Monika Weiss
email: monika.weiss@awi.de

Sven Thatje

Olaf Heilmayer

H.-D M Franke

Weiss

Temperature eVects on zoeal morphometric traits and intraspeciWc variability in the hairy crab Cancer setosus across latitude

Keywords: Brachyura, Early ontogeny, Morphological variability, El Niño, Humboldt Current, Chile

Phenotypic plasticity is an important but often ignored ability that enables organisms, within species-spe-ciWc physiological limits, to respond to gradual or sudden extrinsic changes in their environment. In the marine realm, the early ontogeny of decapod crustaceans is among the best known examples to demonstrate a temperature-dependent phenotypic response. Here, we present morphometric results of larvae of the hairy crab Cancer setosus, the embryonic development of which took place at diVerent temperatures at two diVerent sites (Antofagasta, 23°45Ј S; Puerto Montt, 41°44Ј S) along the Chilean Coast. Zoea I larvae from Puerto Montt were signiWcantly larger than those from Antofagasta, when considering embryonic development at the same temperature. Larvae from Puerto Montt reared at 12 and 16°C did not diVer morphometrically, but sizes of larvae from Antofagasta kept at 16 and 20°C did, being larger at the colder temperature. Zoea II larvae reared in Antofagasta at three temperatures (16, 20, and 24°C) showed the same pattern, with larger larvae at colder temperatures. Furthermore, larvae reared at 24°C, showed deformations, suggesting that 24°C, which coincides with temperatures found during strong EL Niño events, is indicative of the upper larval thermal tolerance limit. C. setosus is exposed to a wide temperature range across its distribution range of about 40° of latitude. Phenotypic plasticity in larval oVspring does furthermore enable this species to locally respond to the inter-decadal warming induced by El Niño. Morphological plasticity in this species does support previously reported energetic trade-oVs with temperature throughout early ontogeny of this species, indicating that plasticity may be a key to a species' success to occupy a wide distribution range and/or to thrive under highly variable habitat conditions.

Introduction

Phenotypic plasticity in larval oVspring of invertebrates has often been discussed as an important mechanism to respond to sudden changes in their habitat, namely temperature and food conditions (e.g. [START_REF] Criales | Experimental studies on the larval development of the shrimps Crangon crangon and C. allmanni[END_REF][START_REF] Shirley | Latitudinal variation in the Dungeness crab, Cancer magister: zoeal morphology explained by incubation temperature[END_REF]). However, quantitative experimental studies on this subject are scarce and most insight is probably available from experimental laboratory studies of decapod crustaceans. There is evidence that phenotypic conditions of larvae may be indicative of their energetic status or conditions experienced during oogenesis like contrasting habitats [START_REF] Silva | Reproductive traits of an estuarine crab, Neohelice (=Chasmagnathus) granulata (Brachyura: Grapsoidea: Varunidae), in two contrasting habitats[END_REF]) and including across geographic temperature gradients [START_REF] Wehrtmann | Larval development of Nauticaris magellanica (Milne Edwards, 1891) (Decapoda: Caridea: Hippolytidae), reared under laboratory conditions[END_REF][START_REF] Wehrtmann | Changes in volume, biomass, and fatty acids of developing eggs in Nauticaris magellanica (Decapoda: Caridea): a latitudinal comparison[END_REF][START_REF] Thatje | Morphological variability in larval stages of Nauticaris magellanica (A. Milne-Edwards, 1891) (Decapoda: Caridea: Hippolytidae) from South American waters[END_REF][START_REF] Giménez | EVects of prehatching salinity and initial larval biomass on survival and duration of development in the zoea 1 of the estuarine crab, Chasmagnathus granulata, under nutritional stress[END_REF]). Most of this knowledge is limited to studies of caridean shrimp larvae (i.e. [START_REF] Criales | Experimental studies on the larval development of the shrimps Crangon crangon and C. allmanni[END_REF][START_REF] Wehrtmann | How important are starvation periods in early larval development for survival of Crangon septemspinosa larvae?[END_REF], which may express high variability in, for example, larval developmental pathways, number of instars, and size and energy contents in response to food and temperature condition. In comparison, and despite known energetic trade-oVs in crab eggs, phenotypic plasticity in crab larvae is generally assumed to be more conservative; a view that might be largely driven by the lack of suYcient information available to date [START_REF] Anger | The biology of decapod crustacean larvae. A.A. Balkema[END_REF]) and that has already been disproved, for example, for Neohelice (formerly Chasmagnathus) granulata [START_REF] Pestana | Occurrence of an alternative pathway in the larval development of the crab Chasmagnathus granulata Dana, 1851 under laboratory conditions[END_REF][START_REF] Cuesta | Morphology of the megalopal stage of Chasmagnathus granulatus Dana, 1851 (Crustacea: Decapoda: Brachyura: Varunidae), with comments on morphological anomalies[END_REF].

The hairy crab Cancer setosus (Molina 1782; synonymous C. polyodon Poeppig 1836) ranges in its distribution from Guayaquil in Ecuador (2°13Ј S, 79°53Ј W) to the Peninsula of Taitao in Southern Chile (46°00Ј S, 75°00Ј W) [START_REF] Garth | Brachyura the PaciWc coast of America, Brachyrhyncha: Portunidae. Allan Hancock monographs in marine biology[END_REF] and is of commercial value to local artisanal Wsheries (WolV and Soto 1992; SERNAP-ESCA 2006; [START_REF] Thatje | Climate variability and El Niño Southern Oscillation: implications for natural coastal resources and management[END_REF]). The early ontogeny, which in C. setosus consists of Wve planktotrophic zoeal and one megalopa stage before reaching the Wrst crab stage [START_REF] Quintana | The complete larval development of the Edible Crab, Cancer setosus Molina and observations on the prezoeal and Wrst zoeal stages of C. coronatus Molina (Decapoda: Brachyura, Cancridae)[END_REF], is considered the most delicate part within the life cycle of brachyuran and in particular cancrid crabs [START_REF] Anger | The biology of decapod crustacean larvae. A.A. Balkema[END_REF]Weiss et al. 2009a, b). A unique physiological plasticity to respond to latitudinal and seasonal changes in temperature has been observed in reproductive traits of C. setosus. Among the most conspicuous characteristics is a synchronisation of a single egg batch release with seasonality at the species southernmost distribution boundary in central southern Chile (Puerto Montt, 41°44Ј S), contrasted by multiple annual ovipositions in northern Chile (Antofagasta, 23°45Ј S) [START_REF] Fischer | Temperature-induced oviposition in the brachyuran crab Cancer setosus along a latitudinal cline: aquaria experiments and analysis of Weld-data[END_REF]. In addition, a correlation of changes in egg energy contents and temperature was observed along latitude as well as in subsequent ovipositions in the same female [START_REF] Fischer | Temperature-induced oviposition in the brachyuran crab Cancer setosus along a latitudinal cline: aquaria experiments and analysis of Weld-data[END_REF]Fischer et al. 2009a). These Wndings are consistent with the concept of a latitudinal gradient in energy provisioning into oVspring of a broad variety of marine invertebrates (for review see [START_REF] Thatje | On the origin of Antarctic marine benthic community structure[END_REF]. The underlying pattern of larger eggs with better energy provisioning at colder temperatures (higher latitudes) is discussed to be an adaptive response to limited food supply and longer pelagic life stage phases [START_REF] Thatje | On the origin of Antarctic marine benthic community structure[END_REF]. The latitudinal cline in energy provisioning of eggs therewith corresponds with the energy demand of the embryo. Egg development is generally faster at higher temperatures, but causes an altered metabolic eYciency often resulting in a size reduction of later life stages. This has been shown for the Dungeness crab, Cancer magister [START_REF] Shirley | Latitudinal variation in the Dungeness crab, Cancer magister: zoeal morphology explained by incubation temperature[END_REF], the spider crab, Hyas araneus [START_REF] Kunisch | Variation in development and growth rates of larval and juvenile spider crabs Hyas araneus reared in the laboratory[END_REF] as well as for caridean shrimps like Nauticaris magellanica [START_REF] Wehrtmann | Changes in volume, biomass, and fatty acids of developing eggs in Nauticaris magellanica (Decapoda: Caridea): a latitudinal comparison[END_REF], Betaeus emarginatus [START_REF] Wehrtmann | EVects of temperature on the embryonic development and hatchling size of Betaeus emarginatus (Decapoda: Caridea: Alpheidae)[END_REF], and Pandalus borealis [START_REF] Brillon | Egg survival, embryonic development, and larval characteristics of northern shrimp (Pandalus borealis) females subject to diVerent temperature and feeding conditions[END_REF] in laboratory cultures, and for Nauticaris magellanica from the Weld [START_REF] Thatje | Morphological variability in larval stages of Nauticaris magellanica (A. Milne-Edwards, 1891) (Decapoda: Caridea: Hippolytidae) from South American waters[END_REF]. In C. setosus eggs from a southern Chilean population were larger containing higher lipid content, resulting in bigger larvae than eggs from northern Chile (Fischer et al. 2009b). Egg development in the southern population is primarily based on the metabolism of energy rich lipids, which Wnally enhances the energetic Wtness of the hatching larvae, and which has been discussed as an adaptive response to colder temperatures and prolonged egg and larval developmental time (Fischer et al. 2009b).

