Springer-VerlagTokyohttp://www.springer.de102650918-94401618-0860Journal
of Plant ResearchJ
Plant ResLife Sciences18510.1007/s10265-004-0185-z
J Plant Res (2005) 118:1–11 Digital Object Identifier (DOI) 10.1007/s10265-004-0185-z
© The Botanical Society of Japan and Springer-Verlag Tokyo 2005
ORIGINAL ARTICLE Jao-Ching Huang • Wei-Kuang Wang • Ching-I Peng • Tzen-Yuh Chiang
Phylogeography and conservation genetics of Hygrophila pogonocalyx (Acanthaceae) based on atpB–rbcL noncoding spacer cpDNA
Received: April 30, 2004 / Accepted: November 2, 2004 / Published online: January 13, 2005
Abstract Genetic variation in the atpB–rbcL intergenic spacer region of chloroplast DNA (cpDNA) was investigated in Hygrophila pogonocalyx Hayata (Acanthaceae), an endangered and endemic species in Taiwan. In this aquatic species, seed dispersal from capsules via elasticity is constrained by gravity and is thereby confined within populations, resulting in limited gene flow between populations. In this study, a total of 849 bp of the cpDNA atpB–rbcL spacer were sequenced from eight populations of H. pogonocalyx. Nucleotide diversity in the cpDNA is low (q = 0.00343 ± 0.00041). The distribution of genetic variation among populations agrees with an “isolation-bydistance” model. Two geographically correlated groups, the western and eastern regions, were identified in a neighborjoining tree and a minimum-spanning network. Phylogeographical analyses based on the cpDNA network suggest that the present-day differentiation between western and eastern groups of H. pogonocalyx resulted from past fragmentation. The differentiation between eastern and western populations may be ascribed to isolation since the formation of the Central Mountain Range about 5 million years ago, which is consistent with the rate estimates based on a molecular clock of cpDNA. Key words atpB–rbcL intergenic spacer · Conservation · Hygrophila pogonocalyx · Nested clade analysis · Past fragmentation · Phylogeography
J.-C. Huang Division of Botany, Taiwan Endemic Species Research Institute, Chi-Chi, Taiwan, 551 W.-K. Wang · T.-Y. Chiang (*) Department of Life Sciences, Cheng-Kung University, Tainan, Taiwan, 701 Tel. +886-6-2757575; Fax +886-6-2752483 e-mail:
[email protected] C.-I Peng Institute of Botany, Academia Sinica, Taipei, Taiwan, 115 Huang JC and Wang WK equally contributed to this work.
Introduction Geographic patterns of genetic variation within and among populations have long been of interest to evolutionary geneticists and conservation biologists (Avise 2000). Organisms live in environments that are dynamic over time. Both past evolutionary history and current population processes affect the distribution of genetic variation; their relative significance is often difficult to distinguish based on contemporary observations. For example, genetic homogeneity among populations of a species can be due to natural selection, recent common ancestry, or contemporary gene flow. Phylogeography, a term first coined by Avise et al. (1987), deals with the genealogy of lineages and their geographical distribution at the intra- or interspecific level. In comparative phylogeography, the phylogeographic patterns among populations or species are compared in order to find general patterns of evolutionary history and to reveal evolutionary processes (Bermingham and Avise 1986; Hewitt 2000). Comparative phylogeography can be used for conservation purposes to identify areas of high biodiversity and thus of high conservation value (Moritz and Faith 1998). A central goal of conservation biology is to understand the levels and partitioning of genetic variation across populations and geographical regions of endangered species. Information about genetic relationships among organisms and populations can help managers focus efforts on truly distinctive evolutionary lineages. This approach has become increasingly popular in recent years, with the development of analytical methods to take phylogenetic distinctiveness into account when setting conservation priorities (Moritz 1995; Moritz and Faith 1998). During the past few decades, the theoretical framework of population genetics and empirical data gathered with the help of molecular genetic methods have been widely used in conservation biology. Hygrophila pogonocalyx Hayata, an aquatic plant in the Acanthaceae, is an endangered species endemic to
