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Molecular Phylogenetics and Evolution Vol. 19, No. 1, April, pp. 105-113, 2001 doi:10.1006/mpev.2000.0901, available online at

http://www.idealibrary.com on

The Phylogenetic Relationships of "Predatory Water-Fleas" (: , Haplopoda) Inferred from 12S rDNA

Stefan Richter,* Anke Braband,* Nikolai Aladin,** and Gerhard Scholtz*

*Vergleichende Zoologie, Institut für Biologie, Humboldt-Universität zu Berlin, Philippstr. 13, 10115 Berlin, Germany **Zoological Institute, Russian Academy of Science, Universitetskaya nab., 1, 199034 St. Petersburg, Russian Federation

Received April 25, 2000 Within the Cladocera, the water-fleas, four major taxa can be distinguished: , , Haplopoda, and Onychopoda. Haplopoda and Onychopoda are called "predatory water-fleas." The Haplopoda is monotypic; its only representative, kindtii, is common in palearctic and nearctic freshwater bodies. The Onychopoda show a remarkable geographic distribution. Most of the described are restricted to the , the , and peripheral areas of the Black Sea, including the -all remnants of the Eastern Paratethys. The remaining onychopods are either freshwater inhabitants or marine , widespread in the world . We present molecular evidence for a sister group relationship between Haplopoda and Onychopoda within the Cladocera. The Onychopoda and its three families are monophyletic. We suggest an independent invasion into the Ponto-Caspian basin at least three times, twice originating in the palearctic freshwater bodies and once starting from the world oceans. © 2001 Academic Press

Introduction The Cladocera, the water-fleas, is a highly diverse group which plays an important role in the ecology of freshwater habitats. In contrast to other branchiopod taxa (, , and Conchostraca) whose representatives are inhabitants mainly of temporary waters, most cladoceran species live in permanent freshwater pools, and few species live in the sea. Four higher taxa within the Cladocera can be distinguished: Ctenopoda (e.g., , ), Anomopoda (e.g., , Chydorus, ), Haplopoda (Leptodora kindtii), and Onychopoda (e.g., , Bytho-trephes, ). The monophyly of the entire Cladocera has been doubted (Schminke, 1981, 1996; Fryer, 1987) but some recent analyses based on morphological and molecular data support a monophyletic Cladocera (Walossek, 1993; Martin and Cash-Clark, 1995; Olesen, 1998; Negrea et al, 1999; Spears and Abele, 2000; Braband et al., 2000). The phylogenetic relationships within the Cladocera are also a matter of debate. Martin and Cash-Clark (1995) suggested a sister group relationship between the Onychopoda and the mono-typic Haplopoda. To this taxon the Anomopoda is the sister group and the Ctenopoda is the sister group to all other Cladocera. A sister group relationship between Haplopoda and Onychopoda, united as Gymnomera (originally introduced in 1867 by G.O. Sars), is also favored by Olesen (1998) and is supported by 16S rDNA data (Schwenk et al., 1998) and by 18S rDNA data (Taylor et al., 1999). This is in contrast to the view that the Haplopoda with free-living larvae (metanau-plius) is separated from all other cladocerans with direct development, which are united as Eucladocera (e.g., Eriksson, 1934; Wingstrand, 1978; Bowman and Abele, 1982; Gruner, 1993; Negrea et al, 1999). Interestingly enough, even this view has been supported by 18S rDNA data (Spears and Abele, 2000). Here we want to focus on the relationships of and within the Onychopoda. Two groups of the Cladocera, the monotypic Haplopoda and the Onychopoda, are often called "predatory" in contrast to filter-feeders (e.g., Rivier, 1998), although the term "macrophagous" would be more appropriate because some Onychopoda prefer to "predate" algae. The monophyly of the Ony- chopoda has been suggested based on morphological data (Martin and Cash-Clark, 1995; Olesen, 1998) but no molecular analysis so far comprises at least one representative of each of the three families within the Onychopoda (Polyphemidae, , ). Within the Onychopoda, Martin and Cash-Clark (1995) suggested a sister group relationship between Cercopagididae and Podonidae. Many representatives of the Onychopoda possess a remarkable general appearance with extensions of head and carapace valves (Fig. 1) but the geographic distribution of the entire taxon is equally remarkable. Thirteen of 14 species of the family Cercopagididae, 1 of 2 species of the Polyphemidae, and 9 of 17 species of the Podonidae (species numbers according to Rivier (1998)) are restricted to the Ponto-Caspian basin, comprising the Black Sea, Caspian Sea, and Aral Sea, all remnants of the Eastern Paratethys. The remaining species of the Cercopagididae, Bythotrephes longima-nus, and the Polyphemidae, , are inhabitants of holarctic freshwater bodies (Bythotrephes was just recently introduced into ; see Martin and Cash-Clark, 1995), whereas the remaining Podonidae are marine (with one exception in lake Hazar Gölü in Turkey; Spandl, 1923), common in most parts of the world oceans (distribution according to Mordukhai-Boltovskoi and Rivier, 1987; Rivier, 1998; Onbe, 1999). The distribution of the Onychopoda in the Ponto-Caspian basin is complex. Simplified, one group of species occurs in the Caspian Sea, Sea of Azov, and other peripheral parts of the Black Sea with relatively low salinity, e.g., the Danube, Dniester, and Dniepr estuaries; a second group is common either only in the Caspian Sea or also in the Aral Sea. In the central part of the Black Sea with a relatively high salinity, the same podonid species occur as in the Med- iterranean Sea (Fig. 2) (Mordukhai-Boltovskoi and Rivier, 1987; Rivier, 1998). More recently, Pleopis polyphemoides also invaded the Caspian Sea via the Volga-Don canal (Mordukhai- Boltovskov, 1962).

