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Plant Species Biology (2014) 29, 2–15

doi: 10.1111/j.1442-1984.2012.00381.x

Genetic diversity and conservation of Ipomoea microdactyla (Convolvulaceae): an endemic vine from the Bahamas, Cuba, and southeastern Florida psbi_381

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JOHN H. GEIGER,1*† ALAN W. MEEROW,‡ CARL LEWIS,* RAMONA OVIEDO§ and JAVIER FRANCISCO-ORTEGA*† *Department of Biological Sciences, Florida International University, University Park, Miami, Florida 33199, †Center for Tropical Plant Conservation, Fairchild Tropical Botanic Garden, Coral Gables, Miami, Florida 33156, ‡National Germplasm Repository, US Department of Agriculture, Agricultural Research Service, Miami, Florida 33158, USA; and §Botany Division and National Herbarium, Institute of Ecology and Systematics, Ministry of Science, Technology, and the Environment, Havana, A.P. 8029, 10800, Cuba

Abstract Ipomoea microdactyla Griseb. (Convolvulaceae) is restricted to the Bahamian archipelago, Cuba, and southeastern Florida. The species is listed as a state endangered species in Florida, where it is mostly restricted to the hyperfragmented pine rockland of MiamiDade County. Using seven DNA microsatellite loci, we assessed levels of genetic diversity for 12 populations of this species from Andros Island in the Bahamas (six sites), Cuba (one site), and Florida (five sites). We found significantly greater mean numbers of alleles, and higher mean values for both observed and expected heterozygosity in populations from the continuous forest on Andros than those from the habitat fragments in Florida. It is unknown if these patterns of genetic diversity in the Florida populations are the result of habitat fragmentation or founder effects. The population from Cuba exhibited relatively high levels of genetic variation, suggesting that this island is a major center of diversity and dispersal for this species. It appears that hybrid introgression for I. carolina alleles within I. microdactyla individuals occurred at a single site on Andros Island. Overall, the mean inbreeding coefficient value was 0.089, suggesting low levels of inbreeding. The highest inbreeding coefficient values were mostly recorded in Florida. Two groups were revealed, one containing the populations from Florida, and the second one encompassing those from the Bahamas and Cuba. Our results highlight the negative genetic consequences of habitat fragmentation and support initiatives recently established to establish corridors to connect the remnants of the pine forest of the Miami-Dade County. Keywords: biodiversity hotspots, Caribbean Basin, conservation genetics, habitat fragmentation, tropical islands. Received 2 January 2012; revision received 11 May 2012; accepted 15 May 2012

Introduction The Caribbean is one of the 34 global biodiversity ‘hot spots’ (sensu Mittermeier et al. 2004). This insular system

Correspondence: John H. Geiger Email: [email protected] 1 Present address: Department of Biology, Health and Wellness, Miami Dade College, Kendall Campus, Miami, Florida 33176, USA. © 2012 The Society for the Study of Species Biology

is poised to lose a large amount of its biodiversity (Maunder et al. 2011) as conservation endeavors have grown weaker and biological studies fewer across the region in general (Maunder et al. 2008). Researchers have suggested that projects aimed at identifying the factors imperiling the Caribbean’s singular biodiversity, especially investigations of the genetic variation among populations of threatened plant species, are vital for conservation efforts (Kress & Horvitz 2005). The present study seeks to use DNA microsatellite data (SSRs) to

IPOMOEA MICRODACTYLA GENETIC DIVERSITY reveal the genetic structure of a species endemic to this region with conservation concerns in southern Florida. In addition, this research augments our plant conservation genetic program for the Caribbean Islands and Florida. Our previous and current DNA microsatellite studies have mostly focused on palms and cycads (Meerow & Nakamura 2007; Meerow et al. 2007, in press; Namoff et al. 2010, 2011; Calonje et al., in press). For this study, we chose a morning glory restricted to southeastern Florida and the Caribbean Islands. As far as we are aware, this is the first ever population genetic study using microsatellite data that focuses on a plant species restricted to southern Florida and the West Indies. The conservation genetics paradigm broadly frames many investigations into preserving biodiversity by considering the link between the loss of population-level genetic diversity and the increased risk of population extinction (Ouborg et al. 2006; DeSalle & Amato 2009). While controversy still exists regarding the relationship between plant population size, fitness, and genetic variation (Oostermeijer et al. 2003), several reviews have shown the association to be generally positive (e.g. Reed & Frankham 2003; Leimu et al. 2006). From the conservation genetics perspective, threats to biodiversity are perceived via properties of the populations themselves (e.g. reduced size, increased isolation from conspecific populations) rather than abiotic aspects of their habitat (e.g. reduced habitat quality). A suite of evolutionary forces such as genetic drift and gene flow, with subsequent inbreeding, can be affected indirectly by habitat fragmentation, leading to negative consequences for these populations (Galeuchet et al. 2005). Genetic drift becomes more severe in small populations, leading to a loss of genetic variation over time (Hartl 2000; Allendorf & Luikart 2007). Inbreeding depression may lower fitness and negatively affect the viability of populations (Young et al. 1996; Höglund 2009). This, in turn, may lead to population extinction (Mills & Smouse 1994; Newman & Pilson 1997). Few population genetic studies have focused on plants from the Caribbean Islands and southern Florida. Most of them have been based on isozymes (e.g. Husband & Barrett 1991, Walters & Decker-Walters 1991; Negrón-Ortiz & Hickey 1996; Ivey & Richards 2001; Dunphy & Hamrick 2007; Pinares et al. 2009) and DNA dominant markers such as RAPDs (e.g. Thornton et al. 2008) or ISSRs (e.g. Cariaga et al. 2005; Davis et al. 2007; Maschinski et al. 2010). Other population genetic studies have used phylogenetic analyses of nucleotide sequences (e.g. Dertien & Duvall 2009), or RFLPs of PCR products (Maskas & Cruzan 2000; Cattell & Karl 2004). Among these works, those by Dunphy and Hamrick (2007), Thornton et al. (2008), and Pinares et al. (2009) have addressed questions pertinent to the effect of habitat Plant Species Biology 29, 2–15