The size and elemental composition of eggs and hatchlings itself has been subject to some studies, but fewer studies have addressed the question of phenotypic eVects on larvae [START_REF] Shirley | Latitudinal variation in the Dungeness crab, Cancer magister: zoeal morphology explained by incubation temperature[END_REF][START_REF] Thatje | Morphological variability in larval stages of Nauticaris magellanica (A. Milne-Edwards, 1891) (Decapoda: Caridea: Hippolytidae) from South American waters[END_REF][START_REF] Wehrtmann | Larvae of Nauticaris magellanica (Decapoda: Caridea: Hipploytidae) reared in the laboratory diVer morphologically from those in nature[END_REF]. Following the assumption that larval size will follow the known latitudinal cline in egg size, we hypothesise that larvae from Puerto Montt have larger sizes and tested whether the temperature experienced during embryonic development triggers phenotypic plasticity of the zoeal stages in this species.

Materials and methods

All samples are reference samples of parallel experiments. For further explanations of the experimental setup see Table 1. [START_REF] Fischer | Temperature eVects on reproduction and early lifehistory traits in the brachyuran crab Cancer setosus in the Humboldt Current System[END_REF]. Puerto Montt is located close to the southern limit of this species. In Antofagasta Bay, SST is signiWcantly higher than in the surrounding Humboldt Current upwelling system (+2-3°C) due to the bay's particular oceanographic conditions [START_REF] Piñones | Nearshore surface temperatures in Antofagasta bay (Chile) and adjacent upwelling centers[END_REF], and thus SST is comparable to the temperature encountered by C. setosus at its northern distributional limit oV Ecuador (Fischer et al. 2009b). Two females (IDs A, B) (inXuence of temperature encountered during larval development) in Antofagasta were maintained individually in Xow-through seawater aquaria (12 l) at ambient temperature »16.0°C, while four females (IDs C, D, E, and F) (inXuence of temperature encountered during embryonic development on larval morphology) were held in 3,200 l Xow-through aquaria (•12 ind./basin) under natural seasonal temperature conditions (up to 10 months; 16-23°C) in Antofagasta. Larval samples of females C and D were taken immediately after hatching at 16°C in November 2005 and larval samples E and F were taken immediately after hatching at 20°C in January 2006. In Puerto Montt, four females (IDs G, H, I, and J) were held in 500 l aquaria (•9 ind./basin). Here, temperatures were kept constant at 12 (IDs G, H) and 16°C (IDs I, J) in order to simulate the temperature conditions at oviposition and embryonic development from Northern and Southern Chile. The whole embryonic development of the eggs from the early blastula stage on took place at the respective experimental temperature. All females were kept in seawater at a salinity of 34, in a 12:12-h light/dark cycle and were fed ad libitum with living Perumytilus purpuratus. The aquaria were cleaned and water temperature was recorded daily.

Sampling and maintenance of adults

Rearing of larvae

Freshly hatched larvae were collected in Wlters receiving water from the overXow of the aquaria. Since most larvae hatched at night, samples were taken every morning. Filters were cleaned every evening to ensure daily larval age did not vary by more than 12 h (after [START_REF] Lovrich | Changes in biomass and chemical composition during lecithotrophic larval development of the southern king crab, Lithodes santolla (Molina)[END_REF]. For the experiment on the inXuence of temperature encountered during embryonic development on larval morphology, samples of freshly hatched, actively moving larvae were taken in Antofagasta (females A-F) and Puerto Montt (females G-J) (Table 1). For the experiment of the inXuence of temperature on larval development, solely actively moving larvae of females A and B (Antofagasta) were transferred to bowls with 16°C Wltered seawater and afterwards allowed to acclimate to the experimental temperatures (16, 20, 24°C). Samples were taken at the Wrst day of each available instar. We were only able to get Zoea II and Zoea III larvae from each experimental temperature, because unfavourable temperatures lead to high mortality rates of C. setosus larvae (Weiss et al. 2009a, b). Zoea IV and V larvae were only available at the optimum temperature of 20°C (Weiss et al. 2009a). Larvae were transferred into Eppendorf caps with 4% formalin buVered seawater. Samples were transferred to the Alfred Wegener Institute, Bremerhaven, for further analyses.

Larval morphometry

Digital pictures (Colour view soft imaging system) of each larva were taken using a stereo-microscope (Leica MZ12 5 ). Images were processed with the programme Cell* imaging software for life sciences microscopy (Olympus Soft Imaging Solutions GmbH). All measurements (Fig. 1) were taken along the dorsal surface of the morphological feature. Zoeal total length (TL) was measured from the telson furca along the dorsal curvature to the tip of the rostral carapace spine. The antenna was measured from its tip to the point of attachment at the carapace. The rostral carapace spine was measured from its tip to its basal indentation of the spine between the eyes. The dorsal and lateral spines were measured from the point of attachment of the carapace to the distal tip. The carapace length (CL) was measured from the Additionally, the distance between the tip of the rostral and the tip of the dorsal spine was measured (r-d distance). The number of measured individuals per feature is given in Tables 2 and3.

Statistical analyses

All data were tested with the Mahalanobis distances test [START_REF] Mahalanobis | On the generalised distance in statistics[END_REF] to exclude outliers from analysis. Ratios were transformed with an arcsin transformation prior analysis. The eVects of temperature and location on instar characteristics were analysed with analysis of variance (ANOVA). Normal distribution of the dataset was tested with a Saphiro-Wilk W test. Post-hoc tests were conducted with the student's t-test.

Results

Geographic diVerences

Freshly hatched Zoea I larvae of females reared at 16°C in Puerto Montt (southern Chile) were signiWcantly larger in all measured parameters than Zoea I larvae of females reared at 16°C in Antofagasta (northern Chile; Fig. 2). In Puerto Montt, TL of freshly hatched Zoea I larvae was 13% longer (p = 0.0031, f = 9.55); the rostral spine was 5% longer (p = 0.0093, f = 7.24), the dorsal spine was 7% longer (p < 0.0001, f = 18.98), the lateral spine was 4% longer (p = 0.0088, f = 7.37), the r-d was 17% longer (p < 0.0001, f = 52.86), the carapace was 10% longer (p < 0.0001, f = 26.68) and the antenna was 4% longer (p = 0.0007, f = 12.76) than in Antofagasta (Fig. 2). The ratio of the measured parameters versus the carapace length (CL) revealed signiWcant diVerences between the two locations. The TL:CL (p = 0.0001, f = 17.09), the r-d:CL (p < 0.0001, f = 2129.98), the RS:CL (p = 0.0003, f = 14.66), the DS:CL (p < 0.0345, f = 4.70) and the Antenna:CL (p < 0.0001, f = 1335.30) were signiWcantly longer in larvae from the Antofagasta region (Table 4).

Temperature eVect during embryonic development Freshly hatched Zoea I larvae which experienced embryogenesis at 16 and 20°C in Antofagasta (Fig. 3a) revealed that larvae with embryonic development at colder temperatures have signiWcantly (16%) longer total length

123

= 0.0082, f = 7.50), a 7% longer rostral spine (p = 0.0279, f = 5.09), a 8% longer r-d distance (p = 0.0049, f = 8.59) and a 3% longer dorsal spine (p = 0.0226, f = 5.49). The ratio of appendages and CL showed that freshly hatched Zoea I reared at 20°C had slightly shorter spines than larvae reared at 16°C; this eVect was signiWcant (p = 0.0203, f = 5.71) in the TL:CL ratio (Table 4).

Freshly hatched Zoea I larvae from Puerto Montt in contrast, which experienced embryogenesis at 12 and 16°C did not show any signiWcant diVerences in the measured parameters (total length, r-d distance, rostral spine, dorsal spine, lateral spine, and antenna) (Fig. 3b) or in the calculated appendages versus carapace length ratios.

Temperature eVect during larval development

In Antofagasta, Zoea II larvae reared at 16, 20, and 24°C showed signiWcant diVerences in larval TL (p = 0.0468, f = 3.40), the length of the rostral (p = 0.0009, f = 8.76) and dorsal spine (p = 0.0014, f = 8.13), the r-d distance (p = 0.0001, f = 12.07) and the CL (p = 0.0072, f = 5.64) (Fig. 4a). In those diVering parameters larvae reared at 24°C were signiWcantly smaller than larvae at 16 and 20°C, except for the larval TL at 16°C, which showed an intermediate TL to that found at 16 and 24°C (Fig. 4a). The appendage versus carapace length ratios showed that the DS:CL was signiWcantly smaller (p = 0.023, f = 4.24) in Zoea II larvae reared at 24°C (Table 4). Additionally, those larvae showed deformations of the carapace and the dorsal spine; the dorsal spine is not broken, but reduced and the carapace shows lateral bulging (Fig. 5). Zoea III larvae were only available at 16 and 20°C, because all larvae maintained at 24°C died during ecdysis, as this is known to be the most critical point during the larval cycle [START_REF] Anger | The biology of decapod crustacean larvae. A.A. Balkema[END_REF]Weiss et al. 2009a). Zoea III larvae reared at 20°C were signiWcantly smaller than those reared at 16°C, as evident in total length (p = 0.0159, f = 8.39), r-d distance

123

(p = 0.0162, f 8.33) and rostral spine (p = 0.0011, f = 20.46; Fig. 4b). The appendage versus carapace length ratios showed that larvae reared at 20°C have shorter appendages, but this was only signiWcant in the RS:CL (p = 0.0196, f = 6.64).

