comparative genetic structure between tropical colombian and north american drosophila pseudoobscura...

BlOTROPlCA 34(1): 81-92 2002

Comparative Genetic Structure between Tropical Colombian and North American Drosophila pseudoobscura Populations’

Diana Alvarez Unidad de Genetica (Grupo de Biologia Evolutiva, Laboratorio de Bioquimica, Biologia y Genetica Molecular de Poblaciones), Departamento de Biologia, Facultad de Ciencias, Pontificia Universidad Javeriana, CRA 7A No 43-82, Bogota DC, Colombia

Mohamed A. F. Noor Department of Biological Sciences, Life Sciences Building, Louisiana State University, Baton Rouge, Louisiana 70803, USA.

and

Manuel Ruiz-Garcia* Unidad de Genetica (Grupo de Biologia Evolutiva, Laboratorio de Bioquimica, Biologia y Genetica Molecular de Poblaciones), Departamento de Biologia, Facultad de Ciencias, Pontificia Universidad Javeriana, CRA 7A No 43-82, Bogota DC, Colombia

ABSTRACT Since the discovery of Drosophila pseudoobscura in the tropical highlands of the Colombian Andes during the 1960s, this population has been studied by many evolutionary biologists because of its geographical isolation from the main North American range of this species. We used five highly variable microsatellite loci (DPSX001, DPS2001, DPS3001, DPS3002, and DPS4001) to analyze the genetic structure of three Colombian populations and the genetic relationships with four North American populations. We found that the average heterozygosity was consistent among the three tropical Colombian populations (H = 0.665-0.675), but they had less variability than their North American coun- terparts. Nonetheless, the genic diversity found in the Colombian populations was higher than that found previously using other genetic markers. The average genic heterogeneity estimate among the Colombian populations (RST = 0.042), although statistically significant, was substantially lower than that found among the North American populations (RST = 0.088). We identified alleles in the Colombian populations not reported in North American populations, suggesting further divergence between the populations. We estimated that the populations on the two continents diverged ca 80,000 years ago, consistent with independent sequence analyses of these populations but contrary to some suggestions in the literature. Finally, we estimated an average effective population size of the Colombian populations to be on the order of 100,000.

Key word: Drosophila pseudoobscura; microsatrllite loci; North America; population genetics; tropical Colombia.

SINCE DOBZHANSKY ET AL.’s (1963) KARYOTYPIC

STUDY of a relict Drosophila psedobscura population on the tropical high plateau of the Colombian Andes near Bogoth, many evolutionary biologists have expressed an interest in this divergent population (Prakash et a/. 1969, Ayala & Dobzhansky 1974, Orr 1987, Schaeffer & Miller 1991, Wang et al. 1997). This population is geographically isolated from the primary range of D. pseudoobscura in North and Central America by almost 2400 km. Only two third-chromosomal arrangements, Santa Cruz and Treeline (SC and TL), are present in this population out of more than 50 rearrangements surveyed in the central distribution range of this species (Guzman et af. 1994), although several new

Received 28 June 2000; revision accepted 1 May 2001. Corresponding author. e-mail: mruiz@javercol.

javeriana.edu.co

chromosomal rearrangements in the Colombian populations have been discovered recently (Ruiz- Garcia et al. 2001). Furthermore, the males of this tropical relict population have only one of the four types of Y-chromosomes described by Dobzhansky (1937). Prakash et al. (1969) detected the loss of genetic variability in 24 allozyme loci in the Co- lombian population relative to North American populations. Further evidence of divergence was demonstrated by Ayala and Dobzhansky (1974), Singh et al. (1976), and Coyne and Felton (1977), who identified rare but unique alleles at the Xfh, Est-5, and Adh allozyme loci in the Colombian populations. Prakash (1972) also documented that hybrid males from crosses between Colombian females and North American males were sterile, and he suggested that the peripatric Colombian population was the result of a temporally recent genetic revolution (perhaps in the 1960s) following the model proposed by Mayr (1954); however, Dobz-

81

82 Alvarez, Noor, and Ruiz-Garcia

hansky (1 974) and Ayala and Dobzhansky (1 974) argued that the depauperate chromosomal arrange- ment number in the tropical population could suggest an ancient origin of more than one million years ago.

