Comparative Phylogeography of Ponto-Caspian Mysid Crustaceans

Molecular Ecology (2006) 15, 2969–2984 doi: 10.1111/j.1365-294X.2006.03018.x

ComparativeBlackwell Publishing Ltd phylogeography of Ponto-Caspian mysid crustaceans: isolation and exchange among dynamic inland sea basins

ASTA AUDZIJONYTE,*† MIKHAIL E. DANELIYA*‡ and RISTO VÄINÖLÄ* *Finnish Museum of Natural History, POB 26, FI-00014 University of Helsinki, Finland, †Department of Biological and Environmental Sciences, FI-00014 University of Helsinki, Finland, ‡Department of Zoology, Rostov State University, B. Sadovaya 105, Rostov-on-Don, 344006, Russia

Abstract The distributions of many endemic Ponto-Caspian brackish-water taxa are subdivided among the Black, Azov and Caspian Sea basins and further among river estuaries. Of the two alternative views to explain the distributions, the relict school has claimed Tertiary fragmentation of the once contiguous range by emerging geographical and salinity barriers, whereas the immigration view has suggested recolonization of the westerly populations from the Caspian Sea after extirpation during Late Pleistocene environmental perturbations. A study of mitochondrial (COI) phylogeography of seven mysid crustacean taxa from the genera Limnomysis and Paramysis showed that both scenarios can be valid for different species. Four taxa had distinct lineages related to the major basin subdivision, but the lineage distributions and depths of divergence were not concordant. The data do not support a hypothesis of Late Miocene (10–5 Myr) vicariance; rather, range subdivisions and dispersal from and to the Caspian Sea seem to have occurred at different times throughout the Pleistocene. For example, in Paramysis lacustris each basin had an endemic clade 2–5% diverged from the others, whereas Paramysis kessleri from the southern Caspian and the western Black Sea were nearly identical. Species-specific ecological characteristics such as vagility and salinity tolerance seem to have played important roles in shaping the phylogeographic patterns. The mitochondrial data also suggested recent, human-mediated cryptic invasions of P. lacustris and Limnomysis benedeni from the Caspian to the Sea of Azov basin via the Volga-Don canal. Cryptic species-level subdivisions were recorded in populations attributed to Paramysis baeri, and possibly in P. lacustris. Keywords: Caspian Sea, cytochrome oxidase I (COI), Limnomysis, Mysida, Paramysis, zoogeography Received 26 January 2006; revision received 29 April 2006; accepted 9 May 2006

ancient lakes has been a topic of much discussion (Rossiter Introduction & Kawanabe 2000). For the Ponto-Caspian, potential factors The history and diversity of aquatic biota in the Ponto- promoting species divergence involve its wide environ- Caspian basin, which encompasses the Caspian, Azov and mental fluctuations and the intermittent interbasin subdivi- Black seas, is attracting scientific interest at least for two sions and connections (Dumont 1998). More recently, the reasons. The Caspian Sea is one of the ancient lakes of the Ponto-Caspian brackish-water fauna has come into focus world; it has been effectively separated from oceans for as a major source of aquatic invading species in Europe c. 7 million years (Myr) and is currently characterized by and North America (Ricciardi & MacIsaac 2000; Leppäkoski 50–80% endemism of the fauna (Martens 1997; Dumont et al. 2002; Jazdzewski et al. 2004). Molecular tools have 2000). The geography and mechanisms of speciation in been invoked to trace the sources and pathways of invasions (Cristescu et al. 2001, 2004), but their successful Correspondence: Asta Audzijonyte, Fax: +358-9-19144430; E-mail: application is contingent on proper taxonomy and genetic [email protected] characterization of the populations in the native ranges.

2970 A. AUDZIJONYTE, M. E. DANELIYA and R . VÄINÖLÄ

Recent invasions also threaten the Ponto-Caspian region Caspian zoogeography between proponents of the immigra- itself, particularly the Caspian Sea, which since the opening tion and relict views. of the Volga-Don canal in the 1950s has received a wave The immigration view emphasizes the role of drastic of nonindigenous species (Grigorovich et al. 2003; Orlova Pleistocene environmental perturbations that must have et al. 2004; Therriault et al. 2005). caused repeated local extirpation in the western basins but The southern and central parts of the Black and Caspian also facilitated faunal exchange (Birshtein 1935; Mordukhai- seas have markedly different faunas — the Black Sea (18‰ Boltovskoi 1960, 1979; Dedyu 1967). For example, during S) is inhabited by typical marine (Mediterranean) taxa, the last 1 Myr the Black Sea salinity has fluctuated from whereas the Caspian Sea (13‰) has many endemic groups nearly oceanic during contacts with the Mediterranean, (Zenkevitch 1963; BÅnÅrescu 1991). In contrast, the diluted to almost freshwater during glaciation maxima and these northern areas of the seas and the lower reaches of their events were coupled with c. 150 m changes in the water rivers contain a largley shared brackish-water fauna, level; the shallow Sea of Azov was nearly dry during comprising, among others, numerous species of crusta- the low stands (Fig. 1b) (Alekseev et al. 1986; Svitoch et al. ceans (in the Mysida, Amphipoda, Cumacea, Cladocera) 2000). During the cold climatic phases of the Middle and and molluscs (in Dreissenidae, Cardiidae, Pyrgulidae). Late Pleistocene the Caspian Sea experienced extensive These taxa typically occur in salinities lower than 6‰ and transgressions, which caused overflow of water to the Azov show relatively little taxonomic differentiation among basin via the Manych depression; the latest event was the three basins (Fig. 1a, Table 1) (Mordukhai-Boltovskoi c. 15 000 years ago (Mangerud et al. 2004; Bahr et al. 2005). 1979; Komarova 1991; Daneliya 2003). The history of their Repeated colonizations from the Caspian basin during the disjunct distributions has been an issue of debate in Ponto- Pleistocene were believed to be the prime source of the

Fig. 1 (a) Map of the Ponto-Caspian region indicating sampling sites and their corresponding codes as listed in Table 2. (b) Schematic presentation of Late Pleisto- cene history of the Black, Azov and Caspian seas (according to Mamedov 1997; Ryan et al. 2003; Mangerud et al. 2004). Dashed lines depict limits of the lakes and rivers during the latest regression stages. Limits of the last extensive Caspian tran- sgression are also shown together with the direction of the outflow to the Azov basin along the Manych Strait. Saline water intru- sions into the Black Sea occurred from the Marmara Sea in the course of a repeatedly established connection through the Bosporus Strait. The Volga-Don canal, opened in 1952, is recognized as a current route of faunal exchange and an invasion corridor.

