Arts. Sci. 13:1-180. BROOKS, J. L., and S. I. DODSON. 1965. Predation, body size and composition .... of the Pomeranian Lakeland by hybridization in the past.
Interspecific Hybridization in Daphnia: Distinction and Origin of Hybrid Matrilines’ Klaus Sch wed Zoologisches
Institut,
J. W. Goethe-Universitat
Three coexisting Duphnia species belonging to the D. longispina group (D. galeuta, complexes by producing interD. hyulina, and D. cucullata) form species-hybrid specific hybrids in several lakes in Germany and The Netherlands. To evaluate the genetic consequences of interspecific hybridization, I studied the patterns of mitochondrial DNA (mtDNA) sequence variation. The directionality of interspecific hybridization and divergence of hybrids from parental species was tested, using the DNA sequences of a segment of mtDNA. Via the polymerase chain reaction, it was possible to investigate single animals and even single resting eggs. A speciesspecific marker was established, using restriction patterns of amplified cytochrome b segments. mtDNA genotypes of hybrids revealed unidirectional mitochondrial gene flow for two hybrids, which were investigated by using multiple clones. No evidence for introgression of mtDNA was found. On the basis of a phylogenetic analysis, the species exhibit considerable distinctness, whereas differences between clones within species and between hybrids and maternal species tend to be very low. These results indicate a recent origin of hybrids and suggest that the radiation of the D. longispina group occurred >5 Mya.
Introduction Information on interspecific hybridization among animals is largely based on studies of terrestrial organisms (e.g., , see Barton and Hewitt 1985; Hewitt 1988; Harrison 1990; Grant and Grant 1992). In comparison, the occurrence and consequences of hybridization among marine or freshwater species has received only limited attention (e.g., see Bert and Harrison 1988; Forbes and Allendorf 199 1; Avise et al. 1992). In the past decade, however, several species-hybrid complexes have been described in ecologically dominant components of freshwater zooplankton (Shan and Frey 1983; Hebert 1985; Wolf and Mort 1986; Hann 1987; Lieder 1987; Taylor and Hebert 1992). In particular, cladocerans of the genus Daphnia are an intriguing choice for investigation of the evolutionary genetics of interspecific hybridization, primarily because of their ability to reproduce either sexually or asexually via ameiotic parthenogenesis (Hebert and Ward 1972). This capability may promote speciation processes (Lynch 1985), because hybrids can (by reproducing parthenogenetically) circumvent deleterious effects of reduced sexual fertility (known as hybrid breakdown). Previous allozyme studies have revealed both interspecific hybridization in the D. longispina group (D. galeata, D. hyalina, and D. cucullata) as well as fluctuating seasonal abundances of hybrid and parental clones (Wolf and Mort 1986; Gieljler 1. Key words: interspecific hybridization, Daphnia, mitochondtial
DNA, PCR-RFLP,
gispina group.
Address for correspondence Limnology,
Rijksstraatweg
and reprints: Klaus Schwenk, Netherlands 6, 363 1 AC Nieuwersluis, The Netherlands.
Institute
Daphnia lon-
of Ecology, Centre for
Mol. Bid. Evol. lO(6): 1289- 1302. 1993. 0 1993 by The University of Chicago. All rights reserved. 0737-4038/93/1006-0011$02.00
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1987; Wolf 1987; Hebert et al. 1989). Compared with parental taxa, hybrids are generally intermediate in morphology (Fliil3ner and Kraus 1986; fig. 1) and ecology (Weider and Wolf 199 1; Weider and Stich 1992; Weider 1993). The phenotypic differences between species and hybrids may affect their susceptibility to predation (Brooks and Dodson 1965; Gliwicz 1990; Spitze 1992). Life-history traits, such as fecundity and resource utilization, show marked differences among Daphnia species and their hybrids (Mort et al. 1989; Mort 1990; Weider and Wolf 199 1; Weider 1993). Furthermore, the hybrids of D. cucullata and D. galeata seem to combine advantageous traits from both parental species, i.e., a relatively low predation risk by fish because of small size (from D. cucullata) and a relatively high reproductive rate (from D. galeata) (P. Spaak, personal communication). These morphological and life-history differences could contribute to differences in the response to selection among species and hybrids and could thus help to elucidate the ultimate causes of temporal (seasonal) and spatial heterogeneity of parental and hybrid abundances. In contrast with the wealth of ecological data (for review, see Peters and De Bernardi 1987), only limited information about the processes influencing the genetic
FIG. 1.- Daphnia longispina species complex. from Wolf and Mort 1986).
