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Molecular markers and conservation of plant species in the Latin-America: the case of Phaedranassa viridiflora (Amaryllidaceae).

Introduction

For nearly two decades, DNA microsatellites have been used as a preferred method to address population genetic inquiries in plants (Morgante & Olivieri, 1993; Aldrich et al., 1998; Ouborg et al., 2010). They are popular in part because SSRs are highly reproducible among laboratories. Because they are codominant, it is possible for SSRs to distinguish homozygotes and heterozygotes. They are also highly variable and relatively easy to analyze, and they occur regularly throughout the genome (Frankel et al., 1995; Cruzan, 1998; Susol et al., 2000; Zane et al., 2002; Squirrell et al., 2003). Microsatellies are two to six nucleotide motifs that are repeated many times in tandem and display high dissimilarity in repeat number among individuals. Mutation rates in microsatellite are estimated to be around [10.sup.-2] to [10.sup.-6] mutation per locus per generation (Schlotterer, 2000; Lai & Sun, 2003). One of the previous limitations for a wider use of microsatellites in non-model plant species was the high cost of developing primers. Commercial companies offer ~10 polymorphic primers with an average cost between 5,000 to 10,000 USD in 1 to 3 months (Selkoe & Toonen, 2006; Csencsics et al., 2010). Recently a new direct sequencing microsatellite discovery technique was found to deliver the same number of loci with a cost of less than 1,500 USD (Abdelkrim et al., 2009).

Once primers have been designed, different type of studies can be accomplished with this molecular tool. In natural populations of plant species, microsatellites have the potential to estimate genetic diversity and infer reproductive patterns (Morgante & Olivieri, 1993); i.e., inbreeding (Aldrich et al., 1998; Liu et al., 2003; Michalski & Durka, 2007), inter and intra-specific hybrids (Lexer et al., 2010), degree of clonality (Ainsworth et al., 2003). One of the rare examples with non-cultivated tropical American plant species showing the prospective use of microsatellite is the CITES protected timber tree Mahogany (Swietenia humilis Zucc. and S. macrophylla). Microsatellite primers were designed from S. humilis in the late 90's (White & Powell, 1997) and from S. macrophylla King (Lemes et al., 2002). These loci were subsequently used broadly to assess genetic structure in S. macrophylla, (Lemes et al., 2003; Lowe et al., 2003; Roth Novick et al., 2003), investigate genetic variation in fragmented populations of S. humilis (White et al., 1999), estimate pollen-mediated gene flow (White, 2002), study mating system (Lemes et al., 2007), measure gene diversity after restoration in former pastures areas (Cespedes et al., 2003) and phylogeographic patterns (Lemes et al., 2010).

Molecular markers are powerful tools for species conservation because they can be used to estimate levels of genetic variation among and within populations (Frankham et al., 2002). These methods allow the evaluation of the impact of genetic drift, levels of inbreeding and amount of gene flow among populations (Ouborg et al., 2010). Molecular markers changed the scenery of population genetic studies and in conjunction with the neutral theory (Kimura, 1991); allowed a more detailed understanding of isolation and migration in populations. Such data are important to develop conservation strategies to preserve genetic diversity, which provides species with the means to evolve and adapt in changing environment (Geffen et al., 2006). Plant conservation genetics' ultimate goal is to minimize extinction risk by guiding and monitoring conservation and restoration efforts (Kramer & Havens, 2009).

The tropical Andes is the most diverse hotspot in the world, comprising 7 % of all plant species (Myers, et al., 2000). Within the hotspot, Ecuador is highly diverse, with more than 4,000 endemic plant species (Valencia et al., 2000). Sixty percent of the critically endangered flora of Ecuador is restricted to the Andes (Pitman et al., 2002). The conservation status of plant species in the Ecuadorian Andes is of concern because elevated extinction-rates of plant species have been reported recently (Pitman et al., 2002) and more than 40 % of the original vegetation has been lost in the country (Baquero et al., 2004). Despite the contribution that molecular markers can offer for conservation purposes, the amount of research on population genetics in non-cultivated plants species from the tropical Andes is minimal, and mostly using allozymes (Aragundim et al., 2011; Figueredo et al., 2010; Nassar et al, 2007). There are two studies with microsatellites of threatened plant species in the tropical Andes. One concerns the microevolution of the wax palm Ceroxylon echinulatum Galeano, (Arecaceae) (Trend et al., 2008), and another addressed mating patterns and their role in genetic variation of the Andean Oak (Quercus humboldtii Bonpl.) (Fernandez & Sork, 2005). Neither addresses the conservation management of these species.

