Molecular phylogeny of pelagic Sargassum

Ethan Alley1, 2, Alesia Hunter1, 3, Walter Hutcheson1, 4, Kate Petersen,1, 5, Katherine Running1, 6

SEA Semester: Marine Biodiversity and Conservation Class C266 April 18-May 19, 2016

1- Sea Education Association, Woods Hole, Massachusetts, 2- Harvard College, Cambridge, Massachusetts, 3- Beloit College, Beloit,

Wisconsin, 4- New York University, New York, New York, 5- The Evergreen State College, Olympia, Washington, 6- American University,

Washington, District of Columbia

Acknowledgements

We wish to express our sincere gratitude to Amy Siuda, Laura Cooney, and Matt Hirsch

for their invaluable research advice and lab assistance. Additionally, we extend thanks to Robbie

Smith for sample collection and moral support at sea. We are grateful to the C266 staff and crew

for their companionship, mentorship, and support of our work aboard the SSV Corwith Cramer.

We are indebted to the past SEA Semester research cruises and Sea Education Association for

their sample collection and preservation. We also would like to express our appreciation to

Annette Govindarajan for her guidance with analyzing our molecular results. Special thanks to

the Virginia Wellington Cabot Foundation for contributing funding towards the Marine

Biodiversity and Conservation program at Sea Education Association.

Abstract

Pelagic Sargassum specimens were collected and preserved at eight stations on four

separate Sea Education Association (Woods Hole, Massachusetts, USA) research cruises

undertaken between April 2015 and April 2016 in the tropical Atlantic Ocean, Caribbean, and

Sargasso Sea. Collected specimens included Sargassum natans I, S. natans VIII, S. fluitans III,

and two morphologically distinct samples, Sargassum sp. Deoxyribonucleic acid (DNA) was

extracted from the specimens and the mtsp and rbcL-S genetic marker regions were amplified

and sequenced. S. fluitans III and S. natans (I and VIII) are shown to be 1.5% genetically distant

at the concatenated sequence of these loci. Mtsp had 10 single nucleotide polymorphisms (SNPs)

(2.4%) and rbcL-S had 2 SNPs and a 9 basepair insertion (1.6%). S. fluitans III and S. natans (I

and VIII) showed consistent allelic differences at both loci. No variability was found between S.

natans I and S. natans VIII. Sargassum sp. was found to be molecularly distinguished from both

S. fluitans III and S. natans (I and VIII) at the mtsp locus. The phylogeny showed that S. natans I

and S. natans VIII are more closely related to each other than they are to S. fluitans III, with

Sargassum sp. as an intermediate taxa. Our results do not support species delineation between S.

natans I and S. natans VIII. These results support but do not confirm S. natans and S. fluitans

species delineations. The analysis of Sargassum sp. suggests the possibility of cryptic diversity

in pelagic Sargassum.

Introduction

As a foundation taxon, Sargassum provides otherwise rare deep ocean surface substrate

to complex localized communities of epibiont and motile fauna (Stoner and Greening 1984;

Casazza and Ross 2008). Over 200 marine species associate with pelagic Sargassum including

endemic species such as the Sargassum frogfish (Histrio histrio), Sargassum crab (Planes spp.),

and Slender Sargassum shrimp (Latreutes fucorum), which exhibit color matching and plant

mimicry adaptive strategies (Coston-Clements 1991; Hacker and Madin 1991; Trott et al. 2011).

These pelagic communities provide food, shelter and nursery grounds for a variety of threatened

and economically important taxa such as sea turtles (Carr and Meylan 1980; Carr 1987; Luschi et

al. 2003), tuna (Coston-Clements 1991), and dolphinfish (Oxenford and Hunte 1999).

