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American Journal of Botany 88(7): 12301239. 2001.

BIOGEOGRAPHY AND ORIGIN OF LILIUM LONGIFLORUMAND L . FORMOSANUM (LILIACEAE) ENDEMIC TO THE

RYUKYU ARCHIPELAGO AND TAIWAN AS DETERMINED

BY ALLOZYME DIVERSITY1

MICHIKAZU HIRAMATSU,2,5 KAORI II,3 HIROSHI OKUBO,3

KUANG LIANG HUANG,4 AND CHI WEI HUANG4

2Laboratory of Agricultural Ecology, Faculty of Agriculture, Graduate School, Kyushu University, Kasuya 811-2307 Japan;3Laboratory of Horticultural Science, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan; and

4Department of Horticulture, National Chiayi University, Chiayi, Taiwan, Republic of China

Allozyme diversity on 13 isozyme loci was investigated for two bulbous species, Lilium longiflorum and L. formosanum, endemic

to the subtropical archipelago of continental origin located in East Asia. Degrees of allozyme variability and divergence for L.

longiflorum were very high for insular endemic species, indicating relatively longtime persistence of the present widespread distribution

across many islands in this phenotypically little-changed species. Lilium formosanum exhibited rather lower variability and divergence

than did L. longiflorum and was genetically close to the southern peripheral populations of L. longiflorum with 0.978 as its highest

genetic identity value. Combined with other biological and insular geohistorical information, our results suggest that L. longiflorum

was established around the end of the Pliocene when the current distribution area was still a continuous part of the ancient Asian

continent, and L. formosanum was derived from southern populations of L. longiflorum around the late Pleistocene when the mainland

of Taiwan was completely separated from the adjacent islands and the main continent. Depauperization of allozyme variability in some

L. longiflorum populations was found on islands with lower altitudes. This reflects bottleneck effects after the complete or almost

complete submergence of such low islands during the archipelagos development.

Key words: allozyme diversity; biogeography; continental island; insular endemic; Liliaceae; Lilium; Taiwan; the Ryukyu Ar-

chipelago.

Island biotas have attracted evolutionary biologists since thetime of Darwin (see Adsersen, 1995 as a review). A basictheory holds that genetic depauperization of organisms pop-ulations and genetic differentiation among them, respectively,are promoted by bottlenecks created by diminishing popu-

lation size and restricted gene flow from and into outer pop-ulations (Wright, 1931, 1943; Hartl, 1981; Kimura, 1983; Nei,1987). Thus, it can be easily assumed that disjunct geographicdistributions in islands, along with factors regarding migration(the founder principle) and isolation, greatly affect an organ-isms genetic structure in terms of its association with histor-ical events. In fact, information on genetic structure obtainedusing molecular techniques has accumulated recently in regardto flowering plants, further clarifying the way current biogeo-graphic structures have been established in relation to histor-ical events in islands (e.g., Witter and Carr, 1988; Inoue andKawahara, 1990; Crawford et al., 1992; DeJoode and Wendel,1992; Westerberg and Saura, 1994; Kim et al., 1996; Weller,Sakai, and Straub, 1996; Affre, Thompson, and Debussche,1997; Ito, 1998; Baldwin et al., 1999; Kim et al., 1999).

These intensive studies simultaneously generated importantfindings, which are now widely accepted regarding the popu-lation and evolutionary biology of insular plants. First, withinan isolated island environment, evolution expressing extraor-dinary morphological and ecological divergence, namely,

1 Manuscript received 25 April 2000; revision accepted 21 December 2000.The authors thank to M. Maki, Tohoku University, and Y. Ozaki, Kyushu

University, for their comments on an early version of the manuscript. Thiswork was supported in part by a Grant-in-Aid for Encouragement of YoungScientists from the Ministry of Education, Science, Sports and Culture ofJapan (No. 09760036).

5 Author for correspondence.

adaptive radiation, can frequently occur with relatively littlemolecular divergence within a short time (see Crawford, 1990,for a review). Second, endemic island taxa often possess rel-atively limited amounts of genetic variation (see DeJoode andWendel, 1992; Weller, Sakai, and Straub, 1996; Frankham,

1997, Gemmill et al., 1998 for reviews), though some re-searchers recently exhibited notable exceptions (Weller, Sakai,and Straub, 1996; Francisco-Ortega et al., 2000). These find-ings, however, have been concerned mostly with the taxa en-demic to oceanic islands, upon which organisms are estab-lished only through migration events from remote continents.

The archipelago running from Ryukyu to Taiwan consistsof nearly 200 islands forming an arc-array in the subtropicalarea between Kyushu, the southwestern district of mainlandJapan, and the southeastern part of China. Since this archi-pelago is considered geologically to be of continental origin(Kimura, 1996), unlike oceanic islands such as Hawaii, its bi-ota is largely comprised of relict taxa, which presumably dif-ferentiated from their relatives in the adjacent continent ormainland (Kizaki and Oshiro, 1977). Thus, comparative phy-

logeographic study between biota in this archipelago and thatin oceanic islands may potentially provide significant new in-sights into island biology. Studies focusing on combinationsof genetic population structures in relation to the biogeographyof relict organisms in the Ryukyu Archipelago and Taiwan,however, have almost entirely been devoted to animals suchas Plecoglossus altivelis (Nishida, 1985), Iriomote cats (Ma-suda and Yoshida, 1995), Gekko hokouensis (Toda, Hikida,and Ota, 1997), wood-feeding cockroaches (Maekawa et al.,1999), Indian rice frogs (Toda, 1999), and pit vipers (Toda etal., 1999). To our knowledge no such studies have been con-ducted regarding plants.