The purpose of this study is to exemplify the used of microsatellites in non-cultivated plant species of the tropical Andes for conservation purposes. With this objective in mind, we focused on P. viridiflora Baker, an endemic, endangered species in Ecuador (Oleas, 2000), with occurrences in the north, central and southern Andes of the country. Phaedranassa is a monophyletic genus in Amaryllidaceae tribe Eucharideae (Meerow et al., 2000; Meerow, 2010). This genus is mostly endemic to the Northeast Andes and includes ten species: one from Costa Rica, two are restricted to Colombia, one is found in both Ecuador and Colombia and the six remaining are restricted to Ecuador (Meerow, 1990). Originally, these species inhabited what corresponds to Cloud Forest and Dry and Wet Inter-Andean Shrubs (Valencia et al., 1999); these areas are now dedicated to agriculture, mining, or increasing residential development. The species of this genus inhabit a diverse array of habitats, ranging from dry inter-andean valleys to wet mountain slopes (Meerow, 1990). The species of this genus are geophytic bulbous plants, with petiolate lanceolate leaves and scapose, pseudo-umbellate inflorescences of tubular flowers with different patterns of red, pink or yellow and green (Meerow, 1990). Two key features allow this genus to survive in low numbers: ability to reproduce clonally by bulbs (all the species in the genus N. Oleas, pers. observ.) and self-compatibility (N. Oleas, pers. observ.). The flowers of this genus are protandrous and produce nectar (N. Oleas pers. observ.). There are no studies of the reproduction and the pollination on this genus; however, in our previous fieldwork a single hummingbird visit was observed in P. dubia (red flowers), and euglosine bees and unidentified butterflies were observed visiting P. viridiflora (yellow flowers) (N. Oleas pers. obs.).

Phaedranassa viridiflora is the only species of the genus with mostly yellow flowers. It was described by Baker (1877) from cultivated material, allegedly from Peru, from where no true Phaedranssa has ever been confirmed (Meerow, 1990). Ravenna (1984) described a second green and yellow taxon as P. viridilutea, which Meerow (1990) placed into synonymy with P. viridiflora. Because of the small number of known populations of P. viridiflora and the restricted geographic area where is locate, P viridiflora is categorized as Endangered under the IUCN criteria (Oleas, 2000). Phaedranassa viridiflora is known in three areas distributed along the Ecuadorean Andes, which is different from the other Phaedranassa species that have clustered geographic distribution (Fig. 1, see "Methods" below). All populations of P. viridiflora are apparently sympatric with other Phaedranassa species of the more typical red--pink flower form. An orange floral variety, which we hypothesized could represent a hybrid between P. viridiflora and P. dubia, has been observed only in the Northern population at the Pululahua Crater.

As with the majority of the tropical plant species, there is a lack of knowledge of the natural history of P. viridiflora. With the use of molecular markers as indirect indicators, we aim to better understand the biology of P. viridiflora. In this study we are specifically interested in: a) describing the population genetic structure of P. viridiflora, and b) testing our hypothesis of natural hybridization between it and other species of Phaedranassa.

Methods

Sample Collection

There are three known geographic areas of sympatry in Phaedranassa, all of them involving P. viridiflora. Within each area, the sampling populations were separated by distances of 3 to 12 km (Fig. 1, Table 1). Area A is located in northern Ecuador in the Pululahua Crater and involves three sampled groups: P. viridiflora (yellow flower), P. dubia (red flower) and the putative hybrid between them (orange flower) (Fig. 2). Area B is situated in the central region of Ecuador where, besides P. viridiflora, P. schizantha (pink-salmon flowers) is found (Figs. 1 and 3). In this area four different patches were sampled. Area C is in the south of the country and involves P. cinerea (red flower) and P. viridiflora (Fig. 3). Three collecting sites of the former and two of the later were sampled (Fig. 1).

Sample collection was completed in August 2003 for the southern localities, for the central locations in December 2005, and in August 2006 for the northern sites. During the first two field collections, approximately 2 g of fresh leave material per individual were fast-dried in silica gel. This sample comprises a total of 277 individuals collected in 10 different sites. The latest collection was at the Pululahua crater located north of Quito. In this location, a total of 85 individuals of P. dubia, P. viridiflora and a putative hybrid between both were sampled (Fig. 2). Because it is easier to differentiate both species by flower color, in this case we only collected flowers in silica gel.

Genotyping Procedures

Genomic DNA extraction protocols were the same methods described in Oleas et al. (2005). Quantification of DNA followed procedures in Livingstone et al. (2009). Polymerase Chain Reaction conditions and genotyping techniques followed the methods described by Oleas et al. (2005, 2009). The PCR products were genotyped by capillary gel electrophoresis in an ABI 3730 Genetic Analyzer (Applied Biosystems Inc. Carlsbad, CA, USA). Allele calls were made in GeneMapper 4.0 (Applied Biosystems Inc. Carlsbad, CA, USA). All individuals were genotyped with 13 microsatellite primers: eight developed from P. tunguraguae: locus pt 14, pt21, pt32 and pt39 (Oleas et al" 2005); pt43, pt48, pt49 and pt61 (Oleas et al., 2012), and five developed from P. schizantha: ps2, psl3, psl6, ps27 and ps28 (Oleas et al., 2009). Both species are phylogenetically close to P. viridiflora and P. cinerea based on ITS sequences (Oleas et al., unpubl.).

