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American Journal of Botany 100(4): 722–734, 2013 ; http://www.amjbot.org/ © 2013 Botanical Society of America

American Journal of Botany 100(4): 722–734. 2013.

Oceanic islands have long been viewed as natural laborato-ries for the study of evolution. Adaptive radiation, a process whereby speciation occurs through rapid evolution into differ-ent ecological zones, is a well-known feature of oceanic island biotas. This type of speciation produces a phylogenetic profi le that is best described as cladogenetic, in which the founder un-dergoes several splitting events into distinct independent evolu-tionary lineages. Numerous examples of this phenomenon in the plant world exist, such as the speciose Lobelia complex in the Hawaiian Islands ( Givnish et al., 1995 ), the genus Echium in the Canary Islands ( Böhle et al., 1996 ), Scalesia (Compositae) in the Galápagos Islands ( Eliasson, 1974 ; Schilling et al., 1994 ), and Dendroseris and Robinsonia (Compositae) in the Juan Fernández Archipelago ( Crawford et al., 1998 ). Although this mode of speciation is common in oceanic islands, it also occurs in some rapidly evolving groups of continental regions (e.g., Lupinus in the Andes of South America; Hughes and Eastwood, 2006 ).

1 Manuscript received 12 October 2012; revision accepted 28 January 2013.

The authors thank the Corporación Nacional Forestal of Chile (CONAF) for collecting permits for Myrceugenia in the Juan Fernández Archipelago National Park, FWF (Austrian Science Fund) for fi nancial support (grant P21723-B16 to T.F.S.), the Japan Society for the Promotion of Science (JSPS) for a postdoctoral fellowship (no. 526 to K.T.), the guides of CONAF in the Juan Fernández Archipelago National Park (especially B. López, O. Chamorro, R. Schiller, J. Angulo, D. Arredondo, A. Andauer, M. Recabarren, G. Araya), our informal guides (E. Paredes, M. González, D. Arredondo Jr.), Sr. I. Leiva, Chief of the Juan Fernández Archipelago National Park, for his very capable administrative assistance in all aspects of our research work, and the Armada de Chile for sending materials and research equipment to Robinson Crusoe Island. They also thank D. Bacho, a trained nurse and present medical student, who kept us healthy during the expedition to Alejandro Selkirk Island.

11 Author for correspondence (e-mail: tod.stuessy@univie.ac.at)

doi:10.3732/ajb.1200541

GENETIC VARIATION (AFLPS AND NUCLEAR MICROSATELLITES) IN TWO ANAGENETICALLY DERIVED ENDEMIC SPECIES OF MYRCEUGENIA

(MYRTACEAE) ON THE JUAN FERNÁNDEZ ISLANDS, CHILE 1

PATRICIO LÓPEZ-SEPÚLVEDA 2,5 , KOJI TAKAYAMA 2 , JOSEF GREIMLER 2 , PATRICIO PEÑAILILLO 3 , DANIEL J. CRAWFORD 4 , MARCELO BAEZA 5 , EDUARDO RUIZ 5 , GUDRUN KOHL 2 ,

KARIN TREMETSBERGER 6 , ALEJANDRO GATICA 7 , LUIS LETELIER 8 , PATRICIO NOVOA 9 , JOHANNES NOVAK 10 , AND TOD F. STUESSY 2,11

2 Department of Systematic and Evolutionary Botany, Biodiversity Center, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; 3 Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, 2 Norte 685, Talca, Chile; 4 Department of Ecology and Evolutionary Biology and the Biodiversity Institute, University of Kansas, Lawrence, Kansas 60045 USA; 5 Departamento de Botánica, Universidad de Concepción, Casilla 2407, Concepción, Chile; 6 Institute of Botany, Department of Integrative Biology and Biodiversity Research, University of Natural Resources and Life Sciences, Gregor Mendel Straße 33, A-1180 Vienna, Austria;

7 Laboratorio de Ecofi siología Vegetal, Departamento de Biología, Facultad de Ciencias, Universidad de La Serena, Casilla 599, La Serena, Chile; 8 Centro de Investigaciones en Ecosistemas, Universidad Nacional Autónoma de México,

C.P. 58190 Morelia, Michoacán, México; 9 Jardín Botánico de Viña del Mar, Corporación Nacional Forestal, Camino El Olivar 305, Viña del Mar, Chile; and 10 Institute for Applied Botany and Pharmacognosy,

University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria

• Premise of the study: Anagenesis (or phyletic evolution) is one mode of speciation that occurs in the evolution of plants on oceanic islands. Of two endemic species on the Juan Fernández Islands (Chile), Myrceugenia fernandeziana and M. schulzei (Myrtaceae), believed to have originated anagenetically from different continental progenitors, the fi rst is endemic to Robinson Crusoe Island and has no clear tie to continental relatives; the last is endemic to the younger island, Alejandro Selkirk Island, and has close affi nity to M. colchaguensis in mainland Chile.

• Methods: Using AFLPs and six nuclear microsatellites from 381 individuals representing 33 populations, we determined pat-terns of genetic variation within and among populations on both islands and between those of the islands and mainland.

• Key results: Considerable genetic variation was found within populations on both islands. The level of gene diversity within M. schulzei was equivalent to that of its close continental relative M. colchaguensis . Genetic diversity was not partitioned geographically in M. fernandeziana and was weakly so and nonsignifi cantly in M. schulzei .

• Conclusions: The high genetic variation in both taxa is most likely due to anagenetic speciation. Subsidence of the older island Robinson Crusoe, landscape erosion, and restructuring of communities have severely reduced the overall island population to a single panmictic system. On the younger and less modifi ed Alejandro Selkirk Island, slightly stronger patterns of genetic divergence are seen in M. schulzei . Because both species are genetically diverse and number in the thousands of individuals, neither is presently endangered in the archipelago.