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July 2001] 1231HIRAMATSU ET AL.BIOGEOGRAPHY AND ORIGIN OF LILIUM

Fig. 1. Geographic distribution of the Lilium longiflorum () and L. formosanum () populations studied. Closed areas of a small map are enlargedAbbreviations of population names are the same as those in Table 1.

Lilium longiflorum and L. formosanum are bulbous plantsof the Liliaceae endemic to this archipelago and are widelyregarded as species of great importance for world horticulture(Miller, 1993; Okazaki, 1996; McRae, 1998). Both specieshave been taxonomically classified into the subsection of thesection Leucolirion by Comber (1949), and their interspecificfertile hybrid cultivars, L. formolongi imply that the twospecies are genetically close. Lilium longiflorum is geograph-ically distributed from the northernmost islands of the RyukyuArchipelago to the mainland seacoast and to small islands inthe eastern part of Taiwan, exhibiting a disjunct distributionfollowing a pattern of arc-arrayed steppingstones (Wilson,1925; Shimizu, 1987). On the other hand, L. formosanum is

natively distributed solely, but widely, within the mainland ofTaiwan (Wilson, 1925; Shii, 1983). The combined distributionof the two species thus covers the entire archipelago acrossmany islands within an 1300 km range.

Based on the abovementioned characteristics regardingstudy sites and plants, we expected that an analysis of thegenetic structure of these species would be significant not onlyin terms of what it might reveal regarding the phylogeneticrelationship of the two species, but also for verifying this re-lationships association with the historical geography in theRyukyu to Taiwan archipelago arc and the widely acceptedgeneralization regarding insular evolutionary biology assessed

mostly in oceanic islands. Allozyme analysis is often employed for studying types of microevolution such as speciationand conspecific population differentiation (Crawford, 1990)Thus, we estimated allozyme diversity of L. longiflorum andL. formosanum in order to address the following questions: (1)When and how are the species established? (2) Does the ge-netic structure of their present populations reflect their ecological nature and/or the historical geography of the archipelago?(3) Are there any properties regarding allozyme diversity ex-pressed by other insular plant taxa?

MATERIALS AND METHODS

Plant materialsThe almost entire native distribution of 19 natural populations of L. longiflorum and eight natural populations of L. formosanum

were studied (Fig. 1, Table 1). These two species are relatively similar in

terms of their appearance but are easily distinguishable by their leaf mor

phology: Lilium formosanum has willowy leaves, which are narrower and

longer than those of L. longiflorum (Wilson, 1925; M. Hiramatsu, unpublished

data). Capsules were collected in the study sites, and plant materials for en

zyme analysis were grown in a greenhouse from air-dried seeds. A single

progeny individual from each maternal genotype was used for the analysis

Samples of each population comprised 1055 individuals (33 for L. longiflo

rum and 25 for L. formosanum, on average).

Isozyme electrophoresisApproximately 200 mg of young fresh leaf sam


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1232 [Vol. 88AMERICAN JOURNAL OF BOTANY

TABLE 1. Study sites ofLilium longiflorum and L. formosanum populations indicated with number of individuals examined, population size (S,50; M, 500; L, 500), population altitude (m), and maximum island peak altitude (m).

SpeciesPopulation

abbreviation Locality

No. ofindividualsexamined

Populationsize

Populationaltitude (m)

Maximumisland peakaltitude (m)

L. longiflorum LYALKILAM1LAM2LTO

Yaku Shima, RyukyuKikai Jima, RyukyuAmami O Shima, RyukyuAmami O Shima, RyukyuTokuno Shima, Ryukyu

3043232740

SLLMM

1010202010

1935224694694645

LOELYRLOK1LOK2LOK3

Okino Erabu Jima, RyukyuYoron To, RyukyuOkinawa, RyukyuOkinawa, RyukyuOkinawa, Ryukyu

3451111310

MLSSS

2020201010

24697

498498498

LKULMILIS1LIS2LIR

Kume Shima, RyukyuMiyako Jima, RyukyuIshigaki Jima, RyukyuIshigaki Jima, RyukyuIriomote Jima, Ryukyu

4329555337

LLLLM

1020

1101010

310109526526469

LYOLPILFULLA

Yonaguni Jima, RyukyuPitouchiao, mainland of TaiwanFulung, mainland of TaiwanLanyu, Taiwan

41372930

LLSL

2060

2050

23139973997

552L. formosanum FWU

FLIFHOFWS

Wulai, mainland of TaiwanLishan, mainland of TaiwanHohuanshan, mainland of TaiwanWushe, mainland of Taiwan

21183320

MMLM

600160030001100

3997399739973997

FSHFTAFPAFCH

between Shuili and Ershui, mainland of TaiwanTatachia, mainland of TaiwanPaolai, mainland of Taiwannear Chihpen, mainland of Taiwan

12334122

SLLL

4002600

450700

3997399739973997

ples was placed into cooled mortars and homogenized with a pestle in 2 mL

of the Tris-HCl grinding buffer (Soltis et al., 1983) with a sprinkle of poly-

vinylpolypyrolidone and sea sand. Crude extracts were soaked by paper wicks

(11 3 mm), and the wicks were inserted into a slit in a starch gel.