Typical phenotypes
of species and hybrid forms (redrawn
Interspecific
Hybridization
in Duphnia
129 1
structure of Daphnia populations in large lakes is available. Recent investigations suggest that interspecific hybridization and diapause are potential factors that affect population structure (Wolf and Mort 1986; Mart 199 1). Allozyme studies revealed hybrid clones that were produced via either backcrossing or sexual reproduction of hybrids (Hebert et al. 1989; Taylor and Hebert 1992), but the limited number of species-specific alleles prevented discrimination between the two potential processes. However, as long as the identification of backcrosses and F2 hybrids is barely feasible and the breeding system (obligate or cyclic parthenogenesis) is not verified, the actual extent of introgression remains largely unknown. To evaluate the mechanisms and phylogenetic implications of interspecific hybridization, I investigated interspecific hybrids in the D. Zongispina species complex by using molecular methods. I addressed the question of directionality of interspecific hybridization (determination of maternal species), and I estimated the evolutionary age of hybrid clones: Are hybrids more or less continuously produced, or are they maintained by ameiotic parthenogenesis after one or a few initial hybridization events in the past? These problems were investigated by both employing a molecular mitochondrial marker and taking advantage of the maternal inheritance of mitochondrial DNA (mtDNA) in animals (Harrison 1989). Standard mtDNA restriction fragment analyses using Southern blot techniques are not suitable for screening large numbers of samples in species with minute body size and those that are difficult to rear in the laboratory. New technical advances such as the polymerase chain reaction (PCR) facilitate genetic analyses by using unpurified mtDNA from nanogram samples (Kocher et al. 1989). The lack of DNA sequence information on Daphnia mitochondrial genes caused us to use universal primers to amplify a segment of mtDNA (Kocher et al. 1989). A segment of the cytochrome b gene has been selected because of its demonstrated usefulness as a phylogenetic and species-specific marker (Kocher et al. 1989; Meyer et al. 1990; Crozier et al. 199 1). The first application of this genetic analysis to Daphnia individuals and single resting eggs enabled us to elucidate the maternal species of any given hybrid clone. b gene that Furthermore, we present sequence data of a segment of the cytochrome were used to estimate sequence divergence between hybrids and parental species and to reconstruct phylogenetic relationships in the genus Daphnia. Material and Methods Specimens Three different Daphnia species (D. galeata, D. hyalina, and D. cucullata) and their hybrids were collected from several lakes in Holstein, northern Germany (Edebergsee, Hoftsee, Kellersee, Schohsee, and Stocksee). Starting with single parthenogenetic females, clones were reared in the laboratory. Other clones investigated were obtained from The Netherlands (Tjeukemeer) and other localities in Germany (Lake Constance, Meerfelder Maar, Hegbachsee, and Neuhof). These samples, preserved in 70% ethanol, were sent to our laboratory, and their species were determined by using morphological characteristics and allozyme electrophoresis. Allozyme
Electrophoresis
The clones were identified by using morphological characters (Flol3ner and Kraus 1986) and informative allozyme loci (Wolf and Mort 1986). Each clone was assayed electrophoretically for two enzymes [aldehyde oxidase (Ao), E.C. 1.2.3.1; and glutamate
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oxaloacetate transferase (Got), E.C.2.6.1.11. The enzymes were stained on cellulose acetate gels (Hebert and Beaton 1989, pp. 5-9), using a Tris-glycine buffer (3 g Trisma Base/liter, 14.4 g glycine/liter). On the basis of previous allozyme studies (Wolf and Mort 1986; Wolf 1987), D. hyalina species are known to be fixed for the s allele, D. galeata species are fixed for the f allele, and D. cucullata species are fixed for the sallele, at the Got locus. In addition, D. hyalina species are fixed for the s allele, at the Ao locus, whereas the D. galeata and D. cucullata species are fixed for the f allele. F1 hybrids are marked distinctly by heterozygous electromorphs at these loci (table 1). The species-specific alleles at the allozyme loci Got and Ao enabled discrimination of the species and hybrids. PCR Amplification
and Sequencing