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fragmentation on the genetic structure of Caribbean plants. Dunphy and Hamrick (2007) studied the impact of habitat fragmentation on population genetic structure of the neotropical tree Bursera simaruba (L.) Sarg. (Burseraceae) in southwestern Puerto Rico. Their study showed that habitat fragmentation has not had an impact in gene flow. The low levels of population divergence and relatively high genetic diversity values within populations supported these conclusions. Jacquemontia reclinata House (Convolvulaceae), is a Florida state endangered species restricted to coastal sand dunes of southeastern Florida (Coile & Garland 2002). Thornton et al. (2008) found a positive correlation between population size and genetic diversity for this species; however, still, most of the variation was found within populations. It was suggested that low genetic differentiation was due to the recent fragmentation of the habitat where this species occurs. In contrast, Pinares et al. (2009) found significant differentiation among populations of the Cuban cycad Microcycas calocoma (Miq.) A. DC. (Zamiaceae) and attributed these results primarily to the fragmentation of the original habitat and to the concomitant isolation of the populations of this critically endangered species (Bosenberg 2010). We examined genetic diversity from 12 populations of Ipomoea microdactyla Griseb. (Convolvulaceae) across its geographic range using seven polymorphic microsatellite loci. Specifically, we focused on the following questions. (i) What is the population genetic diversity of I. microdactyla across its range-wide area? (ii) Is there significant population genetic structure and differentiation among populations from the three sampled regions? (iii) Do populations within habitat fragments show lower levels of genetic variation than populations within continuous habitat? (iv) What are the conservation implications of these findings for I. microdactyla? (v) What are the colonization patterns involving populations from Cuba, the Bahamas, and Florida?

Materials and methods

Plant species Ipomoea microdactyla is a relatively common Caribbean species in the understory of pine forests, evergreen dry forests, and coastal scrubs. The species is restricted to Cuba, most of the islands of the Bahamian archipelago, and southeastern Florida. However, there is a marked contrast in habitat between populations found in the USA, Cuba and the Bahamas (Geiger 2007). In the USA, I. microdactyla is listed as a Florida state endangered species (Coile & Garland 2002) and is restricted to Everglades National Park and to 36 of the remaining ca 400 pine forest fragments in Miami-Dade County (Gann et al. 2002). Most © 2012 The Society for the Study of Species Biology

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of these fragments are less than 10 ha in size and are embedded within the agricultural, suburban and urban landscape of the populous and expanding Miami metropolitan area (O’Brien 1998). Population size of most fragments is less than 50 individuals (Geiger 2007). In contrast, I. microdactyla is found in high densities across most of the pristine, non-fragmented habitat sites in the Bahamas. The species has been reported for all the provinces of Cuba, but it is uncertain to what extent the Cuban populations have similar high densities as seen in the Bahamian sites. The single Cuban population included in our study had only 21 plants in an area of ca 5000 m2 (see habitat description below). Ipomoea microdactyla seems to have a preference for frequently burned pinelands because of its life history traits (e.g. rapid, post-fire vegetative regeneration, long-lived underground tubers resistant to fire mortality, and the necessity for synchronous flowering because of its selfincompatible breeding system) (Geiger 2007). The species is a perennial vine with woody, twining stems to over 10 m in length. Plants are long-lived and have underground tubers much like the congeneric sweet potato (I. batatas (L.) Lam.). Several stems often grow from the same tuber, so without excavating the stems from the substrate, it is difficult to differentiate among different genotypes, particularly when the ‘individuals’ are in close proximity. Plants produce several to hundreds of hermaphroditic light red flowers in discrete flushes during

Population† Florida BLW (17) CAM (18) CRF (15) MON (21) TPK (19) Mean The Bahamas ANT (16) ATL (18) ECO (17) FOR (20) RDG (17) RZM (13) Mean Cuba CUB (21) Overall mean

the blooming season from May to December. Flowers are visited by a diverse group of insects including Hymenoptera and Lepidoptera, but the predominant flower visitors are hummingbirds (Trochiliformes). As with most of its congeners, I. microdactyla is selfincompatible and requires pollen vectors for fruit/seed production (Geiger 2007). Capsular fruit contain a maximum of four seeds covered with hairs 1 cm long. Seeds weigh ca 1.5 mg and are dispersed by wind, but are capable of floating on water; they generally germinate immediately after release in late autumn or early winter (Geiger 2007).

