Xiang Tu

Yonghui Song
email: songyh@craes.org.cn

Zhiyong Tian

Bin Li

Wang Xin

Stephan Brosillon

Marie-Helene Manero

Pilot-scale integrated process for the treatment of dry-spun acrylic fiber manufacturing wastewater

Keywords: Dry-spun acrylic fiber manufacturing wastewater, Pilot scale, Integrated process, Pollutants removal, Excitation-emission matrix spectroscopy

An integrated process of membrane bioreactor (MBR)-advanced ozonation (AO)-biological aerated filters (BAFs) was developed for the treatment of dry-spun acrylic fiber manufacturing wastewater in pilot scale. The results show that the removal efficiencies of chemical oxygen demand (COD) and ammonia nitrogen (NH þ 4 -N) could exceed 90.0 and 95.0%, respectively. The COD concentration in the total effluent was maintained at 80.0-100 mg/L, and the NH þ 4 -N concentration was below 10.0 mg/L. Both the MBR and BAFs could adapt to the influent of practical wastewater after a period of sludge acclimatization. The removal efficiencies of COD and NH þ 4 -N in the MBR were maintained at 65.8-71.9 and 59.4-67.5%, respectively. Excitation-emission matrix spectroscopy confirmed that most of the easy degradable organics were removed in the MBR process. After the AO treatment, COD concentration in the wastewater was reduced by 106-157 mg/L and the biodegradability was enhanced. Simultaneous nitrification-denitrification was performed in the biofilm of BAFs, and the removal efficiency of total nitrogen was in the range of 58.2-71.6% after the entire integrated process.

Introduction

Over the past few decades, the acrylic fiber industry has rapidly progressed in many developing countries. China acquired the technology of dry-spun acrylic fiber (DAF) manufacturing from the American Dubos in the 1990s. The products of this technology are known for their high levels of quality and versatility. However, the effluents of DAF manufacturing contain organic and inorganic contaminants [START_REF] Zhang | Anodic treatment of acrylic fiber manufacturing wastewater with boron-doped diamond electrode: A statistical approach[END_REF]. Some of these pollutants are toxic and biorefractory, which may have serious environmental effects if discharged directly into receiving waters without appropriate treatment.

To improve its biodegradability, the DAF manufacturing wastewater was primarily mixed with domestic wastewater and then subjected to anaerobic, aerobic, and activated carbon (AC) biological treatments. However, highly toxic compounds are recalcitrant towards biodegradation, and high concentrations of inorganic salts (e.g. sulfates and sulfites) decrease the efficiency of anaerobic digestion [START_REF] Li | Organics, sulfates and ammonia removal from acrylic fiber manufacturing wastewater using a combined Fenton-UASB (2 phase)-SBR system[END_REF]. High quantities of polymers and colloids in effluents are also difficult to biodegrade or precipitate naturally. Effluents of biological treatments still contain high concentrations of chemical oxygen demand (COD), ammonia nitrogen (NH þ 4 -N), total nitrogen (TN), and toxic compounds. The biochemical process cannot efficiently remove organic pollutants to meet increasingly strict environmental standards. Thus, hybrid systems are often studied, particularly the combination of powerful ozone degradation, AC catalysis and membrane filtration [START_REF] Khirani | Effect of periodic backwash in the submerged membrane adsorption hybrid system (SMAHS) for wastewater treatment[END_REF], or AC with membrane bioreactor (MBR) [START_REF] Lesage | Study of a hybrid process: Adsorption on activated carbon/membrane bioreactor for the treatment of an industrial wastewater[END_REF] to remove biorefractory organics [START_REF] Merle | Enhanced bio-recalcitrant organics removal by combined adsorption and ozonation[END_REF][START_REF] Reungoat | Oxidation of nitrobenzene by ozone in the presence of faujasite zeolite in a continuous flow gas-liquid-solid reactor[END_REF].

In this study, an integrated process is proposed for the treatment of DAF manufacturing wastewater generated from a petrochemical plant in China. It combines physical, chemical, and biological treatments, which include MBR, advanced ozonation (AO), and biological aerated filters (BAFs). The objective of this integrated process is firstly to remove biodegradable compounds with MBR, and then the ozonation successively decomposes the lower amount of pollutants that are mainly biorefractory. Finally, another biological treatment removes carbon compounds previously transformed into biodegradable compounds. In addition, an improvement of the denitrification is also expected. COD, NH þ 4 -N, and TN removal efficiencies were analyzed to evaluate the pollutant treatment throughout the process. Excitation-emission matrix (EEM) spectroscopy was used to characterize the evolution of organics in the wastewater. To our knowledge, this is the first time that the treatment of real DAF manufacturing wastewater in pilot scale is studied.

Materials and methods

Pilot plant system

A schematic diagram of the pilot system for DAF manufacturing wastewater is presented in Fig. 1. All the pilot reactors were made of stainless steel, and the whole system consisted of three successive units which were MBR, AO, and BAFs, respectively. The DAF manufacturing wastewater was pumped through these three units in sequence and the influent flux of the wastewater during overall operation was 4.8-6.7 m 3 /d.

The effective volume of MBR was 3.0 m 3 , and the available depth was 2.25 m and the hydraulic retention time (HRT) was 32.0 h. The MBR had a compact structure made up of six chambers. Firstly, the wastewater was pumped into the anaerobic chamber, and then flowed through five aerobic chambers in sequence. Hollow-fiber microfiltration membrane modules made of polyvinylidene fluoride were installed at the last aerobic chamber. The membrane had a total surface area of 12.0 m 2 and a nominal pore size of 0.22 μm (Tianjin Motian Membrane Engineering & Technology Co. Ltd, China). Effluent from the MBR was pumped into the AO unit. The sludge separated by the membrane flowed back into the anaerobic chamber at the reflux ratio of 50.0%.

The MBR was operated under the constant flux ranging from 16.7 to 23.3 L/h•m 2 throughout the study. Trans-membrane pressure (TMP) was monitored along operation with values under 15.0 kPa. Nevertheless, in order to prevent and control membrane fouling, once a week, manual cleaning of the membrane was carried out by rinsing fibers with tap water. No further chemical cleaning was needed. The TMP could be restored to below 5.0 kPa after membrane cleaning, the TMP then increased gradually to 12.6-14.2 kPa until the start of the next cleaning cycle.

The AO reactors were composed of an oxidation tower and a retention tank; their effective volumes were 0.9 and 1.0 m 3 , respectively. Ozone was generated from pure dry oxygen using an ozonator (model 3S-OW-40, Tonglin Technology Co. Ltd, China) at concentrations of 50.0-80.0 mg/L. The ozone was fed into the reactor at a flow rate of 480 L/h through a micro-porous plate gas sparger located at the base of the oxidization tower. An ozone trap containing 4.0% potassium iodide solution was connected to the top of the reactors to collect all remaining ozone gas. Six ultrasonic vibrators (60 W each) were fixed in the oxidation tower to make the ozone sufficiently dispersed and dissolved in wastewater, thereby enhancing utilization efficiency of the ozone. Nine ultraviolet (UV) light lamps (120 W each) were set in the retention tank to catalyze the ozonation. The available depth of the retention tank was 2.6 m and the HRT was 6.8-9.5 h.

The BAFs were composed of two tanks in series. Effluent from the AO process was pumped into the bottom of the first tank (tank A) of the BAFs. Tank A was partially aerated and the volume ratio of anoxic/aerated zones was 1:2. The second tank (tank B) was completely aerated and the water flow was also upward. The dimension of each tank was 1.3 × 0.8 × 3.5 m, and the available depth was 3.2 m for each tank. Volcanic granular media with a diameter of 2.0-5.0 mm were packed into each tank with an available depth of 2.0 m, and the HRT for each tank was 10-15 h. The air-towater flow rate ratio was controlled at 5:1, and the BAFs were backwashed every 15 d to remove excess biomass to avoid filter clogging.

