- Email: [email protected]
Patterns of Evolutionary Change in Baikalian Gammarids Inferred from DNA Sequences (Crustacea, Amphipoda)
MOLECULAR PHYLOGENETICS AND EVOLUTION
Vol. 10, No. 2, October, pp. 160–167, 1998 ARTICLE NO. FY970482
Patterns of Evolutionary Change in Baikalian Gammarids Inferred from DNA Sequences (Crustacea, Amphipoda) D. Yu. Sherbakov,* R. M. Kamaltynov,* O. B. Ogarkov,* and E. Verheyen† *Limnological Institute, Siberian Branch of Russian Academy of Sciences, P.O. Box 4199, 664033 Irkutsk, Russia; and †Taxonomy and Biochemical Systematics Section, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium Received December 6, 1996; revised October 28, 1997
The Baikalian gammarids (Crustacea, Amphipoda) are the most widely known and most spectacular example of an adaptive radiation among contemporary freshwater invertebrates. To study the phylogeny of the Baikalian gammarids we sequenced a 622-bplong fragment of the nuclear gene coding for 18S rRNA from species of 18 endemic Baikalian genera and Gammarus pulex—a non-Baikalian taxon. Some important morphological characters appear independently in both lineages and suggest parallelism in the development of gigantism and body armament. The first lineage comprises benthic, mostly unarmed taxa. The second lineage contains predominantly armed taxa, most of which are detrivorous or carnivorous. r 1998 Academic Press
INTRODUCTION Lake Baikal is the largest, deepest, and most ancient (ca. 28 MY old) of all existing great freshwater lakes (Mats, 1992; Logatchev, 1993). Its fauna is characterized by the presence of speciose and endemic species assemblages (Brooks, 1950; Kozhov, 1963, Martin, 1994). The Baikalian gammarids (Crustacea, Amphipoda) are the most widely known and most spectacular example of adaptive radiation among contemporary freshwater invertebrates (Brooks, 1950; Kozhov, 1963). About 30% (259 described species) of all known freshwater gammarid species of the world are endemic to Lake Baikal (Barnard and Barnard, 1983). Most of the 45 genera to which they are currently allocated are also endemic (Kamaltynov, 1993). This systematic diversity reflects the dramatic morphological differentiation within this species assemblage, which contrasts with the low amount of variation in the morphology of non-Baikalian freshwater gammarids. This group contains taxa that posess highly derived and diverse morphologies, some of which are ‘‘armed’’ with spiny structures (called body teeth) of different sizes, shape, and position on the body or appendages. Almost all known armed freshwater amphipods are found in Lake Baikal (Barnard and 1055-7903/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
Barnard, 1983). The Baikalian gammarids occupy the widest variety of ecological niches and are characterized by many highly specialized forms, ranging from the dwarfed herbivorous Micruropus to giant deepwater Abyssogammarus and Garjajewia, from the parasitic genera Pachyschesis and Spinacanthus to the pelagic Macrohectopus. The latter monotypic genus is unique for the whole order and was considered a separate family (Dorogostajski, 1923; Timoshkin et al., 1995). Although Baikalian gammarids have been studied for more than a century, the phylogenetic relationships within this group, as well as their relationships with non-Baikalian gammarids, remain unclear (Dybowsky, 1874; also references in Barnard and Barnard, 1983). Comparative morphological analysis (Bazikalova, 1945) and attempts to use allozymes (Yampolsky et al., 1994; Mechanikova pers. comm.) and mitochondrial DNA sequences (Ogarkov et al., 1997) did not result in a phylogenetic hypothesis for this species flock as a whole. This lack of phylogenetic knowledge has severely impeded studies of gammarid comparative biology and evolution. Despite the morphological diversity of Baikalian gammarids, few reliable characters exist for resolving their phylogeny (Kamaltynov, unpublished results). Since such phylogenies inferred from scanty and insufficiently understood morphological data create tautologies for studies attempting to understand the evolution of these characters, a phylogenetic hypothesis based either upon more morphological characters or on an independent set of characters is required. In order to obtain an overall picture of phylogenetic relationships among Baikalian endemic gammarids the present study uses partial 18S rRNA sequences. This gene is known to be evolutionary conservative and thus to be a potentially suitable tool for molecular phylogenetic analysis at generic and higher taxonomic levels (Hillis and Dixon, 1991; Gerbi, 1985), as illustrated by a recent study of Baikalian flatworms, which represent another major Baikalian invertebrate species assemblage (Kuznedelov and Timoshkin, 1993, 1995).
160
161
GAMMARID EVOLUTION IN LAKE BAIKAL
MATERIALS AND METHODS Species Studied and Gene Sequenced We amplified and sequenced a 622-bp-long fragment of 18S rRNA from 18 Baikalian amphipod species, belonging to 18 genera. These gammarids were selected to represent the whole taxonomical and ecological range of this group in Lake Baikal. All major groups of Baikalian gammarids, as described in the most recent taxonomical reviews (Kamaltynov, 1993, 1995), are represented in our dataset by at least one genus, and as a rule each genus is represented by one species (Table 1). Orthologous DNA sequences were also determined for Gammarus pulex, which is widely distributed throughout nearly the whole of northern Eurasia, but does not occur in Lake Baikal, and Tryphosella murrayi, a marine Antarctic nongammarid lysianassoid amphipod, which we used as outgroup. The Baikaian gammarids were collected during expeditions of the Baikal International Center for Ecological Research in 1995 and species determined using identification keys in Bazikalova (1945) and fixed in 75% ethanol. G. pulex was collected in Belgium (the Ardennes) by B. Goddeeris and determined by G. Petre. The T. murrayi specimen was provided by C. Debroyer. DNA Methods Whole animals or legs (depending on body size) were used for DNA extractions according to the CTAB/ chloroform procedure (Doyle and Sickson, 1987). The primers used for the amplification of the 18S rRNA fragment were 18S I: 58atgaccatcctagttggtccat-38; and 18S II: 58-ccacggtcgtcggcgccatta-38 (Kuznedelov, 1995). Approximately 0.15–0.20 µg of double-stranded PCR product was cycle-sequenced according to the manufacturer’s protocol (Pharmacia), using 0.8 µM primers and 2.5 units of Taq polymerase. Amplified DNA was sequenced from both ends with the same primers. The temperature profile for cycle sequencing was denaturation at 95°C for 36 s, annealing at 52°C (50°C for 18S I primer) for 36 s, and extension at 72°C for 80s and after 25 cycles the samples were kept at 72°C for 5 min. The obtained cycle-sequencing reaction products were analyzed on an ALF DNA sequencer (Pharmacia). The 18S rRNA sequences as well as their alignment have been submitted to EMBL under Accession numbers Z98982– Z99004. Phylogenetic Analyses All sequences were aligned using CLUSTAL W (Thompson et al., 1994), with adjustments by eye. Phylogenetic inferences based upon 18S rRNA sequences were obtained using parsimony and maximum likelihood methods since 18S rRNA sequences are known to evolve with uneven rates of nucleotide substitutions in different lineages, whereas mutation rates are also nonuniform between different positions in this gene (Hillis and Dixon, 1991).