This distribution provokes the question of the evolutionary origin of the onychopod cladocerans. The Ponto—Caspian basin—and in particular the Caspian Sea—has been claimed to be the center of adaptive radiations for many taxa such as molluscs, , and (Dumont, 1998b). For instance, an origin in the Ponto-Caspian basin followed by multiple immigrations into freshwater has been suggested for the European freshwater crayfish species (Astacidae) (Karaman, 1962; but see Scholtz, 1995, for a different opinion). Also for the Onychopoda—and in particular for the Podonidae—the role of the Ponto—Caspian basin for the recent distribution has been discussed over the last 100 years (Sars, 1902; Gibitz, 1922; Mordukhai-Boltovskoi, 1966; Mordukhai-Boltovskoi and Rivier, 1987; Potts and Durning, 1980; Aladin, 1995; Rivier, 1998; Dumont, 1998a). In the present study we analyzed the relationships within and of the Onychopoda using a partial sequence of the 12S rDNA. The monophyly of the Gymnomera, Onychopoda, Cercopagididae, and Podonidae is supported. The results are discussed with respect to geological changes in the area of the Paratethys.

Materials and Methods

Taxonomic Sampling We included 11 representatives of the Cladocera Onychopoda, 2 of the Cladocera Ctenopoda, 2 of the Cladocera Anomopoda, the only recent representative of the Cladocera Haplopoda, and 1 representative of the Conchostraca Spinicaudata as definitive outgroup in this study (Table 1). The Ponto-Caspian onychopods were collected in the northern part of the Caspian Sea, off Zhemtschuzhnyi Island, the marine podonid species (Podon leuckarti, P. intermedius, nordmanni) in the North Sea, off Sylt and Helgoland, and all other cladocerans in freshwater bodies in Berlin or the environments of Berlin. Imnadia yeyetta was collected in the Marchauen, close to Vienna, Austria. All specimens were preserved in 100% ethanol and stored at 4°C.

Sample Preparation Genomic DNA from up to 20 individuals per species was isolated using a 4% СТАВ extraction (Doyle and Doyle, 1990) in a declined procedures. The homogeni-zation procedure was performed directly in СТАВ buffer with grains of sea sand. Selective amplification was carried out for a portion of the mitochondrial gene 12s rDNA (SSU) by polymerase chain reaction (PCR) using the primers 12CR3 (5'-ACTTTGTTACGACT-TATCTC-3') and 12s-5c (5'- GCCAGCCGTCGCGGT-TAGAC-3'). Both PCR primers were extended at the 5' end with the sequences of the primers M13 forward and M13 reverse. PCR conditions were 40 cycles with 94°C/30 s denaturing, 40-42°C/45 s annealing, and 72°C/45 s extensions. PCR products were gel-purified and concentrated using microconcentrators (Microcon 50). PCR products were directly cycle-sequenced by dideoxy chain termination using fluorescein-labeled M13 forward and M13 reverse primers. Sequencing gels were run on ALF sequencers (Pharmacia).