Wastewater

The influent of the pilot experiment was the DAF manufacturing wastewater from an acrylic fiber factory in China. The wastewater quality is shown in Table 1. The inoculated sludge for the biological pilot reactors (MBR and BAFs) was excess sludge collected from the wastewater treatment plant in the acrylic fiber factory, and the mixed liquor suspended solid (MLSS) concentration was 3.0 g/L.

Both MBR and BAFs were started separately, and fed with synthetic wastewater for sludge acclimatization and biofilm growth. The composition of synthetic wastewater was listed in Table 2.

Analytical methods

SS, MLSS, COD, BOD 5 , NH þ

4 -N, TN, NO À 3 -N, and NO À 2 -N analyses were conducted according to standard methods [START_REF]APHA, Standard methods for the examination of water and wastewater[END_REF] and pH was measured by a pH meter (Starter 3C, Ohaus, US). TOC was measured by a TOC analyzer (TOC-VCPN 5000A, Shimadzu, Japan). EEM spectroscopy was made using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). Excitation and emission slit widths were set to 5 nm and PMT voltage to 700 V. The excitation wavelength was scanned from 200 to 400 nm and the corresponding emission wavelength was recorded from 260 to 550 nm. The scan speed was set at 1,200 nm/min and the blank scans were performed at intervals of 10 analysis using Milli-Q water.

Results and discussions

Sludge acclimatization in pilot MBR

After sludge inoculation, the MBR was fed with synthetic wastewater to start the experiment. The 4 -N were maintained at 65.8-71.9 and 59.4-67.5%, respectively. Due to the high MLSS concentration in the MBR, micro-organisms were abundant and active, which could tolerate higher toxicity and influent load than some other bioreactors such as sequencing batch reactor (SBR) [START_REF] Lin | Sludge properties and their effects on membrane fouling in submerged anaerobic membrane bioreactors (SAnMBRs)[END_REF][START_REF] Meng | Recent advances in membrane bioreactors (MBRs): Membrane fouling and membrane material[END_REF][START_REF] Chang | Feasibility study of a cyclic anoxic/aerobic two-stage MBR for treating ABS resin manufacturing wastewater[END_REF]. Furthermore, long SRT in the MBR helped to enrich the ammonia oxidizing bacteria that had longer generation time for growth and multiplication than heterotrophic bacteria [START_REF] Espinoza | Analysis and design of a pilot system for the treatment of industrial wastewaters using membrane processes[END_REF]. Thus, effluent NH þ 4 -N concentration could be stabilized in a short time.

Start and operation of pilot BAFs

Pilot BAFs were started based on the same procedure as the pilot MBR. Synthetic wastewater was used as feed for 40 d until the biofilm was formed on the surface of media in the BAFs. After sludge acclimatization in MBR and biofilm formation in BAFs were completed, the entire pilot system operated continuously for the DAF manufacturing wastewater treatment. The effluent of AO mixed with synthetic wastewater was used as feed for the BAFs. During the first 20 d, the proportion of synthetic wastewater in the influent gradually decreased until the biofilm was completely acclimated to the practical wastewater. As shown in Fig. 3, due to the variation of influent during the first 20 d, influent COD concentration decreased from 800 to 270 mg/L, while influent concentration of NH þ 4 -N was maintained in the range of 58.0-68.0 mg/L. Effluent COD concentration remained stable in the range of 65.0-91.0 mg/L after day 20, while effluent NH þ 4 -N concentration increased gradually and fluctuated for 24 d. It then started to decrease and was maintained below 10.0 mg/L after 30 d of operation. Since day 37, white layer of biofilm had abundantly grown on the surface of the media, and the removal efficiencies of COD and NH þ 4 -N had reached 75.3 and 95.5%, respectively. Because the BAFs provided high total porosity and large surface area for microbe inhabitance by the volcanic granular media, abundant biomass could attach to the surface of the media. Thus, the degradation of pollutants was active in the BAFs, which contributed to mitigating the loading shock [START_REF] Qiu | Performances and nitrification properties of biological aerated filters with zeolite, ceramic particle and carbonate media[END_REF]. These results would confirm that the BAFs had a strong adaptability to the influent of practical wastewater.

COD and NH þ

4 -N removals during steady operation Fig. 4 displays the evolutions of COD and ammonia nitrogen concentration when the whole system operated at steady state. During the 60 d of continuous operation, in spite of the fluctuation of water quality in the influent, the removal efficiencies of COD and NH þ 4 -N could exceed 90.0 and 95.0%, respectively. The COD concentration in the total effluent was maintained at 80.0-100 mg/L and the NH þ 4 -N concentration was below 10.0 mg/L. The BOD 5 /COD ratio decreased from 0.3 to 0.1 after the MBR process, which indicated that the MBR had removed most of biodegradable organics from the DAF manufacturing wastewater, leaving the biorefractory substances transmitted into the AO unit. The COD concentration decreased by 106-157 mg/L after the AO process, whereas the BOD 5 /COD ratio increased from 0.1 to 0.6, which demonstrated that the biodegradability of wastewater had been enhanced after the AO treatment.

The MBR, one of the biological processes used in variety of wastewater treatments, was adopted in this study as pretreatment process prior to the AO unit because of its economic advantages and strong resistance to impact load [START_REF] Hoinkis | Membrane bioreactor (MBR) technology-A promising approach for industrial water reuse[END_REF]. The DAF manufacturing wastewater still contained some biodegradable substrates, which could be utilized by micro-organisms during the MBR process. Thus, the MBR would reduce the ozone dosage for pollutants oxidation and cut down some running costs in the next AO unit.

Ozone is known as one of the most powerful oxidizing agents with a standard redox potential of 1.24 V in alkaline solutions [START_REF] Barriga-Ordonez | Cyanide oxidation by ozone in a steady-state flow bubble column[END_REF]. In this study, the UV light catalyzed the ozone to form hydroxyl radicals that oxidized organic compounds at higher redox potential and reaction rate than ozone itself [START_REF] Shu | Degradation of commercial azo dyes in water using ozonation and UV enhanced ozonation process[END_REF]. In addition, the ultrasonic made the ozone efficiently contact and react with target contaminants by enhancing the mass-transfer efficiency of the ozone between gas-liquid phases. Some organic contaminants were mineralized into carbon dioxide and water after the AO process, while the others were dissociated into small molecules, which therefore reduced the toxicity and improved wastewater biodegradability.

The NH þ 4 -N concentration did not decrease as expected as it is well known that ozone does not react on ammonia. On the contrary, the effluent NH þ 4 -N concentration of the AO process was even higher than that of the influent (Fig. 4). In addition, the pH value also decreased from 7.1 to 6.3 after the AO process. These results indicate that some of the amino groups were broken away from the organics and thus made the NH þ 4 -N concentration increased [START_REF] Wang | Advanced oxidation processes for wastewater treatment: Formation of hydroxyl radical and application[END_REF][START_REF] Wert | Evaluation of enhanced coagulation pretreatment to improve ozone oxidation efficiency in wastewater[END_REF][START_REF] Kidak | Catalysis of advanced oxidation reactions by ultrasound: A case study with phenol[END_REF]. Meanwhile, some organic acids with small molecules might be formed after the AO treatment, which further contributed to the decrease in pH value.

The effluent COD concentration in tank A of the BAFs was in the range of 140-190 mg/L, and higher quantity of COD decreased in tank A than in tank B (Fig. 4). This is because that large number of organics were preferentially utilized as carbon source for the denitrification in the anoxic zone of tank A, which therefore decreased the COD concentration. Considering the evolution of NH þ 4 -N concentration during the entire process, most of the ammonia nitrogen was removed in the MBR process and tank B of the BAFs. The effluent NH þ 4 -N concentration in tank A was in the range of 31.1-42.8 mg/L and the removal efficiency of NH þ 4 -N in tank B was 68.8-88.4%. During the BAFs process, tank A preferentially degraded the residual organics derived from the AO process, leaving the ammonia nitrogen flowing into tank B. mg/L of TN, in which the ammonia nitrogen was also removed. Table 3 shows the variation of NO À 2 -N and NO À 3 -N concentrations throughout the entire process when the system was in steady state. NO À 2 -N concentration was stable in the range of 2.6-5.5 mg/L during the entire operation, and NO À 3 -N concentration obviously increased to 52.1-60.7 mg/L after the MBR process, which was due to the effect of nitrification. Thereafter, the NO À 3 -N concentration constantly decreased to 20.5-25.1 mg/L after the BAFs. Moreover, tank B decreased the NO À 3 -N concentration by 14.7-19.8 mg/L, which was consistent with the decrease of TN concentration. This condition was probably caused by the partial denitrification that occurred in the internal part of the biofilm due to the lack of oxygen, thus it can be assumed that simultaneous nitrification-denitrification was performed in the biofilm of BAFs [START_REF] Qi | Coupling of biological methods with membrane filtration using ozone as pre-treatment for water reuse[END_REF].