Sampling, DNA isolations, and PCR amplifications We surveyed 12 populations across the entire geographic range of I. microdactyla to gauge genetic diversity and population genetic structure (Table 1, Figs 1,2). This included: six sampling sites in one of the largest extant continuous pine forest in the Caribbean on Andros Island (the Bahamas Archipelago) (Fig. 1); five habitat remnant sites in the hyper-fragmented pine rockland of MiamiDade County (Fig. 2); and one scrub forest site (on serpentine soils) in western Cuba (Fig. 1). The Cuban population is part of a highly fragmented and disturbed habitat surrounded by extensive farming. Within this area, the few remaining sites with natural vegetation have been extensively colonized by ‘marabú’ (Dichrostachys cinerea (L.)

Locality

P

np

A

HO

HE

F§

Nig

Null

Bill Sadowski Campbell Drive Coral Reef Moon/Schaffer Turnpike 152

71 100 86 100 100 91.4

1 0 1 0 0 2‡

3.4 4 3 4.3 3.3 3.6

0.429 0.516 0.327 0.456 0.476 0.441

0.422 0.599 0.378 0.559 0.531 0.498

0.042 0.109***¶ 0.179*¶ 0.198***¶ 0.039**¶ 0.113***¶

2 0 1 0 1

1 2 1 2 2

Ant Coppice Atala Coppice Ecotone Forfar Station Coast Ridge Rhizophora

100 100 100 100 100 100 100

0 5 0 0 0 1 6

4.9 5.6 4.1 6.4 4.9 4.7 5.1

0.598 0.540 0.597 0.750 0.681 0.637 0.634

0.563 0.674 0.571 0.705 0.629 0.682 0.638

-0.054 0.244***¶ 0.011 0.033 -0.087 0.112**¶ 0.032***¶

0 0 0 0 0 0

1 2 1 1 0 1

Cuabales de Galindo

100

4

6.3

0.571

0.627

0.123***¶

1

2

12‡

4.6

0.548

0.578

0.089***¶

96.4

Table 1 Ipomoea microdactyla population genetic statistics. See Figures 1 and 2 for geographical distribution of populations. P = percentage of polymorphic loci; np = number of private alleles; A = average number of alleles per locus; HO = observed heterozygosity; HE = expected heterozygosity; F = mean inbreeding coefficient; Nig = number of identical genotype pairs; Null = number of loci in which null alleles may be present as suggested by MICROCHECKER because of a general excess of homozygotes

*P < 0.05; **P < 0.01; ***P < 0.001. †Number of sampled individuals are given inside parentheses. ‡Pooled values. Total number of alleles across all loci for all the populations = 66. §Significant departures from Hardy-Weinberg equilibrium at levels indicated by the asterisks. ¶Heterozygote deficiency (nonsignificant values were obtained for heterozygote excess). © 2012 The Society for the Study of Species Biology

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Fig. 1 Populations from the Bahamas (Andros) and Cuba that were included in this study. The open square shows the area that was sampled on Andros.

Fig. 2 Populations from southeastern Florida that were included in this study. The open square shows the area that was sampled in Florida.


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Locus BM878757 Ib-286 Ib-318 IBSSR01 IBSSR11 IBSSR13 IBSSR14

Repeat motif

Primer reference

Allele size

Number of alleles

(CT)8TA(TC)5 (CT)12 (CT)3C(CT)8 (GA)14 (AC)21 (GA)7N(GA)14 (GA)9

Hu et al. (2004) Buteler et al. (1999) Buteler et al. (1999) Hu et al. (2004) Hu et al. (2004) Hu et al. (2004) Hu et al. (2004)

104–120 82–102 108–130 158–186 240–267 160–186 177–185

8 11 6 13 9 14 5

Wight & Arn.), an aggressive legume from Africa whose invasiveness represents one of the most important challenges for the conservation of xeric ecosystems in Cuba (Mesa Lago 2009). We were unable to study the populations found in Everglades National Park due to the strict permit regulations required to conduct research on Federal lands. Likewise, we could not carry out additional field studies in Cuba because of legislatively imposed limitations for faculty and students from the Florida State University System to travel and perform research in this country. Fresh or silica-gel dried leaf samples were collected from 13 to 21 adults at each site (Table 1). Overall, 212 individuals were sampled. The entire population was sampled at sites BLW, CRF, and TPK in Florida and at CUB in Cuba. Therefore because of complete sampling at these four sites, we have the ability to distinguish genets from ramets of the same genet for the closely spaced stems at these low population density sites (Table 1). On Andros Island, plant densities were relatively uniform and the plant sampling areas were roughly 10 000 m2 at the six collection sites. A minimum distance of 20 m was maintained between collections to avoid resampling the same genet. DNA was extracted using the FastDNA Spin Kit BIO 101 System with FastPrep instrument (Q-BIOgene: Qbiogene, Carlsbad, CA, USA) following the manufacturer’s protocols. We used seven SSR loci originally developed for sweet potato to measure population genetic structure at microsatellite loci in I. microdactyla: BM878757, Ib-286, Ib-318, IBSSR11, IBSSR01, IBSSR13, and IBSSR14 (Buteler et al. 1999; Hu et al. 2004) (Table 2). To genotype individuals, polymerase chain reaction (PCR) amplifications were performed in 10-mL volumes with 25 ng genomic DNA, 0.5 mmol each of the fluorescent dye-labeled forward primer and of the reverse primer, 200 mmol of each dNTP, 1 ¥ reaction buffer (10 mmol Tris-HCL, pH 8.3, 50 mmol KCL, 1.5 mmol MgCl2), and 1 unit GoTaq DNA polymerase (Promega: Promega, Madison, WI, USA). PCR amplifications were performed in a PT-100 thermal cycler (MJ Research: Promega, Madison, WI, USA) using the following protocol: 96°C for 1 min (one cycle), 94°C for 40 s, 55°C for 1 min, 72°C for 1 min (30 cycles), and 72°C © 2012 The Society for the Study of Species Biology