Key words: anagenesis; biogeography; Juan Fernández Islands; Myrtaceae; Myrceugenia ; population genetics; speciation.

723April 2013] LÓPEZ-SEPÚLVEDA ET AL.—ANAGENETIC SPECIATION IN MYRCEUGENIA

An appropriate genus in the Juan Fernández Archipelago for examining genetic consequences of anagenetic speciation is Myrceugenia (Myrtaceae). This genus contains 39 species cen-tered in Chile, Argentina, and Brazil ( Landrum, 1981a , b ), with two species being endemic in the Juan Fernández Archipelago ( Fig. 2 ). Myrceugenia fernandeziana is a major forest tree re-stricted to Robinson Crusoe Island, and M. schulzei is a common tree species on Alejandro Selkirk Island. Recent phylogenetic studies by Murillo-Aldana et al. (2012) reveal these two species to be unrelated. The former is completely isolated within subtribe Myrtiinae, so much so that close generic ties cannot be deter-mined. It has been suggested, in fact, that this species should be removed into its own genus, Nothomyrcia ( Murillo-Aldana and Ruiz, 2011 ). The other species, M. schulzei , relates phylogeneti-cally to M. colchaguensis ( Murillo-Aldana et al., 2012 ) from continental Chile. This system, therefore, provides the opportu-nity to examine the genetic consequences of parallel anagenetic speciation in two species originating from different lineages in islands of different geological ages.

A number of different molecular markers have been used to estimate patterns of genetic variation within and among plant populations. One of the most successfully employed markers has been amplifi ed fragment length polymorphisms (AFLPs), a dominant marker, which provides information from a large number of loci for a sensitive overall measure of genetic varia-tion among individuals ( Vos et al., 1995 ; Tremetsberger et al., 2003a ; Gaudeul et al., 2012 ). More direct allelic information on a smaller number of loci can be obtained from nuclear micro-satellites, a codominant marker that permits the inference of the allelic basis of the phenotypes, but primer design can be time-consuming and costly before population surveying can be done ( Maguire et al., 2002 ; Squirrell et al., 2003 ; Nybom, 2004 ). Use of newer 454 pyrosequencing techniques has made locating mi-crosatellite regions easier ( Takayama et al., 2011 ), and this has been the approach used in the present study. Here both AFLP and nuclear microsatellite data have been obtained and evalu-ated for insights on genetic consequences during anagenetic speciation in Myrceugenia .

The objectives of this paper, therefore, are to (1) assess the range of genetic variation within and among populations of M. fernandeziana on Robinson Crusoe Island and M. schulzei on Alejandro Selkirk Island; (2) determine genetic variation within and among populations of M. colchaguensis , the conti-nental relative of M. schulzei ; (3) compare and contrast the different genetic patterns in the context of islands of different ages and in relation to continental relatives; and (4) offer per-spectives on interpretation of observed genetic variation within and among populations of plants that occur on oceanic islands of differing ages.

Another type of speciation in oceanic islands, anagenesis, recently has been emphasized as another important driver of island endemism ( Stuessy et al., 2006 ). In this process, a founder population establishes itself and begins to accumulate genetic variation in a relatively uniform environment via mutation and recombination, but without any splitting events. The initial im-migrant species, therefore, is simply transformed into another as assessed by observable differences in morphology and genet-ics from the continental progenitor. In the past, this process has also been called phyletic evolution ( Simpson, 1953 , p. 384) or simply transformational speciation. Recent studies have sug-gested that anagenetic speciation in oceanic islands also is rela-tively common, accounting for as much as 25% of all endemic plant species ( Stuessy et al., 2006 ).

The genetic consequences that occur during cladogenetic or anagenetic speciation in oceanic islands are of considerable evo-lutionary interest. For the former, many studies have revealed that low levels of genetic variation occur within and among popula-tions of species that have originated by cladogenesis during adaptive radiation ( Ito et al., 1997 ; Emerson, 2002 ). Because the process is rapid, driven by directional selection within diverse environments, the degree of genetic change (observable through standard presumably neutral or near-neutral molecular markers) is low, despite conspicuous morphological differences accruing among species as evolutionary adaptations within divergent habitats ( Crawford and Stuessy, 1997 ; Stuessy et al., 2005 ).

Few studies have been conducted on the genetic consequences of anagenetic speciation in oceanic islands, in part because this involves also examining genetic variation in continental pro-genitors with the same molecular markers. Anagenesis is a type of progenitor-derivative speciation, recently emphasized also to be important for understanding genesis of biodiver-sity in continental regions ( Crawford, 2010 ). Two studies have documented genetic populational changes during anagenesis in islands, in Dystaenia (Umbelliferae, Pfosser et al., 2005 ) and in Acer (Sapindaceae, Takayama et al., 2012 ), both endemic to Ullung Island, Korea. In both cases, negligible geographic structuring of genetic variation within the endemic island spe-cies was seen, and the total genetic variation among populations on the island was equivalent to that found in populations of con-tinental source populations, from Japan in the case of Dystaenia and from Japan and Korea in the case of Acer . For a general model of genetic change associated with both anagenetic and cladogenetic speciation, see Stuessy (2007) .

A suitable oceanic island archipelago in which to examine further the genetic consequences of anagenetic speciation is the Juan Fernández Archipelago. Situated 667 km west of the coast of continental Chile at 33 ° S latitude are two major islands both approximately 50 km 2 ( Stuessy, 1995 ; Fig. 1 ): Robinson Crusoe (Masatierra) Island, the island closest to the continent and also the older at approximately four million years of age; and Alejandro Selkirk (Masafuera) Island, 180 km farther west and younger at ca. 1–2 Myr ( Stuessy et al., 1984 ). The biogeo-graphic simplicity of this island system ( Stuessy et al., 2005 ), plus a small fl ora of only 423 endemic and native vascular plant species ( Marticorena et al., 1998 ), offers opportunities for phy-logenetic studies to be done, which allow inferences to be made on modes of speciation ( Stuessy et al., 1990 ). In many cases, these studies have also identifi ed closest continental progeni-tors, many being from southern South America ( Bernardello et al., 2006 ). Both anagenetically and cladogenetically derived endemic species are known to occur in the vascular fl ora of this archipelago ( Stuessy et al., 1990 ).