TN removal during steady operation

EEM analysis of the entire system

EEM spectra have been widely used for the characterization of organic compounds in water or wastewater [START_REF] Zhao | Transformation characteristics of refractory pollutants in plugboard wastewater by an optimal electrocoagulation and electro-Fenton process[END_REF][START_REF] Chen | Fluorescence excitation-emission matrix regional integration to quantify spectra for dissolved organic matter[END_REF][START_REF] Bosco | Simultaneous analysis of the photocatalytic degradation of polycyclic aromatic hydrocarbons using three-dimensional excitation-emission matrix fluorescence and parallel factor analysis[END_REF]. In this study, when the pilot system was in stable operation, water samples were collected from the three treatment units for EEM analysis.

As shown in Fig. 6(a), two fluorescence peaks (B 1 and T 1 ) were observed in the EEM spectra of the raw wastewater. Peak B 1 was located at the Ex/Em wavelengths of 295 /335 nm and was related to protein-like substances in the raw wastewater. Generally, this peak was induced by biodegradable substances that might be monocyclic or bicyclic aromatics, such as benzene, phenol, ethyl, benzene, and xylene [START_REF] Bosco | Simultaneous analysis of the photocatalytic degradation of polycyclic aromatic hydrocarbons using three-dimensional excitation-emission matrix fluorescence and parallel factor analysis[END_REF]. In addition, peak T 1 was detected at the Ex/Em of 330 /405 nm. This peak was due to fulvic-like compounds which probably contained three to five aromatic rings, such as phenanthrene, pyrene, and furan [START_REF] Jiji | Excitation-emission matrix fluorescence based determination of carbamate pesticides and polycyclic aromatic hydrocarbons[END_REF]. In the effluent of MBR (Fig. 6(b)), peak B 1 completely disappeared which indicated that monocyclic or bicyclic aromatic compounds were fully decomposed by the bioreaction of MBR. However, a new fulvic-like peak (T 2 ) was formed in the MBR effluent. This peak was probably related to the microbial by-products in the MBR [START_REF] Ni | Microbial products of activated sludge in biological wastewater treatment systems: A critical review[END_REF]. The intensity of peaks T 1 and T 2 significantly decreased after AO treatment (Fig. 6(c)), confirming the efficiency of this process to remove these biorefractory compounds. After the BAF process, the intensity of these peaks further decreased (Fig. 6(d)), but in lower proportions.

These results indicate that most of the fluorescence compounds with complex structures could be decomposed and transformed during the ozonation process. Furthermore, peaks T 1 and T 2 were red shifted in both excitation and emission wavelengths after the ozonation. From this result, it can be concluded that the complex aromatics were oxidized by the hydroxyl radical generated from the ozone, and the carbonyl and carboxyl groups were formed in the aromatic structure [START_REF] He | PAEs and BPA removal in landfill leachate with Fenton process and its relationship with leachate DOM composition[END_REF][START_REF] Wu | The oxidation study of 2-propanol using ozone-based advanced oxidation processes[END_REF].

Conclusions

The real wastewater from DAF manufacturing was treated by an integrated process (MBR, AO, and BAFs) in pilot scale. Both of the MBR and BAFs could adapt to the influent of practical wastewater after a period of sludge acclimatization, and this integrated process had high COD and NH þ 4 -N removal efficiencies. During the steady operation, the removal efficiencies of COD and NH þ 4 -N could reach above 90.0 and 95.0%, respectively. The COD concentration in the effluent was maintained at 80.0-100 mg/L and NH þ 4 -N concentration was below 10.0 mg/L. Most of the easy degradable organics were removed by the MBR; complexstructured organics were greatly decomposed and/or transformed into more biodegradable compounds by the AO treatment. Most of the NH þ 4 -N and TN were removed during the MBR and BAFs. Simultaneous nitrification-denitrification was performed in the biofilm of BAFs, and the TN removal efficiency reached 58.2-71.6% after the entire treatment.

Fig. 1 .

 1 Fig. 1. Schematic diagram of the pilot system.

  FeSO 4 •2H 2 O MgSO 4 •7H 2 O NaCl Peptone Concentration (mg/L) 550COD concentration was 600-800 mg/L, and NH þ 4 -N concentration was 60.0-70.0 mg/L. After 35 d of continuous operation, the effluent COD and NH þ 4 -N concentrations were maintained below 50.0 and 1.0 mg/L, respectively. The MLSS concentration increased to 8.0 g/L with the sludge retention time maintained at 50 d. Thereafter, the MBR was fed with the mixture of DAF manufacturing wastewater and synthetic wastewater for sludge acclimatization. During the first 15 d, the proportion of synthetic wastewater gradually decreased until the influent was completely DAF manufacturing wastewater. The evolutions of COD and NH þ 4 -N concentrations during sludge acclimatization are described in Fig. 2. Influent concentrations of COD and NH þ 4 -N increased until day 15 and remained nearly constant from days 16 to 28. Effluent COD concentration increased gradually even after day 15, and reached 330 mg/L on day 21. Thereafter, the effluent COD concentration was kept almost constant at 310-400 mg/L. The NH þ 4 -N concentration in the effluent increased to 103.6 mg/L on day 14, which was almost as high as the concentration of the influent. It then decreased gradually and became stable in the range of 40.7-52.0 mg/L after day 24. The effluent COD and NH þ 4 -N concentrations became stable since day 25 when sludge acclimatiza-tion in the MBR was fully completed. The removal efficiencies of COD and NH þ

Fig. 2 .

 2 Fig. 2. Evolutions of COD and ammonia nitrogen removals during sludge acclimatization in MBR.

Fig. 3 .

 3 Fig. 3. Evolutions of COD and ammonia nitrogen removals during sludge acclimatization in BAFs.

Fig. 4 .

 4 Fig. 4. Evolutions of COD and ammonia nitrogen concentrations during steady operation of the whole system.

Fig. 5

 5 Fig. 5 shows the evolution of TN concentration during the steady operation of the entire system. It can be observed that the TN concentration in the total effluent was maintained in the range of 70.0-110 mg/ L, corresponding to 58.2-71.6% in TN removal efficiency. The MBR could remove 19.8-31.3% of TN concentration from the influent, and the AO had no effect on TN removal. Tank A in the BAFs could decrease the TN concentration by 40.0-79.0 mg/L, possibly due to the denitrification process in the anoxic zone of tank A. However, tank B could also remove 23.7-59.4

Fig. 5 .

 5 Fig. 5. Evolution of TN concentration during steady operation of the whole system.

Fig. 6 .

 6 Fig. 6. EEM spectra of the samples collected after the different unit operations. (a) Raw wastewater; (b) after MBR; (c) after AO; and (d) after BAFs.

Table 1 Wastewater

 1

	1,000-1,200	350-400	300-350	120-150	250-290	≤80	7.0-7.5	30-35
	Table 2
	Compositions of synthetic wastewater
	Compounds	C 6 H 12 O 6 (NH 4 ) 2 SO 4 KH 2 PO 4 NaHCO 3

quality of the influent COD (mg/L) BOD 5 (mg/L) TOC (mg/L) NH þ 4 -N (mg/L) TN (mg/L) SS (mg/L) pH T (˚C)

Table 3 NO

 3

Acknowledgements This work was financially supported by the National Key Scientific and Technological Project for Water Pollution Control and Management (2008ZX07208-003 & 2012ZX07202-002) and the Sino-French International Scientific and Technological Cooperation Project for Petrochemical & Pharmaceutical Industrial Water Conservation and Reduction Technical Cooperative Research (2010DFB90590).