2
Taiwan (Hsieh and Huang 1974). Due to human disturbance, many habitats for H. pogonocalyx, mostly natural and semi-natural freshwater wetlands at low elevations, have been destroyed in recent decades (Peng et al. 1999). Because of habitat loss, misapplication of herbicides, and competition with invasive plants, the species is now severely threatened. Only eight disjunct populations remain in eastern and western Taiwan. Populations of the two geographical regions have probably been isolated since the formation of the Central Mountain Range (altitude: 3,000 m) some 5 million years ago (Teng 1990; Liu et al. 2000). Today, all extant plants of H. pogonocalyx are restricted to several small ponds from four localities on private lands. In total, fewer than 1,000 individuals were documented in nature by Wang et al. (2000). Since 1996, H. pogonocalyx has been listed by the IUCN (1997), as “critically endangered.” Plants of Hygrophila pogonocalyx, up to 1 m in height, are larger than most congenerics. Plants of this species are perennials pollinated by honeybees (Wang et al. 2000). Gene flow between populations and geographical regions via pollen dispersal is constrained by the migratory capacity and foraging pattern of pollinators. In addition, dispersal of seeds from capsules via elasticity, which is constrained by gravity, is also confined to short distances, although some occasional seed dispersal across populations via incidental transport by migratory birds may have also occurred (cf. Wang et al. 2000). With limited gene flow of seeds and pollen, genetic differentiation between populations and geographical areas has been detected (Huang et al. 2001a). Molecular markers of organelle DNA that have a low frequency of genetic recombination are useful for resolving phylogeographic patterns, conservation genetics, and assessing the migratory routes of species (e.g., Ouborg et al. 1999; Provan et al. 2001). Although chloroplast DNA (cpDNA) evolves relatively slowly, moderate to high levels of genetic variation have frequently been detected in noncoding spacers within and among species (e.g., Ohsako and Ohnishi 2000; Chiang et al. 2001; Huang et al. 2001b; Kanno et al. 2003). Because one or both organelle genomes are usually maternally inherited in plants (Birky 1995), they are particularly suitable for investigating processes associated with seed dispersal, such as range expansions (Cruzan and Templeton 2000) and the contribution of seed movement to total gene flow (e.g., McCauley 1994, 1995; Orive and Asmussen 2000). For species with limited geographical ranges and small or declining populations, historical patterns of demography and hierarchical genetic structure are important not only for determining population structure, but also for developing an effective and sustainable management plan (Moritz 1994). In this study, we investigate genetic variation, population structure, and phylogeography of Hygrophila pogonocalyx. Several aims are pursued: (1) to examine the levels of genetic variation within and between populations, (2) to reconstruct phylogeographical patterns and thereby to examine the extent of genetic differentiation among populations and between geographical regions, and (3) to
identify units for long-term conservation and management based on genetic evidence.
Materials and methods Population sampling Hygrophila pogonocalyx was sampled from ponds in Touchen (TC) in northeastern Taiwan, as well as Da-an (DA), Ching-shui (CS-1 to CS-3), and Long-ching (LC-1 to LC-3) in western Taiwan (Fig. 1, Table 1). The two geographic regions, about 140 km apart, are separated by the Central Mountain Range, which is composed of about 100 peaks higher than 3,000 m in elevation. In total, eight populations were included in this study. About 10% of individuals were sampled from each population. Sampling from the same clonal individuals was avoided. In total, 70 individuals were sampled during May and July 2002. Young healthy leaves were collected, rinsed with tap water, and dried in silica gel immediately. All samples were stored at -70°C until they were processed. Sampled plants were dug up and cultured in the greenhouse of the Taiwan Endemic Species Research Institute at a constant temperature of 25°C.