PONTO-CASPIAN MYSIDS 2971

Table 1 Sizes at maturity and some ecological characteristics of the analysed species. Abbreviations: BLA, Black Sea; AZO, Sea of Azov; CAS, Caspian Sea; RIV, indicates natural occurrences in rivers > 1000 km upstream (records from the early 20th century). Data from Buchalova (1929), Mordukhai-Boltovskoi (1957), Komarova (1991), Daneliya (2003)

Natural distribution Size, Taxon mm BLA AZO CAS RIV Salinity, ‰ Habitats

Limnomysis benedeni 6–12 + + + – 0–12 estuaries, river deltas, limans; common (Czerniavsky, 1882) among vegetation Paramysis lacustris 8–17 + + + – 0–2 estuaries, river deltas, limans; common (Czerniavsky, 1882) Paramysis sowinskii 8–17 – + + – 0–5 estuaries, river deltas; mid-channel; Daneliya, 2002 oxyphilic Paramysis baeri 13–31 + + + + 0–9 rivers, estuaries; oxyphilic Czerniavsky, 1882 sensu lato Paramysis ullskyi 12–23 + + + + 0–4 rivers, estuaries; sandy bottoms; Czerniavsky, 1882 oxyphilic Paramysis kessleri 15–52 + – + – 0–9 estuaries; deep waters of central and G.O. Sars, 1895 southern Caspian and western Black Sea Paramysis intermedia 6–13 + + + – 0–12 estuaries, river deltas; rare (Czerniavsky, 1882)

current brackish-water fauna of the Black Sea and Sea 1976), whereas in mysid crustaceans the earlier taxonomic of Azov basins, thus referred to as the Caspian fauna splitting of the disjunct populations (Martynov 1924) has (e.g. Mordukhai-Boltovskoi 1979; Reid & Orlova 2002). been dismissed (Derzhavin 1939; Daneliya 2003). Throughout the Holocene, until recently, the basins were The alternative zoogeographical hypotheses — relict or again hydrographically isolated, the Caspian being an inland vicariance vs. immigration or dispersal — seem to be readily lake with no outlet (now 28 m below world sea level). The testable by molecular characters. In a pure vicariance Sea of Azov represents a common estuary of the Don and scenario, the disjunct populations should contain deeply Kuban rivers, and geographically actually makes a part of diverged endemic clades, whereas in a pure dispersal case the Black Sea basin. As regards the zoogeographical ques- the overall degree of differentiation should be small. tions in this study, the sea is however, hydrographically Further, if both vicariance (survival in isolated refugia) and isolated from the estuaries of the large western Black intermittent dispersal took place, sympatric occurrences Sea rivers (Danube, Dnestr, Dnepr), has a special position of distinct lineages should be expected. Previous studies of in its past and current contacts with the neighbouring amphipod and cladoceran crustaceans have indeed revealed Caspian basin, and also has its own biogeographical distinct mitochondrial clades in the Caspian and Black seas, characteristics, which justify its treatment here as a distinct and their divergence seems to contradict the hypothesis zoogeographical unit. of faunal exchange in Late Pleistocene times (2–11% in the The relict zoogeographical view claims that the current COI gene; Cristescu et al. 2003, 2004). The divergence distributions of the brackish-water fauna mark the estimates were consistently smaller in cladocerans than in extent of the ancient Sarmatian (c. 10 Myr) or Pontian amphipods, suggesting that besides palaeoenvironmental (c. 6 Myr) inland seas, which later were fragmented into changes the dispersal ability of organisms was also important the distinct basins. Accordingly, the faunal element has in shaping their phylogeographies (Cristescu et al. 2003). been coined as Sarmatian or Pontian relicts (Sars 1907; Comparing molecular divergences across phylogenetically Martynov 1924; Ekman 1953; Weish & Türkay 1975). River distinct taxa is however, risky, as rates of molecular deltas and lagoons are seen as long-term refugia, and evolution may vary considerably in species with distinctly deep evolutionary subdivisions among the populations different generation times and reproduction modes. in different basins are anticipated (Starobogatov 1970; Brackish-water Ponto-Caspian mysids provide a good Grigoriev & Gozhik 1976). The different schools of zooge- model system for a comparative phylogeographic approach ography are also reflected in the current taxonomy and in within a relatively coherent group of taxa. Most of the an uneven degree of endemism recognized in various authochtonous Ponto-Caspian mysid species are from a animal groups. Thus a number of endemic Azov and Black single genus Paramysis, but exhibit a range of ecological Sea species have been described in gastropod and bivalve characteristics (Daneliya 2003). Mysid crustaceans in molluscs (Golikov & Starobogatov 1966; Grigoriev & Gozhik general have poor dispersal abilities — they carry their eggs

2972 A. AUDZIJONYTE, M. E. DANELIYA and R . VÄINÖLÄ and developing young in a brood pouch, cannot withstand changes in the Pleistocene. As the conditions in the Caspian desiccation, and typically cannot actively disperse upstream; Sea are assumed to have been more stable, we anticipate that their continental distributions have therefore been largely genetic structures of taxa from this basin will be closer to defined by direct (lentic) water-way connections (BÅnÅrescu that expected in equilibrium populations. In a comparative 1991). Yet, from distributional evidence, at least two Ponto- framework, from the congruence and differences in the Caspian brackish water mysids do have dispersed upstream geographical structuring in the codistributed mysids in rivers (Buchalova 1929), which provides a good basis to and in other invertebrate taxa, we can address the general compare their intraspecific, interbasin subdivisions to those importance of the vicariance and dispersal scenarios in in more sedentary congeners. Ponto-Caspian mysids also Ponto-Caspian biogeography, and the role of the biological exhibit a variety of salinity tolerances, involving both strictly properties of the individual taxa (e.g. vagility, salinity stenohaline (< 3‰) and relatively euryhaline (0–12%) taxa tolerance) in their response to the environmental history. (Komarova 1991; Daneliya 2003). Given the dynamic history of salinity conditions in the Ponto-Caspian, euryhalinity Materials and methods should have been an important ecophysiological property controlling the dispersal of species. Samples and laboratory analyses In this study we explore the mtDNA diversity and phylogeography in seven opossum shrimp species Mysid samples for molecular analyses were collected in (Crustacea: Mysida) distributed across the Ponto-Caspian 1991, 2000–2004 and stored in 80–96% ethanol. Most samples region (Fig. 1, Table 1). With these data, we test the afore- are deposited at the Finnish Museum of Natural History. mentioned biogeographical hypotheses about the history Six currently recognized species of the genus Paramysis of the brackish-water fauna, i.e. (i) pure vicariance; (ii) pure and one Limnomysis species were studied (Table 2). All these dispersal; and (iii) vicariance and dispersal. Further, we species are endemic to the Ponto-Caspian region. Limnomysis assess the presence of genetic signals of past demographic is a monotypic genus, whereas Paramysis (20 species in changes in populations from the Black Sea and Sea of all) also comprises four taxa in the Mediterranean and the Azov basins, which were affected by drastic environmental Atlantic. All together 165 specimens from 18 localities were

Table 2 Number of specimens analysed from each locality for different taxa. Abbreviations: LB, L. benedeni; PL, P. lacustris; PS, P. sowinskii; PB, P. baeri sensu lato; PU, P. ullskyi; PK, P. kessleri; PI, P. intermedia

