Genetic Variation, Differentiation, and Evolution in a Species Complex of Tropical Shrubs Based on...

Evolution, 39(3), 1985, pp. 582-593

GENETIC VARIATION, DIFFERENTIATION, AND EVOLUTION IN A SPECIES COMPLEX OF TROPICAL SHRUBS

BASED ON ISOZYMIC DATA

KENNETH J. SYTSMA' AND BARBARA A. SCHAAL

Biology Department, Washington University, St. Louis, MO 63130

Abstract. -An isozyme investigation of the Lisianthius skinneri (Gentianaceae) species complex in central Panama assayed levels of genetic variation within and among isolated populations and was used to reconstruct phylogenetic relationships within the complex. The widespread and low elevation L. skinneri and one derived cloud forest endemic species, L. habuensis, are depauperate in genetic variation. Three other endemic cloud forest species exhibiting larger population sizes and apparently more outcrossed breeding systems have higher levels of heterozygosity but retain low levels of allelic diversity. More than 90% of the genetic variation in the species complex is confined to among-population differentiation rather than within-population variation. Isozyme-based relationships within the species complex using both genetic divergence values (Fitch and Margoliash algorithm) and shared allelic states (Nelson and VanHorn algorithm) are identical. This network is not entirely congruent with a previous DNA-based network. Geographical isolation, small population size, low allelic diversity, and high levels of among-population differentiation suggest that repeated instances of founder events and genetic drift have been important in the evolution of this tropical shrub complex.

Received July 2, 1984. Accepted February 1 1, 1985

Actively evolving complexes of populations and closely related species provide rare opportunities to study the evolutionary mechanisms that operate in plant populations. At the same time, these species complexes are often troublesome when assigning taxonomic ranks or reconstructing phylogenetic relationships. Morphological studies alone are usually inadequate to determine evolutionary relationships in these complexes. Integra- tive studies utilizing data from breeding systems, macromolecules, and allozyme variability in addition to morphological comparisons are most powerful in such complex groups.

In plants, isozyme electrophoresis has been used almost exclusively to quantify genetic variation; it has found a more limited use in reconstructing phylogenetic relationships in closely related taxa. Unfortunately, woody angiosperms and tropical species have been largely exclud- ed in such studies. Only a handful of iso-

zyme studies on tropical groups have been attempted. Two Malaysian woody species in the Dipterocarpaceae were analyzed for isozyme variation in a limited survey (Gan et al., 1977; Gan et al., 1981). More detailed isozyme studies examined genetic variation in a large number of South American species of Lycopersicon (So- lanaceae) (Rick and Fobes, 1975a, 1975b; Rick et al., 1976, 1977) and three species of Bulnesia (Zygophyllaceae) (Hunziker and Schaal, 1983). A detailed isozyme- based phylogeny was also constructed for Solanum sect. Lasiocarpa (Solanaceae) of northern South America (Whalen and Caruso, 1983).

*Here we present an isozyme electrophoretic investigation of populations comprising the Lisianthius skinneri (Gentianaceae) species complex of central Panama. This species complex is a monophyletic group of shrubs composed of at least five well-demarcated species and was the subject of a recent biosystematic study integrating classical taxonomic approaches with analyses of specific DNA sequences (Sytsma, 1983; Sytsma and Schaal, 1985). The DNA

I Present address: Department of Botany, Uni- versity of Wisconsin, Madison, Wisconsin 53706.

582

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PHYLOGENETICS: ISOZYMES 583

analysis was particularly useful in pro- viding a detailed and unambiguous phylogeny for this group of tropical shrubs. The isozyme study described here ex- amines genetic variation within and between populations and analyzes genetic relationships among the populations of the L. skinneri species complex. The populations and individuals sampled are the same ones used for the DNA analysis, thus allowing a direct comparison of the utility of these two systematic techniques. Four specific questions are ad- dressed in this isozyme investigation: 1) How much genetic variation is contained in these populations and how do levels of variation compare in populations dif- fering in floral and inflorescence characters and thus pollinators? 2) How is the genetic variation partitioned within and among the isolated populations? 3) What phylogenetic relationships can be de- duced from the isozyme data and how congruent are they with DNA-based relationships? and 4) How does the isozyme investigation contribute to our knowledge of evolution in this tropical group of shrubs?