Discussion

In the present study, the intraspeciWc variability of morphological characteristics of Cancer setosus early zoeal stages in relation to rearing temperature and geographical origin is ). Latitudinal variation (accompanied by variation in temperature) is generally known to lead to higher growth eYciencies at lower temperatures (e.g. [START_REF] Clarke | Life in cold water: the physiological ecology of polar marine ectotherms[END_REF][START_REF] Clarke | Costs and consequences of evolutionary temperature adaptation[END_REF][START_REF] Heilmayer | Growth eYciency and temperature in scallops: a comparative analysis of species adapted to diVerent temperatures[END_REF]). Animals from colder regions have to invest less energy in the maintenance of the standard metabolism and therefore can direct more energy into growth. Fischer et al. (2009a) suggested that blastula eggs of C. setosus at Puerto Montt at 12°C were provided by 30% more fatty acids as blastula eggs produced in Antofagasta at 20°C, and larvae utilise a higher percentage of fatty acids at colder temperatures. Indeed, these Wndings may indicate that a metabolism based on fatty acids might produce larger larvae. Surprisingly, the appendages of freshly hatched Zoea I larvae from the Antofagasta region were relatively longer than those in Puerto Montt. This might be explained by a thermal acclimation of larvae. In the Antofagasta region 16°C can already be regarded as a cold temperature, where the formation of longer spines can serve as a protection against predators during the elongated pelagic life phase. Another explanation would be that instead of an increase in size, warm temperatures might result in the rapid formation of morphological structures due to a certain imbalance between development and growth (Wehrtmann and López 2003 and publications cited therein).

A comparison of the morphology of freshly hatched Zoea I larvae of females from Puerto Montt reared at 12°a nd 16°C did not show any diVerences in total length, r-d distance, rostral spine, dorsal spine, lateral spine, carapace length, antenna or in the calculated ratios of appendages versus carapace length. The energy provisioning with higher lipid content of larvae from the south seems to act as a protection shield not only for unfavourable conditions like food shortage but also makes larvae more tolerant to temperature changes.

Freshly hatched Zoea I larvae from the northern region in contrast show a morphological variability when embryonic development took place at comparable temperature ranges. Again, larvae reared at colder temperatures were larger indicating that development that is mainly based on protein depletion may be more sensitive to temperature changes. DiVerences in the morphological characteristics are especially pronounced in TL, the rostral spine, and the r-d distance. All these parameters include the rostral spine. [START_REF] Shirley | Latitudinal variation in the Dungeness crab, Cancer magister: zoeal morphology explained by incubation temperature[END_REF] showed that a subtraction of the rostral spine from parameters measured prohibit signiWcant diVerences. This is not the case in the present study, but nonetheless the characteristic of the rostral spine seems to be sensitive to temperature.

A diVerentiation of two diVerent species of Pachygrapsus was not possible by means of morphological traits in larvae, but through adult morphology and genetic analyses [START_REF] Schubart | Phylogeography of Pachygrapsus transversus (Gibbes, 1850): the eVect of the American continent and the Atlantic Ocean as gene Xow barriers and recognition of Pachygrapsus socius Stimpson, 1871 as a valid species[END_REF]. Larvae in this study from diVerent oceans and coasts (East PaciWc, West Atlantic east Atlantic) but from similar temperature regimes showed highest similarity in morphometric traits. These examples may suggest that within limits temperature has a more pronounced eVect on larval morphology than genetic deviation. This is supported by the circumstance that C. setosus exhibits very little genetic diversity along its entire distribution range [START_REF] Gomez-Uchida | Allozyme and AFLP analysis of genetic population structure in the hairy edible crab Cancer setosus from the Chilean coast[END_REF].

The comparison between Zoea II larvae reared at diVerent temperatures showed that warmer temperatures lead to signiWcantly smaller larvae, independent of the instar, at 24°C, a temperature typically found during strong El Niño events (SHOA 2009). These observations coincide with the approximate upper thermal tolerance threshold suggested for larvae of the species (Weiss et al. 2009b).

Regarding the r-d distance measured by [START_REF] Quintana | The complete larval development of the Edible Crab, Cancer setosus Molina and observations on the prezoeal and Wrst zoeal stages of C. coronatus Molina (Decapoda: Brachyura, Cancridae)[END_REF] from larvae of the region of Concepción it is apparent that although Concepción is 13° south of Antofagasta and water temperatures are lower (11-14°C), larvae measured in that study seem to be generally smaller. This might be due to the measurement technique used in their study. In the present study the r-d distance was measured along the curvatures, whereas [START_REF] Quintana | The complete larval development of the Edible Crab, Cancer setosus Molina and observations on the prezoeal and Wrst zoeal stages of C. coronatus Molina (Decapoda: Brachyura, Cancridae)[END_REF] measured the straight distance between both spine tips (see [START_REF] Quintana | Desarollo larval de tres especies de cancridae bajo condiciones de laboratorio (Decapoda, Brachyura)[END_REF]. A comparison with cancrid decapods from the North Sea, Cancer pagurus, shows that larvae of this hatch with much larger sizes (»2.5 mm r-d distance) [START_REF] Ingle | The larval and post-larval development of the Edible crab, Cancer pagurus Linnaeus (Decapoda: Brachyura)[END_REF][START_REF] Hartnoll | Laboratory growth of the larvae of six British crabs[END_REF] than larvae of C. setosus over its distributional range, but the Wnal zoeal instar (Zoea V) of larvae reared at 20°C in the present study has nearly the same size in both species (»4.83 mm). This is also reXected in the cumulative growth rates measured in carbon gain (from ZI to ZV) (Weiss et al. 2009a, b). Those are exceptionally high for C. setosus (2,940%) and comparably low for C. pagurus (876%).

Morphological variability in decapod larvae has been frequently discussed as an ecological strategy and adaptation to changing environmental conditions [START_REF] Anger | The biology of decapod crustacean larvae. A.A. Balkema[END_REF]. Those energy saving traits of producing larger eggs and early larval instars at higher latitudes (colder temperatures) once more support the controversially discussed but often supported "Thorson's rule" [START_REF] Mileikovsky | Types of larval development in marine bottom invertebrates, their distribution and ecological signiWcance: a re-evaluation[END_REF][START_REF] Clarke | Egg size and egg composition in polar shrimps (Caridea; Decapoda)[END_REF][START_REF] Thatje | Morphological variability in larval stages of Nauticaris magellanica (A. Milne-Edwards, 1891) (Decapoda: Caridea: Hippolytidae) from South American waters[END_REF] that appears to be particularly useful for the evaluation of energetic trait-oVs in decapods [START_REF] Thatje | Developmental trade-oVs in Subantarctic meroplankton communities and the enigma of low decapod diversity in high southern latitudes[END_REF]. Fischer et al. (2009b) found more energy rich fatty acids in the southern than in the northern population eggs, independent of the incubation temperature. This might indicate a genetic adaptation to colder temperatures and might be a selection in the direction of non-feeding larvae and hence towards lecithotrophic development, which is indicating the beginning of an abbreviated development [START_REF] Thatje | On the origin of Antarctic marine benthic community structure[END_REF]. It is widely accepted, that a development with many larval stages represents a phylogenetically ancestral state, while an abbreviation of the larval stage is said to be derived [START_REF] Strathmann | The evolution and loss of feeding larval stages of marine invertebrates[END_REF][START_REF] Anger | The biology of decapod crustacean larvae. A.A. Balkema[END_REF]. Especially in an unfavourable habitat for planktotrophic, extended larval phase (short period of food availability, temperatures close to the lower limit, and high predation pressure) an abbreviated larval development would enhance survival rates. [START_REF] Quintana | The complete larval development of the Edible Crab, Cancer setosus Molina and observations on the prezoeal and Wrst zoeal stages of C. coronatus Molina (Decapoda: Brachyura, Cancridae)[END_REF] showed that zoeal development of C. setosus larvae of a population from Coliumo Bay (Concepción, central Chile) takes about 60 days under natural temperature conditions (13.5-14.6°C), while larvae from the Antofagasta region have a 1.5 times exceeded zoeal development at 16°C and show higher survival rates (Weiss et al. 2009b), and larvae from Concepcion are capable to develop through all zoeal instars at lower temperatures. These Wndings indicate a better cold adaptation of larvae originating from a southern population (see also [START_REF] Fischer | Temperature-induced oviposition in the brachyuran crab Cancer setosus along a latitudinal cline: aquaria experiments and analysis of Weld-data[END_REF]Fischer et al. 2009a) and from an evolutionary point of view could be considered ancestral to further abbreviated larval developments [START_REF] Strathmann | The evolution and loss of feeding larval stages of marine invertebrates[END_REF], which is favourable for larvae from colder regions.

Although acclimation to colder temperatures leads to a relatively short larval development, the pelagic phase might still be longer in the southern population, where larvae are subject of predation for a longer period of time. However, a synergetic eVect of a larger total length and elongated spines in colder regions might be that larvae stand better chances for survival by being protected from certain predators.

The herein reported phenotypic plasticity in early zoeal larvae enables this species to locally respond to some extent to the inter-decadal warming induced by El Niño, but it should be underlined that strong EN events exceed the upper temperature threshold in this species and are lethal for larval instars.

