Biomechanical Limitations of Developing Pneumatocysts in the Bull Kelp (Nereocystis Luetkeana, Phaeophyceae)

This article isprotected rights by All copyright. reserved. 10.1111/jpy.12776doi: lead todifferences version between this andtheof Pleasec Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer zonation Keywords: Running Head: Editorial Responsibility: A. 2 1 typeArticle : Regular Article MISS LAURAN M LIGGAN (Orcid :ID 0000 Bamfield Marine Sciences Centre, 100Pachena Road, Bamfield, AcceptedAuthor for correspondence: email: Received 28,2018.Accepted June July 16,2018 Article Under pressure: ofdevelopingthe bullkelp biomechanical limitations pneumatocysts in , macroalgae

Buoyancy, Department Botany of and Beaty Biodiversity Research Centre, University British of biomechanical limitations of kelp pneumatocysts

Columbia, Vancouver, British Columbia, Canada V6T1Z4 gas bladder,

Buschmann (Associate Editor) ( Lauran N ereocystis luetkeana

hydrostatic pressure, development, stress,

M. [email protected]

Ligga - 0002 n 2

and Patrick T.Martone -

0404 , .

P - haeophyceae) 388X)

British Columbia, Canada

modulus,

ite this articleite this as depth

V0R1B0

This article isprotected rights by All copyright. reserved. σ t r r P P σ V V P P P h water depth g ρ density ESF environmental safety factor kPa atm ofList symbols and abbreviations V P o o i

buckle buckle avg i o gauge Pn mi mi Pn

atm

Accepted Article

pneumatocyst volume average material stress initial manometerinitial initial manometer volume outer pneumatocyst radius

hydrostatic pressure gauge pressure i internal pneumatocyst pressure total pneumatocyst pressure atmospheric pressure gravitational acceleration k atmosphere nternal pneumatocyst radius buckling material stress

ilopascal pneumatocyst wall thickness

volume manometer the of and pneumatocyst

pressure

This article isprotected rights by All copyright. reserved. Acceptedcolumn single gas Upright endemic Introduction also define the previous measures of lower limits due lightto attenuation, suggesting that hydrostatic buckling reduce wall stress buckling.of Although were demonstrated haveto elevated wall stresses under high compressiv depths gradient Articlepressures less than atmospheric pressure, ensuring that thalli resistto pressure pneumatocyst develop across hydrostatic a gradient. pneumatoc potentially causing hydrostatic pressure kelps kelps Abstract

grow to achieve upright an stature and compete for light .

Small pneumatocysts to accessto light for photosynthesis and to avoid benthic herbivores The Maintaining buoyancy with gas

Nereocystis and three

- filled floatfilled ysts of

up through the water column

b compressional loads

ull - geometry across a range of

dimensional habitats along lower limit - k

induced induced mechanical failure.

Nereocystis as as they grow elp elp them , which

. thalli s When t

Nereocystis

small called to to

are supported bynot rigid stipes as in of rupture could could cause complications

kelps

pneumatocysts Nereocystis collected from depths between 1and 9m

sted luetkeana , they about are 3. ,

, luetkeana indicating that they do not adjust

, flood, and pneumatocysts We measuredWe -

, pneumatocysts are exposed to filled floatsfilled (pneumatocysts) essentialis for resist biomechanical stress

in thein field. thallus sizes and collection dept the

, (

which hereafter Contrary expectations, to a west lose

pneumatocyst 4

buckle times stronger

coast buoyan

keep internal pressure, material properties, and

are moreare likely buckle to as internalas Nereocystis

used for photosynthesis of Northof America d slender,

cy around 35m which depth, are .

In study, this w material properties geometryor

some always

tha gases may expand contract, or flexible thall )

and maintain buoyancy they as b n they to need be uilds unique

other subtidal kelps, but by

( exposed to apositive pressure substantial inner radius 0.4 = e loads and greatest are at risk ll

hs identifyto strategies used (

hydrostatic pressure pneumatocysts had internal Koehl and Wainwright (Springer et al. 20 e i

investigate upright thein water tha . However, as and naturally n rupture changes in some agrees with to resist

– subtidal how

1.0 cm) at all 07

these to to may

) .

This article isprotected rights by All copyright. reserved. tear,to releasing gas internally and creating p the Acceptedgermination (Nicholson 1970). During development, medullary cells along the transition zone begin begin to form within the transition zone between the stipe and the blade about youngspecies, and grow rapidly toward the surface Nereocystis reproduc Brackenbury potentially resist avoid leaks because and, compressible gases are susceptible volumetricto change pressure, with must 2016 Koehl 1994, hydrostatic pneumatocyst) from breaking extreme under physical adjust Articlepneumatocysts resistto breaking during their ascent is remarkable and worthy further of investigation. whil most other species, they inflate at depth and ascend through water the column toward surfacethe 2012 Lane et al. 2006 porra pneumatocysts produced by kelps (i.e., spp known frommany species, seaweed especially within the brown algae (e.g., 1977 ., Sargassum e experiencing adecreasing grad ). Pneumatocysts). of , ). ).

) evolved) independently from of those other macroalgae at least once ing Johnson Johnson and Koehl 19 In Previous studies have shown that tion in Falltion in (Sept

particular structural and mechanical properties preventto organs (i.e.