MATERIALS AND METHODS

The Lisianthius skinneri Species Complex

Of the six well-demarcated species of Lisianthius that occur in Panama, five (excluding L. seemannii [Griseb.] Kuntze) form an unusual and distinctive inter- related species assemblage, hereafter to be referred to as the Lisianthius skinneri species complex. Lisianthius seemannii is clearly not related to the L. skinneri species complex based on foliar, floral, and inflorescence characters, although it is placed in the same series Longifolii of subsection Fruticosa to which the L. skinneri species complex belongs (Weav- er, 1972). Lisianthius skinneri (Hemsl.) Kuntze, ranging from Guatemala to Pan- ama, is the most widespread species in this genus of approximately 29 species. Lisianthius skinneri is usually confined to mid-elevational, moist or wet tropical

forests; is markedly patchy and local in occurrence throughout its range; and ex- hibits extensive morphological variation.

A dramatic divergence in Lisianthius has occurred in the central portion of Panama, topographically defined by isolated mountain peaks and more extensive intervening lower-elevation forest. A number of higher-elevation cloud forest Lisianthius taxa are each endemic to a single or a series of isolated cloud forest peaks in this region. These taxa show mi- nor to major departures from L. skinneri in morphology, breeding system, and habitat. These central Panamanian taxa along with the lower elevation variable L. skinneri populations share several morphological characters, most notably the inflated corolla tube which is con- stricted both apically and distally, strong- ly suggesting a monophyletic origin for the species complex. All taxa in the complex are composed of small, widely separated populations. The taxa display floral and inflorescence features designed for outcrossing, but all are self-compatible. Artificially crossed and selfed flowers produce capsules with similar seed sets in all species (Sytsma, 1983). Variation in levels of inbreeding or outcrossing is, thus, a function of floral and inflorescence structure and pollination behavior rather than of genetically based incom- patability systems. The entire complex is apparently tetraploid with a gametic chromosome number n = 18.

Isozyme Extraction and Electrophoresis Seven populations in the Lisianthius

skinneri species complex were selected for analysis of isozymic variation (Table 1). These included three populations of L. skinneri (El Llano-Carti, Cerro Jefe, Cascajal), and one each of L. jefensis, L. habuensis, L. peduncularis, and L. aurantiacus. Populations sampled consisted of 30-70 individuals. Twenty-five re- productively mature plants were sampled at each site, therefore representing a major portion of the total population. Sam- ples consisted of a single leaf from each



584 K. J. SYTSMA AND B. A. SCHAAL

TABLE 1. Lisianthius populations in Panama examined by isozyme electrophoresis.

Species (Designation) Location Collection no.

L. skinneri (LS4) Panama, Panama, El Llano-Carti Sytsma 4432 (Hemsl.) Kuntze

L. skinneri (LS6) Panama, Panama, Cerro Jefe Sytsma & Knapp 4795 (Hemsl.) Kuntze

L. skinneri (LS7) Panama, Cocle, Cascajal Sytsma 3939 (Hemsl.) Kuntze

L. jefensis (LJ1) Panama, Panama, Cerro Jefe Sytsma 1399 Robyns & Elias

L. habuensis (LH1) Panama, Panama, El Llano-Carti Sytsma 4002 sp. nov. Sytsma

L. peduncularis (LP1) Panama, Cocle, Cerro Carocoral Sytsma 3815 Williams

L. aurantiacus (LAl) Panama, Cocle, Cascajal Sytsma et al. 4379 sp. nov. Sytsma

individual. Leaf tissue was placed on wet ice, flown back to the laboratory in St. Louis, and stored in a -70?C freezer.