Code Location (year of collection) LB PL PS PB PU PK PI

Caspian Sea basin GAM Ilmen Gamta, Volga river basin (2003) 6 4 — ———— VOL Damchik, Volga river delta (2003) 8 6 — 3 3 — — DAG1 Krainovka, Dagestan (2004) — 4 — 2 — 1 — DAG2 Sulak Bay, Dagestan (2004) — 4 — 3 2 — — DAG3 Staroterekskoe, Dagestan (2004) 3 2 — — 3 — — CCA/SCA Central/Southern Caspian Sea (1991, 2004) — — — 1 2 2 — Sea of Azov basin TSI Tsimlyanskoe water reservoir (2004) 6 2 — ———— MAN Lake Manych (2004) — 4 — ———1 SOL Lake Solenoe (2004) — 5 — ———— MDO Middle Don (2000, 2004) — 5 — 1 — — — DON Dead Don, Don river mouth (2003) 5 5 3 3 3 — 3 MIU Miuskii liman (2003) — 5 4 ———— EIS Eiskii liman (2003) — — 3 ———— KUB Akhtanizovskii liman, 2 2 2 3 7 — — Kuban river delta (2003) —— ABR Lake Abrau (2003) — 6 — ———— Black Sea basin DNP Kherson, Dnepr delta (2004) 4 6 — — — 3 3 DNS Dnestr delta (1999) 3* — — ———— DAN Danube delta (1998–2001) 5* 2 — 3 — 3* 2* Total 42 62 12 19 20 9 9

*includes sequences from Cristescu & Hebert (2005) (GenBank Accession nos LB: AY529017–AY529122; PK: AY529034; PI: AY539032).

PONTO-CASPIAN MYSIDS 2973 sequenced (Fig. 1, Table 2); eight additional sequences from 0.01. A likelihood score for a tree with the same topology four taxa were obtained from published data (Cristescu & but with the clock enforced was then calculated, and the Hebert 2005). two likelihood scores compared with a LRT (α = 0.01). The DNA extraction, amplification and sequencing Limnomysis benedeni was strongly diverged from all procedures were as described in Audzijonyte et al. (2005). Paramysis taxa (c. 25% uncorrected and 40% K2P+Γ corrected A part of the mitochondrial COI gene was amplified and divergence) and was not included in the analyses of rate sequenced using either the universal primers LCO1490 constancy. Bootstrap support for the ML topology without and HCO2198 (Folmer et al. 1994) or new specific Paramysis molecular clock was estimated using 500 bootstrap replicates. primers ParaHatD 5′-TGTACTTTGTRTTTGGGGCTT- Because molecular clock assumption was rejected by the GRGC-3′ and ParaHatR 5′-GTGYTGRTABAGRATAG- LRT (see Results), the relative rates of evolutionary change GGTC-3′. The final mtDNA segment used for the analyses along the branches were estimated using nonparametric rate was 605 bp long and had no length variation. smoothing method (NPRS) (Sanderson 1997) implemented All specimens analysed for molecular variation were in treeﬁnder (Jobb 2005). A distinct Mediterranean also assessed morphologically to ensure proper identi- Paramysis species, P. arenosa was used as outgroup for fication and to screen for morphological differences rooting the ML tree, but was excluded from the further corresponding to the lineage subdivisions found in the NPRS procedure (Jobb 2005). The monophyly of the studied molecular data. For this assessment, reference material Ponto-Caspian Paramysis in respect of P. arenosa was verified from additional collections made by MED in 1996–2005 by a preliminary phylogenetic analysis from COI, 18S and and material from the Zoological Institute, Russian 28S sequences (A. Audzijonyte, unpublished data). Academy of Sciences (ZIN) was also examined. Specimens Haplotype (h) and nucleotide (π) diversity indices were first inspected under a stereomicroscope; a geographi- (Nei 1987) were calculated in arlequin 2.000 (Schneider cally representative subset was then fully or partially et al. 2000) for each species and then separately for popu- dissected and mounted for light microscopy. Further lations from the Caspian, Black Sea and Sea of Azov basins. details on taxonomically important characters are given in Diversity level differences between the regions were Daneliya (2004). evaluated with Student’s t test and the standard deviation estimates of the indices provided by arlequin. Molecular divergences among haplotypes from the three basins were Data analysis calculated in terms of the K2P+Γ distance (α = 0.25); both Phylogenetic analyses for intraspecies gene trees were con- the overall mean and net distances (corrected for intrabasin ducted using maximum parsimony (MP) and neighbour- variability) were estimated, using mega (Kumar et al. 2004). joining (NJ) approaches, and for interspecies relationships The partitioning of nucleotide diversity among the three using MP and maximum likelihood (ML), all as imple- basins, among populations within the basins, and among mented in paup* 4.0b10 (Swofford 2003). Tree searches individuals within populations was assessed from the were conducted with 10 random taxa addition replicates, analysis of molecular variance (amova) (Excoffier et al. 1992) followed by the tree-bisection–reconnection (TBR) branch as implemented in arlequin; alternative groupings were swapping; all characters were weighted equally. NJ trees also tested for some taxa. Statistical significance was were constructed using K2P+Γ corrected distances that evaluated with 1000 random permutations. account for the transition/transversion bias and heterogeneity Tajima’s (1989) D and Fu’s (1996) F statistics, and mismatch in mutation rates among sites. The α parameter of rate distributions (i.e. distributions of pairwise nucleotide heterogeneity for within-taxon comparisons was estimated differences; Rogers & Harpending 1992) were used to assess using paup from a NJ tree; the estimates were 0.1–0.5 for the deviations of the observed patterns of molecular diversity different taxa, and an average of 0.25 was then used for all. from that expected in equilibrium populations and selective For both NJ and MP analyses, support for the nodes was neutrality. Statistical significance was assessed from 1000 calculated from 1000 bootstrap replicates. Further, reticula- permutations; the critical P value was 0.05 for Tajima’s tions among haplotypes were assessed using 95% statistical D and 0.02 for Fu’s F (Schneider et al. 2000). Mismatch parsimony networks (Templeton et al. 1992) as implemented distributions illustrate the depths of coalescence within in tcs 1.13 (Clement et al. 2000). a sample of haplotypes in mutation-generation units. The molecular clock assumption among Paramysis taxa Distinctly unimodal distributions are expected in a case of was tested using a likelihood ratio test (LRT). An ML tree a past sudden demographic expansion at a time approximat- search of all unique Paramysis haplotypes, assuming no ing the peak coalescence (τ) (Rogers & Harpending 1992). molecular clock, was first made in paup, under the HKY+Γ+I The goodness-of-fit of the observed data to that expected model of nucleotide substitution, selected from alterna- under the sudden expansion model was assessed from tive models with aid of the modeltest software (Posada & simulations, and the 95% confidence intervals of τ under Crandall 1998) and using LRT and significance level (α) of the expansion model calculated from 1000 parametric

