University of Connecticut
DigitalCommons@UConn
Master's Theses
University of Connecticut Graduate School
12-13-2013
Effects of Increasing Temperature and Ocean
Acidification on the Microstages of two
Populations of Saccharina latissima in the
Northwest Atlantic
Sarah Redmond
[email protected]
Recommended Citation
Redmond, Sarah, "Effects of Increasing Temperature and Ocean Acidification on the Microstages of two Populations of Saccharina
latissima in the Northwest Atlantic" (2013). Master's Theses. 515.
http://digitalcommons.uconn.edu/gs_theses/515
This work is brought to you for free and open access by the University of Connecticut Graduate School at DigitalCommons@UConn. It has been
accepted for inclusion in Master's Theses by an authorized administrator of DigitalCommons@UConn. For more information, please contact
[email protected].
Effects of Increasing Temperature and Ocean Acidification on the
Microstages of two Populations of Saccharina latissima
in the Northwest Atlantic
Sarah Rose Redmond
B.S., University of Maine, 2003
A Thesis
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Science
At the
University of Connecticut
2013
APPROVAL PAGE
Master of Science Thesis
Effects of Increasing Temperature and Ocean Acidification on the Microstages of two
Populations of Saccharina Latissima in the Northwest Atlantic
Presented by
Sarah Rose Redmond, B.S.
Major Advisor
Charles Yarish
Associate Advisor
Gene Likens
Associate Advisor
Senjie Lin
Associate Advisor
George P. Kraemer
University of Connecticut
2013
ii Abstract
Saccharina latissima (Linnaeus) C.E.Lane, C.Mayes, L.D. Druehl and G.W.Saunders, is the
most widely distributed species of kelp in the western North Atlantic, occurring from the Arctic to
Long Island Sound. The effects of global climate change on these ecologically and economically
important cold temperate species at the southern range of their distribution are unknown. This
study investigated the impact of the combined stressors of increased temperature (16, 19, 22,
25 & 28°C) and reduced pH (7.9, 7.8, 7.7, & 7.6) on the gametophyte and juvenile sporophyte
stages of sugar kelp populations from Maine and Long Island Sound. Spore germination and
growth, male and female ratio, fecundity, reproductive success of female gametophytes, and
growth of juvenile sporophytes were investigated on crossed gradient temperature tables with
CO2-adjusted pH levels. The upper critical thermal limit for gametophytes in all trials for both
populations was 22°C, with full mortality of gametophytes occurring at all temperatures tested
above this limit (i.e. 25° and 28°C). Gametophyte survival, growth, and male and female ratios
were similar in all trials for both populations at 16° and 19°C, but gametogenesis was
suppressed at temperatures above ca. 17°C. There were no consistent effects of pH in any
trials, though the lower pH values (7.6-7.7) did result in slightly larger gametophytes (primary
cell diameter & gametophyte length) than the highest value (7.9) at 16° and 19°C in some of the
trials. These results support the hypothesis that the predicted increase in seawater
temperatures will shift the distributional boundary of these cold temperate seaweeds northward,
resulting in the loss of populations at the southernmost boundary.
iii Acknowledgements
I am so grateful for the encouragement and guidance of the members of the Yarish research
family, especially Dr. Charles Yarish, Jang Kim and Rui Pereira. I would like to acknowledge
and thank all of my advisors for their invaluable input and ideas, and William Schoellkopf for
expert assistance with data analysis. This project would not have been possible without the help
of Christian Fox and the dive team at Avery Point, or without support from our NOAA National
Sea Grant College Program. I am thankful for the inspiration provided by Tollef Olson and Paul
Dobbins at Ocean Approved, LLC.
iv Table of Contents
Abstract ................................................................................................................................................................. iii List of Tables ....................................................................................................................................................... vi List of Figures ..................................................................................................................................................... vi Introduction .......................................................................................................................................................... 1 Biology and Ecology of Saccharina latissima ................................................................................................................ 1 Effects of Temperature on Saccharina latissima .......................................................................................................... 6 Ocean Climate Change .............................................................................................................................................................. 8 Methods and Materials ................................................................................................................................... 13 Statistical Analysis .................................................................................................................................................................. 16 Results .................................................................................................................................................................. 17 Germination ............................................................................................................................................................................... 17 Female Gametophyte Diameter ......................................................................................................................................... 19 Female Gametophyte Length .............................................................................................................................................. 20 Cell Number of Female Gametophytes ........................................................................................................................... 23 Male: Female Ratio ................................................................................................................................................................. 25 Fecundity ..................................................................................................................................................................................... 27 Reproductive Success ............................................................................................................................................................. 29 Sporophyte Length .................................................................................................................................................................. 30 Discussion .......................................................................................................................................................... 31 Conclusions ................................................................................................................................................................................. 40 References ................................................................................................................................................................................... 41 v List of Tables
Table 1.
Table of rate of geometric mean growth rate of female gametophytes
22
Table 2.
Kruskal-Wallis Test for Male: Female ratio results
26
Table 3.
ANOVA results for fecundity
27
Table 4.
Observed critical limits for female gametogenesis
28
Table 5.
Comparison of reported critical temperature limits (°C) for S. latissima
32
List of Figures
Figure 1.
Saccharina latissima life cycle
Figure 2.
NOAA National Data Buoy Center data for 2012
12
Figure 3.
Map of Northeast Sea Surface Temperatures for 08/02/2013
16
Figure 4
Percent germination by temperature grouped by pH for all three trials
18
Figure 5
Female gametophyte diameter (µm) at one week after germination.
19
Figure 6
Female Gametophyte Length (µm) for Maine summer trial at weeks 1-3
21
Figure 7
Female Gametophyte Length (µm) for Maine fall trial, 1-3 weeks
21
Figure 8
Female gametophyte length (µm) for Long Island Sound trial
22
Figure 9
Cell number of female gametophytes, Maine summer trial
24
Figure 10
Cell number of female gametophytes, Maine fall trial
24
Figure 11
Cell number of female gametophytes, Long Island Sound fall trial
25
Figure 12.
Male: Female sex ratio plot (Male/Female gametophytes per 100)
26
Figure 13.
Fecundity values plotted with observed gradient table temperature values
28
Figure 14.
Reproductive success for all trials at 2 and 3 weeks
29
Figure 15.
Geometric mean of juvenile sporophyte length
30
3
vi Introduction
Biology and Ecology of Saccharina latissima
Saccharina latissima (Linnaeus) C.E.Lane, C.Mayes, L.D. Druehl and G.W.Saunders
(Lane et al., 2006) belongs to a larger group of the large brown cold temperate kelps, which are
the most ecologically and economically important seaweeds in the Northern Hemisphere
(Lüning et al., 1990; Yarish & Pereira, 2008). Saccharina species are major components of
underwater ecosystems in the North Atlantic, forming extensive, highly productive beds (Mann,
1973; Egan & Yarish, 1990) that can correspond to terrestrial forests in terms of physical
structure and habitat generation (Graham, 2004; Mann, 1972). The three dimensional character
of kelp forests provide substrate, shelter, shade, and oxygen for other organisms (Steneck et
al., 2002), including commercially important fish and invertebrates (Bologna & Steneck, 1993;
Egan & Yarish, 1990; Lazzari & Stone, 2006) and contribute significantly to coastal primary
production and local food webs through release of detritus, dissolved carbon, and meiospores
(Graham, 2004; Duggens et al., 1989; Van Patten & Yarish, 1993).
Kelps are cultivated and wild harvested worldwide for food, medicinal, agricultural, and
industrial products. Kelp can be cultured as the bioextractive component of integrated multitrophic aquaculture systems (Yarish & Pereira, 2008; Chopin et al., 2008), and could represent
a possible carbon-neutral source of biomass for fuel alternatives. Development of global kelp
culture systems could be part of a mitigation strategy for problems of excess atmospheric CO2
and ocean eutrophication, while providing a source of energy, food, and biochemical products
for the demands of a rapidly growing global human population (Turan & Neori, 2010; Holdt &
Kraan, 2011).
The family Laminariaceae, order Laminariales, is widely distributed with great
morphological and physiological plasticity, lending itself to taxonomic confusion. Sporophytes of
1 the Laminariaceae consist of a branched holdfast, simple stipe, and smooth lamina, which can
be either simple or digitate. The simple lamina group in the Northwest Atlantic includes two
morphologically similar species, Saccharina latissima (formerly Laminaria saccharina), and
Saccharina longicruris (formerly Laminaria longicruris) (Lane et al., 2006). These both exhibit
high levels of morphological plasticity controlled mainly by environmental factors (Gerard &
Mann, 1979; Fowler-Walker et al., 2006), with some level of genetic control (Chapman, 1974;
Lüning et al., 1978; Yarish et al., 1990; Egan et al., 1990). These two species are interfertile
(Lüning et al., 1978; Chapman, 1974), and exhibit no phylogenetic evidence of genetic
difference (Neefus et al., 1993; McDevit & Saunders, 2010), While Saccharina longicruris is still
a recognized species name (Lane et al., 2006; Guiry & Guiry, 2013), it is treated here as
conspecific with Saccharina latissima, with the understanding that distinct genetic strains or
races probably exist throughout the North Atlantic as part of a large, very plastic species
complex (Bartsch et al., 2008; Cho et al., 2000; Chapman, 1974; Kain, 1979; Egan et al., 1990;
Lüning, 1975).
Saccharina latissima has a heteromorphic life cycle that consists of an alternation of a
macroscopic diploid sporophyte stage with a microscopic haploid dioecious oogamous
gametophyte stage (fig. 1). Sporophytes can develop sorus tissue and release spores
throughout the year (Parke, 1948; Chapman, 1984), with peak reproductive seasons occurring
in the spring and fall (Egan & Yarish, 1990; Van Patten & Yarish, 1993). Haploid, flagellated
meiospores (~5 µm) are released from unilocular sporangia in an equal ratio of males and
females (Schreiber, 1930). Once attached to a substrate, meiospores shed flagella, germinate,
and move spore cell contents into the primary cell (Lüning, 1980). After a resting stage that can
be a few days to several weeks, the primary cell increases in diameter to form the “dumbbell
stage”, at which point it could potentially enter gametogenesis as a male or female unicellular
gametophyte, or vegetatively divide to form multicellular filaments (Harries 1932; Lüning, 1980).
2 Figure 1. Saccharina latissima life cycle
Female gametogenesis is controlled by interacting environmental variables, including
temperature (Kain, 1964; Lüning, 1980), light (Lüning & Dring, 1979) and availability and
concentration of nutrients (Hsiao & Druehl, 1973; Dayton, 1985), and can be delayed indefinitely
in the vegetative state (Druehl et al., 2005), existing as unicellular females (Lüning, 1980) or
extensive multicellular filaments. After induction of gametogenesis, a single egg is extruded
from mature female oogonia along with the hormone lamoxirene, which signals release and
attraction of the flagellated spermatozoids from nearby males (Lüning & Muller, 1978; Marner et
al., 1984). The diploid zygote immediately begins to divide longitudinally to develop into the
juvenile sporophyte.
Sporophytes exhibit a seasonal growth pattern, with a general increase in growth rates
in the late fall to winter and a rapid increase from late winter to spring as temperatures and light
3 levels increase (Kain, 1979; Chapman & Craigie, 1977). Sporophytes have a broad growth
optimum (10-15°C), a characteristic shared by other seaweed species located at the southern
boundaries of cold temperate zones (Fortes & Lüning, 1980). Growth declines throughout
summer and early fall with the increase in water temperature and decrease in nutrient
availability. Severe blade erosion occurs at 16°C and above in Long Island Sound (Brinkhuis et
al., 1983; Egan & Yarish, 1990) with high rates of mortality occurring at 20°C and above
(Chapman & Craigie, 1977).
The alternation of generations in the life cycle may be a means for populations to survive
unfavorable or stressful environmental conditions. The microscopic gametophyte stage is
considered a ‘survival stage’, a perpetuation phase exhibiting ecophysiological differences from
the large sporophyte stage (Carney & Edwards, 2006; Ladah et al., 1999). Microscopic
gametophytes and juvenile sporophytes are physiologically better adapted than the sporophyte
stage to withstand adverse environmental conditions including pollution (Hopkins & Kain, 1978),
long periods of darkness (Lüning, 1980; Kain, 1964), and higher temperatures, up to 1-7°C
(Ladah & Zertuche, 2007; Kain, 1969; Bolton & Lüning, 1982; tom Dieck, 1993). While
gametophytes may be able to withstand higher summer temperatures than sporophytes,
gametogenesis is inhibited at higher summer temperatures (Lüning, 1980; Egan et al., 1989);
possibly creating a vegetative “resting stage” (Chapman, 1964) that might allow populations at
the southern limit to persist through periods of adverse temperatures. Gametophytes may be
able to exist in a dormant vegetative state indefinitely (Dayton, 1985), surviving up to 30 years in
the lab under vegetative conditions while still retaining sexual capacity (Neushul, 1987).