Horizontal starch gel electrophoresis was carried out according to the pro-

cedures described by Wendel and Weeden (1989). Two combinations of gel

and electrode buffers were used to resolve 11 enzymes: aspartate aminotrans-ferase (AAT), catarase (CAT), diaphorase (DIA), glucose-6-phosphate isom-

erase (GPI), glutamate dehydrogenase (GDH), and malic enzyme (ME) were

resolved using System 6, and fluorescent esterase (FEST), isocitrate dehydro-

genase (IDH), malate dehydrogenase (MDH), phosphoglucomutase (PGM),

and phosphogluconate dehydrogenase (6PGD) were determined using System

2. Staining protocols were also carried out according to the method of Wendel

and Weeden (1989), except for a modification for FEST by dilution of the

substrate with 1/20th volume of acetone.

Statistical analysisAllele frequencies in each population of the species

were calculated for 13 loci encoding the 11 enzyme systems. The following

parameters concerning genetic diversity were estimated at the population and

species level in the manner described by Hamrick and Godt (1990): the pro-

portion of polymorphic loci (Pp) at a 95% criterion, the number of alleles per

polymorphic loci (Ap), the number of alleles per locus (A), and expected het-erozygosity (h), where h was an unbiased estimate (Nei and Roychoudhury,

1974; Nei, 1978).

To estimate genetic differentiation among populations, Neis (1973) gene

diversity statistics, namely, total genetic diversity (HT), genetic diversity with-

in populations (HS), and proportion of the total diversity among populations

(GST), were determined. In addition, Wrights (1951) fixation index ( Fis) was

estimated at each polymorphic locus as unbiased following the method of Nei

and Chesser (1983). Chi-square analyses were performed to determine the

heterogeneity of allelic frequencies among populations (Workman and Nis-

wander, 1970) and to determine deviations from genotypic frequencies ex-

pected under the Hardy-Weinberg equilibrium (Li and Horvitz, 1953).

Unbiased genetic identity and genetic distance were calculated based on

allele frequencies in accordance with the formula derived by Nei (1978). The

resulting distance matrix among all populations of the two species was then

used to construct a neighbor-joining tree (Saitou and Nei, 1987) using the

NEIGHBOR and DRAWTREE programs of PHYLIP (Felsenstein, 1993).

RESULTS

Genetic variability at the species levelThirteen loci listedin Table 2 were consistently resolved. Of a total of 48 allelesdetected across the two species, 14 (Aat-2c, Aat-3c, Cat-1b,Fest-2a, Fest-2d, Gdh-1a, Gpi-2f, Mdh-1a, 6Pgd-1a, 6Pgd-1d,6Pgd-2a, Pgm-1a, Pgm-1b, and Pgm-1e) and 3 (Fest-2b, Fest-2g, and Gpi-2e), respectively, were unique for L. longiflorumand L. formosanum. In other words, 31 alleles (65%) werecommon to both species. The most frequent alleles of eachspecies were the same for ten loci (Aat-2b, Aat-3b, Cat-1a, Dia-1b, Gdh-1c, Idh-2a, Mdh-1c, Me-1a, 6Pgd-1c, and 6Pgd-2c),whereas they were different for the remaining three loci (Gpi-2d, Fest-2c, and Pgm-1c in L. longiflorum and Gpi-2c, Fest-2f,and Pgm-1d in L. formosanum). The two species were not dis-tinguishable by the different alleles, because they were not

species specific.All (100%) of 13 resolved loci were polymorphic in at least

one population in L. longiflorum, whereas 10 (77%) of 13 lociwere polymorphic in L. formosanum (Table 3), in which Aat-2, Cat-1, and Idh-2 were judged monomorphic (Table 2). Theother genetic diversity parameters, A, Ap, and h for L. longi-florum were 1.22.2 times larger than those for L. formo-sanum.

Genetic variability at the population levelMean values ofPp, A, Ap, and h at the population level for L. longiflorum werealso substantially higher than those for L. formosanum (Table


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TABLE 2. Allele frequencies for 13 isozyme loci summarized in Liliumlongiflorum and L. formosanum.

