Mitochondrial DNA Variation and the Evolutionary History of Cryptic Gammarus fossarum Types

Molecular Phylogenetics and Evolution Vol. 15, No. 2, May, pp. 260–268, 2000 doi:10.1006/mpev.1999.0740, available online at http://www.idealibrary.com on

Mitochondrial DNA Variation and the Evolutionary History of Cryptic Gammarus fossarum Types Jakob Mu¨ller Department of Population Biology, Zoological Institute, University of Mainz, D-55099 Mainz, Germany Received May 5, 1999

The evolutionary history of the cryptic Gammarus fossarum species complex (Crustacea, Amphipoda) in Central Europe was approached by investigating the genetic variation in populations of a natural contact zone. Nucleotide sequence variation of a 395-bp segment of the mitochondrial 16S rRNA gene was compared to that of six nuclear allozyme loci. Three major mtDNA lineages were found, the eastern clade being consistent with the former allozyme type A. The two western clades (types B and C) were not distinguished previously. Strong sequence divergence and correlation with nuclear genetic isolation in syntopic populations, however, justifies the specific status of the three G. fossarum types. The common speciation event is believed to be very old (Miocene). The within-type mtDNA variation is probably molded by the ice ages, with type B populations being most affected. Moreover, the patch-like distribution of mtDNA type B lineages in an area near the contact zone corroborates the hypothesis of a recent colonization. r 2000 Academic Press

INTRODUCTION Species boundaries can be regarded as turning points for evolutionary histories. Above the species level phylogenetic relationships are important, whereas below interbreeding relationships dominate (Hennig, 1966). To describe evolutionary histories it is therefore of primary importance to diagnose species limits. Several species concepts have been put forward (Ereshefsky, 1992). Of these the phylogenetic species concept (Cracraft, 1983) seems very appealing, with easy application to extant molecular data. The essence of this concept is concerned with the recognition of diagnosable distinct clades, which can easily be revealed, for example, by nonrecombining mitochondrial DNA markers. The resulting species system may often be different from those revealed by biological species concepts (Mayr, 1963). Diagnosably distinct allopatric populations will be regarded as separate species or diagnosably nondistinct sibling species will be considered as one species (Claridge et al., 1997). The phylogenetic species identi1055-7903/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.

fication depends greatly on the polymorphic level (variability) of the selected marker system and is thus not as widely accepted. Through its focus of searching for diagnostic characters, it can, however, help to reveal potential cryptic species. If such species live in sympatry, they then can be tested for reproductive isolation, for example, by recombining nuclear markers. This combined phylogenetic approach resembles that of Avise and Ball (1990) in recognizing species by the evidence of concordant genealogies of independent genetic attributes in sympatry (i.e., concordance generated by intrinsic reproductive barriers). Here, we provide data on the mitochondrial DNA variation of closely related Gammarus fossarum types across their natural contact zones. G. fossarum belongs to the artificial Gammarus pulex group of morphologically related species (Karaman and Pinkster, 1977). In Central Europe two genetically different forms of G. fossarum have been reported based on allozyme data (Scheepmaker and van Dalfsen, 1989). The taxonomic status of these two forms, referring to the eastern form as type A and to the western form as type B, has been corroborated by an investigation of syntopic populations, i.e., where both G. fossarum forms cooccurred (Mu¨ller, 1998). Our aim was to assess the extent of mtDNA divergence between the G. fossarum types in relation to the geographical variation within the types. Such a comparative approach between the ‘‘deep and shallow evolutionary history’’ is recommended to reliably evaluate the phylogenetic significance of closely related taxonomic units (Hillis et al., 1996; Riddle, 1996). Major discontinuities should help to define species limits and will be compared to the allozyme clustering. Species boundaries will further be corroborated by the inspection of the distribution of allozyme genotypes in syntopic populations. Moreover, the same local populations have been investigated as in the allozyme study of Mu¨ller (1998). It could therefore be tested whether the mtDNA variation reflects or supports the previously proposed hypotheses about the historical and presentday population structure of the G. fossarum contact zone. It has been stated that type B recently colonized

260

MOLECULAR EVOLUTIONARY HISTORY IN CRYPTIC GAMMARIDS

an area near the contact zone. The absence of recombination and the rapid sequence evolution make mtDNA particularly appropriate for tracing recent evolutionary history and thus additional information is expected (Harrison, 1989). METHODS

261

remaining populations belong to the Rhine system. Adult individuals from all available microhabitats in a population were collected with a sieve and stored in liquid nitrogen. About six individuals per subpopulation were analyzed for both genetic characters, allozymes and mtDNA. As an outgroup, eight G. pulex individuals from the populations SAM, NOR, KLE, and SEM were included in the analysis.