Phylogenetic Analysis In general, alignment is the first crucial step in the analysis of rDNA sequences that has a major influence on the results of the phylogenetic analysis. In a cladis-tic framework the best alignment is that which yields the most parsimonious cladogram. We used the direct optimization procedure (Wheeler, 1996) as implemented in the program Poy (Gladstein and Wheeler, 1996-2000) using parsimony as optimality criterion. Direct optimization combines both alignment and phylogenetic analysis in one step, which guarantees a consistent use of analysis assumptions in the entire analysis (Philipps et al., 2000). The key difference between direct optimization and multiple alignment is that evolutionary differences in sequence length are accommodated not by the use of gap characters but by allowing insertion—deletion events. As preferred weighting scheme we weighted substitutions 1 (transitions and transversions equally weighted) and indels 2. In a sensitivity analysis (Wheeler, 1995) six other weighting schemes were also used (transition:transversion:in-dels, 1:2:2, 1:1:4, 1:2:4, 1:2:8, 1:1:7, 1:1:10). Each analysis was carried out using the options "norandomize-outgroup," "maxtrees 10," "fitchtrees," "multibuild 10," "checkslop 10," and "random 100." This includes 10 sequence random additions without branch swapping, SPR and TBR branch swapping based on the shortest tree of these 10 calculations, and an extra TBR branch swapping round, checking all cladogram lengths that are within 1% of the current minimum value. Using the option "impliedalignment," a topology-specific multiple alignment was generated under the preferred weighting scheme (see Appendix). This alignment was transformed into a Phast-file (Goloboff, 1993-2000) and calculated using "autocal," "hold 1000," "mult* 20" (same weighting scheme as in Poy). Also, the decay index (Bremer, 1994) was calculated with Phast ("hold 1000," "bs 20"). A bootstrap analysis (Felsenstein, 1985) was carried out with Nona (Goloboff, 1993-2000) using the same alignment (1000 replications, "mult* 10," "hold/10," "tbr"). Winclada (Nixon, 1999-2000) was used as shell program. Results The calculation under our preferred weighting scheme (ts:tv:indel = 1:1:2) resulted in one most- parsimonious tree (amb-) with a length of 646 steps (181 parsimony informative characters counted in Winclada) (Fig. 3). The calculation of Phast resulted in the same most-parsimonious trees with 646 steps. All clades apart from (Simocephalus-Daphnia) (not supported under assumptions 1:1:7 and 1:1:10) are also supported by all other weighting schemes used.

Our analysis is in agreement with the Gymnomera concept, which suggests a sister group relationship between Haplopoda and Onychopoda but the support is relatively weak (decay index 2; bootstrap value 57%). The monophyly of the Onychopoda and the monophyletic origin of the Cercopagididae and Podonidae are well supported. Only one representative of the Polyphemidae was included in our analysis. Cercopagididae and Podonidae are sister groups and the Polyphemidae is the sister group of both. Within the Podonidae, the two species of the exclusively marine Podon form the most basal clade. The three Podonevadne species (P. trigona, P. camptony, and P. angusta) and Cornigerius maeoti-cus form a monophyletic clade that cannot be resolved with the gene fragment studied. The sister group of this clade is represented by the two Evadne species. Most of the clades within the Podonidae are well supported, except for the Evadne clade, which shows a low decay index (2) and a relatively low bootstrap value (73%).

Discussion In addition to morphological cladistic studies (Martin and Cash-Clark, 1995; Olesen, 1998) and molecular analyses based on 16S rDNA data (Schwenk et al., 1998) and 18S rDNA data (Taylor et al., 1999), our 12S rDNA data also support a sister group relationship between Haplopoda (Leptodora kindtii) and Onychopoda and therefore a monophyletic Gymnomera. Although the Sida-Diaph-anosoma and Daphnia-Simocephalus clades are well supported, it is not justified to infer monophyly for the Ctenopoda or Anomopoda. The taxon sampling is too restricted and some crucial taxa of these groups are not considered (e.g., Holopedium for the Ctenopoda or the for the Anomopoda). The monophyly of the Onychopoda and of its three families is supported by our analysis. The sister group relationship between Cercopagididae and Podonidae, as has been proposed by Martin and Cash-Clark (1995), is also supported by our molecular data.