Parsimony analysis was carried out with PAUP using unweighted characters (version 3.1.1, Swofford, 1993). Settings were: heuristic search, MULPARS and ACCTRAN options in effect, and gaps treated as the fifth character. The robustness of the phylogeny obtained was tested by bootstrapping (Felsenstein, 1985) after 100 replications, using ‘‘heuristic search’’ with the option ‘‘simple addition of taxa.’’ The Bremer support index for each node (Bremer, 1988; Kallersjo et al., 1992) was calculated with PAUP in connection with the program AUTODECAY, version 3.0 (Eriksson and Wikstro¨m, 1996). Phylogenetic relationships of the Baikalian gammarids were also estimated with the maximum likelihood algorithm using the FastDNAml program (Olsen et al., 1994). The transition/transversion ratio was set to 2.0 and global rearrangements were allowed. The input order of taxa was changed until the tree topology with maximal support function value was obtained for three different sets. The branching order was verified with maximum likelihood quartet puzzling using the program PUZZLE (Strimmer and von Haeseler, 1996; Strimmer et al., 1997) with base frequencies and the transition/transversion ratio estimated from the dataset. We applied the Hasegawa et al. (1985) model of base substitution and increased the number of puzzling steps until the resulting topology did not change (5000 puzzling steps). All the maximum likelihood algorithms treat alignment gaps as missing data. RESULTS Sequence Variation Of the 622 base pairs (bp) of the studied 18S rRNA fragment analyzed, 123 sites are variable within the Baikalian gammarids studied and G. pulex, whereas 50 positions are parsimony informative (Table 1). The highest (lowest) uncorrected sequence divergence between two gammarids is 8% (1%). Several base pair insertion/deletion events are inferred to align the sequences. As is typical for this gene, the mutation sites in the sequenced 18S rRNA DNA region are not randomly distributed, some regions containing more variable sequences. Pairwise comparison of transitions versus transversions indicates a bias toward transversions (TS/TV ratios are generally lower than 1). Finally, the overall base composition within the gammarids studied shows no bias in the 18S rRNA DNA region (A, 23.1%; T, 24.2%; C, 25.8%; and G, 26.9%). 18S rRNA Phylogeny of Baikalian Gammarids A single most parsimonious tree of 198 steps was obtained without character weighting (CI 5 0.485, RC 5 0.216, Fig. 1). The topologies of the majority rule consensus tree that are at least four steps longer than the most parsimonious tree is identical to the latter one (199 steps: 58 trees, CI 5 0.482; 200 steps: 1066 trees,
162
SHERBAKOV ET AL.
TABLE 1 Parsimony Informative Sites of the Partial 18S rRNA Sequences of the Baikalian Gammarids Gammarus pulex and Tryphosella murrayi 111111111 1111111222 2222222233 3333333344 4444445555 556 5002223567 7778889000 0037778881 1245568901 1233454577 770 0692340513 7890232124 5872461570 2295864100 5989264412 693 Ceratogammarus Pachysches Parapallasea Eulimnogammarus Spinacanthanthus Brandtia Pallasea Baikalogammarus Micruropus
CGATTAGAAT .........C .C.......C T........C ...A..C..C ...A.....C ...A.....C ...A.....C ...A......
Macrohectopus Gammarus
...A...TTC GG..GC.--. C.T....... T...G.C..C .G.GT.C.G. .C. ...A.....C GG...G.CT. C..A...T.- T.GTG.C..C .G.G..C... .C?
Odontogammarus Ommatogammarus Abyssogammarus Plesiogammarus Paragarjajewia Garjajewia Acanthogammarus Boeckaxellia
.T..AT...C ...--....C ..T.AT...C .C......TC .C.....TTC ......C..C ...A.....C .C.A.....C
Tryphosella
T.TA.....- --A..--G.. .......... -....TCT.. .GG.G....C -C.
ATTTTTTTGA T.AC...CTC G....GCG.. G..GGG.C.G G.AG..-..G G....G-..G G.....C..G GG...G.CT. C.G.--..T.
G..G..CG.G G.AGGG.... G....G.... .....G..T. T...?C.CAG G.......T. .....G...G G.........
TCCTCTTGGA C...G.C..T C...G.C... C...G..... C...G..... C......... C...G..... C.T.GC..AT CT.CGC..AT
C......C.. C......C.. ..T....C.. C......C.. C..C...... .......... .......... CT........
CCCCTGACCG .T....C.GA ..G...C.G. ...G..C.G. ......C.G. ......CTG. ..G...C.G. ..G...CGG. ......CTG.
..GGG.C..C .TGGG..... ....G.C... T..G..C... T.GG.TC... ...GG.C... ..GGG.C... ...GG.C...
GACCAGTGCG C.T.GT.... ..T.....GC .GT...C..C ..T.T..... ..T.T..... .GT.T..... ....T..... ..?.T.....
CG..T.C.GC TGT.T.C... CGT.TTC..C CGG.G.CC.A TGT.T.C..C CGT.T.CC.C CGT.T..-.C CGG.T..CGC
TGG ..? ... ..? .C. .C. .C? .C. .C.
.CT .CT ACT AC. AC? AC. .C. ...
Note. The length of the studied fragments including gaps introduced by alignment is 622 bp. The total alignment (including the outgroup taxon) contains 53 parsimony informative and 169 variable sites.
CI 5 0.480, 201 steps: 11988 trees, CI 5 0.478; 202 steps: 28461 trees, CI 5 0.475). Bremer decay indices, which are shown below branches, as well as bootstrap values, shown above branches, indicate low support for the two major lineages of gammarids. Nevertheless, the same branching pattern is obtained with respect to the split of the taxa into two major groups if maximum likelihood is applied (Fig. 2). To evaluate if this result supports the existence of these two lineages, the maximum likelihood tree was verified using quartet puzzling with the substitution model parameters estimated from the dataset and by applying the gamma correction for the differential base substitutions. The program employed retains only a tree(s) in which all partitions are significantly supported, otherwise it collapses nodes so that a multifurcated tree is constructed. The phylogeny obtained with this approach (Fig. 3) also contains the two lineages and a separate branch of M. branickii/G. pulex, and the ratio of trees where the two major lineages appear as separate clades becomes significant. DISCUSSION Molecular Analyses The low bootstrap values obtained for the lineages of the Baikalian gammarids species flock in the 18S rRNA parsimony tree (Fig. 1) are clearly too weak to give
significant support for the presence of the two major lineages. When the maximum likelihood algorithm implemented in the FastDNAml program is applied to our dataset, it yields essentially the same topology as the two major lineages (Fig. 2), the only exception being the relative position of the G. pulex/M. branickii lineage. The low bootstrap values as well as the low Bremer support index (51) are the result of the fact that relatively few substitutions define the branching order of these arising lineages. This is a particular problem in studies based upon genes or gene segments— such as the 18S rRNA gene fragment studied here— that contain slow and fast evolving sites. Although the slowly evolving sites of such genes are less prone to homoplasy, the number of substitutions per time unit may be insufficient to allow the resolving of rapidly evolving lineages. In contrast, the more rapidly evolving part of the same gene fragment may significantly increase the risk of homoplasies, another factor that may explain the low bootstrap values. The position of the lineage G. pulex/M. branickii differs in the maximum likelihood and the maximum parsimony trees. The results of the maximum likelihood quartet puzzling analysis (as well as the low bootstrapping values and decay indices) suggests that the branching order in the basal part of the tree cannot be resolved with our dataset. At the current level of
GAMMARID EVOLUTION IN LAKE BAIKAL
163
FIG. 1. The single most parsimonious 18S rRNA tree obtained for 18 Baikalian gammarids and one Palearctic species, using Tryphosella murrayi as outgroup. The numbers above the branches are bootstrap values after 100 replications. A number of important morphological and ecological characters appear independently in both lineages and suggest parallelism in the evolution of some of these features. The food preferences of Baikalian gammarids are not always very restricted; nearly all genera contain taxa with different food preferences (Kamaltynov, personal observation). The size classes listed here refer to the majority of species in the genus of the taxon studied.
resolution it is only possible to suggest that there are three lineages descending from the common root: the Eulimnogammarus lineage, the Macrohectopus/G. pulex lineage, and the Paragarjajewia lineage. As argued earlier, the two major lineages are well supported when G. pulex and M. branickii are excluded from the analysis. However, in analyses including these two taxa, they appear to represent a significantly distinctive lineage (Bremer index 5 4). We suggest that separation of these three groups took place approximately simultaneously and should be considered a ‘‘crown’’ radiation. Phylogenetic Inference and Evolutionary Patterns in Baikalian Gammarids The first lineage (group 1, Fig. 1) contains more unarmed or ‘‘smooth’’ taxa lacking body teeth. However, four genera in this group comprise only armed species
that form a single cluster (Figs. 1–3). Since they are separated from the rest of armed taxa by several nodes on both the parsimony and maximum likelihood trees, and this separation is supported by maximum likelihood quartet puzzling (Fig. 3), it is likely that they have acquired their armament independently. All littoral species in our dataset belong to this lineage. The majority of species that comprise the rest of the genera in the first group occurs at depths of less then 200 m (Bazikalova, 1945). Only Ceratogammarus and Parapallasea are predominantly deep-water taxa. They belong to separate lineages in this group, indicating that adaptive radiation into deep-water habitats may have occurred independently several times in different lineages. Giant and dwarfed forms are peculiar to the Baikalian gammarid fauna. Except for Ceratogammarus and some species of the genus Pallasea, species
164
SHERBAKOV ET AL.