Collection of Samples Samples were evenly collected from populations on both sides of and within the suggested contact zone (see Mu¨ller et al., 1999) (Fig. 1). The study area covers several drainage systems: populations SAM, LOM, and SOM belong to the Meuse system; population NWH belongs to the Fulda-Weser system; populations PIN and REI belong to the Danube system; and all the

FIG. 1. Sampling locations and their abbreviations: d, type A; types A and B.

Allozyme and mtDNA Procedures One half of each individual was used for allozyme electrophoresis. The following six polymorphic enzyme loci were selected for the analysis: PGI, MPI, Glycin– Leucin PEP, GOT-I, GOT-II, and PGM-s. All of them are coded by the nuclear genome. For full details of procedures see Mu¨ller (1998).

, type B; and

, type C; shaded area indicates the contact zone between

262

¨ LLER JAKOB MU

With the other half of the individuals, mtDNA sequencing was performed. Animals were homogenized with a microspatula and total DNA was extracted by a silica gel-based spin column procedure according to the protocol of the Quiagen tissue kit. Each 25 µl of the following PCRs consisted of 1 µl DNA template, reaction buffer (Boehringer Mannheim), 2.2 mM MgCl2, 0.1 mM each dNTP, 0.5 µM each primer, and 1 unit Taq DNA polymerase. A 392- to 393-bp-long fragment of the 16S rRNA gene was amplified with newly designed primers LR-J-Gf (58-AAGGTTGAACAAACCCTCTACT38) and LR-N-Gf (58-AAGTAAAACCTGCCCGGTGCTT38). These primers match with conserved regions within previously obtained G. fossarum sequences using the slightly modified universal primers LR-J-12887 (alias 16Sbr) and LR-N-13398 (alias 16Sar) (Simon et al., 1994). The PCR temperature profile consisted of the following steps: initial denaturation at 93°C for 3 min; 35 cycles of 45 s at 92°C, 45 s at 45°C, and 44 s at 72°C; and 7 min at 72°C. The products were purified using the microspin purification kit of Quiagen and sequenced using the dye terminator cycle sequencing premix kit of Amersham. Electrophoresis was carried out on an ABI 377 sequencer. Both strands of every individual were sequenced, the electropherograms were compared, and the consensus sequence was submitted to the EMBL/GenBank/DDBJ database (Accession Nos. AJ269587–AJ269627).

allele distributions of the respective group. Because mtDNA variation defined artificial groups including many local populations, a fixation index (Weir, 1996) is described for each locus in each group and is used in the calculation of the genotypic frequencies. This is done to cope with the Wahlund effect (Hartl and Clark, 1989) in nonpanmictic groups. Before calculation the tested individual was added to the group, to avoid the rejection of this individuum just because it carries a rare allele that has not been observed in that group. Likelihood ratio tests were performed to determine whether an individual is being rejected (on a 5% significance level) from stemming from a certain group. The population structure of mtDNA variation within each newly defined taxonomic unit was analyzed by the program ARLEQUIN 1.1 (Schneider et al., 1997) using Kimura’s two-parameter distances (Kimura, 1980) among all haplotypes. Pairwise FST values of molecular mtDNA variation, calculated according to Weir and Cockerham (1984), were modified to obtain linearized distance measures for population divergence time, according to Reynolds et al. (1983). These matrices of coancestry coefficients among all populations were used to construct trees of genetic relationship according to the method of Fitch and Margoliash (1967) with the program PHYLIP (Felsenstein, 1993).