Locus Allele L. l on gi flo ru m L. f or mo sa nu m

Aat-2 abc

0.0440.9470.008

0.0030.9970.000

Aat-3 a

bc

0.097

0.9020.002

0.024

0.9760.000

Cat-1 ab

0.9960.004

1.0000.000

Dia-1 abc

0.0150.8390.146

0.0100.9590.031

Gdh-1 abc

0.1430.7630.095

0.0000.9900.010

Gpi-2 abcdef

0.0200.2870.1010.5300.0000.062

0.0160.3430.6280.0020.0110.000

Fest-2 a

bcdefg

0.167

0.0000.4790.1720.0930.0880.000

0.000

0.0190.1130.0000.2890.5510.028

Idh-2 ab

0.9740.026

0.9940.006

Mdh-1 abc

0.2000.0130.787

0.0000.0110.989

Me-1 ab

0.9810.019

0.9690.031

6Pgd-1 abcd

e

0.0240.1070.8460.021

0.002

0.0000.0860.9090.000

0.0056Pgd-2 a

bcd

0.1860.0690.6380.107

0.0000.0630.9230.014

Pgm-1 abcde

0.1020.0800.6470.1550.016

0.0000.0000.1150.8850.000

3). The variations of those values within L. longiflorum pop-ulations, however, exhibited considerably wide ranges from23.1 to 76.9 for Pp, 1.31 to 2.15 for A, 2.00 to 3.33 for Ap,and 0.077 to 0.315 for h. Thus, the range of each population

diversity parameter for L. formosanum overlapped with thatfor L. longiflorum. The most highly diverged populations werepresent in Okinawa and Ishigaki Jima. It was noticeable thatthe diversity values, conspicuously those of h, for L. longiflo-rum populations located on islands with relatively lower peakaltitudes (LKI, LOE, LYR, LMI, and LYO) and on the easternsatellite island of the mainland of Taiwan (LLA) were lowerthan those on their adjacent islands with higher peak altitudes.

Genetic population structure and intraspecificdifferentiationFixation indices (Fis) varied greatly amongpopulations of each species, although no significant excess of

heterozygotes was observed (Table 3). Fifty-five loci (44%out of 119 loci tested for L. longiflorum showed significandeviation from 0. Relatively high frequencies of the deviatedloci within a population occurred in LYA, LAM1, LAM2LOE, LYR, LOK2, and LOK3, which are located in the rel-atively northern part of the archipelago. For L. formosanumten loci (30%) out of 33 loci tested were significant.

Chi-square analyses for heterogeneity indicated significan(P 0.01) allele frequency differences among populations inall and seven loci for L. longiflorum and L. formosanum, respectively (Table 4). On average, the indices of genetic dif-ferentiation (GST) were prominently different between the twospecies. The total gene diversity was moderately (35%) apportioned among populations of L. longiflorum, whereas themajority (92%) was apportioned within populations of L. formosanum.

Neis (1978) unbiased genetic identity (I) and standard genetic distance (D) values within and between species are sum-marized in Table 5. The I values between populations within L. longiflorum ranged widely from 0.592 to 1.000 with themean of 0.850, whereas those within L. formosanum rangedmuch more narrowly from 0.946 to 0.997 with the mean of

0.977.The high correlation between genetic and geographic dis

tance among all populations (r 0.791; P 0.001) was detected. Thus, branches of a neighbor-joining tree were combined nearly in the geographic order, showing that the popu-lations of L. formosanum was clustered with southernmost Llongiflorum populations in Taiwan with the highest I value(0.978) between LFU and FSH (Fig. 2, Table 5). The neighbor- joining tree roughly generated four major clusters: (1) LYAand LKI, (2) LOK1, LAM1, LAM2, and LTO, (3) LOK2LOK3, LOE, LYR, LKU, LMI, LIS1, LIS2, LIR, and LYOand (4) the remainder.

DISCUSSION

Allozyme diversity and origin of the two speciesLiliumlongiflorumFrankham (1997) demonstrated that insular endemic species tend to have lower genetic variability than thecontinental taxa. DeJoode and Wendel (1992) summarized theallozyme variability of 55 insular endemic plant taxa, whichin general was seen to express relatively limited amounts ofvariability at the species or infraspecific taxon with Pp 25.0(0.057.0), A 1.32 (1.001.93), and h (HT) 0.064 (0.0000.195) as an average (range) across them. On the other handnotable exceptional cases indicating relatively high estimatedvalues were reported recently. For example, in HawaiianSchiedea and Alsinidendron, respective Pp, A, and h value(and ranges) averaged across 25 populations from 20 specieswere 43.1 (088.9), 1.80 (1.003.00), and 0.183 (00.371

(Weller, Sakai, and Straub, 1996); in Hawaiian Metrosiderosthe latter two values (and ranges) averaged across 14 popu-lations from three species were 3.0 (2.73.3) and 0.371(0.2960.470), respectively (Aradhya, Mueller-Dombois, andRanker, 1991), and in endemics of the Canary Islands, the h(HT) value (and range) averaged across 69 species from 18genera was 0.186 (0.0000.456) (Francisco-Ortega et al.2000). Compared with the variability values of the reportedinsular taxa, those of L. longiflorum (Pp 100, A 3.46, andh 0.312 at the species level, and Pp 48.2, A 1.72, andh 0.187 at the population level) are comparable to higheror the highest ones.


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TABLE 3. Genetic diversity estimates and fixation indices of Lilium longiflorum and L. formosanum at the population and species level based on13 loci examined. N mean sample size per locus, Pp percentage polymorphic loci at 95% criterion, A mean number of alleles per locus,

Ap mean number of alleles per polymorphic locus, h mean expected heterozygosity (Neis [1978] unbiased estimate), Fis fixation index(unbiased estimate of Nei and Chesser [1983]). Results of tests for deviations from Hardy-Weinberg (H-W) equilibrium genotypic frequenciesare also shown (tested number of loci tested out of a total of 13, ns number of loci without significant [P 0.05] deficiency from H-Wequilibrium, number of loci with significant [P 0.05] deficiency of homozygotes).