Saccharina latissima is a widely distributed species in the Northwest Atlantic, ranging
from the Arctic in the North to around the 20°C August seawater isotherm in the south (van den
Hoek, 1982; Egan & Yarish, 1988), which stretches across the Atlantic from Long Island Sound
to the Iberian Peninsula. The Gulf of Maine represents the middle latitudinal range of the
distribution, experiencing lower summer temperatures than Long Island Sound, occurring
4 approximately where the 15°C summer isotherm stretches across the Atlantic to England
(Lüning, 1990). The distribution range of Saccharina latissima is unique among other northern
macroalgal species, in that it extends further south than Cape Cod, a major phytogeographic
boundary on the East Coast of the U.S. (Mathieson, 1979).
Widely distributed species experience different temperature and light regimes throughout
the year, and this can create distinct differences among populations (Lüning & Neushul, 1978).
The large-scale temporal variations in temperature, light (including irradiance and photoperiod),
and nutrient availability (Dayton et al., 1999) are more important in determining kelp
demography than small-scale local processes of competition and grazing (Rinde & Sjotun,
2005). Latitudinal variation can influence seasonal growth and life history patterns, upper lethal
temperatures, and reproduction, and these differences can be seen in Saccharina latissima
populations from the southern and middle ranges of their distribution. The cooler waters of the
more northerly middle latitudes typically allow for a longer growing season for sporophytes
(January-July), and plants can persist through summer temperatures, where they can be
perennial up to 3 years of age (Parke, 1948; Kain, 1979; Johnston et al., 1977). Reproductive
sorus tissue forms on blades that are at least six months of age in the mid-range, and new
spring juvenile sporophytes have higher rates of attachment and survival than new winter plants
(Parke, 1948).
Southern range populations experience a shorter growing season (January-May), with
plants disintegrating or disappearing in late summer due to high water temperatures (Brinkhuis
et al., 1983; Lee & Brinkhuis, 1986) and low levels of nitrogen (Egan and Yarish, 1990). The
majority of sporophytes are annuals, but a percentage of the population are biennials (Egan
&Yarish, 1990), recovering from severe blade erosion and continuing growth the following fall if
enough of the meristematic tissue survives (Lee & Brinkhuis, 1988; Wong, 1989; BradyCampbell et al., 1984). The frequency of annuals increases south of Cape Cod, suggesting
more extreme environmental conditions (Coleman & Mathieson, 1975; Sears & Wilce, 1975).
5 Reproductive sorus tissue can develop earlier, on tissue that is only four months old and as
small as 15 cm in length. New spring plants in Long Island Sound rarely survive through the
summer, while new winter plants have higher survival rates and can persist into the following
year (Parke, 1948). Brinkhuis et al. (1984) suggested that the late spring sporogenesis period
was an adaptation to the higher temperatures in Long Island Sound, since other mid and
northern range populations had been observed to release spores mainly in winter (Kain, 1979).
Seaweeds exposed to climactic stressors (e.g. glaciation cycles) and unique local
conditions (e.g. temperature, nutrient levels) are subject to strong selection pressure and local
adaptation. Periods of isolation and gene transfer of Saccharina latissima populations have
resulted in a genetically diverse species across the northern oceans, with distinct and hybridized
clusters found in the Eastern and Western Atlantic, Arctic, and Eastern Pacific (McDevit &
Saunders, 2010). Regionally, populations have adapted to different environmental conditions
(Lüning & Neushul, 1978), including different light regimes (Gerard, 1988; 1990), nitrogen
supply (Gerard, 1997; Espinoza & Chapman, 1983), and temperature (Gerard & Du Bois, 1988).
The differential responses of gametophytes and sporophytes between middle range and
southern range Saccharina latissima populations in the Northwest Atlantic have been attributed
to ecotypic differentiation (Russell, 1986, Breeman & Pakker, 1994).
Effects of Temperature on Saccharina latissima
Seaweeds with heteromorphic life stages often experience seasonal shifts in optimal
environmental conditions that can vary with development and age (Lüning, 1984; Lobban &
Harrison, 1997; Fortes & Lüning, 1980). Microscopic stages of Saccharina latissima cultivated at
different times of the year have exhibited different light intensity and temperature requirements
for optimal growth (Brinkuis et al., 1984; Egan et al., 1989). Shifts in temperature optima and
maximum survival temperatures have been observed in southern-range populations, indicating
acclimation to warmer summer temperatures (Lee & Brinkhus, 1988; Egan et al., 1989; Kain,
6 1979). Sporophytes of S. latissima can tolerate temperatures up to 18°C, with seasonal shifts of
up to 5°C (Lüning, 1984), and similar shifts have been observed in gametophyte temperature
tolerance in summer months (Egan et al., 1989; Lee & Brinkhuis, 1988). Laminarian
gametophytes have exhibited upper tolerance temperatures 1-7°C above tolerance limits for
sporophytes (tom Dieck, 1993).
Several studies have observed distinct ecotypical differences in physiological response
to environmental variables of light, temperature, and nutrients between populations from the
Gulf of Maine and Long Island Sound. Sporophytes collected from Maine exhibited negative
growth rates at 20°C after two weeks, and did not survive three weeks at this temperature, while
individuals collected from Long Island Sound survived and experienced positive growth rates for
up to 6 weeks at 20°C (Gerard & Du Bois, 1988). Sporophytes from Long Island Sound also
exhibited better growth rates under low light conditions (Gerard, 1990), which might be a result
of the increase in the major light harvesting pigments (chlorophyll a, chlorophyll c, and
fucoxanthin) that occurs under higher temperature conditions (Davison et al., 1991). These
plants also accumulated more tissue nitrogen than sporophytes from Maine, supporting
resilience of the photosynthetic apparatus and possibly contributing to heat shock proteins,
which are important in high temperature stress survival (Gerard, 1997). Other studies have also
observed increased survival of kelps in the presence of adequate nutrients (Dayton, 1985),
including a study of a southern-limit population of Saccharina japonica in China that was found
to survive summer temperatures up to 25°C in the presence of increased levels of nitrogen,
presumably contributing to formation of heat stress proteins and strengthening of the
photosynthetic machinery (Liu & Pang, 2010).
Long Island Sound gametophytes and juvenile sporophytes have exhibited higher
temperature tolerance and more sustained positive growth than individuals obtained from
northern populations (Egan et al., 1990) (Table 5). While the lethal limit for meiospores is 22°C
for populations from higher latitudes (tom Dieck, 1993), meiospore germination has been
7 successful at temperatures as high as 25°C in July and October in one study (Egan et al.,
1989), but not in other months. Gametophytes from Long Island Sound have exhibited a shift in
optimal growth temperature from 10-15°C in March to 15-20°C in July, and successfully
produced young sporophytes at 20°C in all months except January (Egan et al., 1989).
Gametophytes from the middle range also have a broad optimal range for growth, from 10-15°
(Bolton & Lüning, 1982), to 10-19°C (Lüning, 1980), but have lower temperature limits for
gametogenesis, producing sporophytes at temperatures below 16° (Harries, 1932), or 18°C
(Evans, 1965: Bolton & Lüning, 1982: Lüning, 1984). Lethal temperatures for Long Island Sound
gametophyte and juvenile sporophyte populations have also been reported to be higher than
mid-range populations, at 25°C (Egan et al., 1989), compared to reported values of 22° (Lüning,
1980,1984: Kain, 1969) and 23-24°C (Bolton & Lüning, 1982: Breeman, 1988). Juvenile
sporophytes from the mid-range exhibited optimal growth at 10-15°C, reduced growth at 20°C,
and disintegration of plants at 23°C (Bolton and Lüning, 1982).
Ocean Climate Change
Anthropogenic alterations of the carbon cycle through fossil fuel combustion and
changes in land use have resulted in the rapid increase of atmospheric CO2 from pre-industrial
levels, with predictions of a 2-3 fold increase of current levels by year 2100 (IPCC, 2007, 2013),
and a 5-fold increase by year 2300 (IPCC, 2013, Caldeira & Wickett, 2003). Increased levels of
CO2 along with other greenhouse gases, including methane and nitrous oxide, have resulted in
a warming of the global mean surface air and ocean temperatures (Levitus et al., 2000), with a
predicted increase of overall ocean temperatures of about 0.2°C or more per decade (IPCC,
2007, 2013). Global scale temperature increases are expected to impact coastal ecosystems
through sea level rise, increased storm events, changes in hydrologic cycles, and changes in
ocean circulation (IPCC, 2007, 2013; Scavia et al., 2002).
8 The ocean acts as a major inorganic carbon reservoir in the global carbon cycle; slowing
the accumulation of atmospheric CO2 by absorbing ca. 30-50% of all anthropogenically released
CO2 (Sabine et al., 2004; Doney et al., 2009). Atmospheric CO2 dissolves into seawater directly
or reacts with water (H20) to form carbonic acid (H2CO3). Carbonic acid is a weak acid that
readily disassociates into hydrogen (H+) and bicarbonate (HCO3-) ions. Carbonate (CO32-) ions
act to buffer this increase in hydrogen ions by reacting with some of the available H+ ions to
form more bicarbonate (HCO3-). The increased absorption of CO2 from the atmosphere results
in a net increase of carbonic acid, free hydrogen, and bicarbonate ions, but a decrease in
overall carbonate ion concentration. This shift of the carbonate chemistry of seawater decreases
seawater pH and reduces the buffering capacity of seawater (Sabine et al., 2004). Overall
surface pH levels are predicted to decrease between 0.14 and 0.35 units during this century
(IPCC, 2007, 2013), with a maximum pH reduction at the ocean’s surface of 0.77 units at levels
near 2000ppm, predicted by year 2300, with potential profound ecological shifts in marine
ecosystems (Doney et al., 2009).
The steep changes in atmospheric CO2 and global temperatures over relatively short
time scales are expected to have extensive effects on the physical and biological dynamics of
the world’s oceans. These changes could impact ocean temperature, weather patterns,
biogeochemistry, salinity levels, sea level, UV radiation, current circulation patterns,
stratification, upwelling, ocean chemistry, and oxygen levels. Increased ocean temperature and
acidification can affect all marine species’ physiology, including reproduction, growth and
survivorship. The biological and physical changes can act together to affect marine ecological
communities by impacting species distribution, abundance, biogeography, and species
interactions (Rilov & Treves, 2010).
It is unclear how a shift in carbonate chemistry will affect non-calcifying photosynthetic
organisms like kelp, but the increase in carbon dioxide and bicarbonate is likely to increase the
concentration of carbon available for photosynthesis. Most marine autotrophs are characterized
9 by C3 carbon fixation (Koch et al., 2013), which allows organisms to utilize inorganic carbon
from both dissolved carbon dioxide (CO2) gas and the much more abundant bicarbonate (HCO3) ions. Bicarbonate ions are utilized through a set delivery of inorganic carbon to the Rubisco
enzyme by a carbon concentrating mechanism (CCM) (Lucas, 1983; Badger & Price, 1994).
Carbon concentrating mechanisms work with carbonic anhydrase to form CO2 from HCO3through a dehydration reaction, allowing algae to draw from a much larger carbon pool for
photosynthesis.
Both gametophytes and sporophytes of kelp species have detectable intracellular and
extracellular carbonic anhydrase activity, and both are capable of utilizing bicarbonate, though
sporophytes have a greater capacity for HCO3- use than gametophytes (Zhang et al., 2006).
External carbonic anhydrase activity allows Saccharina latissima to maintain photosynthetic
rates over a wide range of pH (7.0-9.5) (Axelsson et al., 2000), though rates begin to decline at
high pH, at values above 8.5 (Blinks, 1963). While CCM’s allow most macroalgae to utilize the
greater HCO3- carbon pool, an increase in availability of the preferred CO2 is expected to
somewhat increase photosynthetic and growth rates (Koch et al., 2013).
While the increase in available and preferred dissolved CO2 could enhance
photosynthetic rates, the accompanying change in pH could affect macroalgae in other ways.
