Comparative phylogeography of Ponto-Caspian mysid …webpages.icav.up.pt/PTDC/BIA-BEC/104097/2008/11.pdf · 2009-02-10 · Finnish Museum of Natural History, POB 26, FI-00014 University

Molecular Ecology (2006)

15

, 2969–2984 doi: 10.1111/j.1365-294X.2006.03018.x

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

Blackwell Publishing Ltd

Comparative 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 subdividedamong the Black, Azov and Caspian Sea basins and further among river estuaries. Ofthe two alternative views to explain the distributions, the relict school has claimed Tertiaryfragmentation of the once contiguous range by emerging geographical and salinity barriers,whereas the immigration view has suggested recolonization of the westerly populationsfrom the Caspian Sea after extirpation during Late Pleistocene environmental perturba-tions. A study of mitochondrial (COI) phylogeography of seven mysid crustacean taxa fromthe genera

Limnomysis

and

Paramysis

showed that both scenarios can be valid for differentspecies. Four taxa had distinct lineages related to the major basin subdivision, but thelineage distributions and depths of divergence were not concordant. The data do not supporta hypothesis of Late Miocene (10–5 Myr) vicariance; rather, range subdivisions and dispersalfrom 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 fromthe others, whereas

Paramysis kessleri

from the southern Caspian and the western BlackSea were nearly identical. Species-specific ecological characteristics such as vagility andsalinity tolerance seem to have played important roles in shaping the phylogeographicpatterns. The mitochondrial data also suggested recent, human-mediated cryptic invasionsof

P. lacustris

and

Limnomysis benedeni

from the Caspian to the Sea of Azov basin via theVolga-Don canal. Cryptic species-level subdivisions were recorded in populations attributedto

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

Introduction

The history and diversity of aquatic biota in the Ponto-Caspian basin, which encompasses the Caspian, Azov andBlack seas, is attracting scientific interest at least for tworeasons. The Caspian Sea is one of the ancient lakes of theworld; it has been effectively separated from oceans for

c

. 7 million years (Myr) and is currently characterized by50–80% endemism of the fauna (Martens 1997; Dumont2000). The geography and mechanisms of speciation in

ancient lakes has been a topic of much discussion (Rossiter& Kawanabe 2000). For the Ponto-Caspian, potential factorspromoting species divergence involve its wide environ-mental fluctuations and the intermittent interbasin subdivi-sions and connections (Dumont 1998). More recently, thePonto-Caspian brackish-water fauna has come into focusas a major source of aquatic invading species in Europeand North America (Ricciardi & MacIsaac 2000; Leppäkoski

et al

. 2002; Jazdzewski

et al

. 2004). Molecular tools havebeen invoked to trace the sources and pathways of inva-sions (Cristescu

et al

. 2001, 2004), but their successfulapplication is contingent on proper taxonomy and geneticcharacterization of the populations in the native ranges.

Correspondence: Asta Audzijonyte, Fax: +358-9-19144430; E-mail:[email protected]

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Recent invasions also threaten the Ponto-Caspian regionitself, particularly the Caspian Sea, which since the openingof the Volga-Don canal in the 1950s has received a waveof nonindigenous species (Grigorovich

et al

. 2003; Orlova

et al

. 2004; Therriault

et al

. 2005).The southern and central parts of the Black and Caspian

seas have markedly different faunas — the Black Sea (18‰S) is inhabited by typical marine (Mediterranean) taxa,whereas the Caspian Sea (13‰) has many endemic groups(Zenkevitch 1963; B

Å

n

Å

rescu 1991). In contrast, the dilutednorthern areas of the seas and the lower reaches of theirrivers contain a largley shared brackish-water fauna,comprising, among others, numerous species of crusta-ceans (in the Mysida, Amphipoda, Cumacea, Cladocera)and molluscs (in Dreissenidae, Cardiidae, Pyrgulidae).These taxa typically occur in salinities lower than 6‰ andshow relatively little taxonomic differentiation amongthe three basins (Fig. 1a, Table 1) (Mordukhai-Boltovskoi1979; Komarova 1991; Daneliya 2003). The history of theirdisjunct distributions has been an issue of debate in Ponto-

Caspian zoogeography between proponents of the immigra-tion and relict views.

The immigration view emphasizes the role of drasticPleistocene environmental perturbations that must havecaused repeated local extirpation in the western basins butalso facilitated faunal exchange (Birshtein 1935; Mordukhai-Boltovskoi 1960, 1979; Dedyu 1967). For example, duringthe last 1 Myr the Black Sea salinity has fluctuated fromnearly oceanic during contacts with the Mediterranean,to almost freshwater during glaciation maxima and theseevents were coupled with

c

. 150 m changes in the waterlevel; the shallow Sea of Azov was nearly dry duringthe low stands (Fig. 1b) (Alekseev

et al

. 1986; Svitoch

et al

.2000). During the cold climatic phases of the Middle andLate Pleistocene the Caspian Sea experienced extensivetransgressions, which caused overflow of water to the Azovbasin via the Manych depression; the latest event was

c

. 15 000 years ago (Mangerud

et al

. 2004; Bahr

et al

. 2005).Repeated colonizations from the Caspian basin during thePleistocene were believed to be the prime source of the

Fig. 1 (a) Map of the Ponto-Caspian regionindicating sampling sites and their cor-responding codes as listed in Table 2.(b) Schematic presentation of Late Pleisto-cene history of the Black, Azov andCaspian seas (according to Mamedov 1997;Ryan et al. 2003; Mangerud et al. 2004).Dashed lines depict limits of the lakes andrivers during the latest regression stages.Limits of the last extensive Caspian tran-sgression are also shown together with thedirection of the outflow to the Azov basinalong the Manych Strait. Saline water intru-sions into the Black Sea occurred from theMarmara Sea in the course of a repeatedlyestablished connection through the BosporusStrait. The Volga-Don canal, opened in 1952,is recognized as a current route of faunalexchange and an invasion corridor.

P O N T O - C A S P I A N M Y S I D S

2971


current brackish-water fauna of the Black Sea and Seaof Azov basins, thus referred to as the Caspian fauna(e.g. Mordukhai-Boltovskoi 1979; Reid & Orlova 2002).Throughout the Holocene, until recently, the basins wereagain hydrographically isolated, the Caspian being an inlandlake with no outlet (now 28 m below world sea level). TheSea of Azov represents a common estuary of the Don andKuban rivers, and geographically actually makes a part ofthe Black Sea basin. As regards the zoogeographical ques-tions in this study, the sea is however, hydrographicallyisolated from the estuaries of the large western BlackSea rivers (Danube, Dnestr, Dnepr), has a special positionin its past and current contacts with the neighbouringCaspian basin, and also has its own biogeographicalcharacteristics, which justify its treatment here as a distinctzoogeographical unit.