Very little isozyme activity was detected by standard extraction and electrophoretic techniques, presumably because large mature leaves with high levels of enzyme-binding phenolics were selected. The grinding technique was mod- ified to obtain consistent results in a number of enzyme systems. A slight modification of the grinding buffer of Yeh and O'Malley (1980) gave the best results (13.5 mM Tris, 4.3 mM citric acid, 0.75 mM NAD, 0.15 mM NADP, 1.0 mM ascorbic acid, 1.0 mM EDTA, 1.0 mM MgCl2, 0.1% BSA w/v, and 10% sucrose w/v; pH 7.4). Leaf tissue was ground in plastic vials with a ground glass rod in 0.1 ml grinding buffer, to which 0.05 ml 1.4 M mercaptoethanol and 5 mg PVPP were added. The resulting slurry was ab- sorbed onto Whatman #3 filter paper wicks (6 x 9 mm). Wicks were loaded into 12% starch gel slabs (14 cm x 18 cm x 9 mm). A Tris-versene borate gel buffer system gave the most consistent isozyme resolution for all enzyme systems except glucose-6-phosphate dehydrogenase (G6PDH) for which a Tris-citrate buffer was utilized. Gel buffer systems are those of Schaal and Ander- son (1974). Tris-versene borate gels were

electrophoresed at constant 200 V and Tris-citrate gels at constant 170 V. Both buffer systems were run for 5 hours at 40C.

Six enzyme systems were assayed: al- cohol dehydrogenase (ADH); aspartate aminotransferase (AAT); glucose-6- phosphate dehydrogenase (G6PDH); phosphoglucose isomerase (PGI); phos- phoglucomutase (PGM); and 6-phos- phogluconate dehydrogenase (6PGDH). Staining recipes are those of Schaal and Anderson (1974). Additional enzyme systems were initially surveyed (esterase, glutamate dehydrogenase, hexokinase, leucine amino peptidase, and malic dehydrogenase) but were discontinued because the banding patterns could not be adequately resolved.

Inferring Genotypes Genotype frequencies were inferred di-

rectly from isozyme phenotypes. Genetic studies utilizing artificially produced F1 hybrids were not possible because of the perennial growth habit and the very poor germination encountered in these Lis- ianthius. Simplifying the process of inferring genotypes from the electrophoretic phenotypes were 1) the general agreement of enzyme substructure in higher plants (Gottlieb, 1981); 2) the general conservation of number of loci cod-




TABLE 2. Allele frequencies for twelve loci in seven populations of the Lisianthius skinneri species complex.

Population

Locus1 Allele2 LS4 LS6 LS7 LiI LHI LPI LAI

Aat-l a 0 0 0 0 1.00 1.00 0 b 1.00 1.00 1.00 0 0 0 1.00 c 0 0 0 1.00 0 0 0

Aat-2 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Aat-3 a 0 0 0 0 0 0 0.90

b 0 0 0 0.56 0 0.94 0.10 c 1.00 1.00 1.00 0.44 1.00 0.06 0

Adh-l a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G6pdh-1 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G6pdh-2 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Pgi-l a 1.00 1.00 1.00 1.00 0.90 0 0

b 0 0 0 0 0.10 0.80 0.58 c 0 0 0 0 0 0.20 0.42

Pgi-2 a 0 0 0 0 1.00 0.02 0 b 1.00 1.00 1.00 1.00 0 0.98 1.00

Pgm-l a 0 0 1.00 0 0 0 0 b 0 0 0 1.00 1.00 1.00 0.04 c 1.00 1.00 0 0 0 0 0.96

Pgm-2 a 0 0 0 0 0 0 0.18 b 0 1.00 1.00 0.86 1.00 0 0.82 c 1.00 0 0 0 0 0 0 d 0 0 0 0 0 0.62 0 e 0 0 0 0.14 0 0.38 0

6Pgdh- I a 0 0 1.00 0 1.00 1.00 1.00 b 1.00 1.00 0 0 0 0 0 c 0 0 0 1.00 0 0 0

6Pgdh-2 a 0 0 0 0 0 1.00 1.00 b 0 0 0 1.00 0 0 0 c 1.00 1.00 1.00 0 0 0 0 d 0 0 0 0 1.00 0 0

1 Locus "1" represents the fastest anodally migrating locus for an enzyme system. 2 Allele "a" represents the fastest anodally migrating allele.

ing enzymes and compartmentalization of these isozymes (Gottlieb, 1982); 3) the known polyploid level of Lisianthius; and 4) the lack of excessive variation within most of the populations sampled.