Population name N Pp A Ap h Fis

H-W deviations

Tested ns

LYALKILAM1LAM2LTO

29.842.522.826.839.5

46.246.246.253.846.2

1.461.621.621.851.77

2.002.332.332.572.67

0.2050.1590.1700.2210.152

0.5710.1880.4650.4510.108

66676

04135

62541

LOELYRLOK1LOK2LOK3

33.950.811.013.0

9.9

69.261.576.969.269.2

1.852.001.922.151.85

2.222.632.202.672.22

0.1860.1970.2990.3150.284

0.4730.3720.2910.6450.460

98

1099

23824

75275

LKULMILIS1LIS2LIR

42.928.354.453.036.8

46.230.846.253.838.5

1.771.382.082.001.69

2.672.253.332.862.80

0.2000.1600.2080.2040.162

0.1190.0860.156

0.0570.219

64675

44563

20112

LYOLPILFULLA

40.536.928.830.0

30.830.830.823.1

1.381.461.541.31

2.252.502.752.33

0.1040.1340.1230.077

0.0770.042

0.0140.291

4443

3442

1001

Mean of L. longiflorumL. longiflorum at the species level

33.3 48.2100.0

1.723.46

2.503.46

0.1870.312

FWUFLIFHOFWSFSHFTAFPAFCH

21.017.933.020.012.033.040.622.0

7.723.130.830.846.246.238.530.8

1.081.231.461.381.541.771.541.46

2.002.002.502.252.172.672.402.50

0.0440.0710.1140.1490.1220.1700.1750.128

0.9010.4830.3470.3310.0320.2280.0430.365

13446654

02136452

11310202

Mean of L. formosanumL. formosanum at the species level

24.9 31.776.9

1.432.46

2.312.90

0.1210.142

TABLE 4. Gene diversity statistics (Nei and Chesser, 1983) for 13 iso-zyme loci in Lilium longiflorum and L. formasanum. HT totalgene diversity, HS gene diversity within population, GST pro-portion of the total gene diversity among populations.

Locus

L. longiflorum

HT HS GSTa

L. formosanum

HT HS GSTa

Aat-2Aat-3Cat-1

Dia-1Fest-2Gdh-1Gpi-2

0.1000.178

0.0090.2740.6970.3900.623

0.0340.107

0.0080.2100.4360.2450.373

0.662**0.397**

0.039**0.233**0.374**0.370**0.402**

0.0060.048

0.0000.0800.6000.0210.489

0.0060.047

0.0000.0740.4450.0200.448

0.0000.018

0.0000.072**0.259**0.056**0.084**

Idh-2Mdh-1Me-16Pgd-16Pgd-2Pgm-1Mean

0.0500.3400.0380.2720.5430.5400.312

0.0450.2030.0250.2200.2020.3340.188

0.102**0.405**0.337**0.192**0.628**0.381**0.348

0.0110.0210.0610.1670.1440.2040.142

0.0110.0210.0480.1570.1160.1980.122

0.0210.0130.206**0.060**0.199**0.0290.078

a Chi-square analysis of heterogeneity of allelic frequency amongpopulations: ** P 0.01.

The both species-level and population-level allozyme vari-ability of L. longiflorum exceeded that averaged across thespecies in various ecological categories, which mainly com-prised continental species, such as monocotyledonous species(Pp 59.2, A 2.38, and h 0.181 at the species level for111 species, and Pp 40.3, A 1.66, and h 0.144 at thepopulation level for 80 species), endemic species (Pp 40.0,A 1.80, and h 0.096 at the species level for 81 species,and Pp 26.3, A 1.39, and h 0.063 at the populationlevel for 100 species) and outcrossing animal-pollinated spe-cies (Pp 50.1, A 1.99, and h 0.167 at the species levelfor 172 species, and Pp 35.9, A 1.54, and h 0.124 at

the population level for 164 species) (Hamrick and Godt,1990), though the wide range of variations was observedamong population-level values of L. longiflorum. Among Lil-iaceous species, the species-level allozyme variability in L.longiflorum is almost comparable to the highest one reportedin Hemerocallis hakuunensis (Pp 83.0, A 6.08, and h 0.279), which is native to the Korean Peninsula and a bulbousplant with similar life history to L. longiflorum (Kang andChung, 1997). Thus, L. longiflorum is a plant species withremarkably high allozyme variability.

Comparison in genetic identity value (I) within a speciesalso revealed that L. longiflorum is highly diverged as a single


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TABLE 5. Summaries of Neis (1978) unbiased genetic identity (I) and standard genetic distance (D) within Lilium longiflorum and L. formosanum

and between them.

