| Foraminifer is a kind of single-celled protozoa which is living mostly in the marine environment.They constitute the most diverse shelled microorganism group in the modern ocean.Most of them secrete a calcium-carbonate shell while a few have agglutinated or organic shell.Benthic foraminifera are the main group of the modern foraminifera.Moreover,only about 40 to 50 species are planktonic foraminifera.Benthic foraminifera are not only more diverse than planktonic foraminifera,they also have a longer geological record.The oldest fossils of benthic foraminifera appeared in the Cambrian period,while those of planktonic foraminifera appeared in the Jurassic period.The establishment of the quantitative relationship between benthic foraminiferal shell elements and marine environmental factors(temperature,salinity,pH,etc.)has become one of the most important methods to reconstruct the palaeoenvironment of bottom seawater.With the global warming,more and more attention has been paid to the influence of temperature on organisms.Foraminifera is one of the most important biological indicators for the study of the palaeotemperature of ocean.It is of great scientific significance to explore the response of benthic foraminifera to temperature for the reconstruction of palaeoclimate and palaeoocean.In this study,benthic foraminifera were used as the research object.The sediment samples of the continental shelf in Jiaozhou bay(2017,2018),the intertidal zone of Qingdao bay(2018),the “Haima” cold seep in the south China sea(2019),and the subarctic region(2019)were collected.Combining the traditional morphological method with the next-generation sequencing technology(high-throughput sequencing technology),we used the field investigation and laboratory culture experiment to analyze the response of the abundance,diversity,shell type,and dominant species of benthic foraminifera to temperature in different marine areas.This study tries to reveal the internal mechanism of the effect of temperature on the benthic foraminifera and establish the relationship between the biological indicators of benthic foraminifera and temperature.The specific research contents are divided into the following five parts:1.A preliminary experiment to study the response of the benthic foraminifera community from the continental shelf to temperature in laboratory cultureAs one of the most important marine environmental indicators,benthic foraminifera play an important role in the reconstruction of the paleotemperature of the bottom seawater.However,the mechanism of the effect of temperature on the benthic foraminifera is not clear.The surface sediment samples from the Jiaozhou bay(3-26 m)of the continental shelf were collected.Sediment samples with living benthic foraminifera communities were cultured without extra food under different temperatures(6℃,10℃,12℃,18℃,and 24℃)in the laboratory.The whole culture experiment lasted for 8 weeks.This study tried to explore the response of the benthic foraminifera communities to different temperatures.A total of 2,331 benthic foraminifera were identified in the experiment,including476 living foraminifera.The abundance of living foraminifera ranged from 6.80 ind/g to 13.25 ind/g dry weight of the sample.The results showed that the abundance of living foraminifera was negatively correlated with temperature.With the increase of temperature,the Margalef index and Shannon-Wiener index of foraminifera increased first and then decreased while the evenness index was stable.Moreover,all the three diversity index had no significant correlation with temperature.The response of dominant species of foraminiferal communities were specific to different temperatures.The study indicated that the change of ambient temperature would affect the foraminiferal community parameters,diversity,and community structure under the temperature range and culture conditions of this experiment.The difference of dominant species in the different temperatures suggested a succession of the foraminiferal community.2.A study on the safe use of commonly used marker for foraminiferal shell in temperature culture experimentsThe foraminiferal shell Mg/Ca is one of the most common paleotemperature indicators.The method of laboratory culture has been widely used to establish the relationship between Mg/Ca and temperature of different foraminiferal species.Calcein has been usually used as an effective marker for tagging newly formed biogenic calcite in shell structure of foraminifera in culture experiments.Whether calcein has any effect on the biological characteristics of foraminifera,such as abundance and size,is unclear.The surface sediment samples from Qingdao bay were collected.The sediment samples with living benthic foraminiferal communities were cultured under four calcein concentration(0 mg/L,1 mg/L,5 mg/L,and 10 mg/L)and three incubation time(2 weeks,4 weeks,and 6 weeks)in the laboratory experiment.This study aimed to explore the impact of calcein on living benthic foraminifera community under different calcein concentration and incubation time.A total of 10,393 living benthic foraminifera were