FIG. 2. The phylogenetic relationships of the Baikalian gammarids as estimated using maximum likelihood are identical to the phylogeny inferred from parsimony analyses of the same sequences (see Fig. 1). The bar below the tree corresponds to 0.1% substitutions per site. The outline drawings of the gammarids shown here (all studied taxa except Baikalogammarus) are not on the same scale and were modified and redrawn from Barnard and Barnard (1983), Kozhov (1963), or from references in Barnard and Barnard (1983).
from the first group never grow so big. Interestingly, the three dwarfed genera (Micruropus, Baikalogammarus, and Pachyschesis) also belong to different lineages within this group. Finally, it is interesting to note that the range of ecological specialization of the first group is exceptionally wide. It includes the two parasitic taxa in our dataset (Spinacanthus and Pachyschesis) as well as herbivorous and warm water resistant Micruropus (Bazikalova, 1962). Almost all genera appearing in the second major group are deep-water (.200 m) taxa (Fig. 1). Five of eight genera are characterized by their large body size
and the presence of highly developed and diverse armament of different shape, size, and position on the appendages (Plesiogammarus, Paragarjajewia, Garjajewia, Acanthogammarus, and Boeckaxellia). The members of this group share a set of distinctive morphological traits which support the 18S rRNA phylogeny. All members of this group have a cylindrical (often extremely prolonged) body shape and prolonged heads, sometimes with a sharp anterior head lobe. Plesiogammarus and Garjajewia share sharp carinae and secondary spines. Together with Paragarjajewia they bear medial tubercules. All former genera and Abyssogamma-
GAMMARID EVOLUTION IN LAKE BAIKAL
165
bers of a third distinctive clade of gammarids. It is surprising that the closest relative of the strictly pelagic M. branickii within our dataset appears to be not a Baikalian, but a palearctic taxon (confirmed by mitochondrial gene sequences; Ogarkov, unpublished results). The very specific adaptation of M. branickii to such a peculiar lacustrine habitat, and the fact that no transitory forms are known, suggests that M. branickii originated within the current Lake Baikal from a common ancestor with G. pulex. The earliest time that the typical Baikalian oxygenated pelagic zone appeared has been dated at the end of Pliocene (2.5 MYR, Mats, 1992; Logachev, 1993). It is therefore unlikely that this pelagic gammarid originated before this time. Extant freshwater Gammarus species are very tolerant to diverse environmental conditions and occur in water bodies adjacent to Lake Baikal, but not in the lake itself (Beckman, 1954). However, it is plausible that Gammarus-like species were able to invade the lake under much milder conditions, which lasted for a long period before the upper Pliocene (Mats, 1992). Gammarid Evolution and the History of Lake Baikal
FIG. 3. The phylogenetic relationships of the Baikalian gammarids were estimated with the maximum likelihood algorithm using the FastDNAml program (Olsen, 1994). The transition/ transversion ration was set to 2.0 and global rearrangements were allowed. The input order of taxa was changed until the tree topology with maximal support function value was obtained for three different sets. The branching order was evaluated with maximum likelihood quartet puzzling using program PUZZLE (Strimmer and von Haeseler, 1996; Strimmer et al. 1997) with base frequencies and transition/ transversion ratio estimated from the dataset. We applied the Hasegawa et al. (1985) model of base substitution and increased the number of puzzling to 5000.
rus have narrow bases of the first article in the peduncle of antenna 1 and short coxal plates (the coxal plates of Ommatogammarus are short or mediumsized). The three small and unarmed genera of this group (Ommatogammarus, Odontogammarus, and Abyssogammarus) form a single cluster. Two of these three taxa consist of few species which are highly specialized scavengers or fast swimming nectobenthic dwellers which may have lost their armament as an adaptation to their peculiar trophic preference or to a higher hydrodynamic requirements. Unlike the first group, the second group contains a distinctive lineage adapted to swimming rather than crawling, which is the typical mode of locomotion of most gammarids. In our study this lineage is represented by species of the genera Garjajewia, Paragarjajewia, and Plesiogammarus, all of which possess morphological adaptations for swimming or sliding above the bottom surface. M. branickii and G. pulex appear as the only mem-
The Baikalian gammarid fauna appears to be a complex species assemblage, whose age may be approximately as old as Lake Baikal itself. The Baikalian gammarid fauna—as represented by our dataset— probably consists of at least two evolutionarily independent lineages containing taxa with an exceptionally wide range of morphological features, some of which appear to have developed independently in both groups (Figs. 1 and 2). The evolutionary importance of the impact of dramatic changes of the Baikalian ecosystem in late Oligocene–early Pleistocene in the formation of current Baikalian fauna remains unclear. At that time Lake Baikal cooled and became superdeep and oxygenated throughout its whole water column. An evolutionary scenario suggests that these environmental changes caused the extinction of all (or nearly all) species that inhabited Lake Baikal at that time (Dorogastajski, 1923; Lukin, 1986). If this hypothesis is correct, the recent Baikalian fauna has evolved from the descendants of taxa that colonized the lake relatively recently. Indeed, age estimates, based on mitochondrial DNA sequences, show that the within-lake radiation of the Baikalian endemic sculpin fishes happened within the last 2.5 MY (Kirilchic et al., 1995; Slobodyanyuk et al., 1994). It is interesting to note that this group of fishes contains a pelagic species (Cottocomephorus) that— within 1 million years—differentiated to such an extent from other sculpins that it is regarded as belonging to a different family (Kirilchic et al., 1995). An alternative evolutionary scenario assumes that a number of taxa survived the changed environmental conditions in Lake Baikal and that these relict faunae gave rise to the extant species diversity (Vereshchagin, 1940; Kohzov,
166
SHERBAKOV ET AL.
1963). The genetic diversity, found in endemic Baikalian turbellarians, supports this hypothesis (Kuznedelov and Timoshkin, 1993, 1995). If future data allow us to establish a valid molecular clock, we can test the hypothesis that the Baikalian gammarid species assemblage also originated before the late Oligocene and early Pleistocene changes in the lake. In that case, M. branickii, which our data suggest is a relatively young species, must have evolved from a secondary introduction. Patterns of Intralacustrine Evolution It is interesting to note that the genus Gammarus— here represented by a single palearctic freshwater taxon—appears in the phylogeny inferred from our dataset as a member of the Baikalian gammarids. It is clear that the implications for the taxonomy of this genus are serious enough to merit further study. More important, however, is the fact that our data suggest that the degree of morphological divergence achieved over a comparable period of time differs dramatically between different Baikalian lineages. These two observations—which seem to characterize the evolution of the gammarid species flock of Lake Baikal—have also been made based upon studies on lacustrine species flocks of cichlid fishes. Indeed, as reported here, uneven rates of morphological evolutionary changes have been suggested to take place in lacustrine cichlid lineages (i.e., the more extensive morphological divergence among extremely young haplochromine cichlid fishes from Lake Victoria versus the ‘‘so-called’’ morphological stasis in the much more ancient cichlid genus Tropheus from Lake Tanganyika, see Sturmbauer and Meyer, 1992). Second, the short basal branches that characterize the molecular phylogenies obtained in the Baikalian gammarid species flock are similar to patterns observed in molecular phylogenies of Tanganyikan cichlid fishes (Sturmbauer et al., 1994). In both species flocks, these short basal branches might be indicative of rapid formation of lineages at the early stages of the intralacustrine evolution of these faunas. Finally, we suggest that the Baikalian gammarids may become a very suitable model group for studying adaptive radiation and morphological evolution in invertebrates. Once their phylogenetic relationships are fully established, the Baikalian gammarids will provide an attractive invertebrate model for studying mechanisms of morphological evolution to complement similar results on more extensively investigated vertebrate species flocks such as the Cichlidae of the Great Lakes of East Africa (e.g., Kocher et al., 1993; Meyer, 1993). ACKNOWLEDGMENTS We thank T. Backeljau, G. Chapelle, C. Debroyer, B. Goddeeris, M. A. Grachev, K. Martens, A. Meyer, L. Ru¨ber, C. Sturmbauer,
R. Va¨inola¨, and three anonymous referees for their constructive criticisms on earlier versions of this paper. D.S., R.K. and O.O. were partially supported by an INTAS-94-4465 grant; during his stay in Belgium, D.S. was supported by the Belgian Ministery of Science and Technology. During this study E.V. was funded by FKFO-MI Project 30-35. This study was carried out in the framework of the Baikal International Centre for Ecological Research (BICER).