Fishermen

  caught ovigerous Cancer setosus by scuba diving at diVerent sites around Antofagasta, Northern Chile (23°45Ј S, 70°27Ј W; November 2005, January 2006,

Fig. 1

 1 Fig. 1 Cancer setosus: Zoea I larvae. Thick black lines indicate measured parameters. TL total body length, r-d rostral-dorsal distance, RS rostral spine, DS dorsal spine, LS lateral spine, A antenna

Fig. 2

 2 Fig. 2 Cancer setosus: morphometric measurements of Zoea I larvae hatched from eggs incubated at 16°C in Antofagasta (23°45Ј S) and Puerto Montt (41°44Ј S), Chile. Measures given are: total length (TL), the distance between the tip of the rostral and the tip of the dorsal spine

Fig. 3 Fig. 4

 34 Fig.3Cancer setosus: morphometric measurements of Zoea I larvae hatched from eggs incubated at a 16 and 20°C in Antofagasta (23°45Ј S), and b 12 and 16°C in Puerto Montt (41°44Ј S), Chile. Measures given are: total length (TL), the distance between the tip of the rostral and the tip of the dorsal spine (r-d), lengths of the rostral, dorsal, and lateral spine, the carapace length (CL) and the antenna length. For details on measurements see "Materials and methods" section (p < 0.05, *signiWcant diVerence)

Fig. 5

 5 Fig. 5 Cancer setosus: Zoea III larvae reared at 24°C, showing a reduced dorsal spine and lateral bulging of the carapace

Table 1

 1 Cancer setosus: origin of samples with reference to related works

	Female ID	Experimental site	Rearing	Date	Experiment	Reference samples for
			temperature (°C)
	A + B	A	16	02/2007	Larval development	Weiss et al. (2009a)
	C + D	A	16	11/2005	Embryonic development	Fischer et al. (2009a)
	E + F	A	20	01/2006	Embryonic development	Fischer et al. (2009a)
	G + H	PM	12	09+11/2006	Embryonic development	Fischer et al. (2009a)
	I + J	PM	16	09+11/2006	Embryonic development	Fischer et al. (2009a)
	A Antofagasta, PM Puerto Montt

Table 2

 2 Cancer setosus: female identiWcations, and morphometric measures of Zoea I larvae hatched at diVerent incubation temperatures in Antofagasta (A) (23°45Ј S), and Puerto Montt (PM) (41°44Ј S), Chile Measures given are: total length (TL), the distance between the tip of the rostral and the tip of the dorsal spine (r-d), lengths of the rostral, dorsal, lateral spine, carapace length (CL) and antenna length. For details on measurements see "Materials and methods" section; n = 10 in each treatment

	Location	T	Female	TL (mm)	r-d (mm)	Rostral	Dorsal	Lateral	CL	Antenna
		(°C)	ID			spine (mm)	spine (mm)	spine (mm)	(mm)	(mm)
				Mean § SD	Mean § SD	Mean § SD	Mean § SD	Mean § SD	Mean § SD	Mean § SD
	A	16	A/B/C/D	2.56 § 0.26	1.71 § 0.05	0.80 § 0.07	0.83 § 0.05	0.32 § 0.03	0.49 § 0.01	0.45 § 0.03
		20	E/F	2.70 § 0.14	1.78 § 0.10	0.84 § 0.06	0.85 § 0.09	0.34 § 0.05	0.50 § 0.01	0.45 § 0.05
	PM	12	G/H	2.85 § 0.20	1.96 § 0.07	0.87 § 0.06	0.94 § 0.06	0.39 § 0.05	0.57 § 0.01	0.50 § 0.06
		16	I/J	2.85 § 0.23	1.96 § 0.09	0.89 § 0.07	0.94 § 0.06	0.37 § 0.05	0.59 § 0.01	0.49 § 0.04

Table 3

 3 Cancer setosus: morphometric measures of Zoea II (ZII), Zoea III (ZIII), Zoea IV (ZIV) and Zoea V (ZV) larvae reared at diVerent incu-

	Instar T	n	Incubation	TL (mm)	r-d (mm)	Rostral	Dorsal	Lateral	CL	Antenna
		(°C)		time (days)			spine (mm)	spine (mm)	spine (mm)	(mm)	(mm)
	ZII	16	16	10-23	3.43 § 0.26 2.27 § 0.17 1.01 § 0.08 1.03 § 0.16 0.38 § 0.07 0.70 § 0.02 0.50 § 0.06
		20	18	7-17	3.43 § 0.36 2.10 § 0.26 0.99 § 0.01 0.92 § 0.20 0.42 § 0.09 0.68 § 0.02 0.52 § 0.03
		24	21	6-18	3.15 § 0.32 1.74 § 0.29 0.88 § 0.01 0.63 § 0.30 0.36 § 0.07 0.61 § 0.02 0.52 § 0.04
	ZIII	16	8	23-34	4.39 § 0.30 2.84 § 0.27 1.34 § 0.10 1.29 § 0.33 0.42 § 0.07 0.88 § 0.04 0.70 § 0.06
		20	3	16-28	3.80 § 0.31 2.29 § 0.34 1.04 § 0.10 1.08 § 0.28 0.48 § 0.07 0.80 § 0.06 0.62 § 0.08
	ZIV	20	14	25-33	5.85 § 0.49 3.94 § 0.34 1.71 § 0.10 1.83 § 0.24 0.44 § 0.10 1.26 § 0.14 0.82 § 0.11
	ZV	20	6	27-39

bation temperatures in Antofagasta (A) (23°45Ј S), Chile Measures given are: total length (TL), the distance between the tip of the rostral and the tip of the dorsal spine (r-d), lengths of the rostral, dorsal, lateral spine, carapace length (CL) and antenna length. For details on measurements see "Materials and methods" section Mean § SD Mean § SD Mean § SD Mean § SD Mean § SD Mean § SD Mean § SD 7.69 § 0.34 4.83 § 0.31 2.01 § 0.10 2.21 § 0.21 0.42 § 0.13 1.47 § 0.16 0.99 § 0.13

Table 4

 4 Cancer setosus: ratios of spine lengths and antenna versus carapace length

		Location	Instar Temperature	TL:CL	r-d:CL	RS:CL	DS:CL	LS:CL	Antenna:CL
				(°C)
		A	ZI	16	5.51	3.63	1.71	1.74	0.68	0.92
		PM	ZI	16	4.86	3.34	1.51	1.60	0.63	0.84
	A Antofagasta, PM Puerto	A	ZI	16	5.51	3.63	1.71	1.74	0.68	0.92
	Montt, ZI, ZII, ZIII: zoeal	A	Z I	2 0	5.15	3.44	1.61	1.66	0.65	0.91
	stages, total Length (TL), the	PM	ZI	12	4.99	3.44	1.53	1.65	0.68	0.87
	distance between the tip of the rostral and the tip of the dorsal spine (r-d), lengths of the rostral	PM A	ZI ZII	16 16	4.86 5.00	3.34 3.23	1.51 1.44	1.60 1.47	0.63 0.53	0.84 0.71
	(RS), dorsal (DS), lateral spine	A	ZII	20	5.06	3.10	1.45	1.36	0.63	0.77
	(LS), antenna and carapace	A	ZII	24	5.14	2.84	1.44	1.03	0.58	0.85
	length (CL). SigniWcantly shorter spines are given in bold	A	ZIII	16	4.99	3.24	1.52	1.47	0.48	0.80
		A	ZIII	20	4.75	2.86	1.30	1.35	0.60	0.77

Acknowledgments

We would like to thank Marcelo Oliva (Universidad Antofagasta, Chile) for providing workspace in his laboratory and Aldo Pacheco for help with larval culture maintenance. Special thanks are due to Sönke Fischer for providing the samples from Puerto Montt and for contributing to this manuscript. This study is conducted in the frame of the EU-FP6-INCO project CENSOR (Climate variability and El Niño Southern Oscillation: Implications for natural coastal resources and management) (Contract no. 511071), and received additional funding from the Marine Biodiversity and Ecosystem Functioning Network of Excellence MarBEF (Contract no. GOCE-CT-2003-505446). Monika Weiss was supported by a travel grant of the DAAD (contract no. 415 D/07/47120). The study is CENSOR publication No. 0386.


Robert Rutkowski
email: robertrut@miiz.waw.pl

Łukasz Rejt

Anna Tereba

Alicja Gryczyńska-Siemiątkowska

Bartosz Janic

W Lutz

Population genetic structure of the European kestrel Falco tinnunculus in Central Poland

Keywords: Falco tinnunculus, Population genetic Genetic structure, Urban populations, Microsatellites

We analysed the genetic structure of the European kestrel population of Central Poland using nine highly polymorphic microsatellite loci. Samples were collected in two urban locations (Warsaw and Łódź) and two rural areas. Sampling locations were at nearly equal distances from each other along an east to west line. We performed genotyping in a total of 99 birds. The results revealed the presence of a genetic structure in the population investigated. Bayesian clustering indicated that samples originated from more than one population. Genetic differentiation was less pronounced among the birds nesting in Warsaw and in the two rural sites, whereas all pairwise comparisons with the Łódź population indicated moderate and significant genetic differentiation. The observed pattern of differentiation might have been caused by two factors: changes in allele frequency between seasons and/ or the founding of the urban population of Łódź from a different source population than the urban population from Warsaw. Additionally, we found a rather high gene flow among kestrels from the Warsaw urban area and the two investigated rural areas.