In the population surveys, electromorphs were resolved in two distinct regions of the gels for four enzyme systems (G6PDH, PGI, PGM, and 6PGDH). ADH had one electromorph and AAT had two well-separated regions of electromorphs along with an intermediate, overlapping, but consistently monomorphic electromorph. The electromorph pattern for each region in polymorphic populations was consistent with expectations based on genetic control of each

region by a single locus. Twelve loci were thus inferred from the electromorph banding patterns (Table 2).

Individuals from different populations were run together on the same gel to determine whether corresponding electromorphs had similar mobilities. These composite samples were also run in gel buffers other than Tris-versene borate to detect variation in mobilities of other- wise similar electromorphs. Electro- morphs from different populations were scored as identical if no mobility differ- ences could be detected in any of the buffer systems. A total of 30 alleles for the 12 loci was inferred from the electromorph pattern (Table 2).




TABLE 3. Percentage of polymorphic loci (P), mean number of alleles per polymorphic locus (kp), mean number of alleles per locus (k), mean observed loci heterozygous per individual (H0), and heterozygosity (HET) in populations of the Lisianthius skinneri species complex.

Popula- tion p2 kp3 k3 o HET

LS4 0 - 1.00 0 0 LS6 0 - 1.00 0 0 LS7 0 - 1.00 0 0 LJ1 16.7 2.00 1.17 0.030 0.061 LH1 8.3 2.00 1.08 0.017 0.015 LP1 33.3 2.00 1.33 0.073 0.082 LAl 33.3 2.00 1.33 0.106 0.079

Mean 13.1 2.00 1.13 0.032 0.035 1 N = 25 for all populations. 2 Values are given for loci in which any alternative allele was

detected. 3 Values are given for any allele detected.

RESULTS Isozyme Variability within Populations The resulting data (Table 2) were ana-

lyzed by various methods to characterize genetic variability within the Lisianthius skinneri species complex. Both within- and between-population variability were analyzed. Each locus containing an alternate allele was considered polymorphic. A minimum alternate allele frequency of 0.02 defines a polymorphic locus, based on 25 individuals surveyed. Four loci (Aat-2, Adh-1, G6pdh-1,2) were monomorphic for the same allele in all populations. Pgi-2 was monomorphic in all populations except one, which contained a single heterozygous individual. The percentage of polymorphic loci (P) ranges from 0-33.3%; the mean number of alleles per polymorphic locus (kp) is 2.00; and the mean number of alleles per locus (k) ranges from 1.00-1.33. The mean proportion of loci heterozygous per individual (Ho) ranges from 0.00-0.106, and heterozygosity (HET = 1 - XI2' averaged over all loci; xi = frequency of ith allele) ranges from 0.00-0.082 (Table 3).

The three populations of Lisianthius skinneri are monomorphic at all loci examined. LH 1 is polymorphic only at one locus. Populations of the cloud forest

species, L. jefensis, L. peduncularis, and L. aurantiacus, are the most polymorphic in the species complex. These three species exhibit the largest population sizes, whereas the L. skinneri and L. habuensis populations have the smallest population sizes. The 25 individuals sampled represent well over 50% of the LS4 and LS6 populations and almost the entire population of both LS7 and LH1. The LS4, LS6, and LH1 populations examined are confined to roadside habitats and, presumably, must have been found- ed relatively recently because the road sites are less than 50 years old. Most likely, the lack of variation can be attributed to some type of founder event from more scattered individuals encountered in the forest itself. The lack of isozyme variability in Lisianthius skinneri is consistent with data from species that exhibit restricted distributions and extensive inbreeding (e.g., Clarkiafranciscana [Gott- lieb, 1973]; Lycopersicon cheesmanji [Rick and Fobes, 1 975b]; Sullivantia spp. [Soltis, 1982]; also see review in Hamrick et al., 1979).