Polymerase Chain Reaction procedures were repeated up to three times when the sample did not amplify. Samples with alleles showing one base pair (bp) difference rather than a repeat unit variation, as expected for microsatellite mutation, were also replicated. When the sample still showed a difference non-consistent with repeat unit size, the particular allele was dropped. Three types of errors could occur during the processes of identification and isolation using primers and amplification by PCR: null alleles (one or more alleles fail to amplify during PCR), stuttering (slight changes occur in the allele sizes during PCR) and large allele dropout (large alleles do not amplify as efficiently as small alleles) (Van Oosterhout et al., 2004). Estimation of the presence of any of these type of errors was performed with Micro-Checker 2.2.0 (Van Oosterhout et al., 2004).

Statistical Analysis

Descriptive population genetic statistics were estimated with GENALEX 6.4 (Peakall & Smouse, 2006) and they included number of alleles per locus, effective number of alleles per locus, number of private alleles, total heterozygosity, observed heterozygosity, fixation index and F-statistics. Linkage disequilibrium (LD) was estimated with GENEPOP 4.0.10 (Raymond & Rousset, 1995; Rousset, 2008); with the following parameters for the Markov chain: 10,000 batches of 10,000 iterations per batch and 10.000 dememorisation. Sequential Bonferroni correction (Rice, 1989) was applied to avoid Type I statistical error. As a measure of degree of clonality, we calculated the genotype discovery rate between genotypes (G) and number of individuals sampled (N) (G/N) (Ellstrand & Rose, 1987; Silvertown, 2008) and the unbiased probability of identity P(ID), which corrects for deviations from HW and LD (Waits et al., 2001) using GIMLET 1.3.3 (Valiere, 2002). Probability of identity is the likelihood that two individuals taken at random will have the same multilocus genotypes (Waits et al., 2001). Analysis of Molecular Variance (AMOVA) was carried out in ARLEQUIN 3.5.1.3 (Excoffier et al., 2005). We also calculated F-statistics and AMOVA excluding loci that showed null alleles with MICROCHECKER.

Tests for recent bottleneck events were carried out with BOTTLENECK 1.2.02 (Comuet & Luikart, 1996; Piry et al., 1999). The collecting site C3 was excluded from the analysis because it has less than 20 individuals. A low number of individuals compromise the tests (Comuet & Luikart, 1996). The estimation was calculated with 100.000 replications, using the sign test and Wilcox on sign-rank test to determine if populations were in mutation-drift equilibrium (Comuet & Luikart, 1996). Both tests were performed under two models of microsatellite mutation (IAM and SMM). We reported results of both types of mutation models as suggested by Comuet and Luikart (1996). It is unlikely that microsatellite loci follow a strict IAM or SMM, and these two models represent the extremes. The mode-shift test was also reported. This test is an indicator of shift of the allelic frequency distribution (Luikart et al., 1998).

Genetic relationships among populations were estimated in POPULATIONS 1.2.30 (Langella, 1999). A phenogram was generated with chord distance Dc (Cavalli-Sforza & Edwards, 1967), and Neighbor Joining (NJ) as the clustering method. Chord distance was used because it is less influenced by the presence of null alleles (Chapuis & Estoup, 2007), but a second analysis was conducted excluding putative nulls. Bootstrap values across loci were based on 10,000 permutations by locus. The tree was visualized with TREE EXPLORER in MEGA 4 (Kumar et al., 2004).

Individual assignment was performed with the program STRUCTURE 2.2. (Pritchard et al., 2000), as implemented in BIOPORTAL (Kumar et al., 2009). To identify the most likely number of genetic groups (k) among P. viridiflora samples, a series of analyses were done by collecting area and by species. The analysis was done with a range of k 1 to 10 for the North area collections and 1 to 15 for the central and southern area. All the analyses were made following the admixture model, with 1,000,000 repetitions after a bum-in of 500,000 and replicated 20 times per k value. The results were loaded into STRUCTURE HARVESTER (Earl, 2011), which calculates the most probable k value following Evanno et al. (2005) method. We summarized the results of the independent runs for the optimal k in CLUMPP 1.1.2 (Jakobsson & Rosenberg, 2007) using the Greedy option with random input order and 100,000 replications. The results were visualized in D1STRUCT 1.1 (Rosenberg, 2004).