Recently, Schaeffer and Miller (1 99 1, 1992) estimated that the divergence time between the Co- lombian and the North American populations could be ca 155,000 and 500,000 years (with higher probability for the first estimate), analyzing sequences of the Adh and Adh-Dup genes. Adding credence to these estimates, Jenkins et al. (1996), studying the srRNA mitochondrial gene sequence, suggested that the divergence time between these populations was ca 109,000 years, although the results of this study were questioned by Noor and Larkin (2000). In contrast, Hoenigsberg (1986) suggested more extreme estimates of the antiquity of the Colombian population. First, he suggested that the Neotropical and U.S.A. population split may have a Pleistocene origin, with a divergence time ca 2-3 million years ago. Later, the same author claimed, "With such antecedents we feel rea- sonably certain that the high altitude Drosophikz psardoobscura postulated ancestor could have, with the proper evolutionary stimulus, adapted to the lower land 'milieu' prevalent in the Central Amer- ican Cordillera during the first part of the Tertiary. From then on, we can have a South American ver- sion of the North American Drosophikzpsardoobs- cura postulated ancestor." Therefore, this suggestion would date the divergence among the North American and the Colombian populations to be ca 40-70 million years ago. This suggestion was used (Hoenigsberg 1988) to extend an existing demic theory of speciation (Gilmor & Gregor 1939, Carson 1975). An unsubstantiated aspect claimed by this theory was that the diverse tropical D. pseudoobscura demes in Colombia were strongly isolated from each other by an absence of gene flow.

Here, we used five highly variable microsatellite loci to analyze (a) the antiquity of the Colombian D. pseudoobscura population and (b) levels of gene flow among demes of this population. To study the genetic structure of this species in the Colombian tropics, we analyzed variability in three Colombian populations and compared the data to a previous study of four D. pseudoobscura populations surveyed in the United States (Noor et al. 2000). Mi- crosatellites are widely distributed along the chromosomes of Drosophila species (Goldstein & Clark 1995, Michalakis & Veuille 1996). Indeed, Schug et al. (1998~) described 1289 microsatellite loci with di-, tri-, and tetranucleotide repeat motifii in

COLOMBIAN AMPLED AREA I. -Magdalena River

CJ

FIGURE 1. Drosophila pseudoobscura were sampled.

Map of the Colombian Andes where three

Drosophikz melanogaster, and these loci have been used to study population genetic structure and the effects of natural selection in different taxa (Schlot- terer et al. 1997, Schug et al. 1998a).

MATERIALS AND METHODS Throughout 1997, we intensively sampled three Colombian D. pseudoobscura populations: Toroba- rroso (04"5513N, 74'0151W; 2600 m elev.; average temperature 13.9"C; 39 km from Bogoti), Susa (05"2719N, 73'4901W; 2580 m elev.; average temperature 14°C; 120 km from Bogoti), and Su- tatausa (05"1505N, 73'5122R 2625 m elev.; average temperature 13°C; 88 km from Bogoti; Fig. 1). Flies were sampled from these localities weekly using fermented banana traps. Flies caught directly from nature were used for all microsatellite assays.

Thirty flies were used for each marker in the microsatellite analyses, hence giving a sample size of 60 chromosomes for autosomal markers. DNA extractions of each individual were done in 60 mM NaCI, 5 percent sucrose, and 1.25 percent SDS, and DNA was resuspended in 50 11.1 of TE buffer. PCR was executed with 2 p1 DNA, 2.5 mM MgC12, 1 rnM dNTPs, 0.5 pM of each primer, and 1 unit of Taq polymerase. The reaction was done in a Perkin-Elmer 9600 thermal cycler with an initial denaturation of 95°C for 5 minutes, fol-

Drosophila Population Genetic Structure 83

lowed by 40 cycles of 94°C for one minute, 60.5"C for one minute, 72°C for one minute, and a final extension of 72°C for five minutes. The annealing temperature was lowered to 58.2"C for the marker DPS3001. The products were visualized on 6 percent denaturanting polyacrylamide gels under the same conditions used for sequencing gels in a Hoeffer SQ3 Chamara Sequencer. Bands were re- vealed with silver staining. The gels were dried for one day, and genotypes were scored manually for each individual. Five microsatellite loci were scored: DPSXOOl, DPS2001, DPS3001, DPS3002, and DPS4001. All microsatellite markers employed had dinucleotide repeat motifs. Details of the isolation and genomic locations of these microsatellites are presented in Noor et al. 2000.