The three polymorphic cloud forest species are near the upper range of isozyme variability found in a recent survey of plant populations (Gottlieb, 1981). P and Ho (Gottlieb's HET) for Lisianthius peduncularis (33.3%, 0.073) and L. aurantiacus (33.3%, 0.106) are similar to average values for populations of outcrossing species (37%, 0.086). All three polymorphic populations, however, are depauperate in terms of mean number of alleles per polymorphic loci (k0). Lisian- thius aurantiacus probably has the great- est amount of outcrossing in the species complex because of its highly reduced inflorescence of one or two flowers. None of the three polymorphic species is pol- linated by the small bees that contribute to selfing in L. skinneri and L. habuensis (Sytsma, 1983).

F, the Fixation Index (Wright, 1969; Jain and Workman, 1967), which measures deviation of heterozygote proportions from Hardy-Weinberg expectations, was determined for each polymorphic lo-



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TABLE 4. Fixation indices (F) of the polymorphic loci in the Lisianthius skinneri species complex.t

Population

Locus Li I LHI LPI LAI

Aat-3 +0.512* - -0.064 -0.333 Pgi-l -0.111 +0.250 -0.232 Pgi-2 - -0.020 - Pgm-l - - - -0.042 Pgm-2 +0.502* - +0.066 -0.220

* Heterozygote proportions significantly different from Hardy-Weinberg expectations based on a Chi-square test (P < 0.05). t Populations LS4, LS6, and LS7 were monomorphic at these loci.

cus (Table 4). F can range from - 1.0 to 1.0, with the former indicating an excess and the latter a deficiency of heterozy- gotes, respectively, when compared to Hardy-Weinberg proportions. Signifi- cance of deviations at each locus was tested by a x2 test against Hardy-Wein- berg expectations. All populations containing polymorphic loci conform to Hardy-Weinberg equilibrium except Lis- ianthius jefensis, which has significant heterozygote deficiencies at both polymorphic loci.

Apportionment of Genetic Diversity The apportionment of genetic diver-

sity within the Lisianthius skinneri species complex was determined using the genetic diversity analysis (Nei, 1975). Total gene diversity (HT) is subdivided into gene diversity within populations (HS) and gene diversity among populations (DsT) where

HT= HS + DST.

HT is calculated by obtaining the weighted average allele frequencies over all populations (HT = 1 - X i2). HS is equal to the weighted average over all populations of the values of 1 - E x12 for each population. DST is obtained by subtraction. Differentiation among populations is calculated as

GsT = DST/HT,

where GST can vary between 0 (HS = HT) and 1 (HS = 0, i.e., populations fixed for different alleles).

There is significant genetic variation between populations in the Lisianthius skinneri species complex (Table 5). Ge-

netic diversity for three loci is due totally to the among-population component. For two additional loci (Pgi-2, Pgm-1), almost all of the diversity is due to among- population variation rather than within- population variation. The mean of 0.903 for differentiation among populations indicates that a large portion of the genetic variability in Lisianthius is not present among the individuals of a population. The data are consistent with extensive differentiation due to isolation and genetic drift. Many of the populations are fixed for alternative alleles at a number of loci, which is evident even between populations of L. skinneri.

Isozyme-Based Species Relationships Extensive isozyme divergence among

species is further indicated by their low Nei genetic identity values (Table 6). The highest identity values are found among the three Lisianthius skinneri populations. The mean of their I values (I = 0.833), however, is lower even than the

TABLE 5. Genetic diversity among populations in the Lisianthius skinneri species complex.

Differentiation among populations

Locus1 HT HS DST (DST/HT)

Aat-l 0.592 0 0.592 1.000 Aat-3 0.518 0.112 0.406 0.784 Pgi-1 0.457 0.141 0.316 0.691 Pgi-2 0.249 0.006 0.243 0.976 Pgm-l 0.612 0.011 0.601 0.982 Pgm-2 0.518 0.144 0.374 0.722 6Pgdh-1 0.571 0 0.571 1.000 6Pgdh-2 0.694 0 0.694 1.000

Mean 0.526 0.052 0.475 0.903 1 Values are given only for polymorphic loci.




