J. Jpn. Bot. 91 Suppl.: 83–98 (2016)

Genetic consequences of anagenetic speciation in endemic angiosperms of Ullung Island, Korea

a, b c,d Koji Takayama *, Byung-Yun Sun and Tod Falor Stuessy

aMuseum of Natural and Environmental History, Shizuoka, 5762, Oya, Suruga-ku, Shizuoka, 422-8017 JAPAN; bDepartment of Life Science, Chonbuk National University, Jeonju, 54896 KOREA; cHerbarium and Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, 1315, Kinnear Road, Columbus, OH 43212, U.S.A.; dDepartment of Botany and Biodiversity Research, University of Vienna, 14, Rennweg, A-1030, Vienna, AUSTRIA *Corresponding author: [email protected]

(Accepted on March 3, 2016)

Two major modes of speciation exist in the evolution of endemic plants of oceanic islands: cladogenesis and anagenesis. The former is where an immigrant population becomes established and then disperses into different ecological zones on the same, or neighboring, island. Over time and in isolation, these different lineages diverge and adapt to their new environments, such that eventually they are regarded as distinct species. These cladogenetic radiations have been chronicled in considerable detail in Hawaii, Galápagos, and the Canary Islands. Anagenesis is a process of transformation of species. After establishment, an immigrant population grows in size but does not split into divergent lines due to a lack of ecological opportunity. Gene flow is maintained among subpopulations, and genetic variation accumulates due to mutation and recombination. The highest known level of anagenesis in the endemic flora of any oceanic island occurs in Ullung Island, Korea. This small island lies 137 km east of the Korean Peninsula. It is of volcanic origin, with no known connections to the mainland. Here, ca. 33 endemic species occur that represent transformational evolution from original immigrants during the past not more than 1.8 million years. A number of studies in this island have been done on isozymic genetic variation in natural populations and more recently also using AFLP and SSR molecular markers. The results from isozymes show a pattern of very limited genetic variation within these endemic species. More comprehensive AFLP and SSR markers, however, reveal much higher levels of variation in some species, even to the extent of approximating that seen in progenitor populations on the continent. The studies in Ullung Island show that anagenetic species can be expected to harbor high levels of genetic variation similar to that of the progenitor population. The individuals in subpopulations on the island appear to be held together genetically by gene flow such that no geographic partitioning of the genetic variation occurs over the landscape.

Key words: AFLP, evolution, isozymes, population genetics, SSR, Ullung-do.

It has long been recognized that oceanic plant evolution (Carlquist 1974, Whittaker archipelagos provide opportunities for and Ferrández-Palacios 2009, Bramwell and understanding patterns and processes of Caujapé-Castells 2011). Islands are especially

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encompassing some 110 woody endemic species (Wagner et al. 1999), all apparently from a single founding event (Givnish et al. 2009). In the Canary Islands, genera such as Echium (Boraginaceae; Böhle et al. 1996), Dendrosonchus (Asteraceae; Kim et al. 1996a, b), and Aeonium (Crassulaceae; Mort et al. 2002, Jorgensen and Olesen 2001) have been intensively studied. In the Galápagos Islands, the largest genus Scalesia (Asteraceae) has also been examined (Eliasson 1974, Schilling et al. 1994, Nielsen 2004). Endemic plants of smaller oceanic Fig. 1. Location of Ullung Island. archipelagos, however, have also been investigated for processes of speciation. attractive as study sites of the origin of plant Important in this regard are the Juan Fernández diversity because they are surrounded by water Archipelago and the Bonin (Ogasawara) Islands. and hence isolated from other land masses, In the former, speciation has been studied in often hundreds or thousands of kilometers the largest genera (all Asteraceae), Dendroseris distant. It is compelling to ask questions as to (Sanders et al. 1987, Sang et al. 1994), how the colonizers arrived to the islands, from Robinsonia (Sanders et al. 1987, Crawford et where they came, and how they proliferated and al. 1993a, Sang et al. 1995, Takayama et al. diversified after arrival. 2015a), and Erigeron (Valdebenito et al. 1992, Among the evolutionary processes of interest López-Sepúlveda et al. 2015). Likewise, in the in plants of islands is speciation. Studies have Bonin Islands, speciation has been examined shown numerous different modes of speciation in Crepidiastrum (Ito and Ono 1990), Ilex in continental regions, such as allopatric (Setogouchi and Watanabe 2000), and others. speciation, peripatric speciation, allopolyploidy, In nearly all of the above examples, the autopolyploidy, catastrophic speciation, and principal type of speciation examined has progenitor-derivative speciation, among others been allopatric, whereby individuals from the (Grant 1981, Crawford 2010). In oceanic islands, founding lineage disperse into different habitats the options appear to be fewer, especially due and adapt to new environments. Usually the to a near absence of mechanisms involving morphology of each lineage transforms to such chromosome change during speciation (Carr an extent that a new species is recognized. This 1998, Stuessy and Crawford 1998, Weiss et al. process, therefore, is one of splitting of the 2002). original immigrant population and hence has As might be expected, many investigators been referred to as cladogenesis (Rensch 1947, have focused on speciation in large endemic 1960, Grant 1985). It is also an evolutionary groups of the Hawaiian Islands, the Galápagos radiation based on rapid adaptation to new Islands, and the Canary Islands. Hawaii has environments, and hence, it is commonly also particularly received much attention due to known as adaptive radiation (Schluter 2000, its high level of endemism at the specific Givnish 2015). level (89%, Wagner et al. 1999). Perhaps But despite these examples of groups in most spectacular has been the evolution of oceanic islands originating by adaptive radiation, the lobelioid complex (Campanulaceae), this does not explain the origin of all endemic December 2016 Takayama et al.: Endemic angiosperms of Ullung Island 85