Table 2 Microsatellite loci used in this study

for 5 min (one cycle). We combined 2 mL of the PCR product with 10 mL of a 20:1 solution of formamide and GeneScan-500(ROX) size standard (Applied Biosystems: Promega, Madison, WI, USA) mix. Samples were run on an ABI Prism 3100 Genetic Analyser and allele sizes were scored using GeneScan version 3.1.12 (Applied Biosystems).

Data analysis Descriptive statistics for populations across all loci included: percent of polymorphic loci (P), number of private alleles (np), average number of alleles per locus (A), observed heterozygosity (HO), expected heterozygosity (HE), inbreeding coefficients (F), and number of identical shared multilocus genotypes (MLGs). They were computed using GenAlEx version 6 software (Peakall & Smouse 2006). We tested for significant values of F using SPAGeDi (Hardy & Vekemans 2002), with 20 000 gene and individual permutations. Tests for Hardy-Weinberg equilibrium (HWE) and the U-test (Rousset & Raymond 1995) for heterozygote excess or deficiency were run using GenePop (Raymond & Rousset 1995; Rousset 2008), with 10 000 Monte Carlo Markov Chain (MCMC) iterations (Guo & Thompson 1992). Linkage disequilibrium (LD), the nonrandom pairwise association of loci, was tested for each population using ARLEQUIN (Excoffier et al. 2005), with a likelihoodratio test (Slatkin & Excoffier 1996). A MCMC method was applied with 100 000 iterations and a 10 000-iteration burn-in period, with the significance level set at P < 0.001. The program Populations (Langella 1999) was used to calculate the chord distance of Cavalli-Sforza and Edwards (1967) among populations. We created a neighbor-joining tree from this genetic distance matrix and substantiated the branching pattern by performing 10 000 bootstrap permutations (over loci) with Populations. The resulting trees were visualized using Tree Explorer as implemented in MEGA 3 (Kumar et al. 2004). The Bayesian clustering program STRUCTURE v.2.3.3 (Pritchard et al. 2000) was used to estimate the underlying genetic structure among our populations. The STRUCTURE analyses were carried out on the freely available Plant Species Biology 29, 2–15

IPOMOEA MICRODACTYLA GENETIC DIVERSITY University of Oslo Bioportal (http://www.bioportal. uio.no). K-values of 1–20 were simulated across 20 replicate runs of 1 000 000 iterations after a burn-in of 100 000. The DK method of Evanno et al. (2005) as implemented in STRUCTURE HARVESTER (Earl 2011) was used for determining the ‘true’ value of K across our samples. After the likely level of K was estimated, a consensus Q-matrix from the 20 runs was constructed using the program CLUMPP (Jakobsson & Rosenberg 2007) for visualization with DISTRUCT (Rosenberg 2004). A Euclidean geographic distance matrix was generated from latitude and longitude coordinates using GenAlEx. The geographical and chord genetic distance (see above) matrices were used for permuted (10 000 iterations) Mantel (1967) tests for isolation by distance, following the methods of Smouse et al. (1986) and Smouse and Long (1992) as implemented in GenAlEx. Using FST, we analyzed the hierarchical distribution of genetic variation of the three regions, among populations nested within the regions, and among all populations with an analysis of molecular variance (amova) (Excoffier et al. 1992), as performed in GenAlEx, with 10 000 permutations across the full data set. FST pairwise comparisons between populations (also with 10 000 permutations across the full data set) were computed with GenAlEx. The number of migrants (Nm) was calculated using the private allele method (after correction for size) of Slatkin (1985), Barton and Slatkin (1986), and Slatkin and Barton (1989), as implemented in GenePop. Four population genetic diversity statistics (i.e. A, HO, HE, and F) for the fragmented populations in Florida and the continuous sites of Andros were contrasted with t-tests. We decided not to include Cuba in these statistical analyses as a single population was sampled on this island. These analyses were performed with SPSS 15.0 (SPSS, Chicago, IL). The program MICRO-CHECKER (Van Oosterhout et al. 2004) was used to evaluate the presence of stuttering patterns, null alleles and large allele dropouts, employing 3000 randomizations.