We compared our data regarding the Colom- bian populations with results obtained in four populations in the United States (Cheney, Washington; Goldendale, Washington; Abajo, Utah; and Flag- st&, Arizona) using the same molecular markers (Noor et al. 2000). The former two were from the northwestern part of D. psetldoobscura's North American range, and the latter two were from more southeastern parts of its range. Population genetic parameters were analyzed as follows:

(1) The expected average heterozygosity (genic diversity) for each population was calculated as: H = n(1 - 2 q2)/(n - I), where q is the ith allele frequency and n is the number of chromosomes analyzed.

(2) Wright's (1965) F-statistic was applied to determine whether there was deviation from Har- dy-Weinberg equilibrium expectations, at an individual population level and for the composite of the three Colombian populations studied. For each estimate of E the variance was estimated with Ras- mussen's (1 964) procedure.

(3) Estimates of the genetic heterogeneity and gene flow among Colombian or North American populations were obtained using Slatkin's (1995) RST diversity analysis.

(4) Matrices of Nei's (1 978) unbiased distance measure as well as the DA (Nei et al. 1983) and 6p2 (Goldstein et al. 1995) genetic distances were produced to compare pairs of these seven populations. From these matrices, UPGMA and neighbor- joining (Saitou & Nei 1987) hierarchical algo- rithms were applied with 1000 bootstrap replica- tions to determine support for the clusters found. Following the suggestion of Alan Templeton (pers. comm.), the cophenetic correlation was calculated for each tree to determine whether they contained branches that represent truly isolated populations.

Correlations below 0.8 would suggest the populations were interconnected to some extent by gene flow and therefore did not represent branches of a real evolutionary tree. A principal coordinates analysis (PCA) with the Gower (1966) procedure was also used to determine the genetic relationships among the seven populations studied. A minimum spanning tree was superimposed on the PCA to determine the distortion grade for the dimensional reduction. The use of these diverse genetic distance measures was motivated to analyze more precisely the phylogenetic relationships among the Colom- bian and North American D. pseudoobsnrra populations.

( 5 ) Finally, the effective sizes (N,) of the Co- lombian populations were calculated from the levels of heterozygosity (H) using the infinite alleles model, where N, = H/(4p(1 - H) Kimura 1964), as well as using the stepwise mutation model, where N, = 1 - (1 - H)2/(8p(1 - H2) (Ohta & Kimura 1973).

These estimates are long-term effective population sizes, which represent the harmonic mean of the N, values over all generations if those values change over time rather than the N, value of a population in a determinate moment. The estimated mutation rates per generation (p) for microsatellite loci with dinucleotide repeat motifs in D. mekanogaster is ca 9.3 X mutations per generation (Schug et al. 1998a,b), and D. pseu- doobrnrra is suggested to have a similar microsatellite mutation rate (Noor et al. 2000).

RESULTS Table 1 presents the allele frequencies observed for the five molecular markers employed and the average allele sizes by marker and by population. The three Colombian populations exhibited very similar average heterozygosities (genic diversity): H = 0.665-0.675. This value is substantially lower than that observed in the two Washington State populations of the northwestern United States: (Cheney, H = 0.904; Goldendale, H = 0.914) and slightly lower than the two southern U.S.A. populations (H = 0.710-0.763) (Noor et al. 2000).

Wright's F-statistics applied to the Colombian populations are presented in Table 2. We observed that only the DPSXOOl marker deviated strikingly from Hardy-Weinberg expectations (in females) within each of the three Colombian populations studied (Torobarroso: F = 0.794, x2 = 12.603, df = 3, P = 0.0056; Susa: F = 1.000, x2 = 11, df = 1, P = 0.0009; Sutatausa: F = 0.747, x2 =

TABLE 1. Microsatellite alkh fiqucncies and average sizes offive markers (DPS2001, DPS3001, DP.3002, DPS4001, and DPSXOOI) in three Colombian and f.ur U.S.A. Drosophila pseudoobscura populations.