Table 1. Features of island systems, number of endemic species, and levels of anagenetic vs. cladogenetic speciation. Modified from Stuessy et al. (2006) Number Size Distance from Age Elevation Number of Anagenetic Cladogenetic Island system of islands (km2) mainland (km) (Myo) (m) endemic species speciation (%) speciation (%) Hawaii 8 16885 3660 5 4250 828 7 93 Canary 7 7601 100 21 3710 429 16 84 Tristan da Cuhna 4 208 2580 18 2060 27 33 67 Juan Fernández 3 100 600 4 1319 97 36 64 Cape Verde 12 4033 570 10 2829 68 37 63 Galápagos 16 7847 930 5 1707 133 43 57 Madeira 3 792 630 14 1862 96 48 52 Ogasawara 12 99 800 Tertiary 916 118 53 47 St Helena 1 123 1850 15 826 36 53 47 Ullung 1 73 130 2 984 37 88 12 species. Some immigrants never split and of the results of studies on speciation in Ullung diverge adaptively. Instead, after arrival they Island, especially at the genetic level. This is an increase in population size, accumulate genetic important island, because it represents a case of diversity through mutation and recombination, speciation so very different from the dramatic and slowly transform morphologically into adaptive radiations in other archipelagos. The what is recognized as a new species distinct purposes of this paper, therefore, are to: (1) from continental progenitors. Such a process is survey the genetic patterns within and among gradual and has been called anagenesis (Stuessy populations of endemic plant species on et al. 2006). Ullung island; (2) summarize these patterns To date, the oceanic island that has shown for an evaluation of the genetic consequences the highest level of anagenesis is Ullung Island, of anagenesis that have taken place; and (3) Korea (Fig. 1). Located 137 km E of the Korean compare these anagenetic trends with those seen Peninsula, it is a single oceanic island of 73 km2 in other oceanic archipelagos. reaching 984 m tall, and dated radiometrically as 1.8 myo (Kim 1985). It is also quite uniform Population genetics of endemic plant species ecologically (Kim 1988). The level of anagenesis on Ullung Island among the 36 endemic species of the island is A number of investigations on genetic at least 88% (Stuessy et al. 2006). Noteworthy diversity in endemic species of Ullung Island is that nearly all of the endemic species are the have been done previously using isozymes only endemics in their respective genera. The (Table 2). Park et al. (2010) investigated only exceptions are in Acer (Aceraceae), Carex genetic variation using eight enzymes within (Cyperaceae), and Viola (Violaceae), with two Scrophularia takesimensis, which is an species each. It is known, however, that in the endangered endemic species. High genetic case of Acer, the two species have each evolved diversity between populations was found. 79 anagenetically from different continental individuals were examined in four populations, progenitors (Pfosser et al. 2002a). Anagenetic which are the only known populations. All four speciation in Ullung Island, therefore, is the populations gave 11.1% polymorphic loci, an dominant form of speciation in the endemic average of 1.18 alleles per locus, mean observed vascular flora, and it is the highest level of heterozygosity in each of the populations (0.021, anagenesis known in any oceanic island (Table 1). 0.000, 0.006, 0.032; overall mean, 0.015), and What is needed at this time is a summary mean expected heterozygosity (0.020, 0.063, 86 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Table 2. Genetic diversity based on isozymes in endemic species of Ullung Island (bold) plus continental relatives Number of Number of Species populations individuals PR HE HO A AE Reference sampled analyzed Campanula takesimana 6 300 7.14 0.021 0.014 1.07 1.03 Chung et al. 1998 C. punctata (Japan, mainland) 7 ca. 325 72.2 0.117 2.39 Inoue and Kawahara 1990 C. punctata (Japan, Izu Islands) 10 ca. 235 61.1 0.044 1.89 Inoue and Kawahara 1990 Corydalis filistipes 4 120 61.2 0.167 0.201 1.73 1.20 Seok et al. 2006 Fagus multinervis 6 300 57.14 0.253 0.211 1.93 1.57 Chung et al. 1998 F. multinervis 5 152 85.44 0.373 0.383 2.13 1.78 Ohkawa et al. 2006 F. japonica (Japan) 2 70 0.444 0.456 2.88 1.89 Ohkawa et al. 2006 Hepatica maxima 6 300 8.70 0.032 0.021 1.08 1.05 Chung et al. 1998 H. maxima 1 52 50.0 0.058 1.56 Woo et al. 2002 H. asiatica (Korea) 4 79.0 2.76 Chung et al. 1998 H. asiatica (Korea) 10 337 94.12 0.276 4.06 Kim and Lee 1994 H. insularis (Korea) 10 508 82.4 0.255 3.35 Kim and Lee 1994 Scrophularia takesimensis 4 79 11.1 0.036 0.015 1.18 Park et al. 2010

PR. Per cent polymorphic loci. HE. Expected proportion of heterozygotes. HO. observed proportion of heterozygotes. A. Mean number of alleles per locus. AE. Mean effective alleles per locus.