Results For the 212 plants sampled, we recorded between five and 14 alleles per locus for a total of 66 alleles at the seven loci. We detected a total of five pairs of individuals with identical shared MLGs (Table 1) within three of the five populations from Florida (BLW, CRF, and TPK) and the Cuban site; we did not find shared MLGs between different populations. These identical genotypes were removed from subsequent analyses because we were uncertain if they belonged to different individuals. Therefore, the final data matrix had 207 individuals. Plant Species Biology 29, 2–15

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The MICRO-CHECKER analysis did not suggest any errors from stutter or large allele dropout, but one to two possible null alleles were indicated in 11 of the populations (Table 1). We ultimately evaluated the apparent homozygote excess as real. The most prominent evidence is that for these populations, loci without any indication of null alleles also deviated from HWE. Moreover, repeat amplifications of a sampling of homozygotes from these populations did not reveal additional undetected alleles. Three loci were monomorphic in two of the Florida populations (loci Ib-318 and IBSSR14 for BLW and locus IBSSR01 for CRF). Polymorphism within populations ranged from 71 to 100% (mean % of polymorphic loci = 96.43, SE = 2.56). The Cuban and all of the Bahamian populations were polymorphic for all the loci (Table 1). The mean number of alleles for all populations was 4.6 (SE = 0.22), with average values of 3.6 (SE = 0.236) for the Florida sites, 5.1 (SE = 0.318) for the Bahamian populations, and 6.3 (SE = 0.94) for the Cuban locality). We detected 12 private alleles among the populations, nearly half of them (five alleles) at the ATL site in the Bahamas. None of the 12 populations showed significant levels of pairwise LD. Average observed and expected heterozygosity for all populations were 0.548 (SE = 0.03) and 0.578 (SE = 0.021), respectively (Table 1). However, these values were larger in the Bahamas (mean HO and HE values of 0.634 (SE = 0.042) and 0.637 (SE = 0.024), respectively) and Cuba (HO and HE values of 0.571 (SE = 0.103) and 0.627 (SE = 0.051), respectively) than in Florida (mean HO and HE values of 0.441 (SE = 0.042) and 0.498 (SE = 0.036), respectively). Overall, the 12 populations had a mean inbreeding coefficient of 0.073 (SE = 0.036, P < 0.001). This coefficient had higher mean values for the overall populations of Florida (F = 0.116, SE = 0.058, P < 0.001) and Cuba (F = 0.123, SE = 0.123, P < 0.001) than for those for the Bahamas (F = 0.032, SE = 0.050, P < 0.001) (Table 1). Therefore, there is evidence that the populations from Cuba and Florida were more inbred than those from the Bahamas. On the basis of the U-tests, seven populations departed significantly from HWE (Table 1). Four of these populations were in Florida, two in the Bahamas, and the one from Cuba. These seven populations showed heterozygote deficit, whereas no population showed significant heterozygote excess. Overall population analyses for Florida and the Bahamas showed highly significant heterozygote deficits for these two regions (Table 1). An unrooted neighbor-joining tree of the 12 populations across all seven loci based on genetic distance resolved two major clusters (Fig. 3) with one outlier. The first cluster (supported with a bootstrap value of 90%) had all of the populations from Florida. The second cluster comprised five of the six sites from the Bahamas and the © 2012 The Society for the Study of Species Biology

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J. H. GEIGER ET AL.

Fig. 3 Neighbor joining dendrogram (based on chord distance of Cavalli-Sforza & Edwards (1967)) showing the genetic relationship among populations. Branch lengths are proportional to distances and bootstrap support for identified clusters are also shown. Circles = populations from Florida. Diamond = population from Cuba. Squares = populations from the Bahamas.

single population from Cuba (supported with a bootstrap value of 87%). The ATL population from the Bahamas was an outlier located between these two main clusters; however, it exhibited a shorter genetic distance to the Bahamas–Cuba group than to the Florida cluster. The DK method indicated that K = 2 was the most likely number of clusters (Fig. 4). The two-cluster solution showed that all of the individuals from Florida belong primarily to one of these two clusters. The Cuban population and five of the six Bahamian populations were mostly assigned to the second cluster, while a few individuals from CUB and FOR exhibited admixture. The ATL site (Bahamas) had the highest levels of admixture. Approximately two-thirds of the individuals of this population were primarily assigned to the cluster that was predominant in Florida. The rest of them were mostly allocated to the other cluster. The amova tests showed that most of the genetic variation was found within populations (83%), as opposed to among regions (10%) or among populations within regions (7%), for all 12 populations (P < 0.001). When the analyses were restricted to only the Florida or Bahamas sites, we found that most of the genetic variation was found within populations (93% for Florida (P < 0.001) and 93% for the Bahamas (P < 0.001)). The low levels of differentiation among populations were also supported by the FST values (mean FST of 0.148 for all the populations, mean © 2012 The Society for the Study of Species Biology

FST of 0.087 for the Bahamas sites, and mean FST of 0.082 for the Florida localities) (Table 3). The two highest FST values (0.306 and 0.292) were found between one population from Florida (CRF) and two Bahamian populations (ECO and RDG, respectively). Within Florida, TPK and CRF exhibited the largest FST values (0.118). Within the Bahamas, the highest FST pairwise values were 0.171 and 0.172, both involved the ATL population. The population from Cuba displayed the highest FST values (between 0.136 and 0.248) with the five Florida sites. All of the FST comparisons were either significant (P < 0.05) or highly significant (P < 0.001) (data not shown). Overall Nm values were equal to 0.51 for all the samples, 0.74 for the Florida sites, and 2.29 for the Bahamian populations. None of the Nm values within Florida were greater than 0.89. In contrast, eight of the 14 Nm values within the Bahamas were greater than 1.4 (Table 2). The highest Nm value (4.41) was found between the RDG and ECO sites in the Bahamas. The Cuban population displayed Nm values higher than 0.85 only with sites from the Bahamas (Table 3). Three of the genetic diversity statistics (i.e. A, HO, and HE) were significantly different between the fragmented populations in Florida and the nonfragmented populations on Andros (P < 0.002). In contrast, no significant differences were detected for the inbreeding coefficient between these two data sets (P = 0.285). Results of the Mantel test indicated a significant correlation between population geographic distance and genetic distance across all 12 populations (Rxy was 0.609 and R2 was 0.371, P < 0.005). However, the Florida (Rxy = 0, R2 = 0.0001, P = 0.396) and Bahamian populations (Rxy = -0.052, R2 = 0.115, P = 0.166) did not exhibit significant isolation by distance.