Introduction

The majority of animal species exhibit some level of genetic population structuring caused by a variety of factors. Dispersal ability, dietary specialisation, phenological asynchrony, discrete differences in breeding biology and mating systems as well as environmental barriers and the spatial structure of habitat may all influence the genetic structure of populations (e.g. [START_REF] Peterson | The influence of dispersal and diet breadth on patterns of genetic isolation by distance in phytophagous insects[END_REF][START_REF] Höglund | Microsatellite markers reveal the potential for kin selection on black grouse leks[END_REF][START_REF] Donnelly | Evidence for extensive genetic differentiation among populations of the malaria vector Anopheles arabiensis in Eastern Africa[END_REF][START_REF] Hendry | Population structure attributable to reproductive date: isolation-by-time and adaptation-by-time[END_REF][START_REF] Keyghobadi | Genetic differentiation and gene flow among populations of the alpine butterfly, Parnasisus smintheus, vary with landscape connectivity[END_REF]. The processes of anthropogenic changes of natural habitats have, especially recently, led to the artificial fragmentation of many populations. Despite the intensive expansion of urbanised areas, only a few studies, up until now, have investigated the relationship between urban landscapes and population genetic structure [START_REF] Rubin | Population genetic structure of Blanding's turtles (Emydoidea blandingii) in an urban landscape[END_REF][START_REF] Wood | Persistence of species in a fragmented urban landscape: the importance of dispersal ability and habitat availability for grassland butterflies[END_REF]. These studies demonstrated the effects of isolation and reduced gene flow between habitat fragments within urban areas. However, the genetic relationships between populations of the same species living in urban and rural environments have seldom been studied. Moreover, the majority of these studies were focused on animal species which were trapped in habitat fragments due to urbanisation: small patches of wetland habitats in the case of turtles [START_REF] Rubin | Population genetic structure of Blanding's turtles (Emydoidea blandingii) in an urban landscape[END_REF], remnant forest fragments in the case of beetles [START_REF] Desender | Rural-urban gradients and the population genetic structure of woodland ground beetles[END_REF] or grassland patches in the case of butterflies [START_REF] Wood | Persistence of species in a fragmented urban landscape: the importance of dispersal ability and habitat availability for grassland butterflies[END_REF], and hence dealt with populations that were isolated by patches of less suitable or unsuitable habitat. Meanwhile, some species actively colonise cities with foxes as one of the best known examples [START_REF] Gloor | The rise of urban fox populations in Switzerland[END_REF][START_REF] Wandeler | The city-fox phenomenon: genetic consequences of a recent colonization of urban habitat[END_REF]. In such cases, the influence of the urban environment on the population structure and on the genetic differentiation between groups from urbanised areas and rural habitats may be much more complex. For example, urban areas, as most species are unlikely to be adapted to them, might function as dispersal sinks maintained by constant immigration from rural populations. Hence, source and sink populations may become indistinguishable even if they are partially isolated [START_REF] Rousset | Genetic differentiation within and between two habitats[END_REF]. On the other hand, some groups of individuals may adapt to local urban conditions and resources. Such adaptation may then result in reproductive isolation leading to a pronounced genetic structure strengthened by the effects of the founder event and random genetic drift especially when a colonising group is small [START_REF] Gloor | The rise of urban fox populations in Switzerland[END_REF].

The European kestrel, widely distributed in the Western Palearctic region, lives in different types of habitats, mainly in vast open spaces, but is also commonly observed in urban-industrial environment areas-the species has been present in European cities for more than 150 years (e.g. Village 1990, Śliwa andRejt 2006). The Polish population of the species is partially migratory, hence, composed of migrants and resident individuals. Despite its relative abundance in the whole country, the kestrel population is rather patchily distributed especially during the breeding season. Birds tend to gather in built-up areas and nest on high buildings or, if accessible, in nesting boxes. In rural regions, tree-based nests have been systematically disappearing over the last few decades. Similarly, observation data suggest that the number of kestrels in many rural areas seems to have decreased in the last decades [START_REF] Śliwa | Kestrel. Wyd. Klubu Przyrodników[END_REF], whereas they appear to become increasingly abundant in cities. It is not clear whether this patchy distribution, caused mainly by gathering within urban areas during breeding season, affects the population genetic structure of the species. However, some level of genetic isolation among urban and rural groups of birds was seen in the Warsaw area [START_REF] Rejt | Genetic variability of urban kestrels in Warsaw-preliminary data[END_REF][START_REF] Rutkowski | Analysis of microsatellite polymorphism and genetic differentiation in urban and rural kestrels Falco tinnunculus (L.)[END_REF].

In Warsaw, the European kestrels have been observed regularly since the 1970s, and presently their population is estimated at ca. 70 breeding pairs [START_REF] Rejt | Breeding biology of urban Kestrel Falco tinnunculus in Warsaw[END_REF]. These breeding pairs are evenly distributed across the city-they nest in the centre of Warsaw, as well as in suburban areas, with numerous open spaces and more similar to natural habitat. Observations suggest that these birds exhibit features which are characteristic of the so-called synurbic populations (e.g. [START_REF] Luniak | Synurbization-adaptation of animal wildlife to urban development[END_REF])-they tend to be resident, re-occupy their former nests and start breeding earlier than rural populations [START_REF] Rejt | Breeding biology of urban Kestrel Falco tinnunculus in Warsaw[END_REF]. In Warsaw, European kestrels produce 4.4-5.4 successfully reared offspring per breeding pair-slightly higher than rural populations (about four successfully reared offspring per breeding pair). The mortality rate of the offspring of urban birds is about 50% in the first year and increases in subsequent seasons (review in [START_REF] Śliwa | Kestrel. Wyd. Klubu Przyrodników[END_REF]. In Warsaw, bulk of prey is composed by mammals (mostly voles "Microtus" sp). Their proportion reached more than 85%. Other prey groups were significantly less numerous-birds composed about 12% of prey; reptiles less than 4% (e.g. [START_REF] Śliwa | Kestrel. Wyd. Klubu Przyrodników[END_REF].

In Łódź, the European kestrel was described as a new breeding species for central parts of the city in the 1980s [START_REF] Markowski | Fauna of Lodz-the state of knowledge, changes, protected and threatened species[END_REF]). Kestrel pairs most willingly nest in the strongly urbanised centre of the town and in large industrial complexes. Presently, the population size is estimated at [40][41][42][43][44][45]unpublished data).

In this paper, we applied highly polymorphic microsatellite loci to investigate if the patchy distribution of European kestrels across rural and urban (Warsaw and Łódź cities) areas causes a genetic structure in the population of this species.

Materials and methods

Study area and sample collection

During a 6-year period (2002)(2003)(2004)(2005)(2006)(2007), we collected material from 99 individuals of the European kestrels from four sampling sites (Fig. 1): an urban area of Warsaw (52°13′N 21°E; n=44); an urban and suburban area of Łódź (51°55′N 19°25′E; n=15); a rural area near Łowicz town (52°07′N 19°56′E) about 50 km east from Łódź and 80 km west from Warsaw (n=18) and a rural area near Siedlce town (52°13′N 22°16′E) about 90 km east from the geographical centre point of Warsaw (n=22).

Blood and feather samples were collected from nestlings. To avoid losses among chicks (resulting from blood loss and/or escaping from nests), all manipulations were done between the 10th and 15th day of their lives according to the Ethic Committee decision No. 157/2002. In the majority of cases, we collected material from two randomly chosen individuals per nest (Table 1).

Blood samples (57.5% of total sample) were collected and stored on FTA cards (Whatman BioScience). Feathers (42.5% of total sample) were stored dry in Eppendorf tubes. In Warsaw, all nests were situated on buildings. In Łódź, kestrels also nested on buildings or in nesting boxes. In Łowicz, nests were built on top of the roof of a church that was located within the open agricultural environment separated from other build-up areas. Samples from Siedlce were from nesting boxes placed on high trees.

DNA isolation and analysis of microsatellite markers Deoxyribonucleic acid (DNA) from blood stored on FTA cards was extracted according to the manufacturer's protocol. DNA from feathers was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Only root ends of feathers, approximately 0.5 cm long, were used for extraction. They were crushed, placed in 180 μl of ATL buffer (Qiagen) and incubated at 56°C overnight with 20 μl of proteinase K (20 μg/ ml). Then, extraction followed the standard protocol. Finally, DNA was eluted in 200 μl of elution buffer (Qiagen).

By PCR, we amplified nine microsatellite loci as described by [START_REF] Nesje | Genetic relationships in the peregrine falcon (Falco peregrinus) analysed by microsatellite DNA markers[END_REF]: NVHfp79-4, NVHfp13, NVHfp31, NVHfp5, NVHfp46-1, NVHfp89, NVHfp79-1, NVHfp82-2 and NVHfp86-2. Microsatellite DNA from feathers was amplified in a 25-μl reaction mix which contained 10 pmol of each primer, 10-50 ng of template DNA, 12.5 μl of REDTaq PCR ReadyMix (Sigma-Aldrich) and 7.5 μl of PCR-grade water (Sigma-Aldrich). For the microsatellite amplification, from blood DNA, we placed a little piece of blood containing an FTA card (ca. 2 mm in diameter) into a tube with 30 μl of PCR reaction mix, containing 15 μl of Red Taq Ready Mix (Sigma-Aldrich), 5 pmol of each primer and 14 μl of PCR-grade water (Sigma-Aldrich). Forward primers were 5′-labelled with a fluorescent dye: Dye2, Dye3 or Dye4 (ProOligo). Prior to PCR, all reagents, tubes and pipettes were exposed to ultraviolet light for 15 min. The following PCR profiles were used in a Techne Touchgene thermocycler: initial denaturation for 3 min at 94°C, followed by 34 cycles of 1 min at 94°C, 45 s at 55°C, 45 s at 72°C and one cycle of 1 min at 94°C, 45 s at 55°C and 5 min at 72°C.

