Mitochondrial Gene Trees Support Persistence of Cold Tolerant Fairy Shrimp Throughout the Pleistocene Glaciations in Both Southern and More Northerly Refugia

Hydrobiologia DOI 10.1007/s10750-013-1533-6

PRIMARY RESEARCH PAPER

Mitochondrial gene trees support persistence of cold tolerant fairy shrimp throughout the Pleistocene glaciations in both southern and more northerly refugia

Jane Reniers • Bram Vanschoenwinkel • Nicolas Rabet • Luc Brendonck

Received: 25 October 2012 / Revised: 16 April 2013 / Accepted: 28 April 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Fairy shrimp (Crustacea: Anostraca) are data, the Eastern European subspecies C. diaphanus specialist inhabitants of temporary aquatic habitats. In romanicus deserves species status while the species many parts of the world and particularly in Western status of two Italian chirocephalids, C. salinus and C. Europe, however, populations are declining while the ruffoi is questionable. Results indicate European C. development of adequate conservation strategies is diaphanus lineages diverged well before the last impeded by a poor knowledge of the genetic structure glacial maximum and survived the Pleistocene glaci- and taxonomic status of remaining lineages. We ations in multiple (sub)refugia along the Iberian, reconstructed a phylogeography of the species Chiro- Italian, and Balkan peninsula. Northern Europe was cephalus diaphanus Pre´vost, 1803 using partial subsequently recolonized from Southern France, sequences of the mitochondrial COI gene and discuss resulting in high levels of cryptic diversity around the importance of different Pleistocene refugia to glacial refugia but also in more widespread haplotypes explain current diversity patterns. In addition to 20 C. in mainland Europe. diaphanus populations, we also included populations of six presumably closely related chirocephalids to Keywords C. diaphanus Cryptic diversity Long evaluate their taxonomic status. Based on molecular distance dispersal Temporary pools Zooplankton Refugia

Jane Reniers and Bram Vanschoenwinkel contributed equally to this manuscript. Introduction

Handling editor: Diego Fontaneto Members of the crustacean order Anostraca, which Electronic supplementary material The online version of includes fairy shrimps and brine shrimps, are charac- this article (doi:10.1007/s10750-013-1533-6) contains teristic inhabitants of temporary aquatic systems supplementary material, which is available to authorized users. worldwide (Brendonck et al., 2008). Most anostracans are omnivorous ﬁlter feeders that are characterized by J. Reniers (&) B. Vanschoenwinkel L. Brendonck Laboratory of Aquatic Ecology and Evolutionary Biology, obligate sexual reproduction. They produce resistant KULeuven, 3000 Leuven, Belgium resting eggs that ensure survival of populations when e-mail: [email protected] the habitat dries out or freezes solid (Brendonck & De Meester, 2003). In addition, these eggs represent the N. Rabet UMR BOREA, MNHN-UPMC-CNRS-IRD, dominant life stage that is passively dispersed by wind, 43 rue Cuvier, 75005 Paris, France water, and animal vectors (Sanchez et al., 2007; 123 Hydrobiologia