Discussion

Genetic diversity and hybridization The statistics for genetic diversity (e.g. total number of alleles, HO, and HE) for I. microdactyla fall within the intervals found in a review of intraspecific genetic diversity in plants (Nybom 2004). These measures are similar to those reported for other species within the family Convolvulaceae (Kim & Chung 1995; Wolf et al. 2000). We obtained greater numbers of alleles per locus for this wild species than the cultivated sweet potato for which the primers we used were originally developed (Buteler et al. 1999; Hu et al. 2004). Three loci were monomorphic at the Florida sites (BLW and CRF) that have the smallest population sizes. As we genotyped all individuals at these sites, no alleles were missing due to statistical sampling (Weir 1996). Rather, this appears to be the result of the fixation of alleles via Plant Species Biology 29, 2–15

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Fig. 4 STRUCTURE analyses. (a) DKvalues for K ranging between 2 and 19. (b) Proportion of individuals per population for K = 2. Color and box sizes indicate the cluster type of each individual and the number of plants sampled per population. The vertical lines indicate the probability that each individual belongs to an inferred cluster.

Table 3 Genetic differentiation and estimates of migration for Ipomoea microdactyla populations. Above diagonal = Nm values. Below diagonal = FST estimates BLW BLW CAM CRF MON TPK ANT ATL ECO FOR RDG RZM CUB

0.073 0.104 0.071 0.081 0.228 0.154 0.258 0.178 0.240 0.206 0.174

CAM

CRF

MON

TPK

ANT

ATL

ECO

FOR

RDG

RZM

CUB

0.75

0.68 0.34

0.96 0.84 0.45

0.64 0.85 0.39 0.67

0.52 0.46 0.31 0.59 0.42

0.52 0.63 0.49 0.58 0.54 0.52

0.23 0.22 0.14 0.34 0.21 2.06 0.49

0.65 0.68 0.49 0.66 0.66 2.4 0.75 2.3

0.3 0.45 0.26 0.6 0.3 4.31 0.52 1.43 1.61

0.39 0.28 0.25 0.31 0.29 1.45 0.45 0.81 1.52 1.62

0.67 0.59 0.46 0.7 0.6 1.47 0.92 0.98 0.69 1.42 0.89

0.096 0.024 0.042 0.157 0.096 0.174 0.107 0.158 0.131 0.153

0.105 0.118 0.263 0.192 0.292 0.208 0.306 0.245 0.248

0.041 0.156 0.102 0.185 0.121 0.184 0.150 0.136

0.200 0.135 0.224 0.156 0.211 0.168 0.175

genetic drift. It is well known that genetic variation is lost over time through genetic drift for small, isolated populations (Young et al. 1996) and empirical support has been reported for many species (Raijmann et al. 1994; Fischer & Matthies 1998; Luijten et al. 2000; Galeuchet et al. 2002; Paschke et al. 2002; Hooftman et al. 2003). This genetic erosion is enhanced through habitat fragmentation (Leberg et al. 2010). The four populations that had individuals with identical shared MLGs were located in Florida (BLW, CRF, and TPK) and Cuba. All individuals of these populations were sampled; therefore the number of genets in these sites Plant Species Biology 29, 2–15

0.171 0.048 0.031 0.035 0.042 0.080

0.172 0.115 0.151 0.132 0.128

0.022 0.036 0.030 0.130

0.030 0.007 0.064

0.003 0.095

0.089

ranged between 14 for CRF and 20 for CUB. This has implications for conservation priorities and management as these sites not only have the fewer number of individuals but have ramets of the same genet. The importance of genetic drift and inbreeding in shaping the patterns of genetic diversity in most of the populations from Florida, in a few of the sites from the Bahamas and in the Cuban locality is illustrated by the results of the exact tests for heterozygote deficiency. They indicated significant departure from HWE for these populations. Both prolonged inbreeding and genetic drift are likely to increase the frequency of homozygotes with deleterious recessive alleles. © 2012 The Society for the Study of Species Biology