The relict zoogeographical view claims that the currentdistributions of the brackish-water fauna mark theextent of the ancient Sarmatian (

c

. 10 Myr) or Pontian(

c

. 6 Myr) inland seas, which later were fragmented intothe distinct basins. Accordingly, the faunal element hasbeen coined as Sarmatian or Pontian relicts (Sars 1907;Martynov 1924; Ekman 1953; Weish & Türkay 1975). Riverdeltas and lagoons are seen as long-term refugia, anddeep evolutionary subdivisions among the populationsin different basins are anticipated (Starobogatov 1970;Grigoriev & Gozhik 1976). The different schools of zooge-ography are also reflected in the current taxonomy and inan uneven degree of endemism recognized in variousanimal groups. Thus a number of endemic Azov and BlackSea species have been described in gastropod and bivalvemolluscs (Golikov & Starobogatov 1966; Grigoriev & Gozhik

1976), whereas in mysid crustaceans the earlier taxonomicsplitting of the disjunct populations (Martynov 1924) hasbeen dismissed (Derzhavin 1939; Daneliya 2003).

The alternative zoogeographical hypotheses — relict orvicariance vs. immigration or dispersal — seem to be readilytestable by molecular characters. In a pure vicariancescenario, the disjunct populations should contain deeplydiverged endemic clades, whereas in a pure dispersal casethe overall degree of differentiation should be small.Further, if both vicariance (survival in isolated refugia) andintermittent dispersal took place, sympatric occurrencesof distinct lineages should be expected. Previous studies ofamphipod and cladoceran crustaceans have indeed revealeddistinct mitochondrial clades in the Caspian and Black seas,and their divergence seems to contradict the hypothesisof faunal exchange in Late Pleistocene times (2–11% in theCOI gene; Cristescu

et al

. 2003, 2004). The divergenceestimates were consistently smaller in cladocerans than inamphipods, suggesting that besides palaeoenvironmentalchanges the dispersal ability of organisms was also importantin shaping their phylogeographies (Cristescu

et al

. 2003).Comparing molecular divergences across phylogeneticallydistinct taxa is however, risky, as rates of molecularevolution may vary considerably in species with distinctlydifferent generation times and reproduction modes.

Brackish-water Ponto-Caspian mysids provide a goodmodel system for a comparative phylogeographic approachwithin a relatively coherent group of taxa. Most of theauthochtonous Ponto-Caspian mysid species are from asingle genus

Paramysis

, but exhibit a range of ecologicalcharacteristics (Daneliya 2003). Mysid crustaceans ingeneral have poor dispersal abilities — they carry their eggs

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 fromBuchalova (1929), Mordukhai-Boltovskoi (1957), Komarova (1991), Daneliya (2003)

TaxonSize, mm

Natural distribution

Salinity, ‰ HabitatsBLA AZO CAS RIV

Limnomysis benedeni 6–12 + + + – 0–12 estuaries, river deltas, limans; common(Czerniavsky, 1882) among vegetation

Paramysis lacustris (Czerniavsky, 1882)

8–17 + + + – 0–2 estuaries, river deltas, limans; common

Paramysis sowinskii 8–17 – + + – 0–5 estuaries, river deltas; mid-channel;Daneliya, 2002 oxyphilic

Paramysis baeri Czerniavsky, 1882 sensu lato

13–31 + + + + 0–9 rivers, estuaries; oxyphilic

Paramysis ullskyi 12–23 + + + + 0–4 rivers, estuaries; sandy bottoms;Czerniavsky, 1882 oxyphilic

Paramysis kessleri G.O. Sars, 1895

15–52 + – + – 0–9 estuaries; deep waters of central and southern Caspian and western Black Sea

Paramysis intermedia (Czerniavsky, 1882)

6–13 + + + – 0–12 estuaries, river deltas; rare

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and developing young in a brood pouch, cannot withstanddesiccation, and typically cannot actively disperse upstream;their continental distributions have therefore been largelydefined by direct (lentic) water-way connections (B

Å

n

Å

rescu1991). Yet, from distributional evidence, at least two Ponto-Caspian brackish water mysids do have dispersed upstreamin rivers (Buchalova 1929), which provides a good basis tocompare their intraspecific, interbasin subdivisions to thosein more sedentary congeners. Ponto-Caspian mysids alsoexhibit a variety of salinity tolerances, involving both strictlystenohaline (< 3‰) and relatively euryhaline (0–12%) taxa(Komarova 1991; Daneliya 2003). Given the dynamic historyof salinity conditions in the Ponto-Caspian, euryhalinityshould have been an important ecophysiological propertycontrolling the dispersal of species.

In this study we explore the mtDNA diversity andphylogeography in seven opossum shrimp species(Crustacea: Mysida) distributed across the Ponto-Caspianregion (Fig. 1, Table 1). With these data, we test the afore-mentioned biogeographical hypotheses about the historyof the brackish-water fauna, i.e. (i) pure vicariance; (ii) puredispersal; and (iii) vicariance and dispersal. Further, weassess the presence of genetic signals of past demographicchanges in populations from the Black Sea and Sea ofAzov basins, which were affected by drastic environmental

changes in the Pleistocene. As the conditions in the CaspianSea are assumed to have been more stable, we anticipate thatgenetic structures of taxa from this basin will be closer tothat expected in equilibrium populations. In a comparativeframework, from the congruence and differences in thegeographical structuring in the codistributed mysidsand in other invertebrate taxa, we can address the generalimportance of the vicariance and dispersal scenarios inPonto-Caspian biogeography, and the role of the biologicalproperties of the individual taxa (e.g. vagility, salinitytolerance) in their response to the environmental history.

Materials and methods

Samples and laboratory analyses

Mysid samples for molecular analyses were collected in1991, 2000–2004 and stored in 80–96% ethanol. Most samplesare deposited at the Finnish Museum of Natural History.Six currently recognized species of the genus

Paramysis

and one

Limnomysis

species were studied (Table 2). All thesespecies are endemic to the Ponto-Caspian region.