Effects of reduced seawater pH on macroalgae have so far been variable; with minimal to no
differences as well as positive and negative responses (Israel & Hophy, 2002). In areas of
natural acidification near underwater vents, large brown macroalgae (Ochrophyta) remain the
dominant canopy forming species across a CO2 gradient with a pH range from 8.20-6.07 (Porzio
et al., 2011). In laboratory studies, very high levels of CO2 (3000ppm) inhibited growth of the
sporophytes of Saccharina latissima, but enhanced growth of kelp species Nereocystis
luetkeana (Swanson & Fox, 2007). There appears to be a difference in response according to
the level of dissolved inorganic carbon (DIC) present, where net apparent productivity of N.
luetkeana sporophytes was enhanced at two times ambient CO2, but reduced at five times the
10 ambient levels (Thom, 1996). The red seaweed Lomentaria articulata responded in a similar
way, with increased growth at two times ambient levels, but not at five times (Kubler et al.,
1999). Macrocystis pyrifera (giant kelp) gametophytes were 32% larger at moderate (7.86) pH,
but only 10% larger at low pH levels (7.61) (Roleda et al., 2012). High levels of dissolved
inorganic carbon (1200 ppm) and resultant low seawater pH (7.61) had minimal effects on
meiospore germination rate, gametophyte sex ratio and size of M. pyrifera gametophytes,
though very high levels decreased spore germination by 6-9% (Roleda et al., 2012).
Kelp population structure and dynamics are determined by successful life stages, which
include spore output, dispersal, recruitment of meiospores, development and survival of
gametophytes, successful reproduction, and development and survival of the juvenile
sporophytes. These stages are primarily controlled by environmental factors including
temperature, light, photoperiod, and nutrient availability, all of which could be affected by a
changing ocean. Very little is known about species-specific responses to interacting variables of
increasing temperature and CO2 (Koch et al., 2013). An increase of CO2 can offset high
temperature stress in some terrestrial C3 plants, but increased temperatures may reduce
availability of dissolved CO2 in seawater, due to a decrease in solubility (Beardall et al., 1998).
Little is known about the duration of the ‘dormant’ microscopic stages of the kelp, or how ocean
acidification might affect gametogenesis and successful production of sporophytes.
The southern distribution limits of cold-water algal species can be characterized by
summer isotherms (Setchell, 1915, 1920), where critical maximum summer temperatures limit
growth, reproduction, or survival (van den Hoek, 1975; 1982; Yarish et al., 1984; 1986;
Breeman, 1988). Seaweed populations located at the edge of their distribution can adapt to
stressful summer conditions (Humm, 1969), but the existence of populations at these
boundaries depends on individual temperature tolerances or reproductive requirements
(Hutchins, 1947). North Atlantic summer isotherms show considerable interannual variability
11 with a general northward trend since 1993, occurring at northern limits with an increasing
frequency (Hobson et al., 2008).
Long Island Sound currently experiences summer surface temperatures up to 23-25°C
(2012, see fig. 2), with sustained temperatures above 20°C for 6 weeks or more, while the Gulf
of Maine has a lower maximum summer temperature, reaching up to 18-22°C. Projected
increases of sea surface temperature will push the summer maximum above upper survival
temperatures for gametophytes and sporophytes of Saccharina latissima in Long Island Sound,
and could stress populations in the Gulf of Maine. The upward trend of higher summer
temperatures could impact the distribution of Saccharina latissima in the Northwest Atlantic,
Sea Surface Temperature (°C) shifting the southern distributional limit north of Long Island Sound.
2012 30 ME (Buoy 44007) 25 LIS (Buoy 44039) 20 15 10 5 0 J F
M
A
M
J J A
S O
N
D
Figure 2. NOAA National Data Buoy Center data for 2012. Graph shows actual daily afternoon
recorded water temperatures. Gulf of Maine (GOM) buoy 44007, 12 NM Southeast of Portland, ME
(http://www.ndbc.noaa.gov/station_page.php?station=44007). Long Island Sound (LIS) buoy 44039,
Central Long Island Sound (http://www.ndbc.noaa.gov/station_page.php?station=44039).
The objectives of this study were twofold; to compare two distinct populations from
different seasons from the Gulf of Maine and Long Island Sound to determine if there were any
ecotypic differences in upper survival temperature thresholds for gametophytes and juvenile
12 sporophytes, and to investigate effects of increased carbon dioxide and reduced pH on the
gametophyte and juvenile sporophyte stages.
The microscopic stages of these two populations from summer and fall were compared
to investigate differences in response to projected increases of pCO2 and temperature. Impacts
on meiospore settlement, germination, growth, male to female ratios, and reproductive success
were investigated under multifactorial temperature (16, 19, 22, 25 & 28°C) and dissolved
inorganic carbon (400, 800, 1200, & 1600ppm) conditions.
Methods and Materials
Three separate trials were conducted with spores obtained from wild-collected
sporophytes from Maine and Long Island Sound, and are represented as; Maine summer
(MES), Maine Fall (MEF), and Long Island Sound Fall (LISF). All three trials were successful,
but a fourth, the Long Island Summer population trial, was not successful after two separate
attempts, due to contamination in each trial by predatory protozoa.
Reproductive plants were collected by snorkeling at low tide at approximately 5-7 meter
depths near Simonton Cove in South Portland, Maine, (coordinates 43.6415; -70.2236) in July
and September of 2011, and by SCUBA at Black Ledge (coordinates 41.3088; -72.0648) in
Long Island Sound in October of 2011 (fig. 3). Reproductive fronds were immediately processed
by excising reproductive sorus tissue and scraping, rinsing, scrubbing, and wiping clean, and
then placed between layers of clean damp paper towels. Plants were transported to the lab on
ice and kept in the dark at 10°C overnight.
Sorus tissue was submerged in 10°C sterilized seawater 20-23 hours after sorus
processing for spore release. Three hundred cut glass microscope slides (25x25mm) were
inoculated by pipetting 10 mL of spore solution into petri dishes containing 25mL of half-strength
Provasoli’s Enrichment Seawater (PES/2) (Provasoli, 1968). The spores were allowed to settle
undisturbed and germinate on the slides for 48 hours at 15°C.
13 Temperature gradient tables at the UCONN Stamford Marine Biotechnology Lab (Yarish
et al., 1979) were used to create 20 different environmental conditions of temperature and pCO2
values. Dishes were placed on five rows of temperature levels that were set at 16, 19, 22, 25,
and 28°C, and four levels of pCO2 (at 1x, 2x, 3x and 4x ambient atmospheric levels) were
bubbled in the dishes. Each condition of temperature and pCO2 had three total replicates,
randomly arranged on the gradient table, for a total of 60 dishes.
Each dish was aerated with the appropriate pCO2 level of air, which was mixed in 13-liter
glass stoppered Pyrex carboy bubbling bottles by combining filtered ambient air and varying
levels of CO2. Approximate levels of pCO2 were set at 400 ppm, which is an average ambient
atmospheric concentration, and 800ppm, 1200ppm, and 1600ppm. These four levels
corresponded to average pH values of 7.6, 7.7, 7.8, and 7.9. Air-CO2 mixtures were determined
with an Extech CO250 CO2 analyzer, and pH and temperature determined by an Accumet pH
meter.
Lighting above the tables consisted of cool white high output T-12 fluorescent bulbs set
at a photon fluence rate of 20 µmol photons m-2 s-1 during the first week, 40 µmol photons m-2 s-1
during the second week, and 60 µmol photons m-2 s-1 the third week, based on investigations on
light requirements for growth and induction of fertility of gametophytes (Lee & Brinkhuis, 1988,
Lüning, 1980, Egan et al., 1989) and optimal development of juvenile sporophytes (Lee &
Brinkhuis, 1988, Fortes & Lining, 1980). Photon flux density was measured with a LI-250 light
meter (LI-COR, Nebraska). Photoperiod was day neutral at 12:12; L: D.
Percent germination under each condition was determined by pipetting 20mL of spore
solution onto cut glass slides in 150mL PES/2 sterilized seawater in deep petri dishes at each
condition of temperature (16, 19, 22, 25, 28°C) and pCO2 (400, 800, 1200 & 1600 ppm). The
deep petri dishes were gently aerated at the surface of the water in each dish so that the pH
levels could be established without greatly agitating the water. Spores were allowed to settle
and germinate in each condition on the gradient table for 48 hours and percent germination was
14 determined on each slide on the basis of germ tube production per 100 randomly selected
spores. All of these slides were removed and new dishes were prepared for the culture of
gametophytes that were previously germinated at 15°C. These slides were rinsed with sterilized
seawater and placed in deep storage petri dishes (300 mL) on the gradient table at each
condition of temperature and pH in PES/2. Germanium dioxide (GeO2) was added for the
inhibition of diatoms (Lewin, 1966).
Seven days after placing germinated spores on the gradient table, one slide was
randomly chosen from each dish and photographed under a compound microscope to record
growth. This was done again at day 14 (week 2), day 21 (week 3), and day 28 (week 4).
Survival, growth, sex ratio, fertility, and sporophyte production and growth were monitored for
each condition of temperature and atmospheric CO2. Temperature and pH measurements were
taken at the end of each week with an Accumet pH meter. The meter was calibrated with 6, 8,
and 10 pH buffers before measurements were taken. All dishes were changed once per week
and slides were transferred into fresh PES/2 + GeO2 seawater media after each dish had
equilibrated to the appropriate temperature and pH level.
Photographic images were analyzed with ImageJ software (Abramoff et al., 2004) for
germination, primary cell diameter, female total length, fertility and reproductive success, and
sporophyte lengths. 50-100 measurements from each dish were taken for germination, primary
cell, length and width measurements.
15 46 44 42
ME
Gulf of Maine
NY
CT
40 Long Island Sound
Summer Sea Surface temperatures in degrees Celsius, 08/02/2013
11.5 13.6 15.7 17.8 20.0 22.1 24.2 26.3 28.4 Figure 3. Map of Northeast Sea Surface Temperatures for 08/02/2013, showing sample collection
sites (stars). Adapted from NOAA/NESDIS Geo-Polar Blended 5km SST Analysis for the North Atlantic,
at http://www.ospo.noaa.gov/data/sst/contour/natlanti.cf.gif.
Statistical Analysis
For each population measure full factorial Analysis of Variance (ANOVA) models were
estimated to identify any interactions between factors of pH, temperature, and weeks where
applicable. To correct for potential pseudo-replication, the average of all measurements taken
within each dish were calculated and used for analysis. Only results from temperature levels of
16°, 19°, and 22°C were analyzed because there were no results at temperature levels 25° and
28°C due to mortality. Model-derived least square (adjusted) means and 95% confidence
intervals were subsequently estimated. Variables were log transformed to meet assumptions
when necessary. For variables that were log-transformed, least square means were reported as
16 geometric means after exponentiation. Residual analysis was conducted for all statistical
models to verify the assumptions of normality and homogeneity of variance. Analysis of
Covariance (ANCOVA) was used to adjust for nuisance variables, when initial results indicated
that covariates were obscuring the results. For variables where a suitable transformation could
not be found, the Kruskal-Wallis non-parametric ANOVA was used when there were more than
two groups being compared and the t-test (assuming unequal variance) was used when
comparing two groups. Least square means are plotted as scatter plots, where the individual
dots are the estimated means and the error bars are the 95% confidence intervals. All analyses
were performed using SAS® 9.3 (Cary, NC) and two sided statistical significance was
considered at p<0.05 unless otherwise noted.
Results
Germination
Percentage germination for each replicate was determined by counting the total number
of germinated spores, which are meiospores that have attached and developed a germ tube,
per 100 randomly selected individuals 48 hours after inoculation of dishes on the gradient table.
Temperature effects on germination were highly significant in all trials (P<0.0001). Data
from all three trials were analyzed for normality and variance using residual diagnostics. There
were no violations of the assumptions of normality or variance for trials 1 (Maine summer) and 3
(Long Island Sound Fall), but there was substantial heterogeneity of variance for trial 2 (Maine
fall). To further investigate this finding, a t-test for unequal variances was conducted, comparing
values at 16° and 19° together to 22°C, indicating a significant effect of temperature on
germination (P=0.001).
Average germination rates were similar at temperature levels 16°C and 19°C, at around
95%, with no effects of pH. Germination rates dropped drastically at 22°C, with great variability
17 between replicates, pH levels, and trials, and rates were 0% at 25 and 28°C due to mortality (fig.
4).
Effects of different pH levels on germination were significant only in the Maine summer
trial (P = 0.021), but not significant for either the Maine fall (P=0.92) or Long Island Sound fall
(P=0.55) trials. Interactive temperature and pH effects were significant for the Maine summer
trial (P=0.02), but not for the Maine or Long Island Sound fall trials (P=0.78 and P=0.73).
Results from all three trials show similar temperature effects on germination (fig. 3). The
first trial (Maine-summer) had the least variability between pH levels at 22°C, and also the
lowest germination success, all which fall below 40%. Trials 2 (Maine-fall) and 3 (Long Island
Sound-Fall) had higher germination success at 22°C (between 30-90%), with increased
variability among the four different levels of pH. There were no significant effects of pH on
germination rates for any of the trials.