REFERENCES Barnard, J. L., and Barnard, C. M. (1983). ‘‘Freshwater Amphipoda of the World,’’ Hayfield Assoc. Bazikalova, A. (1945). Les Amphipodes du Baikal. Acad. Sci. USSR, Proc. Baikal Limnol. Stn. 11: 1–440. Bazikalova, A. (1962). Sistematika, ekologiia i rasprostranenie rodov Micruropus Stebbing i Pseudomicruropus nov. gen. (Amphipoda, Gammaridae). Siberian Branch Acad. Sci. USSR, Proc. Limnol. Inst. 2: 3–140. Beckman, M. Y. (1954). The biology of Gammarus lacustris Sars. In ‘‘Water Bodies near Lake Baikal,’’ Vol. XIV., pp. 263–311, Siberian Branch Acad. Sci. USSR, Proc. Limnol. Inst. Bremer, K. (1988). The limits of amino acid sequence data in angiosperm phylogenetic reconstruction. Evolution 42: 795–803. Brooks, J. L. (1950). Speciation in ancient lakes. Q. Rev. Biol. 25: 30–60, 131–176. Dorogostajski, V. C. (1923). Vertical and horizontal distribution of fauna in Lake Baikal. In ‘‘Collected Works of Professors and Teachers of the Irkutsk University,’’ Vol. 4, pp. 103–131, Irkutsk. Doyle, J. J., and Sickson, E. (1987). Preservation of plant samples for DNA restriction endonuclease analysis. Taxon 36: 715–722. Dybowsky, B. N. (1874). ‘‘Beitrage su¨r naheren kenntnis der in dem Baikal-See vorkommenden niederen krebse aus der gruppe der gammariden.’’ Besobrasoff, Herausgegeben von der Russischen Entomologischen Gesellschaft zu St. Petersburg. Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791. Gerbi, S. A. (1985). Evolution of ribosomal DNA. In Molecular Evolutionary Genetics’’ (R. J. MacIntyre, Ed.), Plenum, Chap. 7, pp. 419–517, New York. Hasegawa, M., Kishino, H., and Yano, K. (1985). Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22: 160–174. Hillis, D. M., and Dixon, M. T. (1991). Ribosomal DNA: Evolution and phylogenetic inference. Q. Rev. Biol. 66: 411–453. Huelsenbeck, J. P. (1995). Performance of phylogenetic methods in simulation. Syst. Biol. 44: 17–48. Kallersjo, M., Farris, J. S., Kluge, A. G., and Bult, C. (1992). Skewness and permutation. Cladistics 8: 275–287. Kamaltynov, R. M. (1993). On the present state of amphipod systematics. Hydrobiol. J. 26: 82–92. Kamaltynov, R. M. (1995). ‘‘Phylogenetic Interrelationships of Baikalian Amphipods, p. 75, Abstracts of 2nd Vereshchagin Baikal Conf., Irkutsk. Kirilchik, S. V., Slobodyanyuk, S. Y., Belikov, S. L., and Pavlova, M. E. (1995). Phylogenetic interrelatedness of 16 species of the Baikal lake Cottoidei bullhead fishes deduced from partial nucleotide sequences of mtDNA cytochrome b gene. Mol. Biol. 29: 817–825. Kocher, T., Conroy, J., McKaye, K., and Stauffer, J. R. (1993). Similar morphologies of cichlid fishes in lakes Tanganyika and Malawi are due to convergence. Mol. Phylogenet. Evol. 2: 158–165. Kozhov, M. (1963). Lake Baikal and its life. In ‘‘Monogr. Biol.’’ (W. W. Weisbach and P. van Oye, Eds.), 11, Junk, The Hague, The Netherlands. Kuznedelov, K. D. (1995). ‘‘Phylogenetic Analysis of the Lake Baikal
GAMMARID EVOLUTION IN LAKE BAIKAL Turbellarians Based on the Comparison of 58-End Sequences of Gene Coding for 18S Ribosomal RNA.’’ Ph.D. thesis, Institute of Cytology and Genetics, Novosibirsk. Kuznedelov, K. D., and Timoshkin, O. A. (1993). Phylogenetic relationships of Baikalian species of Prorhynchidae turbellarian worms as inferred by partial 18S rRNA sequence comparisons. Mar. Mol. Biol. Biotechnol. 2: 300–307. Kuznedelov, K. D., and Timoshkin, O. A. (1995). Molecular phylogenetics of Turbellaria deduced from the 18S rRNA sequencing data. Mol. Biol. 29: 318–325. Logatchev, N. A. (1993). History and geodynamics of the Lake Baikal rift in the context of the Eastern Siberia rift system: A review. Bull. Cent. Rech. Explor. Prod. Elf Aquitaine 17: 353–370. Lukin, E. I. (1986). Fauna of the open waters in the Lake Baikal: Its peculiarities and origin. Zool. Zh. 65: 666–675. Martin, P. (1994). Lake Baikal. Adv. Limnol. 44: 3–11. Mats, V. D. (1992). ‘‘The Structure and Development of the Baikal Rift Depression’’ (D. E. Williams, Ed.), Baikal International Centre of Ecological Research, preprint. Meyer, A. (1993). Phylogenetic relationships and evolutionary processes in East African cichlid fishes. Trends Ecol. Evol. 8: 279–284. Ogarkov, O. B., Kamaltynov, R. M., Belikov, S. I., Sherbakov, D. Yu. (1997). Phylogenetic relatedness of the Baikal lake endemical amphipods (Crustacea, Amphipoda) deduced from partial nucleotide sequences of the cytochrome oxidase subunit III genes. Mol. Biol. 31(1): 24–29. [English translation] Olsen, G. J., Mastuda, H., Hagstrom, R., and Overbeek, R. (1994). FastDNAml, a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comp. Appl. Biosci. 10: 41–48. Slobodyanyuk, S. Y., Kirilchik, S. V., Pavlova, M. E., Belikov, S. I. and
167
Novitsky, A. L. (1994). The evolutionary relationships of two families of cottoid fishes of Lake Baikal (East Siberia) as suggested by analysis of mitochondrial DNA. J. Mol. Evol. 39: 1–8. Strimmer, K., and von Haeseler, A. (1996). Quartet puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13: 964–969. Strimmer, K., Goldman, N., and von Haeseler, A. (1997). Bayesian probabilities and quartet puzzling. Mol. Biol. Evol. 14: 210–211. Sturmbauer, C., and Meyer, A. (1992). Genetic divergence, speciation and morphological stasis in a lineage of African cichlid fishes. Nature 358: 578–581. Sturmbauer, C., Verheyen, E., and Meyer, A. (1994). Mitochondrial phylogeny of the Lamprologini, the major substrate spawning lineage of cichlid fishes from Lake Tanganyika in Eastern Africa. Mol. Biol. Evol. 11(4): 691–703. Swofford, D. L. (1993). ‘‘Phylogenetic Analysis Using Parsimony Version 3.1.1.’’ Illinois Natural History Survey, Champaign. 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 penalities and weight. Nucleic Acids Res. 22: 4673–4680. Timoshkin, O. A., Mekhanikova, I. V., and Shubenkov, S. G. (1995). Morphological peculiarities of Macrohectopus branickii. Pp. 485– 511. In ‘‘Guide and Key to Pelagic Animals of Baikal’’ (O. A. Timoshkin, Ed.), pp. 485–511, Nauka, Novosibirsk. Vereshchagin, G. Yu. (1940). Theoretical questions connected with the elaboration of the problem of the origin and history of Lake Baikal. T. Baik. Limnol. Stn. Acad. Sci. USSR 10: 1–772. Yampolsky, L. Y., Kamaltynov, R. M., Ebert, D., Filatov, D. A., and Chernyck, V. V. (1994). Variation of allozyme loci in endemic gammarids in Lake Baikal. Biol. J. Linn. Soc. 53: 309–323.