Vanschoenwinkel et al., 2008a, b). Chirocephalus is with a lack of clear geographical structure—on the the second largest genus within the Anostraca and is other hand, is explained in terms of a rapid range distributed throughout the Palearctic region (Brtek & expansion after the last glacial maximum (LGM) (e.g., Mura, 2000). Within this genus, Chirocephalus di- in the water flea Daphnia magna; De Gelas & De aphanus is the most widespread and most common Meester, 2005). Little is known, however, about the European anostracan (Brtek & Thie´ry, 1995). C. exact location of glacial refugia in European freshwa- diaphanus is a eurytopic species (Fonseca et al., ter zooplankton (but see Go´mez et al., 2000, 2007;Xu 2008) occurring in a wide range of freshwater habitats et al., 2009). For anostracans, phylogeographic studies in both lowland and highland areas up to 2,300 m are either restricted to halophilic species and fresh- above sea-level (Alonso, 1996; Defaye et al., 1998; water species of the subtropics, Mediterranean or Mura, 1999; Demeter & Stoicescu, 2008). Its habitat North America which often occupy relatively small includes flooded fields, temporary ponds, ditches and geographic areas, or only consider subfragments of occasionally permanent waters in alpine areas that species ranges (Ketmaier et al., 2008, 2012; Munõz freeze solid in winter, excluding fish predators. The et al., 2008; Aguilar, 2011; Vanschoenwinkel et al., species has even been observed living under ice sheets 2011; Pinceel et al., 2012, but see Ketmaier et al., in late winter in lowland areas (Brtek & Thie´ry, 1995; 2005). Czeczuga & Czeczuga-Semeniuk, 1998; Zarattini & The anostracan genus Chirocephalus is character- Mura, 2007; own observation). ized by substantial intraspecific morphological varia- Studies of the genetic structure of different organ- tion. Consequently, species are often not clearly isms in Western Europe typically report important delineated what prevents reliable demarcation of effects of the Pleistocene glaciations with extant taxonomic units based on external traits (Brtek & northern populations representing relatively recent Thie´ry, 1995; Mura et al., 2002). Hitherto, only one colonization events from at least one of the southern phylogenetic study and one phylogeographic study refugia in the Apennine peninsula (Italy), Anatolia, the have attempted to clarify the evolutionary relation- Balkans, and Iberia (Taberlet et al., 1998—review; ships within the genus; they consider one population Hewitt, 1999—review; Beebee & Rowe, 2000—frog; of each of the six nominal species that inhabit of the Habel et al., 2005—butterfly; Munõz et al., 2008— Apennine peninsula (Ketmaier et al., 2003) and all brine shrimp). The relative contribution of these seven extant populations of C. kerkyrensis (Ketmaier refugia to the postglacial colonization often varies, et al., 2012). Within the widespread C. diaphanus however, even among closely related species (Weider some authors recognize several subspecies based on et al., 1999; Micheaux et al., 2005). In addition, recent morphological differences (Daday, 1913; Pesta, 1921; evidence indicates that more northerly regions, Stoicescu, 1992). Thus far, however, no consensus has including areas in central and eastern Europe, also been reached about the taxonomic status of these taxa supported small populations of cold tolerant plants and due to substantial intraspecific morphological vari- animals (Bhagwat & Willis, 2008; Sommer & Zachos, ability and a lack of sequence data from populations 2009) throughout the glaciations (Willis & Whittaker, across subspecies ranges (Brtek & Thie´ry, 1995; 2000; Stewart et al., 2010; Schmitt & Varga, 2012). Marincˇek & Petrov, 1995; Brtek, 1997; Mura, 2001; Phylogeographic studies on freshwater crustaceans Mura et al., 2002). Given the fact that many anostracan in the Western Palearctic (De Gelas & De Meester, lineages are rare and declining (Eng et al., 1990; 2005;Go´mez et al., 2007; Zierold et al., 2007; Petrov & Petrov, 1997; Damgaard & Olesen, 1998; Vanschoenwinkel et al., 2012) generally reveal two Mura, 1999; Eder & Ho¨dl, 2002), molecular markers non-mutually exclusive (e.g., Zierold et al., 2007) can present an important complementary source of patterns. The first pattern—a relatively high degree of information which may not only aid to further resolve genetic differentiation and local endemism of extant the taxonomy and delineate evolutionary significant lineages—is generally attributed to isolation and units (ESU’s) (Moritz, 1994), but also allows recon- persistence in glacial refugia leading to a long structing the impact of the Pleistocene glaciations and independent evolutionary history (e.g., in the rotifer explain the current distribution of genetic lineages. Brachionus manjavacas,Go´mez et al., 2007). The The main goal of this article is to investigate the second pattern—low genetic diversity in combination genetic structure of the Western Palearctic 123 Hydrobiologia

Chirocephalus diaphanus lineages based on the mito- are provided in Table 1 and Fig. 1c. The number of chondrial cytochrome oxidase subunit I gene (COI) sequences varied per population, ranging from one up using model based phylogenetic methods. To be able to a maximum of 10 individuals. Sequences of one C. to reliably delineate meaningful taxonomic units and diaphanus (AY188996) and five additional Chiro- assess genetic distances indicative of species level cephalus species were retrieved from Genbank: C. differentiation, six presumably closely related Wes- kerkyrensis (AY188997; JN246503–JN246529; hap- tern Palearctic Chirocephalus lineages were included lotype codes for C. kerkyrensis used in this article and in the analyses: C. shadini, C. kerkyrensis, C. ruffoi, C. corresponding haplotype codes in Ketmaier et al., marchesonii, C. salinus, and C. sybillae. Based on tree 2012 are given in the Appendix—Supplementary topologies and sequence divergence based methods material), C. marchesonii (AY188998, JN246530), we first of all discuss molecular support for the C. sibyllae (AY189001), C. salinus (AY198000), C. taxonomic status of different nominal species and ruffoi (AY188999). lineages, and define conservation units. This allows us to assess whether current nominal species adequately DNA extraction, gene amplification, reflect genetic diversity in this group or whether and sequencing representatives could be considered part of a larger species complex. In addition, using a molecular clock Genomic DNA was extracted from thoracal segments constrained tree, we reconstruct the timing of diver- using the NucleoSpin Extraction Kit (Machery-Nagel sification in C. diaphanus to discuss the likelihood of GmBH, Germany). A fragment of the COI gene was different phylogeographic scenarios explaining the amplified using a forward—LCO1490 50-GGTCAAC current distribution of genetic lineages. Ultimately, AAATCATAAAGATTGG-30 and reverse primer these analyses permit to test the specific hypothesis HCO2198 50-TAACTTCAGGGTGACCAAAAAAT that, although current Chirocephalus diversity is likely CA-30 (Folmer et al., 1994). In addition, species- to be concentrated around traditional southern refugia, specific primers were developed for the populations the cold tolerant C. diaphanus may have persisted in BRil and RLap (forward 50-CTGAATTAGGGCAAC- more northerly refugia throughout the Pleistocene CAGGA-30; reverse 50-AAATAAAGGCATTCGG glaciations. TCCA-30) using Primer 3 v.0.4.0 software on con- served regions of COI sequence based on preliminary sequence data. Polymerase chain reactions (PCR) Methods were carried out on a Biometra TGRADIENT thermocycler (Westburg) and GeneAmp PCR System Sampling procedure 2700 thermocycler (Applied Biosystems) in a final volume of 25 ll containing 2 ll template DNA, PCR