Nereocystis

pressur thalli

Utter andDenny 1996 et al. 2006 individuals ) and) are developmentally distinct spp rupturing

can be found e ,

., Durvillaea pneumatocysts due to tidal fluctuations

thalli are annuals, starting growth in spring (March ) when) Nereocystis -

Oc are composedare of (as gases(as expand) buckling or gases compress (as 94, t; Riggt; 1912 thalli move growing maximum at depths between

Denny et al spp ,

must remain ‘air Koehl 1999 at a rate of

ient of hydrostatic pressure . are particularly interesting because, unlike gas ; Dromgoole; 1981b Nereocystis luetkeana, Macrocystis pyrifera, Pelagophycus , Maxell , Maxell and Miller 1996 brown algae through the water column.

. a

( 1997 Dromgoole 1981a, Dromgoole 1981b, simple holdfast, stipe blade and about 14 cm ,

neumatocyst (Dromgoole 1981 Hale 2001, Harder et al , (Yoon et 2001al. Springer et al. 2007) - tight’ stress can can resist mechanical forces as they develop , Brackenbury

( , such waveas forces or Rigg andSwain1941 per

day ( (Fig. ). During). the spring, y , ,

Lane et al. 2006,

holdfast, stipe, blade, 30 Fig. 1; 1)

. et al.et 2006 . . - Gas

The ability of

- 2006, 35 m35 ( April) andApril) ending ( Thiel and Gutow2005 ; Dromgoole; 1981b Ascophyllum - Kain 1987 , filled structures are 2 and

a weeks after Starko andMartone Spalding ). Once , ) p Foreman 1976) , however - changes in neumatocysts Johnson andJohnson filled floatsfilled in

Charrier ). Like). allkelp Nereocystis oung spp et al.et .,

Fucus et et al. 2003 , , to to by )

This article isprotected rights by All copyright. reserved. Acceptedsize appliedbeen to 10, the structure is ten approaches before breaking Generally, ESF is the ratio between the environmental safety factor (ESF (Dromgoole 1981b pneumatocysts resist applied that would otherwise pneumatocyst at risk breaking of pneumatocyst Article externalthe pneumatocystthe a is thick mechanical failure and documented Changes in internal volume, extending into the stipe heavier, with blades that can weigh upto 20 kg air (Denny in al. et 1997) from Duncan 1973). pneumatocysts have formed, (Johnson and Koehl 1994). four The ability pneumatocyst of Internal

to to

1 , hydrostatic one , the and hydrostatic s

influence the move upward through water the column, t From 30 , Nereocystis

pressure produced by and the a

understanding these changes structure is tm, ) . pressure volume and during

equilibrate one a - times strongertimes than it needs to be, maintain

pressure and the internal m deep theto surface, thalli experience a working stress, the stress directly - tm 10m per walled walled structure stipes accumula thalli performing

. buoyancy (see Fig. 1) internal ; ,

Because p Because

Johnson and Koehl 1994 grow upward and reach the surface in as little 4monthsas ( to to investigate s

to gas tion material of stress and , and t

breaking stress, the (405.3 kPa(405.3 to 101. resist breaking and external near near es across a

neumatocyst load it experiences day to day. is is (1 to he capacity, an - exerted on the in will help clarify increas 10 mm

pneumatocyst development

geometry, material properties, and deformation the the risk breaking of pneumatocyst hydrostatic pressure gradient pressure e ;

can be can Foreman 197 buoyancy

and his force differential wall d 4 , Stewart 2006 maximum an load object can withstand

failure kPa

risk s s

assessed by calculating

are rigid and how ner ner surface of the pressure ( ; Fig.; Liggan 2016 critical breaking pressure

is lessenedis reduction and may be Nereocystis 6 as as sporophytethe increases in

1 ) kee , the force ).

causes

, As thalli become larger , Koehl al. et 2008 p heavy pneumatocyst

imminent For example,

in in have resist . ) ESF previously has , wall material must could hydrostatic

material stress . thalli

pneumatocyst

not not

differential between volumetric changes thalli put put the ; been

when resist an

s upright.

as increase

pressure

ESF ESF ) . . Fig. As . Since

is and

1; 1;

This article isprotected rights by All copyright. reserved. Acceptedpneumatocyst depth pneumatocyst based on the 0 m datum), and time were recorded for each sample. This was used to calculate the depth of pneumatocyst from depths up 9m to BreakwaterPoint and varying length (0.15 Methods pressure) by hydrostatic pressure on developing pneumatocysts and to predict when pressure Articlehelp pressure maintained, then pneumatocysts could thicken their pressure maintain and a lo ascent adjust gas production, material properties, morphology or reduce to the risk of breaking allowing However, stipeelongation the rate of hydrostatic pressure changes by adjusting the rateof stipe growth

pneumatocysts pneumatocysts . For example, pneumatocysts could change the rate of gas production controlto internal Sample collection Theoretically, t -

in in the field induced tissueinduced stress.

blade individuals can grow and Materials

In t s s

his study his were

to maximto

( Victoria, British Columbia, Canada . resist mechanical failure as as follows fully intact. Collection depth of the holdfast, day, height tidal (at 0m chart We are at greatest . All . All samples were detached from the o minimize wall stress ,

calculate – we ize . w force differential.

rates 12 m) Nereocystis

investigate light light availability.