Total genomic DNA was isolated from single animals by using standard procedures, for both living animals and ethanol-preserved specimens (methods available from the author on request). This DNA preparation (nuclear and mtDNA, - lo-20 ng/animal) was used for a single PCR (Saiki et al. 1988; White et al. 1989). To amplify b gene, the conserved primers H 15 149 a segment of the mitochondrial cytochrome 5’-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3’ and L 1484 1 5’AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3’ (Kocher et al. 1989) were chosen. The target segment was amplified by using 1 U Vent polymerase (New England Biolabs; NEB) 1 U of 7.5 mM MgS04, 10 X AMP buffer (NEB), 0.5 PM of each dNTP, 0.1 pg bovine serum albumin/pi, and 0.8 PM of each primer/reaction (reaction vol = 50 ~1). Each cycle of the PCR consisted of denaturation for 1 min at 93OC, annealing for 1 min at 5O”C, and extension for 2 min at 72°C. This cycle was repeated 45 times (IHB Thermal Reactor; Hybaid). b segment, starting with single It was also possible to amplify the cytochrome resting eggs obtained from an ephippium (thick-shelled dormant eggs). Adult individuals of laboratory cultures that produced resting eggs were identified by using morphological traits and allozyme analysis. Released ephippia were opened, and single
Table 1 Allozyme and mtDNA Patterns of 46 Daphnia Clones ALLOZYME LOCI TAXA D. D. D. D. D. D.
galeata hyalina . . . . cucuilata . galeata X hyalina cucullata X galeata cucullata X hyalina
mtDNA
GENOTYPES @p)
N”
Got
Ao
Tag1
14(2) 11 (1) 6 (2) 9 (1) 5(l) 1
ff ss s-ssf s-f ...
ff ss
199/150/27 Uncut Uncut 199/l 50127 Uncut Uncut
‘sf ... ...
DdeI Uncut 222/99/21/18/16 222/99/21/18/16 Uncut 222/99/21/18/16 222/99/21/18/16
Al241 Uncut Uncut 252/l 1816 Uncut 252/l 1816 252/l 1816
‘N = no. of investigated clones. Nos. in parentheses are no. of clones from which single resting eggs were used for PCR. b The mtDNA genotypes are characterized by three discriminating restriction enzymes (Tuql, DdeI, and AM). Restrictionfragment patterns are given in base pairs. Uncut segments had a length of 376 bp (307-bp cytochrome b + 69-bp primers).
Interspecific
Hybridization
in Daphnia
1293
resting eggs were removed. Eggs were homogenized (10 mM Tris-HCl, pH 8.3, 0.1 mg gelatine/ml, 0.45% Tergitol NP-40, 0.45% Tween 20, 0.06 I.tg proteinase K&l) and incubated for 2-3 h at 5O”C, and proteinase K was denatured at 95°C for 10 min. The homogenates (each 20-30 ~1) were subsequently used for the amplification reaction. Nine amplification products representing at least one clone of each species-hybrid group were sequenced. The amplified products were cloned into the plasmid pUC 18 and were sequenced by using the Sequenase kit (USB). Sequence analysis was performed with the program PC Gene (IntelliGenetics) by using the EMBL sequence database. Sequence divergence and parsimony trees were estimated by the programs DNABOOT and DNADIST (PHYLIP, version 3.3; Felsenstein 1985). The sequence information was used to find restriction enzymes that differentially cut the amplified products of the three species. Three discriminating restriction enzymes (TaqI, AluI, and DdeI) were found and applied to the PCR products [digests as recommended by the suppliers (Boehringer Mannheim and NEB)]. DNA was electrophoresed in 2% and 3% 1 X TAE (Tris-acetic acid-EDTA) agarose gels (SeaKem LE agarose; FMC) and was visualized after staining with ethidium bromide. Results Directionality
of Hybridization
The PCR-restriction-fragment-length polymorphism analysis revealed speciesspecific restriction patterns for Daphnia galeata, D. hyalina, and D. cucullata (table 1). These patterns did not vary within species, regardless of geographic origin of clones (northern Germany, The Netherlands, or southern Germany). For example, the cytochrome b segment amplified from D. galeata was cut by the restriction enzyme TaqI into three fragments (199, 150, and 27 bp), whereas the cytochrome b segment of D. hyalina was not cut by TaqI. To test whether the uncut segments occurred because of (a) insufficient restriction conditions or (b) the expected absence of restriction sites, a control cut was performed. A second restriction enzyme (DdeI), which cuts the D. hyalina segment but not the D. galeata segment (as predicted from sequence information), was applied. In all cases these tests confirmed the results of prior restriction cuts (table 1). Without exception, restriction patterns of hybrid clones were identical to the species-specific pattern of one of the parental species. For instance, all hybrids between D. galeata and D. hyalina exhibited the same restriction pattern as D. galeata ( TaqI cut: 199, 150, and 27 bp) . Thus, D. galeata can be