Due to its primary and significant ecosystem functions and vulnerability to damage from

pollution (Powers et al. 2013) and commercial extraction (Laffoley et al. 2011), pelagic

Sargassum is of conservation concern (South Atlantic Fishery Management Council 2002;

United Nations Environment Programme 2012; Sargasso Sea Commission 2014). Further, a

small study suggested that the species composition of Sargassum associated communities may

have changed over the last 40 years (Huffard 2014), with unknown ramifications for pelagic

ecology. The authors pointed out that ocean temperature and acidity increased concurrently and

suggested more research to investigate this correlation. Additional research is required to verify

Sargassum associated community changes.

Øjvind Winge (1923) and Albert Parr (1939) developed the taxonomic classification

system currently used to describe pelagic Sargassum (Genus Sargassum, subgenus Sargassum,

section Sargassum), which is based on morphological characters such as the presence or absence

of stem thorns and the size and shape of bladders and leaf blades. Parr (1939) described two

species, Sargassum natans and Sargassum fluitans, which have 6 morphological forms: S. natans

I, S. natans II, S. natans VIII, S. natans IX, S. fluitans III, and S. fluitans X. These form

variations are not commonly addressed in modern field guides (Schneider and Searles 1991;

Littler and Littler 2000; Dawes and Mathieson 2008) or scientific literature (Stoner and Greening

1984; Calder 1995) which tend to only refer to S. natans or S. fluitans (most likely referring to S.

natans I and S. fluitans III). This is probably due to the seemingly ubiquitous nature of S. natans

I and S. fluitans III throughout their range (A. N. S. Siuda pers. comm.).

Distinguishing between Sargassum forms in scientific research is necessary because

Sargassum species and form identity appears to be a driver of species abundance and diversity in

Sargassum-associated pelagic ecosystems. For example, a study of S. natans and S. fluitans

found that these species support distinct hydroid species assemblages (Calder 1995). In another

study, Litiopa melanostoma, a common snail, was found to be more abundant on S. natans than

on S. fluitans (Stoner and Greening 1984). No form distinctions were made in either study.

Winge (1923) also observed prolific hydroid colonization on S. fluitans III and very limited

colonization on S. natans II. The only ecological study addressing S. natans I, S. natans VIII, and

S. fluitans III found species diversity differences between the three forms, with S. natans VIII

having the lowest diversity (L. M. Martin unpubl.).

Unprecedented and economically disruptive Sargassum beach inundation events were

reported in 2011, 2012, 2014, and 2015 in the western Caribbean and Gulf of Mexico (Higgins

2011; MercoPress 2015). Some news outlets suggested that the inundations came from the

Sargasso Sea (Boodram 2015; Miller 2015), an area of the north Atlantic Ocean named for its

Sargassum population. However satellite data and backtracking analysis suggested a previously

unknown equatorial Atlantic source (Johnson et al. 2012; Gower et al. 2013). Both S. natans and

S. fluitans (without form descriptions) were implicated in the inundations by various sources

(Johnson et al. 2012; Economist 2015; Fardin et al. 2015). However, verified morphological

identifications and voucher specimen collections were sparse and not always accurate. For

instance, Szechy et al. (2012) reported an equatorial Atlantic S. natans bloom in 2011, but

voucher photographs clearly show S. fluitans specimens, which can be conclusively identified by

the presence of thorns on the stem (Parr 1939). Further complicating the investigation of the

beach inundations was the identification of huge quantities of floating S. natans VIII in 2014

(Schell et al. 2015), a form that had not been treated in the literature since Parr (1939). Schell et

al. (2015) subsequently documented S. natans VIII throughout the tropical Atlantic Ocean,

Caribbean, Antilles Current, and southern Sargasso Sea, a phenomena not previously observed

during their 20 years of shipboard research.

The confusion surrounding the Sargassum beach inundation events underlines the need to

clarify ambiguities in species and form definitions, which complicate Sargassum research at a

time when form distribution and abundance may be changing and original baseline data is

limited. The lack of widespread treatment of the different morphological forms in the literature

challenges the development of functional ecological narratives regarding pelagic Sargassum. The

degree to which existing ecological data for pelagic Sargassum could be confounded by incorrect

or incomplete species or form identification is unknown.