B U.S.A. populations z

Abajo -2 Colombian populations Cheney Goldendale Flagstaff Alleles All Torobarroso Susa Sutatausa Alleles All (WA) (WA) (Az) (UT) z

DPS2001 176 pb

184 pb 186 pb 188 pb

0.137 0.146

0.242 0.188 0.379 0.458 0.137 0.042

193 pb 0.073 0.083 200 pb 0.016 0.042 201 pb 0.016 0.042

Average size (pb) 186.042 DPS3001

131 pb

133 pb

0.465 0.357

0.535 0.643

Average size (pb) 132.286 DPS3002

265 pb 0.029 0.067 270 pb 0.058 0.100

0.125

0.325 0.300 0.200

0.050

184.850

0.618

0.382

131.765

0.024 0.048

0.139

0.222 0.361 0.194

0.083

185.140

0.375

0.625

132.250

0.031

176 pb 180 pb 182 pb 184 pb 186 pb 188 pb 189 pb 190 pb 191 pb 192 pb

125 pb 127 pb 131 pb 132 pb 133 pb 135 pb 136 pb 137 pb 138 pb 139 pb

265 pb 270 pb 272 ob

0.067 0.022 0.067 0.378 0.289 0.089 0.022 0.022 0.022 0.022

0.044 0.022 0.044 0.022 0.022 0.133 0.044 0.622 0.022 0.022

0.025 0.025 0.050

0.167

0.333 0.250 0.083

0.083

0.083

184.667

0.111

0.333 0.333 0.111 0.111

135.667

0.083

0.077 0.077

0.308 0.385 0.077 0.077

184.539

0.071 0.143 0.071 0.071 0.143 0.143 0.357

134.357

0.143 0.167 P 0.286 0.167 P

0.333 3 E'

0.500 0.333 ? 0.143 0.167 P 0.286 0.167 P

0.333 3 E'

0.500 0.333 ?

0.071

184.786 185.333

0.067

0.067

0.867 1 .ooo

136.067 137.000

0.143 0.143 0.143

TABLE 1. Continued.

U.S.A. populations

Cheney Goldendale Flagstaff Abajo Alleles All Torobarroso Susa Sutatausa Alleles All (WA) (WA) (Az) (UT)

Colombian populations

Average size (pb) DPS4001

273 pb 0.125 0.033 0.143 0.188 274 pb 0.029 0.048 0.031 274 pb 0.025 0.067 276 pb 0.154 0.033 0.167 0.250 276 pb 0.050 0.067 0.143 277 pb 0.192 0.100 0.238 0.219 278 pb 0.279 0.300 0.286 0.250 278 pb 0.050 0.167 0.143 280 pb 0.058 0.200 280 pb 0.175 0.167 0.167 0.200 0.143 282 pb 0.067 0.167 0.048 282 pb 0.125 0.083 0.167 0.200

283 pb 0.025 0.083

286 pb 0.025 0.083 290 pb 0.025 0.083 291 pb 0.025 0.067 293 pb 0.050 0.067 0.143 295 pb 0.025 0.083 297 pb 0.025 0.067 299 pb 0.075 0.167 0.133 301 pb 0.025 0.067

284 pb 0.010 0.031 284 pb 0.175 0.333 0.333 0.067

277.067 276.024 276.160 283.667 284.500 286.667 276.286

P 261 pb 0.041 0.071 0.071 8 262 pb 0.020 0.071 3 263 pb 0.041 0.071 0.143 iii

B 0.020 0.071 E

268 pb 0.020 0.071 5 269 pb 0.020 0.071 3

272 pb 0.061 0.071 0.143 5 273 pb 0.082 0.214 0.071 2

274 pb 0.037 0.083 274 pb 0.071 0.071 0.286 2

277 pb 0.280 0.208 0.361 0.227 0.020

z 264 pb 0.020 0.071 266 pb 0.061 0.071 0.143 267 pb B

270 pb 0.020 0.143 271 pb 0.061 0.071 0.071 0.071 !z

2 275 pb 0.071 276 pb 0.171 0.167 0.139 0.227 0.082 $

a, VI

9 .z 2 TABLE 1. Continued.

i5 Cheney Goldendale Flagstaff Abajo -2

2 z F'

U.S.A. populations Colombian populations

Alleles All Torobarroso Susa Sutatausa Alleles All (WA) (WA) (a) (UT) n 279 pb 0.041 0.071 0.143