collected.The abundance of the living foraminifera under different treatments ranged from 122.72 ± 13.69 to458.65 ± 234.29 ind./g dry weight of sample.The highest abundance was found in the5 mg/L concentration group at 2 weeks,and the lowest value was found in the treatment with the longest incubation time(6 weeks)and the highest concentration(10 mg/L).The results of statistical analysis showed that the concentration of calcein significantly affected the community parameters(foraminiferal abundance,Margalef index,and Shannon-Wiener index)while the incubation time only had a significant effect on the abundance of foraminifera.As for the shell type of foraminifera,the proportion of the porcelanous foraminifera in the experimental groups(1 mg/L,5mg/L,and 10 mg/L)was significantly higher than that in the control group(0 mg/L)while the proportion of hyaline foraminifera was lower than that in the control group.In addition,three dominant species were obtained: Ammonia aomoriensis,Cribrononion gnythosuturatum,and Quinqueloculina seminula.The abundance of different dominant species had different responses to calcein concentration and incubation time.The abundance of C.gnythosuturatum and Q.seminula decreased significantly with the increase of calcein concentration.However,the abundance of A.aomoriensis did not change significantly.Moreover,only the abundance of Q.seminula decreased significantly with the increase of incubation time.The shell size and development of dominant species were different under different experimental treatments.The average size of the dominant species in the control group reached the maximum value in the second week while the average size of the dominant species in the experimental group did not reach the maximum value until the fourth or sixth week.The results of this study suggested that calcein might have a positive effect on foraminifera community by enhancing the porcelanous foraminifera at the set range of the calcein concentration and incubation time in the experiment.In addition,considering the negative effects of high calcein concentration and long incubation time on foraminifera,we suggested a relatively safe method of calcein use: Calcein(≈5 mg/L)was added as an initial marker at the beginning of the culture experiment,and natural seawater was substituted halfway through the experiment.About 4 weeks before the end of the culture experiment,calcein can be added to the seawater culture medium again.3.A study to reveal the effect of temperature on benthic foraminifera and its internal mechanism based on morphological and molecular methodsWith the global warming,more and more attention has been paid to the influence of temperature on organisms.Foraminifer is one of the most important environmental indicators in oceanography.Thus,it is very important to establish the relationship between temperature and foraminifera.However,the mechanism of the effect of temperature on foraminifera is still unclear.The surface sediment samples from the stations(A5,9.2m;C4,6m;D6,26m)of Jiaozhou bay continental shelf were collected.The sediment samples with living benthic foraminifera were cultured under different temperatures(6℃,12℃,18℃,24℃,and 30℃)in the laboratory.The whole culture experiment lasted for 80 days.The effects of different temperatures on benthic foraminiferal communities and its internal mechanisms were studied by combining traditional morphological identification and next-generation sequencing technology.In the experiment,4,162 living foraminifera were identified by morphological identification,and 3,871,615 effective tags were obtained by molecular sequencing technology.The results showed that the abundance of living foraminifera at A5,C4,and D6 stations decreased with the increase of culture temperature,and Spearmen correlation analysis showed that the abundance of living foraminifera was significantly negatively correlated with temperature.However,the correlation between foraminiferal reads and temperature was not significant.The Margalef index of foraminiferal community at A5,C4,and D6 stations obtained from morphological and molecular data all increased with the increase of temperature.Correlation analysis showed that the Margalef index of foraminiferal community was significantly positively correlated with temperature.However,the Shannon-Wiener index of foraminifera obtained from morphological and molecular data had no significant correlation with temperature.The abundance of hyaline foraminifera and agglutinated foraminifera decreased significantly with increasing temperature.The reads of hyaline foraminifera and agglutinated foraminifera increased with increase of temperature,but the correlation test was not significant.The reads of porcelanous foraminifera and single-chambered foraminifera at different stations varied with temperature.The dominant species obtained based on the morphology were as follows: Ammonia aomoriensis,A.beccarii,Buccella frigida,Cribrononion subincertum,and Trochammina inflata.The dominant species obtained from the molecular method were not consistent with those from the morphology,and different