Statistics

Differentiation of mtDNA Sequences

Mitochondrial DNA sequences were aligned by the program Clustal W ver.1.74 according to the algorithm described in Thompson et al. (1994). Most-parsimonious trees of all different haplotypes were found using 500 replicates of random addition sequences for heuristic searches with TBR branch swapping by use of the program PAUP (Swofford, 1993). Gaps were treated as fifth base and all mutations were weighted equally. Decay indices were calculated with the program AutoDecay 3.0 of Eriksson and Wikstro¨m (1997) using the reverse constraint option in PAUP. They are synonymous to the branch support indices of Bremer (1994). Bootstrap frequencies were obtained by 1000 replicates of heuristic searches with simple addition sequences. Assignment of individual allozyme genotypes to a list of mtDNA-defined groups was tested with G-Stat 3.2 (Siegismund, 1997) similar to Paetkau et al. (1995). The test shows how indicative an individual’s genotype was of the group in which it was sampled. The procedure involved calculating the expected frequency of each individual’s multilocus genotype in each of the predefined groups and subsequently assigning each individual to the group in which its expected genotype frequency (likelihood) was highest. The expected multilocus genotype frequencies were calculated as the products of expected single-locus frequencies based on the

A total of 395 bp (after alignment) of the 16S rRNA gene were compared for the 181 typed G. fossarum individuals. In the combined data set of all G. fossarum types, 86 nucleotide sites (21.8%) were variable, yielding 39 unique haplotypes. In addition, 2 different haplotypes were obtained from 8 individuals in the outgroup taxa G. pulex. Figure 2 shows the strict consensus tree of 188 equally most-parsimonious trees found in the random taxon addition analysis. Bootstrap values over 70%, which are supposed to indicate significance (Hillis and Bull, 1993), are shown. The decay values representing the length differences between the most-parsimonious trees and the trees lacking the specified branch (Bremer, 1994) are also indicated. In addition to the expected strong differentiation between G. pulex and G. fossarum, three major lineages of G. fossarum are apparent. The upper lineage in Fig. 2 is consistent with the previously described type A clade, whereas both the lower two lineages refer to the former type B (see Mu¨ller, 1998). The splitting of the former type B is supported by high bootstrap percentages (99.8 and 99.7%) and relatively high decay values (8 and 5). Hereafter, I will thus refer to the mitochondrial clade that occur in the populations LUX and SOM as type C. The phylogenetic position of the type C clade is not clearly resolved: In the majority of the random addition

RESULTS

MOLECULAR EVOLUTIONARY HISTORY IN CRYPTIC GAMMARIDS

263

FIG. 2. Strict consensus tree of 188 most-parsimonious trees based on mtDNA haplotypes (EMBL/GenBank/DDBJ identification labels are given) from different G. fossarum populations (see Fig. 1) compared to G. pulex haplotypes; in parentheses are numbers of typed individuals; the numbers on the branches represent the numbers of mutational steps .6; bootstrap percentages .70 are given above the branches in boldface; and decay values .3 are given below the branches in italics.

¨ LLER JAKOB MU

264

and bootstrap trees it splits off after the division of types A and B and lies on the same branch as type B (55 and 53%, respectively) and less frequently it splits off before types A and B separate (45 and 44%, respectively). The trichotomy of the consensus tree reflects this uncertain resolution. Table 1 lists the mean Kimura two-parameter distances, assuming equal evolutionary rates of variable sites and excluding gaps and missing values. Within types of G. fossarum, haplotype divergence is in the range of 0.003 to 0.043 with a mean of 0.017. Among G. fossarum types distances vary between 0.078 and 0.147 with a mean of 0.110. The distances between G. fossarum in general and G. pulex are between 0.209 and 0.268 with a mean of 0.239. In general, the transition/transversion ratio decreased as pairwise sequence divergence increased. Among and within G. fossarum types two to three times more transitions than transversions occur, whereas between G. pulex and G. fossarum the evolution of the transition/ transversion ratio seems to be more saturated. Assignment of Allozyme Genotypes to mtDNA Clades An assignment test was performed to check for correspondence in the clustering of allozyme genotypes and mtDNA haplotypes. The results in Table 2 show that most individuals correctly assign to their mtDNAdefined group. A strict separation of types A and B genotypes is apparent, mutually rejecting all individuals from the contrary group. Some of the type A (40%) and type B (47%) individuals, however, falsely assign to the type C group. This indicates weak allozyme differentiation between type C and both types A and B; 34% of type A individuals and only 10% of type B individuals are significantly rejected to stem from the type C group. It has to be noted, however, that the test has a general bias in assigning individuals to small groups such as the type C or G. pulex groups. This probably accounts for the result that no type C individual is reversely assigned to the type A or B group.