0.033, 0.028; overall mean, 0.036). The mean population, giving a rather large sample in each FST value of the nine loci was 0.158. Nei’s genetic species of 300 individuals. Thirteen enzyme identity (Nei 1978) among the populations systems were successfully scored. In Campanula ranged from 0.980 to 0.999. takesimana, an herbaceous species, the data Seok et al. (2006) examined genetic variation show 7.14% polymorphic loci, 0.021 mean in four populations (“subpopulations”) of the expected heterozygosity, 0.014 mean observed outcrossing endemic Corydalis filistipes using heterozygosity, 1.07 mean number of alleles seven enzyme systems. The results gave 61.2% per locus, and 1.03 mean effective number of polymorphic loci, 0.167 expected proportion alleles per locus. This is a relatively low level of heterozygotes, 0.201 observed proportion of genetic variation among the endemic species. of heterozygotes, 1.73 mean number of alleles Analyses of the close continental relative, per locus, and 1.20 mean effective alleles per Campanula punctata, from seven populations locus. In addition, the analysis of fixation indices from mainland Japan, and 10 from the Izu gave an excess of heterozygotes (mean FIS, Islands (Inoue and Kawahara 1990), revealed −0.1889; FIT, −0.1226), and approximately only percent polymorphic loci of 72.2% and 61.1%, 5.6% of the variation was distributed among the respectively, which is much higher than in C. populations (FST, 0.0557). Nei’s genetic identity takesimana of Ullung Island. The mean expected among the four populations ranged from 0.967 heterozygosity is 0.123, which is considerably to 1.000, emphasizing the lack of geographic higher than in C. takesimana. In an earlier partitioning of the genetic variation. study, Inoue and Kawahara (1990) examined 10 Chung et al. (1998) analyzed six flowering Japanese populations of C. punctata from Izu plant species on Ullung Island, of which three Islands, and these gave a much reduced 0.088 are endemic and of interest for this review: expected proportion of heterozygotes. Campanula takesimana, Hepatica maxima, In Hepatica maxima, Chung et al. (1998) and Fagus multinervis. Six populations of each found that the percentage of polymorphic loci species were sampled with 50 plants from each was 8.70, mean expected heterozygosity 0.032, December 2016 Takayama et al.: Endemic angiosperms of Ullung Island 87 observed mean heterozygosity 0.021, mean in this endemic species, therefore, is quite number of alleles per locus 1.08, and mean high. Ohkawa et al. (2006) also investigated effective number of alleles per locus 1.05. genetic variation in Fagus multinervis using 11 Genetic variation in this herbaceous species, allozymes within 152 plants in five populations. therefore, is somewhat low. In comparison, Woo The results gave 85.44% mean polymorphic et al. (2002) examined a population of Hepatica loci, 0.373 expected proportion of heterozygotes, maxima in Ullung Island, sampling 52 plants, 0.383 observed proportion of heterozygotes, and their data yielded 50% polymorphic loci, 2.13 mean number of alleles per locus, and 0.058 expected proportion of heterozygotes, and 1.78 mean effective alleles per locus. Wright’s 1.56 mean number of alleles per locus. Total (1922) inbreeding coefficient (mean) yielded gene diversity was low. Kim and Lee (1994) FIS , −0.013, which suggests a high level of also sampled and analyzed 10 populations with gene flow among the populations. Furthermore, 10 enzyme systems of the Korean peninsular GST, the proportion of genetic diversity residing H. asiatica, which gave 94% polymorphic loci, among populations, was calculated as 0.036. a 4.06 mean number of alleles per locus, and a In a related study and for comparison with 0.276 expected proportion of heterozygotes in the island endemic, Ohkawa et al. (2006) also this species. They also investigated the closely examined two populations of F. japonica, one related H. insularis, which is confined primarily of the closest relatives from Japan (Oh 2015), to the southern end of the Korean peninsula. The and found 0.444 to be the expected proportion values obtained were 82.4% polymorphic loci, of heterozygotes, 0.456 the observed proportion a 3.35 mean number of alleles per locus, and a of heterozygotes, 2.88 the mean number of 0.255 expected proportion of heterozygotes. In alleles per locus, and 1.89 the mean effective Woo et al. (2002) the same data were reanalyzed alleles per locus. GST was estimated to be 0.083, and provided a range of genetic identities among showing a somewhat higher subdivision of populations within H. asiatica of 0.9313–0.9637 genetic variation between the two populations (mean 0.9480) and within H. insularis 0.6500– studied. The level of genetic variation in the 0.9838 (mean 0.7794). Between the two species endemic species, therefore, is slightly lower than the values averaged 0.7514, which partially in the Japanese relative. At the nucleotide level supports recognition of these populational in F. multinervis, however, Oh (2015) recently systems as distinct species. The relationship examined psbA–trnH chloroplast sequences and between them, however, is not entirely clear found very low haplotype diversity. (Pfosser et al. 2011), and this might be due to These isozyme studies are useful in hybridization after secondary contact during providing a general view of patterns of genetic the Pleistocene. Molecular phylogenetic studies variation within these species, but the results are have indicated that H. asiatica is the closest not precise enough to allow careful comparisons relative of the endemic H. maxima (Pfosser et al. among species nor between types of speciation. 2011). Molecular markers that reveal much more of the Chung et al. (1998) also investigated genotype, such as RAPDs, AFLPs and SSRs, isozymic variation in Fagus multinervis, a large are needed for these evaluations. As an example dominant forest tree in the island, which resulted of this problem, we mention the isozyme studies in 57.14% polymorphic loci, 0.253 mean and SSR investigations done on the endemic expected heterozygosity, 0.211 mean observed Lactoris fernandeziana in the Juan Fernández heterozygosity, 1.93 mean number of alleles Archipelago. With isozymes (Crawford et al. per locus, and 1.57 mean effective number of 1994) almost no genetic variation was detected alleles per locus. The level of genetic variation within and among populations on Robinson 88 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Table 3. Genetic diversity based on AFLPs and SSRs in anagenetically derived species of Ullung Island (bold) and close continental relatives Number of Number of AFLPs SSRs Species populations individuals Reference sampled analyzed PR SDI HE HO AR FIS TA Acer okamotoanum 7 145 0.72 6.60 0.18 57 Takayama et al. 2012b A. mono (Korea and Japan) 7 134 0.80 8.37 0.27 72 Takayama et al. 2012b A. takesimense 7 130 0.53 0.38 3.82 0.28 35.3 Takayama et al. 2012a A. pseudosieboldianum (Japan) 7 133 0.61 0.40 4.60 0.33 42.3 Takayama et al. 2012a Dystaenia takesimana 6 62 96.13 33.13 Pfosser et al. 2006 D. ibukiensis (Japan) 6 64 86.73 29.13 Pfosser et al. 2006