inferred to be the maternal species of D. galeata X hyalina hybrids. All hybrids between D. cucullata and D. galeata showed the same pattern as D. cucullata. These results imply unidirectional hybridization for any given species pair in this complex. Because of the low abundance of D. cucullata X hyalina clones in nature, we could investigate only one individual, which proved to have a restriction pattern identical to that of D. cucullata. Comparison of restriction patterns of the amplified partial sequence of the cytochrome b gene from adults and single eggs of ephippia showed the same speciesspecific patterns (table 1). This was checked by analyzing at least one egg of each species and hybrid except the D. cucullata X D. hyalina hybrid clone. The direct amplification of cytochrome b (without DNA extraction) from single resting eggs revealed no negative effects for the PCR and yielded a sufficient amount of amplified DNA to perform restriction digests.
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Schwenk
Origin of Hybrids Nine individuals of different Daphnia species and hybrids were analyzed by comparing sequence information of a part of the cytochrome b gene (fig. 2). These amplified segments were 307 bp in length (with primers, 376 bp in length), which is in agreement with data from other animal taxa (e.g., see Kocher et al. 1989). As expected, for mitochondrial protein-coding genes, a significantly higher number of transitions (most of them at synonymous sites) than transversions were found (table 2). In contrast, among the more distantly related taxa (D. magna vs. clones of the D. longispina group) the transversions were more numerous (38-40) than they were between species of the D. longispina group (6- 10). The outgroup D. magna (subgenus Ctenodaphnia) differs significantly from all other species investigated (26%-27% sequence divergence). The sequence divergence within members of the D. longispina group (D. galeata, D. hyalina, and D. cucullata) is 11%-l 5% but among clones is only 0.3%-0.65%. For example, two D. galeata clones from different locations (Kellersee and Schiihsee) differed by only one nucleotide substitution. e The same magnitude of sequence divergence (0%-0.6%) characterizes the hybrids D. galeata X hyalina, D. cucullata X galeata, D. cucullata X hyalina, and their respective maternal species. A more detailed analysis of the DNA sequences by using a twoparameter model of nucleotide substitutions (Kimura 1980) leads to the same conclusion (fig. 3). The highest values (0.339-0.354) were generated by the comparison of D. magna to each clone of the D. longispina group, and intermediate values (0.1200.167) were produced by a comparison within the D. longispina group. The lowest values (o-0.006) were obtained by comparing within species and hybrids versus maternal species. To visualize the phylogenetic relationships within the genus Daphnia, an evolutionary tree was generated by using a bootstrap method based on the parsimony approach (DNABOOT, PHYLIP, version 3.3; Felsenstein 1985). The major groups were separated significantly ( 100 times in 100 replicates), exclusive of the branch of D. hyalina and D. galeata/cucullata. This branch was generated 60 times in 100 replicates, by bootstrap analysis ( fig. 4). All sequences of the D. longispina group were derived from clones sampled from two lakes near Plan (northern Germany), Kellersee and Schiihsee. The sole exception was the D. cucullata X hyalina hybrid clone, D. ch (M), which was collected from a maar lake of volcanic origin in the Eifel mountains (Meerfelder Maar). The cytochrome b sequence of this hybrid, despite the different geographic origin, showed no higher divergence than other members of the D. cucullata group. Discussion Evidence for the prevalence of unidirectional hybridization in the Daphnia longispina species complex is presented-namely, that D. galeata and D. cucullata are always the females that pass on their mtDNA to the D. galeata X hyalina and D. cucullata X galeata hybrids, respectively. These results suggest that unidirectional hybridization is a general pattern in this species complex, as has been documented for several other hybridizing taxa, e.g., fishes and lizards (Avise et al. 1992; McGowan and Davidson 1992), frogs (Lamb and Avise 1986), insects (Harrison et al. 1987; Sperling and Spence 199 1 ), and marine crabs (Bert and Harrison 1988). Although our data suggest unidirectional hybridization in Daphnia, it might be argued that, because of the limited sample size, we did not find the hybrids that may have been