Species or pair of species

I

Mean Range

D

Mean Range

Within speciesL. longiflorumL. formosanum

0.8500.977

0.5921.0000.9460.997

0.1690.024

0.0000.5240.0030.056

Between speciesL. longiflorum vs. L. formosanum 0.816 0.6380.978 0.208 0.0220.450

Fig. 2. Phenogram for 19 populations of Lilium longiflorum and eightpopulations of L. formosanum constructed using a neighbor-joining methodbased on Neis (1978) standard genetic distance. Abbreviations of populationnames are the same as those in Table 1.

species (see Crawford, 1990, for a review). The minimum Ivalue between populations within L. longiflorum (0.592),which was recorded between northernmost (LYA) and south-ernmost (LLA) populations, has rarely been reported withinthe flowering plant. For example, extensive minimum Ivalueshave been reported within a selfing insular species, Bidensdiscoidea (0.688; Roberts, 1983), in Limnanthes floccosa(0.575; McNeill and Jain, 1983), and between subspecies pairsof Lens culinaris (0.65; Pinkas, Zamir, and Ladizinsky, 1985).

Generally, among insular plants, lower minimum I valueshave been rarely recorded in very limited congeneric orintergeneric population pairs in a large complex of mor-phologically and ecologically highly radiating taxa; e.g., Al-sinoideae in Hawaii (0.242; Weller, Sakai, and Straub, 1996),silversword alliance in Hawaii (0.426; Witter and Carr, 1988),woody Sonchus alliance in the Canary Islands (0.490; Kim etal., 1999), and Robinsonia in the Juan Fernandez Islands(0.560; Crawford et al., 1992). Unlike morphological and eco-logical phenotypes, protein molecules such as allozymes areassumed to evolve much more consistently because of theirneutral relationship to natural selection, as described by theneutral theory of molecular evolution (Kimura, 1983). The factthat the amount of allozyme divergence in L. longiflorum isclose to that in the maximally radiating insular plant taxa in-

dicates that they originated, roughly, at the same time. Nev-ertheless, they show a great contrast in terms of phenotypicdivergence.

A large number of reports for insular plants have been con-cerned with the highly radiating taxa in their morphologicaland ecological phenotypes in combination with very little mo-lecular divergence (see Crawford, 1989, 1990; Gemmill et al.,1998; Ito, 1998, for reviews). The opposite pattern, in which

genetically highly diverged insular taxa showed little divergence in their morphological and ecological phenotypes, wasrarely observed until the present study on L. longiflorum. Although it may be difficult to conclude the reason why suchcontrasting evolution occurs under insular environments, icould be attributed to differences in the environmental andecological properties of oceanic and continental islands. Di-verse ecologically unoccupied niches, which is presumably amajor factor affecting radiating evolution, are expected to berather few and small even at the birth of continental islandssuch as the Ryukyu Archipelago and Taiwan.

Nei (1987) demonstrated a method for estimating divergence time based on allozyme data, given certain assumptionabout mutation rates and the operation of a molecular clocktime (t) D/2a, where D is the standard genetic distance anda is the substitution rate per locus per year. Usually, a is assumed to be 107 per locus per year. Then, t may be calculated as (5 106)D. Using this formula, the initiation ofdivergence has been estimated in the aforementioned highlyradiating insular taxa, e.g., 3.6 106 yr ago (MYA) for thewoody Sonchus alliance in the Canary Islands (Kim et al.1999) and 2.9 MYA for Robinsonia spp. in the Juan FernandezIslands (Crawford et al., 1992). Since the maximum D within L. longiflorum was 0.524, initiation of divergence is assumed

to be 2.62 MYA. As pointed out by Nei (1987), however, thisvalue may sometimes be an overestimate because the geneticdistance tends to increase when the population experiencebottlenecks. This is likely to occur under insular environ-ments and for colonizing plants like L. longiflorum.

From the geological point of view, Kimura (1996) describedthat at the end of the Tertiary Period (1.72.0 MYA), the areaaround Ryukyu and Taiwan was a continuous coastal marginin East Asia. The archipelago had developed during the Pleis-tocene Era. It is, therefore, realistic to presume that L. longiflorum existed as early as around at the end of the TertiaryPeriod, and then experienced the Quaternary dynamics thagenerated the current Ryukyu to Taiwan arc. The extremelyhigh allozyme variability and divergence in L. longiflorum presumably reflect the relict endemism with the relatively long-

time persistence of the present distribution in this species.

Lilium formosanumLilium formosanum possessed a subseof L. longiflorum alleles for 11 (85%) loci and exhibited lessallozyme variability than L. longiflorum (76.9 vs. 100 for P2.46 vs. 3.46 for A, and 0.142 vs. 0.312 for h at the specielevel). These facts agree with previous data describing progenitor-derivative species pairs (e.g., Gottlieb, 1973, 1974Crawford, Ornduff, and Vasey, 1985; Rieseberg et al., 1987Loveless and Hamrick, 1988; Pleasants and Wendel, 1989Maki et al., 1999). The three species-specific alleles (Fest-2bFest-2f, and Gpi-2e) were detected for L. formosanum. How