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This can lead to reduced fitness and to increased divergence between populations (Carr & Dudash 2003; Hartl 2000; Leberg et al. 2010). Overall, the mean within-population F-value was 0.089 (range -0.087 to 0.244), which suggests low levels of inbreeding. Low F-values are expected for sporophytic self-incompatible species such as I. microdactyla that are obligate outcrossers. High F-values were mostly recorded in Florida. In Miami-Dade County, the most common floral visitors were native solitary bees (Halictidae), while a single species of a migratory hummingbird (rubythroated, Archilochus colubris) was rarely seen visiting flowers in this area. The lowest F-values were noted at sites on Andros where two species of resident hummingbirds (Bahamas woodstar, Calliphlox evelynae, and Cuban emerald, Chlorostilbon ricordii) occurred at high densities, and they were the most frequent visitors to flowers (Geiger 2007). While the territorial behavior of these resident hummingbirds (S. Latta, pers. comm. 2012) would tend to limit pollen flow between high floral display patches, recent research findings suggest that pollen flow from outside patches might be augmented by territorial invaders attracted to these high floral display patches (Justino et al. 2011). The delivery of pollen from adjacent patches would tend to keep the populations/patches connected via gene flow, reflected in the low F-values for the Bahamian populations sampled. Hummingbirds have been shown to be effective pollinators of many species and are capable of traveling relatively long distances (Singer & Sazima 2000; Kraemer 2001). On Andros, they may promote larger effective population sizes in this continuous forest, and therefore we would expect lower levels of biparental inbreeding (Stiles 1978; Feinsinger et al. 1982). We detected private alleles in all three regions, with nearly 50% (5) found in the ATL population on Andros Island and 25% found at the Cuban site. This is the only sampling site in the Bahamas where we found I. microdactyla co-occurring with the congeneric I. carolina (L.) Pursh. All five of these private alleles exist in high allelic proportions among the I. carolina individuals that we have genotyped, and in low allelic proportions among the I. microdactyla individuals (Geiger 2007). In a separate study, we have shown the two species to be crosscompatible and capable of producing viable hybrid seed (Geiger 2007). These two direct lines of proof point to the likelihood of hybrid introgression in our detection of predominately I. carolina alleles within I. microdactyla individuals at this single site on Andros. Cross-compatibility within other members of this genus has been shown previously (Diaz et al. 1996). Ipomoea carolina is a species endemic to Cuba and the Bahamas. It is readily distinguishable from I. microdactyla by having compound palmate leaves and fragrant, purple-burgundy flowers © 2012 The Society for the Study of Species Biology

with broad corolla tubes, whereas I. microdactyla has simple, lobed leaves and unscented, light-red flowers with narrow-tubed, salverform corollas (Correll & Correll 1982). These two species are clearly defined taxonomic units. We believe that the large number of private alleles identified in the Cuban population is not the result of hybrid introgression between I. microdactyla and other Cuban congenerics as no other species of this genus occurs at this site. Molecular markers have provided two additional examples of introgression among congeneric species occurring in the region of our study. This has been recently documented for the native Lantana depressa Small (Verbenaceae) and the introduced L. camara L. in MiamiDade County (Maschinski et al. 2010), and between the native species Borrichia arborescens (L.) DC. (Asteraceae) and B. frutescens (L.) DC. along the Florida Keys.

Biogeographical patterns The landscape of the Bahamas and Florida has changed dramatically in the past 125 000 years because of sea-level changes associated with climate change. The current distribution of I. microdactyla in these regions was below sea level ca 125 000 years ago (Lidz & Shinn 1991) whereas many areas of Cuba were not covered by the ocean during this period (Gutiérrez-Domech & Rivero-Glean 1997). Despite these fluctuations, there is agreement among geologists and paleogeographers that no land bridges have existed between the dry land areas of the Bahamas, Florida, and Cuba in the past 35 million years (Draper & Barros 1994; Gutiérrez-Domech & Rivero-Glean 1997; Haq et al. 1987; Iturralde-Vinent & MacPhee 1999). Under this paleogeographical scenario, a stepping-stone dispersal route from Cuba towards the Bahamas and from this archipelago to Florida might have provided a biogeographical avenue for the evolutionary history of I. microdactyla. The only locality from Cuba had a small population size and was part of a highly fragmented landscape. We were unable to sample extensively in Cuba; therefore, additional population sampling from this island will be needed to confirm this biogeographical scenario. However, phylogeographic studies with other groups support a dispersal route from Cuba/Bahamas to Florida. Phylogenetic analyses within Rhodogeron and Sachsia (Asteraceae) show Cuba as an ancestral area for species occurring in the Bahamas and Florida (Liu et al. 2004). Likewise, Maskas and Cruzan (2000) proposed at least three historical cases of long-distance seed dispersal from Grand Bahama and Abaco to the southeastern coast of Florida for the Piriqueta caroliniana (Walter) Urb. (Turneraceae) complex. Unfortunately, this study did not include any populations from Cuba. An ‘Out of Cuba’ biogeoPlant Species Biology 29, 2–15

IPOMOEA MICRODACTYLA GENETIC DIVERSITY graphical pattern involving lizard populations from this island, the Bahamas, and Florida has also been supported by molecular data (Glor et al. 2005). We anticipate that future studies focusing on species restricted to this region will find Cuba, the largest island in the West Indies, as a source area for the Bahamian and subtropical Florida biota.