Principal Coordinates Analysis (PCA) was performed in GENALEX 6.4 (Peakall & Smouse, 2006) and graphic was generated in SAS 9.1 (SAS Institute, Cary NC). Pairwise genetic distance was calculated in POPULATIONS 1.2.30 (Langella, 1999) using Chord distance Dc matrix (Cavalli-Sforza & Edwards, 1967). Chord distance was calculated with all the individuals and loci.

Results

A total of 362 samples collected in 13 locations were successfully scored with 13 microsatellite loci (Table 1). No evidence of large allele dropout or stuttering was found with MICROCHECKER. Null alleles were identified in a range of one to 12 SSRs loci (Table 1 and Appendix Table 4). After Bonferroni correction, linkage disequilibrium (LD) was found in three populations (Table 1).

The average of alleles per locus ranged from 1.54 to 8.08 and the average effective allele number ranged from 1 to 4 (Table 1). The number of alleles is differed by species and collecting site. Overall, Phaedranassa viridiflora showed the lowest number of alleles (average of 1.39 effective allele number) in all the collecting sites (Table 1). With the exception of most populations of P. viridiflora, all of the species show evidence of inbreeding (positive F values, Table 1). Three populations (VI, V4, C2) that previously had evidence of inbreeding, had negative F values when the fixation index was calculated with only the five loci that did not test positively for possible null alleles (Appendix Table 4). Number of private alleles was also lower in P. viridiflora (Table 1) with the exception of the populations from Area A (north; Table 1). In total five populations showed evidence of inbreeding, after analysis of five loci (Appendix Table 4). The mean Fst was 0.47 and 0.55 with all 13 loci and five loci, respectively. Based on the AMOVA, the majority of the variance was among populations within groups, when the groups represented the three collecting areas (Table 2). When the groups were structured by species identity (n=5), the majority of the genetic variance was among groups (Table 2).

Significant evidence of clonality was found in P. viridiflora (Table 1). In terms of identical multilocus genotypes (MLG), the highest number was found in populations of P. viridiflora from the central and southern collecting sites (Table 1). Both genotype of discovery rate (G/N) and probability of identity (PI) were higher these populations samples (Table 1).

None of the populations showed evidence of recent bottlenecks with all three tests: Wilcoxon, sign statistic test and the mode-shift test (Comuet & Luikart, 1996) (Table 3). The majority of populations showed evidence of bottlenecks under the sign test with the SMM, whereas only one did under IAM using the same test. With the Wilcoxon test, two populations tested positively for a bottleneck with the IAM, and only one under the SMM (Table 3). Only one population showed evidence of bottleneck with the modeshift test (Table 3). None of the Phaedranassa viridiflora populations from the south showed evidence of a recent bottleneck (Table 3).

The NJ tree showed two different clusters, one for the central and one for the southern P. viridiflora populations without inclusion of other Phaedranassa species (Fig. 4). Boostrap values based on 10,000 repetitions were higher than 50 in all nodes (Fig. 4). In the north, P. viridiflora (yellow flower type), P dubia (red flower type) and a putative hybrid between the two (orange flower type) formed a separate cluster (Figs. 2 and 4). When the analysis was performed with five loci, the relationships among populations were still primarily geographical (Fig. 4). The two exceptions were D1 and C1, but they still cluster with populations from the same region (Figs. 4 and 5).

The PCA of all the genotypes resolved three groups, one formed by the central populations (V4, V5, SI, V3), a second with most of the southern populations (V8, C2, V6, V7) and another with populations of P. cinerea from the south and all the north populations (C1, C2, C3, VI, V2, Dl) (Fig. 6). The individual assignment with Bayesian analysis also retrieved three clusters: one formed by the central P. viridiflora populations, another by P. viridiflora from the south and the third one combining populations from the north and south (Fig. 5).

The individual assignment of the collections in the north area (A) retrieved three genetically differentiated groups (k=3). Based on a posterior probability distribution >90 %, one group was formed by most of the individuals of yellow-flowered P. viridiflora in this area. Another group included a little more than half of the P. viridiflora of orange flowers (15 individuals). The third group included a mixture of all P. dubia individuals (red flowers), few (5) individuals of P viridiflora of yellow flower and 12 individuals of P. viridiflora of orange color. Only two individuals showed a posterior probability distribution <90 % to a particular group (Fig. 5). Furthermore, three groups could be distinguished in the PCA: one formed by orange flowers only, another formed by yellow flowers only and one formed by all red flowers and some yellow and orange flowers (Fig. 7).

In the central (B) and south (C) collections, most of the individuals were not in flower at the time of collection, thus the floral color identity of all the samples was unknown. The species identity for the collecting site was assigned based on the individuals with flowers in the patch. The analysis of the central collections showed an optimal population number of k= 2, and the results showed no evidence of extensive admixture among collecting sites of P. viridiflora and P. schizantha (Fig. 5). In the southern region (Area C) the collecting sites were separated at a distance of three to 12 km. The individual assignment shows that these groups belong to three genetically defined populations (Fig. 5). There was no evidence of admixture among the individuals assigned to P. viridiflora and individuals of P. cinerea (Fig. 5). There was evidence of genetic admixture among populations of P. cinerea in the south (Figs. 4 and 5).