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ever, such numbers of unique alleles are not uncommon inrecently derived species (08 in the 11 species; see Pleasantsand Wendel, 1989). The mean I value of population pairs be-tween L. formosanum and L. longiflorum (0.816) is close tothe lowest values among those 11 progenitor-derivative speciespairs listed by Pleasants and Wendel (1989). This result canbe undoubtedly attributed to the unusually extreme genetic dif-

ferentiation within a progenitor species, L. longiflorum. Re-stricted among populations within the mainland of Taiwan,where the speciation between the two species presumably oc-curred, estimation of the mean I between L. formosanum and L. longiflorum was 0.954 (0.9250.978). This indicates thatthe two species are genetically very close in the manner typicalof progenitor-derivative species pairs. Selfing is considered akey characteristic necessary for the rapid expansion of a spe-cies (Maki et al., 1999), and the occurrence of selfing naturesshown by several recently derivative species such as Stephan-omeria malheurensis (Gottlieb, 1973), Polygonella articulata,P. americana (Lewis and Crawford, 1995), and Tricyrtis nana(Maki et al., 1999) are very similar to the case of derivationof self-compatible L. formosanum from self-incompatible L.longiflorum. The accumulated evidence above demonstratesthat L. formosanum could be a recent local derivative from thesouthern peripheral populations of L. longiflorum.

Naturalized populations ofL. formosanum are found in veg-etation dominated by relatively tall grasses with wide geo-graphical ranges in often-disturbed inland areas of the main-lands of Japan, and sometimes they comprised thousands ofindividuals (M. Hiramatsu, personal observation). In contrast, L. longiflorum populations are never seen in such contexts. InSouth Africa, L. formosanum is widely naturalized under veg-etation similar to that found in the mainlands of Japan (Wal-ters, 1983). These facts imply that unlike L. longiflorum, L.formosanum can be distributed rapidly and widely in adaptablecompetitive and disturbed environments. Similar adaptable en-vironments develop in adjacent regions such as the Ryukyu

Archipelago and the Chinese continent. Nevertheless, nativepopulations of L. formosanum persist solely within the main-land of Taiwan. Thus, it is assumed that L. formosanum hasbeen prevented from migrating to adjacent regions because ofits isolation on the mainland of Taiwan prior to species initi-ation. The isolation of the mainland of Taiwan is assumed tohave occurred during the late stage of the archipelagos de-velopment as early as the last glaciation at the end of thePleistocene Era (Kimura, 1996). Examples of recent derivativespecies whose initiation times are assumed to be around thePleistocene glaciation include Cirsium pitcheri (Loveless andHamrick, 1988) , Erythronium propullans (Pleasants and Wen-del, 1989) , Polygonella articulata, and P. americana (Lewisand Crawford, 1995).

The progenitor and derivative relationship between L. lon-giflorum and L. formosanum based on our results contradictsthe speculation made by Dubouzet and Shinoda (1999), whodemonstrated only a sister relationship between the two spe-cies based on the internal transcribed spacer sequences of thespecies nrDNA and regarded L. longiflorum as a species de-rived from L. formosanum. Since our results demonstrate thatL. longiflorum is highly diverged as a single species, the ac-curacy of resolution for the two species phylogenetic rela-tionships will depend on the sample size used in the study.Thus, the very small number of samples (presumably one foreach species) in the study by Dubouzet and Shinoda (1999) is

assumed to be a cause of their inaccurate determination of thephylogenetic relationship between the two species.

The biogeographic structure of L. longiflorum involvinginsular historical eventsDetailed comparisons of allozymediversity among L. longiflorum populations revealed biogeo-graphic structures highly associated with the historical geog-

raphy of the Ryukyu to Taiwan archipelago arc, which hasbeen assumed based on geology and the biogeography of otherorganisms.

First, the depauperization in allozyme variability for somepopulations closely correlated with the maximum altitude oftheir islands, i.e., the populations that did not exhibit as muchallozyme variability as did the adjacent island populations,LKI, LOE, LYR, LMI, and LYO, were located on islands low-er than 231 m (Tables 1, 3). Because the sea level was at onetime 200 m higher than that at present, lower islands weresubmerged largely or completely and then pushed upward dur-ing the late Pleistocene Era (0.41.0 MYA) (Kimura, 1996).This evidence suggests that those L. longiflorum populationshad recently experienced very severe bottlenecks either by di-minishing population size or by subsequent migration from

relict populations on adjacent islands not highly submergedand with higher altitudes. Similarly, another substantially ge-netically eroded population on the small but high volcanic is-land southeast of the mainland of Taiwan (LLA) seems to havealso experienced severe bottlenecks, although the initiatingtime of this island is not known. A similar geohistory-asso-ciated biogeographic hypothesis regarding this archipelago hasbeen proposed to explain the mosaic distribution pattern of pitvipers (Trimeresurus spp.), whether they are present or absentin each island (Takara, 1962). However, no evidence has beenbased on population genetic diversity until the present study.