dominant species at different stations showed diverse responses to temperature.This study revealed that temperature significantly affected the abundance,diversity,shell type,and dominant species of the foraminiferal community.After correlation analysis and function fitting,the linear relationships between foraminiferal abundance,Margalef index,abundance of different shell types,and some dominant species and temperature were established.It will provide first-hand data and function reference for the reconstruction of palaeotemperature and palaeo-ocean.In addition,the presence of small cryptic species and single-chambered foraminifera(most without shell)could be detected by molecular methods,which resulted in the increase of species and diversity of foraminifera obtained based on the molecular methods.Moreover,the differences in the species composition resulted in different responses of foraminifera to temperature between morphological and molecular methods.4.A study to explore the response of foraminifera to cold seep(low temperature and low oxygen environment)using high-throughput sequencing technologyCold seep is a kind of rare deep-sea system,which is closely related to the distribution of hydrocarbon resources(such as natural gas and oil)in the ocean.Benthic foraminifera have attracted much attention as a potential indicator of cold seep activity.However,almost all the studies on foraminifera in cold seep area are limited to traditional morphological classification.In this experiment,we collected sediment samples from the 4 stations(SY186-1,SY186-3,SY186-5,and SY186-7;depth > 1380m)in the “Haima” cold seep area.The total DNA was extracted and a region of the small subunit(SSU)r RNA gene was amplified with the foraminiferal specific primers.Then we analyzed the molecular diversity and community composition of foraminifera in the cold seep.In total,3,109,553 Effective tags and 432 OTUs were gained after strict quality control.The results showed that the foraminiferal community parameters(reads,OTUs,Margalef index,and Shannon-Wiener index)at SY186-1 and SY186-3(closer to the cold seep activity center)were higher than those at SY186-5 and SY186-7(farther away from the center).In addition,we found that the majority of foraminifera were Rotaliida(>85%),followed by a small amount of Textulariida and Monothalamida,without Miliolida(Identity value > 97.7%).Moreover,three ecological foraminiferal groups were found: cold-water benthic foraminifera,denitrifying benthic foraminifera,and planktonic foraminifera.Our study revealed that low temperature and low oxygen conditions in the cold seep center caused foraminiferal reads and diversity higher than that in the non-seep sites;The diversity of foraminifera in the cold seep was higher than that in the general sea area but lower than that in some high nutrient zone;The cold seep foraminifera are characterized by high proportion of Rotaliida;Denitrifying foraminifera is a characteristic group in the cold seep,among which Uvigerina is the most common foraminifera.Thus,foraminifera can be used as an effective environmental indicator of cold seep.5.A study to explore the response of foraminifera to subarctic area(low temperature environment)using high-throughput sequencing technologyThe arctic ocean is an important part of the global marine climate system,which is of great significance for the study of global climate change(e.g.global warming).However,our understanding of the ocean’s past and present is still very limited.Calcareous foraminifera are considered to be one of the most useful proxies to reconstruct the marine paleoenvironment of the arctic ocean.In order to investigate the molecular diversity and community composition characteristics of foraminifera in subarctic sediments,total genomic DNA was extracted from surface sediments collected from 11 stations in the subarctic.Then the foraminiferal DNA was amplified using foraminiferal specific primers.After strict quality control process and data processing,a total of 2,170,762 effective tags and 1,046 OTUs were obtained in the experiment.The result showed BR10 had the highest reads of foraminifera.The highest values of foraminiferal Margarlef index and Shannon-Wiener index were obtained at BR06.The spearman correlation test showed that the foraminiferal community parameters(reads,OTU,Margarlef index,and Shannon-Wiener index)had no significant correlation with environmental factors(temperature,salinity,and water depth).In the foraminiferal community,hyaline foraminifera accounted for the highest proportion,followed by agglutinated and single-chambered foraminifera,then porcelanous foraminifera accounted for the lowest proportion.The results indicated that the molecular diversity of foraminifera in the subarctic was lower than those in other areas.High proportion of hyaline foraminifera and low proportion of porcelanous foraminifera were the important characteristics of subarctic foraminifera.Nonionella labradorica was the characteristic dominant species of the subarctic foraminifera,with the genera Reophax,Globobulimina,and Elphidium as the characteristic groups. |