Vol. 10, No. 2, October, pp. 160–167, 1998 ARTICLE NO. FY970482
Patterns of Evolutionary Change in Baikalian Gammarids Inferred from DNA Sequences (Crustacea, Amphipoda) D. Yu. Sherbakov,* R. M. Kamaltynov,* O. B. Ogarkov,* and E. Verheyen† *Limnological Institute, Siberian Branch of Russian Academy of Sciences, P.O. Box 4199, 664033 Irkutsk, Russia; and †Taxonomy and Biochemical Systematics Section, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium Received December 6, 1996; revised October 28, 1997
The Baikalian gammarids (Crustacea, Amphipoda) are the most widely known and most spectacular example of an adaptive radiation among contemporary freshwater invertebrates. To study the phylogeny of the Baikalian gammarids we sequenced a 622-bplong fragment of the nuclear gene coding for 18S rRNA from species of 18 endemic Baikalian genera and Gammarus pulex—a non-Baikalian taxon. Some important morphological characters appear independently in both lineages and suggest parallelism in the development of gigantism and body armament. The first lineage comprises benthic, mostly unarmed taxa. The second lineage contains predominantly armed taxa, most of which are detrivorous or carnivorous. r 1998 Academic Press
INTRODUCTION Lake Baikal is the largest, deepest, and most ancient (ca. 28 MY old) of all existing great freshwater lakes (Mats, 1992; Logatchev, 1993). Its fauna is characterized by the presence of speciose and endemic species assemblages (Brooks, 1950; Kozhov, 1963, Martin, 1994). The Baikalian gammarids (Crustacea, Amphipoda) are the most widely known and most spectacular example of adaptive radiation among contemporary freshwater invertebrates (Brooks, 1950; Kozhov, 1963). About 30% (259 described species) of all known freshwater gammarid species of the world are endemic to Lake Baikal (Barnard and Barnard, 1983). Most of the 45 genera to which they are currently allocated are also endemic (Kamaltynov, 1993). This systematic diversity reflects the dramatic morphological differentiation within this species assemblage, which contrasts with the low amount of variation in the morphology of non-Baikalian freshwater gammarids. This group contains taxa that posess highly derived and diverse morphologies, some of which are ‘‘armed’’ with spiny structures (called body teeth) of different sizes, shape, and position on the body or appendages. Almost all known armed freshwater amphipods are found in Lake Baikal (Barnard and 1055-7903/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
Barnard, 1983). The Baikalian gammarids occupy the widest variety of ecological niches and are characterized by many highly specialized forms, ranging from the dwarfed herbivorous Micruropus to giant deepwater Abyssogammarus and Garjajewia, from the parasitic genera Pachyschesis and Spinacanthus to the pelagic Macrohectopus. The latter monotypic genus is unique for the whole order and was considered a separate family (Dorogostajski, 1923; Timoshkin et al., 1995). Although Baikalian gammarids have been studied for more than a century, the phylogenetic relationships within this group, as well as their relationships with non-Baikalian gammarids, remain unclear (Dybowsky, 1874; also references in Barnard and Barnard, 1983). Comparative morphological analysis (Bazikalova, 1945) and attempts to use allozymes (Yampolsky et al., 1994; Mechanikova pers. comm.) and mitochondrial DNA sequences (Ogarkov et al., 1997) did not result in a phylogenetic hypothesis for this species flock as a whole. This lack of phylogenetic knowledge has severely impeded studies of gammarid comparative biology and evolution. Despite the morphological diversity of Baikalian gammarids, few reliable characters exist for resolving their phylogeny (Kamaltynov, unpublished results). Since such phylogenies inferred from scanty and insufficiently understood morphological data create tautologies for studies attempting to understand the evolution of these characters, a phylogenetic hypothesis based either upon more morphological characters or on an independent set of characters is required. In order to obtain an overall picture of phylogenetic relationships among Baikalian endemic gammarids the present study uses partial 18S rRNA sequences. This gene is known to be evolutionary conservative and thus to be a potentially suitable tool for molecular phylogenetic analysis at generic and higher taxonomic levels (Hillis and Dixon, 1991; Gerbi, 1985), as illustrated by a recent study of Baikalian flatworms, which represent another major Baikalian invertebrate species assemblage (Kuznedelov and Timoshkin, 1993, 1995).
160
161
GAMMARID EVOLUTION IN LAKE BAIKAL
MATERIALS AND METHODS Species Studied and Gene Sequenced We amplified and sequenced a 622-bp-long fragment of 18S rRNA from 18 Baikalian amphipod species, belonging to 18 genera. These gammarids were selected to represent the whole taxonomical and ecological range of this group in Lake Baikal. All major groups of Baikalian gammarids, as described in the most recent taxonomical reviews (Kamaltynov, 1993, 1995), are represented in our dataset by at least one genus, and as a rule each genus is represented by one species (Table 1). Orthologous DNA sequences were also determined for Gammarus pulex, which is widely distributed throughout nearly the whole of northern Eurasia, but does not occur in Lake Baikal, and Tryphosella murrayi, a marine Antarctic nongammarid lysianassoid amphipod, which we used as outgroup. The Baikaian gammarids were collected during expeditions of the Baikal International Center for Ecological Research in 1995 and species determined using identification keys in Bazikalova (1945) and fixed in 75% ethanol. G. pulex was collected in Belgium (the Ardennes) by B. Goddeeris and determined by G. Petre. The T. murrayi specimen was provided by C. Debroyer. DNA Methods Whole animals or legs (depending on body size) were used for DNA extractions according to the CTAB/ chloroform procedure (Doyle and Sickson, 1987). The primers used for the amplification of the 18S rRNA fragment were 18S I: 58atgaccatcctagttggtccat-38; and 18S II: 58-ccacggtcgtcggcgccatta-38 (Kuznedelov, 1995). Approximately 0.15–0.20 µg of double-stranded PCR product was cycle-sequenced according to the manufacturer’s protocol (Pharmacia), using 0.8 µM primers and 2.5 units of Taq polymerase. Amplified DNA was sequenced from both ends with the same primers. The temperature profile for cycle sequencing was denaturation at 95°C for 36 s, annealing at 52°C (50°C for 18S I primer) for 36 s, and extension at 72°C for 80s and after 25 cycles the samples were kept at 72°C for 5 min. The obtained cycle-sequencing reaction products were analyzed on an ALF DNA sequencer (Pharmacia). The 18S rRNA sequences as well as their alignment have been submitted to EMBL under Accession numbers Z98982– Z99004. Phylogenetic Analyses All sequences were aligned using CLUSTAL W (Thompson et al., 1994), with adjustments by eye. Phylogenetic inferences based upon 18S rRNA sequences were obtained using parsimony and maximum likelihood methods since 18S rRNA sequences are known to evolve with uneven rates of nucleotide substitutions in different lineages, whereas mutation rates are also nonuniform between different positions in this gene (Hillis and Dixon, 1991).