As a complex and morphologically plastic taxa (Kilar 1992), molecular techniques are

necessary to corroborate and refine morphological species and form delineations in Sargassum.

Genetic markers used previously to delineate taxa in Sargassum and other brown algae groups

include the chloroplastic Ribulose bisphosphate carboxylase large chain + spacer + partial

Ribulose bisphosphate carboxylase small chain (rbcL-S) (N. E. Phillips unpubl.; Phillips et al.

2005; Dixon et al. 2014), the nuclear Internal Transcribed Spacer 2 (ITS-2) (Stiger et al. 2000,

2003; Mattio et al. 2010), and the mitochondrial cytochrome oxidase 3 subunit (cox3) (Mattio et

al. 2008, 2009; Dixon et al. 2014). In these studies, the rbcL-S, ITS-2, and cox3 markers were

successfully used to obtain consistent phylogenetic resolution at the subsections level or higher,

but resulted in shallow unresolved trees below the subsection level.

An additional marker, the mitochondrial intergenic spacer region (mstp) between the

mitochondrial 23S gene and transcription ribonucleic acid Valine (tRNA-Val), was proposed for

use within Sargassum and other Sargassaceae by Draisma et al. (2010) after development for use

in the Fucus genus by Coyer et al. (2006). This non-coding region was shown to be the most

variable of those studied, but could not be aligned for a multi-genus data set (Draisma et al.

2010). A subsequent marker assessment analysis of ITS-2, the chloroplast partial RubisCO

operon (rbcL-S), cox3, mtsp and the mitochondrial cytochrome oxidase unit 1 gene (Co1) by

Mattio and Payri (2010) identified mtsp as a potentially useful barcoding marker, but also found

difficulties in alignment.

Although analyses of these loci have produced important taxonomic insights across the

genus in benthic species, molecular phylogeny of pelagic Sargassum is highly understudied. A

2015 study of S. fluitans III, S. natans I, and S. natans VIII at the CO1 marker found a single

nucleotide difference between S. fluitans III and S. natans (I and VIII), but no variation between

S. natans I and S. natans VIII at this locus (A.N.S. Suida pers.comm.). Camacho et al. (2015)

included two samples identified as S. natans and two identified as S. fluitans in a molecular

phylogeny of Sargassum subgenus Sargassum from the Caribbean and western tropical Atlantic

using ITS-2, rbcL-S, and cox3. Form distinctions were not made in this study. As a whole, the

phylogeny constructed from these markers did not have sufficient resolution within Sargassum

sect. Sargassum. When the pelagic species’ sequences were retrieved from GenBank and

examined, only rbcL-S appeared to be variable between S. natans and S. fluitans. Camacho et al.

(2015) also sequenced mtsp but did not include these sequences in the phylogeny due to

alignment difficulty. However, mtsp was one of the few markers to show any variability within

Sargassum sect. Sargassum (O. Camacho pers.comm.).

In this study, we amplify and sequence rbcL-S and mtsp loci of pelagic Sargassum which

represent the predominant morphological forms collected in the Sargasso Sea, western tropical

Atlantic and Caribbean, S. natans I, S. natans VIII, and S. fluitans III, as well as two specimens

of Sargassum sp. (provisionally identified as S. natans II) collected in the south Sargasso Sea.

We then construct a molecular phylogeny based on these markers.

Methods

Pelagic Sargassum specimens were collected and preserved at eight stations on four

separate Sea Education Association (Woods Hole, Massachusetts, USA) research cruises (C259,

C263, C264, and C266) undertaken between April 2015 and April 2016 in the tropical Atlantic

Ocean, Caribbean, and Sargasso Sea (Table 1). Sampling from a broad geographic area was

necessary due to the minimally overlapping ranges of pelagic Sargassum forms (Schell et al.