Fig. 1. Hygrophila pogonocalyx sample locations and distribution. Frequency of clades of cpDNA in each population is indicated with pie diagrams. See Fig. 3 for the clade names. CS Ching-shui, LC Longching, DA Da-an, TC Tou-chen
3 Table 1. Numbers of individuals sampled and site coordinates of the different populations of Hygrophila pogonocalyx, and the estimates of haplotype diversity (h) and nucleotide diversity (q) within populations based on DNA sequences. Possible minimum recombination events within populations were inferred using software DnaSP Population
Western region Long-ching LC-1 LC-2 LC-3 Ching-shui CS-1 CS-2 CS-3 Da-an DA-1 Eastern region Tou-chen TC-1 Overall
Site coordinate
120°32¢E 24°12¢N
120°34¢E 24°17¢N
Sample size
Number of haplotypes
Polymorphic sites (S)
h ± SD
q ± SD
Minimum recombination (Rm)
60 15 3 5 7 25 2 12 11
24 8 2 3 5 11 2 8 6
34 15 3 3 12 12 1 6 9
0.827 ± 0.046 0.733 ± 0.124 0.667 ± 0.314 0.700 ± 0.218 0.857 ± 0.137 0.823 ± 0.067 1.000 ± 0.500 0.909 ± 0.065 0.727 ± 0.144
0.00237 ± 0.00031 0.00270 ± 0.00079 0.00243 ± 0.00114 0.00146 ± 0.00060 0.00419 ± 0.00134 0.00192 ± 0.00040 0.00122 ± 0.00061 0.00182 ± 0.00029 0.00218 ± 0.00084
1 0 0 0 0 1 0 1 0
20 10
13 9
13 21
0.911 ± 0.054 0.978 ± 0.054
0.00263 ± 0.00039 0.00647 ± 0.00128
1 1
10 70
9 33
21 52
0.978 ± 0.054 0.870 ± 0.036
0.00647 ± 0.00128 0.00343 ± 0.00041
1 2
120°36¢E 24°22¢N
121°49¢E 24°58¢N
DNA extraction and PCR amplification
Sequence alignment and phylogenetic analysis
Leaves were powdered in liquid nitrogen and stored in a -70°C freezer. Genomic DNA was extracted from the powdered tissue following a CTAB procedure (Doyle and Doyle 1987) and was gel-quantified. The intergenic spacer between the atpB and rbcL genes of the chloroplast DNA was amplified using a pair of universal primers (Chiang et al. 1998). Each 100-ml PCR contained: 10 ng template DNA, 10 ml 10 ¥ reaction buffer, 10 ml MgCl2 (25 mM), 10 ml dNTP mix (8 mM), 10 pmole of each primer, and 4 U of Taq polymerase (Promega, Madison, WI, USA). The reaction was programmed on an MJ Thermal Cycler with first cycle of denaturation at 95° for 2 min, then 30 cycles of denaturation at 94° for 45 s, annealing at 48° for 1 min 15 s, and extension at 72° for 1 min 30 s, followed by 72° extension for 10 min and 4° for storage.
Chloroplast DNA sequences were aligned with the program CLUSTAL X 1.81 (Thompson et al. 1997). Indels were excluded from the data analysis. Neighbor-joining (NJ) analysis based on Kimura’s (1980) two-parameter distance was performed using the software MEGA 2.0 (Kumar et al. 2001). To evaluate clade support, 1,000 replicates of bootstrap analysis (Felsenstein 1985) were performed. Nested clade analysis of Templeton et al. (1995) provides a statistical framework for examining associations between the geographical distribution of haplotypes and their genealogical relationships (reviewed by Templeton 1998; Avise 2000). Pairwise comparisons between DNA haplotypes were calculated using MEGA 2.0 (Kumar et al. 2001). These were used to construct a minimum spanning network in a hierarchical manner with the aid of the MINSPNET (Excoffier and Smouse 1994; Chiang et al. 2001).
T-vector cloning and nucleotide sequencing Population genetic analysis of the cpDNA All PCR products were purified from an agarose gel using the PCR product purification kit (Viogene, Sunnyvale, CA, USA) and cloned into a pGEM-T easy cloning vector (Promega). For each cpDNA atpB–rbcL fragment, four randomly selected clones were sequenced to detect possible amplification error of Taq polymerase. Both DNA strands were cycle-sequenced using the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystem, Foster City, CA, USA). Products of the cycle sequencing reactions were run on an ABI 377XL automated sequencer (Applied Biosystem). Cloned PCR products were sequenced using universal T7 forward (5¢-TAATACGACT CACTATACGGG-3¢) and SP6 reverse (5¢-TATTTAGGT GACACTATAG-3¢) primers located on p-GEM-T easy vector termination sites.