The length of amplified fragments was estimated using a CEQ8000 Beckman Coulter automated sequencer (Beckman Coulter, Fullerton, CA, USA). Data were analysed using Beckman Coulter Fragment Analysis Software (v. 9.0).

Statistical analysis

For each locus and each location investigated, we assessed number of alleles (A), allelic richness (R; [START_REF] Petit | Identifying populations for conservation on the basis of genetic markers[END_REF], observed heterozygosity (H O ) and unbiased expected heterozygosity (H E ; [START_REF] Nei | Sampling variances of heterozygosity and genetic distance[END_REF]. The inbreeding fixation index (F IS ) was calculated for each sampling location, and its significance was tested using 360 randomisations. A Bonferroni correction was applied for multiple comparisons. To assess whether our sampling scheme (sampling of two nestlings from one nest) influences the level of observed heterozygosity and deviation of heterozygosity from values expected under Hardy-Weinberg equilibrium, we repeated the calculation of F IS with random sample sets containing only one sample from each nest. For each population, we repeated this random sampling ten times, calculating F IS for each sample set. These analyses were performed using GenAlEx (v. 5.04; Peakall and Smouse 2001) and FSTAT (v. 2.9.3;[START_REF] Goudet | FSTAT V2.9.3, a program to estimate and test gene diversities and fixation indices[END_REF]. Genotypic linkage disequilibrium among all pairs of loci was evaluated using Genepop (v.3.1b; [START_REF] Raymond | GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism[END_REF]. We also applied the software Micro-Checker [START_REF] Van Oosterhout | Microchecker: software for identifying and correcting genotyping errors in microsatellite data[END_REF]) to test for the presence of "null alleles" as well as for possible scoring errors. The latter problem was excluded by the analysis; however, presence of "null alleles" was suggested for some loci in all populations except for loci from the Łódź population (see Results). Thus, we estimated the frequency of these alleles using the algorithm of van Oosterhout et al. ( 2004), and all subsequent analyses were performed based on corrected genotypes. Presence of a genetic structure in the kestrel population and pattern of genetic differentiation among different groups of birds was tested using a variety of methods. First, we estimated significance of genotypic differentiation among the investigated groups of birds using Genepop (v.3.1b;[START_REF] Raymond | GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism[END_REF]. We also estimated measures of population differentiation with pairwise F ST by using an analysis of molecular variance procedure of the software Arlequin (v. 2.0, [START_REF] Schneider | ARLEQUIN: a software for population genetic data analysis[END_REF], which calculates F ST as described by [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF]. P values for F ST were calculated using 1,000 permutations. Then, we used SAMOVA software (v. 1.0; [START_REF] Dupanloup | A simulated annealing approach to define the genetic structure of populations[END_REF] to estimate the proportion of variance at different levels of population subdivision. The following indices were estimated: F CT , a measure of differentiation between groups of locations; F SC , a measure of sub-structuring within groups and F ST , a measure of differentiation among locations after accounting for regional structure. The significance of the observed variance components was evaluated by means of a non-parametric permutation method.

Then, we estimated Nei's standard genetic distance D S [START_REF] Nei | Genetic distance between populations[END_REF]) using an online calculator (http://www.biology. ualberta.ca/jbrzusto) and Slatkin's (1995) R ST , an analogue of F ST developed for analysing microsatellite data using RstCalc [START_REF] Goodman | RstCalc: a collection of computer programs for calculating estimates of genetic differentiation from microsatellite data and determining their significance[END_REF]. To get unbiased estimate of R ST , the data set was standardised for sample size and allelic length, and the variance components were averaged over loci before calculating R ST . P values for R ST were calculated using 1,000 permutations.

Finally, we used the Bayesian clustering method (Structure v. 2; [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] to examine how well predefined "populations" (=sampling locations) corresponded to genetic groups (K). To choose an appropriate run length, we performed ten runs for each value of K with different values of burn-in and run length (from 10,000 to 1,000,000). Results of these initial simulations were checked for variation across runs for each value of K to assess contingency across simulation. We found that the likelihoods have converged at burn-in of 50,000 and 100,000 iterations of the total data set. Thus, we performed additional three runs for each user-defined K (1-4) with an initial burn-in of 50,000 and 100,000 iterations. Default values were maintained for all other parameters. We used the admixture model of ancestry and the correlated model of allele frequencies. The proportion of membership of each predefined "population" within each genetic group was estimated for K with the highest likelihood. Sampling location was not used as prior information.

Although reliable estimate of a significant association between geographical and genetic distances requires comparison of at least five populations, we decided to perform Mantel test to check whether there is any suggestion of isolation by distance among populations investigated. This analysis was performed with Genepop v.3.1b using the pairwise F ST as a measure of genetic differentiation between groups.

Results

We successfully amplified nine microsatellite loci in 95 samples. We failed to amplify NVHfp79-1 in four individuals from Siedlce. All loci analysed were polymorphic with numbers of alleles ranging from five (NVHfp82-2 and NVHfp79-1) to 14 (NVHfp79-4) (Table 2). The test for significance of linkage disequilibrium over all the loci and locations, as well as for each sampling location separately, showed no significant linkage disequilibrium among loci. In general, diversity indices, which were based on microsatellite polymorphisms, showed similar values in the locations investigated (Table 2). The mean value of A across all loci was the highest in Warsaw, but it was caused by a high number of alleles in just one locus -NVHfp46-1. After correction for sample size, allelic richness (R) was the highest in Siedlce. In the locations, except Łódź, F IS was significantly greater than zero, which indicates heterozygote deficiency. In the analysis of random sample sets containing only one sample from each nest, the same three locations showed F IS values greater than zero; however, the differences were not statistically significant after Bonferroni correction (Table 2). In one case (Siedlce), the mean F IS value estimated for the group with one sample per nest (n= 11) was even greater than the one estimated for the group with two samples per nest (Table 2). The global test for heterozygote deficiency also showed significant deviation (P<0.0001) even after an analysis of a data set containing only one randomly chosen sample per nest.

Analysis in Micro-Checker indicated no scoring errors but indicated the presence of "null alleles" in all populations investigated except that in Łódź. In Łowicz, "null alleles" were present in loci NVHfp13 and NVHfp86-2 (frequency 0.23 and 0.17, respectively; P<0.05), and in Warsaw in loci NVHfp13 (frequency 0.18), NVHfp5 (0.09), NVHfp46-1 (0.22) and NVHfp86-2 (0.13; P<0.05 for all these loci). In Siedlce, presence of "null allele" was indicated only for locus NVHfp79-1 (frequency 0.27, P< 0.01). There was no relation between presence of "null alleles" and sample type (feathers vs. blood samples). For example, Micro-Checker suggested presence of "null alleles" in loci NVHfp13 and NVHfp86-2 in Łowicz and Warsaw, whereas in the former, feathers had been used as a source of DNA, and in the latter, blood stored on FTA cards. Using Micro-Checker, we generated corrected genotypes for each locus with "null alleles", which were subsequently used in analysis of population differentiation.

Genotypic differentiation was significant for all the comparisons among locations (Table 3). Small but significant differentiation among locations was indicated by an overall F ST value of 0.058 (P<0.001). Significant values of F ST were observed for all pairwise comparisons except Warsaw-Łowicz. For all comparisons with Łódź, F ST suggested moderate genetic differentiation (Table 3), whereas values for comparison among Warsaw, Łowicz and Siedlce indicated small genetic differentiation. Similarly, SAMOVA indicated the only significant F CT when populations were grouped into two groups (group 1, Łódź; group 2, Łowicz, Warsaw and Siedlce; F CT =0.10669, P< 0.0001; F SC = 0.02926, P < 0.0001; F ST = 0.13282, P < 0.0001). More than 10% of the genetic variation was explained by differentiation between groups of populations; 1.7% by variation among populations within groups and nearly 88% by within-population variation. Pairwise Nei's standard genetic distance and R ST showed a pattern similar to F ST values. In both cases, the smallest value was observed between Warsaw and Łowicz, whereas the highest values were observed for pairwise comparisons with Łódź (Table 4). Overall R ST was 0.167 (P<0.001) indicating moderate and significant population structure.

The Bayesian analysis in structure revealed the highest posterior probability for three genetic clusters under admixture model [mean value for three runs ln P(X│K)= -2707.16; P(K│X)≈0.99]. Cluster I was nearly exclusively composed by individuals from Łódź: 14 of individuals from this locality were assigned to this cluster, whereas one was assigned to cluster II and another one to cluster III. Cluster II and III were composed of individuals from Łowicz, Warsaw and Siedlce, symmetrically distributed between those two clusters (Fig. 2).

Mantel test revealed that F ST pairwise comparisons were not related to geographical distance (r=0.34; P=0.1). However, after the exclusion of the Łódź population, correlation was stronger but still insignificant (r=0.87; P=0.09).