In Europe, Chirocephalus diaphanus (Fig. 1a, b) is buffer, 2 mM MgCl2, 0.2 mM dNTPs, 0.4 mMol of distributed from Poland to Britain and from the Iberian each primer, 1 U TAQ-polymerase (Silverstar Euro- peninsula across the European mainland as far as gentec), and MQ-H2O. The PCR profile was: three Greece and the borders of the Black Sea (Belk & cycles of an initial denaturation at 94°C for 10,an Brtek, 1995). For this study, we obtained sequences annealing step at 45°C for 1.50, and an elongation at from 18 C. diaphanus populations that cover the range 72°C for 1.50 followed by 35 cycles of denaturation at of the species in Europe and two additional popula- 94°C for 10, annealing at 50°C for 1.50 and elongation tions from North Africa. New sequences were also at 72°C for 1.50 followed by a final elongation of 60 at obtained for the nominal species C. salinus and C. 72°C. All PCR products were visualized on agarose shadini. All newly generated sequences were depos- gel (1%) using Gelred (Biotium). PCR products were ited in GenBank (accession codes JX990335– purified using the GFX-PCR DNA and gel band JX990362). purification kit (GE Healthcare). The PCR products Specimens were collected in the field using simple were sequenced in both directions using the BigDye dip nets or bred from larvae which were hatched in the Terminator v. 3.1 Cycle Sequencing Kit on an ABI lab from pond sediments. All specimens were pre- 3130 automated capillary DNA Sequencer (Applied served on 100% ethanol. Details of sampling locations Biosystems). 123 Hydrobiologia

Fig. 1 a Male and female Chirocephalus diaphanus (photo Bayesian inference (BI), neighbor joining (NJ), and quartet courtesy of Kris Hermans). b Male C. diaphanus (photo puzzling (QP) algorithms, separated by a ‘/’. Unsupported courtesy of Aline Waterkeyn). c Geographic origin of the groupings are indicated using a ‘–’. Population codes corre- Chirocephalus populations in this study. Locality numbers spond to population ID’s in Table 1. Numbers between brackets correspond to reference numbers shown in Table 1. Color codes are locality numbers corresponding to population entries in as well as symbols correspond to main genetic lineages as Table 1, letters between brackets are haplotype codes corre- provided in d consensus phylogram of COI sequences. Numbers sponding to haplotype entries in Table 1. Arrows indicate at nodes represent statistical support values indicated based on haplotype sharing. C. diaphanus subclades are labeled F1, F2, maximum likelihood (ML), maximum parsimony (MP), and F3

Alignment and genetic analysis 2009). Index of substitution saturation (Iss) was found to be signiﬁcantly smaller than the critical index of Sequences were checked by eye using SEQSCAPE substitution saturation (Iss c) in both cases, indicating version 2.1 and consensus sequences were constructed little substitution (i.e., similarities between sequences afterwards. All obtained DNA sequences were aligned are not biased by multiple substitutions). Maximum using CLUSTALW multiple alignment in Bioedit likelihood (ML) and maximum parsimony trees (MP) Alignment editor (Hall, 1999) and collapsed into were constructed in PaupUp version 1.0.3.1 (Swof- haplotypes prior to genetic analysis using DnASP ford, 2000). The MP tree was constructed using the version 5 (Librado & Rozas, 2009). Phallocryptus heuristic search option with the Tree Bisection and spinosa was aligned to the sequence data as outgroup, Reconnection (TBR) algorithm. The adequate base as it was also used in a previous phylogenetic study of substitution model of DNA evolution for ML analysis this genus (Ketmaier et al., 2003). Substitution was determined in JModeltest (Posada, 2008) using saturation on the third codon position was tested in ML-optimized likelihood calculations for the likeli- DAMBE v5.2.57 (Xia & Xie, 2001; Xia & Lemey, hood scores. Corrected Akaike (AICc) and Bayesian

123 Hydrobiologia

Table 1 Population codes (format XYyy: X: ﬁrst letter of country of origin, Yyy: ﬁrst three letters of site of origin), number of specimens (n), reference numbers and sampling locations (site) and corresponding latitude and longitude