TABLE 1 Kimura Two-Parameter Distances (in Normal Typeface) and Transition to Transversion Ratios (in Italics) within (Diagonal) and among Taxonomic Units of G. pulex and G. fossarum (Gf ); mean 6 sd Gf type A Gf type A Gf type B Gf type C G. pulex

0.015 6 0.008 2.984 6 2.524 0.115 6 0.010 2.636 6 0.366 0.102 6 0.007 3.308 6 0.490 0.235 6 0.006 0.922 6 0.080

Gf type B

0.021 6 0.012 2.814 6 2.118 0.095 6 0.011 2.772 6 0.380 0.248 6 0.012 1.020 6 0.092

Gf type C

0.007 6 0.003 1.967 6 0.968 0.225 6 0.015 1.127 6 0.091

G. pulex

0.074 1.450

TABLE 2 Assignment of Individual Allozyme Genotypes to Four mtDNA-Defined Groups (Three Groups of G. fossarum (Gf ) Types and One Outgroup of G. pulex) Assigned group Source group

Gf type A

Gf type B

Gf type C

G. pulex

Sum

Gf type A Gf type B Gf type C G. pulex

32 (9) 0 (86) 0 (4) 0 (7)

0 (53) 45 (7) 0 (4) 0 (7)

21 (18) 40 (9) 4 (0) 0 (7)

0 (52) 1 (84) 0 (4) 7 (0)

53 86 4 7

Note. Values are the number of individuals of the row group assigned to the column group; in parentheses are numbers of individuals significantly rejected to stem from the respective column group.

Differentiation of Populations within Types The trees of genetic relationships among populations based on mtDNA variation are shown in Fig. 3 for each G. fossarum type. Most of the underlying pairwise FST values are significant by permutation tests (P , 0.05; 1000 replicates), indicating significant population structure for each type. Population differentiation is stronger for type B (and probably type C) than for type A. The type A populations are relatively homogeneous in distribution and show some correlation with geography and allozyme variation (see Fig. 3 in Mu¨ller, 1998). There are, however, two distinct type B population clades which do not correspond to the clusters found with allozymes. Populations GAI and KAP, for example, belong to different mtDNA clades but form a unique branch in the allozyme analysis. The same holds true for the other population cluster inferred from the allozyme study: HUB, WOL-B, HOM, GAU, WAE, NWH, DOR-B, DOE-B, and STJ. From the mtDNA differentiation a geographic pattern is apparent. The upper clade of type B in Fig. 3 comprises populations located in the western part of the study area, which contrasts to the lower more eastern located clade. The only exceptions are the populations WAE and GAI, belonging to the upper clade, but lying in the eastern part. DISCUSSION Taxonomic Significance of mtDNA Variation Maximum-parsimony analysis identified three major reciprocal monophyletic groups of mtDNA haplotypes in G. fossarum (types A, B, and C). These groups are strongly supported by bootstrap percentages and decay indices. Furthermore, they show high levels of fixed diagnostic differences in their nucleotide sequences: on average 6.8% with a range of 6.6 to 7.1%. One of these clades corresponds perfectly with the previously re-

MOLECULAR EVOLUTIONARY HISTORY IN CRYPTIC GAMMARIDS

265

FIG. 3. Phenogram of mtDNA variation among populations within G. fossarum types.

ported type A group based on allozyme data, whereas the other two clades could not be distinguished by allozyme criteria (Scheepmaker and van Dalfsen, 1989; Mu¨ller, 1998). The two populations containing type C haplotypes were previously allocated to type B (population SOM) or described as intermediate between types A and B (population LUX) (Mu¨ller, 1998). The weak differentiation of type C allozyme genotypes from the genotypes of type A or B was amply illustrated by the assignment tests. In the total study area there are no fixed diagnostic allozyme alleles at the analyzed loci for each G. fossarum type. Only the allele frequency distributions show some differences between type A and type B (or C). If geographic variation is excluded, that is only local syntopic populations are considered separately, diagnostic alleles are found. In such a way the populations DOR, DOE, WOL, and SEM have diagnostic alleles at the loci PGI, MPI, and PEP for type A and type B, corresponding with the mtDNA split. Reinspection of population MUN (Mu¨ller et al., 1999) revealed only one presumably diagnostic locus (PGI), but sample size for this population was too small to find both mtDNA haplotypes. The mitochondrial DNA variation suggests a subdivision of the population SOM into types B and C. The allozyme criteria for population subdivision in Mu¨ller