2015). Every 12 hours along these cruise tracks, at 0000 h and 1200 h, a one-meter wide, half-

meter high Neuston net with 333-micrometer mesh was deployed. The net was towed at a speed

of two to three knots for 30 minutes, for a total approximate sampling distance of one nautical

mile. Concurrent sampling using a dip net with 333-micrometer mesh occurred at morning

stations. Sargassum was preserved in silica for molecular analysis when at least seven

individuals of a particular morphological form were collected at a single station. All specimens

were identified using morphological characters described by Parr (1939). Morphological

vouchers for all samples were collected and are kept at Sea Education Association.

A 5-10 cm sample was clipped from the apical end of each Sargassum replicate and

persistent epiphytes were removed with a razor blade. The samples were then rinsed in

freshwater and dried in silica desiccant for at least 48 hours before extraction. Two to four leaf

blades or an approximately equivalent volume of floats and stems from the dried samples were

ground in 1.5 ml microcentrifuge tubes using disposable pellet pestles.

DNA was extracted using MoBio Power Plant Pro Kit (Carlsbad, California, USA)

according to manufacturer protocols as modified by Wilson et al. (2016) to replace the bead

beating step with 24hrs of heating at 65°C with periodic vortexing. Extractions were cleaned

using MoBio Power Clean Pro Kit according to manufacturer protocols. The clean genomic

DNA was profiled using a Nanodrop ND-1000 Spectrophotometer.

A mitochondrial spacer (mtsp), and the chloroplast-encoded partial Ribulose

bisphosphate carboxylase large chain + spacer + partial Ribulose bisphosphate carboxylase small

chain (rbcL-S) regions were amplified using Polymerase Chain Reaction (PCR). PCR products

were amplified using site-specific primers (Table 2) and reaction protocols were optimized from

those developed by Coyer et al. (2006) for mtsp and Mattio et al. (2008) for rbcL-S. The cycling

conditions included: (i) a 4 minute initial denaturation at 95°C, (ii) 40 cycles of a 40 second

denaturation at 95°C, (iii) a 30 second annealing at 50°C (mtsp), 45°C (rbcL-S), (iv) a 45 second

extension at 68°C, and (v) a 7 minute final extension at 68°C. New England Biolabs HotTaq 2x

Master Mix with Standard Buffer (Ipswich, Massachusetts, USA) was used for all reactions.

PCR products were verified with agarose gel electrophoresis. PCR products were purified

with Qiagen Qiaquick PCR Cleanup Kit (Germantown, Maryland, USA). The DNA

concentration of purified PCR products was quantified with a Nanodrop ND-1000

Spectrophotometer. Purified PCR products were sequenced by Eurofins MWG Operon

(Huntsville, Alabama, USA) in both directions with primers from the amplification step (Table

2) using the Sanger sequencing method. In total, this analysis included 13 replicates of

Sargassum natans VIII, 15 replicates of Sargassum natans I, 14 replicates of Sargassum fluitans

III, and two replicates of Sargassum sp., which were morphologically distinct specimens

suggestive of Sargassum natans II.

Results

This study produced 65 sequences for mtsp and 49 for rbcL-S which are available on

Genbank (Table 3). Sequences were aligned with ClustalW (Larkin et al. 2007) in Geneious

9.1.7 software (Biomatters Ltd.) to an appropriate outgroup obtained from Genbank via the

National Center for Biotechnology Information’s Basic Local Alignment Search Tool (NCBI

BLAST). The mtsp alignment was 195 base pairs (bp) long including a two and three bp gap

introduced by the outgroup comparison. This is significantly shorter than previously reported

(Coyer et al. 2006, Draisma et al. 2010). The rbcL-S alignment was longer, at 687 bp, including

a 9 bp gap. Both alignments were examined and it was determined that all sequences from

Sargassum natans I and Sargassum natans VIII were identical to one another at both the mtsp

and rbcL-S loci. All S. fluitans III sequences were also identical to one another at each loci.