Parsimony analysis was carried out with PAUP using unweighted characters (version 3.1.1, Swofford, 1993). Settings were: heuristic search, MULPARS and ACCTRAN options in effect, and gaps treated as the fifth character. The robustness of the phylogeny obtained was tested by bootstrapping (Felsenstein, 1985) after 100 replications, using ‘‘heuristic search’’ with the option ‘‘simple addition of taxa.’’ The Bremer support index for each node (Bremer, 1988; Kallersjo et al., 1992) was calculated with PAUP in connection with the program AUTODECAY, version 3.0 (Eriksson and Wikstro¨m, 1996). Phylogenetic relationships of the Baikalian gammarids were also estimated with the maximum likelihood algorithm using the FastDNAml program (Olsen et al., 1994). The transition/transversion ratio was set to 2.0 and global rearrangements were allowed. The input order of taxa was changed until the tree topology with maximal support function value was obtained for three different sets. The branching order was verified with maximum likelihood quartet puzzling using the program PUZZLE (Strimmer and von Haeseler, 1996; Strimmer et al., 1997) with base frequencies and the transition/transversion ratio estimated from the dataset. We applied the Hasegawa et al. (1985) model of base substitution and increased the number of puzzling steps until the resulting topology did not change (5000 puzzling steps). All the maximum likelihood algorithms treat alignment gaps as missing data. RESULTS Sequence Variation Of the 622 base pairs (bp) of the studied 18S rRNA fragment analyzed, 123 sites are variable within the Baikalian gammarids studied and G. pulex, whereas 50 positions are parsimony informative (Table 1). The highest (lowest) uncorrected sequence divergence between two gammarids is 8% (1%). Several base pair insertion/deletion events are inferred to align the sequences. As is typical for this gene, the mutation sites in the sequenced 18S rRNA DNA region are not randomly distributed, some regions containing more variable sequences. Pairwise comparison of transitions versus transversions indicates a bias toward transversions (TS/TV ratios are generally lower than 1). Finally, the overall base composition within the gammarids studied shows no bias in the 18S rRNA DNA region (A, 23.1%; T, 24.2%; C, 25.8%; and G, 26.9%). 18S rRNA Phylogeny of Baikalian Gammarids A single most parsimonious tree of 198 steps was obtained without character weighting (CI 5 0.485, RC 5 0.216, Fig. 1). The topologies of the majority rule consensus tree that are at least four steps longer than the most parsimonious tree is identical to the latter one (199 steps: 58 trees, CI 5 0.482; 200 steps: 1066 trees,
162
SHERBAKOV ET AL.
TABLE 1 Parsimony Informative Sites of the Partial 18S rRNA Sequences of the Baikalian Gammarids Gammarus pulex and Tryphosella murrayi 111111111 1111111222 2222222233 3333333344 4444445555 556 5002223567 7778889000 0037778881 1245568901 1233454577 770 0692340513 7890232124 5872461570 2295864100 5989264412 693 Ceratogammarus Pachysches Parapallasea Eulimnogammarus Spinacanthanthus Brandtia Pallasea Baikalogammarus Micruropus
CGATTAGAAT .........C .C.......C T........C ...A..C..C ...A.....C ...A.....C ...A.....C ...A......
Macrohectopus Gammarus
...A...TTC GG..GC.--. C.T....... T...G.C..C .G.GT.C.G. .C. ...A.....C GG...G.CT. C..A...T.- T.GTG.C..C .G.G..C... .C?
Odontogammarus Ommatogammarus Abyssogammarus Plesiogammarus Paragarjajewia Garjajewia Acanthogammarus Boeckaxellia
.T..AT...C ...--....C ..T.AT...C .C......TC .C.....TTC ......C..C ...A.....C .C.A.....C
Tryphosella
T.TA.....- --A..--G.. .......... -....TCT.. .GG.G....C -C.
ATTTTTTTGA T.AC...CTC G....GCG.. G..GGG.C.G G.AG..-..G G....G-..G G.....C..G GG...G.CT. C.G.--..T.
G..G..CG.G G.AGGG.... G....G.... .....G..T. T...?C.CAG G.......T. .....G...G G.........
TCCTCTTGGA C...G.C..T C...G.C... C...G..... C...G..... C......... C...G..... C.T.GC..AT CT.CGC..AT
C......C.. C......C.. ..T....C.. C......C.. C..C...... .......... .......... CT........
CCCCTGACCG .T....C.GA ..G...C.G. ...G..C.G. ......C.G. ......CTG. ..G...C.G. ..G...CGG. ......CTG.
..GGG.C..C .TGGG..... ....G.C... T..G..C... T.GG.TC... ...GG.C... ..GGG.C... ...GG.C...
GACCAGTGCG C.T.GT.... ..T.....GC .GT...C..C ..T.T..... ..T.T..... .GT.T..... ....T..... ..?.T.....
CG..T.C.GC TGT.T.C... CGT.TTC..C CGG.G.CC.A TGT.T.C..C CGT.T.CC.C CGT.T..-.C CGG.T..CGC
TGG ..? ... ..? .C. .C. .C? .C. .C.
.CT .CT ACT AC. AC? AC. .C. ...
Note. The length of the studied fragments including gaps introduced by alignment is 622 bp. The total alignment (including the outgroup taxon) contains 53 parsimony informative and 169 variable sites.
CI 5 0.480, 201 steps: 11988 trees, CI 5 0.478; 202 steps: 28461 trees, CI 5 0.475). Bremer decay indices, which are shown below branches, as well as bootstrap values, shown above branches, indicate low support for the two major lineages of gammarids. Nevertheless, the same branching pattern is obtained with respect to the split of the taxa into two major groups if maximum likelihood is applied (Fig. 2). To evaluate if this result supports the existence of these two lineages, the maximum likelihood tree was verified using quartet puzzling with the substitution model parameters estimated from the dataset and by applying the gamma correction for the differential base substitutions. The program employed retains only a tree(s) in which all partitions are significantly supported, otherwise it collapses nodes so that a multifurcated tree is constructed. The phylogeny obtained with this approach (Fig. 3) also contains the two lineages and a separate branch of M. branickii/G. pulex, and the ratio of trees where the two major lineages appear as separate clades becomes significant. DISCUSSION Molecular Analyses The low bootstrap values obtained for the lineages of the Baikalian gammarids species flock in the 18S rRNA parsimony tree (Fig. 1) are clearly too weak to give
significant support for the presence of the two major lineages. When the maximum likelihood algorithm implemented in the FastDNAml program is applied to our dataset, it yields essentially the same topology as the two major lineages (Fig. 2), the only exception being the relative position of the G. pulex/M. branickii lineage. The low bootstrap values as well as the low Bremer support index (51) are the result of the fact that relatively few substitutions define the branching order of these arising lineages. This is a particular problem in studies based upon genes or gene segments— such as the 18S rRNA gene fragment studied here— that contain slow and fast evolving sites. Although the slowly evolving sites of such genes are less prone to homoplasy, the number of substitutions per time unit may be insufficient to allow the resolving of rapidly evolving lineages. In contrast, the more rapidly evolving part of the same gene fragment may significantly increase the risk of homoplasies, another factor that may explain the low bootstrap values. The position of the lineage G. pulex/M. branickii differs in the maximum likelihood and the maximum parsimony trees. The results of the maximum likelihood quartet puzzling analysis (as well as the low bootstrapping values and decay indices) suggests that the branching order in the basal part of the tree cannot be resolved with our dataset. At the current level of
GAMMARID EVOLUTION IN LAKE BAIKAL
163
FIG. 1. The single most parsimonious 18S rRNA tree obtained for 18 Baikalian gammarids and one Palearctic species, using Tryphosella murrayi as outgroup. The numbers above the branches are bootstrap values after 100 replications. A number of important morphological and ecological characters appear independently in both lineages and suggest parallelism in the evolution of some of these features. The food preferences of Baikalian gammarids are not always very restricted; nearly all genera contain taxa with different food preferences (Kamaltynov, personal observation). The size classes listed here refer to the majority of species in the genus of the taxon studied.
resolution it is only possible to suggest that there are three lineages descending from the common root: the Eulimnogammarus lineage, the Macrohectopus/G. pulex lineage, and the Paragarjajewia lineage. As argued earlier, the two major lineages are well supported when G. pulex and M. branickii are excluded from the analysis. However, in analyses including these two taxa, they appear to represent a significantly distinctive lineage (Bremer index 5 4). We suggest that separation of these three groups took place approximately simultaneously and should be considered a ‘‘crown’’ radiation. Phylogenetic Inference and Evolutionary Patterns in Baikalian Gammarids The first lineage (group 1, Fig. 1) contains more unarmed or ‘‘smooth’’ taxa lacking body teeth. However, four genera in this group comprise only armed species
that form a single cluster (Figs. 1–3). Since they are separated from the rest of armed taxa by several nodes on both the parsimony and maximum likelihood trees, and this separation is supported by maximum likelihood quartet puzzling (Fig. 3), it is likely that they have acquired their armament independently. All littoral species in our dataset belong to this lineage. The majority of species that comprise the rest of the genera in the first group occurs at depths of less then 200 m (Bazikalova, 1945). Only Ceratogammarus and Parapallasea are predominantly deep-water taxa. They belong to separate lineages in this group, indicating that adaptive radiation into deep-water habitats may have occurred independently several times in different lineages. Giant and dwarfed forms are peculiar to the Baikalian gammarid fauna. Except for Ceratogammarus and some species of the genus Pallasea, species
164
SHERBAKOV ET AL.