PR. Percent polymorphic fragments. SDI. Mean Shannon Diversity Index. HE. Expected proportion of heterozygotes. HO. Observed proportion of heterozygotes. AR. Allelic richness. FIS. Inbreeding coefficient.T A. Total number of alleles.

Crusoe Island. With RAPDs (Brauner et al. Korean peninsula, and the genetic variation in 1992) and ISSRs (Crawford et al. 2001), the island was not partitioned geographically. however, considerable variation was detected. Results from Pfosser et al. (2006) with Hence, it is nearly impossible to compare Dystaenia reveal that in D. takesimana the directly the results of AFLPs, SSRs and percentage of polymorphic fragments varies from isozymes in this archipelago or in any island 93.33 to 98.39 (mean 96.13), and in D. ibukiensis region. this value varies from 84.62 to 88.98 (mean RAPD population markers were used by 86.73). The Shannon Diversity Index gave 31.91 Ku et al. (2004) to analyze the only two known to 33.74 (mean 33.13) in D. takesimana and populations of the Ullung endemic Bupleurum 27.04 to 30.48 (mean 29.13) in D. ibukiensis. latissimum Nakai. This is a rare outcrossing, The number of polymorphic fragments, the entomophilous perennial herb. One population percent of polymorphic fragments, the Shannon contained 45 individuals that gave 0.146 Nei’s Diversity Index, and the average gene diversity gene diversity and 62.5% polymorphic bands. over loci are all higher in D. takesimana. As The second population with 61 individuals for analysis of molecular variance (AMOVA), showed 0.151 Nei’s gene diversity and 56.0% the FST among populations was 0.014 in D. polymorphic bands. The AMOVA for the species takesimana vs. 0.19 in D. ibukiensis, suggesting within populations was 65.9% and between little genetic variation among the populations populations 34.1%. The overall conclusion, in Ullung Island. These results reveal a quite therefore, is that this species harbors a relatively high level of genetic variation within Dystaenia high level of genetic variation, and that most takesimana in comparison to the Japanese D. of the variation is found within the populations ibukiensis. It is of interest that morphological rather than between them. studies conducted by Sun et al. (1997) have AFLP investigations have been completed on shown that, in contrast to the genetic results, two endemic species of Ullung Island: Dystaenia leaves are less variable in D. takesimana than in takesimana (Pfosser et al. 2006) and Hepatica D. ibukiensis. This anomaly might be explained maxima (Pfosser et al. 2002b, 2011, unpubl.) by the absence of selection for diverse habitats (Table 3). Hepatica maxima has been examined in the relatively uniform island environment in in detail with AFLPs, but these data are not yet contrast to the broader ecological differences fully published. In an abstract (Pfosser et al. within mainland Japan. 2002b), a high number of private alleles was As for microsatellites (SSRs), studies reported in comparison to H. asiatica on the have been completed on Acer takesimensis December 2016 Takayama et al.: Endemic angiosperms of Ullung Island 89