Claudio A González-Wevar

Mathias Hüne

Sebastián Rosenfeld

Thomas Saucède

Jean-Pierre Féral

Andrés Mansilla

Elie Poulin

Patterns of genetic diversity and structure in Antarctic and sub-Antarctic Nacella (Patellogastropoda: Nacellidae) species

Keywords: southern ocean, biogeography, phylogeography, Antarctic Circumpolar Current, Last Glacial Maximum, Nacella

The biogeography of the Southern Ocean reflects complex interactions between major macro-evolutionary forces and biotic elements. Major gateway openings, the establishment of the Antarctic Circumpolar Current and climate cooling are deeply connected to the composition, abundance and distribution of the Southern Ocean marine benthic fauna. Glacial episodes of the Quaternary heavily impacted the distribution of the genetic variation of the Southern Ocean biota. The genus Nacella includes 12 nominal species in different provinces of the Southern Ocean. In this study, we compared patterns of mitochondrial DNA diversity in three Nacella species from Antarctic Peninsula, Kerguelen Island and Patagonia. Low levels of genetic diversity and absence of genetic structure characterise each one of them showing the strong impact of ice advances and retreats over their respective demographics. Haplotype diversity, short genealogies and demographic inference recorded suggest the occurrence of a more dramatic demographic process in Antarctic Peninsula than in the sub-Antarctic.

Introduction

The Southern Ocean encircling the Antarctic continent covers an area of approximately 35 million km 2 , represents 8% of the world's ocean surface and is considered as a major driver of global oceanic circulation [START_REF] Rintoul | The Antarctic Circumpolar Current System[END_REF]. This vast region harbours approximately a 5% of the marine diversity [START_REF] Barnes | Geographic Range Shift Response to Climate Change by Antarctic Benthos: Where to Look[END_REF][START_REF] Barnes | Vulnerability of Antarctic Shelf Biodiversity to Predicted Regional Warming[END_REF]. The distribution of the marine benthic biota in Antarctic and sub-Antarctic provinces largely reflects the interaction of tectonics, climate, oceanography and biotic elements at different temporal and spatial scales [START_REF] Aronson | Climate Change and Invasibility of the Antarctic Benthos[END_REF][START_REF] Crame | An Evolutionary Perspective on Marine Faunal Connection between Southernmost South America and Antarctica[END_REF][START_REF] Griffiths | Towards a Generalized Biogeography of the Southern Ocean Benthos[END_REF][START_REF] Pierrat | Comparative Biogeography of Echinoids, Bivalves and Gastropods from the Southern Ocean[END_REF]). According to Mackensen (2004) three main periods dramatically affected the biogeography of the Southern Ocean. First, the Eocene/Oligocene boundary (~34 Ma) marks the onset of the Antarctic isolation by the formation of major gateway openings and the initiation of the Antarctic Circumpolar Current (ACC; [START_REF] Barker | 143-162. of the Genus Nacella (Patellogastropoda) in the South-Western Atlantic Coast[END_REF]Barker et al. 2007;[START_REF] Pfuhl | Evidence for Late Oligocene Establishment of the Antarctic Circumpolar Current[END_REF][START_REF] Scher | Onset of Antarctic Circumpolar Current 30 Million Years Ago as Tasmanian Gateway Aligned with Westerlies[END_REF][START_REF] Zachos | Trends, Rhythms, and Aberrations in Global Climate 65 Ma to Present[END_REF]. This current represents the major oceanographic system of the planet delimited by two boundaries, the Antarctic Polar Front (APF) and the sub-Antarctic Front (SAF; Barker et al. 2007;[START_REF] Rintoul | The Antarctic Circumpolar Current System[END_REF]. The positions of these fronts have major biogeographical implications for the Southern Ocean biota [START_REF] González-Wevar | Molecular Phylogeny and Historical Biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]2012a;[START_REF] Griffiths | Towards a Generalized Biogeography of the Southern Ocean Benthos[END_REF][START_REF] Pierrat | Comparative Biogeography of Echinoids, Bivalves and Gastropods from the Southern Ocean[END_REF][START_REF] Poulin | Divergence between Antarctic and South American Marine Invertebrates: What Molecular Biology Tells Us about Scotia Arc Geodynamics and the Intensification of the Antarctic Circumpolar Current[END_REF][START_REF] Strugnell | The Thermohaline Expressway[END_REF]). The ACC acts as an effective barrier for many invertebrate taxa, especially between Antarctic and sub-Antarctic provinces [START_REF] Barnes | Incursion and Excursion of Antarctic Biota: Past, Present and Future[END_REF][START_REF] Clarke | How Isolated is Antarctica?[END_REF]González-Wevar et al. 2012a;[START_REF] Poulin | Divergence between Antarctic and South American Marine Invertebrates: What Molecular Biology Tells Us about Scotia Arc Geodynamics and the Intensification of the Antarctic Circumpolar Current[END_REF][START_REF] Shaw | Genetic Structuring of Patagonian Toothfish Populations in the Southwest Atlantic Ocean: The Effect of the Antarctic Polar Front and Deep-Water Troughs as Barriers to Genetic Exchange[END_REF]. In contrast, across the sub-Antarctic the ACC acts as an important carrier of organisms between geographically distant provinces, especially for those species currently distributed in ice-free rocky areas along maritime Antarctica and some peri-Antarctic islands inside the APF (South Georgia, South Sandwich and Bouvet). South America harbours the greatest diversity for the genus with nine nominal species described, eight of them found along the Patagonian coast [START_REF] Powell | The Patellid Limpets of the World (Patellidae)[END_REF][START_REF] Valdovinos | Nacellidae Limpets of Southern South America: Taxonomy and Distribution[END_REF]. Nevertheless, recent phylogenetic reconstructions and divergence time estimations based on mitochondrial DNA (mtDNA) markers indicate that this Patagonian radiation of the genus represents the most derived one [START_REF] González-Wevar | Molecular Phylogeny and Historical Biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]) and only four of the nominal species (N. magellanica, N. deaurata, N. mytilina and N. flammea) constitute significant evolutionary units (SEUs; [START_REF] González-Wevar | Concerted Genetic, Morphological and Ecological Diversification in Nacella Limpets in the Magellanic Province[END_REF]). Among them, N. magellanica exhibits the broadest distribution from Chiloé Island in the Pacific to Río Negro Province in the Atlantic including the Patagonian fjords, the Strait of Magellan, Tierra del Fuego, Cape Horn and the Falkland/Malvinas Islands [START_REF] Powell | The Patellid Limpets of the World (Patellidae)[END_REF][START_REF] Valdovinos | Nacellidae Limpets of Southern South America: Taxonomy and Distribution[END_REF].

One of the main purposes of this study was to evaluate the levels of molecular divergence among congeneric broadcaster-spawners Nacella species from Kerguelen Island (N. edgari), maritime Antarctica (N. concinna) and Patagonia (N. magellanica). The information contained in their DNA sequences will permit us to determine the rhythms and trends in the biogeography of this important marine benthic group. Secondly, we estimated patterns of genetic diversity and structure in each one of the analysed species/provinces in order to further understand the impact of the Quaternary glacial processes along a latitudinal gradient.