FIG. 2. The phylogenetic relationships of the Baikalian gammarids as estimated using maximum likelihood are identical to the phylogeny inferred from parsimony analyses of the same sequences (see Fig. 1). The bar below the tree corresponds to 0.1% substitutions per site. The outline drawings of the gammarids shown here (all studied taxa except Baikalogammarus) are not on the same scale and were modified and redrawn from Barnard and Barnard (1983), Kozhov (1963), or from references in Barnard and Barnard (1983).
from the first group never grow so big. Interestingly, the three dwarfed genera (Micruropus, Baikalogammarus, and Pachyschesis) also belong to different lineages within this group. Finally, it is interesting to note that the range of ecological specialization of the first group is exceptionally wide. It includes the two parasitic taxa in our dataset (Spinacanthus and Pachyschesis) as well as herbivorous and warm water resistant Micruropus (Bazikalova, 1962). Almost all genera appearing in the second major group are deep-water (.200 m) taxa (Fig. 1). Five of eight genera are characterized by their large body size
and the presence of highly developed and diverse armament of different shape, size, and position on the appendages (Plesiogammarus, Paragarjajewia, Garjajewia, Acanthogammarus, and Boeckaxellia). The members of this group share a set of distinctive morphological traits which support the 18S rRNA phylogeny. All members of this group have a cylindrical (often extremely prolonged) body shape and prolonged heads, sometimes with a sharp anterior head lobe. Plesiogammarus and Garjajewia share sharp carinae and secondary spines. Together with Paragarjajewia they bear medial tubercules. All former genera and Abyssogamma-
GAMMARID EVOLUTION IN LAKE BAIKAL
165
bers of a third distinctive clade of gammarids. It is surprising that the closest relative of the strictly pelagic M. branickii within our dataset appears to be not a Baikalian, but a palearctic taxon (confirmed by mitochondrial gene sequences; Ogarkov, unpublished results). The very specific adaptation of M. branickii to such a peculiar lacustrine habitat, and the fact that no transitory forms are known, suggests that M. branickii originated within the current Lake Baikal from a common ancestor with G. pulex. The earliest time that the typical Baikalian oxygenated pelagic zone appeared has been dated at the end of Pliocene (2.5 MYR, Mats, 1992; Logachev, 1993). It is therefore unlikely that this pelagic gammarid originated before this time. Extant freshwater Gammarus species are very tolerant to diverse environmental conditions and occur in water bodies adjacent to Lake Baikal, but not in the lake itself (Beckman, 1954). However, it is plausible that Gammarus-like species were able to invade the lake under much milder conditions, which lasted for a long period before the upper Pliocene (Mats, 1992). Gammarid Evolution and the History of Lake Baikal
FIG. 3. The phylogenetic relationships of the Baikalian gammarids were estimated with the maximum likelihood algorithm using the FastDNAml program (Olsen, 1994). The transition/ transversion ration was set to 2.0 and global rearrangements were allowed. The input order of taxa was changed until the tree topology with maximal support function value was obtained for three different sets. The branching order was evaluated with maximum likelihood quartet puzzling using program PUZZLE (Strimmer and von Haeseler, 1996; Strimmer et al. 1997) with base frequencies and transition/ transversion ratio estimated from the dataset. We applied the Hasegawa et al. (1985) model of base substitution and increased the number of puzzling to 5000.
rus have narrow bases of the first article in the peduncle of antenna 1 and short coxal plates (the coxal plates of Ommatogammarus are short or mediumsized). The three small and unarmed genera of this group (Ommatogammarus, Odontogammarus, and Abyssogammarus) form a single cluster. Two of these three taxa consist of few species which are highly specialized scavengers or fast swimming nectobenthic dwellers which may have lost their armament as an adaptation to their peculiar trophic preference or to a higher hydrodynamic requirements. Unlike the first group, the second group contains a distinctive lineage adapted to swimming rather than crawling, which is the typical mode of locomotion of most gammarids. In our study this lineage is represented by species of the genera Garjajewia, Paragarjajewia, and Plesiogammarus, all of which possess morphological adaptations for swimming or sliding above the bottom surface. M. branickii and G. pulex appear as the only mem-
The Baikalian gammarid fauna appears to be a complex species assemblage, whose age may be approximately as old as Lake Baikal itself. The Baikalian gammarid fauna—as represented by our dataset— probably consists of at least two evolutionarily independent lineages containing taxa with an exceptionally wide range of morphological features, some of which appear to have developed independently in both groups (Figs. 1 and 2). The evolutionary importance of the impact of dramatic changes of the Baikalian ecosystem in late Oligocene–early Pleistocene in the formation of current Baikalian fauna remains unclear. At that time Lake Baikal cooled and became superdeep and oxygenated throughout its whole water column. An evolutionary scenario suggests that these environmental changes caused the extinction of all (or nearly all) species that inhabited Lake Baikal at that time (Dorogastajski, 1923; Lukin, 1986). If this hypothesis is correct, the recent Baikalian fauna has evolved from the descendants of taxa that colonized the lake relatively recently. Indeed, age estimates, based on mitochondrial DNA sequences, show that the within-lake radiation of the Baikalian endemic sculpin fishes happened within the last 2.5 MY (Kirilchic et al., 1995; Slobodyanyuk et al., 1994). It is interesting to note that this group of fishes contains a pelagic species (Cottocomephorus) that— within 1 million years—differentiated to such an extent from other sculpins that it is regarded as belonging to a different family (Kirilchic et al., 1995). An alternative evolutionary scenario assumes that a number of taxa survived the changed environmental conditions in Lake Baikal and that these relict faunae gave rise to the extant species diversity (Vereshchagin, 1940; Kohzov,
166
SHERBAKOV ET AL.
1963). The genetic diversity, found in endemic Baikalian turbellarians, supports this hypothesis (Kuznedelov and Timoshkin, 1993, 1995). If future data allow us to establish a valid molecular clock, we can test the hypothesis that the Baikalian gammarid species assemblage also originated before the late Oligocene and early Pleistocene changes in the lake. In that case, M. branickii, which our data suggest is a relatively young species, must have evolved from a secondary introduction. Patterns of Intralacustrine Evolution It is interesting to note that the genus Gammarus— here represented by a single palearctic freshwater taxon—appears in the phylogeny inferred from our dataset as a member of the Baikalian gammarids. It is clear that the implications for the taxonomy of this genus are serious enough to merit further study. More important, however, is the fact that our data suggest that the degree of morphological divergence achieved over a comparable period of time differs dramatically between different Baikalian lineages. These two observations—which seem to characterize the evolution of the gammarid species flock of Lake Baikal—have also been made based upon studies on lacustrine species flocks of cichlid fishes. Indeed, as reported here, uneven rates of morphological evolutionary changes have been suggested to take place in lacustrine cichlid lineages (i.e., the more extensive morphological divergence among extremely young haplochromine cichlid fishes from Lake Victoria versus the ‘‘so-called’’ morphological stasis in the much more ancient cichlid genus Tropheus from Lake Tanganyika, see Sturmbauer and Meyer, 1992). Second, the short basal branches that characterize the molecular phylogenies obtained in the Baikalian gammarid species flock are similar to patterns observed in molecular phylogenies of Tanganyikan cichlid fishes (Sturmbauer et al., 1994). In both species flocks, these short basal branches might be indicative of rapid formation of lineages at the early stages of the intralacustrine evolution of these faunas. Finally, we suggest that the Baikalian gammarids may become a very suitable model group for studying adaptive radiation and morphological evolution in invertebrates. Once their phylogenetic relationships are fully established, the Baikalian gammarids will provide an attractive invertebrate model for studying mechanisms of morphological evolution to complement similar results on more extensively investigated vertebrate species flocks such as the Cichlidae of the Great Lakes of East Africa (e.g., Kocher et al., 1993; Meyer, 1993). ACKNOWLEDGMENTS We thank T. Backeljau, G. Chapelle, C. Debroyer, B. Goddeeris, M. A. Grachev, K. Martens, A. Meyer, L. Ru¨ber, C. Sturmbauer,
R. Va¨inola¨, and three anonymous referees for their constructive criticisms on earlier versions of this paper. D.S., R.K. and O.O. were partially supported by an INTAS-94-4465 grant; during his stay in Belgium, D.S. was supported by the Belgian Ministery of Science and Technology. During this study E.V. was funded by FKFO-MI Project 30-35. This study was carried out in the framework of the Baikal International Centre for Ecological Research (BICER).