Levels of inter- and intrapopulation genetic diversity were quantified by indices of haplotype diversity (h) (Nei and Tajima 1983) and estimates of nucleotide diversity (q) (Jukes and Cantor 1969) using DnaSP (version 3, Rozas and Rozas 1999). Patterns of geographical subdivision and gene flow were also estimated hierarchically with the aid of DnaSP. Gene flow within and among regions or populations was approximated as Nm, the number of female migrants per generation between populations. Nm was estimated using the expression FST = 1/(1 + 2 Nm), where N is the female effective population size and m is the female migration rate (Slatkin 1993). Genetic recombination among nucleotide sequences was detected using partial likelihoods assessed through optimi-
4
zation (PLATO) (Grassly and Holmes 1997), a computer program developed for detecting gene regions that do not fit with a “global” phylogenetic topology based on Monte Carlo simulations. Using maximum-likelihood, the likelihood for each site of a sequence can be calculated independently, and the likelihood of an anomalous region occurring by chance can then be tested. Since the tests are carried out for each region from 5 nt up to half of the sequence length, the Bonferroni adjustment is applied, whereby the alpha level used for each test of significance is divided by the number of region size classes considered (cf. Wu et al. 1999). A model of “isolation by distance” was assessed by plotting pairwise Nm values against geographical distance (cf. Slatkin 1993). The correlation between Nm and distance was determined by a regression of F-test values over distances using SPSS (Noruis 1994). Geographical associations of haplotypes and clades within the minimum-spanning network were tested using the program GeoDis (Posada et al. 2000). Two statistics were calculated: (1) the clade distance, Dc, a measure of the geographical spread of a clade, and (2) the nested clade distance, Dn, a measure of the geographical distribution of a clade relative to other clades in the same, higher-level nesting category. These measures of geographical distribution were used to infer historical processes (restricted gene flow, past fragmentation, and range expansion) following the methods of Templeton et al. (1995) (cf. Maskas and Cruzan 2000).
Relative rate tests and molecular dating The hypothesis of a molecular clock (Zuckerkandl and Pauling 1965) was tested by a relative rate test (Sarich and Wilson 1973; Wu and Li 1985). The total number of nucleotide substitutions (K), which is the number of transitional and transversional substitutions summed over all sites, was calculated for each lineage. Number and ratio of transversions versus transitions between sequences was obtained from MEGA2 (Kumar et al. 2001). The null hypothesis of a molecular clock predicts that the number of nucleotide substitutions between sister lineages will be the same. Based on the assumption of a normal distribution of nucleotide substitutions (Wu and Li 1985), the hypothesis of a molecular clock will be rejected with 95% significance when the difference in substitution rates between two lineages is greater than 1.96 times the standard error (cf. Chiang and Schaal 2000a). For estimating divergence between populations or species, a well-documented evolutionary rate is needed. In this study, an evolutionary rate for the chloroplast atpB–rbcL spacer of 2.32 ± 0.018 ¥ 10-10 substitutions per site per year estimated from mosses (Chiang and Schaal 2000b) was used as a reference. Divergence time estimates between western and eastern populations of H. pogonocalyx were then calculated using penalized likelihood, as implemented in the r8s program (Sanderson 2002). The semi-parametric method does not assume clocklike molecular evolution, but allows different evolutionary rates for each branch. In addition, there is an element of rate autocorrelation in penalized
likelihood based on the idea that descendants inherit their evolutionary rate from their ancestors. A smoothing value determines the relative importance of the likelihood score and the autocorrelation penalty for the optimality score in penalized likelihood. The optimal smoothing value is determined through a cross-validation procedure (Sanderson 2002). A 95% confidence interval was calculated for two relevant nodes using an algorithm included in r8s (Sanderson 2002) with a cutoff value of 4. Maximum likelihood was used in PHYLIP 3.6a2 (Felsenstein 1993) to estimate branch lengths with the general time-reversible model. The best tree found was input into r8s. An unconstrained penalized likelihood analysis (Sanderson 2002) was conducted with the Powell algorithm.