ID (n) Reference no. Site Latitude Longitude No. of haplotypes (haplotype code)

C. diaphanus NVij (10) 1 The Netherlands: Vijlen 50°4701900 5°5705000 1 (10J) FNor (3) 2 France: Villers Bocage (Normandy) 49°6043.5700 0°4208.8200 2 (2H,1J) FLoi (3) 3 Lions en Sullias, Loiret (Centre) 47°51041.000 2°04035.9’’ 1 (3A) FRig (3) 4 Rigny Usse´ (Centre) 47°150 0°180 1 (3C) FCha (3) 5 Chaillevette (Poitou-Charentes) 45°4303000 -1°303000 3 (1A,1B,1D) FTou (2) 6 Toulouse (Midi Pyre´nees) 43°360 1°270 1 (2A) Fleu (2) 7 Leucate (Languedoc) 42°54019.0100 3°101300 2 (1F,1G) FRoq (2) 8 Roque Haute (Languedoc) 43°1802000 3°2105800 1 (2E) FVal (2) 9 Valliguie`re (Languedoc) 44°004200 4°3404200 2 (1L,1M) FCam (3) 10 Arles (Provence-Camargue) 43°2902400 4°4004200 1 (3M) FVar (2) 11 Flassans sur Issole (Provence) 43°2005900 6°1402100 2 (1I,1K) FLac (2) 12 Lac des Rives (Languedoc) 43°5104200 3°1505200 1 (2A) SPla (2) 13 Spain: Planillar de Abajo 40°1806000 0°180000 1 (2R) SDon (2) 14 Don˜ana 36°5605200 -6°24057.916800 2 (1T,1U) PSag (3) 15 Portugal: Sagres 37°302400 -8°570200 2 (1N,2O) MBen (2) 16 Maroc: Ben Slimane 33°370 -7°701300 1 (2S) MMoy (1) 17 Moyen Atlas 33°5901800 -5°2802900 1 (P) BRil (3) 18 Bulgaria: Rila 42°1102700 23°350200 2 (2V,1X) RLap (5) 19 Romania: Laptici Bucegi 46°48059.99700 25°30059.99800 2 (1W,4Y) ICas (3) 20 Italy: Castel Porziano 41°4603100 12°240800 1 (1Q) C. kerkyrensis (27) 20–27 Ketmaier et al. (2012) 27 (A-Z2)—Table 3 C. marchesonii (4) 28 Pilato Lake-Marche 2 (A,B) C. sybillae (3) 29 Palazzo Borghese-Marche 1 C. salinus I (2) 30 Ozieri-Sardinia 1 (A) C. ruffoi (3) 24 Pollino National Park-Calabria 1 C. salinus F (2) 31 France: Suartone 41°2705300 -9°1403000 1 (B) C. shadini (2) 32 Romania: Ditro 46°4805900 25°2905000 2

Number of haplotypes per site are provided, haplotype codes and their frequency are indicated between brackets