One of the most interesting questions of our analysis concerns the habitat of the different onychopod taxa. Unfortunately, different possibilities are equally parsimonious under the assumption of equal probability of changing the habitat; i.e., the interpretation depends on the optimization mode. Therefore, our suggestions are based not only on the here-presented phylogenetic relationships but also on a tentative morphological phylogenetic analysis including the podonid genera Pleopis and Pseudoevadne (unpublished) and geological data. We suggest several independent invasions into the Ponto—Capian basin—separate in each of the onychopod families (Fig. 4). Quite probable is that there were two invasions from freshwater. One of these took place within the Cercopagididae. As a freshwater inhabitant, represents the ancestral condition. The ancestor of the representatives of Cercopagis invaded the Ponto-Caspian basin. A second invasion from freshwater is suggested for the ancestor of the Caspian polyphemid species P. exiguus. Although this species is not included in our analysis, a sister group relationship to the freshwater species Polyphemus pediculus is strongly suggested based on morphological correspondences (Mordukhai-Boltov-skoi, 1968); as a matter of fact, it cannot be excluded that P. exiguus is only a different morphotype from P. pediculus (Aladin et ah, 1999). The interpretation of phylogenetic relationships within the Podonidae with respect to habitats is more complicated. There is no doubt that the early ancestors of the Podonidae lived in freshwater like almost all other Cladocera. The crucial question is whether the freshwater ancestors invaded the Ponto—Caspian, and then later the world oceans, or whether they directly conquered the world oceans from freshwater and later the Ponto-Caspian basin. Both possibilities have been suggested by several authors. Due to the already mentioned fact that almost all other cladocerans are freshwater inhabitants, it has been claimed that freshwater ancestors immigrated into the Ponto-Caspian basin and that much later, after a species radiation, the most salt-tolerant species expanded to the world oceans through the Bosporus (Aladin, 1995; Dumont, 1998a). Dumont (1998a) supposed that the latter happened during its most recent opening in the Holocene. On the other hand, Gibitz (1922), Mordukhai-Boltovskoi and Rivier (1987), and Potts and Durning (1980) argued for an origin of the Ponto-Caspian Podonidae in the world oceans. We think that the latter is the more plausible explanation. A good candidate for the time of the podonid invasion into the Ponto-Caspian is the Agchagylian transgression (3.4-2 myr ago) when Eastern Paratethys was reconnected with the world oceans (Degens and Paluska, 1979; Jones and Simmons, 1996). During this time marine was introduced into the Ponto—Caspian basin (Jones and Simmons, 1996). However, after this event, the Ponto— Caspian basin freshened again and only a brackish water fauna could survive (Jones and Simmons, 1996). Some of these fauna representatives were possibly the ancestors of the Ponto—Caspian podonids of the genera Cornigerius, Podonevadne, Caspievadne, and the Cas- pian Evadne species. After this major Agchagylian transgression period, beginning with the Aspheronian (2-0.7 myr ago, which was according to Jones and Simmons (1996) essentially regressive in character), several high and low stands of the Ponto-Caspian basin were linked to the glaciation cycles (Dumont, 1998b). The invasions from freshwater within the Cer- copagididae and Polyphemidae probably occurred in one of these transgression periods when smaller freshwater bodies (lakes and rivers) were flooded by the Caspian and Black Seas. At this time, both "seas" were freshwater bodies at least in their major parts (Dumont, 1998b). Due to the fact that occurs in the Caspian Sea and the Sea of Azov, this invasion probably happened before these water bodies became separated. This was about 77,000 years ago (Yakovlev, 1956). One could argue that this period (between about 2 myr and some 100,000 years ago) is not long enough for a radiation of the 13 Cercopagis species, but the species number is very likely to be overestimated (Aladin, 1995; Aladin et al., 1999). Only very recently, long after the loss of the connection between the Black Sea and the Caspian Sea, the connection between the Mediterranean Sea and the Black Sea opened again. This Holocenian event—recently interpreted as Noah's flood (Ryan and Pitman, 2000)— about 7150 years ago, resulted in a rapid increase in the water level of the Black Sea (Ryan et al., 1997; Demirbag et al, 1999). Also, the salinity of the central part of the Black Sea increased and marine species from the Mediterranean Sea invaded this region (Schrader, 1979). We suggest that podonid species (our group 3) also were involved in this invasion. The brackish water Ponto—Caspian endemics, which had lived in the freshened Black Sea before, were displaced to the Sea of Azov and other peripheral parts of the Black Sea with a lower salinity.

Acknowledgments We are grateful to Dr. Vasili Petuchov, Dr. R. Saborowski, and E. Eder for theis assistance in collecting specimens. We are thankful to Replicon GmbH, which allowed the complete DNA work to be carried out in its lab. We thank Dr. B. Schröder for support with geological interpretations and for providing literature, Dr. J. Olesen for comments on the manuscript, Dr. S. Rogaschewski for support during the SEM study, and I. Drescher for translations from Russian into German. The financial support by the Deutsche Forschungsgemein-schaft to S.R. and G.S. (436 RUS 18/15/98; DFG Scho 442/6-1) and the financial support to N.A. (Russian Fund for Basic Research 96-04-48114) is gratefully acknowledged.

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