Results Genetic diversity and cpDNA phylogeny of Hygrophila pogonocalyx No intra-individual variation was detected in the noncoding spacer between atpB and rbcL genes of the chloroplast DNA. Identical sequences were obtained from clones derived from the same amplification reaction, indicating few PCR artifacts caused by Taq polymerase and sequencing errors. The atpB–rbcL intergenic region of cpDNA in Hygrophila pogonocalyx varied from 817–830 bp in length. The cpDNA sequences were aligned with a consensus length of 849 bp, of which 52 sites (6.1%) were variable. The chloroplast spacer was A/T rich (mean A/T content = 69.2%), which is consistent with the nucleotide composition of most noncoding spacers and pseudogenes because of low functional constraints (Li 1997). In total, 33 haplotypes were identified from 70 individuals of Hygrophila pogonocalyx, with an estimated haplotype diversity of h = 0.870 ± 0.036 (Table 1). Haplotype diversity was high and varied across populations, ranging from 0.667 (LC-1) to 1.000 (CS-1). Low levels of nucleotide diversity were detected within the species as a whole (q = 0.00343 ± 0.00041) and within populations, ranging from q = 0.00122 (CS-1) to 0.00647 (TC-1). At the geographical level, the nucleotide diversity and haplotype diversity in the eastern region (q = 0.00647 ± 0.00128, h = 0.978) were both higher than those of western populations (q = 0.00237 ± 0.00031, h = 0.827). Two recombination events were detected in the cpDNA sequences with the PLATO software. One involved a small fragment, sites 840–843, similar to recombination in organelle DNA of other plants (Lonsdale et al. 1988; Kubo et al. 1995). A second large fragment crossover between sites 539–840 was detected in Hygrophila pogonocalyx. A neighbor-joining tree obtained using MEGA, recovered four cpDNA clades, A–D (Fig. 2). Most of the clades were well supported, except for clade B. However, phylogenetic relationships among clades A, B, and C remain unresolved. Clades D and A–C correspond to eastern and western geographical regions, respectively. Clades of the
5 Fig. 2. Neighbor-joining tree of Hygrophila pogonocalyx based on sequences of the atpB–rbcL intergenic spacer of cpDNA. Numbers at nodes are bootstrap values. See Table 1 for the acronyms of population names
western group, including the most abundant clade A (50%), were widespread in all populations of CS, LC, and DA. That is, all populations were highly heterogeneous in their genetic composition. Relative rate tests and estimation of substitution rates Relative rate tests via pairwise comparisons of the substitutions between cpDNA haplotypes indicated that the evolution in atpB–rbcL intergenic spacer in Hygrophila pogonocalyx is congruent with the hypothesis of a molecular clock, except for long branches of lineages of LC-3-02, CS-3-01, and TC1-01. Nevertheless, no significant difference was observed in the rate of substitutions between haplo-
types. The total number of nucleotide substitutions (K), which is the number of transitional and transversional substitutions summed over all sites, was calculated. The total nucleotide substitution between the two major lineages of western and eastern regions was estimated to be K = 0.002295. Nested-clade analysis, phylogeography and population differentiation A nested-clade analysis was accomplished by linking nucleotide haplotypes in a hierarchical manner based on mutational changes. After linking the haplotypes into a clade, closely related clades were linked further to form a higher
6
was further grouped with clades 2-2 to 2-4 into a higher level clade 3-1. Likewise, clades 2-5 and 2-6, which consisted of clades 1-11 to 1-12 and 1-13 to 1-14, respectively, were clustered and nested in the higher level clade 3-2. Clades 3-1 and 3-2 corresponding to clades A–C and clade D of the NJ
level group; via such hierarchical linking, a nested network was drawn (Fig. 3). In total, 33 haplotypes (H1–H33) and 14 clades, 1-1 to 1-14, were identified. The distribution of haplotypes and clades in populations is indicated in Tables 2 and 3. Clades 1-1 to 1-4 were clustered into clade 2-1, which Fig. 3. Minimum-spanning network based on mutations between haplotypes of the atpB–rbcL noncoding spacer of cpDNA of Hygrophila pogonocalyx. 0 represents hypothetical ancestral nodes that were not detected or became extinct in populations