Discussion

Until now, at least three different processes have been described to cause population genetic structure in kestrels. The first one is spatial separation as demonstrated in kestrels from the Cape Verde archipelago [START_REF] Hille | Genetic structure of kestrel populations and colonization of the Cape Verde archipelago[END_REF]. There, a genetic structure evolved through spatial isolation by water belts, and genetic differentiation was correlated with geographical distances among islands. The second one is breeding asynchrony, which generates genetic differentiation between early-and late-breeding groups of kestrels even within a sympatric population [START_REF] Casagrande | Genetic differences between early-and late-breeding Eurasian kestrels[END_REF]. The third process is a reduced gene flow between birds nesting in urban and rural environments [START_REF] Rutkowski | Analysis of microsatellite polymorphism and genetic differentiation in urban and rural kestrels Falco tinnunculus (L.)[END_REF]; however, this observation should be considered as preliminary. The results suggested the presence of a genetic structure in the population, and different measures of genetic distance show a very similar pattern of differentiation among populations. However, we did not find any clear pattern of differentiation between urban and rural populations. All pairwise measures of genetic differentiation, as well as Bayesian clustering and SAMOVA, clearly indicated the presence of two groups of birds. The first group contained kestrels from the urban area of Warsaw and two adjacent rural areas, whereas the second group was composed of birds from the urban area of Łódź. This pattern could be an outcome of our sampling scheme: samples from Łódź were collected during a single season separated by 4 years from the season in which the majority of samples from Warsaw were collected. The mean lifespan of kestrels, based on ringing data, was assessed to be 1.3-1.9 years, but for birds in the second year of life, it increases to 2.9 years (reviewed in [START_REF] Śliwa | Kestrel. Wyd. Klubu Przyrodników[END_REF]. Most kestrels breed for the first time in their first year of life [START_REF] Village | The kestrel. Poyser, London Village A (1998) Kestrel[END_REF](Village , 1998)). However, among breeders, the proportion of first-year breeders may vary between 60% and 6% depending on the phase of vole cycle or seasonal migration (e.g. Village 1998;[START_REF] Laaksonen | Sex-specific recruitment and brood sex ratios of Eurasian kestrels in a seasonally and annually fluctuating northern environment[END_REF]). Hence, in the case of kestrel, the shift in allele frequencies among seasons can be considerable. On the other hand, we found a rather small differentiation between the Warsaw and Łowicz populations despite sampling of these populations in different years, whereas both of them showed an almost identical value of F ST in comparison with Łódź. This suggests that changes in allele frequencies among seasons may not be very significant. For example, philopatry could prevent such changes. Unfortunately, there is no precise data on the level of philopatry in the populations studied. In other rural kestrel populations, the philopatry seems to be low but varies simultaneously with the varying abundance of voles: in Fennoscandia, only 8% of the females and about 25% of the males return to their breeding territories the following year [START_REF] Lundvall | Mating and hunting behaviour in European kestrels (Falco tinnunculus)[END_REF] while in Holland, between 10% and 70% of birds do return according to the vole index [START_REF] Cave | The breeding of the kestrels, Falco tinnunculus L., in the reclaimed area Oostelijk Flevoland[END_REF]). Ringing data from Berlin suggests that birds, which had hatched in urban areas, tend to return to their sites of birth (S. Kupko, unpublished data). Nonetheless, this issue requires further investigation especially the comparison of samples from different populations collected in one season as well as among different seasons in separate populations. Differentiation between the two investigated urban areas could also be explained by another factor. It is possible that the urban populations in Łódź and Warsaw were formed by different groups of individuals. Then, founder effect and subsequent genetic drift could generate significant genetic differentiation between them. Indeed, the urban populations of Łódź and Warsaw differ in some aspects. For example, the first breeding pairs were observed (in the centre of Łódź) in 1980 (B. Janic, unpublished data), whereas in Warsaw, kestrels have been regularly observed since the 1970s, and the number of pairs breeding within city borders is now much higher than in Łódź [START_REF] Rejt | Breeding biology of urban Kestrel Falco tinnunculus in Warsaw[END_REF].

Genetic structure within the group of the other three localities, Łowicz, Warsaw and Siedlce, was weakly pronounced, and pairwise measures of genetic differentiation indicated the presence of intensive gene flow between localities. This group of localities is a rather panmictic unit irrespective of the presence of the urban environment, and genes are frequently exchanged between the city and adjacent areas. This fact stands in contradiction with results obtained for the urban populations of other species. In their study of the fox population living in the city of Zurich and in adjacent rural areas, [START_REF] Wandeler | The city-fox phenomenon: genetic consequences of a recent colonization of urban habitat[END_REF] found significant genetic differentiation between rural and urban groups-pairwise comparisons among rural sampling sites showed no genetic differentiation (estimated by F statistics), whereas differentiation among the urban and rural groups was high and significant. Unfortunately, there are few similar studies on birds, which are much more mobile and thus able to cross natural and artificial barriers better than mammals. However, in the case of European blackbird (Turdus merula), analysis of neutral genetic markers showed no genetic differentiation between urban and a nearby forest-living population [START_REF] Partecke | Is urbanisation of European blackbirds (Turdus merula) associated with genetic differentiation?[END_REF] despite several phenotypic differences between these populations [START_REF] Partecke | Annual cycles of urban and forest-living European blackbirds (Turdus merula): genetic differences or phenotypic plasticity? Dissertation at the Ludwig-Maximilians-University[END_REF][START_REF] Partecke | Annual cycles of urban and forest-living European blackbirds (Turdus merula): genetic differences or phenotypic plasticity? Dissertation at the Ludwig-Maximilians-University[END_REF]. Although the study is based on small number of individuals and only two populations, this result suggests that high mobility create favourable conditions for intensive gene flow between urban and rural populations of birds.

Differentiation between the Łowicz rural area and the Warsaw urban area was small and insignificant suggesting strong gene flow. In the case of Łowicz, since samples were collected from nests placed on a building, this group may represent birds which "prefer" urban landscapes (i.e. buildings); it may even consist of migrants from the urban area of Warsaw. A similar phenomenon was found for the Peregrine-young birds released from cliff-like sites (e.g. buildings) also preferred them as nesting places later on [START_REF] Kirmse | Wiedereinbürgerung baumbrütender Wanderfalken (Falco peregrinus) in Mitteleuropa[END_REF]. For the time being, our data are too weak to confirm or reject such a pattern for kestrels. Additionally, a large number of samples from birds nesting on buildings, in nesting boxes and in high trees are required in order to analyse whether the selection of nesting sites could influence the genetic structure of the species.

In three out of the four localities investigated, we have found heterozygote deficiency, which could be due to the presence of "null alleles", at least at some loci. However, other explanations are also possible. A similar phenomenon was found by [START_REF] Hille | Genetic structure of kestrel populations and colonization of the Cape Verde archipelago[END_REF] in a kestrel population from Austria. Authors ascribed this observation to polygamy, which was known to exist in that population. In kestrels, polgyny reaches 12% and is probably associated with colonial breeding (Glutz von Blotzheim 1971;Village 1998). In seasons with a high vole number, it may even reach 20% [START_REF] Korpimäki | Copulatory behaviour and paternity determined by DNA fingerprinting in kestrels: effects of cyclic food abundance[END_REF]. The level of polyandry in open landscape populations is usually only 5-7% but could be lower in years with poor prey abundance [START_REF] Village | The kestrel. Poyser, London Village A (1998) Kestrel[END_REF]). In addition, frequency of extra-pair fertilisations among birds of prey increases with the growing density of breeding pairs [START_REF] Korpimäki | Copulatory behaviour and paternity determined by DNA fingerprinting in kestrels: effects of cyclic food abundance[END_REF][START_REF] Mougeot | Territorial intrusions and copulation patterns in red kites, Milvus milvus, in relation to breeding density[END_REF], and increased density of breeding pairs is thought to be one of the factors connected with synurbisation [START_REF] Luniak | Synurbization-adaptation of animal wildlife to urban development[END_REF]). Indeed, observation of ringed birds in Warsaw provides evidence that some females copulate with more than one male during single breeding season (Rejt Ł, personal observation). Thus, extra-pair fertilisation could be a possible explanation for the significant heterozygote deficiency in the case of the Warsaw but only if small number of males would participate in the reproduction of a whole population. It is also possible that a similar process took place in the Łowicz group where a few nests were found on one building. Hence, the presence of buildings within the environment changes the nesting behaviour of kestrels may be not only in terms of nesting site selection but also in terms of nesting-related territorial behaviour, which can favour extra-pair copulation and paternity. On the other hand, an F IS value significantly greater than zero was found in Siedlce where the density of nesting pairs did not change.

Another explanation of homozygote excess could be inbreeding within the population. Although such a process is usually the case for small, isolated populations, some plumage aberrations were observed in kestrels from Warsaw [START_REF] Rejt | Nietypowe ubarwienie piskląt pustułki Falco tinnunculus w Warszawie[END_REF]. Based on the present data, it is difficult to judge if such aberrations and significant F IS values are really effects of inbreeding. However, if strong philopatry existed in urban populations, it is possible that breeding could occur between closely related individuals resulting in an increase in homozygosity.