280 pb 0.012 0.028 280 pb 0.082 0.143 0.071 0.071 281 pb 0.159 0.083 0.056 0.409 281 pb 0.082 0.071 0.214

0.071 $ 282 pb 0.341 0.542 0.333 0.136 282 pb 0.020 283 pb 0.041 0.071 0.143 285 pb 0.082 0.071 0.143 0.143 286 pb 0.014 0.143 287 pb 0.020 0.071 290 pb 0.020 0.071

Average size (pb) 279.875 278.583 279.090 271.857 274.143 278.929 275.429 DPSXOOl

184 pb 0.045 0.143 186 pb 0.045 0.091 0.071

190 pb 0.023 0.071

192 pb 0.091 0.214 0.071 193 pb 0.023 0.071 194 pb 0.045 0.091 0.071 195 pb 0.023 0.091 196 pb 0.023 0.071 197 pb 0.068 0.214

200 pb 0.045 0.091 0.071

188 pb 0.182 0.273 0.143 0.143 0.200

191 pb 0.045 0.071 0.200

199 pb 0.277 0.345 0.242 0.250 199 pb 0.068 0.091 0.071 0.200

201 pb 0.691 0.621 0.727 0.719 201 pb 0.182 0.182 0.071 0.286 0.200 202 pb 0.068 0.091 0.071 0.200'

203 pb 0.032 0.034 0.030 0.031 205 pb 0.023 0.071

Average size (pb) 200.379 200.576 200.560 194.727 195.1429 193.429 196.200


TABLE 2. Hardy- Weinberg anaLysis of j h e microsatellite loci applied to three Colombian Drosophila pseudoobscura populations and the global Colombian population. H = expected beterozygosity; df = Agrees ofjeedom; P = probabilig.

H-W Marker H F variance x2 df P equilibrium

DPS2001 DPS3001 DPS3002 DPS4OO 1 DPSXOO 1

DPS2OO 1 DPS300 1 DPS3002 DPS4001 DPSXOOl

DPS2001 DPS3001 DPS3002 DPS4001 DPSXOOl

DPS2001 DPS3001 DPS3002 DPS4001 DPSXOOl

0.761 0.503 0.841 0.758 0.436

0.737 0.476 0.844 0.656 0.511

0.765 0.487 0.826 0.749 0.416

0.778 0.489 0.815 0.757 0.4 16

ALL COLOMBIAN POPULATIONS 0.0593 0.0027 1.309 0.1587 0.0233 1.083 0.4229 0.0021 83.685 0.2510 0.0049 12.914 0.8495 0.0161 44.744

0.0758 0.0069 0.828 0.0667 0.0714 0.062 0.5095 0.0095 27.261 0.2044 0.0278 1.504 0.7938 0.0500 12.603

TOROBARROSO

SUSA 0.129 0.0125 1.331 0.3773 0.0588 2.42 0.3502 0.0068 18.029 0.161 0.01 11 2.333 1 0.0909 11

-0.1020 0.0139 0.75 -0.2444 0.0833 0.717

0.3663 0.0104 12.883 0.1962 0.0278 1.385 0.7468 0.0500 11.155

SUSTATAUSA

21 1

45 15 3

21 1

28 6 3

10 1

28 15 1

10 1

21 6 3

NS 0.2980 0.0000 0.6089 0.0000

NS 0.8030 0.5041 0.9592 0.0056

NS 0.1198 0.9254

NS 0.0009

NS 0.3971 0.9127 0.9667 0.0109

11.155, df = 3, P = 0.0109). Our results showed that the average F was 0.33 2 0.079 in the Toro- barroso population, 0.403 ? 0.098 in Susa, and 0.192 t 0.123 in Sutatausa. When the three Co- lombian populations were pooled, two microsatellites (DPSX001 and DPS3002) failed to corre- spond with Hardy-Weinberg equilibrium expectations due to an excess of homozygotes. This het- erozygous deficit may have resulted from a Wahlund effect or by the presence of null alleles.