(Takayama et al. 2012a), and Acer okamotoanum divergence to occur. Regarding Hepatica (Takayama et al. 2012b) (Table 3). In these cases, maxima, despite the conspicuous morphological comparisons have been made with continental difference between the endemic species and close relatives, i.e., A. pseudosieboldianum, and the Korean peninsula H. asiatica, the former A. mono, respectively. In A. okamotoanum vs. has been treated by some workers as either a A. mono the genetic diversity statistics reveal, form or variety of the latter (Park 1974, Lee respectively: HE as 0.72 vs. 0.80; AR, 6.80 vs. 1976). Some workers have also transferred 8.37; FIS, 0.18 vs. 0.27; and TA, 57 vs. 72. In the species to Anemone (e.g., Sun et al. 2014). A. takesimense vs. A. pseudosieboldianum, the In Acer, the endemic A. takesimense has been genetic diversity statistics reveal, respectively: HE regarded as a good species (Park et al. 1993, as 0.53 vs. 0.61; HO, 0.38 vs. 0.40; AR, 3.82 vs. Pfosser et al. 2002a, Takayama et al. 2012a) 4.60; FIS, 0.28 vs. 0.33; and TA, 35.3 vs. 42.3. All or as a subspecies of A. pseudosieboldianum these data indicate that the mainland Japanese (de Jong 1994, Yang et al. 2015), to which it and Korean related species harbor slightly more is clearly related evolutionarily (Pfosser et al. genetic variation than the Ullung Island endemic. 2002a, Takayama et al. 2012a). Likewise, the AMOVA results show that the genetic variation endemic A. okamotoanum has been relegated is apportioned mostly within populations in A. by Ohashi (2002) to a subspecies of A. pictum okamotoanum (96%) in contrast to A. mono Thunb., although previous molecular studies by with 89.5%. In the other species pair, the island Pfosser et al. (2002a) suggested that it belonged A. takesimense also contains 96.2% of genetic most closely to A. mono. Denk (2003), based variation within populations, and the mainland on morphological analyses, regarded Fagus relative 93.5%. These data indicate considerable multinervis as a synonym of F. engleriana, but gene flow among individuals on the island the isozyme data presented by Ohkawa et al. that prevents geographic structuring. The (2006) would not support that action. mainland species in both cases, especially with Evaluation of patterns of genetic variation for A. pseudosieboldianum, have more variation purposes of understanding genetic consequences accounted for among populations. of anagenetic and cladogenetic speciation within endemic island species is not a simple task, Anagenetic speciation in the endemic flora and many factors may be responsible (Stuessy of Ullung Island et al. 2014). Population size is obviously To properly consider speciation in Ullung significant, with larger populations containing Island, it is necessary to first deal briefly with more variation than smaller ones. The type of taxonomic considerations. A number of recent breeding system is also important, as inbreeders floristic and revisionary studies have attempted will nearly always harbor lower levels of genetic to refine estimates of endemic vascular plants variation than outcrossers (Hamrick et al. 1991). in Ullung Island (Jung et al. 2013, Yoon et al. The age of the species can also affect the degree 2013, Chang and Gil 2014, Sun et al. 2014, Yang of genetic diversity, especially in endemic et al. 2015). These more recent contributions island species. From an initial founder effect have documented 30–33 endemic taxa, fewer upon arrival and establishment in an island, than the 37 species listed previously (Sun and genetic variation accumulates and is channeled Stuessy 1998). The reason for different estimates depending upon whether the ecological setting is due, in part, to different concepts of species. provides avenues for cladogenesis or only Because Ullung is a young volcanic island, anagenesis. The type of speciation, therefore, is being only 1.8 million years old, only a short particularly influential in the pattern of genetic period of time has existed for morphological diversity that results. Added to these natural 90 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue factors is the human disturbance on an island, change that eventually allow designation of which can have a major impact on reduction of distinct species. habitat, loss of populations or population size, Second, the overall levels of genetic variation and loss of genetic diversity. For comparisons can vary from species to species. Dystaenia among species, therefore, care must be exercised takesimensis has relatively high levels, in fact, in drawing conclusions. This is particularly the higher than in its close relative, D. ibukiensis. case when comparing results between species In the two species of Acer, however, the levels on different archipelagos, which themselves of genetic diversity are lower than their close may have had very different geological and continental relatives. In no cases, however, ecological ontogenies (Stuessy 2007). are the levels of genetic diversity in the island The isozyme data available for endemic endemics depauperate. When the original species of Ullung Island (Table 2) clearly show colonists to the island arrived some 1.8 million that the island endemics harbor less genetic or less years ago, surely there was an initial variation than their continental close relatives. founder effect. Over time, however, this strong Particularly conspicuous in this regard is reduction from the continental gene pools was Hepatica maxima. Chung et al. (1998), for no longer evident as the growing population example, obtained a value of 8.70 for percent accumulated genetic variation. polymorphic loci and 79 for H. asiatica. Woo et Third, among the subpopulations on the al. (2002) found values of 50.0 for H. maxima, island, no geographic patterning to the genetic a much higher value, but also a very high 94.12 variation is seen. That is, the profile is always for H. asiatica. The conclusions in these cases a mosaic with the genetic diversity varying emphasized the genetic bottleneck resulting in no particular geographic pattern. This from the initial founder effect, which has often would be in sharp contrast to groups that have been invoked to explain limited genetic variation adaptively radiated, in which each species is in endemic species of oceanic islands. As contained in a small portion of the island in a pointed out by Stuessy et al. (2012), however, particular ecological zone, and each harboring as time continues after initial colonization, and a distinct genetic pattern. Adaptive radiation, speciation ensues (by whichever means), it is however, is unknown in Ullung Island. Genetic unlikely that the restricted genetic signature partitioning does not occur within species from the original founding event would still be diverging anagenetically because the population evident. simply keeps growing and accumulating genetic The data from the molecular markers diversity over time. Gene flow continues among RAPDs, AFLPs and SSRs from species in the subpopulations and promotes homogeneity. Ullung Island, however, reveal a different picture Mutation, recombination and drift are important than that suggested by isozymes. Several trends in establishing the levels of variation seen. can be seen (Table 3). First, the Ullung Island Likewise, the morphology is also uniform over endemics all show clear morphological and the landscape, which would be expected in an genetic differences from their continental close environment lacking distinct ecological zones relatives. This bears on the taxonomic points where radiations could take place. mentioned earlier, and the genetic data provide support for specific recognition in these three Comparison of anagenetic speciation cases. Anagenetic speciation is a slow process in in Ullung Island with other island system which the developing population simply enlarges Despite the numerous studies that have been and accumulates genetic variation over time, completed on adaptive radiation in endemic and in the process some morphological features plant taxa, only a few have examined the December 2016 Takayama et al.: Endemic angiosperms of Ullung Island 91 genetic consequences resulting from anagenesis. however, that has a very different evolutionary Aside from Ullung Island, the archipelago history (Stuessy et al. 1998) and hence is in which similar studies have been carried inappropriate for comparisons of genetic out is the Juan Fernández Archipelago. This consequences during speciation. island system consists of two major islands: Results from the isozyme analyses in the Robinson Crusoe Island and Alejandro Selkirk three anagenetically evolved species in the Island. They are approximately the same size Juan Fernández Archipelago are of interest (50 km2) but the geological ages are different, (Table 4), but they cannot be related directly with ca. 4 myo for the former and 1–2 myo to those from AFLPs and SSRs because of for the latter (Stuessy et al. 1984). Studies on the greater sampling of the genome of the the genetic consequences of anagenesis in this latter. The species level diversity (HES) within archipelago have been completed for: Drimys Dysopsis hirsuta, Myrceugenia fernandeziana, confertifolia (Winteraceae; López-Sepúlveda and Rhaphithamnus venustus are 0.039, 0.091, et al. 2014), Myrceugenia fernandeziana and and 0.028, respectively. These are relatively M. schulzei (Myrtaceae; López-Sepúlveda low values, typical of species in Pacific Islands et al. 2013), Rhaphithamnus venustus (de Joode and Wendel 1992, Frankham 1997). (Verbenaceae; López-Sepúlveda et al. in press), Mean population diversity (HEP) in these species and Robinsonia masafuerae (Takayama et al. is: 0.022, 0.056, and 0.022; the proportion of 2015a). Comparison between anagenetic and diversity residing among populations (GST) cladogenetic consequences in Juan Fernández is: 0.299, 0.232, 0.224; and the proportion of have also been summarized recently (Takayama polymorphic loci (PR) is: 0.30, 0.67, and 0.17. et al. 2015b). The advantage of these studies These results point to very low genetic variation is that they have been done using the same within these species in general. Dysopsis methods in the same island system, and hirsuta is a perennial, wind-pollinated herb therefore, it is easier to compare and contrast (Bernardello et al. 2001) occurring in isolated results. Likewise, these methods are basically populations (patches) in small clearings of the the same as those used for the AFLP and SSR Myrceugenia forests. Rhaphithamnus venustus data from Ullung Island. Hence, comparisons is a gynodioecious (Sun et al. 1996), thin tree to between species in these two island systems are 5 m tall, hummingbird-pollinated (Anderson et more meaningful than with attempts to compare al. 2001), and occurring in small populations. genetic variation examined by other methods in Myrceugenia fernandeziana is a large tree, often species of other archipelagos with very different growing in large populations, and being the geological histories (e.g., Hawaiian Islands). dominant forest tree on Robinson Crusoe Island. As with Ullung Island, isozyme studies It is also apparently wind-pollinated, based on have also been completed on quite a number lack of insects seen in pollination observations of the endemic species of the Juan Fernández (Skottsberg 1928). This species shows the Archipelago (Crawford et al. 2001). In context highest level of genetic diversity among the of anagenetic speciation, however, only three of three taxa. the anagenetically evolved species have been Use of AFLP and SSR molecular markers investigated: Dysopsis hirsuta; Myrceugenia in the Juan Fernández Archipelago also allows fernandeziana; and Rhaphithamnus venustus. greater precision in examining levels of intra- Lactoris fernandezianus is also the only species and interpopulational variation within the of its genus in the islands, and therefore rightly anagenetically derived species. Table 5 presents would fall into the category of an anagenetically genetic diversity statistics for five anagenetic originated species. This is a very old relict taxon, species plus comparisons with four presumptive 92 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Table 4. Genetic diversity in anagenetically derived species of the Juan Fernández Archipelago based on allozymes. From Crawford et al. (2001) Number of Species P H H G populations studied P ES EP ST Dysopsis hirsuta 21 0.30 0.039 0.022 0.299 Myrceugenia fernandeziana 19 0.67 0.091 0.056 0.232 Rhaphithamnus venustus 14 0.17 0.028 0.022 0.224