Genetic structure The patterns of microsatellite variation have a clear geographical signature and this was supported by the clustering analyses. Two groups were revealed both after the neighbor joining and the STRUCTURE analyses. One of the groups had the populations from Florida and the second one those from Bahamas and Cuba. We believe the unique genetic signature of individuals at one of the Bahamas sites (ATL) to be the result of hybrid introgression with I. carolina (see above). This population was shown either as an outlier in the neighbor joining tree or as having high levels of admixture in the STRUCTURE diagram. This geographical division was also supported by the isolation-by-distance analysis as, for the complete data set, there was a highly significant correlation between genetic divergence and geographical distance values. Interestingly, this correlation was not significant within the Bahamas or Florida. Support for these results was also provided by the FST and Nm values. There were lower FST and larger Nm values within the Florida and within the Bahamas–Cuba sites than between populations from these two main regions. Geographically, Cuba is much closer to the Bahamas than to Florida, and the distance between these two insular systems was only 10 km during the latest glacial maximum, approximately 18 000 years ago. It is noteworthy that the average level of amongpopulation genetic variation for I. microdactyla (FST = 0.148) is larger than the mean value reported by Hamrick and Godt (1996) for other long-lived, outcrossing perennial plants (GST = 0.094). This difference can be explained by the effect that the size of the geographic area sampled has on the estimates of FST, where values tend to increase by increasing the geographic extent of sampling (Morjan & Rieseberg 2004). Nevertheless, we obtained much smaller FST estimates when we tested Florida and Andros populations separately, 0.082 and 0.087, respectively. These low FST values suggest that most of the variation is found within populations as supported by the amova results. The relatively low Nm values (< 1) detected within Florida suggest limited gene-flow within this region and might be one of the outcomes of habitat fragmentation in this region. In contrast, most of the Nm values for Andros are > 1, supporting higher levels of genetic migration on this island. Plant Species Biology 29, 2–15

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Habitat fragmentation and conservation implications We found significantly greater mean numbers of alleles, and higher mean values for both observed and expected heterozygosity in populations from the continuous forest on Andros than those from the habitat fragments in Miami-Dade County. Unfortunately, our study did not include material from the continuous populations of Everglades National Park. Therefore, we are not certain if our finding of lower genetic diversity in fragment populations is the result of the recent arrival of I. microdactyla to Florida, rather than a case of habitat fragmentation/ isolation leading to genetic erosion in these sites. Populations within Miami-Dade County also constitute the expanding range edge for I. microdactyla in the Caribbean region. Other researchers have found lower levels of genetic diversity for populations at the species range edge, both on islands (Maskas & Cruzan 2000; RiveraOcasio et al. 2002) and in continental systems (Jump et al. 2003; Petit et al. 2003; Van Rossum & Prentice 2004). Whether the patterns of genetic diversity in the Florida populations are the result of habitat fragmentation or founder effects, these sites still have low levels of microsatellite variation and very limited gene flow appears to connect them. Fortunately, these sites are officially protected within the network of state/county reserves that exist in Miami-Dade County. However, these localities are still part of a highly fragmented pine forest located within an urban matrix. It is likely that inbreeding and genetic drift will be critical factors in determining the survival of the species at these sites because of the small number of individuals found in these populations. Under these conditions, the endangered taxa cannot escape from the extinction vortex even if their habitats are protected (Fagas & Holmes 2006). Most likely, I. microdactyla is simply one of a suite of Caribbean species with similar life histories whose genetic make-up is being negatively affected by loss of populations and loss of population connectedness as a result of extreme habitat fragmentation. Species found on this list might include dioecious species (e.g. Ilex krugiana Loes. ex Urb. (Aquifoliaceae) and Clusia rosea Jacq. (Clusiaceae)) and those species exhibiting self-incompatiblity (e.g. species within Asteraceae, Convolvulaceae, Malvaceae, Fabaceae, Poaceae, and Solanaceae), as species with these traits are generally most susceptible to genetic erosion via habitat fragmentation (Young et al. 1996). 2001). Future conservation action plans will need to include mechanisms to restore gene flow among populations of these similarly threatened species by the establishment of corridors or by the introduction of genotypes from other populations. Interestingly, separate studies focusing on seed germination and seedling performances found no significant differences between the sites from Andros and Florida; © 2012 The Society for the Study of Species Biology

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suggesting that, at least for these traits, fitness has not been affected by habitat fragmentation (Geiger 2007). The pine forests in Miami-Dade County have been severely fragmented in the past 120 years as urban areas in Miami have expanded from the east coast towards the west. This relatively recent habitat fragmentation coupled with the life history of this species (a long-lived perennial vine with underground tubers) indicates that there has not been enough time for fitness to be reduced via fragmentation and subsequent genetic erosion. This is supported by the fact that the inbreeding coefficient values between the Florida and Andros sites were not significantly different. We cannot anticipate how many more years of continuous genetic drift and inbreeding will lead to inbreeding depression, therefore initiatives like those recently established in Miami-Dade County to establish corridors to connect the remnants of the pine forest will be extremely beneficial for the long-term survival of this species in southern Florida (Goodell et al. 1997; Maschinski et al. 2009).

Acknowledgments We dedicate this paper to Dr Suzanne Koptur in recognition of her mentorship and outstanding career in tropical plant ecology. This is contribution number 220 from the Tropical Biology Program of FIU. Our gratitude to the Forfar Biological Field Station in Andros. We thank Miami-Dade County Parks and Recreation Department (Natural Areas Management), Florida Division of Agriculture and Consumer Services, Palmetto Bay Parks and Recreation Department, and the Florida Department of Transportation for facilitating research in Florida. Daniel Gann helped with the preparation of maps. This project was funded by an EPA MAI STAR grant (number U916090); Fairchild Tropical Botanic Garden; FIU Graduate School Dissertation Year Fellowship; the Garden Club of America; the Florida Native Plant Society; the FIU Graduate Student Association; and the Judith EvansParker Travel Grant at FIU.

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