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd 2974 A. AUDZIJONYTE, M. E. DANELIYA and R . VÄINÖLÄ bootstrapping replicates. All calculations were performed Intraspecific diversities varied greatly among the seven in arlequin. It must be emphasized that unimodal distribu- analysed taxa; the maximum sequence divergences ranged tions may also be caused by selective sweeps or by hetero- from 1.2% in P. kessleri to 14.8% in P. baeri sensu lato (Table 3). geneity in mutation rates (Simonsen et al. 1995; Schneider In the material initially identified as P. baeri, however, two & Excoffier 1999), and generally effects of selection and divergent haplotype groups were observed (Fig. 2a) which demographic changes are indistinguishable in data from a also turned out to differ morphologically in several characters single locus, such as mtDNA (Galtier et al. 2000). The demo- and were therefore judged as separate species. One of these graphic interpretations of the equilibrium statistics are groups was identified in samples from the coast of central therefore only suggestive. Caspian Sea and will below be referred to as P. baeri s. str. Another clade comprised the remaining material from the northern Caspian, Azov and Black seas, and is here Results provisionally called P. cf. baeri I. Moreover, the GenBank sequence of P. baeri (AY529030, northern Caspian Sea, Molecular divergence and diversity Cristescu & Hebert 2005) was similarly distinct from the No stop codons were identified in the analysed COI sequences two groups identified in our data, suggesting even further and no heterogeneity in base composition was detected species level subdivisions in the taxon. This haplotype is using a χ2 test; the mean A+T content in all sequences was referred to as P. cf. baeri II, but it will not be included in c. 55%. Altogether 87 different haplotypes were observed further genetic diversity calculations below. It should in the here analysed taxa (N = 165) (DQ779792–DQ779878) be noted that the taxonomy of P. baeri has even previously (a table of haplotype frequencies in each sample is available undergone changes. P. cf. baeri I will evidently correspond from the authors). All MP, ML and NJ analyses strongly to the taxon Paramysis bakuensis G.O. Sars, 1895, which supported the monophyly of mtDNA lineages in each of the was later synonymized with P. baeri Czerniavsky, 1882 by seven currently recognized species (bootstrap support 90– Derzhavin (1939); the question will be treated more formally 100%). Resolution at the interspecific level was considerably elsewhere (Daneliya et al., in preparation). weaker (bootstrap support < 75% for most nodes; Fig. 2h); Most of the intraspecific nucleotide variation in the seven here molecular divergences among taxa were typically taxa was at silent sites, but four and seven amino acid replace- 30–40% (HKY+Γ+I correction). A closer interspecies ment substitutions were found in Limnomysis benedeni and relationship (13% divergence) was between the sister P. lacustris (Table 3). In contrast, no such substitutions were species Paramysis lacustris and Paramysis sowinskii, only found among 88 variable sites of the three taxa of P. baeri recently taxonomically distinguished (Daneliya 2002). sensu lato. The assumption of a molecular clock during Paramysis Apart from the case of P. baeri s. l., the deepest genealogical evolution was rejected by the likelihood ratio test (P < 0.01), subdivision was within P. lacustris (maximum divergence suggesting that molecular differences cannot necessarily 6.5%). The molecular lineages of P. lacustris were not be interpreted in identical temporal terms across taxa. associated with consistent morphological differentiation Conspicuous rate variation was evident from the branch to corroborate taxonomic distinction however. The lineage lengths in the interspecies tree, rooted by Paramysis arenosa; division in P. lacustris was reflected in the overall estimate the branches of Paramysis kessleri, Paramysis intermedia and of its nucleotide diversity (π ≈ 2.5%), higher than those Paramysis ullskyi were notably longer than in the apparent in remaining taxa (0.5–1.3%; Table 3). Exceptionally high sister group involving P. lacustris and P. sowinskii (Fig. 2h). local intraspecies diversity in absence of lineage division In the NPRS approach, the substitution rates in P. kessleri, was found in P. baeri s. str. from the central Caspian P. intermedia and the central Caspian Paramysis baeri branches Sea, where each of the five analysed specimens had unique were 2.0–2.5 times faster than in the P. lacustris + P. sowinskii haplotypes (h = 1) that differed on average by 12 nucleotide group. Therefore also the comparisons of intraspecies molec- substitutions (π ≈ 2%) (Figs 2a, 3a). ular divergences across taxa must be treated with reservation. Haplotype diversities were generally high across the Anyway, the focus here is on comparisons of phylogeo- analysed taxa (c. 0.9) and about 50% of all sequenced graphic structures (presence vs. absence of subdivisions) specimens had unique haplotypes. The diversities were and on the most extreme differences in levels of divergence. particularly high in P. sowinskii and L. benedeni (Table 3).

Fig. 2 (a–g) Neighbour-joining (NJ) topologies of mitochondrial lineages in seven Ponto-Caspian mysid taxa (K2P + Γ distance). The tree of Limnomysis benedeni was rooted using mid-point rooting, other trees using other Paramysis taxa as outgroups. Only unique haplotypes were used; their geographical origins in the three main basins are illustrated by colour codes and site labels referring to Fig. 1a. All trees are plotted to the same scale. Bootstrap support values (> 50%) from NJ analyses are indicated above branches, from maximum parsimony (MP) — below branches. (h) Maximum likelihood (ML) tree (HKY + I + G) of the main lineages in the Paramysis taxa studied. Bootstrap support values (> 50%) from ML analyses are indicated above branches, from MP — below branches (MP topology was nearly identical to that of ML).

Table 3 Molecular diversity estimates in the seven analysed mysid species and in their intraspecific genealogical/geographical subdivisions. Abbreviations: n, number of specimens analysed; k, number of samples; NH, number of different haplotypes (proportion of the total n given in parentheses); S, number of segregating sites; RS, number of observed replacement substitutions; h, haplotype diversity; π, nucleotide diversity; P, mean number of pairwise differences; SD, standard deviation; other abbreviations as in Table 1. The maximum observed intrataxon divergence is indicated in parentheses next to the taxon name

Species/group n/k NH S (RS) h (SD) π, % (SD) P (SD)

Limnomysis benedeni (max 3.7%) All 42/9 25 (0.60) 42 (4) 0.93 (0.02) 1.35 (0.71) 8.2 (3.9) CASPIAN 23/4 14 (0.61) 21 (4) 0.88 (0.06)* 0.47 (0.29)* 2.9 (1.6) AZOV 7/2 4 (0.57) 9 (0) 0.75 (0.14)* 0.55 (0.36)* 3.3 (1.9) BLACK 12/3 8 (0.67) 23 (0) 0.89 (0.08)* 1.22 (0.69)* 7.4 (3.7) Paramysis lacustris (max 6.5%) All 62/15 24 (0.39) 58 (7) 0.89 (0.03) 2.56 (1.28) 15.5 (7.0) CASPIAN 32/8 10 (0.31) 19 (4) 0.67 (0.09)* 0.41 (0.25)* 2.5 (1.4) AZOV 22/5 10 (0.45) 15 (4) 0.87 (0.05)* 0.37 (0.24)* 2.3 (1.3) BLACK 8/2 4 (0.50) 18 (1) 0.82 (0.10)* 1.27 (0.76)* 7.6 (4.0) Paramysis baeri s. l. ** (max 14.8%) 74 (0) P. baeri s. str. (max 2.8%) 5/2 5 (1.00) 27 (0) 1.00 (0.13) 1.96 (1.25) 11.8 (6.5) P. cf. baeri I (max 1.2%) 14/6 9 (0.64) 14 (0) 0.90 (0.07) 0.49 (0.31) 3.0 (1.7) Paramysis ullskyi (max 2.6%) All 20/6 9 (0.45) 20 (0) 0.86 (0.05) 1.01 (0.56) 6.1 (3.0) C Caspian 7/3 4 (0.57) 7 (0) 0.71 (0.18) 0.33 (0.24) 2.0 (1.3) Other 13/3 5 (0.38) 17 (0) 0.73 (0.10) 0.56 (0.34) 3.4 (1.8) Paramysis kessleri (max 1.2%) CASPIAN+BLACK 9/4 4 (0.44) 8 (1) 0.81 (0.09) 0.53 (0.34) 3.2 (1.8) Paramysis intermedia (max 2.0%) AZOV+BLACK 9/4 6 (0.67) 14 (0) 0.89 (0.09) 0.96 (0.57) 5.8 (3.1) Paramysis sowinskii (max 1.8%) AZOV 12/4 10 (0.83) 18 (1) 0.97 (0.04) 0.80 (0.47) 4.8 (2.5)