PP. Proportion of polymorphic loci. HES. Species level diversity (Hamrick and Godt 1989). HEP. Mean population diversity. GST. Proportion of species diversity residing among populations. progenitors. and Drimys, the anagenetically evolved island Looking at all the anagenetic species in endemic species is considerably more genetically the Juan Fernández Archipelago (Table 5), diverse than the continental progenitor. In the several general trends can be seen. First, case of Robinsonia, R. masafuerae has evolved anagenetically derived endemic species have from R. evenia, a cladogenetically evolved island similar/slightly higher average gene diversity endemic in the older island. This represents an over loci (AGDOL) than that of related anagenetic speciation event that has taken place continental species. Second, the coefficient of during the past 1–2 million years (the maximum inbreeding shows that the lowest values occur age of Alejandro Selkirk Island). Here the two in Robinsonia masafuerae and Rhaphithamnus species are very similar in genetic diversity. This venustus, which are the two species with atypical is an interesting case because R. evenia does breeding systems, dioecious and gynodioecious, not harbor broad diversity, having been derived respectively. The highest values are in the two from splitting events over the past four million species of Myrceugenia and Drimys confertifolia, years (Takayama et al. 2015a). From out of this all large trees occurring in large populations. cladogenetic complex, therefore, has dispersed Third, because data exist for endemic species on propagules to the younger island, when formed both of the Juan Fernández Islands, one older from the hotspot in the ocean, and R. masafuerae than the other, it allows this time dimension to has accumulated genetic variation now nearly be examined in the data. Differences between equaling its progenitor species. Rhaphithamnus the species do occur, and this is particularly seen venustus is another interesting situation. The in D. confertifolia which has greater genetic earlier studies on isozymes showed that the diversity on the older island in six parameters endemic species had much lower genetic (SDI, AGDOL, HE, HO, AR, NA, Table 5). These variation than the continental R. spinosus data suggest that over time, populations on the (Crawford et al. 1993b). The AFLP and SSR older island during the past four million years data (Table 5) also suggest that the island species have been accumulating genetic diversity. These harbors less genetic variation than the mainland levels during the 1–2 million years on Alejandro relative. Some of the microsatellite statistics (AR, Selkirk Island have not yet risen to the levels on FIS, and NA), however, show the reverse trend. the older island. Determination of close continental relatives Conclusions of the endemic island species, putative The data available for anagenetically derived progenitors (or direct descendents of them), species in Ullung Island, plus those species allows comparisons to be made in genetic now also analyzed for the Juan Fernández diversity statistics (Table 5). In Myrceugenia Archipelago, allow a summary to be made December 2016 Takayama et al.: Endemic angiosperms of Ullung Island 93