Limnomysis

is a monotypic genus, whereas

Paramysis

(20 species inall) also comprises four taxa in the Mediterranean and theAtlantic. 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 basinGAM 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 basinTSI Tsimlyanskoe water reservoir (2004) 6 2 — — — — —MAN Lake Manych (2004) — 4 — — — — 1SOL Lake Solenoe (2004) — 5 — — — — —MDO Middle Don (2000, 2004) — 5 — 1 — — —DON Dead Don, Don river mouth (2003) 5 5 3 3 3 — 3MIU 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 basinDNP Kherson, Dnepr delta (2004) 4 6 — — — 3 3DNS 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).

P O N T O - C A S P I A N M Y S I D S

2973


sequenced (Fig. 1, Table 2); eight additional sequences fromfour taxa were obtained from published data (Cristescu &Hebert 2005).

The DNA extraction, amplification and sequencingprocedures were as described in Audzijonyte

et al

. (2005).A part of the mitochondrial COI gene was amplified andsequenced using either the universal primers LCO1490and HCO2198 (Folmer

et al

. 1994) or new specific

Paramysis

primers ParaHatD 5

′

-TGTACTTTGTRTTTGGGGCTT-GRGC-3

′

and ParaHatR 5

′

-GTGYTGRTABAGRATAG-GGTC-3

′

. The final mtDNA segment used for the analyseswas 605 bp long and had no length variation.

All specimens analysed for molecular variation werealso assessed morphologically to ensure proper identi-fication and to screen for morphological differencescorresponding to the lineage subdivisions found in themolecular data. For this assessment, reference materialfrom additional collections made by MED in 1996–2005and material from the Zoological Institute, RussianAcademy of Sciences (ZIN) was also examined. Specimenswere first inspected under a stereomicroscope; a geographi-cally representative subset was then fully or partiallydissected and mounted for light microscopy. Furtherdetails on taxonomically important characters are given inDaneliya (2004).

Data analysis

Phylogenetic analyses for intraspecies gene trees were con-ducted using maximum parsimony (MP) and neighbour-joining (NJ) approaches, and for interspecies relationshipsusing MP and maximum likelihood (ML), all as imple-mented in

paup

* 4.0b10 (Swofford 2003). Tree searcheswere conducted with 10 random taxa addition replicates,followed by the tree-bisection–reconnection (TBR) branchswapping; all characters were weighted equally. NJ treeswere constructed using K2P+

Γ

corrected distances thataccount for the transition/transversion bias and heterogeneityin mutation rates among sites. The

α

parameter of rateheterogeneity for within-taxon comparisons was estimatedusing

paup

from a NJ tree; the estimates were 0.1–0.5 for thedifferent taxa, and an average of 0.25 was then used for all.For both NJ and MP analyses, support for the nodes wascalculated from 1000 bootstrap replicates. Further, reticula-tions among haplotypes were assessed using 95% statisticalparsimony networks (Templeton

et al

. 1992) as implementedin

tcs

1.13 (Clement

et al

. 2000).The molecular clock assumption among

Paramysis

taxawas tested using a likelihood ratio test (LRT). An ML treesearch of all unique

Paramysis

haplotypes, assuming nomolecular clock, was first made in

paup

, under the HKY+

Γ

+Imodel of nucleotide substitution, selected from alterna-tive models with aid of the

modeltest

software (Posada &Crandall 1998) and using LRT and significance level (

α

) of

0.01. A likelihood score for a tree with the same topologybut with the clock enforced was then calculated, and thetwo likelihood scores compared with a LRT (

α

= 0.01).

Limnomysis benedeni

was strongly diverged from all

Paramysis

taxa (

c

. 25% uncorrected and 40% K2P+

Γ

correcteddivergence) and was not included in the analyses of rateconstancy. Bootstrap support for the ML topology withoutmolecular clock was estimated using 500 bootstrap replicates.Because molecular clock assumption was rejected by theLRT (see Results), the relative rates of evolutionary changealong the branches were estimated using nonparametric ratesmoothing method (NPRS) (Sanderson 1997) implementedin

treefinder

( Jobb 2005). A distinct Mediterranean

Paramysis

species,

P. arenosa

was used as outgroup forrooting the ML tree, but was excluded from the furtherNPRS procedure (Jobb 2005). The monophyly of the studiedPonto-Caspian

Paramysis

in respect of

P

.

arenosa

was verifiedby a preliminary phylogenetic analysis from COI, 18S and28S sequences (A. Audzijonyte, unpublished data).

Haplotype (

h

) and nucleotide (

π

) diversity indices(Nei 1987) were calculated in

arlequin

2.000 (Schneider

et al

. 2000) for each species and then separately for popu-lations from the Caspian, Black Sea and Sea of Azov basins.Diversity level differences between the regions wereevaluated with Student’s

t

test and the standard deviationestimates of the indices provided by

arlequin

. Moleculardivergences among haplotypes from the three basins werecalculated in terms of the K2P+

Γ

distance (

α

= 0.25); boththe overall mean and net distances (corrected for intrabasinvariability) were estimated, using

mega

(Kumar

et al

. 2004).The partitioning of nucleotide diversity among the threebasins, among populations within the basins, and amongindividuals within populations was assessed from theanalysis of molecular variance (

amova

) (Excoffier

et al

. 1992)as implemented in

arlequin

; alternative groupings werealso tested for some taxa. Statistical significance wasevaluated with 1000 random permutations.

Tajima’s (1989)

D

and Fu’s (1996)

F

statistics, and mismatchdistributions (i.e. distributions of pairwise nucleotidedifferences; Rogers & Harpending 1992) were used to assessdeviations of the observed patterns of molecular diversityfrom that expected in equilibrium populations and selectiveneutrality. Statistical significance was assessed from 1000permutations; the critical

P

value was 0.05 for Tajima’s

D and 0.02 for Fu’s F (Schneider et al. 2000). Mismatchdistributions illustrate the depths of coalescence withina sample of haplotypes in mutation-generation units.Distinctly unimodal distributions are expected in a case ofa past sudden demographic expansion at a time approximat-ing the peak coalescence (τ) (Rogers & Harpending 1992).The goodness-of-fit of the observed data to that expectedunder the sudden expansion model was assessed fromsimulations, and the 95% confidence intervals of τ underthe expansion model calculated from 1000 parametric

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bootstrapping replicates. All calculations were performedin arlequin. It must be emphasized that unimodal distribu-tions may also be caused by selective sweeps or by hetero-geneity in mutation rates (Simonsen et al. 1995; Schneider& Excoffier 1999), and generally effects of selection anddemographic changes are indistinguishable in data from asingle locus, such as mtDNA (Galtier et al. 2000). The demo-graphic interpretations of the equilibrium statistics aretherefore only suggestive.