The average genic heterogeneity (RsT) among the Colombian populations (RST = 0.042; x2 = 114.083, 46 df, P < 0.001), although statistically significant, was substantially lower than that found among the North American populations ( R ~ T = 0.088; x2 = 601.33, df = 216, P < 0.001). We confirmed this difference with a Fisher-Snedecor F- test comparing both heterogeneities (F&, 46 = 5.27, P < 0.0005). Therefore, the Colombian populations are substantially more homogeneous than the North American ones for the markers consid- ered. The DPS3002, DPS4001, and DPSXOOl microsatellite loci showed significant differentiation among the Colombian populations (Table 3),

whereas the DPS2001 and the DPS3001 loci were less differentiated (range: RST = 0.010 for DPS2001 through RST = 0.081 for DPSX001). In contrast, within the U.S.A. populations, all rnicro- satellite loci were significantly differentiated (range: RST = 0.058 for DPS3001 through RST = 0.219 for DPS3002). If we assume a neutral dynamic for these markers, the gene flow estimate (Nm) among the tropical Colombian populations (Nm = 5.6) was higher than that among the North American populations (Nm = 2.59), consistent with their greater geographic proximity. Comparing the com- bined Colombian and North American populations, genetic differentiation was much greater (overd RST = 0.165; range: 0.055 [DPS3001]- 0.398 [DPS3002]).

We identified several alleles in the Colombian populations that were not sampled from the North American populations (private alleles). The DPS2001 marker possessed three alleles in the Co- lombian populations that were larger in size than all those detected in the U.S.A. populations (193, 200, and 201 bp). DPS3001 possessed two alleles in Colombian populations, and neither was de-


TABLE 3. Overall genetic diversity (= heterozygosity) and Slatkin? (1995) diversity ana&is in three Colombian andfDur U.S.A. Drosophila pseudoobscura populations. Nm = gene flow estimates; Rsr = relative gene daferentiation among the populations; HT = total genetic diversity; HS = average genetic diversity in the subpopulations.

CHENEY GOLDENDALE FLAGSTAFF ABAJO

Average heterozygosity RST HT HS

COLOMBIAN POPULATIONS Nm = 5.6 TOROBARROSO 0.6655 ? 0.0711 DPSXOOl 0.0101 0.4464 0.44 19 SUSA 0.6672 2 0.0836 DPS2001 0.0186 0.7554 0.7413 SUTATAUSA 0.6750 ? 0.0828 DPS300 1 0.0572 0.4950 0.4666

DPS3002 0.0388 0.8358 0.8033 DPS4OO 1 0.0810 0.7500 0.6892 ALL 0.0426 0.6565 0.6285

NORTH AMERICAN POPULATIONS Nm = 2.89 0.9042 ? 0.0282 DPSXOO 1 0.0627 0.8927 0.8367 0.9140 ? 0.0385 DPS2OO 1 0.0580 0.7633 0.7190 0.7637 ? 0.1327 DPS300 1 0.2190 0.5686 0.4441 0.7101 ? 0.1791 DPS3002 0.0718 0.8957 0.8314

DPS4001 0.0731 0.9412 0.8724 ALL 0.0881 0.8123 0.7400

COLOMBIAN-NORTH AMERICAN POPULATIONS DPSXOOl 0.1532 0.7883 0.6675 DPS2OO 1 0.0549 0.7710 0.7286 DPS300 1 0.3979 0.7537 0.4537 DPS3002 0.0980 0.9085 0.8 194 DPS4001 0.1405 0.9237 0.7939 ALL 0.1645 0.829 0.6926

tected in the North American populations. Private alleles of DPSXOOl and DPS4001 were also detected in Colombian populations (Table 1).

The UPGMA tree derived from Nei’s (1978) genetic distance (Fig. 2a) yielded a bootstrap of 100 percent, suggesting unambiguous differentiation between the Colombian and the North American

TOROBARRQSO USA UTATAUSA

B

FLAGSTAFF ABAJO

OROBARROSO

UTATAUSA

FIGURE 2. Two different dendrograms showing the genetic relationships among seven Drosophila pseudoobscura populations (three from Colombia and four from the United States). (A) UPGMA algorithm with the DA genetic distance. (B) UPGMA algorithm with Nei’s (1978) genetic distance.

populations. This bootstrap value was substantially stronger than that identified by Jenkins et al. (1996) between the Colombian and North Amer- ican populations using sequences of the srRIVA mitochondrial gene (cf Noor & Larkin 2000). Among the North American populations, the northwestern populations were significantly divergent (97% bootstrap) from the more southeastern populations (Noor et al. 2000).