Methods

Nacella species were identified following [START_REF] Powell | The Patellid Limpets of the World (Patellidae)[END_REF] and [START_REF] Valdovinos | Nacellidae Limpets of Southern South America: Taxonomy and Distribution[END_REF]. Intertidal and subtidal individuals of N. edgari were sampled at different localities of Kerguelen (Table 1) and were included alongside previously published data on N. magellanica (González-Wevar et al. 2012b) andN. concinna (González-Wevar et al. 2013). For comparison purposes, we included in the analyses a similar number of individuals per locality/ area. All the specimens were fixed in ethanol (95%) and DNA was extracted from the mantle and the pedal muscle using a salting-out method [START_REF] Aljanabi | Universal and Rapid Salt-Extraction of High Quality Genomic DNA for PCR-Based Techniques[END_REF]. We amplified a partial fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene using universal [START_REF] Folmer | DNA Primers for Amplification of Mitochondrial Cytochrome C Oxidase Subunit I from Diverse Metazoan Invertebrates[END_REF]) and specific [START_REF] González-Wevar | Molecular Phylogeny and Historical Biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF] primers. Polymerase chain reaction (PCR) procedures were prepared in a 25 μl reaction containing 2.5 μl 10X Buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.0), 1.0 μl of 50 mM MgCl 2 , 200 mM dNTPs, 0.5 μl of each primer (10 pg/μl), 1 U Taq (Invitrogen), 17.5 μl of double-distilled water and 50 ng of DNA. Thermal cycling parameters with high dispersive potential [START_REF] Díaz | Evolutionary Pathways among Shallow and Deep-Sea Echinoids of the Genus Sterechinus in the Southern Ocean[END_REF][START_REF] Fraser | Kelp Genes Reveal Effects of Subantarctic Sea during the Last Glacial Maximum[END_REF][START_REF] Waters | Driven by the West Wind Drift? A Synthesis of Southern Temperate Marine Biogeography, with New Directions for Dispersalism[END_REF]) and to kelp-dwelling organisms [START_REF] Cumming | Transoceanic Genetic Similarities of Kelp-Associated Sea Slug Populations: Long-Distance Dispersal via Rafting?[END_REF][START_REF] Fraser | Oceanic Rafting by a Coastal Community[END_REF][START_REF] Nikula | Circumpolar Dispersal by Rafting in Two Subantarctic Kelp-Dwelling Crustaceans[END_REF]. Second, the middle Miocene (~14 Ma) associated to an increased southern bottom formation, the intensification of the ACC and the re-establishment of a permanent ice sheet on East Antarctic and along the Pacific margin of West Antarctica [START_REF] Lawver | Evolution of Cenozoic Seaways in the Circum-Antarctic Region[END_REF][START_REF] Verducci | The Middle Miocene Climatic Transition in the Southern Ocean: Evidence of Paleoclimatic and Hydrographic Changes at Kerguelen Plateau from Planktonic Foraminifers and Stable Isotopes[END_REF]. Recent molecular studies between Antarctic and sub-Antarctic congeneric species in different marine invertebrates groups (González-Wevar et al. 2012a;[START_REF] Poulin | Divergence between Antarctic and South American Marine Invertebrates: What Molecular Biology Tells Us about Scotia Arc Geodynamics and the Intensification of the Antarctic Circumpolar Current[END_REF] indicate that their respective separations occurred long after the physical separation of both continents. Molecular divergence between Antarctic and South American lineages seems to be related to important changes in the Southern Ocean including the evolution of the Scotia Arc and a late deepening of the Drake Passage until the middle Miocene [START_REF] Dalziel | A Potential Barrier to Deep Antarctic Circumpolar Flow until the Late Miocene?[END_REF]). Together with these tectonic and oceanographic changes, climate shift has exerted a major role in the Southern Ocean, shaping the abundance, structure and distribution of its biota [START_REF] Aronson | Climate Change and Invasibility of the Antarctic Benthos[END_REF]Clarke andCrame 1989, 2010). Finally, the Quaternary is characterised by the alternation between glacial and interglacial episodes that greatly affected seasonality and intensity of sea-ice formation, as well as the position of the ACC [START_REF] Gersonde | Sea-Surface Temperature and Sea Ice Distribution of the Southern Ocean at the EPILOG Last Glacial Maximum -A Circum-Antarctic View Based on Siliceous Microfossil Records[END_REF][START_REF] Kemp | Migration of the Antarctic Polar Front through the mid-Pleistocene Transition: Evidence and Climatic Implications[END_REF]. Furthermore, the development of large ice sheets during glacial periods exerted major changes in the spatial distribution and demography of populations, species and communities, especially at higher latitudes [START_REF] Hewitt | The Genetic Legacy of the Quaternary Ice Ages[END_REF][START_REF] Provan | Phylogeographic Insights into Cryptic Glacial Refugia[END_REF]. For instance, ice advances and retreats radically altered the geographical range of the species and populations sizes and therefore deeply affected the distribution of intraspecific genetic variation [START_REF] Allcock | Southern Ocean Diversity: New Paradigms from Molecular Ecology[END_REF][START_REF] Maggs | Evaluating Signatures of Glacial Refugia for North Atlantic Benthic Marine Taxa[END_REF][START_REF] Provan | Phylogeographic Insights into Cryptic Glacial Refugia[END_REF]. Near-shore marine benthic communities in the Southern Ocean, and particularly those from Antarctica, would have been especially vulnerable to grounded ice sheets [START_REF] Dambach | Effects of Late-Cenozoic Glaciation on Habitat Availability in Antarctic Benthic Shrimp (Crustacea: Decapoda: Caridea)[END_REF][START_REF] Thatje | On the Origin of Antarctic Marine Benthic Community Structure[END_REF]. Therefore, it is expected a direct relationship between latitude and the impact of continental-and sea-ice formation over the demography of populations and species.

True limpets of the genus Nacella includes at least 11 nominal species distributed in Antarctic and sub-Antarctic provinces where they are dominant shallow macrobenthic invertebrates of inter-and sub-tidal rocky ecosystems [START_REF] González-Wevar | Concerted Genetic, Morphological and Ecological Diversification in Nacella Limpets in the Magellanic Province[END_REF][START_REF] Powell | The Patellid Limpets of the World (Patellidae)[END_REF][START_REF] Valdovinos | Nacellidae Limpets of Southern South America: Taxonomy and Distribution[END_REF]. Almost half of the species of Nacella are endemic to particular isolated oceanic sub-Antarctic islands. For instance, N. delesserti, N. edgari and N. terroris are found at Marion, Kerguelen and Campbell islands, respectively. The Antarctic limpet, N. concinna, is included an initial denaturation step at 94 °C for 5 min, followed by 35 cycles at 94 °C for 1 min, 48 °C for 1 min and 72 °C for 1 min, and a final extension at 72 °C for 5 min. PCR amplification products were purified using QIAquick Gel Extraction Kit (QIAGEN) and sequenced in both directions at Macrogen Inc (Seoul, Korea). Forward and reverse sequences of each gene were assembled independently and edited using Proseq v.3.5 [START_REF] Filatov | Processing and Population Genetic Analysis of Multigenic Datasets with ProSeq3 Software[END_REF]) and aligned with Muscle [START_REF] Edgar | MUSCLE: A Multiple Sequence Alignment Method with Reduced Time and Space Complexity[END_REF]). Sequences were translated to amino acids to check for the presence of pseudogenes and/or sequencing errors in MEGA v6.0 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. Similarly, MEGA was used to determine base composition and we performed a DNA saturation analysis following [START_REF] Roe | Patterns of Evolution of Mitochondrial Cytochrome C Oxidase I and II DNA and Implications for DNA Barcoding[END_REF] to evaluate how saturation of transitions are accumulated in relation to nucleotide divergence in the complete COI data-set.

We estimated the average number of nucleotide substitutions and calculated the pairwise percent of divergence (uncorrected p-distances) among Nacella lineages from different provinces of the Southern Ocean. We determined whether the evolutionary rate was constant in the analysed taxa using a likelihood ratio test [START_REF] Felsenstein | Evolutionary Trees from DNA Sequences: A Maximum Likelihood Approach[END_REF] in DAMBE [START_REF] Xia | DAMBE: Software Package for Data Analysis in Molecular Biology and Evolution[END_REF]. Once the constancy of the evolutionary rate was confirmed we performed divergence time estimations among the analysed lineages using a strict molecular clock hypothesis (MCH) and specific evolutionary rates [START_REF] González-Wevar | Molecular Phylogeny and Historical Biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]. This hypothesis assumes that DNA and protein sequences evolve at a constant rate through time and therefore the genetic divergence between any two species is proportional to the time since they last shared a common ancestor [START_REF] Bromham | The Modern Molecular Clock[END_REF]. We constructed maximum parsimony genealogical relationships among Nacella lineages using Hapview. This methodology allows a simple reconstruction of phylogenies based on intraspecific genetic data [START_REF] Posada | Intraspecific Gene Genealogies: Trees Grafting into Networks[END_REF].