Discussion

Phaedranassa viridiflora showed the least amount of genetic diversity especially in central and south Ecuador (Table 1, Fig. 2). This species had the lowest number of private alleles, the fewest alleles per population and the lowest heterozygosity (Table 1). Our results indicate that there are two different scenarios involving this species. The genetic structure of the P. viridiflora of central-south group presents characteristics of asexual reproduction. There was no evidence of hybridization between P. viridiflora and other species of the genus (Figs. 4 and 5) in this area. In the north, however, there is apparent admixture between P. viridiflora and P. dubia.

Clonality

Clonality seems to be an important feature of the central-south populations of P. viridiflora based on all three estimates: repeated multilocus genotypes (MLG), G/N and Probability of identity [P.sub.(ID)] (Table 1). Two out of the three P viridiflora populations in the central and south region showed significant excess of heterozygotes (Table 1). Four factors have been found to produce heterozygosity excess: small populations, heterosis, assortative mating and asexual reproduction (Balloux et al., 2003; Stoeckel et al., 2006; Schurko et al., 2008; Millar et al., 2010). We cannot rule out any of those four conditions, but our data strongly support asexual reproduction in P. viridiflora. All Phaedranassa species have the potential to reproduce vegetatively (N. Oleas, pers. obs.). The high level of clonality in most populations of P. viridiflora suggests that this species is reproducing mainly asexually. For this study, we maintained at least one meter of separation between collections in an effort to reduce the sampling of the same genet. However, our results show that clonality is much higher in P. viridiflora than in other species of the genus (Oleas, 2011). Besides reproduction by bulbs, the high number of clones of P. viridiflora might be an indication of apomixis (Koltunow & Grossniklaus, 2003). The winged, wind dispersed seed of Phaedranassa would facilitate dispersal of apomictic seed away from the maternal plant. Further research is needed to determine if apomixis is occurring in P. viridilfora.

The population of P. viridiflora in the north showed a degree of clonality (Table 1), higher than the levels of the sympatric P. dubia, but lower than other P. viridiflora populations (Table 1). A reduction in the level of clonality was observed in the putative hybrid group (Table 1). The cluster of yellow flowered individuals showed seven times more clones than the group formed by the orange flowers. This lower degree of clonality might be the result of increased sexual reproduction by orange-flowered individuals and introgression with P. dubia.

Population history also influences the genetic signature found in this group. With the exception of the P. viridiflora populations from the south, all populations showed evidence of recent bottleneck. Recent bottleneck can be a consequence of founder events (Comuet & Luikart, 1996). Founder events are likely in areas as the Andes, where changes in the orography as a result of volcanism may open up areas for colonization, as explained in the following section for the north population at the crater of Pululahua. However, bottlenecks are characteristic of many population of Phaedranassa, including those that do not show significant degrees of clonality (Oleas, 2011).

Hybridization

Our data provide evidence of natural hybridization and introgression between P. dubia and P. viridiflora in the Pululahua crater (Figs. 3, 4 and 5). In this particular collecting site flowers were gathered in an effort to analyze the three-color types separately (red for P. dubia, yellow for P. viridiflora and orange for a presumed hybrid). As opposed to the central and southern P. viridiflora groups described above, Pululahua's populations of P. viridiflora are more diverse, with almost double the effective number of alleles. In a Bayesian analysis to assign individuals to any of the three groups, only red individuals (P. dubia) in all cases showed a consistent assignment (Fig. 4). This pattern was not always evident for the yellow and orange forms, because some individuals showed evidence of admixture (Fig. 5). Our hypothesis of introgression between hybrids and P. dubia in the Pululahua crater is corroborated by the PCA analysis as well (Fig.7). Phaedranassa dubia is known from multiple localities in northern Ecuador as well as Colombia, and there is no evidence that this species is of hybrid origin (Oleas, 2011). Almost half of the putative hybrid individuals of orange flowers were assigned by the posterior probability of the Bayesian analysis to the group formed by P. dubia with red flowers. Because none of the individuals with orange flower were associated genetically with individuals of yellow flowers, we can argue that there is uneven backcrossing towards P. dubia, which suggests introgressive hybridization.