Figure 4. Percent germination by temperature grouped by pH for all three trials.
18 Female Gametophyte Diameter
Female gametophyte diameter was measured as the diameter of the female primary cell
7 days after germination (fig. 5). Data were log transformed and the geometric mean was used
for plotting results, omitting temperature levels 25° and 28°C due to full mortality.
Temperature effects on female cell diameter were highly significant in all trials
(P<0.0001). Maine summer and fall diameters were very similar at 16° and 19°C, where values
were only slightly larger by approximately 1 micron on average at 16°C for both trials. The Long
Island Sound trial produced larger female gametophytes than either of the Maine trials at all
temperature levels, and gametophytes were slightly larger at 19°C than at 16°C. Diameters
were approximately 4 microns smaller in all trials at 22°C than at 16 or 19°C. Maine fall
diameters were slightly larger than the Maine summer trial. There were no significant effects of
pH or pH-temperature interactive effects on gametophyte diameter for any of the trials.
M E Summer
M E Fall
LIS Fall
18
Geometric Mean Diameter (µm)
16
14
12
10
8
16 °C
19 °C
22 °C
16 °C
19 °C
22 °C
16 °C
Temperature
pH
7.6
7.7
7.8
7.9
Figure 5. Female gametophyte diameter (µm) at one week after germination.
19 19 °C
22 °C
Female Gametophyte Length
Female gametophyte length was measured as the total length at the longest point of
randomly selected female gametophytes. Data were log transformed due to slight non-constant
variance and checked for normality. Results are presented using adjusted geometric means,
omitting temperature levels 25° and 28°C due to full mortality (figures 6-8).
Temperature effects on female gametophyte length were highly significant in all trials
(P<0.0001). Total length measurements of female gametophytes were very similar at
temperatures 16° and 19°C for all trials, with values only slightly lower at 19° than at 16°C, a
difference that was more pronounced in the Maine summer trial (fig. 6) than the two fall trials.
Geometric mean growth rates over three weeks were highest at 16°C in all three trials, though
rates were only slightly lower at 19°C (table 1). Females were significantly shorter at 22°C in all
trials, with slower growth rates, the Maine summer trial showing the lowest rates at 14%, and
the two fall trials showing similar rates at 70% and 69%.
There were significant effects of pH (P=0.002) on length in the Maine fall trial (figure 7),
but not in the other two trials. In some cases gametophytes exposed to the lower pH values (7.6
& 7.7) were slightly longer than the higher pH levels (MES at 19°C, weeks 1-3, MEF at 19°C,
weeks 2-3, and LISF at week 2). There were no significant interactive effects of pH and
temperature in any of the trials.
20 population=ME Summer
16 °C
19 °C
22 °C
180
140
Geometric Mean Length (µm)
100
80
60
40
20
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
Weeks
pH
7.6
7.7
7.8
7.9
Figure 6. Female Gametophyte Length (µm) for Maine summer trial at weeks 1-3.
population=ME Fall
16 °C
19 °C
22 °C
180
140
Geometric Mean Length (µm)
100
80
60
40
20
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
1 Week
Weeks
pH
7.6
7.7
7.8
7.9
Figure 7. Female Gametophyte Length (µm) for Maine fall trial, 1-3 weeks.
21 2 Weeks
3 Weeks
population=LIS Fall
16 °C
19 °C
22 °C
180
140
Geometric Mean Length (µm)
100
80
60
40
20
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
Weeks
pH
7.6
7.7
7.8
7.9
Figure 8. Female gametophyte length (µm) for Long Island Sound trial, weeks 1-3.
Table 1. Table of rate of geometric mean growth rate of female gametophytes
Population
Temperature
(°C)
ME Summer
16
19
22
16
19
22
16
19
22
ME Fall
LIS Fall
22 Geometric Mean
Growth Rate
(% Increase/Week)
110%
78%
14%
122%
119%
70%
124%
108%
69%
3 Weeks
Cell Number of Female Gametophytes
Cell number was determined by counting the number of cells of randomly selected
female gametophytes for weeks 1-3. Data were log transformed due to skewed distribution.
Temperature effects on female gametophyte cell number were highly significant in all trials
(P<0.0001). All gametophytes at 16° and 19°C developed an average increase in cell number
from week one to week three, increasing cell number by approximately two cells per week (figs.
9-11). There was very little growth and much less variability at 22°C, with most values at 1 cell
and fewer at 2 cells. Median values increased more from week 1-2, with a slower increase from
week 2-3. Values ranged from 1 to 31 cells per individual, and unicellular gametophytes could
be found at all weeks. Gametophyte size was highly variable, ranging from a unicellular
individual to a short filament to a many-celled cluster of cells at both 16° and 19°C and at all pH
levels.
There were significant effects of pH (P=0.001) on cell number in the Maine fall trial (fig.
9), but not in the other two trials. Maine and Long Island Fall values were very similar at 22°C,
with more cells produced at lower pH values (7.6, 7.7, 7.8) than at 7.9. This trend was not seen
in Maine summer values (fig. 8), where most gametophytes were only one cell. Effects of pH
were variable, with no obvious trend.
23 population=ME Summer
16 °C
19 °C
22 °C
8
6
5
Cell Number
4
3
2
1
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
Weeks
pH
7.6
7.7
7.8
7.9
Figure 9. Cell number of female gametophytes, Maine summer trial.
population=ME Fall
16 °C
19 °C
22 °C
8
6
5
Cell Number
4
3
2
1
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
Weeks
pH
7.6
7.7
7.8
7.9
Figure 10. Cell number of female gametophytes, Maine fall trial.
24 3 Weeks
population=LIS Fall
16 °C
19 °C
22 °C
8
6
5
Cell Number
4
3
2
1
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
1 Week
2 Weeks
3 Weeks
Weeks
pH
7.6
7.7
7.8
7.9
Figure 11. Cell number of female gametophytes, Long Island Sound fall trial.
Male: Female Ratio
Male: female sex ratio was determined by counting total number of males and females
from 100 randomly selected individuals during week 2 of each trial. Total number of males was
divided by total number of females, with a number of 1 representing equal numbers of males
and females, n>1 = more males than females, and n<1 = more females than males. The
Kruskal-Wallis test (table 2) was used to determine if temperature effects for mean values were
significant. Temperature had a significant effect on male to female ratios for the Long Island
Sound trial, but not on the other two trials. Gametophytes at 22°C did not develop enough to
determine sex, and there was full mortality at temperatures of 25 and 28°C, so only ratios at
16°C and 19°C are reported.
There were no consistent effects of pH on Male: Female sex ratios. Average values for
the Maine summer trial clustered around 1.0 at 16°C, and showed a greater range of values at
25 19°C, from 0.8 to 1.2 (fig. 12). Maine fall values ranged from 0.7-1.1 at 16°C, and 0.8-1.2 at
19°C. The LIS fall trial ranged from 0.6-1.1 at 16°C, indicating a trend towards more females
than males, but this result was more equally spread around 1.0 at 19°C. Overall, there was a
tendency towards n<1, indicating slightly more females to males within all trials.
Figure 12. Male: Female sex ratio plot (Male/Female gametophytes per 100)
Table 2. Kruskal-Wallis Test for Male: Female ratio results
Kruskal-Wallis Test
FME
SME
Chi-Square
DF
Pr > ChiSquare
FLIS
10.0833
0.0953
0.0038
1
1
1
0.0015
0.7575
0.9509
26 Fecundity
Fecundity is a measure of reproductive females. Fecundity was determined as the ratio
of total fertile female gametophytes, which included eggs and sporophytes, to the total females
present at 14 and 21 days after germination. The fecundity ratio is a measurement of the
development and degree of gametogenesis, where females become reproductive and begin to
release eggs for fertilization.
Temperature was highly significant for all trials. Gametogenesis was not observed at
temperatures of 19° or 22°C. There were no significant effects of pH or pH and temperature
interactions on fecundity (table 3). Since pH did not have a significant effect on fecundity, all
values were plotted across the range of actual observed temperatures. Observed temperatures
were plotted to visualize temperature effects, to observe effects of fine scale temperature
differences experienced on the gradient table (fig. 13).
The gradient table had a range of values around the set temperature level, and there
was an interesting pattern of decreasing fecundity values with increasing temperatures within
the range of 15-17°C, with no gametogenesis occurring above 17.2°C (table 4). Fecundity
increased with time, from 2 weeks to 3 weeks, for all trials. Maine summer and fall trials
exhibited similar values for both weeks, while the Long Island Sound had lower values of
fecundity overall for both weeks.
Fecundity ANOVA Results
Effect
MES
MEF
LISF
pH
0.9221
0.8602
0.8653
weeks
0.0524
0.0033
0.3141
pH. / weeks
0.9747
0.9662
0.9921
Temperature
<.0001
<.0001
0.0018
pH. / temp
0.9647
0.902
0.9102
Weeks / temperature
0.0089
<.0001
0.2749
Table 3. ANOVA results for
fecundity
P-Value
27 Figure 13. Fecundity values plotted with observed gradient table temperature values
Table 4. Observed critical limits for female gametogenesis
Trial
Week 2 (°C)
Week 3 (°C)
ME Summer
17.1
17.2
ME Fall
16.8
16.9
LIS Fall
16.8
17
28 Reproductive Success
Reproductive success was determined as the ratio of sporophytes to the total number of
female gametophytes 14 and 21 days after germination. Different levels of pH had no consistent
effect on reproductive success, though the lowest pH (7.6) resulted in slightly more sporophytes
than the other pH levels in the LIS fall trial. Reproductive success increased from week 2 to
week 3 at 16°C, and sporophytes were not produced at temperatures above 16°C. All trials
showed similar patterns, with fewer sporophytes produced during the LIS fall trial than the two
other ME trials (fig. 14).
Figure 14. Reproductive success for all trials at 2 and 3 weeks
29 Sporophyte Length
Sporophyte length was determined from temperature level 16°C (there were no
sporophytes produced at 19°C or above) in week three and week four (fig. 15). Values were log
transformed to account for exponential growth from week 3 to week 4. ANCOVA was used to
determine the effect of the actual temperatures on the gradient table, so observed temperature
was initially included as a covariate. Observed temperature did not have significant interaction
effects with weeks or pH, indicating that the observed gradient temperature was not a significant
factor and was dropped from the model. Re-running the analysis without the observed
temperature showed weeks as the only significant factor (P < 0.0001), while pH and the pH x
Weeks interaction were insignificant (P= 0.7886 and P=0.3372 respectively). Geometric growth
rates were calculated to look at relative growth from week 3 to 4 at the four levels of pH at 16°C
(table 4). The lowest pH (7.6) had the highest growth rate, while pH 7.8 had the lowest growth
rate.
Geometric Mean Sporophyte Length (microns)
pH = 7.6
pH = 7.7
pH = 7.8
pH = 7.9
800
600
400
200
0
3
4 3
4 3
Weeks
mean
4 3
4
mean
Figure 15. Geometric mean of juvenile sporophyte length for all values at weeks 3 and 4.
30 Table 4. Geometric mean growth rates for
juvenile sporophytes at 16°C
Geometric Mean Growth Rate
pH
(% Increase/Week)
7.6
7.7
7.8
7.9
345%
147%
63%
171%
Discussion
Temperature is a major environmental factor controlling the success of microscopic
stages of Saccharina latissima, where major changes in survival, growth, and gametogenesis
can occur within just a few degrees of the upper limits, while decreases in seawater pH have
slight and variable to no impacts. Gametophytes were cultured at five temperature levels (16°,
19°, 22°, 25°, 28°C), representing a range of optimal to lethal temperatures for the microstages
of S. latissima. Gametophytes experienced full mortality in all three trials at temperatures of 25°
and 28°C, with 22°C representing an upper critical limit, with significant mortality and little
growth or development of gametophytes. The same patterns of temperature response (slightly
higher or similar values for 16°C compared to 19°C, with a large decrease in values at 22°C)
were observed in all three trials for percent germination, primary cell diameter, and female
length and cell numbers over three weeks.
Germination and male and female growth were very similar at 16° and 19°C, but greatly
reduced at 22°, and full mortality at the higher temperatures of 25° and 28°C. This indicates that
there is a critical shift in the ability of spores to germinate and develop at 22°C, an upper limit
where cells are under significant temperature stress. This value is slightly lower than reported
values within the range of 23-25°C (Egan et al., 1989; tom Diek, 1993), but is within the 18-23°C
range reported by other studies (Lüning, 1980; Bolton & Lüning, 1982; Kain, 1969; Breeman,
1988; Lüning, 1984). Growth rates of female gametophytes at 16° and 19° were similar, but
31 gametogenesis was completely inhibited above 17°C for all trials. This is similar to values
reported by other studies (Egan et al., 1989; Bolton & Lüning, 1982; Yarish & Egan, 1989;
Lüning, 1984; Lee & Brinkhus, 1988, see table 5). All gametophytes increased in size over time.