*in L. benedeni h (AZO) < h (BLA) ≈ h (CAS); in P. lacustris h (CAS) < h (AZO) ≈ h (BLA); in L. benedeni and P. lacustris π (BLA) > π (CAS) ≈ π (AZO), t-test, P < 0.05. **P. cf. baeri II excluded. Samples of L. benedeni from TSI were included into the Caspian group; samples of P. lacustris from TSI, SOL and MDO were included into the Caspian group.

Comparison of molecular diversities across the three basins No evidence of population expansion was inferred from was feasible in two species with more extensive geographical the Black Sea samples of any taxon (not shown). sampling, i.e. L. benedeni and P. lacustris. In both species nucleotide diversities were higher in the Black Sea than Phylogeographic structure in the Azov and Caspian basins (P < 0.05), but this trend was not evident in haplotype diversities. The strongest phylogeographic structure and deepest Significantly negative Tajima’s D values (and in some intraspecific subdivisions were seen in P. lacustris. All three cases Fu’s F) and unimodal mismatch distributions, basins and also the separate river estuaries of the Black suggesting past demographic expansion, were observed Sea basin contained specific haplotype groups (Fig. 2e). only in the Caspian samples of L. benedeni, in the Caspian The deepest genealogical split (c. 5% divergence) separated and Azov samples of P. lacustris and in the two genealogical haplotypes from the geographically intermediate Sea of clades of P. ullskyi (central Caspian, and the remaining Azov from those in the Caspian and Black seas, which in widespread clade) (Fig. 4). A star phylogeny with a turn were 1.7% diverged from each other. When corrected common central haplotype was particularly evident from for the intrabasin variability (net distance) the Azov basin the parsimony network of L. benedeni Caspian Sea clade clade differed from those in the other two basins by 4.4%. (Fig. 3g) and to a lesser extent in P. lacustris (Fig. 3e). Of the two populations from the past contact area between However, rare distinct haplotypes were also identified Caspian and Azov seas (Manych depression), one contained in the Caspian Sea populations of all the three species, Caspian, the other Azov lineage haplotypes. An exception causing secondary peaks in the mismatch distributions. to the genealogical-geographical congruence in P. lacustris

Fig. 3 Statistical (95%) parsimony haplotype networks for the seven mysid taxa. Geogra- phical origins of haplotypes are illustrated by colours shown in Fig. 1a. The size of a circle is proportional to the observed number of the corresponding haplotype. The minimum number of steps needed to connect each of the two clades in Paramysis baeri sensu lato and in Paramysis lacustris is indicated along the dashed lines.

was recorded from two upstream sites of the Don River in TSI on the Don (Azov drainage) had Caspian lineage the Azov basin, i.e. Middle Don (MDO) and Tsimlyanskoe haplotypes (Fig. 2g). water reservoir (TSI). Specimens from these sites contained A different phylogeographic structure was found in Caspian lineage haplotypes, identical to those from the P. baeri s. l. and in P. ullskyi. In each of these taxa the basal Volga river delta (VOL) (Fig. 2e). genealogical split separated central Caspian Sea populations A similarly distinct Caspian Sea clade was identified in from the rest, i.e. from a widespread lineage encompassing L. benedeni, whereas no consistent subdivision was evident the northern Caspian, Azov and Black Sea samples; the between the Azov and Black Sea populations of this species. pattern was broken by a single Volga river haplotype The latter basins contained groups of genealogically distinct connected with the central Caspian clade in P. ullskyi but geographically intermixed haplotypes (Figs 2g, 3g). (Fig. 2a, b). The depths of the principal subdivisions were The mean divergence of the Caspian Sea clade from those however, very different: a species-level subdivision in P. baeri in Black/Azov sea basins was 1.8% (net distance 1.1%). As s. l. (12% divergence) contrasted by a 1.5% distinction in with P. lacustris, L. benedeni from Tsimlyanskoe reservoir P. ullskyi (Table 4). Apart from the distinct central Caspian

Fig. 4 Mismatch distributions, or distributions of pairwise differences in geographical groups of populations, for taxa with larger sample sizes. Dashed lines show 95% confidence intervals of the simulated mismatch distribution (solid line). None of the Black Sea populations conformed to the expecta- tions of demographic expansion and are not shown here. * — for Paramysis ullskyi, divergent haplotypes from the central Caspian Sea coast were excluded. Tajima’s D and Fu’s F statistics are shown.

Table 4 Net (and mean) K2P + Γ corrected sequence divergences (% ± SE) among the three Ponto-Caspian basins for different mysid taxa. Alternative groupings based on phylogenetic analyses are given for Paramysis baeri sensu lato and Paramysis ullskyi and indicated in italics. Proportion of interpopulation molecular variance, partitioned to among-basin and within-basin (among populations) components (from amova), is given for the two species with larger sample sizes; all variance components were highly significant (P < 0.001)

Taxon Caspian — Azov Caspian — Black Azov — Black % among % within

Limnomysis benedeni 1.2 ± 0.4 (1.7 ± 0.5) 1.1 ± 0.4 (2.0 ± 0.5) 0.1 ± 0.0 (1.0 ± 0.3) 56.1 10.8 Paramysis lacustris 4.7 ± 1.1 (5.1 ± 1.1) 0.7 ± 0.3 (1.7 ± 0.4) 3.9 ± 0.9 (4.9 ± 1.0) 85.8 6.2 Paramysis kessleri — 0.2 ± 0.1 (0.6 ± 0.2) — Paramysis intermedia — — 0.3 ± 0.1 (1.1 ± 0.3) Paramysis ullskyi 0.8 ± 0.2 (1.3 ± 0.3) — — C Caspian — other 1.1 ± 0.4 (1.5 ± 0.5) Paramysis cf. baeri I 0.0 ± 0.0 (0.6 ± 0.2) 0.1 ± 0.1 (0.6 ± 0.2) 0.1 ± 0.1 (0.5 ± 0.2) P. cf. baeri I–P. baeri s. str. 11.0 ± 2.2 (12.3 ± 2.3)

clades, neither P. baeri s. l. nor P. ullskyi showed further southern Caspian Sea and the Danube delta in the western phylogeographic structure among or within the northern Black Sea basin were separated by only one nucleotide Caspian, Azov and Black seas. difference (Fig. 3b). The least differentiation and no phylogeographic struc- There was little congruence in the depths of divergence turing were seen in P. kessleri (Fig. 2b) and in P. intermedia among the interbasin clades of different mysid taxa (Table 4) (Fig. 2f). For instance, in P. kessleri specimens from the and those of other invertebrate groups in previously

Black/Azov and Caspian sea basins, or those among all three basins — in Limnomysis benedeni and Paramysis lacustris. Two further taxa, Paramysis sowinskii and Paramysis intermedia, were sampled from the Azov and Black Sea basins only and did not show consistent geographical differentiation.