Results

Molecular divergence and diversity

No stop codons were identified in the analysed COI sequencesand no heterogeneity in base composition was detectedusing a χ2 test; the mean A+T content in all sequences wasc. 55%. Altogether 87 different haplotypes were observedin the here analysed taxa (N = 165) (DQ779792–DQ779878)(a table of haplotype frequencies in each sample is availablefrom the authors). All MP, ML and NJ analyses stronglysupported the monophyly of mtDNA lineages in each of theseven currently recognized species (bootstrap support 90–100%). Resolution at the interspecific level was considerablyweaker (bootstrap support < 75% for most nodes; Fig. 2h);here molecular divergences among taxa were typically30–40% (HKY+Γ+I correction). A closer interspeciesrelationship (13% divergence) was between the sisterspecies Paramysis lacustris and Paramysis sowinskii, onlyrecently taxonomically distinguished (Daneliya 2002).

The assumption of a molecular clock during Paramysisevolution was rejected by the likelihood ratio test (P < 0.01),suggesting that molecular differences cannot necessarilybe interpreted in identical temporal terms across taxa.Conspicuous rate variation was evident from the branchlengths in the interspecies tree, rooted by Paramysis arenosa;the branches of Paramysis kessleri, Paramysis intermedia andParamysis ullskyi were notably longer than in the apparentsister group involving P. lacustris and P. sowinskii (Fig. 2h).In the NPRS approach, the substitution rates in P. kessleri,P. intermedia and the central Caspian Paramysis baeri brancheswere 2.0–2.5 times faster than in the P. lacustris + P. sowinskiigroup. Therefore also the comparisons of intraspecies molec-ular divergences across taxa must be treated with reservation.Anyway, the focus here is on comparisons of phylogeo-graphic structures (presence vs. absence of subdivisions)and on the most extreme differences in levels of divergence.

Intraspecific diversities varied greatly among the sevenanalysed taxa; the maximum sequence divergences rangedfrom 1.2% in P. kessleri to 14.8% in P. baeri sensu lato (Table 3).In the material initially identified as P. baeri, however, twodivergent haplotype groups were observed (Fig. 2a) whichalso turned out to differ morphologically in several charactersand were therefore judged as separate species. One of thesegroups was identified in samples from the coast of centralCaspian Sea and will below be referred to as P. baeri s. str.Another clade comprised the remaining material fromthe northern Caspian, Azov and Black seas, and is hereprovisionally called P. cf. baeri I. Moreover, the GenBanksequence of P. baeri (AY529030, northern Caspian Sea,Cristescu & Hebert 2005) was similarly distinct from thetwo groups identified in our data, suggesting even furtherspecies level subdivisions in the taxon. This haplotype isreferred to as P. cf. baeri II, but it will not be included infurther genetic diversity calculations below. It shouldbe noted that the taxonomy of P. baeri has even previouslyundergone changes. P. cf. baeri I will evidently correspondto the taxon Paramysis bakuensis G.O. Sars, 1895, whichwas later synonymized with P. baeri Czerniavsky, 1882 byDerzhavin (1939); the question will be treated more formallyelsewhere (Daneliya et al., in preparation).

Most of the intraspecific nucleotide variation in the seventaxa was at silent sites, but four and seven amino acid replace-ment substitutions were found in Limnomysis benedeni andP. lacustris (Table 3). In contrast, no such substitutions werefound among 88 variable sites of the three taxa of P. baerisensu lato.

Apart from the case of P. baeri s. l., the deepest genealogicalsubdivision was within P. lacustris (maximum divergence6.5%). The molecular lineages of P. lacustris were notassociated with consistent morphological differentiationto corroborate taxonomic distinction however. The lineagedivision in P. lacustris was reflected in the overall estimateof its nucleotide diversity (π ≈ 2.5%), higher than thosein remaining taxa (0.5–1.3%; Table 3). Exceptionally highlocal intraspecies diversity in absence of lineage divisionwas found in P. baeri s. str. from the central CaspianSea, where each of the five analysed specimens had uniquehaplotypes (h = 1) that differed on average by 12 nucleotidesubstitutions (π ≈ 2%) (Figs 2a, 3a).

Haplotype diversities were generally high across theanalysed taxa (c. 0.9) and about 50% of all sequencedspecimens had unique haplotypes. The diversities wereparticularly 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 treeof Limnomysis benedeni was rooted using mid-point rooting, other trees using other Paramysis taxa as outgroups. Only unique haplotypeswere used; their geographical origins in the three main basins are illustrated by colour codes and site labels referring to Fig. 1a. All treesare 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 supportvalues (> 50%) from ML analyses are indicated above branches, from MP — below branches (MP topology was nearly identical to that of ML).

P O N T O - C A S P I A N M Y S I D S 2975




Comparison of molecular diversities across the three basinswas feasible in two species with more extensive geographicalsampling, i.e. L. benedeni and P. lacustris. In both speciesnucleotide diversities were higher in the Black Sea thanin the Azov and Caspian basins (P < 0.05), but this trendwas not evident in haplotype diversities.

Significantly negative Tajima’s D values (and in somecases Fu’s F) and unimodal mismatch distributions,suggesting past demographic expansion, were observedonly in the Caspian samples of L. benedeni, in the Caspianand Azov samples of P. lacustris and in the two genealogicalclades of P. ullskyi (central Caspian, and the remainingwidespread clade) (Fig. 4). A star phylogeny with acommon central haplotype was particularly evident fromthe parsimony network of L. benedeni Caspian Sea clade(Fig. 3g) and to a lesser extent in P. lacustris (Fig. 3e).However, rare distinct haplotypes were also identifiedin the Caspian Sea populations of all the three species,causing secondary peaks in the mismatch distributions.

No evidence of population expansion was inferred fromthe Black Sea samples of any taxon (not shown).