TABLE 6. Nei genetic identity values and Rogers' divergence coefficients between populations in the Lisianthius skinneri species complex.*

LS4 LS6 LS7 LJ1 LH1 LP1 LA1 LS4 - 0.916 0.750 0.553 0.495 0.437 0.606 LS6 0.083 - 0.833 0.627 0.579 0.437 0.678 LS7 0.250 0.167 - 0.627 0.663 0.524 0.681 LJ1 0.458 0.392 0.392 - 0.623 0.590 0.522 LH1 0.509 0.425 0.342 0.400 - 0.626 0.519 LP1 0.563 0.563 0.479 0.424 0.384 - 0.695 LA1 0.399 0.337 0.307 0.473 0.489 0.321 -

* Genetic identity above the diagonal and divergence below.

mean I value reported for Northern Hemisphere outcrossing species (I = 0.965) (Gottlieb, 1981). The average for all pairwise comparisons in the L. skinneri species complex (I = 0.618) is close to the mean identity value reported for congeneric populations (I = 0.670) (Gottlieb, 1981).

Isozyme analysis can clearly differen- tiate between any species or population pair in the Lisianthius skinneri group, in- cluding L. skinneri and L. jefensis. This is in contrast to DNA analysis in which these two species exhibit no detectable changes in nuclear ribosomal DNA (rDNA) and only one detectable change in chloroplast DNA (cpDNA) (Sytsma and Schaal, 1985). Lisianthius peduncularis and L. aurantiacus, morphologi- cally related, are likewise well separated by isozyme analysis but differ only by a few mutations in rDNA or cpDNA. In order to analyze cladogenesis in the L. skinneri species complex based on isozyme data, the strategies of Fitch and Margoliash (1967) and Nelson and VanHorn (1975) for constructing unrooted networks were employed. These two methods were selected because they represent different approaches for dis- cerning cladogenetic events. The Fitch and Margoliash algorithm is based on overall measures of resemblance or divergence. The Wagner network algorithm of Nelson and VanHorn uses the character state data to construct networks that minimize the number of character state changes (evolutionary steps).

The Fitch and Margoliash (1967) al-

gorithm was utilized to construct unrooted networks estimating relationships in the Lisianthius skinneri species complex based on Rogers' (1972) genetic divergence values (Table 6). The analysis was straightforward except for the last two steps that involved placing LJ 1 and LH 1 on the expanding network. Diver- gence values for the pairs LJ 1-LH 1 and LJ 1-[LS4, LS6, LS7] were sufficiently close (0.400 and 0.414, respectively) to necessitate the construction of two networks (Fig. l a, b). The two networks differ only in the relative placements of LJ 1 and LH 1 on the network. Expected divergence values between species pairs calculated for each network were statistically tested against observed divergence values to obtain a percent standard deviation (% SD) (Fitch and Margoliash, 1967). Network A (Fig. 1 A) and network B (Fig. 1 B) deviate equally from observed divergence values (% SD = 12.1), and thus both equally estimate relationships within the species complex.

Restriction endonuclease analysis of both rDNA and cpDNA has provided an unambiguous phylogenetic tree for the group (Sytsma and Schaal, 1985). This tree is depicted in network form in Figure 1C with the root to the outgroup Lisian- thius seemannii (LSM1) shown by a dashed line. Based on Rogers' genetic divergence values, this network deviates more from the observed data (% SD= 15.8) than do networks A or B, and also contains a negative branch. There is good congruence, however, between the DNA-based network (Fig. 1C) and net-



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work A (Fig. 1A), based on isozyme divergence. The only difference between the two networks is due to a switch of the LJ1 and LS7 branches. The DNA analysis has shown that LJ 1 is very similar to LS4 and LS6 (eastern Panamanian populations of L. skinnerl) and that LS7 (western Panamanian population) has di- verged in a separate lineage (Sytsma and Schaal, 1985). The isozyme divergence data do not support these cladogenetic events.