Secondly, by excluding the islands with genetically erodedpopulations (LKI, LOE, LYR, LMI, LYO, and LLA), threemajor vicariant splits generating large genetic differentiation

on the neighbor-joining tree correspond to interisland splitsbetween Yaku Shima and Amami O Shima, between TokunoShima and Okinawa, and between Iriomote Jima and the main-land of Taiwan, though a northernmost population in Okinawa(LOK1) is included as an exception in the geographically dif-ferent cluster group over the splits (Figs. 1 and 2). The north-ernmost split has long been recognized as the first straitformed in the archipelago land bridge (Kizaki and Oshiro,1977; Kimura, 1996) and as a vicariant border called WatasesLine (Kuroda, 1931; Hotta, 1974; Ono, 1989), since it corre-sponds to distribution borders dividing Japanese biota (Kuro-da, 1931; Inger, 1950; Hotta, 1974; Ono, 1989; Ota, 1998) andto the Tokara Tectonic Strait (Kimura, 1996). Thus, due to thepersistence of such an old strait, the population of L. longiflo-rum on Yaku Shima (LYA) seems to have been isolated from

the other southern populations for a long time. Whereas thevicariant border between Tokuno Shima and Okinawa hadscarcely been recognized based on the distribution patterns oforganisms until the accumulation of recent molecular data re-garding such species as Japanese newts, Cynops ensicauda(Hayashi and Matsui, 1988), semi-aquatic annual ferns, Cer-atopteris thalictroides (Watano and Masuyama, 1994), wood-feeding cockroaches, Salganea taiwanensis (Maekawa et al.,1999), and pit vipers, Trimeresurus flavoviridis (Toda et al.,1999) exhibits considerable genetic differentiation betweenOkinawa and Tokuno Shima. Our results together with thoseregarding other organisms may suggest the possibility that an-


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other noticeable vicariant border limiting gene flow has longpersisted between Okinawa and Tokuno Shima. At present, wedo not know why the remaining major vicariant split existsbetween Iriomote Jima and the mainland of Taiwan, only thatthe separation of this region is assumed to have originatedduring a relatively late stage of the archipelagos development(Kimura, 1996).

Population structure with respect to its relation to reproductive and breeding system, and geographicdistribution of the two speciesIn general, the breeding sys-tem of flowering plant species greatly affects their GST values,e.g., outcrossed and mixed animal-pollinated species have 39and 42% GST values of selfing species, respectively (Hamrickand Godt, 1990). Most Lilium species including L. longiflorumsecrete nectaries to attract pollinating insects (McRae, 1998;M. Hiramatsu, personal observation), and L. longiflorum isgenerally regarded as self-incompatible (Miller, 1993). Thesefacts imply that L. longiflorum is an obligate outcrossed, in-sect-pollinated species. Nevertheless, the GST value of L. lon-giflorum (0.348) was 77% higher than that of the mean across124 outcrossed, animal-pollinated species (0.197) and 61%

higher across 60 mixed, animal-pollinated species (0.216)(Hamrick and Godt, 1990). This shows conspicuously limitedgene flow between L. longiflorum populations. The distribu-tion of L. longiflorum ranges 1300 km between its northern-most and southernmost populations, but is disconnected in themanner of steppingstones (Fig. 1). Since the 19 populationstreated in the present study are located widely across 14 dif-ferent islands, it is highly unlikely that pollen transfer by in-sects and seed dispersal across the sea occurs for these pop-ulations.

The frequent occurrence of the loci with significant excessesof homozygous genotypes in some northern populations fromOkinawa (LYA, LAM1, LAM2, LOE, LYR, LOK2, andLOK3) is also an unexpected result, because L. longiflorum is

a putative self-incompatible, outcrossed species. Because ofthe lack of additional evidence, this result is difficult to inter-pret. For the moment, it could only be said that this is eitherbecause of the random drift of a small specimen as seen inOkinawa (LOK2 and LOK3), the relatively restricted geneflow of the metapopulation structure within a large population,or possibly the lack of random mating within a population,namely, the selfing of self-compatible individuals.

Likewise, the frequency of loci deviating significantly to-ward an excess of homozygotes varied between populationswithin L. formosanum and tended to be high in populationswith relatively low percentages of polymorphic loci (Table 3). Lilium formosanum is generally recognized as self-compatible(Shii, 1983; M. Hiramatsu, unpublished data). These facts thusindicate that facultative breeding occurs in L. formosanum, i.e.,

within some populations, outcrossing dominates, while selfingdominates within others. Selfing must play an important role,particularly in rapidly establishing new colonies from only sin-gle introductions, as described by Bakers law (Baker, 1955,1967; Stebbins, 1957).

The GST value of L. formosanum (0.078) was 60% smallerthan the mean of 124 outcrossed, animal-pollinated speciesand 64% smaller than the mean of 60 mixed, animal-pollinatedspecies (Hamrick and Godt, 1990). This shows frequent geneflow between populations. Unlike in L. longiflorum, L. for-mosanum populations are distributed solely on the mainlandof Taiwan. Further, L. formosanum produces thinner seeds with

a wider winged margin than those of L. longiflorum and hasan advantage in natural seed dispersal by wind (Shii, 1983McRae, 1998). Gene flow between the populations of the spe-cies, therefore, seems to be maintained by frequently repeatedpollen flow by insects and seed dispersal by wind or humanactivities within Taiwan without a major restriction by the sea

Conservational aspectsWe are confident that natural populations of L. longiflorum and L. formosanum are graduallydiminishing because of such human activities as robbery forhorticultural purposes and developmental destruction on islands, even though they are exempted from inclusion in thered data book at present. From the viewpoint of conservationbiology, our present study is quite educational; the diminishment of natural populations and genetic assimilation causedby reckless human activities will eventually erase the naturalhistory written within the genes of these attractive lilies.

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