Fig. 1. Location of the islands of the Juan Fernández Archipelago, Chile.

724 AMERICAN JOURNAL OF BOTANY [Vol. 100

December ( Landrum, 1981a ). The third species in this study is M. colchaguen-sis (Phil.) L.E.Navas, “Colchaguillo”, a small tree (4 m) of continental Chile, with hermaphroditic fl owers in January and February ( Landrum, 1981a ). Only fi ve small populations of this species are known ( Landrum, 1981a ) in perturbed areas ( Zamorano et al., 2008 ).

Plant material and DNA isolation — We collected leaf samples from ran-domly selected individuals in 18 populations of Myrceugenia fernandeziana on Robinson Crusoe Island, from 13 populations of M. schulzei on Alejandro Selkirk Island ( Fig. 3 ), and from two populations of M. colchaguensis from

MATERIALS AND METHODS

Species — Myrceugenia schulzei Johow, “Luma de Masafuera” ( Fig. 2A, B ), is an endemic and dominant tree on Alejandro Selkirk Island reaching 12 m in height. The species has hermaphroditic fl owers and an entomophilous pollina-tion system as reported by Skottsberg (1920b) ; it fl owers from January to April ( Landrum, 1981a ). Myrceugenia fernandeziana (Hook. & Arn.) Johow, “Luma” ( Fig. 2C, D ), is an endemic and dominant tree on Robinson Crusoe Island, up to 25 m tall. It also is hermaphroditic with aromatic fl owers and an apparently ento-mophilous pollination system ( Skottsberg 1920a , b ); it fl owers from August to

Fig. 2. Trees of Myrceugenia in the Juan Fernández Archipelago. (A) M. schulzei on Alejandro Selkirk Island. (B) Detail of infl orescences. (C) M. fernandeziana on Robinson Crusoe Island. (D) Detail of infl orescences. Scale bar = 7 mm.

725April 2013] LÓPEZ-SEPÚLVEDA ET AL.—ANAGENETIC SPECIATION IN MYRCEUGENIA

corresponding to percentage of polymorphic fragments (PPB), total number of AFLP fragments (TNB), and Shannon diversity index (SDI) ( H Sh = − Σ p i ·ln p i ), where p i is the frequency of the i th fragment in the respective population based on all AFLP fragments recorded. The average gene diversity over loci (AGDOL; the probability that two homologous fragment sites, randomly chosen, are dif-ferent) was calculated with Arlequin 3.5.1.2 ( Excoffi er et al., 2005 ). The genetic divergence parameters, rarity index (RI) (“frequency-down-weighted marker values”; Schönswetter and Tribsch, 2005 ), was calculated using R-script AFLPdat ( Ehrich, 2006 ), and the number of private fragments (NPB) was recorded using FAMD ver. 1.25 (Schlüter and Harris, 2006) . To test correlation between these values, Pearson correlations were done with the program SPSS ver. 15.0 (SPSS; IBM, Armonk, New York).

From the AFLP matrix, a Nei-Li distance matrix was calculated and used as an input fi le for an individual network analysis (1000 bootstrap replicates) with the NeighborNet algorithm ( Bryant and Moulton, 2004 ), implemented by the software SplitsTree4 ver. 4.10 ( Huson and Bryant, 2006 ). Genetic differentia-tion among/within populations was estimated by analysis of molecular variance (AMOVA) in Arlequin 3.5.1.2 ( Excoffi er et al., 2005 ). The signifi cance of the variance components was tested by calculating their probabilities based on 1023 permutations. A Bayesian clustering method was employed for assess-ment of population structure in the program Structure 2.3.3 ( Falush et al., 2007 ; Hubisz et al., 2009 ; Pritchard et al., 2000 ). An admixture model with correlated allele frequencies ( Falush et al., 2003 ) was used to assign individuals into K clusters. The number of steps was 20 000, with 10 000 iterations, and 20 repli-cate runs in each K from 1 to 10. The uppermost level of structure was inferred from a posterior probability of the data for a given K and Δ K value ( Evanno et al., 2005 ).

For microsatellites, tests for signifi cant deviation from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between loci in each pop-ulation were carried out with the Markov chain method (10 000 dememoriza-tion steps, 1000 batches, 500 iterations per batch) using the program GENEPOP 4.0 ( Raymond and Rousset, 1995 ). The frequency of null alleles in each marker was calculated following Brookfi eld (1996) using the program Micro-Checker 2.2.3 ( van Oosterhout et al., 2004 ). Allelic richness ( A R ), expected proportion of heterozygotes ( H E ), and number of alleles per locus ( N A ), were calculated for each species and population using the program FSTAT 2.9.3.2 ( Goudet, 1995 ). Allelic richness was standardized for 10 individuals of M. fernandeziana and three individuals of M. schulzei and M. colchaguensis based on the minimum sample size of populations using the rarefaction method ( Hurlbert, 1971 ). The inbreeding coeffi cient ( F IS ) was also calculated using FSTAT 2.9.3.2. The ge-netic divergence parameters, number of private alleles ( N PA ), and number of locally common alleles ( N LCA ) were calculated using the program GENALEX 6 ( Peakall and Smouse, 2006 ).