. :

tide line. tide are rapid ( were

force differential risk of risk of

haphazardly thalli how

External hydrostatic pressure (P Duncan 1973, failing

(n=72) internal pressure, morphology, as as they grow upwardthrough agradient of hydrostatic

and risk breaking of Alternatively Instead, we hypothesize thatpneumatocysts likely . These data allow us clarifyto constraints s

collected walls or stiffen their tissues to help resist of varying experienced by thalli of various sizes ; 48.413542 Kain 1987 , i substratum the at holdfast so that

between f thef pressure differential is not

pneumatocyst ° ,

), ), Nereocystis N the maximumthe depth , a April nd thalli race to the surface - 123.387235 o ) was ) estimated from -

August and to slowto ascent their

volume

thalli

material properties °

2015

could ( 3

) du

each – with SCUBA (i.e.,

from

ring their to 1200 control imposed

predict

Ogden

. cm

3 ) This article isprotected rights by All copyright. reserved. so, And Law: Gas Ideal the and manometer the of dimensions the pneumatocysts were punctured. reading. manometer inside. pressure gas the syringe, a and collection use were conducted using R Institutes of Health) by taking of photos each sample with a replicates. with pneumatocyst depth, and where ρ is the density seawater of (1025 Accepted Article water

Pneumatocyst P

d in the field the in d

o P

Pneumatocyst length andwidth was measured usingImage 64version J 1.49(National (Fig = Measuring internal pressure internal Measuring pn then pouring the water into a graduated cylinder three times, taking an average the of three

(ρgh)

21 V .

pn 2

gauge (0.7 mm) (0.7 gauge ; Studio

n + + P +

P Initial pressure readin

64)

volume wasrecorded by carving ahole in atm to measure internal pressure of pneumatocysts of pressure internal measure to mi

. All needles were lubricated with Vaseline to avoid air leaking during the the during leaking air avoid to Vaseline with lubricated were needles All

P Water manometersWater were attached pneumatocyststo using plastic tubing, a version V

atm

is atmosphericis pressure.

Internal pneumato Internal = = 2.11.1 P

P needle that was used to puncture pneumato puncture to used was that needle gauge gauge

(R Core Development Team). kg . V (V Water manometers manometers Water

gs were recorded no more than 5 gauge ·

pn m

- 3 + V + ), g), acceleration is due gravityto m (9.81

cyst pressure pressure cyst mf )

meter stick. statistical All analyses were

(diameter: 4 each (P pn ) pneumatocyst

was then calculated by using using by calculated then was (at the surface) the (at -

8 mm; length:8 20 seconds after the cysts and measure measure and cysts

(n=72) [Eq.

directly

1 , filling it s ] - 2

- ), h ), is 60 cm)

after

This article isprotected rights by All copyright. reserved. (measured by manometer) depth) hydrostatic pressure applied to outerthe surface of the pneumatocyst wall w (Fung 1993) pneumatocystthe broke first 5 weighted thalli down 50to m seawater, of and conducted determineto which portion of the pneumatocyst experience a differen hollow stipe and a spherical end substantially less manometerthan volume. volume manometer between andpneumatocyst; error was greatest whe move water column the 1mm, given dimensions the theof manometer tubing and difference the in pneumatocyst. theof manometer arm before the pneumatocyst is punctured and pneumatocyst was punctured, P manometer, V where P

here here

Accepted Article

, σ

Pn Calculating wall stress avg i

. Thus, this study focused on the average wall stress accumulated in the spherical region of is the internal pneumatocyst pressure applied to the inner surface of the pneumatocyst wall

is is unknownthe pneumatocyst pressure, P

: is theis average circumferential stress in the pneumatocyst wall,

Manometer measurement was error calculated as the amount pressure of required to

is the is . Average wall stress

t magnitudet of material stress

total

volume manometer the of from water the line theto needle after the

is is the intern .

Older pneumatocysts have two different geometries: a cylindrical (see Fig. 1)

is the pressure the is of the ambient air in manometer,the

in in a pneumatocyst at a specific depth was calculated as follo

al pneumatocyst radius

Because

found that

than the spherical end

gauge

the cylinder portion of the pneumatocyst

is the pressure reading from the would break the spherical end pneumatocysts of

V (calculated from caliper

is the volume of the

first. first. pneumatocyst volume was ,

(calculated from collection a

preliminary study was

In the field, w

is the

external

[Eq. [Eq.

[Eq. [Eq. is volumeis e

dropp 3 ] always would 2

]

ws ed

This article isprotected rights by All copyright. reserved. determine if modulus changed pneumatocyst with volume. stressthe strain (see f measurements modulus of ( cycled through compression (to The movement down) or (up of Instronthe crossbeam was used measure to strain. force applied theto sam sample wassprayed and hydrated with s strain and these values ( measured stiffness, the crosstop beam andthe base block haveto height a (if equipped with an 8 (stiffness) MA, USA). Pneumatocysts werecut into circular varying sizes(n=33; volume =3 determine if material stress changed with pneumatocyst depth. were measured using calipers to the nearest 1mm. Aregression linear analysis was conducted to pneumatocystthe wall. Positive values indicated compressive stres therefore the outer portion pneumatocystof wall experiences different loads than the inner portion of thickness) pneumatocyst radius measurements of pneumatocyst diameter, after subtracting wall thickness) AcceptedCasio exilim. EX Article Compressive modulus -

strain strain curve during 4 the . of of material.the Eq ig. Liggan 7in Young’s 2016). modulus (initial stiffness) was calculated from slope of the u ation -

FH25 ring x

3 (calculated from caliper measurements pneumatocyst of diameter, including wall 8

takes into consideration that the pneumatocyst a is thick were adhesion cm block at the base and a 9 ) and) width (ifcubed) length no less than 8mm. Sampleswereglued onto the ple by crossple its ) .