Table 2. Geographical distribution of haplotypes in populations of Hygrophila pogonocalyx Haplotype
Population LC-1
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 Total number of individuals
Total number of individuals LC-2
LC-3
CS-1
CS-2
CS-3
1
1
DA-1
TC-1
2 1 1 1
1 1 2
3
3 1
4
1 1 6
6
1 1 1 1 1 1
1
1
1 1 2 1 1
4
1 1 1 1
3
5
7
2
1
12
11
1
20
1 2 1 1 1 1 1 1 1 10
4 1 1 1 1 2 1 24 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 3 1 2 1 1 1 1 1 1 1 70
7 Table 3. Number of individuals and geographical distribution of clades in populations of Hygrophila pogonocalyx based on cpDNA data Cladea
Individuals (n)
1-1 1-2 1-3 1-4 2-1 1-5 1-6 2-2 1-7 1-8 2-3 1-9 1-10 2-4 3-1 1-11 1-12 2-5 1-13 1-14 2-6 3-2 Total
1 31 1 1 34 5 5 10 10 1 11 4 1 5 60 6 2 8 1 1 2 10 70
a
TC-1
LC-1
LC-2
LC-3
2
3
1 4 1
(48.6%)
2
1 4
(14.3%)
1 1
(15.7%)
(7.1%) (85.7%)
(11.4%)
(2.9%) (14.3%)
6 2 8 1 1 2 10 10
CS-1
6
1
1
1
1 1
3
5
1 1 7
3
5
7
CS-2
CS-3
6
8
8
6 1 1 2 4
8 1
8 3 3 6 4
4
1
DA-1
1 1 1
4 2
1 2
12
1 11
2 20
2
12
11
20
The clades were inferred from the minimum-spanning network (Fig. 3)
Fig. 4. Result of the nested clade analysis. Clade (Dc) and nesting clade (Dn) distances are given for each level of the nesting design. Superscripts refer to significantly small (S) or large (L) clade and nested clade distances. Inferences of current population structure and population history based on nested clade analysis and the interpretation key given in Templeton et al. (1995) are indicated at the bottom of the figure
tree, respectively, were identified. The former is distributed in western Taiwan, whereas the latter is a clade of the eastern part of the island. Both NJ tree and the minimumspanning network reveal a topology showing geographical division. A nested contingency analysis detected significant geographical associations within clade 3-1 and the whole cladogram. The phylogeographical inferences are listed in Fig. 4. All seven tip clades were restricted to unique regions, whereas five of seven interior clades were widespread (Table 3, Fig. 3). The results agree with the hypothesis of
constrained seed dispersal of the species. The deduced Nm of 0.29–0.52 and FST of 0.49–0.63 indicated high levels of genetic differentiation between TC and all populations of the western region (Table 4). In contrast, in the western region, genetic differentiation between Da-an, Ching-shui, and Long-ching was not significant (Nm = 5.74–121.17, and FST = 0.004–0.10), except for that between Ching-shui 1 (CS1) and other populations (Nm = 0.29–1.19, FST = 0.30–0.63) (Table 4), a result possibly due to the extremely small size of the CS-1 population. An isolation-by-distance model across four major populations of the species was supported
8 Table 4. Pairwise comparisons of Nm (below diagonal) and FST (above diagonal) between populations of Hygrophila pogonocalyx based on the cpDNA TC-1 TC-1 CS-1 CS-2 CS-3 LC-1 LC-2 LC-3 DA-1
CS-1 0.63
0.29 0.47 0.44 0.39 0.47 0.52 0.46
1.19 0.55 0.29 0.75 0.87 0.95
CS-2 0.51 0.30 48.29 6.04 8.94 34.77 22.91
CS-3 0.53 0.47 0.01 14.25 7.75 13.19 121.17
LC-1
LC-2
LC-3
DA-1
0.56 0.63 0.08 0.04
0.52 0.40 0.06 0.07 0.02
0.49 0.36 0.01 0.04 0.04 0.10
0.52 0.35 0.02 0.004 0.02 0.02 0.01
25.50 13.36 19.65
5.74 28.90
34.72
See Table 1 for the acronyms of population names
by a regression test between Nm values and geographical distance (R = 0.745). Relative values of Dc and Dn for each clade representing contemporary distributions of haplotypes were used to interpret historical and contemporary gene flow processes following the key of Templeton et al. (1995) (Fig. 4). Past fragmentation was the primary process responsible for the present-day distribution in western and eastern Taiwan (total cladogram), whereas restricted gene flow with isolation by distance was inferred for the overall western populations (clade 3-1).