(BIC) information criteria were used to select the best was constructed using MrBAYES (Huelsenbeck & model. A neighbor joining (NJ) tree was constructed in Ronquist, 2001; Ronquist & Huelsenbeck, 2003)by MEGA version 4.0 (Tamura et al., 2007) using running the General Time-Reversible (GTR) model of evolutionary distances computed with the maximum DNA sequence evolution with gamma-distributed rate composite likelihood method. In addition, a quartet variation across sites and a proportion of invariable puzzling (QP) tree was constructed using TREE- sites for 60,00,000 generations and a relative burn-in PUZZLE 5.2 (Strimmer & von Haeseler, 1996). period of 25%. Number of puzzling steps for the QP-algorithm was Genetic distances between haplotypes as well as set at 1000. The robustness of the branches for MP and average genetic distances between and within main NJ analysis was assessed by performing 1000 boot- clades were computed in MEGA version 4.0 (Tamura strap replications and 100 replicates for ML analysis. et al., 2007). To facilitate comparison between studies, A Bayesian (BI) tree and clock constrained BI tree we calculated both uncorrected P distances, which do 123 Hydrobiologia not correct for multiple substitutions, and Kimura Results 2-distances (K2P), which take into consideration that transitions occur more frequently than transversions. Sequences and haplotype diversity To test for clocklike evolution and the validity of the molecular clock, a likelihood ratio test was performed A total of 60 Chirocephalus haplotypes were aligned, in TREEPUZZLE 5.2 (Schmidt et al., 2002). Diver- including 28 sequences of 560 bp (C. marchesonii gence rates of known arthropod COI mtDNA used to (B) and C. kerkyrensis) and 23 sequences of 550 bp date the divergence of main clades, range from 1.4 to (C. diaphanus, C. marchesonii (A), C. sybillae, C. 2.6% per my and were calibrated for snapping shrimps salinus (A), C. ruffoi, C. kerkyrensis). For newly (Decapoda: Alpheidae) (Knowlton & Weight, 1998). generated C. salinus haplotype B and C. diaphanus To date evolutionary splits, the time to the most recent haplotypes X, W, Y, G, I, O, U, R, the length of the common ancestor (tMRCA) of various monophyletic obtained sequences was 462, 429, 394, 429, 493, 449, clades was estimated using Bayesian inference (BI) as 544, 395, 549 bp, respectively. When possible, all implemented in BEAST version 1.6.1 (Drummond informative sites were retained using the pairwise- et al., 2012), using the evolutionary model identified deletion option. All haplotypes, along with their by jModeltest, under the assumption of constant frequency in the sampled populations, are listed in population size, with expansion growth and a strict Table 1. molecular clock. Different rates of molecular evolu- The alignment showed no indels or ambiguities. A tion were specified (0.007 and 0.013 mutations/site/ total of 256 polymorphic sites, of which 158 were my for COI which correspond to pairwise sequence parsimony informative, were observed. Of the C. divergence rates of 1.4 and 2.6%). All estimated dates diaphanus populations analyzed in this article, 11 were approximated by sampling parameters at an consisted of a single haplotype. In three instances, a interval of 500 generations over 107 Markov chain haplotype was shared among populations; haplotype A Monte Carlo steps, after discarding 106 burn-in steps. is shared between four populations (FLoi, FLac, FCha, The convergence of the sampled parameters was FTou), haplotype J and M are shared between two subsequently verified in TRACER version 1.5. In populations (NVijl, FNor and FVal, FCam, respec- order to check whether the observed intraspecific tively) (explanation of population codes in Table 1). clades can be considered separate species, we applied The number of substitutions between haplotypes two methods: the ‘‘49 rule’’ (Birky et al., 2005; Birky ranged from one to 53, and showed a maximum & Barraclough, 2009; Birky et al., 2010) and Auto- sequence divergence of 24.3% K2P (between French matic Barcode Gap Discovery (ABGD; Puillandre C. salinus and C. shadini). Among the specimens of et al., 2012). The 49 rule states that a (monophyletic) the nominal species C. diaphanus, a maximum lineage achieves species status when the mean divergence of 12.9% K2P was observed (between sequence divergence between specimens belonging BRil X and ICas), and a minimum divergence of to these lineages (D) is greater than the depth of 0.03% K2P was noted (within population FNor). clades formed by random drift with a 95% confidence Sequence divergence between the Italian C. ruffoi, C. (‘‘4h,’’ where ‘‘h’’ is the nucleotide polymorphism of diaphanus, and C. salinus haplotypes was very small each clade) (Birky et al., 2005). ABGD is a method to (0.012–0.058% K2P). In contrast, the genetic distance delimit species based on the so-called ‘‘barcode gap’’ between the haplotypes of two C. salinus populations in the distribution of pairwise differences. Such a gap was very high, amounting to 17.4% K2P. K2P- can be observed whenever the divergence among distances within C. kerkyrensis ranged from 5.8 to organisms belonging to the same species is smaller 0.3% K2P. than divergence among organisms from different The molecular clock hypothesis was not rejected species (Puillandre et al., 2012). We used the online (P = 0.10) if the most divergent basal lineages C. version to analyze the data (http://wwwabi.snv.jussieu. shadini, and C. marchesonii as well as C. sybillae were fr/public/abgd/). Number of steps was increased to 20, excluded from the dataset, justifying the use of the the gap width (X) was set at 1 while other parameters range of clocks to date major splits within the other were set at default. lineages. For ML analysis, the Tim3? G evolutionary