Phylogeographic structure

The strongest phylogeographic structure and deepestintraspecific subdivisions were seen in P. lacustris. All threebasins and also the separate river estuaries of the BlackSea basin contained specific haplotype groups (Fig. 2e).The deepest genealogical split (c. 5% divergence) separatedhaplotypes from the geographically intermediate Sea ofAzov from those in the Caspian and Black seas, which inturn were 1.7% diverged from each other. When correctedfor the intrabasin variability (net distance) the Azov basinclade differed from those in the other two basins by 4.4%.Of the two populations from the past contact area betweenCaspian and Azov seas (Manych depression), one containedCaspian, the other Azov lineage haplotypes. An exceptionto the genealogical-geographical congruence in P. lacustris

Table 3 Molecular diversity estimates in the seven analysed mysid species and in their intraspecific genealogical/geographicalsubdivisions. Abbreviations: n, number of specimens analysed; k, number of samples; NH, number of different haplotypes (proportion ofthe 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 maximumobserved 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.



was recorded from two upstream sites of the Don River inthe Azov basin, i.e. Middle Don (MDO) and Tsimlyanskoewater reservoir (TSI). Specimens from these sites containedCaspian lineage haplotypes, identical to those from theVolga river delta (VOL) (Fig. 2e).

A similarly distinct Caspian Sea clade was identified inL. benedeni, whereas no consistent subdivision was evidentbetween the Azov and Black Sea populations of this species.The latter basins contained groups of genealogically distinctbut geographically intermixed haplotypes (Figs 2g, 3g).The mean divergence of the Caspian Sea clade from thosein Black/Azov sea basins was 1.8% (net distance 1.1%). Aswith P. lacustris, L. benedeni from Tsimlyanskoe reservoir

TSI on the Don (Azov drainage) had Caspian lineagehaplotypes (Fig. 2g).

A different phylogeographic structure was found inP. baeri s. l. and in P. ullskyi. In each of these taxa the basalgenealogical split separated central Caspian Sea populationsfrom the rest, i.e. from a widespread lineage encompassingthe northern Caspian, Azov and Black Sea samples; thepattern was broken by a single Volga river haplotypeconnected with the central Caspian clade in P. ullskyi(Fig. 2a, b). The depths of the principal subdivisions werehowever, very different: a species-level subdivision in P. baeris. l. (12% divergence) contrasted by a 1.5% distinction inP. ullskyi (Table 4). Apart from the distinct central Caspian

Fig. 3 Statistical (95%) parsimony haplotypenetworks for the seven mysid taxa. Geogra-phical origins of haplotypes are illustratedby colours shown in Fig. 1a. The size of acircle is proportional to the observednumber of the corresponding haplotype.The minimum number of steps needed toconnect each of the two clades in Paramysisbaeri sensu lato and in Paramysis lacustris isindicated along the dashed lines.



clades, neither P. baeri s. l. nor P. ullskyi showed furtherphylogeographic structure among or within the northernCaspian, Azov and Black seas.

The least differentiation and no phylogeographic struc-turing were seen in P. kessleri (Fig. 2b) and in P. intermedia(Fig. 2f). For instance, in P. kessleri specimens from the

southern Caspian Sea and the Danube delta in the westernBlack Sea basin were separated by only one nucleotidedifference (Fig. 3b).

There was little congruence in the depths of divergenceamong the interbasin clades of different mysid taxa (Table 4)and those of other invertebrate groups in previously

Fig. 4 Mismatch distributions, or distribu-tions of pairwise differences in geographicalgroups of populations, for taxa with largersample sizes. Dashed lines show 95% con-fidence intervals of the simulated mismatchdistribution (solid line). None of the BlackSea populations conformed to the expecta-tions of demographic expansion and arenot shown here. * — for Paramysis ullskyi,divergent haplotypes from the centralCaspian Sea coast were excluded. Tajima’sD 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 (fromamova), 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 10.8Paramysis 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 6.2Paramysis 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)



published data (Fig. 5). In most taxa, the Sea of Azov-BlackSea subdivision was weaker than the distinction of theCaspian from these two basins, but in P. lacustris even thispattern did not hold.

Discussion

Three main phylogeographic patterns were identifiedin the analysed mysid taxa: (i) overall homogeneity withclosely related haplotypes across the entire Ponto-Caspianregion — in Paramysis kessleri; (ii) a main genealogicaldivision that separates central Caspian Sea haplotypesfrom a widespread, relatively uniform Black/Azov/Volgariver basin clade — in Paramysis baeri s. l and (with oneexception) in Paramysis ullskyi; and (iii) genealogicalsplits that geographically match the border between the

Black/Azov and Caspian sea basins, or those among allthree basins — in Limnomysis benedeni and Paramysis lacustris.Two further taxa, Paramysis sowinskii and Paramysisintermedia, were sampled from the Azov and Black Seabasins only and did not show consistent geographicaldifferentiation.

Past exchange among basins and rivers of the Ponto-Caspian

The palaeontological record of the Black Sea basin providesevidence of repeated changes between marine and brackish/freshwater faunal communities that tracked the changingsalinity conditions (Fedorov 1978; Zubakov 1988). Whenevera connection to the Mediterranean was established andmarine waters intruded into the Black Sea, the brackish-water fauna was extirpated or confined to the diluted riverdeltas, as today. Low-salinity periods in turn could haveprovided opportunities for dispersal among rivers, butit is also possible that the coastal and estuarine mysids,normally absent from open water habitats, still remainedconfined to river deltas and did not substantially intermix.Yet phylogeographic data from amphipods, cladoceransand fishes (Barbus) with habitat requirements similar to thoseof the mysids have generally shown little differentiationamong distant Black Sea rivers, suggesting that gene flowdid occur (Cristescu et al. 2003; Kotlík et al. 2004).

In contrast, the Pleistocene period within the CaspianSea was marked by relatively stable salinity conditions thatin the open Southern Caspian were within the range of10–13‰, i.e. similar to those today (Fedorov 1980). Waterlevel fluctuations nevertheless spanned c. 100 m amplitude(Svitoch et al. 2000) and repeatedly inundated and exposedlarge areas in the northern parts of the basin (Mamedov1997). The Caspian transgressions coincided with the coldperiods of the Pleistocene; it was at these times, and also asrecently as c. 15 000 years ago, that the Manych depressionwas flooded, enabling downstream dispersal of the Caspianfauna towards the Azov basin (Fig. 1b) (Svitoch et al. 2000;Ryan et al. 2003; Bahr et al. 2005). During regressions,the previous one c. 20 000 years ago (Mamedov 1997), thenorthern coast of the Caspian Sea in turn retreated c. 100 kmsouthwards creating good opportunities for exchangebetween the currently separated northern and centralCaspian coastal populations of stenohaline taxa (Fig. 1b).It remains unclear however, to what extent these changesof the Caspian Sea affected subdivisions of its fauna; so farthere are virtually no data on intrabasin phylogeographyof Caspian Sea organisms.