Results The movement down) or (up of the i.e., i.e.,

compared to .

Material properties of pneumatocyst walls Mullin’s effect). Stress -

integrity 8%) 8%) and tension (to 8%) four times avoid to any spu –

between the two shapes were comparable th using super glue 1200 cm

cy cle at 1% at compression.cle - sectional area eawat

real during 3 ) - er before each Stresstest. was measured by dividing using an Instron 5500R (Model # 1122, Instron Inc. time each rings o

. the

x T

o ensure gluethe did not slip andaffect where wa it 0.8 cm bea cross test wastest verified with a high - measure strain curvesstrain were lin r cubesr measureto the elastic modulus

Instron cross A linear regression was ments analyzed from video the s in contact with the cross ses. All radii and wall thicknesses m atop

, and were beam was used measure to . The Instron was ear from .

- All samples were cut

r walled structure and quantified for thalli of o

All samplesAll were - is theis outer speed cameraspeed riously high - used 8% to 8% 8% to 8% - to to beam .

Each .

This article isprotected rights by All copyright. reserved. pneumatocyst at w then Fig. pressure pneumatocysteach buckled, detected by anaudible “pop.” depthThis was converted to down 50 to m.video A camera (Go weighted basket this size range pneumatocysts (r pneumatocyst’s measured inner radius (r measured outer radius (r estimating the t geometry directly influences average wall stress. takes into consideration that the pneumatocyst is athick thickness was (t) used determineto the inner radius to wall

Accepted here Article 3 estimated ), σ

and E Changes pneumatocystin geometry

buckle and nvironmental safety factor average buckling internal pressure was averaged

were collected to bucklingtest stress and depth. as follows: is is otal pneumatocyst volume (tissue and gas space) of a sphere from pneumatocyst’s the with aSCUBA analogue depth gauge (Scubapro MODEL #28.019.900) and dropped its

the

collection 0.4

buckling - o

1.0 cm) hadelevated wall stress ) ) minus pneumatocyst the gas space spherical volume from the

str

depth stress of of stress ess was -

Pro Hero 3+ Pro Hero silver edition) recorded the sound anddepth when (Eq. 3) and buckling d the then i ). pneumatocyst wall and

. .

across all measured pneumatocysts Measured pneumatocyst inner radius (r

calculated using Pneumatocyst volume tissue was calculated by epth . -

walled walled structure and therefore pneumatocyst C . Therefore alculations suggested that thickness ratio (r Eq

Each thallus was . 3 σ .

a The ESF of pneumatocysts were small vg

pneumatocysts the the i :t), or geometry. wall stress of

(80 placed

kPa; shown in i ) and) wall small

into a hydrostatic [Eq.

(n (n = 8) each 4 Eq. 3 ]

in in This article isprotected rights by All copyright. reserved. P weightedthe internal radius increased from 3.5 1to 0.001, when 6 greatest (between 250 and 400 kPa gas)with F significantly to (up 12 MPa) pneumatocyst as volume increased from modulus small of pneumatocysts ( estimated depth ( hydrosta 200 kPa less than external hydrostatic external hydrostatic pressure (101 kPa), r change with depth ( Results B) Acceptedneumatocysts had an average 1,32 Article , when

ratios of inner radius wallto thickness ( = 25.79, R Fig.

B W V 2 tic = 0.53

olume uckling stress to 3.5to at stress, all tissue volumetissue 4 pressure 8 ; basket

F m and depth minimum P 1,60 )

cm pneumatocysts as grew from 9 and . < 0.001 Pneumatocyst tissue volume increased from 2.3to 200 cm Fig.

pressure at all depths ( stiffness, had an environmental safety factor of above 3 m anging from 100 3to kPa ( internal pressure = 101.2 . 3 , R Small s ;

were largely unchanged F 2 = 1,57 , P

and geometry 0.4

pneumatocysts (r wall stresswall ( < 0.001 Fig

3 = 3 ). Inner). radius increased from 0 )

- 2.796 50 cm when pneumatocysts had 3

) wall stresswall .

pressure below 4m . cm (

Internal pressure ,

R ,

between 2 P 3 Fig.

Fig. . = ) va) = 0. Pneumatocyst wall stress significant

r 0.62 i :t 3 ried ried from 10MPa 0.9to ( Fig. 6 10 i

) ). Internal). pneumatocyst pressure was generally 100

( D = 102 kPa varied from 2.8 ). ). )

). (average volume =2.3± 1.7 cm m

202 0.4 and was consistently 3

Maximum depth depth ). Pneumatocyst internal pressure - and

of pneumatocystsof 1.0 cm) ) was estimated)

3. depth to to 287 kPa ( 4

inne ± 0. ± the surfacethe

wall stresswall , and1

that were – 4

r radir cm

( 4 mean +SD .0 Table 1 3 (before pneumatocyst(before fill -

cm 50 kPa less ( i

Fig. between 0.4 less less than atmospheric Fig.