Table 5. Comparison of reported critical temperature limits (°C) for Saccharina latissima.
Study
Germination
optimal
Egan et al.,
1989
Lee and
Brinkhuis,
1988
max
5-15
25
Growth
range
10 -20
upper
25
Gametogenesis
lethal
25
10 - 20
Lüning, 1980
Bolton &
Lüning, 1982
Yarish &
Egan, 1989
tom
Diek,1993
Kain, 1969
range
5-19
20
Long Island Sound
7-14
17
Long Island Sound
20 - 21
22
10-15
10 -15
18-22
23-24
5-18
<18
5-20
<21,23
22
<22
>13
16
Harries, 1932
23
24
21
22
22
22
Long Island Sound
N. Atlantic
NE Pacific
>16
Breeman,
1988
Lüning, 1984
Helgoland, North
Sea
N. Atlantic,
latitudes 44°N-69°N
NE Atlantic (mid
range)
N. Atlantic
23-25
Schreiber,
1930
Druehl, 1967
This study
max
10 - 19
10 -20
Location
Irish Sea, NE
Atlantic
N. Atlantic
15
18
17
North Sea
Long Island Sound,
Gulf of Maine
Population response differed, most notably between the female primary cell diameters.
Long Island Sound cultures were larger than Maine trials at all temperatures, and gametophytes
at 19°C were slightly larger than at 16°C, where the reverse was true for both Maine trials.
Gametophytes obtained in the summer months had lower germination success, smaller primary
cell diameters, shorter lengths, and fewer cells at 22°C than both fall trials, indicating a seasonal
difference. Altering pH did not result in any obvious or consistent effects though lower pH values
32 (7.6-7.7) did result in slightly better growth (primary cell diameter; MES and MEF at 16 and
19°C, gametophyte length: MES at 19°C, MEF and LISF at 16 and 19°C) than the highest value
(7.9).
Ecotypes are populations within a species that have adapted to local conditions,
displaying differential responses to given environmental variables. Several studies have
attempted to identify Saccharina latissima ecotypes, with results ranging from no evidence of
ecotypic response (Breeman, 1988; Lüning, 1984) to distinct differences in upper survival
temperatures between geographically distinct populations (Gerard, 1990; Bruhn & Gerard,
1996; Lüning, 1975, Lüning et al., 1978; Müller et al., 2008). Seaweed populations from the
southernmost portion of their distribution often exhibit higher temperature tolerance (Breeman,
1988), and this has been observed in gametophytes and sporophytes from Long Island Sound
(Egan et al., 1990; Gerard, 1990), though in my study the upper critical temperature limit was
the same for the Maine and Long Island Sound populations, at 22°C. There were small
differences between the two populations, however, in primary cell diameter, where Long Island
Sound female gametophytes were larger in diameter than the Maine gametophytes at all
temperatures.
Water temperature is the most important factor determining distribution of seaweed
species (Lüning, 1990), so a predicted increase of seawater temperature could shift the lethal,
reproductive, and growth limit isotherms for Saccharina latissima northward (Bartsch, et al.,
2008), though no major latitudinal shifts have been observed to date in the subarctic and boreal
zones in the Western Atlantic in the past century (Merzouk & Johnson, 2011). Southern Maine
kelp populations experience upper summer maximum temperatures of 21-23°C, with sustained
temperatures of 18-20°C (fig. 2). An increase of 2°C above 19°C could be physiologically
stressful or lethal for southern Maine and New England populations of kelp sporophytes.
Vegetative growth of gametophytes will likely increase at temperatures above 15°C, with an
inhibition of gametogenesis above 17°C (this study; Lüning, 1980; Lee & Brinkhuis, 1988; Bolton
33 & Lüning, 1982; Harries, 1932; Druehl, 1967; Yarish et al., 1990), and increase in mortality
above 22°C (this study; Lüning, 1980; Bolton & Lüning, 1980). Growth rates of sporophytes
could increase for kelp populations located in the middle and upper latitudes up to a point
(Coelho et al., 2000), since photosynthetic rates rise with increasing temperatures, but above
optimal temperatures, rates decline rapidly (Druehl, 1967).
Distributional boundaries are coinciding extremes of environmental conditions. A
sustained, viable kelp population depends on water temperatures that do not either exceed
individual critical survival temperatures of sporophytes or gametophytes, or inhibit the
settlement, germination, or reproduction of gametophytes. In Long Island Sound, the warm
temperature Western Atlantic boundary for Saccharina latissima, sea surface temperatures
have already increased an average of 1°C in just over 2 decades (1979-2002), with summer
temperatures peaking at 22-23°C (Keser et al., 2005). Summer seawater temperatures in
Western Long Island Sound are already 2-3 degrees higher than temperatures in Eastern Long
Island Sound, (Howard-Strobel et al., 2000), so it would be interesting to compare the presence
or absence of kelp beds in Western Long Island Sound compared to Eastern Long Island Sound
to evaluate the impact of summer temperatures rising above the 22°C upper critical thermal
limit. As this warming trend is predicted to continue (IPCC, 2013), with an increase of 3°C by the
end of the century, maximum summer temperatures in all of Long Island Sound are likely to
exceed upper survival temperatures for sporophytes and gametophytes.
Sporophytes are annuals or biennials in Long Island Sound (Pedersen et al., 2008),
which means that either sporophytes or gametophytes must survive the summer temperatures
to produce new generations every year (Müller et al., 2009). Upper survival temperatures are 15°C higher for gametophytes than sporophytes, so it is likely that the gametophyte stage is the
dominant survival stage. Lee & Brinkhuis (1986) first suggested that gametophytes were the
over summering stage in Long Island Sound, but later retracted that hypothesis in favor of
sporophytes (1988). They observed that first year sporophytes were most evident in the field in
34 May-June and early autumn, but whether these are from previously existing microscopic stages
or new recruits is unknown. The peak reproductive periods for sporophytes occurs in the fall
(September-October) at periods of decreasing temperatures of 18-8°C (Van Patten & Yarish,
1993), which would produce young sporophytes for the late fall and winter season, but would
not account for the early fall sporophytes.
High summer temperatures may significantly affect meiospore germination, though there
is evidence of seasonal acclimation to germination (Egan et al., 1989). Meiospore germination
success was similar across both Maine and Long Island Sound populations and both summer
and fall trials at temperatures of 16-19° C, but was greatly reduced at 22° C. Both fall trials were
more successful (~60%) than the summer trial (~20%).
Southern range gametophytes would likely be vegetative throughout the summer at
temperatures of 17- 22°C, which typically occurs between June-July and September-November
in Long Island Sound. Gametogenesis was initiated only at temperatures below 17° C in this
study, and high levels of mortality experienced above 22°. Egan et al. (1989) suggested that
exposure to high temperatures in summer could suppress development and growth of
microscopic stages. However, there are other factors that can influence gametogenesis,
including interacting factors of light, temperature, temperature history, and nutrient availability.
Initiation of gametogenesis at higher temperatures (>15° C) requires exposure to higher photon
flux than at lower temperatures (Lüning, 1980), so it is possible that gametogenesis occurs at
summer temperatures above 17° C. There could also be a seasonal temperature acclimation for
fecundity (Costa, 1989), as observed in trials of acclimated vegetative gametophytes cultured in
the lab (Bolton & Lüning, 1982).
For mid range populations, gametogenesis has been observed to occur at all times of
the year in areas of maximum summer temperatures of 16°C (Hsaio, 1972). In these
populations, the establishment of new macroscopic sporophytes is limited not by
gametogenesis but by response of the juvenile sporophytes to environmental factors (Hsaio,
35 1972). Maximum summer temperatures in the Gulf of Maine are consistently above 16°C from
July to September, fluctuating from 16-20°C. Both gametogenesis and vegetative growth
probably occurs throughout the summer in the Gulf of Maine, with the production and success of
macroscopic sporophytes a function of the interaction of physical environmental variables and
other biological variables, such as competition and herbivory.
While male and female gametophytes are released at a 1:1 ratio (Schreiber, 1930), there
have been variable results reported for M:F sex ratios at higher temperatures. More males than
females have been observed at higher temperatures (Lee & Brinkhus, 1988; Kain, 1979; Bolton
& Lüning, 1982), but females had a slightly higher upper survival temperature (tom Dieck,
1993), and have been observed to be more prevalent under stressful conditions (Uller et al.,
2007). The M:F ratio ranged around the typical 1:1 ratio at temperatures of 16-19° C,
temperatures within the “broad range” of S. latissima, though gametophytes at 22° did not
develop enough to determine sex.
Egan et al. (1989) suggested that the summer strategy for gametophytes is to produce
sporophytes rapidly, after observing new recruitments in LIS just one month after setting lines
out in eastern coastal Long Island Sound in June of 1985. Yarish (unpublished data) has also
observed new recruits in western and central Long Island Sound on lines set out in 2012 and
2013. Kelp meiospores have a limited range of dispersal (Dayton, 1985) so new recruits indicate
the presence of a reproductive population somewhere within the vicinity. Summer survival and
presence of new recruits throughout the year may be an indication of possible deep-water kelp
refugia populations within Long Island Sound. Deep water kelp forests have recently been
discovered in tropical waters where warm temperatures and low nutrient levels are not
otherwise adequate for kelp survival (Graham et al., 2007). Mathieson (1979) observed
Saccharina latissima populations at 26 meters depth in New Hampshire, and found that deeper
water sites supported more perennial populations compared to shallower sites with a wider
range of temperatures. Transplantation experiments also observed positive net growth
36 throughout the summer at depths of 6, 9 and 12 m, compared to a net decrease in shallow
water individuals (Boden, 1979). Deep water populations may be able to support sporophyte
populations through stressful summer temperatures with colder, more stable temperatures and
higher levels of nutrients available below the thermocline (van den Hoek, 1982).
Mean surface sea temperatures are a very general measurement of actual water
temperatures, averaged over time. The upper surface layers of the ocean are typically warmer
in the summer than the deeper, colder layers experienced by subtidal populations (van den
Hoek, 1982). While experimental temperatures can provide some insight into critical upper
limits, the ability for southern limit populations to persist above upper limits may be due to lower
actual temperatures experienced by deeper populations, or their ability to withstand short-term
exposure to higher temperatures. Macroalgae can often recover from short term exposure to
stressful super-optimal temperatures, but as overall temperatures rise, there will be increasing
episodes of exposure of longer durations to upper survival temperatures, and populations will
need to adapt to these exposures, or will disappear from these areas.
The increase in dissolved seawater carbon concentrations predicted for this century and
the resultant decreases in pH are not likely to directly impact Saccharina latissima
gametophytes. There were no obvious effects of decreased seawater pH on the microscopic
stages in this study, even up to 4 times higher than current levels. Subtidal kelp populations
already experience local variability and exposure to low levels of pH in coastal areas, due to
physical (e.g. upwelling, freshwater inflows, temperature, tidal exchange) (Hunt et al., 2011),
seasonal (e.g. lower pH values during winter) (Hsaio, 1972), and daily natural cycles from the
biological processes of photosynthesis and respiration (e.g. 2.4 – 1.05 units over one day
(Wootton et al., 2008, Delille et al., 2000)). Indirect effects of decreased pH could include
changes in seawater chemistry and nutrient availability (e.g. increased iron bioavailability
(Breitbarth et al., 2010)) or decreases in herbivory pressure by impacting key shell-forming
predators (i.e. echinoderms and snails).
37 Areas of natural local acidification from underwater volcanic vents show overall shifts in
benthic community composition, with a decrease in overall biodiversity. Algal groups in these
pH-shifted environments are successful, while coralline species (corals and sea urchins) decline
and disappear along gradients toward the vents as CO2 levels increase (804-957µatm pCO2)
and pH levels decrease (7.8-7.4) (Hall-Spencer et al., 2008). Green sea urchins
(Strongylocentrotus droebachiensis) and the herbivorous snail Lacuna vincta are major algal
herbivores of kelp and could be negatively affected by a changing ocean climate. High densities
of sea urchins can reduce productive kelp beds to bare urchin and encrusting coralline algal
communities, while a decrease in urchin population will allow a re-colonization of the kelp
canopy. Kelp forests and urchin barrens have historically been the two major Northwest Atlantic
subtidal community types, the dominant community type controlled by the increase or decrease
in urchin population (Chapman & Johnson, 1990). Ocean acidification and increased
temperatures have negative effects on tropical larval sea urchins, resulting in decreased
biomineralization of the larval skeleton (Brennand et al., 2010). Epizootic events causing
widespread urchin mortality is associated with warmer summer temperatures and more frequent
tropical storm and hurricane activity in Nova Scotia (Scheibling et al., 1999). A decrease in
urchin or snail abundance could reduce grazing pressure on kelp and result in more stable kelp
dominated communities with fewer herbivory-related disturbances.