The weak phylogeographic structuring recorded amongBlack Sea river drainages and between the Azov and Blacksea basins in L. benedeni, P. intermedia and P. cf. baeri I wasin line with the aforementioned observations from othercrustaceans and fishes, suggesting past gene flow, plausibly

Fig. 5 Summary of the average interbasin mitochondrial COIsequence divergence (K2P + Γ distance, not corrected for intrabasindiversity) in mysids (this study) and in other invertebrate taxa(data from Cristescu et al. 2003, 2004; Nikula & Väinölä 2003;Therriault et al. 2004). Comparisons where the Caspian Sea isrepresented only by a central/southern Caspian population areindicated with triangles. Divergences judged to involve distinctspecies are marked with an asterisk (*). Abbreviations: CG,Cerastoderma glaucum; DR, Dreissena rostriformis; CM, Cornigeriusmaeoticus; CP, Cercopagis pengoi; PT, Podonevadne trigona; EI, Echino-gammarus ischnus; OC, Obesogammarus crassus; PM, Pontogammarusmaeoticus; PR, Pontogammarus robustoides; LB, Limnomysis benedeni;PB, Paramysis baeri sensu lato; PI, Paramysis intermedia; PK, Paramysiskessleri; PL, Paramysis lacustris.



during the diluted phases of the Black Sea. Also withinthe Caspian Sea, populations of P. lacustris and L. benedenifrom northern and central coasts contained identical orclosely related haplotypes, supporting faunal exchange.Differences in P. ullskyi and in P. baeri sensu lato, however,suggest continued isolation between populations of thenorthern and central Caspian Sea coasts. In these taxa,clades from the two areas defined the main Ponto-Caspiangenealogical split, which however, involved groups ofevidently different age (1.5 and 12% divergence) anddifferent systematic rank. In both taxa, the coastal centralCaspian clades were endemic to this basin, whereas thenorthern populations contained haplotypes close to thosein the Azov and Black Sea basins. The uniformity of thelatter lineages in P. cf. baeri I and P. ullskyi, as well assimilarity of the Black and Caspian haplotypes in P. kesslerisuggest that the Manych Strait between Caspian and Azovbasins was an efficient dispersal route. The causes for thespatial segregation of the northern and the endemic centralCaspian Sea clades however, remain unclear.

Given the extreme environmental changes in the Blackand Azov seas and their postulated destructive effects onlocal faunas, it has been assumed that the main directionof migration along the Manych Strait was westwards, outof the Caspian Sea (Mordukhai-Boltovskoi 1979; Zubakov1988). On the other hand, there is also evidence of a LatePleistocene/Holocene colonization in the opposite direc-tion, as regards appearance of Mediterranean Cerastodermabivalves in the Caspian Sea (Fedorov 1978; Nikula &Väinölä 2003). From the current mtDNA data, no conclu-sive inferences about the direction of gene flow in P. ullskyi,P. cf. baeri I and P. kessleri can be drawn, as none of thebasins appeared to harbour higher and possibly ancestraldiversity (disregarding the distinct central Caspian groups).Downstream dispersal from the Caspian would seemmore feasible a priori. On the other hand, the presence oftwo distinct lineages of P. ullskyi (and three of P. baeri s. l.)in the Caspian basin could reflect their dual origins in thebasin, i.e. (re)invasion from the Black-Azov basin followingPleistocene interbasin vicariance and divergence.

If such an immigration from the Black-Azov to theCaspian basin occurred via the Late Pleistocene Manychconnection, the mysids possibly had to migrate upstream.Indeed, in contrast to most Mysida, P. ullskyi and P. baeri s.l. are known to be capable of such dispersal (Buchalova1929). For instance, natural occurrences of P. ullskyi wereknown from 1000 to 3000 km upstream in the Volga river(Mordukhai-Boltovskoi 1957). An eastward dispersal ofP. ullskyi and P. cf. baeri I could in fact have occurred evenmore recently. Throughout the Holocene, the Caspian Seahas been an isolated lake with no connection to the west,until a new link with the Sea of Azov basin was establishedin 1952 through the Volga-Don canal. Since then a numberof nonindigenous organisms have colonized the Caspian

Sea, whereas few taxa seem to have dispersed out fromthe Caspian (Grigorovich et al. 2003; Orlova et al. 2004;Therriault et al. 2005). With the current data, we cannotstrictly rule out a possibility that the Black-Azov lineagesof P. ullskyi and of P. cf. baeri I arrived to the Volga River viathe Volga-Don canal. In that case, however, we shouldalso assume that the new invaders or their mitochondriadisplaced local populations (lineages) that were commonin Volga prior to the canal construction (Mordukhai-Boltovskoi 1957). Such a recent immigration to the Caspianalso seems very unlikely for P. kessleri, which is currentlyconfined to the western Black Sea and south-centralCaspian and is similarly homogeneous. Finally, the data onP. lacustris and L. benedeni suggest that mysid dispersalthrough the Volga-Don canal has been out of the Caspianbasin rather than into it (see below).

Although gene flow among estuaries of the Black-Azovbasin rivers was inferred for most taxa, the patterns ofmolecular diversity nevertheless indicated varying impor-tance of local refugia in different species. Populations ofL. benedeni from the Don, Kuban, Danube and Dneprestuaries contained deeply divergent but intermixedhaplotypes, suggesting both persistence of local populations(refugial divergence, no extinctions) and subsequent geneflow. In contrast, the weak overall differentiation withinP. cf. baeri I, P. ullskyi and P. kessleri indicates that in thesespecies either the local populations in different refugia havegone extinct before re-colonizations or that competitivereplacement of mitochondria or of entire populations hastaken place. Yet generally, given the small sample sizes,we cannot exclude the possibility that additional, moredivergent but undetected lineages also exist in the lattertaxa. Although the current data can reject the pure vicariancehypothesis (no migration among refugia) they do nothave power to discriminate between the pure dispersal vs.vicariance + dispersal scenarios.