Table 5. Genetic diversity based on AFLPs and SSRs in anagenetically derived species of the Juan Fernández Islands (bold) and close continental relatives Number of Number of AFLPs SSRs Species populations individuals Reference sampled analyzeda SDI AGDOL HE HO AR FIS NA Robinson Crusoe Island Myrceugenia fernandeziana 18 211 (231) 56.9 0.20 0.47 - 4.01 0.19 4.33 López-Sepúlveda et al. 2013 Alejandro Selkirk Island Myrceugenia schulzei 13 129 (155) 58.6 0.23 0.51 - 2.49 0.28 3.87 López-Sepúlveda et al. 2013 M. colchaguensis 2 19 (12) 55.1 0.21 0.53 - 2.49 0.44 2.92 López-Sepúlveda et al. 2013 Robinsonia masafuerae 5 9 (7) 74.5 0.15 0.21 0.39 1.39 −0.85 1.50 Takayama et al. 2015a R. evenia 6 73 (86) 84.5 0.16 0.19 0.19 1.21 −0.03 1.90 Takayama et al. 2015a On both islands Drimys confertifolia (RC) 16 183 (181) 104.0 0.24 0.62 0.48 4.24 0.21 4.89 López-Sepúlveda et al. 2014 D. confertifolia (AS) 15 96 (80) 90.5 0.22 0.47 0.35 3.30 0.21 3.73 López-Sepúlveda et al. 2014 D. winteri 10 126 (110) 73.3 0.17 0.60 0.47 3.96 0.19 4.71 López-Sepúlveda et al. 2014 Rhaphithamnus venustus (RC) 20 143 (140) 50.4 0.18 0.17 0.16 1.20 −0.02 1.73 López-Sepúlveda et al. in press R. venustus (AS) 4 18 (11) 37.4 0.17 0.28 0.38 1.43 −0.46 1.78 López-Sepúlveda et al. in press R. spinosus 8 116 (129) 60.6 0.18 0.34 0.42 1.43 0.14 3.43 López-Sepúlveda et al. in press

PR. Percent polymorphic fragments. SDI. Mean Shannon Diversity Index. HE. expected proportion of heterozygotes. HO. observed proportion of heterozygotes. AR. allelic richness. FIS. inbreeding coefficient. NA. number of alleles per locus. aFirst value is for AFLPs and in parentheses for SSRs. regarding genetic diversity in species with this is no partitioning of this growing gene pool into type of mode of speciation. The advantage of discrete and isolated populations, such as is the investigating in Ullung Island is that nearly case with cladogenesis. all, if not all, of the endemic species have The second important point is that owing to evolved anagenetically. The situation in the gene flow broadly connecting the subpopulations Juan Fernández Archipelago is useful because on the island, no distinct genetic partitioning can it permits a direct comparison between the two be seen. Rather, a mosaic of genetic variation modes of speciation in the same island system develops. There is no north-south or east-west with the same population genetics techniques. patterning seen in all of the species examined. Furthermore, there are two principal islands of The third point to emphasize is that the differing geological ages. amount of genetic variation that may be The first general point from these studies documented in an anagenetic species should be is that the level of genetic variation in directly related to its age. If genetic variation anagenetically evolved species is normally accumulates over time, the degree of diversity quite high. Despite that the initial founder effect should be correlated with the size of the growing must have greatly reduced the incoming genetic population, which probably relates somewhat to variation in comparison to the range from the the size of the island, and also the geological age progenitor population, over millions of years, of the island. One would expect more genetic the level of genetic diversity has increased. Gene variation in species on an older island rather than flow is maintained over the landscape among on a younger island. The best evidence of this the subpopulations, such that with mutation is seen in a comparison of Drimys confertifolia and recombination, a broader population forms and Rhaphithamnus venustus, which occur on containing considerable genetic variation. There both islands of the Juan Fernández Archipelago 94 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