REFERENCES Barnard, J. L., and Barnard, C. M. (1983). ‘‘Freshwater Amphipoda of the World,’’ Hayfield Assoc. Bazikalova, A. (1945). Les Amphipodes du Baikal. Acad. Sci. USSR, Proc. Baikal Limnol. Stn. 11: 1–440. Bazikalova, A. (1962). Sistematika, ekologiia i rasprostranenie rodov Micruropus Stebbing i Pseudomicruropus nov. gen. (Amphipoda, Gammaridae). Siberian Branch Acad. Sci. USSR, Proc. Limnol. Inst. 2: 3–140. Beckman, M. Y. (1954). The biology of Gammarus lacustris Sars. In ‘‘Water Bodies near Lake Baikal,’’ Vol. XIV., pp. 263–311, Siberian Branch Acad. Sci. USSR, Proc. Limnol. Inst. Bremer, K. (1988). The limits of amino acid sequence data in angiosperm phylogenetic reconstruction. Evolution 42: 795–803. Brooks, J. L. (1950). Speciation in ancient lakes. Q. Rev. Biol. 25: 30–60, 131–176. Dorogostajski, V. C. (1923). Vertical and horizontal distribution of fauna in Lake Baikal. In ‘‘Collected Works of Professors and Teachers of the Irkutsk University,’’ Vol. 4, pp. 103–131, Irkutsk. Doyle, J. J., and Sickson, E. (1987). Preservation of plant samples for DNA restriction endonuclease analysis. Taxon 36: 715–722. Dybowsky, B. N. (1874). ‘‘Beitrage su¨r naheren kenntnis der in dem Baikal-See vorkommenden niederen krebse aus der gruppe der gammariden.’’ Besobrasoff, Herausgegeben von der Russischen Entomologischen Gesellschaft zu St. Petersburg. Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791. Gerbi, S. A. (1985). Evolution of ribosomal DNA. In Molecular Evolutionary Genetics’’ (R. J. MacIntyre, Ed.), Plenum, Chap. 7, pp. 419–517, New York. Hasegawa, M., Kishino, H., and Yano, K. (1985). Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22: 160–174. Hillis, D. M., and Dixon, M. T. (1991). Ribosomal DNA: Evolution and phylogenetic inference. Q. Rev. Biol. 66: 411–453. Huelsenbeck, J. P. (1995). Performance of phylogenetic methods in simulation. Syst. Biol. 44: 17–48. Kallersjo, M., Farris, J. S., Kluge, A. G., and Bult, C. (1992). Skewness and permutation. Cladistics 8: 275–287. Kamaltynov, R. M. (1993). On the present state of amphipod systematics. Hydrobiol. J. 26: 82–92. Kamaltynov, R. M. (1995). ‘‘Phylogenetic Interrelationships of Baikalian Amphipods, p. 75, Abstracts of 2nd Vereshchagin Baikal Conf., Irkutsk. Kirilchik, S. V., Slobodyanyuk, S. Y., Belikov, S. L., and Pavlova, M. E. (1995). Phylogenetic interrelatedness of 16 species of the Baikal lake Cottoidei bullhead fishes deduced from partial nucleotide sequences of mtDNA cytochrome b gene. Mol. Biol. 29: 817–825. Kocher, T., Conroy, J., McKaye, K., and Stauffer, J. R. (1993). Similar morphologies of cichlid fishes in lakes Tanganyika and Malawi are due to convergence. Mol. Phylogenet. Evol. 2: 158–165. Kozhov, M. (1963). Lake Baikal and its life. In ‘‘Monogr. Biol.’’ (W. W. Weisbach and P. van Oye, Eds.), 11, Junk, The Hague, The Netherlands. Kuznedelov, K. D. (1995). ‘‘Phylogenetic Analysis of the Lake Baikal
GAMMARID EVOLUTION IN LAKE BAIKAL Turbellarians Based on the Comparison of 58-End Sequences of Gene Coding for 18S Ribosomal RNA.’’ Ph.D. thesis, Institute of Cytology and Genetics, Novosibirsk. Kuznedelov, K. D., and Timoshkin, O. A. (1993). Phylogenetic relationships of Baikalian species of Prorhynchidae turbellarian worms as inferred by partial 18S rRNA sequence comparisons. Mar. Mol. Biol. Biotechnol. 2: 300–307. Kuznedelov, K. D., and Timoshkin, O. A. (1995). Molecular phylogenetics of Turbellaria deduced from the 18S rRNA sequencing data. Mol. Biol. 29: 318–325. Logatchev, N. A. (1993). History and geodynamics of the Lake Baikal rift in the context of the Eastern Siberia rift system: A review. Bull. Cent. Rech. Explor. Prod. Elf Aquitaine 17: 353–370. Lukin, E. I. (1986). Fauna of the open waters in the Lake Baikal: Its peculiarities and origin. Zool. Zh. 65: 666–675. Martin, P. (1994). Lake Baikal. Adv. Limnol. 44: 3–11. Mats, V. D. (1992). ‘‘The Structure and Development of the Baikal Rift Depression’’ (D. E. Williams, Ed.), Baikal International Centre of Ecological Research, preprint. Meyer, A. (1993). Phylogenetic relationships and evolutionary processes in East African cichlid fishes. Trends Ecol. Evol. 8: 279–284. Ogarkov, O. B., Kamaltynov, R. M., Belikov, S. I., Sherbakov, D. Yu. (1997). Phylogenetic relatedness of the Baikal lake endemical amphipods (Crustacea, Amphipoda) deduced from partial nucleotide sequences of the cytochrome oxidase subunit III genes. Mol. Biol. 31(1): 24–29. [English translation] Olsen, G. J., Mastuda, H., Hagstrom, R., and Overbeek, R. (1994). FastDNAml, a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comp. Appl. Biosci. 10: 41–48. Slobodyanyuk, S. Y., Kirilchik, S. V., Pavlova, M. E., Belikov, S. I. and
167
Novitsky, A. L. (1994). The evolutionary relationships of two families of cottoid fishes of Lake Baikal (East Siberia) as suggested by analysis of mitochondrial DNA. J. Mol. Evol. 39: 1–8. Strimmer, K., and von Haeseler, A. (1996). Quartet puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13: 964–969. Strimmer, K., Goldman, N., and von Haeseler, A. (1997). Bayesian probabilities and quartet puzzling. Mol. Biol. Evol. 14: 210–211. Sturmbauer, C., and Meyer, A. (1992). Genetic divergence, speciation and morphological stasis in a lineage of African cichlid fishes. Nature 358: 578–581. Sturmbauer, C., Verheyen, E., and Meyer, A. (1994). Mitochondrial phylogeny of the Lamprologini, the major substrate spawning lineage of cichlid fishes from Lake Tanganyika in Eastern Africa. Mol. Biol. Evol. 11(4): 691–703. Swofford, D. L. (1993). ‘‘Phylogenetic Analysis Using Parsimony Version 3.1.1.’’ Illinois Natural History Survey, Champaign. 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 penalities and weight. Nucleic Acids Res. 22: 4673–4680. Timoshkin, O. A., Mekhanikova, I. V., and Shubenkov, S. G. (1995). Morphological peculiarities of Macrohectopus branickii. Pp. 485– 511. In ‘‘Guide and Key to Pelagic Animals of Baikal’’ (O. A. Timoshkin, Ed.), pp. 485–511, Nauka, Novosibirsk. Vereshchagin, G. Yu. (1940). Theoretical questions connected with the elaboration of the problem of the origin and history of Lake Baikal. T. Baik. Limnol. Stn. Acad. Sci. USSR 10: 1–772. Yampolsky, L. Y., Kamaltynov, R. M., Ebert, D., Filatov, D. A., and Chernyck, V. V. (1994). Variation of allozyme loci in endemic gammarids in Lake Baikal. Biol. J. Linn. Soc. 53: 309–323.