( at at the surface

( 3 Fig. did not signifi submerged and crushed 375

3 to 1200cm to ). Buckling stress 6

5 as C ). ). Modulus increased

6 kPa ; F ; ; A). pneumatocyst

ly increased with Table Table 1 1,62 3 ) was ) than external ;

Wall stress was Fig. –

wa 1 3 ( = cantly Fig. .0

) (Fig.

6 . 84.09 s less than D)

cm 4 ,

) and 5 varied (Fig. .

ed ;

P< The

in in - This article isprotected rights by All copyright. reserved. 1994 resists compression, and examine the histological differences thatmay between exist ( (mammalian bone, development. Other biolog never tension, which would only occur if internal pressure exceeded hydrostatic pressure during exploding. strategyThis ensures thatpneumatocyst tissue Thus, pneumatocysts always experience apositive pressure gradient consistently throughout development. maintain internal pressures less than atmospheric to ensure that thalli need only resist compression internal pressure, material properties, nor tissue morphology survive to their ascent but, instead, howof they resist stress sporophyte grows to increase light single pneumatocyst can experience change a hydrostatic in pressure from Discussion (Table 1). from Acceptedmussel byssus, Bell and Gosline 1996; aquatic plants, Article

, 7 from Denny et al 8 6 Pneumato Creating to to

hydrostatic pressure as they

1032 kPa less than a .

cyst internal pressure

breaking 1997, gas Reilly andBurstein 1974

, and ambient hydrostatic pressure and - filled cavityfilled that ascends Nereocystis

Hale 200 ical materialsical are known to

pneumatocysts were has beenhas limite 1 interception ,

Denny and Gaylord 2002, stipe whichtissue, known is to resist tension ( . ascend Pneumatocysts d. ; Our data cetacean heart valves, Lillie et 2013al.

towards surface the for photosynthesis observed through

similarly suggests

Etnier andEtnier Vil atmospher

from all thallus sizes the water column

to buckleto can specialize in resisting compression and Nereocystis

Denny and Hale 2003 that specialize in either compression ic pressureic at the ocean’s surface .

P and on average

Nereocystis neumatocysts and are at risk buckling, of not lani 2007 ,

u pneumatocyst tissue, which ntil

4 to 1 4 to is a remarkable a is feat;

now, understandingour

have ) at . Future work should

thalli donot adjust a 35 ± 35 ± tm endure significant

Johnson andJohnson Koehl internal pressure )

). ). as the the as

or tension 4.7

m de ep a

. s

This article isprotected rights by All copyright. reserved. Acceptedlifetime ascend through the water column, generally decreasing overall the risk buckling of developmental hurdl estimated in walls of mature pneumatocysts (Fig. wall stress to increase: wall stress youngin pneumatocysts maymore double be than wallthe stress pneumatocysts initially for increases with greatest bucklingrisk of happen growto too deep. guarantee that they stay in compression at all depths and locations atmospheric pressure, thalli only need to tune th morebeen likely survive to time, Articlegradient, causing rupture, it to flood, sink, andbecome otherwise unviable. Thus, over e eventually switch from compression to tension as ascendedit through the hydrostatic pressure atmospheric selection may to act ensure that pneumatocyst internal pressure never exceeds hydrostatic than atmospheric pressure, despite never 2016) demonstrating that the internal pressure in large individuals only changes diurnally by 2kPa gas production and composition greaterin detail ( varies from presumably Nereocystis . Perhaps even more striking that is . R Internal pressure did not change significantly predictably or

isk ofisk , 3 pressure over the lifetime growing of pneumatocysts. This is remarkable pneumatocystas volume

to to depth buckling 1200

thalli that maintained internal pressures less than atmospheric pressure may have , e, wall stress ; ; that i

young pneumatocysts cm . First, . First, because . 3

Data suggest that y , yet internal pressure is somehow maintained. m, radius inner increases but pneumatocyst wall

s, any pneumatocyst with an internal pressure greater than 101kPa would and reproduce declines t he difference in hydrostatic pressure and internal pressure having j . uvenile and the pressure gradient decrease

Because hydrostatic pressure alwaysis greater than experienc oung, small pneumatocysts

eir internal pressures according to surface pressure to be Langdon 1917 6 Nereocystis en B)

. e exposed to the If pneumatocysts small can su

the greatestthe

have internal pressures consistently less , Rigg a –

compressive

across thalli differentof sizes a good strategy, unless they surface

nd Swain 1941 (r Other studies have i

s = 0.4

. do not thicken, s We propose that

loads. - thalli

1.0 cm)

during kelp’s the rvive th

) Second, w mature volutionary , in onein case or are causing explored is (Liggan

at the and initial hen or,

This article isprotected rights by All copyright. reserved. attributed Gaylord 2002), increase in hydrostatic pressure would have a Nereocystis depth buckling a times stronger than they measured characteristics. First, d predict precisely when pneumatocysts willbuckle by estimating buckling stress modifygeometry, material properties, or internal pressure offsetto the risk buckling, of we can wall stress), to1:1 almost 4 stress and risk of buckling pneumatocyst morphology reduce to stress, then r ma tend getto stiffer with age. volume increase pneumatocyst depth to reduce deformation from elevated wall stress. Contrary to this pneumatocystif would be needed to resist breakage pneumatocyst as volume increased (Dromgoole 198 (modulus) nd hence