(1998), however, were cumulative, with more diagnostic loci giving more support of subdivision. Reinspection of multilocus genotypes in the SOM population reveals only one diagnostic locus (PGI), whose allele distribution corresponds with the mtDNA lineages. Strong Hardy–Weinberg deviations and the scarcity of heterozygotes with both diagnostic alleles justifies the subdivision of population SOM. Further splitting of G. fossarum into genetically isolated types seems to be unsupported by allozyme data. For example, the second greatest mtDNA differentiation with nine mutational steps, a bootstrap value of 98%, and a decay value of 4 was found for the most derived group within type B haplotypes (see Fig. 2), rendering populations HOM and WOL-B as potential candidates for subdivision. However, there was no corresponding allelic separation at any allozyme loci. In general, there is a good agreement of differentiation patterns of nuclear allozyme characters and mtDNA characters across closely related species groups (Knowlton et al., 1993; Taylor et al., 1996, 1998; Marko, 1998). Concordant patterns also have been found in local syntopic populations of G. fossarum types, which was used as a taxonomic criterion according to Avise and Ball (1990). On a large geographic scale, however, no concordance among mtDNA and allozyme variation

266

¨ LLER JAKOB MU

was found for the differentiation between types B and C. The following hypotheses for explaining less structuring in allozymes than in mtDNA can be put forward. (a) MtDNA evolves faster than coding nuclear DNA (Rand, 1994). It does not, however, explain the difference in concordance between the taxa pairs of types A and B and types B and C. (b) The relatively lower resolving power of protein electrophoresis relative to DNA sequencing results in artifactual uniformity of allozymes (Burton and Lee, 1994). This is realistic for the present study because intratype geographic variation of allozymes was strong, probably due to genetic drift in G. fossarum populations, which are confined only to the upper reaches of rivers. Geographic variation within types B and C could thus have swamped the intertype variation. (c) Homogenizing selection on allozyme loci could have hindered differentiation in relation to mtDNA differentiation (Burton and Lee, 1994). Similar selective constraints could have evolved for types B and C because of their supposed common ice age refugium (see below) and their present-day distribution. As shown, only the combined analysis of mitochondrial and nuclear genes can uncover the taxonomic significance within the species complex of G. fossarum. Hints that the nominal G. fossarum comprises a polytypic species group are well reported (Scheepmaker, 1990; Jazdzewski, 1977) but seem to be disproved by hybridization experiments (Pinkster and Scheepmaker, 1994). Only the evidence from nuclear genetic isolation of mitochondrial lineages in natural syntopic populations could reveal the taxonomic status of three different G. fossarum species in Central Europe. They should be given distinct species names, despite their morphological resemblance (Mu¨ller et al., 1999). Mitochondrial introgression among types is not indicated for the study area. Additional taxonomic evidence comes from the high haplotype distance values. While within types only 1.7% divergence was measured, a mean value of 11% was found among types. This last value falls within the range of 4 to 18% 16S rRNA sequence divergence among postulated sibling species of the amphipod Eurythenes gryllus distributed in different depths (France and Kocher, 1996). Similar 16S divergences among congeneric species have been reported for some decapods (Cunningham et al., 1992; Machado et al., 1993; Sturmbauer et al., 1996; Tam et al., 1996; Sarver et al., 1998). Even lower congeneric distances below 4% have been found in other decapods (Geller et al., 1997; Schneider-Broussard et al., 1998; Tam and Kornfield, 1998). The strong divergence between G. pulex and the G. fossarum species complex (24%) normally reflects the among-genus level in decapods (Cunningham et al., 1992). In total, most of the comparisons show similar evolutionary rates. Possible divergent rates of some taxa could be correlated with differential thermal habits or differential numbers of mtDNA generations in the germ line (Rand, 1994).