Among the Colombian sites, the two neigh- boring populations (Susa and Sutatausa) were ge- netically more similar to each other than either was to Torobarroso, consistent with an isolation-by-distance model. The same relationships were detected when using the DA distance (Fig. 2b), also consistent with isolation-by-distance within each main array. In addition, the cophenetic correlation coefficients were higher than 0.96, evidence that these trees contained branches representing truly isolated populations. The PCA also showed clear differenc- es between the North American and Colombian populations (Fig. 3).

We calculated the divergence time among the Colombian populations and between the Colom- bian and North American populations throughout the 6k2 genetic distance, assuming that the D. pseudoobscura generation time is ca 20 days in nature. As such, we estimated that the Colombian


0.520 NEI

LAGSTAFF SUTATAUSA

0 0.080- z

v) TOROBARROSO ““1 0 .am G E Y I I I , Om 0 S W 0 4 0 0 O W O M 0 O l m 0402 O W O S W

FIRSTCOORDENATE

1 4 . m

6.4W -4

CAVAWSFORZA

UTATAUSA

LAGSTAFF

I A B N O

-8.m 6.m -4.m .z.m 0 . m 4 . m 4 . m 6.m n . m FIRST COORDENATE

6 4

BNO 6.m CHENEY

7 f

-4,-i / I -W.W -1s.m .LO.mO -5.W 0- S.wO lO.aX, ILmO W.mO

FIRSTCOORDENATE

FIGURE 3. Principal coordinates analysis with Gower’s (1966) procedure using three different genetic distances and with a minimum spanning tree superimposed. (A) With Nei‘s (1 978) genetic distance. (B) With the Cavalli- Sforza and Edwards chord genetic distance. (C) With the &* genetic distance.

and North American populations diverged 75,000 ? 27,000 years ago. If we exclude the Abajo population (for which the sample size was smaller), this estimate would be 88,700 ? 12,500 years. In contrast, the divergence among the Colombian popu-

lations appeared very recent (1447 5 1 149 yr). As our cophenetic correlation coefficients of the trees obtained were higher than 0.8 in all the cases, the divergence time estimates could represent real divergence times. The estimates obtained among the Colombian populations, however, could represent “effective divergence times” rather “real divergence times” because the possibility of internal gene flow among the Colombian populations cannot be ex- cluded. Using an infinite alleles model, we calculated that the average effective population size for the Colombian populations to be between 54,405 and 77,841 flies, whereas with the stepwise mutational model these values ranged from 109,461 to 156,6 13 flies.

DISCUSSION GENETIC VAWILTY IN COLOMBLW POPULATIONS.-

We surveyed the patterns of variability at five microsatellites in three Colombian populations of D. pseudoobscura and compared the results with published studies of North American populations of this species. We found that the Colombian populations exhibited significantly less genetic diversity than North American populations, consistent with a founder effect (or several bottleneck events) in the origin of the Colombian population. This suggestion is in agreement with previous studies by Schaeffer and Miller (1991) and Jenkins et al. (1996), who found more nucleotide diversity for the Adh and srRNA gene in North American populations than in Colombian ones. Nevertheless, the loss of variability is not as extreme as reported for several isoenzyme markers (Prakash etal. 1969, Or- doiiez et al. 1993). In fact, the observed heterozygosities of the three Colombian populations studied were much higher than those found in an Aus- tralian D. melanogarter population (H = 0.39; England et al. 1996), or in a D. melanogarter population collected in Maryland, U.S.A. (H = 0.38; Schug et al. 1997).

GENE FLOW AMONG COLOMBIAN D. PSEUDOOBSCURA roruLATroNs.-Previously, other authors such as Sokoloff (1965), from a morphological perspective, and Keith (1983), Keith et al. (1985), Riley et al. (1989), and Schaeffer and Miller (1992), from a molecular perspective, showed strong genetic homogeneity among the North American populations of this species. These authors invoked the existence of high rates of gene flow between the populations of this central range. Comparatively, our results suggest that the gene flow among Colombian pop-


ulations should be even higher than that found in the North American populations. Furthermore, in the adverse environments analyzed in North Amer- ica, which were somewhat similar to deforestation conditions found in the Colombian localities studied, individual D. pseudoobscura can move more than 10 km Uones et af. 1981; Coyne et af. 1982, 1987). Our result questions a proposed demic structure of this species in Colombia (Hoenigsberg 1986, 1988).