Accepted Article terial . M Predict . Theoretically, p stiffness to easured Second, p

previously

the of their walls to resist buckling

( suggest 35 n

s young developing

: ESF is oftenis ing 1 could

m; s materials were adjust

. buckling depths (i.e., when increased internal volume constant and wall thickness cause This This pattern consistentis wit

buckling depth Spalding

neumatocysts ing approaching reduce wall stress to lightto

cause pneumatocyst

lowest youngin that pneumatocysts do not adjust their geometry reduce to wall stress. neumatocysts could change geometry their or need be to are are Thus, according our to data,

et al. et attenuation greatest. However,

at at that depth

ata suggest that pn 1

were

tested thein weighted basket .

2003)

and would be barely Given thatthis study found no evidence that pneumatocysts eumatocysts to survive hydrostatic the pressure imposed on them the in field ing

as as they mature remarkably similar to maximumthe depth of observed , deep

. (

Our calculations suggest that thalli growing at to resistto buckling,modulus would need begreatestto at Spalding al. et 2003) . s

It been has suggested that to buckle.to , including big waves passing overhead (Denny

individuals and h Krumhansl et al. (2015), who

tested in this study small i smaller pneumatocysts :t

would be smallest depthat wherematerial wall . Furthermore, if While

pneumatocysts

capable capable of tolerating compressive loads Nereocystis

depth limitation of increase

, buckled at approximately t he present study demonstrates that

were

pneumatocysts donot a

s ( Nereocystis adjust large increase modulusin r

speculation as i

at at = have pneumatocysts

0.4 relatively low the materialthe

variable - noted that kelp tissues

Nereocystis and depth using 1.0 cm)

were adjusting ,

the the r 1b). 1b). modulus

i 35 m are aboutare 3. :t :t ratios elevated 35 ± 4.7 35 ± stiffness age and risk of

adjust

However ha and

depth depth

d .

been Any of from

4 ,

This article isprotected rights by All copyright. reserved. UBC Aqua Society provided Bartomłiej Radziej, Without the SCUBA diving andsnorkeling support of F experience conducting studyThis Acknowledgements field. hydrostatic pressure they needed to be resistto pressure 1.0 cm) material properties switch from re theat surface morphology, bymaintaining but internal pressures gradient pneumatocyst limiting. limits are predominantly defined by pressure with thalli factor has a greater influence. It possible is that l required to clarify the effects of light and pressure on hydrostatic pressure Acceptedieldwork was tedious and required acohort of people collectto useful data a in timelymanner. Article

adapting pressure to only where light isn’t limiting; however, it is equally possible that lower

Conclusion . have .

Risk rupture of is mitigated not by modifying internal However, benefited from , the greatestthe s sisting compression to tension during development ensuring that p

manymeters underwater, which must then ascend through ahydrostatic pressure

or s

young

, not just light attenuation, may and . Nereocystis may

geometry research research in the offield biomechanics

Kyra Janot

also wall stresses

the helpthe and support failure pneumatocysts endless training, SCUBA tanks, and other resources, which made neum

explain to to reduce

in in the field, thalli atocyst , this study would have impossible. been I am very grateful that t

th and are likely at greatest risk of failure due hydrostaticto face biomechanical constraints e lower limit of

the the risk of

collected during this study wall buckl s of of

always experienc ower limits predominantly are defined by with light Dr. Margo Lillie who imparted knowledgeher and thalli

that are consistently

ing help buckling; y thallus

Meagan Abele around 35

adapting light to levels where pressure isn Nereocystis define , particularly with regards pressureto development and to determine which

the pressure, material properties, or tissue oung

. e m depth. P

lower limit compressive loads neumatocyst in the field

, small , Sam were

inflating gas less than atmospheric pressure

3.4 uel Our data suggest th

pneumatocysts

times stronger Starko of . Further studies are s modifydo not

Nereocystis - filled filled , Laura Borden,, Laura

and need not

than i in

at = 0.4

the the . ’t he -

This article isprotected rights by All copyright. reserved. Acceptedorganisms. Denny, & M.W. Denny, determining wave on forces bull the kelp Denny, M., and underlying Charrier, B., LeBail, A. & deReviers, B.2012 Ascophyllum nodosum (Fucales, Phaeophyceae). Brackenbury, A. M., Kang,Article E. J. strength. E Bell, References NSERC Disc Samuel Starko, Jennifer Clark, andAngela Stevenson. research All funds were provided by a many collecting hours data thein lab and field. sub tidal collections possible. I would . & M. &

J. Exp. Biol.

Gosline, J. 1996. Mechanical design mussel of byssus: material yield enhances attachment

Philos Gaylord, B. P. & overy Grant awarded to

Gaylord, B. 2002. mechanicsThe waveof developmental mechanisms.

Hale, B. B. 2003.Cyberkelp: an integrative approach to the .

T .

R 199 oy. :

1005 Soc Cowen, B.1997.Flow and flexibility.II. roles The sizeof shape and in .

- B

17. &

358

Garbary, D. 2006. pressure Air J. regulation in air bladders of

PTM : also 1535 Nereocystis luetkeana

like .