Lastly, the sequences obtained from Sargassum sp. were found to be identical to one another at

both loci.

We discovered 10 polymorphic loci (5.2%) in mtsp at which S. fluitans III and S. natans I

and VIII showed a consistent allelic pattern, which corresponded exactly to the species

delineation (S. natans or S. fluitans). We found 2 polymorphic loci and a 9 bp indel in the rbcL-S

sequences, which also corresponded to the S. natans/S. fluitans delineation and did not vary

between the morphological forms of S. natans (.2%).

Sequences from samples with both rbcL-S and mtsp represented in the alignments were

concatenated for S. natans I (n=15), S. natans VIII (n=13), S. fluitans III (n=14), and Sargassum

sp. (n=2) resulting in a combined alignment of 882 bp. Identical sequences for each form were

removed. Gaps in both sequences were treated according to the simple indel coding method of

Simmons and Ochoterena (2000) to inform the phylogeny.

A consensus Neighbor Joining Tree was constructed from the concatenated, gap-coded

sequences in Geneious 9.1.7 software (Biomatters Ltd.) using the Tamura-Nei genetic distance

model with 10,000 bootstrap replications and a support threshold of 95% (Figure 1). The

concatenated consensus tree showed genetic distance of 1.5% between S. natans (I and VIII) and

S. fluitans III, .07% between S. natans (I and VIII) and Sargassum sp., and .08% between

Sargassum sp. and S. fluitans III. Genetic distances were also calculated for each locus

individually (not concatenated) using the same parameters above. This showed rbcL-S was less

variable, with .4% genetic distance between S. natans (I and VIII) and S. fluitans III, .4% genetic

distance between Sargassum sp. and S. fluitans III, and no distance between S. natans (I and

VIII) and Sargassum sp. Contrarily, mtsp alone produced genetic distances of 5.5% between S.

natans (I and VIII) and S. fluitans III, 5.6% between S. natans (I and VIII) and Sargassum sp.,

and 2.2% between Sargassum sp. and S. fluitans III.

Discussion

We found genetic distance between S. fluitans III and S. natans (I and VIII) at both the

mtsp (5.5%) and rbcL-S (.4%) loci. This finding corroborates the morphologically based species

delineations proposed by Parr (1939) as well as the 1 SNP difference observed in Co1 between

these taxa (A.N.S. Siuda pers. comm.). Our observed variation does not meet the criteria for

species delineations proposed by Mattio and Payri (2010) who conducted the only study of the

effectiveness of various genetic markers as barcodes for Sargassum subgenus Sargassum. This

study included the section Sargassum, to which pelagic Sargassum belongs, along with a number

of additional sections, but did not include any samples from pelagic Sargassum. The authors

reported a range of interspecific genetic distances for mtsp (16-19.5%) and rbcL-S (1-6%)

(Mattio and Payri 2010), much higher than the genetic distances found between our S. natans

group and S. fluitans III at these loci.

Difficulty resolving closely related taxa at or below the section level within the

Sargassum genus is not uncommon (Phillips 2005; Mattio et al. 2010; Dixon et al. 2014). N. E.

Phillips (unpubl.) argues that there is unlikely to be a universal pattern of genetic variation for

spacer regions in macroalgae, because these regions demonstrate distinct patterns of variation

depending on the studied taxa. It may be that the barcode assessment of Mattio and Payri (2010)

was too broad to definitively represent Sargassum sect. Sargassum and that this section has

patterns of genetic variation distinct from other sections in subgenus Sargassum. This hypothesis

is corroborated by the observation that excluding Sargassum sect. Sargassum from barcode

analysis changed the identification success of all markers especially rbcL-S (Mattio and Payri

2010) and the evidence that a molecular phylogeny with the best known markers was unable to

resolve between species within Sargassum section Sargassum (Camacho et al. 2015). The

section has been noted as a primary target for barcode development efforts because “...no good

barcoding marker will be identified for the genus Sargassum before one can genetically

discriminate between the very closely related and morphologically distinct species of S. section

Sargassum” (Mattio and Payri 2010).