123 Hydrobiologia model was selected by jModeltest version 0.0.1 based localities (FVal and FCam). Clade F2 groups a on AICc and BIC selection criteria with the following southern French population (FVar) and the northern parameter settings: base frequencies: A: 0.2971 T: populations from the Netherlands (NVijl) and Nor- 0.3734, C: 0.1942, G: 0.1353, rate matrix: A ? C: mandy (FNor). The single haplotype found in NVijl 0.6227; A ? G: 11.5869 A ? T: 1.0000 C ? G: (J) is shared with FNor. Clade F2 is a sister group of a 0.6227, gamma shape parameter: 0.2180. large monophyletic clade of C. diaphanus haplotypes (F3) from France (FLeu, FTou, FCha, FRig, FLac, Evolutionary relationships FRoq). Within this clade, there is one instance of haplotype sharing (A) among four localities (FLoi, A 50% majority consensus tree was constructed, FLac, LCha, FTou). Uncorrected P- and model based combining results from the different methods of Kimura-2 distances among and within main clades are phylogenetic inference (ML, MP, BI, NJ, and QP) provided in Table 2. A clock constrained tree and and illustrating the link between tree topology and tMRCAs are given in the Appendix—Supplementary geography (Fig. 1c, d). Although phylogenetic meth- material. ods did not converge on a single resolved topology, all Both ABGD (P = 5.01; previous seven partitions methods recognize 14 distinct evolutionary lineages, were identical, further partitioning lumped all embedded in a large polytomy except for the basally sequences) and the 49-rule delimited the Eastern branching C. shadini, C. marchesonii, and C. kerkyr- European clade and C. salinus (France) as candidate ensis. These lineages represent both single haplotypes species and confirmed species status of C. marchesonii, (C. shadini, C. sybillae, C. salinus A, MMoy, SPla) as C. kerkyrensis, C. shadini,andC. sybillae.Inaddition, well as clades of up to 10 haplotypes whose relation- both methods indicate the Italian clade (ICas, C. ships are well resolved and supported. The sequences salinus(A) and C. ruffoi)isnestedwithinC. diaphanus. of the Italian C. diaphanus, C. ruffoi, and C. salinus emerge as a single clade with a maximal within clade K2P divergence of 5.8%. Another clade groups C. Discussion diaphanus individuals from Ben Slimane in Morocco together with specimens originating from Donãna in Taxonomic status of current lineages Southern Spain (MBen-SDon). A similar grouping of relatively spatially proximate C. diaphanus popula- Both ABGD and the 49-rule support the species status tions is found in Eastern Europe, clustering individuals of the traditionally recognized nominal species C. from populations in Romania and Bulgaria (RLap and kerkyrensis,C.shadini and C. marchesonii, and BRil). In addition, a number of populations appear as suggest that the C. salinus lineage from Corsica and unique clades with average pairwise divergence the Eastern European C. diaphanus clade could be values between other clades in the range of 6.3 and considered separate species. Since the observed pair- 13.9% (K2P). Besides the Corsican C. salinus and wise sequence divergence between these clades and all Italian C. sybillae, these include a Catalonian (SPla), other recognized lineages ranges from 11.0 to 24.7%, central Moroccan (MMoy) and a Portuguese (PSag) these finding are also in line with commonly accepted clade. The latter could be a sister group of a large molecular thresholds indicative for species level predominantly French clade that groups the remaining differentiation at COI in branchiopod crustaceans lineages although this hypothesis is only weakly (average sequence divergence of 7–10%: Cox & supported (statistical support value of 0.80 for the Hebert, 2001; Zofkova & Timms, 2009) and interspe- Bayesian analysis and 70 for the QP algorithm). cies sequence divergence values commonly found in Within the large French C. diaphanus clade, three large branchiopods (10–14% for American Branchi- smaller well supported subclades can be distinguished, necta in Vandergast et al., 2009; 15.61–15.89% for which from here on will be referred to as clades F1, F2, Streptocephalus in Daniels et al., 2004; 6.8–18.6% for and F3. All used methods converge on the split of the Limnadopsis, Schwentner et al., 2011). The topology F1-subclade from F2 and F3. F1 consists of haplotypes of the C. kerkyrensis clade, based on sequences from southern France (FVar, FVal, and FCam) and originating from all extant localities for C. kerkyren- includes one haplotype (M) that is shared by two sis, is congruent with results from Ketmaier et al. 123 123

Table 2 Distance matrix between investigated chirocephalid clades based on Kimura 2 parameter (below diagonal) and uncorrected P distances (above diagonal). Within clade divergence is depicted on the diagonal (Kimura parameter 2/uncorrected P distances)

1234567891011121314

1. Spain plan / 0.067 0.088 0.092 0.071 0.088 0.095 0.081 0.101 0.182 0.108 0.123 0.114 0.113 2. Spain–Morocco 0.071 0.0150/0.0160 0.072 0.075 0.065 0.069 0.073 0.084 0.107 0.181 0.117 0.138 0.101 0.146 3. Morocco Moy 0.075 0.068 / 0.063 0.066 0.068 0.064 0.096 0.107 0.165 0.109 0.128 0.115 0.110 4. Portugal 0.096 0.078 0.070 0.003/0.003 0.065 0.020 0.017 0.094 0.101 0.160 0.111 0.138 0.123 0.114 5. F1 0.095 0.073 0.070 0.072 0.004/0.004 0.066 0.068 0.083 0.107 0.169 0.123 0.138 0.111 0.149 6. F2 0.101 0.080 0.069 0.067 0.020 0.001/0.001 0.032 0.096 0.098 0.165 0.111 0.143 0.114 0.148 7. F3 0.105 0.078 0.072 0.069 0.033 0.017 0.010/0.010 0.098 0.105 0.169 0.111 0.143 0.128 0.153 8. Italy (C. ruf, C. dia, C. 0.087 0.090 0.089 0.105 0.104 0.103 0.107 0.039/0.040 0.126 0.184 0.134 0.147 0.121 0.164 sal) 9. Eastern Europe 0.111 0.118 0.118 0.118 0.106 0.110 0.116 0.141 0.011/0.011 0.186 0.119 0.150 0.125 0.157 10. C. shadini 0.210 0.208 0.193 0.187 0.188 0.181 0.194 0.212 0.217 / 0.206 0.203 0.203 0.171 11. C. salinus (F) 0.120 0.131 0.139 0.122 0.123 0.123 0.124 0.152 0.134 0.247 / 0.135 0.151 0.135 12. C. marchesonii 0.137 0.156 0.156 0.142 0.161 0.156 0.161 0.166 0.171 0.243 0.153 0.06/0.057 0.163 0.129 13. C. sibyllae 0.126 0.109 0.121 0.128 0.125 0.137 0.144 0.134 0.140 0.239 0.174 0.188 / 0.125 14. C. kerkyrensis 0.125 0.107 0.111 0.154 0.127 0.133 0.143 0.133 0.146 0.197 0.152 0.145 0.138 0.032/0.031