Fig. 2

 2 Fig. 2 Bayesian assignment of individuals to K=3 clusters without using prior population information. Each bar represents the estimated posterior probability of each individual bird belonging to

Table 1

 1 Description of kestrel samples collected for genetic analysis

	Fig. 1 Map of Poland with sampling location (black dots)	14	15	16	17	18	19	20	21	22	23	24
			54									54
			53									53
											Warsaw
			52										52
												Siedlce
			51										51	25
			50
			14	15	16								50	25
						17 49		18	19	20	21	22	23	49 24
					100 km
		Łódź	Łowicz	Warsaw	Siedlce	Total
	n	15	18	44	22	99
	N	9	1 0	2 2	1 1	5 2
	N (ii)	6	8	22	11	47
	N (i)	3	2	-	-	5
	Years of sampling
	2002	-	-	40	-	40
	2003	-	-	4	-	4
	2004	-	4	-	10	14
	2005	-	5	-	12	17
	2006	15	8	-	-	23

n number of individuals sampled, N number of nests sampled, N (ii) number of nests where two samples were collected, N (i) number of nests where single samples were collected

Table 2

 2 Description of polymorphism of microsatellite loci in the investigated groups of kestrels

	Warsaw Siedlce Total	A R H O H E F IS A R H O H E F IS A R H O H E F IS	13 10.55 0.795 0.833 0.056 9 8.38 0.727 0.845 0.162 14 9.92 0.780 0.872 0.111*	4 3.59 0.591 0.613 0.048 4 3.93 0.682 0.644 -0.036 6 4.82 0.610 0.711 0.147	5 4.88 0.432 0.708 0.400* 6 5.37 0.455 0.632 0.302 6 4.64 0.400 0.639 0.379*	5 3.86 0.545 0.562 0.041 3 3.00 0.455 0.485 0.085 7 5.07 0.570 0.654 0.133	8 7.55 0.682 0.839 0.198 10 9.43 0.909 0.843 -0.055 12 8.39 0.780 0.851 0.089	6 4.80 0.364 0.695 0.457* 6 5.59 0.318 0.726 0.578* 9 5.55 0.400 0.729 0.455*	4 3.99 0.636 0.698 0.099 5 4.93 0.591 0.755 0.240 5 4.74 0.680 0.716 0.055	4 3.36 0.614 0.580 -0.046 4 4.00 0.733 0.679 -0.116 5 4.12 0.620 0.633 0.026	6 4.55 0.477 0.635 0.259 4 3.73 0.455 0.635 0.306 6 4.19 0.440 0.617 0.292*	6.11 5.23 0.57 0.68 0.173* 5.67 5.37 0.59 0.69 0.164* 7.78 5.71 0.58 0.71 0.183*	(0.15) (0.180) (0.163*)	IS inbreeding coefficient sensu Weir and Cockerham (1984)
	Ł owicz	A R H O H E F IS	7 6.99 0.833 0.833 0.029	4 3.89 0.667 0.613 -0.060	3 2.99 0.222 0.551 0.615*	5 4.78 0.667 0.614 -0.057	7 6.98 0.778 0.810 0.068	4 4.00 0.444 0.546 0.214	4 4.00 0.833 0.711 -0.143	5 4.99 0.556 0.656 0.181	4 3.99 0.333 0.653 0.511*	4.78 4.73 0.59 0.66 0.137*	(0.121)
	Locus Łódź	A R H O H E F IS	NVHfp79-4 10 10.00 0.750 0.861 0.161	NVHfp89 4 4.00 0.500 0.635 0.243	NVHfp13 2 2.00 0.438 0.404 -0.050	NVHfp31 3 3.00 0.688 0.623 -0.071	NVHfp5 8 8.00 0.875 0.822 -0.032	NVHfp46-1 6 6.00 0.563 0.729 0.258	NVHfp79-1 4 4.00 0.750 0.590 -0.241	NVHfp82-2 3 3.00 0.500 0.635 0.243	NVHfp86-2 3 3.00 0.438 0.354 -0.207	Mean 4.78 4.78 0.61 0.63 0.058	(0.035)	In parentheses are mean F	*P<0.05	Table 3 P values for genotypic differentiation (above diagonal) and pairwise comparison of F ST values (below diagonal) between the sampling localities Łódź Ł owicz Warsaw Siedlce Łódź 0.0001 0.0001 0.0001 Łowicz 0.1280 0.0096 0.00034 Warsaw 0.1252 0.0133 0.00010 Siedlce 0.1177 0.0488 0.0232 Italicized values of F ST are significant after Bonferroni correction (corrected P=0.00833)

IS values calculated for ten data sets containing one randomly chosen sample per nest

A number of alleles, R allelic richness, H O heterozygosity observed, H E heterozygosity expected, F

Table 4

 4 Nei's standard genetic distance D S (above diagonal) and R ST (below diagonal) among the sampling localities

		Łódź	Ł owicz	Warsaw	Siedlce
	Łódź		0.367	0.389	0.384
	Łowicz	0.2208		0.061	0.140
	Warsaw	0.1869	0.0120		0.088
	Siedlce	0.1863	0.1084	0.0477
	Italicized values of R ST are significant (P<0.05)

Eur J Wildl Res (2010) 56:297-305

Acknowledgements

The study was partly supported by the Ministry of Science and Higher Education grant no. N N304 4078 33. We thank all persons who contributed in the gathering of material for genetic analysis. We also thank two anonymous reviewers for valuable and constructive comments on the paper.

The experiment presented in this paper complies with the current laws of Poland.


Kamal Chaouachi

Errors and misquotations in the study of shisha, blood pressure and heart rate in Jordan

Keywords: Smoking, Shisha, Narghile, Tobacco, Heart rate, Blood pressure

Errors and misquotations in the study of shisha, blood

pressure and heart rate in Jordan

In their introduction, Al-Safi et al. wrongly credit Kiter for mentioning countries such as China, India or Pakistan [START_REF] Sa | Does shisha smoking affect blood pressure and heart rate?[END_REF][START_REF] Kiter | Water-pipe smoking and pulmonary functions[END_REF]. Then, they endorse the idea that "maassel" "simplifies water pipe preparation", whereas this smoking product has actually made shisha smoking more complex than the ones traditionally used for centuries, mainly tumbak ("'ajamy") and jurak. Indeed, besides the well-known and "ritual" complicated procedures described in all anthropological studies, a tin (aluminium) foil has to be carefully prepared by piercing it and placing it over the bowl of the pipe. This innovative tool actually serves as a thermal screen between the smoking mixture and the charcoal, whereby the former is only heated by the latter and not burnt as in cigarettes. This has extremely important consequences for the chemical reactions involved (distillation vs. pyrolysis), which completely differ from those occurring in the tobacco rod of a cigarette [START_REF] Chaouachi | The medical consequences of narghile (hookah, shisha) use in the world[END_REF].

According to the authors, "water pipe" smoking is associated with an increased risk of lung cancer. This statement is in contradiction with the results of comprehensive and critical reviews of the related literature over several decades, as described in a recent study [START_REF] Sajid | Hookah smoking and cancer. Carcinoembryonic antigen (CEA) levels in exclusive/ever hookah smokers[END_REF]. Al-Safi et al. cite a publication that shows "a high increase in heart rate and blood pressure and markedly impaired baroreflux sensitivity" [START_REF] Al-Kubati | The short term effect of water-pipe smoking on the baroreflex control of heart rate in normotensives[END_REF]). However, there was an important unnoticed bias in this study, because only 5 g of the smoking product (moassel) was used for a whole 45-min session, whereas the common amount used in most reliable studies of the Middle East, and particularly Jordan, is 20 g [START_REF] Bacha | Saliva cotinine and exhaled carbon monoxide levels in natural environment waterpipe smokers[END_REF][START_REF] Shafagoj | Levels of maximum end expiratory carbon monoxide and certain cardiovascular parameters following hubble-bubble smoking[END_REF][START_REF] Hadidi | Nicotine content in tobacco used in hubble-bubble smoking[END_REF]. Under such unusual conditions, the smoking mixture may char rapidly with a consequent overproduction of CO, carbonaceous material and tar. Interestingly, shisha smoke contains high proportions of water and glycerol. Its tar differs totally from cigarette tar. On the whole, shisha smoke is much less complex than cigarette smoke, as was established early on by [START_REF] El-Aasar | Studies on jurak smoke. I. The organic constitutents of jurak smoke[END_REF]. These researchers identified only 142 compounds in the former, to which others, related to the presence of charcoal, should be added, however. By comparison, cigarette smoke contains about 4,700 chemical compounds.

Al-Safi et al. state that there is a "significant elevation of the mean SBP, DBP, ABP, and heart rate values in shisha smokers compared to non-smokers". This is above all true for mixed cigarette/shisha smokers. The third column of Table 4 is striking in this respect. The increase of arterial blood pressure in "pure"/exclusive shisha smokers from 92.57±13.90 to 92.62±10.58 (NS) (Table 4) may be "significant" in statistical terms, but remains quite modest, as does the increase in heart rate from 76.40±10.46 to 76.81±10.19 (NS). This is certainly in agreement with [START_REF] Shafagoj | Levels of maximum end expiratory carbon monoxide and certain cardiovascular parameters following hubble-bubble smoking[END_REF], who, in their study, found only a light increase of the above parameters.

Finally, it is surprising to see that the publications of the US-Syrian Centre for Tobacco Studies are cited 80 times in a manuscript of only six pages. Furthermore, 64 of these citations concern one chief author, Maziak, who is the director of that centre. In the first two paragraphs, Maziak is cited 18 times, perhaps even 22 times since it appears that " Asfar et al. 2004a, b, c, d" (line 9) apparently refers to the same author.

Competing interests I have no competing interests. I have never received direct or indirect funding either from pharmaceutical companies (nicotine "replacement" therapies and products) or from the tobacco industry.