Notably, none of the Black Sea populations showed signsof a demographic expansion, which could have happenedif the vast diluted Late Pleistocene lake-sea was overtakenfrom a single refugial source. It therefore seems that diver-sity in the Black Sea basin has been maintained through thehigh and low salinity periods. On the other hand, signals ofpast demographic expansion were observed in the CaspianSea populations of P. lacustris and L. benedeni, an unexpectedresult considering the relatively stable salinity conditionsin that basin. The data nevertheless suggest that the twospecies experienced more or less severe bottlenecks inthe Caspian Sea (a coalescence of haplotypes to a singleancestor before the expansion), but currently no conclu-sions can be drawn whether these were true bottlenecks ofpopulations or only of their mitochondria (e.g. selectivesweep). Clearly, further sampling and markers of higherresolution are needed for more detailed phylogeographicunderstanding.



Local refugia in Paramysis lacustris and Limnomysis benedeni

Although extensive gene flow among Ponto-Caspian basinswas inferred in some taxa, other species showed distinctinterbasin structuring. The Caspian populations of L. benedeniwere clearly separated from those in the Black/Azov seabasins (c. 1.8%), and still deeper geographical subdivisionswere found in P. lacustris, which is the most stenohalineof the taxa studied. The 5% Azov-Caspian divergence isnotably high considering the typical levels of intraspecificdifferentiation (generally < 3%) in other crustaceansand invertebrates (Hebert et al. 2003). Yet, no consistentmorphological differentiation among various populationswas recorded to corroborate a presence of sibling species.Applying the commonly used invertebrate mitochondrialmolecular clock of 2–3% per 1 Myr (Knowlton & Weigt1998; Wares & Cunningham 2001), the 2–5% Azov-Caspiandivergences in L. benedeni and P. lacustris could plausiblybe of Early to Middle Pleistocene age. On the other hand,patterns of molecular diversity in postglacially isolatedpopulations of another mysid species have suggested10-fold higher rates of COI evolution on recent timescales (Audzijonyte & Väinölä 2006). If this also appliesto Ponto-Caspian taxa, the distinction of Azov basin cladescould be as young as 0.1–0.2 Myr. However, comparisonof molecular rates across Paramysis taxa actually suggestedslower than average rates in P. lacustris (Fig. 2h), whichrather emphasizes the relative antiquity of the divergence.Whatever the molecular rate, from palaeogeographicaldata and from the phylogeographical structure ofother mysids it is evident that there should have beenopportunities for intermixing of the Caspian and Azovbasin clades, when interbasin contacts were establishedthroughout the Pleistocene (Svitoch et al. 2000). Theretained Caspian-Azov distinctions within P. lacustris andL. benedeni therefore could indicate reproductive isolationbetween the lineages despite their apparent morphologicaluniformity. The current mtDNA data alone however,do not provide sufficient grounds for definite taxonomicconclusions.

The deep molecular subdivisions in mysids also haveparallels in other weakly dispersing Ponto-Caspian taxa.Comparisons of allegedly conspecific Black Sea vs. Caspianbasin populations of amphipods have revealed COI diver-gences up to 11% (or 16% in terms of the K2P+Γ distanceapplied here) (Cristescu et al. 2003), and up to 4% (c. 5%)between populations from a single river (Cristescu et al.2004) (Fig. 5). However, the phylogeographical distribu-tion of the P. lacustris clades, i.e. a greater separation of theAzov population from the Black+Caspian clade, is excep-tional and unexpected considering the palaeogeographichistory of the region. Repeatedly through the Pleistocene,the shallow Sea of Azov was nearly dry during the Black Sea

low water stands (Ryan et al. 2003). It is therefore thought— and also inferred from the data of other mysid taxa inthis study — that the Azov basin fauna generally consistsof postglacial recolonisers either from the Black Sea (whosesalinity at that time was lower), or from the Caspian basinvia the Manych connection.

The diverged Sea of Azov clade of P. lacustris neverthe-less suggests survival in a local refugium, possibly inthe Don and Kuban rivers, which during the low standsextended far south and drained directly into the Black Sea.The contrasting patterns in P. lacustris and the other mysidsas regards isolation vs. mixing of the Azov and Black Sealineages could be related to the different salinity tolerancesof the taxa in face of the palaeosalinity changes in the BlackSea. The discussion remains unsettled whether duringthe last regression the salinity of the Black Sea decreasedto practically fresh (0–1‰) or to slightly brackish levels (4–5‰) (Ryan et al. 1997, 2003; Mudie et al. 2002). Comparativemysid phylogeography would seem to support the latteroption. The taxa that naturally thrive in salinities of up to5–6‰ are homogeneous, indicating Black-Azov gene flow(i.e. P. intermedia, P. baeri, L. benedeni). In contrast, thestenohaline P. lacustris, typically confined to salinities< 2–3‰, has remained isolated, plausibly due to a salinity–related dispersal barrier.

Also within the Black Sea, distinct allopatric lineageswere found in the Danube and Dnepr estuary populationsof P. lacustris, indicating limited gene flow among the rivers,but the sampling was too limited to exclude possible inter-mixing. Notably, P. lacustris in the Azov basin also showeda signal of a sudden demographic expansion, not seen inother taxa (Fig. 4). The inferred time of population expan-sion c. 150 000 (50 000–300 000) years ago however, wouldnot fit the postglacial refilling of the basin, if a conventionalmolecular rate is assumed (c. 2% per Myr). On the otherhand, with the 10-fold faster postglacial mtDNA ratediscussed above, the data could indeed reflect a postglacialexpansion (22 000–7000 years).

There is also evidence that the Sea of Azov refugium ofP. lacustris was not entirely restricted to rivers, as haplotypesof this lineage are also present in the ‘relict’ Lake Abrau(ABR, Fig. 1). The lake, currently 83 m above the sea leveland 1.8 km from the Black Sea coast, contains a communityof brackish-water Ponto-Caspian taxa and even an endemicclupeid fish species (Mordukhai-Boltovskoi 1964). The lakeprobably originated after an earthquake-induced land-slide(c. 175 m high) dammed the ancient river Abrau from theBlack Sea; subsequent accumulation of sediments lifted thelake level and the inhabitants captured into it (Ostrovskii1970). No geological dating of the event is available, butthe presence of the brackish-water community has beentaken as evidence of isolation during the Late Pleistocenelow-salinity phase of the Black Sea (Mordukhai-Boltovskoi1960). The faunal evidence alone actually does not prove



the Late Pleistocene age of the lake, as desalinizations ofthe Black Sea and expansions of the brackish-water faunahave occurred repeatedly throughout the Pleistocene.The molecular data of P. lacustris however, now points toa recent lake origin, as all the Abrau specimens had ahaplotype also found in the Kuban River. The absence ofmolecular variation in P. lacustris also implies a smalleffective population size, as expected in a small lake(c. 1.5 km2).