The combined effects of increased temperature, increased CO2, and increased nutrient
availability in coastal waters (Connell & Russell, 2010; Russell et al., 2009) are predicted to
contribute to changing algal communities, with an increase in fleshy, ephemeral, foliose and
filamentous macroalgae, and a decrease in calcifying algae. This shift can be seen in areas of
natural acidification near underwater vents (Hall-Spencer et al., 2008), where erect and crustose
calcified species are replaced by non-calcified algae in areas of lowered pH, resulting in an
overall simplification of the macroalgal community (Porzio et al., 2011). Red algal species are
predicted to increase in abundance with an increase in temperature (Schiel et al., 2004), and
38 these effects have been observed in Long Island Sound with declines in intertidal
Phaeophyceae and simultaneous increases in Rhodophytes (Pedersen et al., 2008). There
have already been increasing occurrences of invasive algal species in New England [e.g.
Heterosiphonia japonica, (Newton et al., 2013), Grateloupia turuturu, (Mathieson et al., 2008)
and Codium fragile (Levin et al., 2002)] with broad thermal and salinity tolerances.
Environmental abiotic factors (temperature, currents, nutrient levels, eutrophication, UV
exposure, acidification) and biotic factors (competition, grazing, pathogens) can all affect kelp
distribution and abundance. These manifold factors can work to have antagonistic or synergic
effects on an organism. It is difficult to predict the effects of just one variable on an organism
since one factor will likely occur with others. Survival will depend on mechanisms of acclimation,
including photoinhibition and photoprotection, nutrient uptake strategies, patterns of growth and
reproduction, and morphogenetic responses (Figueroa & Korbee, 2010).
The stress associated with an increase in temperature can act synergistically with other
factors, including pests and pathogens. Kelp pathogens can include bacteria, viruses, fungi,
oomycetes, bryozoans, and endophytic red and brown filamentous algae. Infections in kelp can
weaken plants, reduce fertility, and cause mortality. Increased temperatures could increase
susceptibility to disease and cause increased pathogenic infection due to increased algal stress,
faster pathogenic development, increased winter survival, and increased host susceptibility
(Eggert et al., 2010). Membranipora membranacea is an introduced crust-forming bryozoan that
is significantly affecting kelp populations in the Northwest Atlantic (Lambert et al., 1992) and its
spread and success is correlated with higher sea temperatures. The encrusting parasite can
cover large portions of the blade, resulting in an inhibition of photosynthesis, weakened tissue,
reduced spore output, an increase in blade loss and ultimate kelp deforestation (Saier &
Chapman, 2004; Andersen et al., 2011; Yarish & Egan, 1987).
The coastal zone is a dynamic system that represents an important link between land,
open ocean, and atmosphere. Anthropogenic activities have altered coastal marine systems
39 directly and indirectly through fossil fuel and biomass emissions, land use change, inorganic
fertilizers, and increased nutrient loading. The carbon cycle is linked to other important
biogeochemical cycles, including nitrogen, phosphorus, and sulfur, and is affected by riverine
and atmospheric inputs as well as biological and physical feedbacks (Mackenzie et al., 2002).
The seawater carbonate system will react to changes in any of the individual components, so
increases in total alkalinity could buffer the effects of increased CO2 (Egleston, et al., 2010).
Alkalinity is a function of riverine inputs, calcification, sedimentation, nitrogen uptake, and other
biological and chemical sources and sinks, and riverine inputs to the coastal zone are
increasingly alkaline in the Northeast due to acid deposition and terrestrial weathering (Kaushal
et al., 2013). Increased nitrogen and phosphorus loading to coastal systems could enhance
production, resulting in a downward carbon pump, but organic loading can also cause
eutrophication, which results in anaerobic conditions and a decrease in overall productivity.
Anthropogenic alternation of global biogeochemical cycles will have cascading chemical,
physical, and biological feedback effects that could impact the health of the marine system.
Conclusions
Rapid changes in the ocean environment have unforeseen consequences on marine
organisms and ecosystems. Temperature and pH are both critical biological variables that can
affect survival, growth, reproduction, recruitment, productivity, and biodiversity of a system.
These factors all interact in a natural system, and significant changes in these important
variables will most likely affect the larger marine ecosystem, which could have both direct and
indirect affects on kelp populations worldwide. It can be predicted, however, that increasing
temperatures at the southern boundaries will inevitably shift the distribution northward.
40 References
Abramoff, M.D., Magalhaes, P.J. and Ram, S.J. 2004. Image processing with ImageJ.
Biophotonics International 11(7) 36-42.
Andersen, G.S., Steen, H., Christie, H., Fredriksen, S. and Moy, F.E. 2011. Seasonal patterns
of sporophyte growth, fertility, fouling, and mortality of Saccharina latissima in
Skagerrak, Norway: implications for forest recovery. J. Mar. Biol. Article ID 690375.
Axelsson, L., Mercado, J.M. and Figueroa, F.L. 2000. Utilization of HCO3- at high pH by the
brown macroalga Laminaria saccharina. Eur. J. Phycol. 35: 53-59.
Badger, M.R. and Price, G.D. 1994. The role of carbonic anhydrase in photosynthesis. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 45: 369-392.
Bartsch, I., Wiencke, C., Bischof, K., Buchholz, C., Buck, B.H., Eggert, A., Feuerpfeill, Hanelt,
P., Jacobsen, S., Karez, R., Karsten, U., Molis, M., Rolenda, M.Y., Schubert, H.,
Schumann, R., Valentin, K., Weinberger, F. and Wiese, J. 2008. The genus Laminaria
sensu lato: recent insights and developments. Eur. J. Phycol, 43(1) 1-86.
Beardall, J., Beer, S. and Raven, J. 1998. Biodiversity of marine plants in an era of climate
change: some predictions based on physiological performance. Bot. Mar. 41:113-123.
Blinks, L.R. 1963. The effect of pH upon the photosynthesis of littoral marine algae.
Protoplasma 57(1-4) 126-136.
Boden, G.T. 1979. The effect of depth on summer growth of Laminaria saccharina (Phaeophyta,
Laminariales). Phycologia 18(4) 405-408.
Bologna, P.A.X. and Steneck, R.S. 1989. Kelp beds as habitat for American lobster Homarus
americanus. Mar. Ecol. Prog. Ser. 100:127-134.
Bolton, J.J. and Lüning, K. 1982. Optimal growth and maximal survival temperatures of Atlantic
Laminaria species (Phaeophyta) in culture. Mar. Biol. 66: 89-94.
Brady-Campbell, M.M., Campbell, D.B. and Harlin, M.M. 1984. Productivity of kelp (Laminaria
spp.) near the southern limit in the Northwestern Atlantic Ocean. Mar. Ecol. Prog. Ser.
(18) 79-88.
Breeman, A.M. 1988. Relative importance of temperature and other factors in determining
geographic boundaries of seaweeds: experimental and phenological evidence.
Helogländer Meeresunters. 42: 199-241.
Breeman, A.M. and Pakker, H. 1994. Temperature ecotypes in seaweeds: adaptive significance
and biogeographic implications. Bot. Mar. 37:171-180.
Breitbarth, E., Bellerby, R.J., Neill, C.C., Ardelan, M.V., Meyerhofer, M., Zollner, E., Croor, P.L.,
and Riebesell, U. 2010. Ocean acidification affects iron speciation during a coastal
seawater mesocosm experiment. Biogeosciences 7: 1065-1073.
41 Brennand, H.S., Soars, N., Dworjanyn, S.A., Davis, A.R. and Byrne, M. 2010. Impact of
warming and ocean acidification on larval development and calcification in the sea
urchin Tripneustes gratilla. PLoS ONE 5(6): e11372. doi:10.1371/journal.pone.0011372.
Brinkhuis, B., Breda, V., Sheila, T., and Macler, B.A. 1983. New York Marine Biomass ProgramCulture of Laminaria saccharina. J. World Maricul. Soc. 14(1-4) 360-379.
Brinkhuis, B.E., Mariani, E.C., Breda, V.A., and Brady-Campbell, M.M. 1984. Cultivation of
Laminaria saccharina in the New York Marine Biomass Program. Hydrobiologia, 116117(1) 266-271.
Bruhn, J. and Gerard, V. 1996. Photoinhibition and recovery of the kelp Laminaria saccharina at
optimal and superoptimal temperatures. Mar. Biol. 125: 639-648.
Caldeira, K. and Wickett, M.E. 2003. Anthropogenic carbon and ocean pH. Nature 425:365.
Carney, L.T. and Edwards, M.S. 2006. Cryptic processes in the sea: A review of delayed
development in the microscopic life stages of marine macroalgae. Algae 21: 161-168.
Chapman, A. 1974. The genetic basis of morphological differentiation in some Laminaria
populations. Mar. Biol. 24:85-91.
Chapman, A.R.O. 1984. Reproduction, recruitment and mortality in two species of Laminaria in
Southwest Nova Scotia. J. Exp. Mar. Biol. Ecol. 78: 99-109.
Chapman, A.R.O. and Craigie, J.S. 1977. Seasonal growth in Laminaria longicruris: relations
with dissolved inorganic nutrients and internal reserves of nitrogen. Mar. Biol. 40: 197205.
Chapman, V.J. 1964. The Chlorophyta. Oceanog. Mar. Biol. Ann. Rev. 2: 193-228.
Cho, G., Yoon, H., Boo, S. and Yarish, C. 2000. Atlantic kelp species Laminaria longicruris and
L. saccharina (Laminariales) are conspecific. J. Phycol. 36: 12-13.
Chopin, T., Robinson, S.M.C., Troell, M., Neori, A., Buschmann, A.H. and Fang, J. 2008. Multitrophic integration for sustainable marine aquaculture. In: Jorgensen, S.E and Fath, B.D.
(Editor-in-Chief) Encyclopedia of Ecology 5 vols. Ecological Engineering. Vol. 3, pp
2463-2475, Elsevier, Oxford.
Coelho, S.M., Rijstenbil, J.W., and Brown, M.T. 2000. Impact of anthropogenic stresses on the
early development stages of seaweeds. J. Aquat. Ecosys. Stress Recovery 7(4) 317333.
Coleman, D.C. and Mathieson, A.C. 1975. Investigations of New England marine algae VII:
Seasonal occurrence and reproduction of marine algae near Cape Cod, Massachusetts.
Rhodora 77 (809) 76-104.
Connell, S.D. and Russell, B.D. 2010. The direct effects of increasing CO2 and temperature on
non-calcifying organisms: increasing the potential for phase shifts in kelp forests. Proc.
R. Soc. B. (277) 1409-1415.
42 Costa, F.T. 1989. Use of small-scale temperature perturbations to induce sorus formation in
Laminaria saccharina (L.) Lamour. and effects on subsequent gametophyte growth and
development. M.S. Thesis. SUNY at Stony Brook. 59pp.
Davison, I.R., Green, R.M. and Podolak, E.J. 1991. Temperature acclimation of respiration and
photosynthesis in the brown alga Laminaria saccharina. Mar. Biol. 110(3) 449-454.
Dayton, P.K. 1985. Ecology of Kelp Communities. Ann. Rev. Ecol. Syst. (16) 215-45.
Dayton, P.K., Tegner, M.J., Edwards, P.B. and Riser, K.L. 1999. Temporal and spatial scales of
kelp demography: the role of oceanographic climate. Ecol. Monogr. 69: 219-250.
Delille, B., Delille, D., Fiala, M., Prevost, C. and Frankignoulle, M. 2000. Seasonal changes of
pCO2 over a subantarctic Macrocystis kelp bed. Polar Biol. 23: 706-716.
Doney, S.C., Fabry, V.J., Feeley, R.A. and Kleypas, J.A. 2009. Ocean acidification: the other
CO2 problem. Annu. Rev. Mar. Sci. 1: 169-192.
Druehl, L.D., Collins, J.D., Lane, C.E. and Saunders, G.W. 2005. Evaluation of methods used to
assess intergeneric hybridization in kelp using Pacific Laminariales (Phaeophyceae). J.
Phycol. 41: 250-262.
Druehl, L.D. 1967. Distribution of two species of Laminaria as related to some environmental
factors. J. Phycol. 3(2) 103-108.