Hybridization has been proposed as a major role in plant speciation in general (Soltis & Soltis, 2009). It has been estimated that more than 25 % of plant species are involved in hybridization with 11 % being putative hybrids (Ellstrand et al., 1996; Mallet, 2005). Introgressive hybridization is well known in plants (Anderson, 1953). Even though the degree of hybridization and introgression among Andean plant species is largely unknown, evidence of both has been found across different taxa: e.g., wild tomato species Solanum lycopersicum and S. pimpinellifolium (Solanaceae) (Nakazato & Housworth, 2010), Vasconcellea (Caricaceae) (Van Droogenbroeck et al., 2006) and Puya (Bromeliaceae) (Schulte et al., 2010). An experimental crossability test in the Andean Caiphora (Loasaceae) showed that these species could hybridize even between morphologically divergent species (Ackermann et al., 2008). In Caiphora, species were maintained by ecogeographical isolation and hybrids were a result of second contact by human influence (Ackermann et al., 2008). In general, disturbed habitats can promote hybridization (Schmickl & Koch, 2011). The Pululahua crater has been exposed to disturbances in the past; the crater constitutes the remains of the collapse of the volcano flanks after intensive volcanism during the Quaternary (Hall & Mothes, 2008). The crater of Pululahua is currently heavily utilized by agriculture. Both past and present disturbances might be also the source of bottleneck evidence found in these populations.

Taxonomy and Conservation Implications

All Phaedranassa individuals with yellow flowers were considered as P. viridiflora in the latest taxonomic revision of the Amaryllidaceae in Ecuador (Meerow, 1990), despite the fact that no type specimen or illustration is available for this species. Its exact provenance remains unknown. Ravenna's P. viridilutea was described from a specimen located at the south of the generic range in Ecuador (Ravenna, 1984), and likely represents a population closely related to populations V6-8 in this study. Our data indicates that the yellow forms of Phaedranassa in different regions in Ecuador are genetically distinct and thus likely evolved independently three times across the geographic distribution of the genus in Ecuador. The number of lineages attributed to what is known as P. viridiflora needs to be corroborated by phylogenetic studies. Ultimately, it remains to be determined how best to taxonomically treat these highly clonal, possibly apomictic, forms.

Another taxonomic issue is evident among P. cinerea populations, the samples of which were clustered in two genetically different groups. An evaluation of the morphological differences among these two groups and further genetic and ecological studies are required for a better understanding of this species.

Our findings have several implications for the conservation of Phaedranassa viridiflora. Currently P. viridiflora is listed as endangered under the IUCN criteria, and this category holds for each one of the groups found in this study. Phaedranassa viridiflora is known only in three locations within Ecuador. The type of forest inhabited by this species is classified as Dry mountain shrub vegetation (Matorral seco montano), which has been reduced in half by human disturbances (Baquero et al., 2004). However both number of populations and area of occupancy are greatly reduced if this species is treated as three distinct taxa. Any conservation plan should take into consideration that the north, the central and south groups of the yellow Phaedranassa are genetically divergent. In order to protect the natural demographic process, all three groups of P. viridiflora should be treated individually. In the north, the natural hybridization process should be preserved because hybrid zones are sources of species (Whitman et ah, 1994). The group in the north is located at the Reserva Geobotanica Pululauhua, which enable the conservation of the natural hybridization process described in this study. The protection of the groups in the center and south is more difficult to achieve because these individuals live in agricultural and rural areas. However, several characteristics of the natural history of the genus facilitate the conservation of these species. Bulbs provide the means to survive in disturbed habitats, giving both protection (from fire, Oleas pers. obs.) and capacity for asexual reproduction. Clonal reproduction of diploids over time can generate heterozygosity excess (Balloux et al., 2003; Masel & Lyttle, 2011). Heterozygosity and the lack of genetic drift maintain genetic diversity needed for the long-term survival in a changing environment (Van der Merwe et al., 2010). The evolutionary potential is shown in the Pululahua crater, where sexual reproduction, specifically hybridization with P. dubia, result in a new orange flower phenotype. Finally, Phaedranassa species also have ornamental potential that has not been exploited. In-situ conservation can be easily accomplished by cultivation of these species in their original location, in parks or other open areas. The degree of genetic diversity reported in the present investigation can be used to evaluate the success of conservation initiatives by in-situ propagation.

Our study provides an example of how population genetic data can offer indirect evidence of the reproductive biology of a species, an understanding of which is important to effectively conserve them. This type of information is mostly unknown, and it can be difficult and time consuming to obtain. Molecular markers have the potential to relatively quickly identify reproductive modes such as clonality, inbreeding and hybridization. These three traits are also important for conservation purposes, because genetic diversity provides the means to survive in a changing environment. With the anticipation of continued reduction in cost and time required to obtain molecular markers, it is likely that microsatellites will increasingly contribute to the knowledge and conservation of plant species in Latin America. However it is important to acknowledge that molecular markers are not a complete substitute of detailed natural history and ecological studies, which are necessary to better understand population patterns.