Recent contact between Caspian and Azov basins

Four of our sampling sites were from the region betweenthe Azov and Caspian seas, either from the waters alongthe Manych Strait that formerly connected the two basins(SOL, MAN), or along the Don river (Azov drainage), whichsince 1952 has been connected through a canal also to theVolga river and the Caspian (TSI, MDO). In the 1950s, anumber of water reservoirs were also established on theDon (including TSI, the Tsimlyanskoe reservoir), and thesereservoirs were stocked with P. lacustris and L. benedenifrom the Don delta area for fish-food enrichment purposes(Ioffe 1958). No natural occurrences of these species wereknown from localities this high upstream on the Donuntil then (Martynov 1924; Buchalova 1929). Unexpectedly,however, all P. lacustris and L. benedeni from both the Donsites (TSI downstream of the Volga-Don canal junction,MDO 250 km upstream) had haplotypes identical or closelyrelated to those in the natural habitats in the Volga riverdelta. The data thus suggest recent immigration of theCaspian lineages into the middle reaches of the Don riverbasin, via the Volga-Don canal, whereas no trace of theinitially translocated stock were seen. It remains unclearwhether the recent Caspian invaders have replaced thestocked Azov-Don basin mysids, or whether what wesee is a result of selective introgression of the Caspian-type mitochondria into the Don populations followinginterbreeding.

In two lakes along the former Manych Strait, P. lacustrisboth of the Azov lineage (in MAN) and of the Caspianlineage (SOL) were found. If the populations actuallyrepresent relicts of the Manych Strait, this could reflecttwo-way dispersal in Late Pleistocene times. Alternatively,it could indicate recent immigration of the Caspian lineagevia the Volga-Don canal, or of a transplanted Don River(Azov drainage) population from adjacent, connected waterreservoirs (Kruglova 1959). Study of independentlysegregating nuclear markers could clarify the origins ofthe transition zone populations. Such markers shouldalso help to evaluate the possible reproductive isolationbetween the Azov and Caspian clades in nature, andelucidate the question of apparent long-term isolation ofthe endemic Azov clade despite the evidently good coloniza-tion capacity of the Caspian lineage.

Conclusions

A comparative phylogeographic assessment of seven Ponto-Caspian mysid taxa, and of other codistributed invertebrategroups, suggests a complex history of lineage isolationand interbasin exchange events — vicariance and dispersal— during the dynamic Pleistocene history of the Black, Azovand Caspian seas. The lack of intertaxon congruence inthe geographic distributions of intrataxon lineages, andparticularly in depths of their divergence (Fig. 5), impliesthat similar distributional patterns have arisen at widelydifferent time scales.

Overall, there was no evidence of intraspecific divergenceas old as the Late Miocene period (10–5 Myr); the deepestsubdivision, in Paramysis lacustris (5–6%), could plausiblybe of Early Pleistocene age. In fact, the Tertiary vicariancescenario could better account for the initial divergence ofsome of the Paramysis species themselves, which were 12–40% diverged from each other. At the intraspecific level,all three biogeographical scenarios (vicariance, vicariance+ dispersal, vicariance + extinction + dispersal) seemedapplicable to the histories of different mysid species. Theinterbasin segregation of the deep P. lacustris lineagesand of the Caspian Limnomysis benedeni lineage conformedto the vicariance expectation; the sympatry of distinctlineages of L. benedeni in Azov-Black Sea estuaries to thevicariance and dispersal scenario; whereas the large-scaleuniformity in Paramysis cf. baeri I, Paramysis ullskyi andParamysis kessleri implied local extinctions and relativelyrecent recolonizations.

The phylogeographical histories of species in responseto the oscillating Pleistocene palaeogeographical andpalaeoecolgical conditions have evidently been controlledby their ecological characteristics. This is seen in comparisonsamong mysid species (vagile species being homogeneous,stenohaline species most subdivided), as well as in com-parisons among groups at higher taxonomic levels. Taxaapt for long-distance dispersal, such as bivalve molluscs withplanktonic larvae, or cladoceran crustaceans with restingeggs, show weaker Caspian vs. Black Sea differentiationthan do amphipods (Cristescu et al. 2003; Fig. 5). However,in this broader comparison, average mysid patterns appearcloser to those of cladocerans than of amphipods, whichwould not necessarily be expected from dispersal charac-teristics (Fig. 5).

In some mysid taxa, i.e. P. lacustris and L. benedeni, thegenealogical subdivisions generally matched geographicalborders among the basins, whereas in others, i.e. P. baeri s.l. and P. ullskyi, the deepest subdivisions were seen withinthe Caspian Sea itself. The data thus warn against treatingany of the basins as a uniform area, and show that furthermolecular analyses of the Ponto-Caspian fauna will likelyincrease estimates of species diversity, at least in thosegroups where the taxonomic tradition has preferred



lumping. Finally, the study also has important implicationsfor attempts to trace the origin of recent invaders ofEuropean waters using mitochondrial genes (e.g. Cristescuet al. 2001, 2004). Such an approach would seem promisingin P. lacustris and to some extent also in L. benedeni, giventhe geographical structuring of their mitochondrialdiversity. In other taxa however, mitochondrial data wouldprovide no power to distinguish most of the Caspian, Azovand Black Sea populations, and markers of higher resolutionshould be tested.

Acknowledgements

We thank all those who have contributed to sampling, includingK. J. Wittmann (for samples from the Danube Delta), F. Riedel,N. Mugue, A. Gorbunov, V. Ponomarenko, and T. Aleksenko.The study has been supported by grants from the Walter andAndrée de Nottbeck Foundation, the University of Helsinkiresearch funds and the Academy of Finland.

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Asta Audzijonyte has recently completed her PhD on diversityand zoogeography of continental mysid crustaceans. Her studiesaddress evolution both in the Ponto-Caspian region and in thenorthern glacial lakes. Mikhail Daneliya is a specialist of Ponto-Caspian mysid systematics. The research interests of Risto Väinöläare in the diversity and history of northern aquatic fauna. All threeare interested in speciation processes in Eurasian ancient lakes.

Documents

Comparative phylogeography of Ponto-Caspian mysid …webpages.icav.up.pt/PTDC/BIA-BEC/104097/2008/11.pdf · 2009-02-10 · Finnish Museum of Natural History, POB 26, FI-00014 University