Past exchange among basins and rivers of the Ponto-Caspian The palaeontological record of the Black Sea basin provides evidence of repeated changes between marine and brackish/ freshwater faunal communities that tracked the changing salinity conditions (Fedorov 1978; Zubakov 1988). Whenever a connection to the Mediterranean was established and marine waters intruded into the Black Sea, the brackish- water fauna was extirpated or confined to the diluted river deltas, as today. Low-salinity periods in turn could have provided opportunities for dispersal among rivers, but it is also possible that the coastal and estuarine mysids, normally absent from open water habitats, still remained confined to river deltas and did not substantially intermix. Yet phylogeographic data from amphipods, cladocerans Barbus Fig. 5 Summary of the average interbasin mitochondrial COI and fishes ( ) with habitat requirements similar to those sequence divergence (K2P + Γ distance, not corrected for intrabasin of the mysids have generally shown little differentiation diversity) in mysids (this study) and in other invertebrate taxa among distant Black Sea rivers, suggesting that gene flow (data from Cristescu et al. 2003, 2004; Nikula & Väinölä 2003; did occur (Cristescu et al. 2003; Kotlík et al. 2004). Therriault et al. 2004). Comparisons where the Caspian Sea is In contrast, the Pleistocene period within the Caspian represented only by a central/southern Caspian population are Sea was marked by relatively stable salinity conditions that indicated with triangles. Divergences judged to involve distinct in the open Southern Caspian were within the range of species are marked with an asterisk (*). Abbreviations: CG, Cerastoderma glaucum; DR, Dreissena rostriformis; CM, Cornigerius 10–13‰, i.e. similar to those today (Fedorov 1980). Water maeoticus; CP, Cercopagis pengoi; PT, Podonevadne trigona; EI, Echino- level fluctuations nevertheless spanned c. 100 m amplitude gammarus ischnus; OC, Obesogammarus crassus; PM, Pontogammarus (Svitoch et al. 2000) and repeatedly inundated and exposed maeoticus; PR, Pontogammarus robustoides; LB, Limnomysis benedeni; large areas in the northern parts of the basin (Mamedov PB, Paramysis baeri sensu lato; PI, Paramysis intermedia; PK, Paramysis 1997). The Caspian transgressions coincided with the cold kessleri; PL, Paramysis lacustris. periods of the Pleistocene; it was at these times, and also as recently as c. 15 000 years ago, that the Manych depression was flooded, enabling downstream dispersal of the Caspian published data (Fig. 5). In most taxa, the Sea of Azov-Black fauna towards the Azov basin (Fig. 1b) (Svitoch et al. 2000; Sea subdivision was weaker than the distinction of the Ryan et al. 2003; Bahr et al. 2005). During regressions, Caspian from these two basins, but in P. lacustris even this the previous one c. 20 000 years ago (Mamedov 1997), the pattern did not hold. northern coast of the Caspian Sea in turn retreated c. 100 km southwards creating good opportunities for exchange between the currently separated northern and central Discussion Caspian coastal populations of stenohaline taxa (Fig. 1b). Three main phylogeographic patterns were identified It remains unclear however, to what extent these changes in the analysed mysid taxa: (i) overall homogeneity with of the Caspian Sea affected subdivisions of its fauna; so far closely related haplotypes across the entire Ponto-Caspian there are virtually no data on intrabasin phylogeography region — in Paramysis kessleri; (ii) a main genealogical of Caspian Sea organisms. division that separates central Caspian Sea haplotypes The weak phylogeographic structuring recorded among from a widespread, relatively uniform Black/Azov/Volga Black Sea river drainages and between the Azov and Black river basin clade — in Paramysis baeri s. l and (with one sea basins in L. benedeni, P. intermedia and P. cf. baeri I was exception) in Paramysis ullskyi; and (iii) genealogical in line with the aforementioned observations from other splits that geographically match the border between the crustaceans and fishes, suggesting past gene flow, plausibly