42. Trends

. Plant Proteus: brown algal morphological plasticity

Finally, this manuscript benefited from input of the e

Algae specially thank Sarah Calbick P lant

- 21 swept algae.swept Sci :245 . 17

- . 51. : J. Exp.Biol. 468

77. J. Exp. Biol.

mode 200

for : 3165

205 ling flexible of

volunteering : - 1355 83.

- n 62.

This article isprotected rights by All copyright. reserved. Accepted dissertation, Stanford University, 367pp. Hale, materials: B.2001.Macroalgal foiling forces. fracture and fatiguefrom fluid Ph Business Media Fung, Y. Nereocystis luetkeana Foreman, R.E. 1976.Physiological aspects of carbon monoxide production by the brown alga Bot. aerial petioles of the aquatic plant S.A.Etnier, & Article luetkeana Duncan, M. morphology andresistance hydrostaticto pressure. Dromgoole, F.1981b. Form of andfunction the pneumatocysts marine of algae. II. Variations in pressure and composition of internal gases. Dromgoole, F.1981a. Form of andfunction the pneumatocysts marine of algae. I. in Variations the

94 : 1067

C. 199 . Helgoländer Wissenschaftliche Meeresuntersuchungen

J. 1973. InJ. situ studies growth of andpigmentation of phaeophyceanthe - 72.

Villani, P. J. 2007 , 3.

New York,

Biomechanics: mechanical properties of living tissues . Can .

J 357 pp. .

Bot . Differences mechanical in and structural properties of surface and Nymphaea odorata subsp. tuberosa .

: 352 - 60. Bot

Mar Bot . 24: .

Mar 257 . -

24 66.

: 299

. 24: - 310.

510 (Nymphaeaceae). .

2 - 25. nd

Springer Science & Nere

Am. J. ocystis . D .

This article isprotected rights by All copyright. reserved. and temporal patterns of mechanical stress. Koehl, M. differentto flow regimes: a newwrinkle. Koehl, M. 22: Koehl, M. A.R. 87. Kain, J. 195 factor differentin flow habitats: thallus allometry and material properties of agiant kelp. Johnson, A. & 18:457 Harrison, W. G.1973. Nitrate reductase activity during adinoflagellate bloom. wave Harder, D.

Accepted Article 1067 : 381 - exposed seaweeds.exposed – - - 65. M. 1987. Patterns relativeof growth in 71. 410.

A. A. L.

, R. R.

Hurd, C. Koehl, M. 1994. Maintenance of dynamic similarity strain and environmental stress ,

& 1999. Ecological biomechanics of benthic organisms: history, life mechanical design Silk, W.K.

Wainwright, S.A. 1977. Mechanical adaptations of a giantkelp.

L. Am & ,

. Speck, 2006. T. Comparison mechanical of properties of four large, Liang, H.

Bot.

93: & 1426

Integr Mahadevan, L. 2008.How kelp produce blade shapes suited J . -

Exp 32. . Ne

Comp

. reocystis luetkeana Biol . .

Biol 202 . :

3469 48

: 834 - 76. - 51.

(Pheophyta)

Limnol. Oceanogr . J Limnol .

Phyc J .

. . Exp.

23: Oceanog 181

Biol. . -

This article isprotected rights by All copyright. reserved. Nicholson, N. Costaria Costata B.Maxell, & Biol. design in fin whales: high Lillie, M.A. M Liggan, L.2016. Chem Langdon, S. Phyc. theof kelp (Laminariales, Phaeophyceae) supports Lane, between red algae andkelps influence biomechanical properties. Krumhansl, K. .

AcceptedS Article .

216 dissertation

. 42 C. E., Mayes, C., Druehl, L.D.

Soc : : 493 2548 .

, C. 1917.

- Miller, 1996.K. Demograph Piscitelli, M. A.

512. : L. 1970. Field studies on giantthe kelp

- 149 A. 63. .

Under pressure: biomechanics of buoyancy Bull in Kelp ( (Laminariales, P , University Columbia, British of Vancouver, 78pp.

- Demes, 56.

Carbon monoxi

- stiffness stiffness arteries protect against

K. , Vogl, A. W.,

W. ,

haeophyta) in Carrington,

& Saunders, G. W. 2006.Amulti de, occurrence free kelp in ( ic studiesic of annual the kelps Gosline, M.&J. Shadwick, R. E.2013. Cardiovascular

E. Puget Sound, W & substantial taxonomic re

Harley Nereocystis , adverse pressure gradients at depth. C.

D . 2015. ashington. Am. J.Bot. . Nereocystis luetkeana J .

Phyc ‐ Nereocystis Luetkeana Divergent growth gene molecular investigation ol Nereocystis leutkeana

‐ Bot. Mar. . organization. 102 6 : 177 :1938 - 82.

39 - 44.

). ). :479

J J. strategies

Am - 89.

J. and .

) Exp.

This article isprotected rights by All copyright. reserved. reproductive investment in a drifting macroalga Stewart, H. Phyc avoidance and tolerance strategies wave in Starko, S. Nereocystis luetkeana Springer, Y. water (>30m) ma Spalding, H. alga, Rigg, G. contribution from Puget the Soundmarine station. Rigg, G. S Reilly, Accepted Article urg . ol

Nereocystis luetkeana 56

. D. T. & D. T.