To utilize the Nelson and VanHorn (1975) algorithm, the 30 alleles comprising the 12 loci analyzed were chosen to be the characters. Each allele for each population was assigned the character state "0" if absent and the state "1" if present in the population at any frequency. The application of the algorithm re- sulted in the derivation of seven Wagner networks, each depicting different branching relationships. The derivation of seven, rather than a single Wagner network, was due to instances when the data set presented equal or nearly equal choices and each alternative was separately followed. The networks were tested by the parsimony criterion to determine the best estimation of the branching relationships for the Lisianthius skinneri species complex. The total number of evolutionary steps for each network was determined, and the corresponding consistency index (C.L) of Kluge and Farris (1969) was calculated.

The seven constructed Wagner networks required fewer than 40 evolutionary steps to account for the observed isozyme data of 30 allelic characters (C.I. > 0.75). The three networks requiring the fewest evolutionary steps (34, 35, and 36 steps) are depicted in Figure 1D-F. The minimum-length Wagner network D (Fig. 1D) is identical to network A generated by the Fitch and Margoliash algorithm (Fig. lA). Network E differs from the minimum-length network D by a single extra evolutionary step and is identical to network B utilizing the Fitch and Margo- liash algorithm (Fig. 1B). Network F is two steps longer than the minimum-

length Wagner network and differs only in the relative placement of LS7 and LJ 1. This network is identical to that derived from DNA analysis (Sytsma and Schaal, 1985; see Fig. 1 C). The algorithm of Fitch and Margoliash and the algorithm of Nel- son and VanHorn thus provide the same most likely network based on this isozyme data set. As a possible criticism against the ability of any network constructing algorithm to generate the best network, it should be noted that network F was constructed as the minimum-length network when the Nelson and VanHorn algorithm was strictly followed. The shorter networks D and E could only be obtained by selecting alternative branch placements in an early step of the algorithm.

The networks depicted in Figure 1D-F were converted to Wagner phylogenetic trees by rooting a hypothetical ancestor of the Lisianthius skinneri species complex into the networks. The related-group criterion, which asserts that the primitive character state is more likely to have a wider distribution in related groups and within the group examined, was used to construct a hypothetical ancestor. This ancestor contained the character state " 1 " for only the most widely distributed allele of each locus; all other alleles were assigned a character state of "O." The Lundberg (1972) method was used to find the branch(es) in the networks most similar to this hypothetical ancestor. The roots for networks D, E, and F are indicated by dashed lines in Figure 1D-F.

The minimum-length network D is converted to a phylogenetic tree composed of two distinct lineages. The lineage encompassing all Lisianthius skinneri populations is split apart from the lineage of four cloud forest species. Net- work E has an ambiguous corresponding tree because the hypothetical ancestor can be rooted in either of two branches. The Wagner tree derived from network F places the cloud forest species L. jefensis with the L. skinneri populations. Al- though network F is identical to the most parsimonious network derived from








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DNA analysis (Sytsma and Schaal, 1985; see Fig. 1 C), the corresponding rooted trees differ. Phylogenetic reconstruction using DNA data involving an outgroup species statistically rejected the ancestral root location depicted in network F. In- stead, the ancestral root for the DNA- based phylogeny was placed in the branch separating LJ 1 and LS7. This placement provided strong DNA evidence for 1) the alignment of L. jefensis to the eastern Panamanian populations of L. skinneri (LS4 and LS6) and the alignment of the western Panamanian L. skinneri population (LS7) to the other three cloud forest species; 2) the derivation of at least two lineages of cloud forest species; 3) the paraphyletic nature of L. skinneri; and 4) the likely ancestral nature of the widespread species of L. skinneri to the nar- rowly restricted, cloud forest species (Sytsma and Schaal, 1985). The two shortest isozyme-based Wagner trees do not support these DNA-based conclu- sions.

DISCUSSION

Evolution in the Lisianthius skinneri Species Complex

Isozyme analysis indicates that the presumed ancestral species or its modern derivative, Lisianthius skinneri (Sytsma and Schaal, 1985), is genetically depauperate in all three populations examined. Three of the cloud forest species, however, are highly polymorphic, even re-

sembling highly outcrossing species of North America in the amount of genetic variation they contain. This variation is correlated not only with population size and local extent, but also with more outcrossing types of pollination syndrome. Although all populations are self-compatible and apparently do undergo some amount of selfing, based on observations of pollinator movements (Sytsma, 1983), the three polymorphic cloud forest species exhibit flower and inflorescence forms that minimize selfing.