Empty cells indicate that sequence information was unavailable Hydrobiologia Hydrobiologia

(2012) and will not be discussed in detail. For C. Depending on whether additional markers support marchesonii, the two sampled haplotypes are quite this grouping, the Italian clade (C. diaphanus, C. divergent (6% K2P), considering they originate from ruffoi, C. salinus) could be considered a separate ESU, the same and only known locality of this species which is likely to contain unique genetic variation (Ketmaier et al., 2003, 2012). For C. shadini, obtained worthy of conservation due to its independent evolu- specimens originated from a single locality and tionary trajectory. Analogously, a similar status could constituted a single haplotype. Therefore, no definitive be attributed to C. diaphanus populations from the conclusions about the validity of this taxon can be central Atlas in Morocco, SW Portugal, and Catalunia made. The taxon C. salinus, originally described as a (pairwise divergence between these clades and other subspecies of C. diaphanus, should be revised since C. diaphanus lineages: min 6.7% K2P). pairwise sequence divergence between the two sampled haplotypes was quite large, tree topologies Phylogeographic scenario suggest it may be polyphyletic and species delimitation algorithms tests identify C. salinus of Corsica as a The advance and retreat of ice sheets during multiple separate species. This taxon was first described based glacial episodes in the Quaternary (2.6 mya—present) on basal lamina morphology from specimens collected had a major impact on species’ distributions. Overall, from a saline habitat in le Croisic in Brittany (France) scenarios of temperate species’ responses to glaciations (Daday, 1913). Continental populations of this species in the temperate zone of Europe typically involve were never found again in France (Defaye et al., 1998) interglacial northern expansions, followed by popula- but a similar basal lamina was discovered in freshwa- tion extinctions and a southward retreat to at least one of ter populations from the Mediterranean islands of the three southern European peninsulae of Italy, Iberia, Sardinia (Cottarelli & Mura, 1983) and Corsica and the Balkans during subsequent cold periods (Champeau & Thiery, 1990). Our results suggest that (Hewitt, 1993, 2000; Taberlet et al., 1998;Montaya basal lamina morphology may not be taxonomically et al., 2007;Schmitt;2007). In concordance with this informative. Analyses of nuclear markers, however, scenario, Chirocephalus diaphanus shows clear ende- will be required to exclude the possibility of hybrid- mism linked with each of these different Mediterranean ization with local C. diaphanus lineages and intro- peninsulae. The most recent common ancestors of the gression of mitochondrial genes explaining the Iberian, Italian, and Eastern European lineages precede apparent divergence between the sampled C. salinus the onset of the Pleistocene (4.765–8.840 mya), illus- populations. In this context, the unexpected appear- trating that these populations experienced independent ance of the strongly supported monophyletic clade that evolutionary trajectories, promoting divergence and joins C. ruffoi, an Italian C. diaphanus population, and speciation (Taberlet et al., 1998;Hewitt,2000). Similar the presumed Italian C. salinus population, is note- deep divergences among refugia have been observed in worthy. Although hybridisation may explain the low studies on some zooplankton species (cladocerans, genetic divergence between these populations, the fact notostracans, anostracans), but not in others reflecting that this clade is nested in the large C. diaphanus clade more frequent dispersal and gene flow (De Gelas & De according to ABGD and 49 rule algorithms is Meester, 2005). consistent with the lack of clear morphological The highly divergent haplotypes found in different differences to separate these taxa (except for the basal Mediterranean areas (7.2% in Morocco, 9.8% in lamina morphology of C. salinus, mentioned above). Iberia, 4% in the Apennine peninsula), hint at the Egg morphology was initially considered as the only long term presence of suitable habitat in these areas useful trait to unequivocally distinguish C. ruffoi from throughout the Pliocene and Pleistocene. The exis- its congener C. diaphanus (Mura, 1986; Mura & tence of genetic substructure within the Mediterranean Rossetti, 2002). Later studies, however, discovered refugia has already been reported for other organisms that C. ruffoi is characterized by extreme variation in (Schmitt et al., 2006; Canestrelli et al., 2007;Go´mez egg morphology, with only a small percentage of the et al., 2007; Munõz et al., 2008) and is typically studied eggs conforming to the morphotype of the explained in the context of long distance colonization original description (Zarattini et al., 2001; Mura & or the existence of multiple subrefugia. For instance, Rossetti, 