a whole generation of plant taxonomists. This study was supported by the Mohamed Bin Zayed Species Conservation Fund [grant number 13257600 through Florida International University (FIU)]. Fairchild Tropical Botanic Gardenprovided matching funds to conduct molecular research. The Consejo Nacional de Investigaciones Agropecuarias y Forestales from the Dominican Republic supported conservation assessment studies of Critically Endangered palms (project number CAD/014-05/RN through the Consorcio Ambiental Dominicano and the Jardín Botánico Nacional). This is contribution number 318 from the Tropical Biology Program of FIU. Celio Moya and S. Zona critically read an early version of the manuscript.
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