Levels of polymorphism were calculated using standard mtDNA genetic diversity indices including the number of haplotypes (k), the number of segregating sites (S), haplotype diversity (H), the average number of pairwise differences (Π) and nucleotide diversity (π) at each locality and for each of the analysed species using DnaSP v.5.00.07 [START_REF] Librado | DnaSP V5: A Software for Comprehensive Analysis of DNA Polymorphism Data[END_REF]. We performed neutrality statistical tests (Tajima's D and Fu's F S ) to estimate if COI sequences in deviate from expectations under a neutral model. We determined the level of genetic differentiation among localities for each of the analysed species based on haplotype frequencies (G ST ) and mean pairwise differences (N ST ) following [START_REF] Pons | Measuring and Testing Genetic Differentiation with Ordered versus Unordered Alleles[END_REF] in Arlequin [START_REF] Excoffier | Arlequin (Version 3.0): an Integrated Software Package for Population Genetics Data Analysis[END_REF]. We test the statistical significance of the genetic differentiation using permutation tests (20,000 iterations) of haplotype identities. We inferred the spatial genetic structure for each species by estimating the number and composition of groups that were most differentiated based on sequence data-set with an analysis of molecular variance (AMOVA) in Arlequin. AMOVA allows using multiple spatial scales in statistical methods to characterise the spatial genetic structure by partitioning it into: within populations, among populations within groups and among groups.

We constructed genealogical relationships in the different species of Nacella using maximum parsimony networks in Hapview (http://www.cibiv.at). Finally, we compared the patterns of demographic history in the analysed Nacella species using the distribution of pairwise differences between haplotypes (mismatch distribution) analysed species of Nacella showed low levels of genetic diversity and absence of structure along their respective distributions in Antarctic and sub-Antarctic areas.

Patterns of genetic diversity among the analysed species of Nacella showed higher levels of polymorphism in species from the sub-Antarctic (South America and Kerguelen Island) than in the Antarctic limpet N. concinna (Table 1).

In fact, all the analysed indices detected higher levels of diversity in N. edgari (Kerguelen Island) and N. magellanica (Patagonia), compared to N. concinna from maritime Antarctica. Pairwise N ST and G ST comparisons within each of the analysed Nacella species detected an absence of genetic structure in each of the analysed species.

Haplotype networks (Figure 1(a) and 1(b)) showed similar patterns in the different species of Nacella with typical star-like topologies and short genealogies. However, as previously estimated through standard diversity indices, the number of haplotypes was considerably lower in the Antarctic limpet N. concinna than in their sub-Antarctic relatives from Patagonia (N. magellanica) and Kerguelen Island (N. edgari). The maximum parsimony haplotype network in N. concinna included 12 haplotypes with a central haplotype (H1; Figure 2(b)) distributed at all localities an present in more than 58.42% of the individuals. A second haplotype was also broadly distributed along N. concinna distribution and present in 16.85% of the individuals. All the haplotypes in the Antarctic limpet are related to the dominant one through a branch length of one or two mutational steps (Figure 2(b)). The maximum parsimony haplotype network in sub-Antarctic Patagonian N. magellanica included a total of 25 different haplotypes. The dominant haplotype (H1; Figure 2(c)) was present in 51.54% of the individuals and was recorded along the species' distribution in the Pacific coast of Patagonia. A second haplotype (H2; Figure 2(c)) of medium frequency (14.43%) was also found in all N. magellanica populations. All the haplotypes in N. magellanica are related to the dominant one (H1; Figure 2(c)) through a branch length of no more than three mutational steps. Finally, the maximum parsimony haplotype network in sub-Antarctic N. edgari from Kerguelen Island included a total of 21 different haplotypes. In this species, a dominant haplotype (H1; Figure 2(d)) was present in 45.54% of the individuals and was found in all the analysed populations. Two haplotypes (H2 and H3; Figure 2(d)) of medium frequencies (15.84% and 13.86%, respectively) were recorded in all the analysed N. edgari populations. All the haplotypes in N. edgari are related to the dominant one (H1) through a branch length of no more than six mutational steps (Figure 2(d)). As expected for star-like topologies, general Tajima's and Fu's neutrality tests were both negative and significant for the whole COI data-set of each of the analysed and by comparing it to the expected distribution under the sudden expansion growth model [START_REF] Rogers | Population Growth Makes Waves[END_REF]. This is a common method in demographic inference analyses considering that the amount of nucleotide differences between haplotypes depends on the length of time since they diverged.

Results

COI sequences in the analysed Nacella species were 658 bp long and, as expected working with coding sequences, no indels or stop codons were detected when comparing the alignment to the translated open reading frame. Sequences were not saturated at any position but we recognised a high degree of genetic polymorphism among the analysed Nacella species. 100 positions were variable (15.19%) of which 85 (85%) were parsimoniously informative. We recognise two amino acid changes (positions 106 and 157), one transition (from A to G in site 157) and one transversion (from T to G in site 106). As previously estimated in Nacella species, sequences were A-T rich (60.4%) compared with the mean G-C content.

As previously estimated in the genus, main patterns of genetic divergence among Nacella lineages from maritime Antarctica, Patagonia and sub-Antarctic Kerguelen Island indicate a marked distinction among them (Figure 2(a)). By analysing these results under a phylogenetic framework [START_REF] González-Wevar | Out of Antarctica: Quaternary Colonization of Sub-Antarctic Marion Island by the Limpet Genus Nacella (Patellogastropoda: Nacellidae)[END_REF]), an early separation of Nacella into two main clades is detected. The first one includes lineages from South America and the second one those species from maritime Antarctica and from sub-Antarctic islands. The molecular divergences between Antarctic N. concinna and its sub-Antarctic relatives N. edgari and N. magellanica were 6.9% and 8.3%, respectively. The divergence between sub-Antarctic South American N. magellanica and sub-Antarctic Kerguelen Island N. edgari was 8.0%. Considering a specific substitution rate for nacellids [START_REF] González-Wevar | Concerted Genetic, Morphological and Ecological Diversification in Nacella Limpets in the Magellanic Province[END_REF], the divergence among the analysed lineages of Nacella ranged between 3.5 Ma and 4.2 Ma. The last contact between N. concinna from maritime Antarctica and N. edgari from Kerguelen Island occurred 3.5 Ma. The last contact between N. concinna from maritime Antarctica and N. magellanica from Patagonia occurred 4.15 Ma. Finally, the last contact between N. magellanica from Patagonia and N. edgari from Kerguelen Island occurred 4.0 Ma.

After this Pliocene diversification, each one of the analysed species/lineages of Nacella followed different evolutionary pathways in distinct provinces of the Southern Ocean (South America, maritime Antarctica and Kerguelen Island). Nevertheless, short-term phylogeographic analyses recognised similar demographic signals in the analysed species of Nacella. In general terms, all the Antarctica, South America and Kerguelen Island. In this study, each one of the analysed species, maritime Antarctic N. concinna, South American N. magellanica and Kerguelen Island N. edgari, constitutes a different taxonomic units that have been separated for several millions of years. Divergence time estimations based on a specific substitution rate for nacellids indicate an effective separation among the analysed lineages of Nacella during the Pliocene between 3.5 and 4.2 Ma. Accordingly, the ACC constitutes an effective barrier for the analysed Nacella species between maritime Antarctic and the two sub-Antarctic lineages (southern South America and Kerguelen Island), respectively.