Moreover, differential speciation times for different taxonomic groups or simply a tendency to split wellknown and popular taxonomic groups like decapods more than others would have a strong effect (Hewitt, 1996). The transition/transversion ratios, where indicated, show similar saturation patterns relative to sequence divergences compared to the 12S rRNA gene (Taylor et al., 1996). Evolutionary History and Demographic Structure of mtDNA Clades The present-day genetic structure of species allows some inferences to be made concerning their history. The low resolution of the splitting of type C and similar haplotype distances between all types suggest that all three G. fossarum types originated at about the same time. Conservative estimates about the minimum time of divergence between the three G. fossarum types can be made using proposals of 16S divergence rates in other malacostraceans. For the 16S fragment homologous to the present comparisons, estimates range between 0.4 and 0.9% sequence divergence per million years (Cunningham et al., 1992; Sturmbauer et al., 1996; Schubart et al., 1998). The minimum time since common ancestry for the three G. fossarum types can thus be estimated as between 9 and 19 million years ago. Given the possible uncertainty of similar evolutionary rates among malacostraceans, the estimate can only be compared to plausible scenarios. The time of the speciation event seems very old and would fall into the tertiary epoch of the Miocene, an age on the same order of magnitude as some speciations of Baikalian gammarids (Sherbakov et al., 1998). The formation of these cold-stenothermic G. fossarum types could have happened either during range expansion from the proposed gammarid-rich Siberian area or in situ in Europe during a period of general cooling in the Miocene (Schmidt, 1978; Scheepmaker, pers. comm.). After repeated contractions and expansions of their geographical ranges during Pleistocene climatic oscillations, the G. fossarum types A and B met in postglacial times at a common suture zone (Taberlet et al., 1998; Mu¨ller, 1998). G. fossarum could have survived the cold periods of the Pleistocene near the borders of southern glaciers and the warm periods in the Alps or other mountain ranges (Thienemann, 1950). According to the distributions, it can be inferred that type A had a refugium in an area east of the Alps and types B and C in an area west of the Alps. The taxonomic position within the species-rich G. pulex group of the Mediterranean refugia (Scheepmaker, 1990) will be investigated in the future. According to the above-mentioned molecular clock, the intratype mtDNA variability (shallow splits) would have originated during or before the beginning of the ice ages at about 2.4 million years ago (Webb and Bartlein, 1992). Haplotype diversities may have formed

MOLECULAR EVOLUTIONARY HISTORY IN CRYPTIC GAMMARIDS

through isolation by distance effects before the onset of the ice ages and subsequent vicariance events during climatic fluctuations, as suggested by Taberlet et al. (1998). Periods of vicariances, however, were too short to build up reproductive isolation, leaving this variation as subspecific. Within the study area, type B (and probably type C as well) with its high genetic diversity appears to be strongly influenced by these demographic events. Moreover, it could be explained that the potential refugial zone of type B (and C) is closer than for type A, rendering a higher chance for different haplotype lineages to reach the likely reinvaded study area (Hewitt, 1996). Both this historical factor and current demographic factors, such as less isolation among extant populations of type A, may be invoked to explain the lower population differentiation of type A. The strong differentiation of type B populations is due mainly to two almost completely sorted mtDNA lineages. It is interesting that within the proposed recently colonized area of type B around and east of Mainz (Mu¨ller, 1998), the mtDNA lineages are least geographically sorted (populations WAE and GAI belong genetically to the western clade). It seems likely that long-distance dispersal from different source populations could have created this patch-like design of genetic structure, which will persist for some time (Ibrahim et al., 1996). Such a patchwork could well be a general characteristic of recently colonized areas. ACKNOWLEDGMENTS I am grateful to Stephanie Giesen for laboratory help and Alfred Seitz for general support. Simon J. Hadfield, two anonymous reviewers, and Rob DeSalle made valuable comments on the manuscript. This work was partly supported by the German Science Foundation, Grant No. Se506/4-2.

REFERENCES Avise, J. C., and Ball, R. M., Jr. (1990). Principles of genealogical concordance in species concepts and biological taxonomy. Oxford Surv. Evol. Biol. 7: 45–67. Bremer, K. (1994). Branch support and tree stability. Cladistics 10: 295–304. Burton, R. S., and Lee, B.-N. (1994). Nuclear and mitochondrial gene genealogies and allozyme polymorphism across a major phylogeographic break in the copepod Tigriopus californicus. Proc. Natl. Acad. Sci. USA 91: 5197–5201. Claridge, M. F., Dawah, H. A., and Wilson, M. R. (1997). Practical approaches to species concepts for living organisms. In ‘‘Species: The Units of Biodiversity’’ (M. F. Claridge, H. A. Dawah, and M. R. Wilson, Eds.), pp. 1–15. Chapman & Hall, London. Cracraft, J. (1983). Species concepts and speciation analysis. In ‘‘Current Ornithology’’ (R. Johnston, Ed.), pp. 159–187. Plenum, New York. Cunningham, C. W., Blackstone, N. W., and Buss, L. W. (1992). Evolution of king crabs from hermit crab ancestors. Nature 355: 539–542. Ereshefsky, M. (ed.) (1992). ‘‘The Units of Evolution,’’ MIT Press, Cambridge, MA.