Duggins, D.O., Simentstad, C.A., and Estes, J.A. 1989. Magnification of secondary production
by kelp detritus in coastal marine ecosystems. Science 245: 170-173.
Egan, B. and Yarish, C. 1988. The distribution of the genus Laminaria (Phaeophyta) at its
southern limit in the western Atlantic Ocean. Bot. Mar. 31: 155-161.
Egan, B. and Yarish, C. 1990. Productivity and life history of Laminaria longicruris at its
southern limit in the Western Atlantic Ocean. Mar. Ecol. Prog. Ser. 67: 263-273.
Egan, B., Garcia-Ezquivel, Z., Brinkhuis, B.H. and Yarish, C.1990. Genetics of morphology and
growth in Laminaria from the North Atlantic Ocean- implications for biogeography. In:
Garbary, D. & South, G.R. [Eds.] Evolutionary biogeography of the marine algae of the
North Atlantic. 147-171. Springer-Verlag, Berlin.
Egan, B., Vlasto, A. and Yarish, C. 1989. Seasonal acclimation to temperature and light in
Laminaria longicruris de la Pyl. (Phaeophyta). J. Exp. Mar. Biol. Ecol. 129: 1-16.
Eggert, A., Peters, A.F., Kupper, F.C. 2010. The potential impact of climate change on
endophyte infections in kelp sporophytes. In: Israel, A., Einav, R., Seckbach, J., editors.
Seaweeds and their Role in Globally Changing Environments. Cellular Origin, Life in
Extreme Habitats and Astrobiology. Springer. 15:139-154.
Egleston, E.S., Sabine, C.L. and Morel, F.M.M. 2010. Revelle revisited: Buffer factors that
quantify the response of ocean chemistry to changes in DIC and alkalinity. Global
Biogeochem. Cycles 24, GB1002, doi: 1O.1029/2008GB003407.
43 Espinoza, J. and Chapman, A.R.O. 1983. Ecotypic differentiation in Laminaria longicruris in
relation to seawater nitrate concentration. Mar. Biol. 74:213-218.
Evans, L.V. 1965. Cytological studies in the Laminariales. Ann. Bot. 29: 541-562.
Figueroa, F.L. and Korbee, N. 2010. Interactive effects of UV radiation and nutrients on
ecophysiology: vulnerability and adaptation to climate change. In: Israel, A., Einav, R.,
Seckbach, J., editors. Seaweeds and their Role in Globally Changing Environments.
Cellular Origin, Life in Extreme Habitats and Astrobiology. Springer. 15:157-182.
Fortes, M.D. and Lüning, K. 1980. Growth rates of North Sea macroalgae in relation in
temperature, irradiance and photoperiod. Helogländer Meeresunters. 34:15-29.
Fowler-Walker, M.J., Wernberg, T. and Connell, S.D. 2006. Differences in kelp morphology
between wave sheltered and exposed localities: morphologically plastic or fixed traits?
Mar. Biol. 148: 755-767.
Gerard, V.A. 1990. Ecotypic differentiation in the kelp Laminaria saccharina: phase-specific
adaptation in a complex life cycle. Mar. Biol. 107: 519-528.
Gerard, V.A.1997. The role of nitrogen nutrition in high-temperature tolerance of the kelp,
Laminaria saccharina (Chromophyta). J. Phycol. 33: 800-810.
Gerard, V.A. and Du Bois, K.R.1988. Temperature ecotypes near the southern boundary of the
kelp Laminaria saccharina. Mar. Biol. 97(4) 575-580.
Gerard, V.A. and Mann, K.H. 1979. Growth and production of Laminaria longicruris
(Phaeophyta) populations exposed to different intensities of water movement. J. Phycol.
15(1) 33-41.
Graham, M.H. 2004. Effects of local deforestation of the diversity and structure of southern
California giant kelp forest food webs. Ecosystems 7: 341-357.
Graham, M.H., Kinlan, B.P., Druehl, L.D., Garske, L.E. and Banks, S. 2007. Deep-water kelp
refugia as potential hotspots of tropical marine diversity and productivity. Proc. Natl.
Acad. Sci. 104(42) 16576-16580.
Guiry, M.D. and Guiry, G.M. 2013. AlgaeBase. World-wide electronic publication, National
University of Ireland, Galway. http://www.algaebase.org: searched on 09 October 2013.
Hall-Spencer, J.M., Rodolfo-Metalpa, R., Martin, S., Ransome, E., Fine, M., Turner, S.M.,
Rowley, S.J., Tedesco, D. and Buia, M.C. 2008. Volcanic carbon dioxide vents show
ecosystem effects of ocean acidification. Nature 454: 97-99.
Harries, R. 1932. An investigation by cultural methods of some of the factors influencing the
development of the gametophytes and early stages of the sporophytes of Laminaria
digitata, L. saccharina and L. cloustoni. Ann. Bot. 46: 893-928.
Hobson, V.J., McMahon, C.R., Richardson, A. and Hays, G.C. 2008. Ocean surface warming:
the North Atlantic remains within the envelope of previous recorded conditions. DeepSea Research Part I: Oceanographic Research Papers 55(2)155-162.
44 Holdt, S.L. and Kraan S. 2001. Bioactive compounds in seaweed: functional food applications
and legislation. J Appl. Phycol 23: 543-597.
Hopkin, R. and Kain, J.M. 1978. The effects of some pollutants on the survival, growth and
respiration of Laminaria hyperborea. Estuar. Coast. Mar. Sci. 7: 531-553.
Howard-Strobel, M.M, Bohlen, W.F., Cohen, D.R. and Tebeau, P.A. 2000. Variability in
temperature, salinity and dissolved oxygen measurements from time series observations
in Long Island Sound. In: M. Van Patten (Ed.), The Fifth Biennial Long Island Sound
Research Conference Proceedings Stamford: University of Connecticut. Pp. 77-82.
Hsiao, S. I. 1972. Nutritional requirements for gametogenesis in Laminaria saccharina (L.)
Lamour. Ph.D. Thesis, Simon Fraser University, Burnaby, British Columbia, Canada.
Hsaio, S.I., and Druehl, L.D. 1973. Environmental control of gametogenesis in Laminaria
saccharina. IV. In situ development of gametophytes and young sporophytes. J. Phycol.
9(2) 160-165.
Humm, H.J. 1969. Distribution of marine algae along the Atlantic coast of North America.
Phycologia 7(1) 43-53.
Hunt, C.W., Salisbury, J.E., Vandemark, D. and McGillis, W. 2011. Contrasting carbon dioxide
inputs and exchange in three adjacent New England estuaries. Estuaries and Coasts
34(1) 68-77.
Hutchins, L.W. 1947. The bases for temperature zonation in geographical distribution. Ecol.
Monogr. 17(3) 325-335.
IPCC, 2007. Climate Change 2007: The Physical Science Basis. Contribution of Working Group
I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change
[Solomon, S., D. Qin, M. Manning, Z. Chen, M. Marquis, K.B. Averyt, M.Tignor and H.L.
Miller (eds.)]. Cambridge University Press, Cambridge, United Kingdom and New York,
NY, USA.
IPCC, 2013. Climate Change 2013: The Physical Science Basis. Contribution of Working Group
I to the IPCC Fifth Assessment Report. Summary for Policymakers. [S. Joussaume, J.
Penner, F. Tangang, (review eds.)]. Retrieved from www.ipcc.ch, 15 Oct. 2013.
Israel, A. and Hophy, M. 2002. Growth, photosynthetic properties and Rubisco activities and
amounts of marine macroalgae grown under current and elevated seawater CO2
concentrations. Global Change Biology 8(9) 831-840.
Johnston, C.S., Jones, R.G. and Hunt, R.D. 1977. A seasonal carbon budget for a laminarian
population in a Scottish sea-loch. Helgolander wiss. Meersesunters 30:527-545.
Kain, J.M. 1964. Aspects of the biology of Laminaria hyperborea III. survival and growth of the
gametophytes. J. mar. biol. Ass. UK. , 44: 415-433.
Kain, J.M. 1969. The biology of Laminaria hyperborea. V. Comparison with early stages of
competitors. J. mar. biol. Ass. U.K. 55: 567-582.
45 Kain, J.M. 1979. A view of the genus Laminaria. . Oceanogr. Mar. Biol. Annu. Rev. 17: 101-161.
Kaushal, S.S., Likens, G.E., Utz, R.M., Pace, M.L., Grese, M. and Yepsen, M. 2013. Increased
river alkalinization in the Eastern U.S. Environ. Sci. Technol. 47(18) 10302-10311.
Keser, M., Swenarton, J.T. and Foertch, J.F. 2005. Effects of thermal input and climate change
on growth of Ascophyllum nodosum (Fucales, Phaeophyceae) in eastern Long Island
Sound. J. Sea Res. 54(3) 211-220.
Koch, M., Bowes, G., Ross, C. and Zhang, X. 2013. Climate change and ocean acidification
effects on seagrasses and marine macroalgae. Global Change Biology 19(1) 103-132.
Kubler, J.E., Johnston, A.M and Raven, J.A. 1999. The effects of reduced and elevated CO2
and O2 on the seaweed Lomentaria articulata. Plant, Cell and Environment 22(10) 13031310.
Ladah, L.B. and Zertuche-Gonzalez, J.A. 2007. Survival of microscopic stages of a perennial
kelp (Macrocystis pyrifera) from the center and the southern extreme of its range in the
Northern Hemisphere after exposure to simulated El Nino stress. Mar. Biol. 152: 677686.
Ladah, L.B., Zertuche-Gonzalez, J.A. and Hernandez-Carmona, G. 1999. Giant kelp
(Macrocystis pyrifera, Phaeophyceae) recruitment near its southern limit in Baja
California after mass disappearance during ENSO 1997-1998. J. Phycol. 35:1106-1112.
Lambert, W.J., Levin, P.S. and Berman, J. 1992. Changes in the structure of a New England
(USA) kelp bed: the effects of an introduced species? Mar. Ecol. Prog. Ser. 88: 303-307.
Lane, C.E., Mayes, C., Druehl, L.D., and Saunders, G.W. 2006. A multi-gene molecular
investigation of the kelp (Laminariales, Phaeophyceae) supports substantial taxonomic
re-organization. J. Phycol. 42: 493-512.
Lazzari, M.A. and Stone, B.Z. 2006. Use of submerged aquatic vegetation as habitat by young
of the year epibenthic fishes in shallow Maine nearshore waters. Est. Coast. Shelf Sci.
69: 591-606.
Lee, J.A. and Brinkhuis, B.H. 1986. Reproductive phenology of Laminaria saccharina (L.)
Lamour (Phaeophyta) at the southern limit of its distribution in the northwestern Atlantic
Ocean. J. Phycol 22: 276-285.
Lee, J.A. and Brinkhuis, B.H. 1988. Seasonal light and temperature interaction effects on
development of Laminaria saccharina (Phaeophyta) gametophytes and juvenile
sporophytes. J. Phycol. 24(2)181-191.
Levin, P.S., Coyer, J.A., Petrik, R. and Good, T.P. 2002. Community-wide effects of
nonindigenous species on temperate rocky reefs. Ecology 83(11) 3182-3193.
Levitus, S., Antonov, J.I., Boyer, T.P. and Stephans, C. 2000. Warming of the world ocean.
Science 287: 2225-2229.
46 Lewin, J.C. 1966. Silicon metabolism in diatoms. V. Germanium dioxide, a specific inhibitor of
diatom growth. Phycologia 6: 1-12.
Liu, F. and Pang, S.J. 2010. Performances of growth, photochemical efficiency, and stress
tolerance of young sporophytes form seven populations of Saccharina japonica
(Phaeophyta) under short-term heat stress. J. Appl. Phycol. 22(2): 221-229.
Lobban, C.S. and Harrison, P.J. 1997. Seaweed Ecology and Physiology. NY: Cambridge
University Press. 384pgs.
Lucas, W.J. 1983. Photosynthetic assimilation of exogenous HCO3- by aquatic plants. Ann. Rev.
Plant Physiol. 34: 71-104.
Lüning, K. 1980. Critical levels of light and temperature regulating the gametogenesis of three
Laminaria species. J. Phycol. 16: 1-15
Lüning, K.1984. Temperature tolerance and biogeography of seaweeds: the marine algal flora
of Helgoland (North Sea) as an example. Helogländer Meeresunters. 38:305-317.
Lüning, K. 1975. Kreuzungsexperimente an Laminaria saccharina von Helgoland und von der
Isle of Man. Helgol. Wiss. Meersunters. 27: 108-114.
Lüning, K. and Dring, M. 1972. Reproduction induced by blue light in female gametophytes of
Laminaria saccharina. Planta (Berl.) 104: 252-256.