From the microsatellite matrix, genetic distance among individuals and populations was calculated by the D A genetic distance ( Nei et al., 1983 ) using the program Populations 1.2.30 ( Langella, 1999 ), and a NeighbourNet split graph was generated by SplitsTree4. Hierarchical structuring of genetic varia-tion among/within populations (AMOVA) and among north/south regions on each island was evaluated with Arlequin and Bayesian clustering was done with Structure, along with the AFLP analyses. In microsatellite and AFLP analyses, the Mann-Whitney U test was used for pairwise comparisons with the program SPSS ver. 15.0. On Robinson Crusoe island the high elevations around Cerro el

continental Chile (130 km from each another) ( Table 1 ). Leaf samples were desiccated with silica gel in zip-lock plastic bags until DNA extraction. Voucher specimens of these samples were deposited in the herbarium of the University of Vienna (WU; Table 1 ). Total genomic DNA was extracted from dried leaves using the DNeasy 96 Plant Kit (Qiagen, Hilden, Germany). The number of individuals used for AFLPs and microsatellites in each population is found in Table 1 .

AFLP fi ngerprinting — The AFLP procedure implemented by Vos et al. (1995) was followed, using modifi cations by Tremetsberger et al. (2003b) . A primer trial using four individuals of fi ve populations with 80 selective primer combinations was done fi rst. We then analyzed 211 individuals of M. fernande-ziana with four selected primer combinations: MseI-CTT/EcoRI-ACT (FAM), MseI-CAA/EcoRI-ACT (FAM), MseI-CTT/EcoRI-ATG (NED), MseI-CTG/EcoRI-ATG (VIC). We analyzed 129 individuals of M. schulzei and 19 indi-viduals of M. colchaguensis with the same four, plus one additional combina-tion, MseI-AGC/EcoRI-CAC (NED). The amplifi ed fragments were run on an automated sequencer (ABI 3130xl, Applied Biosystems, Foster City, California, USA) and scored using the program GeneMarker ver. 1.85 (SoftGenetics, State College, Pennsylvania, USA). The range for allele call was 150–510 base pairs. The minimum and maximum relative intensity for peak detection threshold was 120 and 30 000, respectively, using the option stutter peak and plus-a fi lter. Samples with size calibration below 90% were manually adjusted. An automatic panel editor was generated for each selective primer combination ( Curtin et al., 2007 ), after being adjusted manually. For analysis, we combined each of the binary matrices for each primer combination in one matrix ( Wooten and Tolley-Jordan, 2009 ). Thirty individuals were replicated (8.6% of total samples), and the error rate was calculated as the ratio of number of fragment differences/total number of comparisons ( Bonin et al., 2004 ).

Microsatellites — We selected 12 microsatellite markers, isolated from Myr-ceugenia fernandeziana according to repeatability and scoring convenience ( Takayama et al., 2011 ). We analyzed 231 individuals of M. fernandeziana , 138 of M. schulzei , and 12 of M. colchaguensis . We applied the 5 ′ -tailed primer method ( Boutin-Ganache et al., 2001 ) to label PCR amplifi ed fragments for detection in the capillary sequencer, following Takayama et al. (2011) . Four combinations of multiplex PCR amplifi cation using different dyes (MF-A7N9P, MF-A064D, MF-A3UWE with 6-FAM; MF-AP6Q2, MF-A75KK, MF-AUXXC with VIC; MF-ATS7C, MF-AREPO, MF-A2F82 with NED; MF-AZHZD, MF-A425T, MF-AY4D2 with PET) were performed using a slightly modifi ed protocol of the Qiagen Multiplex PCR Kit, following Takayama et al. (2011) . PCR amplifi cations were performed with 0.2 µmol/L of each reverse primer, 0.04 µmol/Lof each forward primer, and 0.6 µmol/L of fl uorescent dye-labeled primer in a fi nal volume of 3 µL, using the touchdown thermal cycling program of Takayama et al., 2011 . The amplifi ed fragments were run on an automated sequencer (ABI 3130xl), and scoring with GeneMarker ver. 1.85.

Data analysis — For AFLPs, the total number of different phenotypes in each population was calculated with the program Arlequin 3.5.1.2 ( Excoffi er et al., 2005 ). In each population, the genetic diversity was estimated using the program FAMD ver. 1.25 ( Schlüter and Harris, 2006 ), with calculated values

Fig. 3. Locations of populations sampled. (A) Myrceugenia schulzei on Alejandro Selkirk Island. (B) M. fernandeziana on Robinson Crusoe Island.

726 AMERICAN JOURNAL OF BOTANY [Vol. 100

The northern populations of M. schulzei on Alejandro Selkirk Island (pops. 19–24; Fig. 3 ) have comparably higher values for all genetic diversity estimates than the southern populations (pops. 25–31); the differences, however, are not signifi cant.

The total genetic variation within species using the AGDOL estimate was 0.23 in M. fernandeziana , 0.27 in M. schulzei , and 0.26 in M. colchaguensis .

Microsatellites — Genetic diversity parameters estimated by mi-crosatellites are shown in Table 1 . Comparisons in Myrceugenia fernandeziana of the genetic diversity measures, i.e., the expected proportion of heterozygotes ( H E ), allelic richness ( R A ), and number of alleles per locus ( N A ), between the northern (1–9; Fig. 3 ) and southern populations (10–18) of M. fernandeziana , show no signifi cant difference. The same analyses in populations of M. schulzei reveals no signifi cant differences (pops. 19–24, and pops. 25–31; Fig. 3 ) for all genetic diversity estimates.

The values of total genetic diversity within species were 0.490 in M. fernandeziana , 0.563 in M. schulzei , and 0.717 in M. colchaguensis. Thus, the highest total diversity is found in M. colchaguensis , although we analyzed only two continental populations from this species (only fi ve are known to exist; Landrum, 1981a ). There is no signifi cant difference in genetic diversity estimates within populations between M. schulzei and M. colchaguensis . F IS values were signifi cantly positive in 14 populations of M. fernandeziana , in 8 of M. schulzei, and in all of M. colchaguensis ( P < 0.05) after Bonferroni correction.