DOI 10.1007/s12229-013-9125-8

Published online: 15 August 2013

Appendix

Table 4 Loci with null alleles and Fixation index (F) calculated with
all [loc.sup.(1)] and with loci with less null [alleles.sup.(2)]

Pop            Loci

               pt14   pt48   pt49   ps2   psl3   ps28   ps27   pt43

V8             -      -      -      -     -      -      -      -
V5             -      -      -      -     +      -      -      -
V6             +      -      +      -     -      -      -      -
V3             -      +      +      -     -      -      -      -
V1             -      +      +      +     +      +      -      -
V4             -      +      +      +     +      -      -      +
C3             -      -      +      +     +      +      +      +
V7             +      +      -      +     +      +      +      -
Cl             +      -      -      -     +      +      +      +
D1             +      +      +      +     +      -      -      +
C2             +      -      +      +     +      +      +      +
V2             +      +      +      +     +      +      +      -
S1             +      +      +      +     +      +      +      +
Total loci     7      7      9      8     10     7      6      6
null alleles

Pop            Loci

               pt[21.     pt[32       pt[39       ps[16       pt[61
               sup.(2)]   .sup.(2)]   .sup.(2)]   .sup.(2)]   .sup.(2)]

V8             -           -           -           -           -
V5             -           -           -           -           -
V6             -           -           -           -           -
V3             -           -           -           -           +
V1             -           -           -           -           -
V4             -           -           -           -           +
C3             -           -           -           -           -
V7             -           -           +           -           -
Cl             -           +           +           -           -
D1             +           +           -           -           -
C2             +           +           -           -           -
V2             +           +           +           +           +
S1             +           +           +           +           -
Total loci     4           5           4           2           3
null alleles

Pop                           [F.sup.(1)]    [F.sup.(2)]
               Total loci
               null alleles

V8             0              -0.45         -0.92
V5             1              -0.41         -0.71
V6             2              -0.22         -0.72
V3             3              -0.02         -0.24
V1             5               0.08         -0.31#
V4             6               0.14         -0.24#
C3             6               0.42          0.07
V7             7              -0.12         -0.80
Cl             7               0.20          0.08
D1             8               0.34          0.23
C2             9               0.26         -0.15#
V2             12              0.66          0.71
S1             12              0.44          0.37
Total loci
null alleles

Numbers in bold are populations not showing inbreeding when
analyzed with loci without putative null alleles

Note: Numbers in bold are populations not showing inbreeding when
analyzed with loci without putative null alleles are indicated with #.


Acknowledgments We thank the USDA-ARS Plant Science lab technicians, the Herbarium QCA and its director Dr. Hugo Navarrete, and the Ministerio del Medio Ambiente of Ecuador for collecting and export permits. We also thank Lou Jost, Marcelo Ayabaca, Tania Sanchez and Cristian Melo for their help during fieldwork. N.O. thanks Dr. Robert Colwell for helpful discussion about Phaedranassa. N.O. also thanks Fairchild Tropical Botanic Garden, the Graduate Student Funding Committee, the College of Art & Sciences and the Department of Biological Sciences of FIU, for supporting her participation at the 10th Latin American Botanical Congress. We also thank to two anonymous reviewers for their helpful comments. This study was supported by National Science Foundation (NSF Grant DEB 0129179 to AWM. This is contribution number 223 from the Tropical Biology Program of Florida International University.

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Nora H. Oleas (1,2,4) * Alan W. Meerow (3,5) * Javier Francisco-Ortega (1,2)

(1) Department of Biological Sciences, Florida International University, 11200 SW Sth St., Miami, FL 33199, USA

(2) Fairchild Tropical Botanic Garden, Center for Tropical Plant Conservation, 11935 Old Cutler Road, Coral Gables, Miami, FL 33156, USA

(3) USDA-ARS-SHRS, National Germplasm Repository, 13601 Old Cutler Rd., Miami, FL 33158, USA

(4) Universidad Tecnologica Indoamerica, Centro de Investigacion en Biodiversidad y Cambio Climatico, Machala y Sabanilla, Quito, Ecuador

(5) Author for Correspondence; e-mail: alan.meerow@ars.usda.gov

Table 1 Descriptive statistics of genetic variation
among Phaedranassa spp

Species                       Location   Col ID   N       Na     Ne

P. dubia                      North      D1       27      8.08   4.13
P. viridiflora                North      V1       31      4.92   2.10
P. viridiflora x P. dubia *   North      V2       27      6.85   2.90
P. schizantha                 Central    S1       28      7.85   3.66
P. viridiflora                Central    V3       29      2.15   1.34
P. viridiflora                Central    V4       30      3.54   1.66
P. viridiflora                Central    V5       30      1.54   1.32
P. cinerea                    South      Cl       27      5.69   2.50
P. cinerea                    South      C2       30      4.08   2.04
P. cinerea                    South      C3       13      3.92   3.10
P. viridijlora                South      V6       29      2.31   1.41
P. viridiflora                South      V7       30      2.77   1.48
P. viridiflora                South      V8       31      1.92   1.29
Mean                                              27.85   4.28   2.23
Stdev                                              4.69   2.25   0.96