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FIG. 2.-DNA sequences of a 307-bp segment of the cytochrome b gene in Daphnia mtDNA (base-pair positions 14842-15148, referring to the mitochondrial genome; Anderson et al. 198 1). Species abbreviations are as follows: D.g = D. galeata; D.h = D. hyalina; D.c = D. cucullata; D.cg = D.ch = D. cucullata X hyalina; D.gh = D. gakata X hyalina; and D.m = D. magna. The lake of origin is given in parentheses,as follows: S = Schiihsee; = Meerfelder Maar; and F = pond of the botanical garden of Frankfurt am Main. Dots indicate sequence identity with D. galeata (S) mtDNA. The D. was obtained by sequencing the amplified product of one resting egg. The boldface letters above the nucleotide sequence represent the amino acid sequence the Drosophila mitochondrial code (Clary and Wolstenholme 1985). Underlined letters represent recognition sites of restriction enzymes TaqI (TCGA), DdeI (CTNAG).
1296
Schwenk
Table 2 Numbers of Transitional Nucleotide Differences
Daphnia galeata
D. galeata
D. galeata X hyalina
(S)
(K)
(K)
2
3 1
Taxa (lake) D. D. D. D. D. D.
galeala (S) galeata (K) galeala X hyalina (K) cucullata (S) cucullata (K) cucullata X galeata (K) D. cucullata X hyalina (M) D. hyalina (K) D. magna (F) NOTE.-Lake comparisons.
(above the Diagonal) and Transversional
abbreviations
0
(below the Diagonal)
D. cucullata
D. cuciillata X galeata
D. cucullata X hyalina
D. hyalina
D. magna
(9
(K)
(K)
(M)
(K)
(F)
31 29 30
30 28 29 1
30 28 29
29 29 30 3 2
31 29 30 36 35
44 44 43 41 40
2
35
40
37
41 43
D. cucullata
0 6 6
0 6 6
6 6
0
6
6
6
0
0
6 8 38
6 8 38
6 8 38
0 10 40
0 10 40
1 0
0 10 40
are as in, fig. 2. Nos. in boldface type refer to interclonal
10 40
40
and hybrid vs. maternal
species
produced via the alternative direction. When bidirectional hybridization was assumed, the probabilities of sampling only one of two possible combinations were 0.06 (0.5 4, for D. cucullata X galeata and 0.004 (0.5 *) for D. galeata X hyalina hybrids. Further investigations with a larger sample from geographically distant locations are necessary to test whether these first results can be generalized. However, the data presented do suggest the existence of premating barriers to bidirectional gene flow or the potential inviability or low fitness of such hybrids (if produced). The allozyme study of Wolf and Mort ( 1986) reported low (~9%) frequencies of backcross and F2 individuals in the Daphnia species complex of northern Germany. Because the methodology was limited to only two fixed loci (Got and Ao) for three species, an accurate estimation of backcross frequency seems unlikely. Moreover, using Got as a marker locus leads to an overestimation of hybrid frequency, because the alleles s (D. hyalina) and f (D. galeata) are not 100% fixed in these parental species (S. Giessler, personal communication). In addition, the availability of only two marker loci allows no reliable discrimination of backcross and F2 hybrid individuals. However, if gene flow is exclusively unidirectional, and if backcross individuals are produced, then asymmetrical introgression of mtDNA is to be expected (Harrison et al. 1987; Aubert and Solignac 1990). Therefore, such a process would generate individuals containing the characteristic phenotype and nuclear genotype of one species but the mitochondrial genotype of the other species. In such a case we should observe individuals that, in terms of their’phenotype and nuclear genes, are D. hyalina but that bear the mitochondrial genotype of D. galeata. None of the investigated clones exhibits such a combination of nuclear and mitochondrial genotypes. Furthermore, the sequence divergence of the cytochrome b segment, between D. galeata, D. hyalina, and D. cucullata, was, on average, 12.5% f 1.5%, a rather high value compared with sequence divergence within the family Drosophilidae (7%-9%; Garesse 1988). The marked genetic separation of Daphnia species, as well as the apparent absence of
Interspecific
Hybridization
in Daphnia
1297
0.35
0.3 % 5
025
2. 9 5 E F:
0.2
0.15
s 0'
0.1
2 0.05
0
I
D. magna versus species of the D. longispina group
between species of the D. longispina group
between clones within species
between hybrids and maternal species
FIG. 3.-Plot of nucleotide sequence comparisons, based on the two-parameter model of Kimura ( 1980) and calculated by DNADIST. The average sequence divergence between Duphnia magna and species of the D. longispinu group, within the D. longispinu group, between clones, and between hybrids and maternal species is shown. Error bars show standard errors. Standard-error bars of ~01s. 3 and 4 are too small for graphic presentation.