Genetic divergence — AFLPs — Genetic divergence values are shown in Table 1 . The number of private fragments (NPF) and rarity index (RI) in populations of Myrceugenia fernandeziana is correlated. The Pearson correlation between them is r = 0.819 ( N = 18, P < 0.001). Comparison of northern (1–9) with southern (10–18) populations on Robinson Crusoe Island did not reveal signifi cant differences. In M. schulzei , only a moderate correla-tion exists between genetic divergence parameters, with Pearson correlation r = 0.772 ( N = 16, P = 0.001). Populations distributed in the northern region of Alejandro Selkirk Island (pops. 19–24) have comparably higher values for all genetic divergence esti-mates than the southern populations (pops. 25–31), but these dif-ferences are not statistically signifi cant. The mean F ST values within populations for each of the species ( M. fernandeziana / M. schulzei / M. colchaguensis ) are 0.1026/0.1646/0.3137, respec-tively (Appendices S5, S6, see online Supplemental Data).

Microsatellites — The two estimates of genetic divergence are shown in Table 1 . Differences in the number of private alleles ( N PA ) and number of locally common alleles ( N LCA ) between northern (1–9) and southern (10–18) populations of Myrceugenia fernandeziana are not signifi cant. The same ten-dency is seen in M. schulzei on Alejandro Selkirk Island (pops. 19–24 vs. pops. 25–31). The mean F ST value for populations of M. fernandeziana was 0.0454, 0.0790 in M. schulzei , and 0.4218 in M. colchaguensis (online Appendices S5, S6).

Genetic structure — Genetic variation among/within popula-tions was examined by AMOVA at different hierarchical levels in the three species ( Table 2 ). For AFLPs and microsatellites in M. fernandeziana and M. schulzei , most of the variation was found among individuals within populations (89.6%, 95.5%; 83.6%, 91.5%), and AFLPs presented larger genetic variation among populations (10.4%, 16.4%) in comparison with micro-satellites (4.5, 8.5). In M. colchaguensis , 68.7% and 46.7% of

Yunque (916 m a.s.l.) and Cordón Central divide the island roughly into north-ern and southeastern sections. On Alejandro Selkirk, the deep and dry valley of Quebrada Pasto provides an obvious divide between the northern populations surrounded mostly by dry grassland and the southern ones in close contact to the tall fern assemblage.

RESULTS

Description of AFLPs and microsatellites — The total num-ber of AFLP fragments found in Myrceugenia fernandeziana was 371, of which 340 (91.6%) are polymorphic ( Table 1 ). For M. schulzei and M. colchaguensis , the total number of AFLP fragments was 432, of which 427 are polymorphic (98.8%; Table 1 ). The number of fragments resulting from each pair of primers was ( M. fernandeziana / M. schulzei - M. colchaguensis ) 104/104 for primers MseI-CTT/EcoRI-ACT, 106/102 for MseI-CAA/EcoRI-ACT, 77/82 for MseI-CTT/EcoRI-ATG, 84/83 for MseI-CTG/EcoRI-ATG, and -/61 for MseI-CTA/EcoRI-ACC (Appendix S1, S2, see Supplemental Data with the online ver-sion of this article). All individuals had unique AFLP pheno-types. The reproducibility of the AFLP fragments was 96%.

Twelve microsatellite loci were successfully genotyped in 231 individuals of M. fernandeziana . In case of M. schulzei and M. colchaguensis , six of the 12 markers resulted in no amplifi -cation or showed complex patterns in some samples. Hence, we used the remaining six markers (MF-A064D, MF-A3UWE, MF-AP6Q2, MF-ATS7C, MF-AREPO, MF-A2F82) for fur-ther population analyses of 150 individuals of M. schulzei and M. colchaguensis (Appendix S3, S4, see online Supplemental Data). An exact test for HWE across populations and loci showed that seven cases of 216 in M. fernandeziana and six of 78 in M. schulzei deviated from HWE ( P < 0.05) after Bonferroni cor-rection. Except for one case found in M. schulzei , all the deviating cases were related to the positive F IS , indicating HWE deviation due to heterozygote defi cit. We estimated the frequency of null alleles across populations and loci using Micro-Checker, re-sulting in the highest frequency of 0.176 (MF-AUXXC), with an average frequency of 0.042 in all of the 12 marker loci in M. fernandeziana , and 0.179 (MF-A064D) and 0.062 in all of the six marker loci in M. schulzei . Signifi cant LD was not found be-tween any pairwise combinations of loci in all populations within each species ( P < 0.05) after Bonferroni correction.

Genetic diversity — AFLPs — Table 1 summarizes the ge-netic diversity among the three species of Myrceugenia . In M. fernandeziana four estimates of genetic diversity are highly correlated: total number of fragments (TNF), percentage of poly-morphic fragments (PPF), Shannon diversity index (SDI), and average genetic diversity over loci (AGDOL). The Pearson cor-relation between TNF and SDI is r = 0.924 ( N = 18, P < 0.001), between PPF and SDI r = 0.955 ( N = 18, P < 0.001), and between TNF and PPF r = 0.971 ( N =18, P < 0.001). The values of genetic diversity vary between populations, the lowest value occurring in population 8. The northern populations (1–9) are not signifi cantly different from the southern populations (10–18) in Robinson Crusoe Island.

In M. schulzei , all indices of genetic diversity are highly correlated. The Pearson correlation between TNF and SDI is r = 0.806 ( N = 15, P < 0.001), between PPF and SDI r = 0.932 ( N = 15, P < 0.001), and between TNF and PPF r = 0.873 ( N = 15, P < 0.001). Population 29 has the lowest value for all parameters of genetic diversity; the highest value is found in population 19.