We found S. natans I and S. natans VIII to be genetically identical at the mtsp and rbcL-S

loci despite the fact that both morphological forms possess distinct morphological characters

(Parr 1939; Schell et al. 2015) and associated ecological communities (L.M. Martin unpubl.).

This data corroborates the previously mentioned Co1 marker study which also found no

difference between these forms (A.N.S. Siuda pers. comm.) However, our results clearly show

that S. natans I and S. natans VIII, traditionally considered to be morphological variants of the

same species, are more closely related to each other than they are to either S. fluitans III or

Sargassum sp. This highlights important areas of future research, both for development of new

genetic markers to resolve evolutionary relationships in pelagic Sargassum and to investigate

how distinct phenotypes and ecological characteristics can arise from yet undiscovered genetic

variation.

We found that Sargassum sp. was .07% distant from S. natans (I and VIII) and .08%

distant from S. fluitans III, suggesting the possibility of cryptic diversity in pelagic Sargassum.

These samples had subtle, but noticeable morphological differences which resembled S. natans II

(Parr 1939). Unfortunately, we were unable to complete our identification before the majority of

our specimens were discarded. More specimens are required to determine whether this form was

described by Parr (1939) and that the observed grouping is not anomalous. This finding

illustrates the importance of further integrative taxonomy research on the less common

morphological forms of pelagic Sargassum, which could clarify evolutionary relationships and

may also contribute to the understanding of inter and intraspecific genetic diversity in Sargassum

sect. Sargassum.

Further research is also necessary to establish new genetic markers within Sargassum

sect. Sargassum to facilitate integrative taxonomy work, with barcoding assessments on large

representative samples from within the section. Lastly, research into life history and reproduction

patterns of pelagic Sargassum could clarify morphological changes that occur during an

individual’s life cycle.

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Table 1: Station summary information for samples of each morphological form.

Date Position Region # Samples

Sargassum natans VIII

26 Nov 2015 16°20’N x 33°20’W E. Tropical Atlantic 9

7 Dec 2015 14°54’N x 53°04’W W. Tropical Atlantic 4

Sargassum fluitans III

7 Dec 2015 14°54’ N x 53°04’W W. Tropical Atlantic 4

21 Feb 2016 17°56’N x 65°06’W E. Caribbean 10

Sargassum natans I

26 Apr 2015 25°00’N x 67°09’W Sargasso Sea 7

11 May 2015 32°55’N x 65°20’W Sargasso Sea 8

Other:

Sargassum sp.

20 Apr 2016 18°40.9'N x 66°9.7'W Tropical Atlantic 1

24 Apr 2016 24°10.7'N x66°44.2'W Sargasso Sea 1

Table 2: Primers used to isolate the selected markers from genomic DNA. AT = PCR annealing

temperature (°C).

Marker Primer Primer sequence Reference AT

mtsp mtsp-F 5′ CGTTTGGCGAGAACCTTACC-3′ Coyer et al. 2006 50

mtsp-R 5′ TACCACTGAGTTATTGCTCCC-3′ Coyer et al. 2006 50

rbcL-S 3F 5′-CATCGTGCTGGTAACTCTAC-3′ Mattio et al.

2008

45

S97R 5′-CATCTGTCCATTCWACACTAAC-3′ Mattio et al.

2008

45

Table 3: GenBank accession numbers for sequenced samples

Figure 1: Consensus Neighbor Joining Tree of the concatenated rbcL-S and mtsp loci. Bootstrap

support is indicated at nodes, with those less than 95% supported collapsed. Number of replicates

of each sequence shown in parentheses. Branch length is unconstrained and corresponds to

genetic distance at a scale of 0.002. Outgroup sequences obtained from GenBank using NCBI

Blast.