In contrast to the evidence of genetic identities recorded between geographically distant sub-Antarctic populations for mytiliids [START_REF] Gérard | Pleistocene Separation of Mitochondrial Lineages of Mytilus Spp. Mussels from Northern and Southern Hemispheres and Strong Genetic Differentiation among Southern Populations[END_REF], crustaceans [START_REF] Nikula | Circumpolar Dispersal by Rafting in Two Subantarctic Kelp-Dwelling Crustaceans[END_REF]) echinoids [START_REF] Díaz | Evolutionary Pathways among Shallow and Deep-Sea Echinoids of the Genus Sterechinus in the Southern Ocean[END_REF]) and pulmonate gastropods [START_REF] Cumming | Transoceanic Genetic Similarities of Kelp-Associated Sea Slug Populations: Long-Distance Dispersal via Rafting?[END_REF], geographical distance and oceanographical patterns in the region seem to represent an unbearable barrier for connectivity in Nacella, particularly between South America and sub-Antarctic islands like Marion (González-Wevar et al. in press) and Kerguelen Island [START_REF] González-Wevar | Molecular Phylogeny and Historical Biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]this study). Similarly, a recent molecular study in the sea spider Austropallene cornigera also recognised clear evidence of genetic differentiation between Antarctic continental shelf and peri-Antarctic Bouvet Island populations suggesting a limited pattern of gene flow between these areas [START_REF] Dömel | Genetic Data Support Independent Glacial Refugia and Open Ocean Barriers to Dispersal for the Southern Ocean Sea Spider Austropallene Cornigera (Möbius, 1912)[END_REF].

Nacella species (Table 1). Similarly, the distributions of pairwise differences between sequences in each of the analysed Nacella species were L-shaped for N. concinna and unimodal for N. edgari and N. magellanica due to the fact that the majority of the individuals shared the same haplotype. Considering a specific substitution rate for nacellids, demographic expansion would have started around 5500 years ago for N. concinna, around 7500 years ago for N. magellanica and around 4000 years ago for N. edgari, assuming the sudden growth model [START_REF] Rogers | Population Growth Makes Waves[END_REF].

Discussion

The emergence of new methodological approaches in molecular biology has renewed the interest on biogeographical studies, particularly in the Southern Ocean biota [START_REF] Downey | Diversity and Distribution Patterns in High Southern Latitude Sponges[END_REF][START_REF] Fraser | Kelp Genes Reveal Effects of Subantarctic Sea during the Last Glacial Maximum[END_REF][START_REF] González-Wevar | Molecular Phylogeny and Historical Biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]2012a;[START_REF] Griffiths | Towards a Generalized Biogeography of the Southern Ocean Benthos[END_REF][START_REF] Linse | Biodiversity and Biogeography of Antarctic and Sub-Antarctic Mollusca[END_REF][START_REF] Pierrat | Comparative Biogeography of Echinoids, Bivalves and Gastropods from the Southern Ocean[END_REF][START_REF] Poulin | Divergence between Antarctic and South American Marine Invertebrates: What Molecular Biology Tells Us about Scotia Arc Geodynamics and the Intensification of the Antarctic Circumpolar Current[END_REF]. The use of diverse sources of data available from palaeogeographical, palaeoclimatic and palaeontological records and those obtained from DNA sequences make possible the interpretation of current distribution patterns in a more precise historical framework [START_REF] Pearse | Brooding and Species Diversity in the Southern Ocean: Selection for Brooders of Speciation within Brooding Clades?[END_REF].

Mitochondrial DNA comparisons in species of the genus Nacella from different provinces of the Southern Ocean recognised clear and marked genetic divergences between lineages from maritime Southern Ocean invertebrate taxa does not appear to be a direct consequence of continental drift processes occurred in the region since the Mesozoic but to more recent oceanographic/climatic processes during the Miocene (Mackensen 2004;[START_REF] Verducci | The Middle Miocene Climatic Transition in the Southern Ocean: Evidence of Paleoclimatic and Hydrographic Changes at Kerguelen Plateau from Planktonic Foraminifers and Stable Isotopes[END_REF][START_REF] Zachos | Trends, Rhythms, and Aberrations in Global Climate 65 Ma to Present[END_REF].

In this context, [START_REF] Dalziel | A Potential Barrier to Deep Antarctic Circumpolar Flow until the Late Miocene?[END_REF] have argued that the full development of a deep ACC was only achieved during the late Miocene (~12 Ma) and not earlier, at the Eocene/Oligocene boundary, as long surmised. The formation of a full deep ACC may have played an important role in the subsequent Antarctic cryosphere expansion [START_REF] Zachos | Trends, Rhythms, and Aberrations in Global Climate 65 Ma to Present[END_REF], as well as in the intensification of the ACC associated with an increased strength of the westerly winds [START_REF] Flower | The Middle Miocene Climatic Transition: East Antarctic Ice Sheet Development, Deep Ocean Circulation and Global Carbon Cycling[END_REF][START_REF] Shevenell | Middle Miocene Southern Ocean Cooling and Antarctic Cryosphere Expansion[END_REF]. Such oceanographic/climatic processes would have promoted the separation between the different lineages of Nacella that are currently distributed in different provinces of the Southern Ocean.

Over the last two million years the climate on Earth has undergone recurring fluctuations that resulted in the Ice Under a phylogenetic framework [START_REF] González-Wevar | Molecular Phylogeny and Historical Biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]in press), the origin and diversification of Nacella in different provinces of the Southern Ocean occurred during the mid-to late-Miocene, long after the physical separation of the continental landmasses estimated between 41 Ma [START_REF] Livermore | The Southern Ocean as a Centre of Origin for Deep-Sea Octopuses[END_REF]) and 23.9 [START_REF] Scher | Timing and Climatic Consequences of the Opening of Drake Passage[END_REF] and to the initiation of the ACC. Such divergence within Nacella is basic agreement with recent molecular studies in notothenioid fishes [START_REF] Near | Ancient Climate Change, Antifreeze, and the Evolutionary Diversification of Antarctic Fishes[END_REF][START_REF] Stankovic | Recent Origin of Sub-Antarctic Notothenioids[END_REF] and in different groups of marine invertebrates [START_REF] Page | More Evidence of Speciation and Dispersal across Antarctic Polar Front through Molecular Systematics of Southern Ocean Limatula (Bivalvia: Limidae)[END_REF][START_REF] Wilson | Ocean Barriers and Glaciation: Evidence for Explosive Radiation of Mitochondrial Lineages in the Antarctic Sea Slug Doris Kerguelenensis (Mollusca, Nudibranchia)[END_REF] showing recent processes of diversification in the Southern Ocean. In this context, levels of pairwise uncorrected p-distances recorded in Nacella are comparable to those detected between Antarctic and South American lineages of Astrotoma [START_REF] Hunter | Evaluating Connectivity in the Brooding Brittle Star Astrotoma agassizii across the Drake Passage in the Southern Ocean[END_REF], Odontaster [START_REF] Janosik | Evolutionary History of Southern Ocean Odontaster Sea Star Species (Odontasteridae; Asteroidea)[END_REF], Sterechinus [START_REF] Poulin | Divergence between Antarctic and South American Marine Invertebrates: What Molecular Biology Tells Us about Scotia Arc Geodynamics and the Intensification of the Antarctic Circumpolar Current[END_REF] and Yoldia (González-Wevar et al. 2012a;[START_REF] Poulin | Divergence between Antarctic and South American Marine Invertebrates: What Molecular Biology Tells Us about Scotia Arc Geodynamics and the Intensification of the Antarctic Circumpolar Current[END_REF]. Therefore, the onset of divergence in different ( [START_REF] Raupach | Genetic Homogeneity and Circum-Antarctic Distribution of Two Benthic Shrimp Species of the Southern Ocean, Chorismus Antarcticus and Nematocarcinus Lanceopes[END_REF], Parbolasia corrugatus [START_REF] Thornhill | Open-Ocean Barriers to Dispersal: A Test Case with the Antarctic Polar Front and the Ribbon Worm Parborlasia Corrugatus (Nemertea: Lineidae)[END_REF] and Sterechinus neumayeri [START_REF] Díaz | Evolutionary Pathways among Shallow and Deep-Sea Echinoids of the Genus Sterechinus in the Southern Ocean[END_REF]). Nevertheless, genetic comparisons of standard genetic diversity indices showed lower levels of variability in the lineages from maritime Antarctica (N. concinna) compared to those from South America (N. magellanica) and Kerguelen Island (N. edgari). Such a result suggests a more pronounced demographic effect of Quaternary glacial cycles over the Antarctic species than the sub-Antarctic ones, and that such impact may be directly related to the latitudinal distribution of taxa. Such hypothesis must be further tested through the study of other genus with similar geographic distribution. Patterns of mismatch distribution in species of Nacella are consistent with the patterns of genetic diversity and recognised differences among the analysed taxa and evidence the recent population expansion process that experienced Antarctic and sub-Antarctic species.