727April 2013] LÓPEZ-SEPÚLVEDA ET AL.—ANAGENETIC SPECIATION IN MYRCEUGENIA

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728 AMERICAN JOURNAL OF BOTANY [Vol. 100

genus reside ( Landrum, 1981a ). Each colonized and subsequently diverged in the archipelago, representing, then, two separate cases of anagenetic speciation, M. fernandeziana in Robinson Crusoe Island and M. schulzei in Alejandro Selkirk Island. It is possible to suggest more complex explanations, such as adap-tive radiation of large complexes in each island followed by extreme extinction down to only a single extant species on each island. Data, however, do not support such scenarios. First, the two species are dominant forest trees on each of the islands, which would not be expected if they were the last survivors af-ter extinction of all congeners. Second, restricted distributions in specialized habitats might also be expected, but this is totally contrary to the observed broad distributions of the two species. Third, if the present species were the last remaining taxa from large adaptively radiated complexes, then the amount of genetic variation within each species would be expected to be extremely low, which is not the case ( Frankham, 1997 ) ( Table 2 ). It seems most probable, therefore, that the two endemic species have originated anagenetically from different continental ancestors. This is a type of progenitor-derivative speciation as reviewed recently by Crawford (2010) .

Anagenetic speciation and patterns of genetic variation within Myrceugenia— Stuessy (2007) hypothesized that the ge-netic correlates of anagenetic vs. cladogenetic speciation in oce-anic islands would be different. During cladogenesis, which is assumed to be a faster process ( Schluter, 2000 ) that splits the original founding populations into many divergent lineages that speciate into notably different ecological zones, each derivative species retains only a limited range of genetic variation. Just how the variation is partitioned will depend on the geographical and ecological constraints of speciation ( Crawford and Stuessy, 1997 ; Baldwin et al., 1998 ). With anagenesis, in contrast, based on lim-ited experimental comparisons ( Pfosser et al., 2005 ; Takayama et al., 2012 ; Yamada and Maki, 2012 ), the immigrant population becomes established, enlarges, and accumulates genetic variation slowly through mutation and recombination. After anagenetic di-vergence yields a new species (i.e., suffi ciently different in cor-related characters from the continental progenitor), the level of genetic variation across all populations can be high, perhaps even approximating that of the parental species. Furthermore, little geographic patterning of the genetic variation would be expected as gene fl ow is maintained in the similar environment among populations within the island.

The results within both Myrceugenia fernandeziana and M. schulzei reveal high levels of genetic variation within and weak differentiation among what we considered populations during

genetic variation within populations was found in AFLPs and microsatellites, respectively.

The NeighbourNet trees among all individuals of M. fernan-deziana and M. schulzei plus M. colchaguensis are shown in Figs. 4 and 5 . In M. fernandeziana ( Fig. 5 ), no obvious separa-tion of populations was found with either marker. There is a separation between populations of M. schulzei and M. colch-aguensis (Bootstrap probability = 80 for AFLP analysis; Fig. 4A ) and also between the two populations of M. colchaguensis , but no coherent separation of populations within M. schulzei . The tree based on AFLPs presents weak populational differentiation in comparison with that from microsatellites, and there is no geo-graphical partitioning. Bayesian clustering analyses conducted in STRUCTURE are shown in Fig. 6 . Based on Δ K values, the uppermost level of structure was at K = 2 in M. fernandeziana in both markers, and K = 3 and K = 4 in M. schulzei with AFLPs and microsatellites, respectively. Here again, AFLPs reveal weak populational differentiation, and generally most populations con-sist of multiple clusters in both markers.

The Mantel test shows no positive correlation between ge-netic divergence [ F ST /(1 − F ST )] ( Rousset, 1997 ) and geographic distance. For AFLP in M. fernandeziana , the value was r 2 = 0.0059 and in M. schulzei r 2 = 0.0063. In the case of microsatel-lites, the values were r 2 = 0.0098 and r 2 = 0.0001 for M. fernan-deziana and M. schulzei , respectively.

DISCUSSION

Modes of speciation in the endemic island species of Myr-ceugenia — Different modes of speciation are associated with different genetic signatures. It is important, therefore, to con-sider the types of speciation involved with the origin of the two endemic species of Myrceugenia before attempting to interpret the observed patterns of genetic variation.

Earlier morphological ( Landrum, 1981b ) and recent molecu-lar phylogenetic studies ( Ruiz et al., 2004 ; Murillo-Aldana et al., 2012 ) in Myrceugenia , including the two island species, M. fernandeziana and M. schulzei , have shown that they are not closely related to each other. In fact, based on DNA sequences (ITS, ETS, matK-trnK , rpl32-trnL , trnQ-5 ′ rps16 , and rpl16 ) that are extremely divergent, it has now been judged ( Murillo-Aldana and Ruiz, 2011 ) that M. fernandeziana does not even belong to Myrceugenia , but is better placed in its own mono-typic genus, Nothomyrcia . This being the case, there obviously have been two independent evolutionary lines deriving from southern South America, where the majority of the species of the

TABLE 2. Summary of analyses of molecular variance (AMOVA) for AFLPs and microsatellites in Myrceugenia fernandeziana , M. schulzei , and M. colchaguensis .

AFLPs Microsatellites

Species Source of variation df SS Variance components Total variance (%) df SS Variance components Total variance (%)

M. fernandeziana Among populations 17 1507.0 4.4 10.4* 17 105.1 0.1 4.5*Within populations 193 7269.0 37.7 89.6* 444 1249.7 2.8 95.5*

M. schulzei Among populations 12 1746.0 9.8 16.4* 12 54.6 0.1 8.5*Within populations 116 5753.0 49.6 83.6* 263 406.5 1.5 91.5*

M. colchaguensis Among populations 1 241 674.0 20.7 31.3* 1 1242.7 99.1 53.3*Within populations 17 773 800.0 45.5 68.7* 22 1907.1 86.7 46.7*

Notes: The total variance contributed by each component (%) and its associated signifi cance ( N = 1023 permutations) are shown; df, degrees of freedom; SS, sum of squares; * P < 0.001.