52 :

B. on 1912.Notes ecology the andeconomic importance of 1001 B.

& :54

L. 2006. Ontogenetic changes buoyancy,in breaking strength, extensibility, and

, Foster, M. S. & Heine, N.J. &

Martone, P. Hays, C. - -

63. 22.

Swain, L.A. 1941.Pressure Burstein, A. H. 1974. mechanicalThe properties of cortical bone. croalg

. ,

Lenfest Ocean Program report Carr, M. ae inae central California

T. .

2016. Evidence of an evolutionary Plant & P Mackey, M. hysio l.

2003. Composition distribution, and abundance of deep -

- 16: swept intertidal kelps (Laminariales, Phaeophyceae). composition relationships of the gas in marine the brown . 361.

J M. 2007. Ecologymanagement and of the Bull Kelp, Turbinaria ornata .

Phyc The WorldPlant

, . 53

39:

pp 273 .

- - developmental trade 84.

(Phaeophyta).

15 Nereocystis luetkeana : 83 - 92.

J J . .

- Phyc B off betweenoff drag one ol J . oint : a : 42 : 43

J - - .

50.

This article isprotected rights by All copyright. reserved. 2. needle. Figure pressure. originaltheir size) that often reach the surface, experiencing only atmospheric (not hydrostatic) increase and exp toward the surface and experience le cycle. pneumatocysts subtidally, up 35 to shaded black; arrows pointing increasing toward parameter values). Young(a) thalli develop small Figure 1: Figure nuclear Laminariaceae, Lessoniaceae and (Phaeophyceae) based onplastid Yoon, H. S.,Lee, (Agardh): field test of a computational model. Utter, B. & substrata. Thiel, M.&

Accepted Article (b 2 - legends Internal pneumatocyst encoded ITSencoded sequence comparisons. : Diagram water of manometer. Pneumatocysts were punctured using alubricated syringe -

c) Overc) time

Schematic of pneumatocyst development in growing Oceanogr. Mar Biol. Denny, M.1996. Wave

Gutow, L.2005. The ecology rafting of in marine the : ands into the stipe. (e) Mature thalli have lar

J. Y.,Boo, S.M.,& Bhattacharya, D. 2001.Phylogeny Alariaceae, of

stipes elongate and pneumatocyst volume increases as pneumatocysts move pressure was calculated 42 :

181 - m where deep, hydro induced forcesinduced on giantthe kelp ss pressure.ss (d) As thalli grow - 264.

Mol. J .

Exp Phylogenet. E .

Bio from change the water in height static pressure g is l.

199 ge pneumatocysts (more 300 than

Nereocystis : vol 2645 environment. I.The floating

- pneumatocyst volume to continues . encoded RuBisCoencoded spacer and

21 Macrocystis pyrifera - 54. : 231

reatest during the life thalli (pneumatocysts - 43.

(h)

using Eq. times This article isprotected rights by All copyright. reserved. part Cin represents linear regression of geometric ratio against inner radius an inner radius between 0.4 and1cm. inner thickness F 25.79, P<0.001, R Figure 101.2, P<0.001, R Figure methods) represent functions of Figure Acceptedig Article ure ure

pneumatocyst radius 4 3 6 5 : ( : Young’s modulus : : . W I variation pressurein calculation from 1mmmeasurement on error ( nternal pneumatocystnternal pressure

A F all stress 1,62 ) pneumatocyst Dotteddepth. line represents atmospheric pressure (101 kPa). Error bars

Collection depth, (B) p = 84.09 P< 0.001,R 2 2 = 0.43 = 0.62 es es calculated , . ,

y =0.01x y =0.03x+3.05 Data points within the (material stiffness) a as function of

Eq. 3 f neumatocyst wall stress +3.5 2

= 0.53 Line Line or pneumatocysts collected (black circles) and hydr ) .

) in partin B represents . ,

y =1.3x +1.4)

shaded boxes represent young pneumatocysts with , , (C) ratio of internal radius wall to

and tissue(D) volume as

pneumatocyst volumes moving ostatic pressure (open circles) as

from

average of wall wall average of stress

0 . -

manometer 9 m 9

depth

a function ( ( F (see F 1,60 1,32

and line = =

of This article isprotected rights by All copyright. reserved. pneumat Table 1.

Accepted ArticleSample 8 7 6 5 4 3 2 1

ocysts. Collection depth, wall stress, buckling stress, ESF, andbuckling depth

Collection depth depth (m) 5.44 5.89 5.33 7.34 7.00 7.32 4.22 7.60

radius (cm) Internal

0.70 0.58 0.40 0.55 0.45 0.55 0.45 0.45

Wall stress 245.60 258.46 287.26 285.11 255.45 284.56 201.86 267.38 (k P

stress stress (kPa) Buckling Average: 1032.13 785.94 797.76 886.83 796.20 930.34 892.76 950.69

3.4 ± 0.43.4 ± 3.20 3.09 3.59 3.11 3.12 3.27 4.42 3.56 ESF of 8young, small

depth depth (m) Buckling 35 ± 4.735 ± 30 30 32 35 35 37 40 43

This article isprotected rights by All copyright. reserved. Accepted Article