2010). deep phylogenetic splits in Iberia have previously 123 Hydrobiologia been reported as possible colonization events from oscillations (Webb & Bartlein, 1992). This implies North Africa (Zierold et al., 2007). Our results may that rather than contributing to the postglacial colo- reflect both scenarios as haplotypes from northern nization of Europe, Mediterranean populations likely Spain are distinct and divergent from those of southern represent long-term isolates, while old splits among Spain, but there are also indications for a potentially northern populations suggests the possibility of long old (2.236–1.199 mya) Moroccan origin of southern term persistence of C. diaphanus populations at least Spanish haplotypes. as high up as Southern France throughout the Pleis- C. diaphanus probably colonized France from the tocene. The fact that C. diaphanus is a eurytopic, cold South, in the lower Pleistocene 2.230–1.320 mya, tolerant species which can be found in high mountain before the onset of major climatic oscillations. Within habitats as well as in lowland areas surviving under ice France, five separate recent long distance dispersal sheets (Brtek & Thie´ry, 1995; Czeczuga & Czeczuga- events were detected at the northern edge of the Semeniuk, 1998; Zarattini & Mura, 2007; Fonseca species range, which is consistent with a scenario of et al., 2008; own observation) supports this hypothesis rapid range expansion. Empirical evidence from an and can explain why similar patterns have thus far array of terrestrial and aquatic organisms suggests that not been reported in other, more thermophilic, range expansion from refugia often occurred quite zooplankton species such as Triops cancriformis and rapidly (Taberlet et al., 1998; Durant et al., 1999; Artemia (Munõz et al., 2008; Vanschoenwinkel Stewart & Lister, 2001) resulting in weak genetic et al., 2012). structure in the European mainland (Verovnik et al., 2005). This pattern was also observed in other branchiopod crustaceans. European populations of Conclusion Daphnia magna diverged not more than 1.74% (De Gelas & De Meester, 2005), while in the clam shrimp Overall, our analyses of the COI gene in Western Leptestheria dahalacensis populations consisted of Palearctic chirocephalids support a number of previ- the same, single haplotype throughout Germany, Italy ously held assumptions about phylogenetic relation- and Austria (Cesari et al., 2007). For the large ships in this group, while contradicting others. branchiopods Triops cancriformis (Notostraca) and Although the current taxonomic status of most main Tanymastix stagnalis (Anostraca), mean sequence lineages was supported, our results suggest the pres- divergence of the COI gene among European popu- ence of significant levels of cryptic diversity. Analyses lations was 0.3 and 1.5%, respectively (Ketmaier revealed European C. diaphanus lineages diverged et al., 2005; Vanschoenwinkel et al., 2012). well before the LGM and survived the Pleistocene Recent studies indicate that glacial distribution glaciations in multiple (sub)refugia along the Iberian, patterns in Europe were considerably more complex Italian and Balkan peninsulae, while recolonizing than previously thought (Bhagwat & Willis, 2008; northern Europe from a refugium in Southern France. Schmitt & Varga, 2012). Hence, it is not surprising This resulted in high levels of cryptic diversity around that little is known about the location of glacial refugia refugia but also in more widespread haplotypes within and subsequent colonization routes for freshwater the European mainland. Unlike previously studied zooplankton species. Exceptions include the Iberian zooplankton species that recolonized Europe from refugia of the rotifers Branchionus plicatilis and refugia in the Mediterranean peninsulae, the ability of Branchionus manjavacas (Go´mez et al., 2000, 2007) C. diaphanus to tolerate frost and cold water condi- and the region north of the Ponto-Caspian basin that tions probably enabled it to persist throughout the harbored populations of Polyphemus pediculus which Pleistocene glaciations in a more northerly refugium subsequently recolonized large parts of the Western in Southern France. Palearctic (Xu et al., 2009). In our study, besides from the recent dispersal events suggested by haplotype Acknowledgments Jane Reniers is currently supported by a sharing, the main French C. diaphanus clade also PhD grant from the Fund for scientific research Flanders (FWO). The authors wish to thank Tom Pinceel for useful comments on reflects deeper genetic substructure (3.3% K2P F3– the manuscript as well as the multitude of researchers that kindly F1) which can be dated to less than a million years ago, contributed samples: Maria Rosa Miracle, Laszlo Demeter, Luis around the onset of the major Pleistocene climatic Cancela da Fonseca, Frank Van de Meutter, Aline Waterkeyn, 123 Hydrobiologia

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