Lüning, K. and Neushul, M. 1978. Light and temperature demands for growth and reproduction
of Laminarian gametophytes in Southern and Central California. Mar. Biol. 45: 297-309.
Lüning, K. and Müller, D.G. 1978. Chemical interaction in sexual reproduction of several
Laminariales (Phaeophyceae): release and attraction of spermatozoids. Z.
Pflanzenphysiol. 89: 333-341.
Lüning, K., Chapman, A. and Mann, K. 1978. Crossing experiments in the non-digitate complex
of Laminaria from both sides of the Atlantic. Phycologia 17:293-298.
Lüning, K. 1990. Seaweeds - Their Environment, Biogeography, and Ecophysiology, IN: Yarish, C.
and H. Kirkman (Editors) Edited Translation of the German language edition
Meeresbotanik: Verbreitung, Okophysiologie und Nutzung der marinen Makroalgen by
Klaus Lüning. John Wiley and Sons, Inc. New York, 527 pp.
Mackenzie, F.T., Ver, L.M. and Lerman, A. 2002. Century-scale nitrogen and phosphorous
controls of the carbon cycle. Chemical Geology 190: 13-32.
Mann, K. 1972. Ecological energetics of the seaweed zone in a marine bay on the Atlantic
Coast of Canada. II. Productivity of the seaweeds. Mar. Biol. 14:199-209.
Mann, K.H. 1973. Seaweeds: Their Productivity and Strategy for Growth. Science, 182: 975981.
47 Marner, F-J., Müller, B. and Jaenicke, L. 1984. Lamoxirene, the Laminaria pheromone:
Structural proof of the spermatozoid releasing and attracting factor of Laminariales. Z.
Naturforsch. (39c) 689-691.
Mathieson, A. 1979. Vertical distribution and longevity of subtidal seaweeds in Northern New
England, USA. Bot. Mar. 30: 511-520.
Mathieson, A.C., Dawes, C.J., Pederson, J., Gladych, R.A. and Carlton, J.T. 2008. The Asian
red seaweed Grateloupia turuturu (Rhodophyta) invades the Gulf of Maine. Biol.
Invasions 10(7) 985-988.
McDevit, D. and Saunders, G. 2010. A DNA barcode examination of the Laminariaceae
(Phaeophyceae) in Canada reveals novel biogeographical and evolutionary insights.
Phycologia 49: 235-248.
Merzouk, A. and Johnson, L.E. 2011. Kelp distribution in the northwest Atlantic Ocean under a
changing climate. J. Exp. Mar. Biol. Ecol. 400(1-2) 90-98.
Müller, R., Laepple, T., Bartsch, I. and Wiencke, C. 2009. Impact of ocean warming on the
distribution of seaweeds in polar and cold-temperate waters. Bot. Mar. 52: 617-638.
Müller, R., Wiencke, C. and Bischof, K. (2008). Interactive effects of UV radiation and
temperature on microstages of Laminariales (Phaeophyceae) from the Arctic and North
Sea. Clim. Res. 37: 203-213.
Neefus, C.D., Allen, B.P., Baldwin, H.P., Mathieson, A.C., Eckert, R.T., Yarish, C. and Miller,
M.A. 1993. An examination of the population genetics of Laminaria and other brown
algae in the Laminariales using starch gel electrophoresis. Hydrobiologia 260/261: 6779.
Neushul, M. and Harger, B.W.W. 1987. Nearshore kelp cultivation, yield and genetics. In:
Seaweed Cultivation for Renewable Resources Bird, K.T. and Benson, P.H. (Eds) pp.
69-93. Elsevier, Amsterdam.
Newton, C., Bracken, M.E., McConville, M., Rodrigue, K. and Thornberg, C. Invasion of the Red
Seaweed Heterosiphonia japonica Spans Biogeographic Provinces in the Western North
Atlantic Ocean. Lin,S. (Ed.) PLoS ONE , 8 (4), e62261.
Parke, M. 1948. Studies on British Laminariaceae. I. Growth in Laminaria saccharina (L.)
Lamour. J. Mar. Biol. Ass. U.K. 27: 651-709.
Pedersen, A., Kraemer, G., & Yarish, C. 2008. Seaweed of the littoral zone at Cove Island in
Long Island Sound: annual variation and impact of environmental factors. J. Appl.
Phycol. 20(5) 869-882.
Porzio, L., Buia, M.C. and Hall-Spencer, J.M. 2011. Effects of ocean acidification on macroalgal
communities. J. Exp. Mar. BIol. Ecol. 400 (1-2) 278-287.
Rilov, G. and Haim, T. 2010. Climate change effects on marine ecological communities. In:
Israel, A., Einav, R., Seckbach, J., editors. Seaweeds and their Role in Globally
48 Changing Environments. Cellular Origin, Life in Extreme Habitats and Astrobiology.
Springer. 15: 51-68.
Rinde, E. and Sjøtun, K.. (2005). Demographic variation in the kelp Laminaria hyperborea along
a latitudinal gradient. Mar. Biol. 146: 1051-1062.
Roldeda, M.Y., Morris, J.N., McGraw, C.M. and Hurd, C.L. 2012. Ocean acidification and
seaweed reproduction: increased CO2 ameliorates the negative effect of lowered pH on
meiospore germination in the giant kelp Macrocystis pyrifera (Laminariales,
Phaeophyceae). Global Change Biol. 18: 854-864.
Russell, B.D., Thompson, J.I., Falkenberg, L.J. and Connell, S.D. 2009. Synergistic effects of
climate change and local stressors: CO2 and nutrient driven change in subtidal rocky
habitats. Global Change Biol. 15: 2153-2162.
Russell, G. 1986. Variation and natural selection in marine macroalgae. Oceanogr. Mar. Biol. A.
Rev. 24: 309-377.
Sabine, C.L., Feeley, R.A., Gruber, N., Key, R.M., Lee, K., Bullister, J.L., Wanninkof, R., Wong,
C.S., Wallace, D.W.R., Tilbrook, B., Millero, F.J., Peng, T.H., Kozyr, A., Ono, T. and
Rios, A.F. 2004. The oceanic sink for anthropogenic CO2. Science 305 (5628) 367-371.
Saier, B. and Chapman, A.S. 2007. Crusts of the alien bryozoan Membranipora membranacea
can negatively impact spore output from native kelps (Laminaria longicruris). Bot. Mar.
47:265-271.
Sears, J.R. and Wilce, R.T. 1975. Sublittoral, benthic marine algae of Southern Cape Cod and
adjacent islands: seasonal periodicity, associated diversity and floristic composition.
Ecol. Monogr. 45(4) 337-365.
Setchell, W.A. 1915. The law of temperature connected with the distribution of the marine algae.
Ann. Missouri Bot. Gard. (2) 287-305.
Setchell, W.A. 1920. The temperature interval in the geographic distribution of marine algae.
Science. (52) 187-190.
Scavia, D., Field, J.C., Boesch, D.F., Buddemeier, R.W., Burkett, V., Cayan, D.R., et al. 2002.
Climate change impacts on US coastal and marine ecosystems. Estuaries 25(2) 149164.
Scheibling, R., Hennigar, A. and Balch, T. 1999. Destructive grazing, epiphytism, and disease:
The dynamics of sea urchin - kelp interactions in Nova Scotia. Canadian Journal of
Fisheries and Aquatic Sciences, 56: 2300-2314.
Schiel, D.R., Steinbeck, J.R. and Foster, M.S. 2004. Ten years of induced ocean warming
causes comprehensive changes in marine benthic communities. Ecology 85(7) 18331839.
Schreiber, E. 1930. Untersuchungen uber Parthenogenesis, Geschlechtsbestimung und
Bastardierungsvermogen bei Laminarien. Planta, 12: 331-351.
49 Steneck, R.S., Graham, M.H., Bourque, B.J., Corbett, D., Erlandson, J.M., Estes, J.A., and
Tegner, M.J. 2002. Kelp forest ecosystems: biodiversity, stablility, resilience and future.
Environ. Conserv. 29(4) 436-459.
Swanson, A.K. and Fox, C.H. 2007. Altered kelp (Laminariales) phlorotannins and growth under
elevated carbon dioxide and ultraviolet-B treatments can influence associated intertidal
food webs. Global Change Biology 13(8) 1696-1709.
Thom, R.M. 1996. CO2-enrichment effects on eelgrass (Zostera marina L.) and bull kelp
(Nereocystis luetkeana (mert.) P & R.). Water, Air, and Soil Pollution 88(3-4) 383-391.
tom Dieck, I. 1993. Temperature tolerance and survival in darkness of kelp gametophytes
(Laminariales, Phaeophyta): ecological and biogeographical implications. Mar. Ecol.
Prog. Ser. 100: 253-264.
Turan, G. and Neori, A. 2010. Intensive seaweed aquaculture: a potent solution against global
warming. In: Israel, A., Einav, R., Seckbach, J., editors. Seaweeds and their Role in
Globally Changing Environments. Cellular Origin, Life in Extreme Habitats and
Astrobiology. Springer. (15) 357-372.
Uller, T., Pen, I., Wapstra, E., Beukeboom, L.W. and Komdeur, J. 2007. The evolution of sex
ratios and sex-determining systems. Trends in Ecology and Evolution 22(6) 292-297.
Van den Hoek, C. 1975. Phytogeographic provinces along the coasts of the Northern Atlantic
Ocean. Phycologia (14) 317-330.
Van den Hoek, C. 1982. The distribution of benthic marine algae in relation to the temperature
regulation of their life histories. Biol. J. Linn. Soc. 18(2) 81-144.
Van Patten, M., & Yarish, C. 1993. Allocation of blade surface to reproduction in Laminaria
longicruris of Long Island Sound (USA). Hydrobiologia 260/261:173-181.
Vandemark, D., Salisbury, J.E., Hunt, C.W., Shellito, S.M., Irish, J.D., McGillis, W.R, Sabine,
C.L. and Maenner, S.M. 2011. Temporal and spatial dynamics of CO2 air-sea flux in the
Gulf of Maine. J Geophys Res 116: C01012, doi:10.1029/2010JC006408.
Williams, L.G. 1949. Growth-regulating substances in Laminaria agardhii. Science (1102850)169.
Wong, S. 1989. Nitrate effect on growth and storage of inorganic nitrogen (nitrate) in two
different age groups of Laminaria saccharina (L.) Lamour. Msc Thesis. Stony Brook
University, New York.
Wooton, J.T., Pfister, C.A. and Forester J.D. 2008. Dynamic patterns and ecological impacts of
declining ocean pH in a high-resolution multi-year dataset. Proc. Natl. Acad. Sci. 105
(48) 18848-18853.
Yarish, C., Breeman, A.M. and van den Hoek, C. 1984. Temperature, light and photoperiod
responses of some Northeast American and West European endemic rhodophytes in
relation to their geographic distribution. Helogländer Meeresunters. 38: 273-304.
50 Yarish, C., & Egan, P.1987. Biological Studies of Laminaria longicruris and its aquaculture
potential in Long Island Sound. In: Halasi-Kun, G.J. (Ed.) Columbia University Seminars
on Pollution and Aquatic Resources. Columbia University Press. 20: 193-220.
Yarish, C. and Egan, B. 1989. The biology of Laminaria longicruris and its potential for
mariculture in Long Island Sound. In: Kanungo,K., Brousseau,K., Singletary, R. and
Liscek, L. (Eds.) The Second Conference on the Aquatic Environment: Problems and
Perspectives. Western Connecticut State University, Danbury, CT. Pp. 55-89.
Yarish, C. and Pereira, R. 2008. Mass production of Marine Macroalgae. In Sven Eriik
Jørgensen and Brian D. Fath (Editor-in-Chief) Ecological Engineering. Vol. [3] of
Encyclopedia of Ecology, 5 vols. pp. 2236-2247. Oxford: Elsevier.
Yarish, C, Brinkhuis, B.H., Egan, B. and Garcia-Ezquivel, Z. 1990. Morphological and
physiological bases for Laminaria selection protocols in Long Island Sound aquaculture.
In Yarish, C., Penniman, C.A., & van Patten, P. [Eds.] Economically Important Marine
Plants of the Atlantic: Their Biology and Cultivation. Connecticut Sea Grant College
Program, Groton, 158pp.
Yarish, C., Lee, K. and Edwards, P. 1979. An improved apparatus for the culture of algae under
varying regimes of temperature and light intensity. Bot. Mar. 22(6) 395-397.
Zhang, X., Hu, H. and Tan, T. 2006. Photosynthetic inorganic carbon utilization of gametophytes
and sporophytes of Undaria pinnatifida (Phaeophyceae). Phycologia 45(6) 642-647.
51