267

Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, Univ. of Washington, Seattle. Fitch, W. M., and Margoliash, E. (1967). Construction of phylogenetic trees. Science 155: 279–284. France, S. C., and Kocher, T. D. (1996). Geographic and bathymetric patterns of mitochondrial 16S rRNA sequence divergence among deep-sea amphipods, Eurythenes gryllus. Mar. Biol. 126: 633–643. Geller, J. B., Walton, E. D., Grosholz, E. D., and Ruiz, G. M. (1997). Cryptic invasions of the crab Carcinus detected by molecular phylogeography. Mol. Ecol. 6: 901–906. Harrison, R. G. (1989). Animal mitochondrial DNA as a genetic marker in population and evolutionary biology. Trends Ecol. Evol. 4: 6–11. Hartl, D. L., and Clark, A. G. (1989). ‘‘Principles of Population Genetics,’’ Sinauer, Sunderland, MA. Hennig, W. (1966). ‘‘Phylogenetic Systematics,’’ Univ. of Illinois Press, Urbana. Hewitt, G. M. (1996). Some genetic consequences of ice ages, and their role in divergence and speciation. Biol. J. Linn. Soc. 58: 247–276. Hillis, D. M., Moritz, C., and Mable, B. K., Eds. (1996). ‘‘Molecular Systematics,’’ Sinauer, Sunderland, MA. Hillis, D. M., and Bull, J. J. (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42: 182–192. Ibrahim, K. M., Nichols, R. A., and Hewitt, G. M. (1996). Spatial patterns of genetic variation generated by different forms of dispersal during range expansion. Heredity 77: 282–291. Jazdzdewski, K. (1977). Remarks on the morphology of Gammarus fossarum Koch, 1835, and Gammarus kischineffensis Schellenberg, 1937. Crustaceana Suppl. 4: 201–211. Karaman, G. S., and Pinkster, S. (1977). Freshwater Gammarus species from Europe, North Africa and adjacent regions of Asia (Crustacea-Amphipoda). Part I. Gammarus pulex-group and related species. Bijdr. Dierk. 47: 1–97. Kimura, M. (1980). A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111–120. Knowlton, N., Weigt, L. A., Solorzano, L. A., Mills, DeE. K., and Bermingham, E. (1993). Divergence in proteins, mitochondrial DNA, and reproductive compatibility across the Isthmus of Panama. Science 260: 1629–1632. Machado, E. G., Dennebouy, N., Suarez, M. O., Mounolou, J.-C., and Monnerot, M. (1993). Mitochondrial 16S-rRNA gene of two species of shrimps: Sequence variability and secondary structure. Crustaceana 65: 279–286. Marko, P. B. (1998). Historical allopatry and the biogeography of speciation in the prosobranch snail genus Nucella. Evolution 52: 757–774. Mayr, E. (1963). ‘‘Animal Species and Evolution,’’ Harvard Univ. Press, Cambridge, MA. Mu¨ller, J. (1998). Genetic population structure of two cryptic Gammarus fossarum types across a contact zone. J. Evol. Biol. 11: 79–101. Mu¨ller, J., Partsch, E., and Link, A. (2000). Differentiation in morphology and habitat partitioning of genetically characterized Gammarus fossarum forms (Amphipoda) across a contact zone. Biol. J. Linn. Soc., in press. Paetkau, D., Calvert, W., Stirling, I., and Strobeck, C. (1995). Microsatellite analysis of population structure in Canadian polar bears. Mol. Ecol. 4: 347–354. Pinkster, S., and Scheepmaker, M. (1994). Hybridization experiments and the taxonomy of Gammarus (Amphipoda): A contribution to