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd 2980 A. AUDZIJONYTE, M. E. DANELIYA and R . VÄINÖLÄ during the diluted phases of the Black Sea. Also within Sea, whereas few taxa seem to have dispersed out from the Caspian Sea, populations of P. lacustris and L. benedeni the Caspian (Grigorovich et al. 2003; Orlova et al. 2004; from northern and central coasts contained identical or Therriault et al. 2005). With the current data, we cannot closely related haplotypes, supporting faunal exchange. strictly rule out a possibility that the Black-Azov lineages Differences in P. ullskyi and in P. baeri sensu lato, however, of P. ullskyi and of P. cf. baeri I arrived to the Volga River via suggest continued isolation between populations of the the Volga-Don canal. In that case, however, we should northern and central Caspian Sea coasts. In these taxa, also assume that the new invaders or their mitochondria clades from the two areas defined the main Ponto-Caspian displaced local populations (lineages) that were common genealogical split, which however, involved groups of in Volga prior to the canal construction (Mordukhai- evidently different age (1.5 and 12% divergence) and Boltovskoi 1957). Such a recent immigration to the Caspian different systematic rank. In both taxa, the coastal central also seems very unlikely for P. kessleri, which is currently Caspian clades were endemic to this basin, whereas the confined to the western Black Sea and south-central northern populations contained haplotypes close to those Caspian and is similarly homogeneous. Finally, the data on in the Azov and Black Sea basins. The uniformity of the P. lacustris and L. benedeni suggest that mysid dispersal latter lineages in P. cf. baeri I and P. ullskyi, as well as through the Volga-Don canal has been out of the Caspian similarity of the Black and Caspian haplotypes in P. kessleri basin rather than into it (see below). suggest that the Manych Strait between Caspian and Azov Although gene flow among estuaries of the Black-Azov basins was an efficient dispersal route. The causes for the basin rivers was inferred for most taxa, the patterns of spatial segregation of the northern and the endemic central molecular diversity nevertheless indicated varying impor- Caspian Sea clades however, remain unclear. tance of local refugia in different species. Populations of Given the extreme environmental changes in the Black L. benedeni from the Don, Kuban, Danube and Dnepr and Azov seas and their postulated destructive effects on estuaries contained deeply divergent but intermixed local faunas, it has been assumed that the main direction haplotypes, suggesting both persistence of local populations of migration along the Manych Strait was westwards, out (refugial divergence, no extinctions) and subsequent gene of the Caspian Sea (Mordukhai-Boltovskoi 1979; Zubakov flow. In contrast, the weak overall differentiation within 1988). On the other hand, there is also evidence of a Late P. cf. baeri I, P. ullskyi and P. kessleri indicates that in these Pleistocene/Holocene colonization in the opposite direc- species either the local populations in different refugia have tion, as regards appearance of Mediterranean Cerastoderma gone extinct before re-colonizations or that competitive bivalves in the Caspian Sea (Fedorov 1978; Nikula & replacement of mitochondria or of entire populations has Väinölä 2003). From the current mtDNA data, no conclu- taken place. Yet generally, given the small sample sizes, sive inferences about the direction of gene flow in P. ullskyi, we cannot exclude the possibility that additional, more P. cf. baeri I and P. kessleri can be drawn, as none of the divergent but undetected lineages also exist in the latter basins appeared to harbour higher and possibly ancestral taxa. Although the current data can reject the pure vicariance diversity (disregarding the distinct central Caspian groups). hypothesis (no migration among refugia) they do not Downstream dispersal from the Caspian would seem have power to discriminate between the pure dispersal vs. more feasible a priori. On the other hand, the presence of vicariance + dispersal scenarios. two distinct lineages of P. ullskyi (and three of P. baeri s. l.) Notably, none of the Black Sea populations showed signs in the Caspian basin could reflect their dual origins in the of a demographic expansion, which could have happened basin, i.e. (re)invasion from the Black-Azov basin following if the vast diluted Late Pleistocene lake-sea was overtaken Pleistocene interbasin vicariance and divergence. from a single refugial source. It therefore seems that diver- If such an immigration from the Black-Azov to the sity in the Black Sea basin has been maintained through the Caspian basin occurred via the Late Pleistocene Manych high and low salinity periods. On the other hand, signals of connection, the mysids possibly had to migrate upstream. past demographic expansion were observed in the Caspian Indeed, in contrast to most Mysida, P. ullskyi and P. baeri s. Sea populations of P. lacustris and L. benedeni, an unexpected l. are known to be capable of such dispersal (Buchalova result considering the relatively stable salinity conditions 1929). For instance, natural occurrences of P. ullskyi were in that basin. The data nevertheless suggest that the two known from 1000 to 3000 km upstream in the Volga river species experienced more or less severe bottlenecks in (Mordukhai-Boltovskoi 1957). An eastward dispersal of the Caspian Sea (a coalescence of haplotypes to a single P. ullskyi and P. cf. baeri I could in fact have occurred even ancestor before the expansion), but currently no conclu- more recently. Throughout the Holocene, the Caspian Sea sions can be drawn whether these were true bottlenecks of has been an isolated lake with no connection to the west, populations or only of their mitochondria (e.g. selective until a new link with the Sea of Azov basin was established sweep). Clearly, further sampling and markers of higher in 1952 through the Volga-Don canal. Since then a number resolution are needed for more detailed phylogeographic of nonindigenous organisms have colonized the Caspian understanding.

low water stands (Ryan et al. 2003). It is therefore thought Local refugia in Paramysis lacustris and Limnomysis — and also inferred from the data of other mysid taxa in benedeni this study — that the Azov basin fauna generally consists Although extensive gene flow among Ponto-Caspian basins of postglacial recolonisers either from the Black Sea (whose was inferred in some taxa, other species showed distinct salinity at that time was lower), or from the Caspian basin interbasin structuring. The Caspian populations of L. benedeni via the Manych connection. were clearly separated from those in the Black/Azov sea The diverged Sea of Azov clade of P. lacustris neverthe- basins (c. 1.8%), and still deeper geographical subdivisions less suggests survival in a local refugium, possibly in were found in P. lacustris, which is the most stenohaline the Don and Kuban rivers, which during the low stands of the taxa studied. The 5% Azov-Caspian divergence is extended far south and drained directly into the Black Sea. notably high considering the typical levels of intraspecific The contrasting patterns in P. lacustris and the other mysids differentiation (generally < 3%) in other crustaceans as regards isolation vs. mixing of the Azov and Black Sea and invertebrates (Hebert et al. 2003). Yet, no consistent lineages could be related to the different salinity tolerances morphological differentiation among various populations of the taxa in face of the palaeosalinity changes in the Black was recorded to corroborate a presence of sibling species. Sea. The discussion remains unsettled whether during Applying the commonly used invertebrate mitochondrial the last regression the salinity of the Black Sea decreased molecular clock of 2–3% per 1 Myr (Knowlton & Weigt to practically fresh (0–1‰) or to slightly brackish levels (4– 1998; Wares & Cunningham 2001), the 2–5% Azov-Caspian 5‰) (Ryan et al. 1997, 2003; Mudie et al. 2002). Comparative divergences in L. benedeni and P. lacustris could plausibly mysid phylogeography would seem to support the latter be of Early to Middle Pleistocene age. On the other hand, option. The taxa that naturally thrive in salinities of up to patterns of molecular diversity in postglacially isolated 5–6‰ are homogeneous, indicating Black-Azov gene flow populations of another mysid species have suggested (i.e. P. intermedia, P. baeri, L. benedeni). In contrast, the 10-fold higher rates of COI evolution on recent time stenohaline P. lacustris, typically confined to salinities scales (Audzijonyte & Väinölä 2006). If this also applies < 2–3‰, has remained isolated, plausibly due to a salinity– to Ponto-Caspian taxa, the distinction of Azov basin clades related dispersal barrier. could be as young as 0.1–0.2 Myr. However, comparison Also within the Black Sea, distinct allopatric lineages of molecular rates across Paramysis taxa actually suggested were found in the Danube and Dnepr estuary populations slower than average rates in P. lacustris (Fig. 2h), which of P. lacustris, indicating limited gene flow among the rivers, rather emphasizes the relative antiquity of the divergence. but the sampling was too limited to exclude possible inter- Whatever the molecular rate, from palaeogeographical mixing. Notably, P. lacustris in the Azov basin also showed data and from the phylogeographical structure of a signal of a sudden demographic expansion, not seen in other mysids it is evident that there should have been other taxa (Fig. 4). The inferred time of population expan- opportunities for intermixing of the Caspian and Azov sion c. 150 000 (50 000–300 000) years ago however, would basin clades, when interbasin contacts were established not fit the postglacial refilling of the basin, if a conventional throughout the Pleistocene (Svitoch et al. 2000). The molecular rate is assumed (c. 2% per Myr). On the other retained Caspian-Azov distinctions within P. lacustris and hand, with the 10-fold faster postglacial mtDNA rate L. benedeni therefore could indicate reproductive isolation discussed above, the data could indeed reflect a postglacial between the lineages despite their apparent morphological expansion (22 000–7000 years). uniformity. The current mtDNA data alone however, There is also evidence that the Sea of Azov refugium of do not provide sufficient grounds for definite taxonomic P. lacustris was not entirely restricted to rivers, as haplotypes conclusions. of this lineage are also present in the ‘relict’ Lake Abrau The deep molecular subdivisions in mysids also have (ABR, Fig. 1). The lake, currently 83 m above the sea level parallels in other weakly dispersing Ponto-Caspian taxa. and 1.8 km from the Black Sea coast, contains a community Comparisons of allegedly conspecific Black Sea vs. Caspian of brackish-water Ponto-Caspian taxa and even an endemic basin populations of amphipods have revealed COI diver- clupeid fish species (Mordukhai-Boltovskoi 1964). The lake gences up to 11% (or 16% in terms of the K2P+Γ distance probably originated after an earthquake-induced land-slide applied here) (Cristescu et al. 2003), and up to 4% (c. 5%) (c. 175 m high) dammed the ancient river Abrau from the between populations from a single river (Cristescu et al. Black Sea; subsequent accumulation of sediments lifted the 2004) (Fig. 5). However, the phylogeographical distribu- lake level and the inhabitants captured into it (Ostrovskii tion of the P. lacustris clades, i.e. a greater separation of the 1970). No geological dating of the event is available, but Azov population from the Black+Caspian clade, is excep- the presence of the brackish-water community has been tional and unexpected considering the palaeogeographic taken as evidence of isolation during the Late Pleistocene history of the region. Repeatedly through the Pleistocene, low-salinity phase of the Black Sea (Mordukhai-Boltovskoi the shallow Sea of Azov was nearly dry during the Black Sea 1960). The faunal evidence alone actually does not prove