EVOLUTION OF MICROSATELLITE ALLELE SIZE IN CO- LOMBIAN PoPmTioNs.-Except for one locus, the Colombian D. pseudoobscura populations did not possess alleles outside of the size distribution found in D. pseudoobscura from North America. This sug- gests that the divergence between these taxa did not occur millions of years ago (Goldstein et al. 1995), in contrast to some suggestions (Hoenigsberg 1986). The finding of new alleles in the Colom- bian populations is consistent with allozyrne studies by Singh (1983), who also detected unique alleles for the Colombian populations. It is also possible that some selective constraints maintained the current allele sizes in the Colombian populations (Garza et al. 1995). Nonetheless, we acknowledge that without sequencing all of these alleles, we cannot determine whether the unique size polymorphisms that we observed resulted from length variation within the microsatellite or within the flank- ing regions. This does not, however, affect our con- clusions about levels of genetic variability or gene flow.

DIVERGENCE TIME BETWEEN COLOMBIAN AND NORTH AMERICAN PoPuLATIoNs.-Divergence time estimates between the Colombian and the North American D. pseudoobscura populations, which we obtained by using five microsatellite loci, were similar to those obtained by Schaeffer and Miller (1991) and Jenkins et al. (1996): 75,000-87,500 years since separation. Schaeffer and Miller (1991), using sequences from the nuclear A& gene and with a 1.7 percent nucleotide substitution rate per million years, estimated a divergence between the Colombian and North American populations of 155,000 years, with a less probable maximum of 500,000 years. Jenkins et al. (1996) estimated, using a substitution rate of 3.2 percent per million years for the srRNA mitochondrial gene, a separation between the Colombian and the Mexican populations of 109,375 years (with 1 SE limits of 87,500 and 131,250 yr). Schaeffer (in Jenkins et al. 1996), by using the 3.2 percent criterion, short-

ened his original estimate to 77,000 years ago. In this way, our independent result agrees quite well with these published results, and these recent find- ings refute the suggestions that the Bogota population could be the product of a very recent colonization (Prakash 1972) or an ancient colonization millions of years ago (Hoenigsberg 1986).

EFFECTIVE POPULATION SIZES IN COLOMBIAN POPULA-

moNs.--In North America, the estimated effective size of D. pseudoobscura populations is ca 4.5 X lo6 (Schaeffer 1995). We have estimated that the Colombian populations have a smaller population size on the order of 54,000 to 157,000, depending on the mutational model used. This finding dis- agrees with that reported by Ordofiez et al. (1993), who claimed that the effective size of the Colom- bian populations was closer to 3 flies per population. Additionally, these authors inadequately used certain population genetic equations to calculate the effective numbers of these populations. Our new values for D. pseudoobscura are only slightly lower than those found by Schug et al. (1998b) for D. melanogaster in a U.S.A. population (65,512 and 216,819 flies for the mutational infinite alleles model and for the stepwise model, respectively) and in an African population (80,823 and 328,278 flies, respectively). These authors also estimated the effective numbers for five D. melanogaster populations from throughout the world obtaining a value of 530,466 flies, although other authors had previously obtained higher values (N, = 3.3 x lo6; Kreitman 1983). The real values of the effective numbers could be intermediate to the extreme values obtained throughout of the mutational models employed (DiRienzo et al. 1994). Note that our effective size values represent overall estimations from a long-term perspective during the history of the Colombian D. pseudoobscura populations rather than momentary and current census numbers. It may prove very informative to study the same microsatellite loci in more North American and Co- lombian populations as well as Mexican and Gua- temalan populations, which may prove to be the origin of the Colombian populations.

ACKNOWLEDGMENTS

The authors heartily thank the dean of the Faculty of Sciences from the Pontificia Universidad Javeriana for help and support. This study was supported in part by grant No. 980024 to D. AIvara and the Banco de la Repliblica (Colombia).


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comparative genetic structure between tropical colombian and north american drosophila pseudoobscura...

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