268

¨ LLER JAKOB MU

the understanding of controversial results. Crustaceana 66: 129– 143. Rand, D. M. (1994). Thermal habit, metabolic rate and the evolution of mitochondrial DNA. Trends Ecol. Evol. 9: 125–131. Reynolds, J., Weir, B. S., and Cockerham, C. C. (1983). Estimation for the coancestry coefficient: Basis for a short-term genetic distance. Genetics 105: 767–779. Riddle, B. R. (1996). The molecular phylogeographic bridge between deep and shallow history in continental biotas. Trends Ecol. Evol. 11: 207–211. Sarver, S. K., Silberman, J. D., and Walsh, P. J. (1998). Mitochondrial DNA sequence evidence supporting the recognition of two subspecies or species of the Florida spiny lobster Panulirus argus. J. Crust. Biol. 18: 177–186. Scheepmaker, M., and van Dalfsen, J. (1989). Genetic differentiation in Gammarus fossarum and G. caparti (Crustacea, Amphipoda) with reference to G. pulex pulex in northwestern Europe. Bijdr. Dierk. 59: 127–139. Scheepmaker, M. (1990). Genetic differentiation and estimated levels of gene flow in members of the Gammarus pulex-group (Crustacea, Amphipoda) in western Europe. Bijdr. Dierk. 60: 3–30. Schmidt, K. (1978). ‘‘Erdgeschichte,’’ de Gruyter, Berlin. Schneider, S., Kueffer, J.-M., Roessli, D., and Excoffier, L. (1997). Arlequin ver. 1.1: A software for population genetic data analysis. Genetics and Biometry Laboratory, Univ. of Geneva, Switzerland. Schneider-Broussard, R., Felder, D. L., Chlan, C. A., and Neigel, J. E. (1998). Tests of phylogeographic models with nuclear and mitochondrial DNA sequence variation in the stone crabs, Menippe adina and Menippe mercenaria. Evolution 52: 1671–1678. Schubart, C. D., Diesel, R., and Hedges, S. B. (1998). Rapid evolution to terrestrial life in Jamaican crabs. Nature 393: 363–365. Sherbakov, D. Y., Kamaltynov, R. M., Ogarkov, O. B., and Verheyen, E. (1998). Patterns of evolutionary change in Baikalian gammarids inferred from DNA sequences (Crustacea, Amphipoda). Mol. Phylogenet. Evol. 10: 160–167. Siegismund, H. R. (1997). G-Stat, version 3.2, Genetical statistical programs for the analysis of population data. Department of Plant Ecology, Univ. of Copenhagen, Oster Farimagsgade 2D, DK-1353 Copenhagen K, Denmark. Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., and Flook, P. (1994). Evolution, weighting, and phylogenetic utility of mitochon-

drial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87: 651–701. Sturmbauer, Ch., Levinton, J. S., and Christy, J. (1996). Molecular phylogeny analysis of fiddler crabs: Test of the hypothesis of increasing behavioral complexity in evolution. Proc. Natl. Acad. Sci. USA 93: 10855–10857. Swofford, D. L. (1993). PAUP: Phylogenetic Analysis Using Parsimony, version 3.1.1. Computer program distributed by the Illinois Natural History Survey, Champaign, IL. Taberlet, P., Fumagalli, L., Wust-Saucy, A.-G., and Cosson, J.-F. (1998). Comparative phylogeography and postglacial colonisation routes in Europe. Mol. Ecol. 7: 453–464. Tam, Y. K., Kornfield, I., and Ojeda, F. P. (1996). Divergence and zoogeography of mole crabs, Emerita spp. (Decapoda: Hippidae), in the Americas. Mar. Biol. 125: 489–497. Tam, Y. K., and Kornfield, I. (1998). Phylogenetic relationships of clawed lobster genera (Decapoda: Nephropidae) based on mitochondrial 16S rRNA gene sequences. J. Crust. Biol. 18: 138–146. Taylor, D. J., Hebert, P. D. N., and Colbourne, J. K. (1996). Phylogenetics and evolution of the Daphnia longispina group (Crustacea) based on 12S rDNA sequence and allozyme variation. Mol. Phylogenet. Evol. 5: 495–510. Taylor, D. J., Finston, T. L., and Hebert, P. D. N. (1998). Biogeography of a widespread freshwater crustacean: Pseudocongruence and cryptic endemism in the North American Daphnia laevis complex. Evolution 52: 1648–1670. Thienemann, A. (1950). ‘‘Verbreitungsgeschichte der Su¨ßwassertierwelt Europas,’’ Band XVIII in ‘‘Die Binnengewa¨sser’’ (A. Thienemann, Ed.). Schweizerbart’sche Verlagsbuchhandlung, Stuttgart. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680. Webb, T., and Bartlein, P. J. (1992). Global changes during the last 3 million years: Climatic controls and biotic responses. Annu. Rev. Ecol. Syst. 23: 141–173. Weir, B. S. (1996). ‘‘Genetic Data Analysis,’’ Vol. II, Sinauer, Sunderland, MA. Weir, B. S., and Cockerham, C. C. (1984). Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370.