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd 2982 A. AUDZIJONYTE, M. E. DANELIYA and R . VÄINÖLÄ the Late Pleistocene age of the lake, as desalinizations of Conclusions the Black Sea and expansions of the brackish-water fauna have occurred repeatedly throughout the Pleistocene. A comparative phylogeographic assessment of seven Ponto- The molecular data of P. lacustris however, now points to Caspian mysid taxa, and of other codistributed invertebrate a recent lake origin, as all the Abrau specimens had a groups, suggests a complex history of lineage isolation haplotype also found in the Kuban River. The absence of and interbasin exchange events — vicariance and dispersal molecular variation in P. lacustris also implies a small — during the dynamic Pleistocene history of the Black, Azov effective population size, as expected in a small lake and Caspian seas. The lack of intertaxon congruence in (c. 1.5 km2). the geographic distributions of intrataxon lineages, and particularly in depths of their divergence (Fig. 5), implies that similar distributional patterns have arisen at widely Recent contact between Caspian and Azov basins different time scales. Four of our sampling sites were from the region between Overall, there was no evidence of intraspecific divergence the Azov and Caspian seas, either from the waters along as old as the Late Miocene period (10–5 Myr); the deepest the Manych Strait that formerly connected the two basins subdivision, in Paramysis lacustris (5–6%), could plausibly (SOL, MAN), or along the Don river (Azov drainage), which be of Early Pleistocene age. In fact, the Tertiary vicariance since 1952 has been connected through a canal also to the scenario could better account for the initial divergence of Volga river and the Caspian (TSI, MDO). In the 1950s, a some of the Paramysis species themselves, which were 12– number of water reservoirs were also established on the 40% diverged from each other. At the intraspecific level, Don (including TSI, the Tsimlyanskoe reservoir), and these all three biogeographical scenarios (vicariance, vicariance reservoirs were stocked with P. lacustris and L. benedeni + dispersal, vicariance + extinction + dispersal) seemed from the Don delta area for fish-food enrichment purposes applicable to the histories of different mysid species. The (Ioffe 1958). No natural occurrences of these species were interbasin segregation of the deep P. lacustris lineages known from localities this high upstream on the Don and of the Caspian Limnomysis benedeni lineage conformed until then (Martynov 1924; Buchalova 1929). Unexpectedly, to the vicariance expectation; the sympatry of distinct however, all P. lacustris and L. benedeni from both the Don lineages of L. benedeni in Azov-Black Sea estuaries to the sites (TSI downstream of the Volga-Don canal junction, vicariance and dispersal scenario; whereas the large-scale MDO 250 km upstream) had haplotypes identical or closely uniformity in Paramysis cf. baeri I, Paramysis ullskyi and related to those in the natural habitats in the Volga river Paramysis kessleri implied local extinctions and relatively delta. The data thus suggest recent immigration of the recent recolonizations. Caspian lineages into the middle reaches of the Don river The phylogeographical histories of species in response basin, via the Volga-Don canal, whereas no trace of the to the oscillating Pleistocene palaeogeographical and initially translocated stock were seen. It remains unclear palaeoecolgical conditions have evidently been controlled whether the recent Caspian invaders have replaced the by their ecological characteristics. This is seen in comparisons stocked Azov-Don basin mysids, or whether what we among mysid species (vagile species being homogeneous, see is a result of selective introgression of the Caspian- stenohaline species most subdivided), as well as in com- type mitochondria into the Don populations following parisons among groups at higher taxonomic levels. Taxa interbreeding. apt for long-distance dispersal, such as bivalve molluscs with In two lakes along the former Manych Strait, P. lacustris planktonic larvae, or cladoceran crustaceans with resting both of the Azov lineage (in MAN) and of the Caspian eggs, show weaker Caspian vs. Black Sea differentiation lineage (SOL) were found. If the populations actually than do amphipods (Cristescu et al. 2003; Fig. 5). However, represent relicts of the Manych Strait, this could reflect in this broader comparison, average mysid patterns appear two-way dispersal in Late Pleistocene times. Alternatively, closer to those of cladocerans than of amphipods, which it could indicate recent immigration of the Caspian lineage would not necessarily be expected from dispersal charac- via the Volga-Don canal, or of a transplanted Don River teristics (Fig. 5). (Azov drainage) population from adjacent, connected water In some mysid taxa, i.e. P. lacustris and L. benedeni, the reservoirs (Kruglova 1959). Study of independently genealogical subdivisions generally matched geographical segregating nuclear markers could clarify the origins of borders among the basins, whereas in others, i.e. P. baeri s. the transition zone populations. Such markers should l. and P. ullskyi, the deepest subdivisions were seen within also help to evaluate the possible reproductive isolation the Caspian Sea itself. The data thus warn against treating between the Azov and Caspian clades in nature, and any of the basins as a uniform area, and show that further elucidate the question of apparent long-term isolation of molecular analyses of the Ponto-Caspian fauna will likely the endemic Azov clade despite the evidently good coloniza- increase estimates of species diversity, at least in those tion capacity of the Caspian lineage. groups where the taxonomic tradition has preferred

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd PONTO-CASPIAN MYSIDS 2983 lumping. Finally, the study also has important implications Daneliya ME (2002) Paramysis sowinskii sp. n. — novyi vid mizid for attempts to trace the origin of recent invaders of (Crustacea: Mysidacea) iz Ponto-Kaspiya. Vestnik Zoologii, 36, European waters using mitochondrial genes (e.g. Cristescu 69–72. Daneliya ME (2003) Mizidy (Crustacea, Mysidacea) basseina Azovskogo et al. 2001, 2004). Such an approach would seem promising morya. Summary of PhD thesis. Rostov State University, Russia. in P. lacustris and to some extent also in L. benedeni, given Daneliya ME (2004) K sistematike mizid roda Paramysis (Crustacea, the geographical structuring of their mitochondrial Mysidacea) iz basseina Ponto-Kaspiya. Zoologicheskii Zhurnal, diversity. 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