729April 2013] LÓPEZ-SEPÚLVEDA ET AL.—ANAGENETIC SPECIATION IN MYRCEUGENIA

derived species of having relatively high levels of genetic varia-tion that is not structured geographically.

Previous isozyme studies within M. fernandeziana also point to relatively high levels of genetic variation. Jensen et al. (2002) examined 25 populations for leaf outline variation and 15 of these same populations for isozyme variability. In general, there was considerable morphological variation that did not correlate

sampling. The patterns ( Figs. 4, 5 ) in both species are similar, with a slight tendency toward genetic structuring observed within M. schulzei , but virtually none within M. fernandeziana . From a genetic point of view, therefore, all individuals in each of the islands form a large population with little genetic struc-ture that does not correlate with geographic structure. These data appear compatible, then, with the concept of anagenetically

Fig. 4. Phylogenetic network (SplitsTree NeighbourNet) showing genetic affi nities among individuals in populations (numbers) of Myrceugenia schulzei and M. colchaguensis . (A) AFLPs. (B) Microsatellite data.

730 AMERICAN JOURNAL OF BOTANY [Vol. 100

populations of this species revealed much lower genetic diver-sity on the specifi c level ( H ES = 0.091) in comparison with the continental M. exsucca ( H ES = 0.214) ( Stuessy et al., 2005 ). Based on isozymes, the proportion of genetic variation residing among populations was lower in M. schulzei ( F ST = 0.16) than in M. fernandeziana ( F ST = 0.23).

with geography or habitats on the Robinson Crusoe Island, but there was some nonsignifi cant covariation between isozyme and leaf variation patterns. Crawford et al. (2001) examined allozymic variation and calculated gene diversity statistics for 30 endemic species in the Juan Fernández Islands, includ-ing M. fernandeziana from Robinson Crusoe. Analysis of 19

Fig. 5. Phylogenetic network (SplitsTree NeighbourNet) showing genetic affi nities among individuals in populations (numbers) of Myrceugenia fernandeziana. (A) AFLPs. (B) Microsatellite data.

731April 2013] LÓPEZ-SEPÚLVEDA ET AL.—ANAGENETIC SPECIATION IN MYRCEUGENIA

(even thousands) of individuals; neither is an endangered spe-cies in the two islands. Both species exist at the same chromo-somal (diploid) level of 2 n = 22 ( Sanders et al., 1983 ). It is likely that the two species are outcrossing (they are clearly protogynous, Bernardello et al., 2001 ), but this has not been confi rmed. Both have white, conspicuous fl owers that give the appearance of being insect-pollinated (as inferred by Skottsberg,

Major factors infl uencing patterns of genetic variation within endemic species of Myrceugenia— To interpret genetic varia-tion within and among any set of plant populations requires also considering their biological attributes. In Myrceugenia , the two species are quite similar morphologically. Both are large forest trees to 12–25 m tall that form a large portion of the dominant vegetation on both islands. Stands exist with literally hundreds

Fig. 6. STRUCTURE analysis showing genetic relationships among populations. (A) Myrceugenia schulzei based on AFLPs ( K = 3, left) and nuclear microsatellites ( K = 4, right). (B) M. fernandeziana based on AFLPs ( K = 2, left) and nuclear microsatellites ( K = 2, right).

732 AMERICAN JOURNAL OF BOTANY [Vol. 100

At the present time, the two endemic species of Myrceugenia exist in many thousands of individuals, and no short-term ex-tinction threat exists for either of them. Vargas et al. (2010) estimate a frequency of at least 800 trees/ha in three points sampled on Robinson Crusoe Island. Direct harvesting by the human population is now rigidly controlled by the national park service (CONAF). Invasive species represent a constant and real danger to the native and endemic fl ora ( Wester, 1991 ; Matthei et al., 1993 ; Swenson et al., 1997 ; Stuessy et al., 1998b , c ; Greimler et al., 2002a , b ; Dirnböck et al., 2003 ), but the im-mediate impact on Myrceugenia is low, due to the large size and long duration of these trees. Similar to other Myrtaceae in conti-nental Chile, the fruits of M. fernandeziana and M. schulzei are dispersal pregerminated (M. Ricci, CONAF Chile, personal communication; Figueroa and Jaksic, 2004 ); it is not possible to store seeds for a long period of time. Very important, therefore, is maintenance of plants in situ in both of the islands.

In M. fernandeziana , there is little geographic partitioning of the observed genetic variation, which means that no particular regions (e.g., valleys or ridge areas) have a higher conservation priority than any of the others from a genetic point of view. In M. schulzei , however, some geographic patterning of the genetic variation is seen. One can make the argument here, too, that there is no compelling need to maintain any particular population within the island. All populations are quite similar to each other genetically for the neutral markers used in this study.

LITERATURE CITED

ANDERSON , G. J. , G. BERNARDELLO , T. F. STUESSY , AND D. J. CRAWFORD . 2001 . Breeding system and pollination of selected plants endemic to Juan Fernández Islands. American Journal of Botany 88 : 220 – 233 .

BALDWIN , B. G. , D. J. CRAWFORD , J. FRANCISCO-ORTEGA , S.-C. KIM , T. SANG , AND T. F. STUESSY . 1998 . Molecular phylogenetic insights on the ori-gin and evolution of oceanic island plants. In D. E. Soltis, P. S. Soltis, and J. J. Doyle [eds.], Molecular systematics of plants II: DNA se-quencing, 410–441. Kluwer, Dordrecht, Netherlands.