Species                       Ho      He              F       LD

P. dubia                      0.45    0.69            0.34    0
P. viridiflora                0.51    0.47            0.08    22
P. viridiflora x P. dubia *   0.20    0.59            0.66    19
P. schizantha                 0.39    0.69            0.44    0
P. viridiflora                0.24   [0.16.sup.2]    -0.01    0
P. viridiflora                0.33    0.31            0.14    0
P. viridiflora                0.31   [0.17.sup.12]   -0.41    0
P. cinerea                    0.41    0.49            0.20    18
P. cinerea                    0.32    0.32            0.27    0
P. cinerea                    0.41    0.62            0.42    0
P. viridijlora                0.33   [0.21.sup.12]   -0.22    0
P. viridiflora                0.31    0.22           -0.12    0
P. viridiflora                0.25    0.1412         -0.45    0
Mean                          0.34    0.39            0.10    4.54
Stdev                         0.09    0.21            0.34    8.67

Species                       NL     PA     MLG

P. dubia                      8      13     0
P. viridiflora                4      3      14
P. viridiflora x P. dubia *   12     6      2
P. schizantha                 12     14     0
P. viridiflora                3      0      19
P. viridiflora                6      0      6
P. viridiflora                1      0      28
P. cinerea                    7      11     4
P. cinerea                    9      2      13
P. cinerea                    6      1      0
P. viridijlora                2      0      18
P. viridiflora                7      2      12
P. viridiflora                0      1      26
Mean                          5.92   4.08   11.00
Stdev                         3.84   5.20   9.74

Species                       G/N    PID

P. dubia                      1.00   7.65 x [10.sup.15]
P. viridiflora                0.61   1.96 x [10.sup.7]
P. viridiflora x P. dubia *   0.96   1.47 x [10.sup.-11]
P. schizantha                 1.00   7.37 x [10.sup.14]
P. viridiflora                0.41   7.05 x [10.sup.3]
P. viridiflora                0.90   4.26 x [10.sup.-5]
P. viridiflora                0.13   1.29 x [l0.sup.-2]
P. cinerea                    0.93   4.20 x [10.sup.9]
P. cinerea                    0.73   5.15 x [10.sup.6]
P. cinerea                    1.00   2.95 x [10.sup.12]
P. viridijlora                0.01   2.51 x [10.sup.3]
P. viridiflora                0.63   1.08 x [10.sup.3]
P. viridiflora                0.26   1.88 x [10.sup.2]
Mean                          0.37   3.25 x [10.sup.3]
Stdev                         0.32   0.006

Collection site (Col ID); Sample number (N); Number of alleles (Na);
Effective Allele number (Ne), Expected and Observed heterogosity
(He, Ho), significant p<0.05 (1); He excess p< 0.5 excluding loci
with null alleles ('); Fixation index (F), Number of pair of locus
with Linkage disequilibrium (LD); Loci with evidence of null
alleles (NL); Number of Private alleles (PA); Repetitive multilocus
genotypes (MLG); Genotype discovery rate (G/N), Probability of
identity (PID); hybrid (*)

Table 2 Analysis of molecular variance (AMOVA) of 13 loci
across 13 populations of Phaedranassa in Ecuador

Source of variation   By location

                             Sum of    Percentage of
                      d.f.   squares   variation

Among groups            2     371.08    26.09 *
Among populations      10     526.57    43.25 *
  within groups
Within populations    349     459.97    30.66 *
Total                 361    1357.62

Source of variation   By location--species

                             Sum of    Percentage of
                      d.f.   squares   variation

Among groups            5     676.24    42.22 *
Among populations       7     221.41    26.31 *
  within groups
Within populations    349     459.97    31.46 *
Total                 361    1357.62

* Significant <0.001

Table 3 Test for recent bottleneck events in seven populations
of Phaedranassa spp. on the basis of three tests

Species   (1) Sign test   (2) Wilcoxon test   (3)Mode-shift

          IAM     SSM     IAM       SMM

C1        0.01#   0.00#   0.98      1.00
C2        0.23    0.02#   0.92      1.00
D1        0.15    0.00#   0.05#     1.00
S1        0.46    0.01#   0.27      1.00
V1        0.50    0.00#   0.77      1.00
V2        0.26    0.00#   0.88      1.00
V3        0.22    0.04#   0.81      0.95
V4        0.10    0.00#   0.93      1.00
V5        0.35    0.45    0.05#     0.05#     +
V6        0.53    0.20    0.63      0.85
V7        0.13    0.08    0.68      0.97
V8        0.39    0.26    0.58      0.95

In bold are significant evidence of bottleneck cases

Note: In bold are significant evidence of bottleneck cases is
indicated with #.
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