mitochondrial gene flow between parental species, suggests no introgression, despite interspecific hybridization. In contrast to these findings and to the often proposed suggestion that introgression plays a negligible role in this Daphnia species complex (Glagolev 1986; Wolf and Mort 1986; Wolf 1987)) backcrossed populations within a D. galeata/ D. Zongispina and within a D. galeata/D. rosea species complex have been observed, even as the dominant group in some lakes (Hebert et al. 1989; Taylor and Hebert 1992). Furthermore, Taylor and Hebert ( 1992) suggest gene flow from D. rosea to D. galeata in the North American D. galeata complex. These observations, as well as the high morphological similarity of these taxa, demonstrate the potential for introgressive hybridization. The absence of any molecular evidence for introgression in populations from northern Germany and The Netherlands might be due to infertility of backcross hybrids. Based on DNA distance data and the phylogenetic tree for the D. longispina species complex, two previously proposed hypotheses concerning the origin of hybrids can be evaluated. The D. cucullata X galeata hybrid had previously the status of a separate species (FlijI3ner and Kraus 1986 ) , but allozyme investigations of Wolf and Mot-t ( 1986) revealed the hybrid nature of these phenotypes. The mtDNA sequence divergence data show that the hybrid populations did not evolve as separate species. Another interpretation has been that the hybrid populations arose via an initial hybridization event in the distant past, with the Fi hybrids subsequently maintained as independent clonal lineages through parthenogenetic reproduction (Wolf 1987 ) . For both hypotheses, one would expect sequence divergence to be of the magnitude seen between species. However, both the concordant interclonal sequence divergence (0.3%0.65%) and sequence divergence between hybrids and their maternal species (O%1% ) suggest relatively recent hybridization events. If the nucleotide substitution rate
1298
Schwenk
D. galeata (S) D. galeata (K) D. galeata x hyalina (K)
D. cucullata (S) D. cucullata (K) D. cucullata x galeata (K) D. cucullata x hyalina (M) D. hyalina (K) D. magna (F)
25
20
Is
10
5
0
Transversions
FIG. 4.-Phylogenetic tree based on parsimony analysis generated by DNABOOT ( PHYLIP, 3.3), using the Daphnia cytochrome b sequences from fig. 2. Bootstrap values are given above each Lake abbreviations are as in fig. 2. The branch lengths of the tree are drawn in proportion to the of transversions in each lineage, with each transition being considered equivalent to 0.1 transversions et al. 1982).