A major portion of the genetic variability within the species complex as detected by isozyme analysis occurs among populations and not within populations, despite the high level of within-population variation in some members of the group. These data, in conjunction with biogeographical distributions and breeding system analyses, support the hypoth- esis that most populations were initiated by founder events. The lack of self-incompatibility systems, the observed movement of pollinators between flowers within an individual, and the lack of active seed dispersal mechanisms (Syts- ma, 1983) would permit populations to derive from a single introduction. Fur- ther evidence that extant populations have grown from a preliminary "bottleneck" is the low average number of alleles per locus. Average number of alleles for the polymorphic populations is below the average for North American temperate selfing species. The loss of alleles rath-

FIG. 1. (A-C) Unrooted networks estimating relationships in the Lisianthius skinneri species complex based on Rogers' genetic divergence values. Networks A and B are the two networks obtained from the algorithm of Fitch and Margoliash (1967). Network C is derived from a DNA-based phylogenetic tree (Sytsma and Schaal, 1985) with its root indicated by a dashed line. Numbers are calculated divergence values for each respective branch. Percent standard deviations of each network from observed divergence data are A: 12.1; B: 12.1; and C: 15.8. (D-F) Unrooted Wagner networks estimating relationships in the Lisianthius skinneri species complex based on the presence or absence of 30 isozyme allelic states in each population. The three Wagner networks were derived as the three shortest networks utilizing the algorithm of Nelson and VanHorn (1975). Nodes identified by letters indicate constructed hypothetical taxonomic units. Values represent the number of evolutionary steps occurring along each branch. Total number of evolutionary steps and corresponding consistency index (Kluge and Farris, 1969) for each network are D: 34 (0.88); E: 35 (0.86); and F: 36 (0.83). Dashed lines indicate the position of the hypothetical root(s) for the Wagner networks based on the Lundberg (1972) algorithm. Networks D, E, and F are identical in topology to networks A, B, and C.








er than reduction in heterozygosity can be a primary result following the large population size reductions implicit in founder events (Nei et al., 1975; Tem- pleton, 1980a). Reduction in heterozygosity can be minimal even following an extreme bottleneck if population size in- creases rapidly after the bottleneck. Av- erage number of alleles per locus, however, is drastically reduced by bottlenecks. Lisianthius populations might well have exhibited such extreme founder bottlenecks.

Geographical isolation and the small area usually occupied by each population also imply that genetic drift may well be an important force in maintaining high levels of genetic variability between populations. All isozyme loci examined contain a large portion of their genetic diversity among populations rather than within populations. Many of the loci are totally monomorphic within populations but with populations fixed for one of several alternative alleles. Genetic identities are also very low for between-population comparisons. Even identity values between Lisianthius skinneri populations are low relative to average conspecific population identity values from North America. It is unlikely that the populations comprising the L. skinneri species complex are more ancient (and thus at different historical divergence levels) than their counterparts in North America. Considering the isolation of each population, their small effective sizes, and lack of self-incompatibility systems, high in- terpopulation variation is best explained by genetic drift operating on an already impoverished allelic diversity.

The theoretical work by Wright ( 1931, 1932, 1940) and later by Templeton (1980a, 1980b, 1981) has dealt with the interaction of genetic drift and selection in allowing founder or small populations to undergo transitions (involving either extensive "revolutions" or, more com- monly, only a few major loci) to new "adaptive peaks." The stochastic nature of this model as applied to the Lisian- thius skinneri species complex and the

interaction of drift with natural selection that must occur in the extreme cloud forest environment, could explain the spe- ciose pattern seen in this group.

ACKNOWLEDGMENTS This research was supported by Na-

tional Science Foundation Grant DEB- 8111312. Grateful acknowledgment is given to Kaius Helernum for suggestions concerning electrophoresis and to George Johnson, Peter Raven, Alan Templeton, and two anonymous reviewers for helpful criticisms on the manuscript.

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Corresponding Editor: J. C. Avise








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