BERNARDELLO , G. , G. J. ANDERSON , T. F. STUESSY , AND D. J. CRAWFORD . 2001 . A survey of fl oral traits, breeding systems, fl oral visitors, and pollination systems of the angiosperms of the Juan Fernández Islands (Chile). Botanical Review 67 : 255 – 308 .

BERNARDELLO , G. , G. J. ANDERSON , T. F. STUESSY , AND D. J. CRAWFORD . 2006 . The angiosperm fl ora of the Archipelago Juan Fernandez (Chile): Origin and dispersal. Canadian Journal of Botany 84 : 1266 – 1281 .

BÖHLE , U. T. , H. H. HILGER , AND W. F. MARTIN . 1996 . Island colo-nization and evolution of the insular woody habit in Echium L. (Boraginaceae). Proceedings of the National Academy of Sciences, USA 93 : 11740 – 11745 .

BONIN , A. , E. BELLEMAIN , P. BROKEN EIDESEN , F. POMPANON , C. BROCHMANN , AND P. TABERLET . 2004 . How to track and assess genotyping errors in population genetics studies. Molecular Ecology 13 : 3261 – 3273 .

BOUTIN-GANACHE , I. , M. RAPOSO , M. RAYMOND , AND C. F. DESCHEPPER . 2001 . M13-tailed primers improve the readability and usability of micro-satellite analyses performed with two different allele-sizing methods. BioTechniques 31 : 24 – 28 .

BROOKFIELD , J. F. Y. 1996 . A simple new method for estimating null allele frequency from heterozygote defi ciency. Molecular Ecology 5 : 453 – 455 .

BRYANT , D. , AND V. MOULTON . 2004 . Neighbor-Net: An agglomerative method for the construction of phylogenetic networks. Molecular Biology and Evolution 21 : 255 – 265 .

CRAWFORD , D. J. 2010 . Progenitor-derivative species pairs and plant spe-ciation. Taxon 59 : 1413 – 1423 .

CRAWFORD , D. J. , E. RUIZ , T. F. STUESSY , E. TEPE , P. AQUEVEQUE , F. GONZÁLEZ , R. J. JENSEN , ET AL . 2001 . Allozyme diversity in endemic fl owering plant species of the Juan Fernandez Archipelago, Chile: Ecological

1920b ). Anderson et al. (2001) and Bernardello et al. (2001) , however, have surmised that they might be wind-pollinated, and Jensen et al. (2002) also suggested that this might be the case. If this were so, there would be even greater potential for gene exchange among populations within each of the islands. Jensen et al. (2002) and Vargas et al. (2006) suggested that the fruits are probably bird-dispersed, which would further con-tribute to gene fl ow within each of the islands.

The two factors that most probably have infl uenced levels of genetic variation within the island endemic species of Myr-ceugenia , therefore, are (1) the processes of anagenetic spe-ciation on both islands, plus (2) loss of surface area and habitat diversity on Robinson Crusoe Island. A high level of genetic diversity can be observed within both species, with very little geographic partitioning of variation. The major difference be-tween the two species on the islands of different ages probably relates to reduction of island surface area and loss habitat of diversity. Vegetational studies on both islands by Greimler et al. (2002a , unpublished manuscript) reveal much clearer eleva-tional separation of the major trees Drimys and Myrceugenia on the younger island, Alejandro Selkirk Island, than on the older one, Robinson Crusoe Island. As erosion and subsidence occurred on Robinson Crusoe Island over the past four mil-lion years, the island surface was perhaps reduced up to 95% ( Stuessy et al., 1998a ). The original vegetation zones, initially clearly elevationally distinct, began to lose their distinctness as the fl ora became refugially compacted. The original distinct plant communities converged during reassembly into a relict-ual mix, and gene fl ow between populations, once separated, became enhanced. The genetic picture from both AFLPs and microsatellites does not show a geographic partitioning of the genetic variation within M. fernandeziana on Robinson Crusoe Island, appearing as a single large panmictic population. Within M. schulzei on Alejandro Selkirk Island, on the other hand, there exists some slight north–south structuring of genetic vari-ation and therefore also more genetic divergence among these populations. The extant populations of this species form a nar-row zone mostly on the eastern side of the island between ca. 200 and 700 m a.s.l. on the slopes of the deep canyons that are separated by ridges with grassland and fern heath. To some extent, this pattern of isolation can be seen in the genetic data ( Figs. 4, 5 ), although only weakly.

Implications for conservation — From a conservation per-spective, human impact on the endemic vegetation, especially on Myrceugenia forests, was particularly intense in the 18th and 19th centuries. For Robinson Crusoe Island, many early maps show symbols representing forest trees on the eastern part of the island, presumably of M. fernandeziana . Excellent detailed views of the forests have been provided by Commodore Anson in 1741 (in Walter and Robins, 1974 ) whereby the “laurel trees” are shown extending down to the seaside. In a plate showing their encampment, Myrceugenia trees can be seen in abundance. The evidence, therefore, suggests that M. fernandeziana once covered the eastern slopes of Robinson Crusoe Island, and that there was huge loss due to cutting over the past centuries. The tree population of the island has been severely reduced during the last two centuries also by alien scrub impact ( Aristotelia chilensis , Rubus ulmifolius ). Despite disturbance, Myrceugenia fernandeziana remains the dominant native forest tree in lower elevations between 250 and 400 m a.s.l. Alejandro Selkirk Is-land, due to having no protected bay, has been visited and dis-turbed much less than Robinson Crusoe Island.

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ITO , M. A. , A. SOEJIMA , AND M. ONO . 1997 . Allozyme diversity of Pittosporum (Pittosporaceae) on the Bonin (Ogasawara) Islands. Journal of Plant Research 110 : 455 – 462 .

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LANDRUM , L. 1981a . A monograph of the genus Myrceugenia (Myrtaceae). New York Botanical Garden Press, Bronx, New York, USA.

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