Ages of the Quaternary. A vast body of evidence clearly demonstrates that many species experienced major and rapid latitudinal shifts as a response to ice advances and retreats during the Pleistocene [START_REF] Marko | The "Expansion-Contraction" Model of Pleistocene Biogeography: Rocky Shores Suffer a Sea Change?[END_REF][START_REF] Provan | Phylogeographic Insights into Cryptic Glacial Refugia[END_REF]. Several studies have demonstrated the impact of Quaternary glacial processes over population dynamics and the demography of different Southern Ocean invertebrates [START_REF] Convey | Exploring Biological Constraints on the Glacial History of Antarctica[END_REF][START_REF] Fraser | Kelp Genes Reveal Effects of Subantarctic Sea during the Last Glacial Maximum[END_REF]2010;[START_REF] Allcock | Southern Ocean Diversity: New Paradigms from Molecular Ecology[END_REF]González-Wevar et al. 2012a;in press). Fossil records and biogeographical data indicate that most of the biota, and particularly cold temperate species, persisted through glacial maxima in lower latitude refugia, where climatic conditions were less extreme [START_REF] Hewitt | The Genetic Legacy of the Quaternary Ice Ages[END_REF][START_REF] Provan | Phylogeographic Insights into Cryptic Glacial Refugia[END_REF]. Hence, ice advances during colder glacial periods generated major reduction of shallow rocky habitat and should have generated reduction in population sizes. Accordingly, a marked decline in genetic diversity through bottleneck effects or founder effects followed by population expansion is expected [START_REF] Slatkin | Pairwise Comparisons of Mitochondrial DNA Sequences in Stable and Exponentially Growing Populations[END_REF]. Relatively little is known on how and where ice-sensitive benthic biota of Antarctic and sub-Antarctic regions would have endured processes of extinction and recolonization as sea ice covered the region [START_REF] Fraser | Kelp Genes Reveal Effects of Subantarctic Sea during the Last Glacial Maximum[END_REF]. Ice shelves during glacial maxima extended even further and summer sea ice extended north of the 60º S [START_REF] Gersonde | Sea-Surface Temperature and Sea Ice Distribution of the Southern Ocean at the EPILOG Last Glacial Maximum -A Circum-Antarctic View Based on Siliceous Microfossil Records[END_REF], and thus restricted light penetration and primary production. However, the benthic marine fauna in the Southern Ocean did survive and new molecular data are helping to further elucidate how and where [START_REF] Allcock | Southern Ocean Diversity: New Paradigms from Molecular Ecology[END_REF].

As general pattern, Antarctic and sub-Antarctic Nacella species showed a strong signal of postglacial population expansion with the presence, in each of the analysed species, of a dominant haplotype surrounded by several unique or low-frequency haplotypes, commonly known as star-like genealogy. Such results are evidence of the impact of glacial processes over the demography of both Antarctic and sub-Antarctic near-shore benthic taxa, resulting in a reduction in their population size and probably their distribution, during the coolest periods of the Quaternary. Together with these results, levels of genetic diversity in Antarctic and sub-Antarctic populations of Nacella are lower than those found in temperate patellogastropod groups [START_REF] Bird | Diversification of Sympatric Broadcast-Spawning Limpets (Cellana Spp.) within the Hawaiian Archipelago[END_REF][START_REF] Nakano | Systematic Revision of Patelloida Pygmaea (Dunker, 1860) (Gastropoda: Lottiidae), with a Description of a New Species[END_REF] but comparable to those detected in higher latitudes ones [START_REF] De Aranzamendi | Limpets of the Genus Nacella (Patellogastropoda) from the Southwestern Atlantic: Species Identification Based on Molecular Data[END_REF], 2011;[START_REF] Goldstien | Comparative Phylogeography of Coastal Limpets across a Marine Barker[END_REF][START_REF] González-Wevar | Concerted Genetic, Morphological and Ecological Diversification in Nacella Limpets in the Magellanic Province[END_REF]. These results further corroborate the impact of Quaternary glacial processes over the patterns of genetic diversity along the whole Antarctic and sub-Antarctic realms. Low levels of genetic diversity as the ones detected in the Antarctic limpet have been also recorded in populations of several Antarctic marine invertebrates including Chorismus antarcticus

Figure 1 .

 1 Figure 1. distribution of Nacella in the southern ocean (in grey) including maritime Antarctica, south America and sub-Antarctic oceanic islands (Marion, Kerguelen, Heard, Macquarie and Campbell). dashed coloured lines indicate the approximate distribution of the analysed Nacella species (red = N. magellanica, blue = N. concinna and green = N. edgari). Underwater photographs courtesy of César Cárdenas (N. magellanica), dirk schories (N. concinna) and elie Poulin (N. edgari).

Figure 2 .

 2 Figure 2. (a) General maximum parsimony haplotype networks in Nacella species showing the divergence (based on uncorrected p-distances) among them. A coloured circle indicating the province where it was collected represents each haplotype. Green = Kerguelen island (N. edgari), blue = maritime Antarctica (N. concinna) and red = southern south America (N. magellanica). the size of each circle is proportional to its frequency in the whole sampling effort. (b) Maximum parsimony haplotype network in N. concinna along maritime Antarctica. (c) Maximum parsimony haplotype network in N. magellanica from southern south America. (d) Maximum parsimony haplotype network in N. edgari along Kerguelen island.

Table 1 .

 1 diversity indices and neutrality tests in Nacella species from different provinces of the southern ocean, including N. edgari (Kerguelen island), N. concinna (maritime Antarctica) and N. magellanica (Patagonia). : n = number of analysed individuals; k = number of haplotypes; S = polymorphic sites; H = haplotype diversity; Π = average number of pairwise differences; π = nucleotide diversity.

	Species and localities	n	k	H	S	Π	π	Tajima's D	Fu's FS
	N. edgari
	Port Christmas	28	7	0.767	8	1.354	0.0019
	Port Aux Francais	32	9	0.633	8	0.869	0.0012
	ile du Prince de Monaco	41	13	0.788	15	1.505	0.0021
	total	101	20	0.750	19	1.287	0.0018	-1.92*	-16.013***
	N. concinna
	Antarctic Peninsula	31	5	0.688	5	0.985	0.0014
	south shetland island	32	6	0.573	6	0.724	0.0010
	south orkney island	26	9	0.625	8	0.757	0.0011
	total	89	12	0.0627	11	0.827	0.0012	-1.84*	-8.07***
	N. magellanica
	Metri	25	9	0.743	10	1.380	0.0020
	Aysén	24	6	0.496	6	0.790	0.0011
	santa Ana	24	10	0.775	11	1.365	0.0020
	tekenika	24	11	0.822	13	1.822	0.0027
	total	97	25	0.714	28	1.409	0.0022	-2.24**	-24.33***

Notes* p < 0.05. ** p < 0.01. *** p < 0.001.

Acknowledgements

We thank the editor and an anonymous reviewer for helpful comments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This study was supported by a Fondecyt Initiation Project 11140087, Chilean Antarctic Institute INACH (Office Project G_04-11) to C.G-W. Projects P05-002 ICM and PFB 023 (Instituto de Ecología y Biodiversidad IEB, Universidad de Chile), to E.P and CG-W. Field access at Kerguelen Island are supported by IPEV programme 1044 Proteker to E.P., J.-P.F. and T.S. We recognise the SCAR AntEco and EBA programmes, and CAML, for encouraging and supporting Antarctic and sub-Antarctic evolutionary biology. This paper also contributes to the SCAR 'State of the Antarctic Ecosystem' (AntEco) programme