Discussion Genetic variation in the atpB–rbcL spacer region in Hygrophila pogonocalyx We investigated the phylogeographical patterns and population structure of the endangered species Hygrophila pogonocalyx. When estimating population structure and gene flow genetically, a certain level of genetic variation is required. Use of markers that lack resolution and variation will inevitably produce ambiguous results, because of difficulties distinguishing co-ancestry from ongoing gene flow (Bossart and Prowell 1998). Although low substitution rates have been reported at this spacer in some plants (e.g., mosses, Chiang and Schaal 2000b; Rubiaceae, Manen and Natali 1995), some genetic variation (q = 0.00343) was detected among individuals in Hygrophila pogonocalyx. The level of genetic variation of cpDNA in this species is close to that of Dunnia sinensis (q = 0.0022) (Ge et al. 2002), and Kandelia candel (q = 0.00051) (Chiang et al. 2001), but is lower compared to other endangered species, e.g., q = 0.01018 for the cpDNA trnD–trnT spacer of Cunninghamia konishii (Lu et al. 2001) and q = 0.01268 for the cpDNA atpB–rbcL spacer of Cycas taitungensis (Huang et al. 2001b). The twofold lower nucleotide diversity in H. pogonocalyx may be associated with its extremely small population size. Recent habitat loss has reduced the number and size of Hygrophila pogonocalyx populations (Huang et al. 2001a). Small populations of narrowly distributed species are expected to exhibit low levels of genetic variation, but high
levels of genetic differentiation among populations (Hamrick and Godt 1989), as shown between western and eastern regions in this study (Table 4), because of effects of genetic drift and restricted gene flow. It is also expected that plants that are narrowly distributed and have small population sizes have a high risk of population extinction (Hanski and Gilpin 1997; Frankham et al. 2002). Interestingly, the western populations possessed fewer haplotypes and lower genetic diversity than the small eastern population at TC (Tables 1 and 2). The depletion of genetic variability was perhaps associated with demographic bottlenecks that the western populations of Hygrophila pogonocalyx experienced due to human disturbance, misapplication of herbicides and competition with invasive plants (Wang et al. 2000; Huang et al. 2001a).
Genetic differentiation and incongruence between cpDNA and RAPD fingerprinting The genes of seed plants can be dispersed during two stages of their life cycle: pollen dispersal prior to fertilization and seed dispersal. Because seed dispersal is constrained by gravity in Hygrophila pogonocalyx, gene flow between geographical regions is limited, as indicated by the low Nm and high FST between TC and western populations (Table 4). A correlation between genetic differentiation and geographic distance agrees with an isolation-by-distance model (Hedrick 1983) between eastern and western populations. In our previous study based on RAPD fingerprinting, significant genetic differentiation was detected between populations in the western region (Huang et al. 2001a). This result contradicts the cpDNA genealogy, which shows high genetic heterogeneity within populations. Lack of genetic differentiation between populations in cpDNA sequences may be due to effects of lower substitution rates or lineage sorting (Chiang 2000). According to the coalescence theory, offspring populations descending from a common ancestral population would eventually attain genetic uniqueness and become differentiated from each other as a result of coalescent processes, if no or low genetic exchange occurs after isolation. In contrast, co-ancestry due to recent common ancestry, short isolation, or exchanging migrants at a constant rate even given long isolation results in paraphyly within populations (cf. Hoelzer et al. 1998; Ray et al. 2003). In Hygrophila pogonocalyx, as current gene exchange hardly exits between populations, lack of population structuring within the western region revealed by cpDNA (Table 4) reflects a scenario of recent colonization or subdivision. Under such circumstances, the allele composition within these newly established populations would have a low probability of attaining homogeneity due to insufficient evolutionary time for coalescence (Hudson 1990; Futuyma 1998). With ancestral polymorphisms maintained within populations due to such effects of lineage sorting, an accurately resolved gene tree may not be consistent with the species/population tree (cf. Moore 1995; Chiang 2000). That is, during the lineage sorting stages, cpDNA genealogy is less likely to reflect population structure and ongoing gene
9
flow (Whitlock and MaCauley 1999). These high Nm values deduced from cpDNA are hardly indicative of ongoing gene flow, but rather of shared ancestral polymorphism within populations. Nevertheless, in contrast to the low levels of differentiation among populations of the western region, the CS-1 population is genetically unique and differentiated from others, except for CS-2 (Table 4). Random genetic drift associated with its small population size (Table 1) resulted in such uniqueness in genetic composition. In contrast to the cpDNA, RAPDs that mostly represent nuclear DNA (Hawkins and Harris 1998) have reached “coalescence” at most loci and resulted in genetic differentiation between DA and LC + CS populations. However, according to coalescence theory, nuclear DNA usually requires a longer period to attain monophyly than organelle DNA. Here, monophyly of RAPDs vs. paraphyly of cpDNA within populations of western region of Hygrophila pogonocalyx is likely derived from the different inheritance modes of the two genomes. With maternal inheritance and haplotype nature, crossover is usually lacking in organelle DNA, whereas genetic recombination occurring frequently between homologous chromosomes may have played a determining role in homogenizing the genetic differences within populations and increasing the heterogeneity between populations.