version branch. number (Brown
of the cytochrome b gene in Daphnia is of the same magnitude as that in Drosophila ( -2.0% /Myr; DeSalle et al. 1987), then the radiation of the D. Zongispina group can of hybrids and their maternal be dated at w-5.7-6.9 Mya, whereas the separation species occurred within the past 0.26 & 0.20 Mya. Since the chosen samples do not represent the complete clonal diversity of hybrid and species groups, our results of comparing a limited number of clones may well lead to an overestimation of sequence divergence. These uncertainties introduced by small sample size, as well as our finding of one hybrid clone bearing the same cytochrome b “allele” as its maternal species, suggest a very recent hybrid origin. Thus, the data suggest perhaps seasonal or annual production of hybrid clones. The relatively slow substitution rate of cytochrome b causes the resolution within species-hybrid groups of both the D. cucullata and D. galeata lineage to be too low to separate them. Further investigations of faster-evolving segments of the mitochondrial genome (e.g., A-T rich region) would facilitate a more accurate estimation of sequence divergence between hybrid clones and their maternal species. Despite the tremendous amount of attention that has focused on the classification of daphnids (e.g., see Brooks 1957; Fliil3ner and Kraus 1986; Hrbacek 1987), the taxonomy of the group is still unsettled. This arises mainly from the frequent occurrence of local races that differ morphologically and show more or less continuous transitions of morphological traits among different forms. These phenomena, as well as hybridizing taxa and the absence of any paleolimnological records, complicate the phylogenetic studies of Daphnia. Field observations of higher abundances of D. galeata X hyalina hybrids when compared with D. cucullata X galeata and D. cucullata X hyalina hybrids suggest a close phylogenetic relationship between D. galeata and D. hyalina, with D. cucullata as the more distantly related taxon (Flijf3ner and Kraus 1986; Wolf and Mot-t 1986). A different pattern was generated by the comparison of mtDNA sequences
Interspecific
Hybridization
in Duphnia
1299
(fig. 4): D. guleata and D. cudata seem to be more closely related to each other than either of them is to D. hyalina. Despite the relatively higher sequence divergence between D. hyalina and D. galeata, compared with D. galeata and D. cucullata, they are morphologically largely identical, even where they occur sympatrically (FliiBner and Kraus 1986). These patterns raise the question of whether speciation and morphological evolution, which usually occur synchronously, may proceed independently within the genus Daphnia (Larson 1989; Sturmbauer and Meyer 1992). On the other hand, it might be argued that alternative branching orders of the branch D. cucuZZata/ D. galeata and D. hyalina are possible because of the relatively low bootstrap value (60). This pattern has to be verified by analyzing mtDNA sequences from numerous individuals from a larger number of locations. According to FliiBner and Kraus ( 1986 ), the radiation of the D. longispina group took place during the late glacial or early postglacial. The sequence data, however, date their radiation time markedly earlier (before glacial), 6.3 -+ I. 1 Mya. One explanation for the origin of hybrid zones in Europe and North America is that they were formed as a result of range expansion from Pleistocene refugia after the retreat of the glaciers (Hewitt 1989). Such a scenario implies that populations were already differentiated when contact was established. This assumption is in concordance with the mitochondrial sequence data and the assumed nucleotide substitution rate, 2%/ Myr, of the cytochrome b gene. The very recent separation of hybrids and maternal species, in conjunction with relatively high sequence divergence between species (and inferred old age), may explain the hybrid complexes as a manifestation of a secondary contact after the glacial period. More information about evolutionary rates of mitochondrial genes, as well as phylogenetic investigations about species and hybrid clones from various locations in the northern temperate region, is needed to test this hypothesis. Acknowledgments I thank Thomas Stadler, Axe1 Meyer, Piet Spaak, Larry Weider, Ramesh Gulati, Steph B. J. Menken, Jos M. M. van Damme, and Peter van Dijk for stimulating discussions and insightful comments that resulted in many improvements in the manuscript. I am grateful to Bruno Streit and J. Feierabend for providing laboratory facilities and to Mona A. Mort for initiating this project. Matthias Schmidt and Matthias Dittmar gave advice regarding PCR and DNA sequencing, and Andrea Ender and Ulrike Kaufer helped with most of the laboratory work. I thank two anonymous reviewers for very helpful comments. I am also grateful to Piet Spaak, Larry Weider, and Jakob Miiller for providing specimens and allozyme data. I thank the Deutsche Forschungsgemeinschaft for providing research funding (MO 492/l-2), and I thank The Netherlands Institute of Ecology (Centre for Limnology) for support during manuscript preparation. LITERATURE
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JAN KLEIN, reviewing
editor
Received March 18, 1993; revision received June 2, 1993 Accepted June 2, 1993