(Table 5). In both cases much more genetic systems, floral visitors, and pollination systems of the variation is found in Robinson Crusoe Island, angiosperms of the Juan Fernández Islands (Chile). Bot. Rev. 67: 255–308. which is twice the geological age of Alejandro Böhle U.-R., Hilger H. H. and Martin W. F. 1996. Island Selkirk Island. A recently evolved species should colonization and evolution of the insular woody habit have less genetic variation than an older one. in Echium L. (Boraginaceae). Proc. Natn. Acad. Sci. Considerable time has to pass, obviously, before U.S.A. 93: 11740–11745. enough genetic and morphological distinctness Bramwell D. and Caujapé-Castells J. (eds.) 2011. The Biology of Island Floras. Cambridge University Press, appears such that the island population is Cambridge. considered a new species. Prior to that point, the Brauner S., Crawford D. J. and Stuessy T. F. 1992. island taxon would be still conspecific with its Ribosomal DNA and RAPD variation in the rare plant progenitor. family Lactoridaceae. Amer. J. Bot. 79: 1436–1439. Carlquist S. 1974. Island Biology. Columbia University It is also important to remember, however, Press, New York. that a counter-trend may be taking place over Carr G. D. 1998. Chromosome evolution and speciation in geological time. Reduction of island size and Hawaiian flowering plants. In: Stuessy T. F. and Ono elevation due to plate subsidence and erosion M. (eds.), Evolution and Speciation of Island Plants. will have a marked impact on lowering habitat pp. 5–47. Cambridge University Press, Cambridge. Chang C.-S. and Gil H. Y. 2014. Sorbus ulleungensis, diversity (Stuessy et al. 2005). An older island, a new endemic species on Ulleung Island, Korea. therefore, may have more restricted original Harvard Papers in Botany 19: 247–255. vegetation zones in comparison to those on a Chung H.-G., Chung J.-M. and Chung M.-G. 1998. younger island, such as can be seen clearly in Allozyme variation in six flowering plant species characterizing Ullung Island, Korea. J. Jpn. Bot. 73: the Juan Fernández Archipelago (Greimler et al. 241–247. 2002, 2013). This change over time may result Crawford D. J. 2010. Progenitor-derivative species pairs in fewer or smaller populations and overall loss and plant speciation. Taxon 59: 1413–1423. of genetic diversity. Crawford D. J., Ruiz E., Stuessy T. F., Tepe E., Aqueveque P., González R., Jensen R. J., Anderson G. J., Bernardello G., Baeza C. M., Swenson U. and Silva It is a pleasure to acknowledge: the kind M. 2001. Allozyme diversity in endemic flowering invitation to participate in this special 100-year plant species of the Juan Fernández Archipelago, Chile: celebration of The Journal of Japanese Botany; ecological and historical factors with implications for conservation. Amer. J. Bot. 88: 2195–2203. financial support from the Austrian Science Crawford D. J., Stuessy T. F., Cosner M. B., Haines D.W. Fund (FWF) to TFS; financial assistance from and Silva M. 1993a. Ribosomal and chloroplast the Japan Society for the Promotion of Science DNA restriction site mutations and the radiation of (JSPS) to KT; and Research Base Construction Robinsonia (Asteraceae: Senecioneae) on the Juan Fund Support Program, funded by Chonbuk Fernández Islands. Pl. Syst. Evol. 184: 233–239. Crawford D. J., Stuessy T. F., Cosner M. B., Haines National University 2011 to BYS. We dedicate D. W., Wiens D. and Peñailillo P. 1994. Lactoris this paper to the memory of Prof. T. Nakai, who fernandeziana (Lactoridaceae) on the Juan Fernández accomplished pioneering taxonomic studies on Islands: allozyme uniformity and field observations. Ullung Island and the Korean flora in general Cons. Biol. 8: 277–280. Crawford D. J., Stuessy T. F., Rodríguez R. and Rondinelli (Nakai 1937, 1952). M. 1993b. Genetic diversity in Rhaphithamnus venustus (Verbenaceae), a species endemic to the Juan Literature Cited Fernández Islands. Bull. Torrey Bot. Club 120: 23–28. Anderson G. J., Bernardello G., Stuessy T. F. and Crawford Crawford D., Tago-Nakazawa M., Stuessy T. F., Anderson D. J. 2001. Breeding system and pollination of selected G. J., Bernardello G., Ruiz E., Jensen R. J., Baeza plants endemic to Juan Fernández Islands. Amer. J. C. M., Wolfe A. D. and Silva M. 2001. Intersimple Bot. 88: 220–233. sequence repeat (ISSR) variation in Lactoris Bernardello G., Anderson G. J., Stuessy T. F. and fernandeziana (Lactoridaceae), a rare endemic of the Crawford D. J. 2001. A survey of floral traits, breeding Juan Fernández Archipelago, Chile. Pl. Spec. Biol. 16: December 2016 Takayama et al.: Endemic angiosperms of Ullung Island 95

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a b c 高山浩司 ，B.-Y. Sun ，T. F. Stuessy ：韓国鬱陵島固有 植物の遺伝的多様性と種分化 系統進化のパターンに注目すると，海洋島での種分化 されている韓国の鬱陵島の植物に着目し，AFLP やマイ には 2 つの様式が見られる．一つは適応放散的種分化に クロサテライトマーカーを用いて，その遺伝的特徴を明 代表される cladogenesis（分岐を伴う種分化）で，島に らかにした．鬱陵島固有種は大陸産の近縁種と同程度の 侵入した祖先が複数の種に分化している様式である．も 遺伝的多様性を保持しており，島内の集団分化もほとん う一つは anagenesis（分岐を伴わない種分化）で，島に ど見られなかった．この結果は，anagenesis に起源する 侵入した祖先系統が分岐せずに時間と共に変化し，母種 固有種が局所適応によらず，大きな集団を保ちながら突 とは異なる種に分化している様式である．anagenesis に 然変異を蓄積し，種分化したことを示唆している． 起源する固有種の遺伝的特徴を調べた研究は極めて少 （aふじのくに地球環境史ミュージアム， なく，cladogenesis に起源する固有種の遺伝的特徴と比 b韓国・全北大学校生命科学科， 較することで，海洋島における種分化過程の全貌が明ら c米国・オハイオ州立大学自然史博物館； かになっていくことが期待されている．著者らは，固有 オーストリア・ウィーン大学植物園） 種のほとんどが分岐を伴わない種分化を遂げたと推測