Phylogeography and conservation of Hygrophila pogonocalyx In this study, two geographical groups of western and eastern populations were identified in the NJ tree and the minimum-spanning network (Figs. 2 and 3). Both geographical isolation (140 km) and topographic barriers of high mountains hinder seed dispersal between the two regions. According to the geological evidence, the formation of the Central Mountain Range in Taiwan occurred ca. 5 million years ago (Teng 1990; Liu et al. 2000). Hence the two geographical regions may have been isolated for a long time. Phylogeographical tests for geographical associations of haplotypes and clades within phylogenies provide further insights into these historical events (Fig. 4). As ancestral populations were divided into two or more isolated populations, restricted levels of Dc and a large Dn can be used to explain the differentiation between clades 3-1 (the western populations) and 3-2 (the eastern population) (cf. Maskas and Cruzan 2000). A past-fragmentation model was therefore inferred in the total cladogram. According to a molecular clock hypothesis (Zuckerkandl and Pauling 1965), if DNA is not influenced by natural selection, the number of nucleotide substitutions within two sister lineages after splitting will be the same. In this study, the relative rate test via pairwise comparisons of sequences of the cpDNA atpB–rbcL spacer in Hygrophila pogonocalyx revealed that most lineages are consistent with a molecular clock. The total nucleotide substitution between two major lineages of western and eastern regions was estimated to be K = 0.002295. Using the evolutionary rate of the chloroplast atpB–rbcL spacer, 2.32 ± 0.018 ¥ 10-10 sub-
stitutions per site per year as a reference (cf. Chiang and Schaal 2000b), based on correction with the r8s program, the divergence between western and eastern regions of H. pogonocalyx was then estimated at 4.91–4.95 million years ago, a time coinciding with the formation time of the Central Mountain Range. Interestingly, abundant rainfall and lower average temperature occurring in eastern Taiwan may have favored differences in flowering timing between populations of the two geographical regions. According to our observations, eastern plants usually flower between late January and April, whereas plants in western Taiwan flower between September and early January. Reproductive barriers due to temporal isolation may be triggering speciation in Hygrophila pogonocalyx. A similar speciation pattern has been documented in Taiwan’s oaks, Lithocarpus formosanus and Lithocarpus dodonaeifolius (Fagaceae; Chiang et al. 2004). Significant genetic differentiation between geographical regions indicated that eastern and western cryptic species might have evolved. There are no detectable phenotypic differences between the cryptic species but highly differentiated genetic composition. Such cryptic speciation has also been documented in the liverwort, Conocephallum conicum (Kim et al. 1996). Genetic variation in Hygrophila pogonocaly will inevitably be lost due to habitat destruction, if the human disturbance continues. The quick loss of genetic diversity in populations can cause reduced adaptive ability and eventual extinction. Our results have implications for future conservation planning. Populations of geographical regions should be considered different evolutionary significant units (ESUs) for long-term management, and populations within the western region should be considered management units (MUs) for short-term management to avoid further drift of rare alleles (Moritz 1994). To avoid rapid loss of genetic variation within populations, habitat conservation that allows a large number of individuals to survive will be of most importance for the conservation of H. pogonocalyx.
Conclusion Compared to other endangered species, genetic variation was relatively low in Hygrophila pogonocalyx based on our cpDNA noncoding spacer survey. Apparently, extremely small population size plays a predominant role. Genetic drift due to small extant populations will inevitably lead to additional losses of genetic variability. Based on the NJ tree and the minimum-spanning network, populations of different geographical regions have become genetically differentiated since their separation, probably after the formation of the Central Mountain Range in the island 5 million years ago. Phylogeographical analyses of the reconstructed network suggest that the present-day H. pogonocalyx populations in western and eastern Taiwan were derived via past fragmentation. The two geographical populations should be treated as different evolutionarily significant units for conservation.
10 Acknowledgments Financial support for this project was provided by the National Science Council and the Council of Agriculture, Taiwan. The authors thank Mei-Jane Fang of Academia Sinica for the technical assistance with nucleotide sequencing. We are indebted to two anonymous reviewers for their critical comments.
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