ANALYSIS OF THE COMPLETE PLASTOMES OF THREE SPECIES OF MEMBRANOPTERA(CERAMIALES, RHODOPHYTA) FROM PACIFIC NORTH AMERICA1

Jeffery R. Hughey2

Division of Mathematics, Science, and Engineering, Hartnell College, 411 Central Ave., Salinas, California 93901, USA

Max H. Hommersand

Department of Biology, University of North Carolina at Chapel Hill, CB# 3280, Coker Hall, Chapel Hill, North Carolina 27599-

3280, USA

Paul W. Gabrielson

Herbarium and Department of Biology, University of North Carolina at Chapel Hill, CB# 3280, Coker Hall, Chapel Hill, North

Carolina 27599-3280, USA

Kathy Ann Miller

Herbarium, University of California at Berkeley, 1001 Valley Life Sciences Building 2465, Berkeley, California 94720-2465, USA

and Timothy Fuller

Division of Mathematics, Science, and Engineering, Hartnell College, 411 Central Ave., Salinas, California 93901, USA

Next generation sequence data were generatedand used to assemble the complete plastomes of theholotype of Membranoptera weeksiae, the neotype(designated here) of M. tenuis, and a specimenexamined by Kylin in making the new combinationM. platyphylla. The three plastomes were similar ingene content and length and showed high genesynteny to Calliarthron, Grateloupia, Sporolithon, andVertebrata. Sequence variation in the plastomecoding regions were 0.89% between M. weeksiae andM. tenuis, 5.14% between M. weeksiae andM. platyphylla, and 5.18% between M. tenuis andM. platyphylla. We were unable to decipher thecomplete mitogenomes of the three species due tolow coverage and structural problems; however, weassembled and analyzed, the cytochrome oxidase I,II, and III loci and found that M. weeksiae andM. tenuis differed in sequence by 1.3%, M. weeksiaeand M. platyphylla by 8.4%, and M. tenuis andM. platyphylla by 8.1%. Evaluation of standardmarker genes indicated that sequences from therbcL, RuBisCO spacer, and CO1 genes closelyapproximated the pair-wise genetic distancesobserved between the plastomes of the threespecies of Membranoptera. A phylogenetic tree basedon rbcL sequences showed that M. tenuis andM. weeksiae were sister taxa. Short rbcL sequenceswere obtained from type specimens of M. dimorpha,M. multiramosa, and M. edentata and confirmed theirconspecificity with M. platyphylla. The data supportthe recognition of three species of Membranoptera

occurring south of Alaska: M. platyphylla, M. tenuis,and M. weeksiae.

Key index words: Ceramiales; Delesseriaceae; holo-type; Membranoptera; Northeast Pacific; phylogeneticsystematics; plastid genome; plastome; rbcL

Freshwater and Rueness (1994) were the first touse gene sequences to address species-level taxo-nomic problems in the Florideophyceae. Since then,other investigators have analyzed nuclear, plastid,and mitochondrial DNA markers to resolve bothhigher level and species-level problems in red algae.The major limitation of current markers is therequirement for complete or nearly complete DNAsequences to infer phylogenetic relationships.Although this is not an issue with fresh or silica gel-dried material that contains large amounts of intactnucleic acids, DNA from historically importantherbarium specimens, including type specimens, isdegraded and present only in low concentrations(Hughey and Gabrielson 2012). The correct applica-tion of any name requires genetic analysis of typematerial (Hughey et al. 2001, 2002, Gabrielson2008a,b, Gabrielson et al. 2011, Lindstrom et al.2011, 2015a,b, Martone et al. 2012, Hind et al.2014a,b, 2015, Sissini et al. 2014, Adey et al. 2015,Hernandez-Kantun et al. 2015, van der Merwe et al.2015). In these studies, however, targeted PCRmethods were used to acquire small hypervariablesequences, which due to their length are not suit-able for phylogenetic analysis. Hughey et al. (2014)proposed a solution to the limitation of working

1Received 3 December 2015. Accepted 12 August 2016.2Author for correspondence: e-mail: jhughey@hartnell.edu.Editorial Responsibility: C. Lane (Associate Editor)

J. Phycol. 53, 32–43 (2017)© 2016 Phycological Society of AmericaDOI: 10.1111/jpy.12472

32

with degraded DNA by analyzing the plastid andmitochondrial genomes from 12 archival herbariumspecimens (including nine type specimens) of Ban-giaceae using next generation sequencing method-ologies. Herein, we extended this methodology toclosely and more distantly related species of North-east Pacific florideophytes in the genus Membra-noptera Stackhouse (Delesserioideae, Delesseriaceae)to evaluate the efficacy of plastid markers, particu-larly the most commonly used marker rbcL.

On the basis of morpho-anatomical characters,Gabrielson et al. (2012) recognized six species ofMembranoptera in the Northeast Pacific: M. dimorphaN.L.Gardner (type locality: Neah Bay, ClallamCounty, Washington, USA); M. multiramosaN.L.Gardner (type locality: Moss Beach, San MateoCounty, California, USA); M. platyphylla (Setchell &N.L.Gardner) Kylin (type locality: Pleasant Beach,Kitsap County, Washington, USA); M. spinulosa(Ruprecht) Kuntze (syntype localities: Sea ofOkhotsk and St. Paul Island, Bering Sea); M. tenuisKylin (type locality: Canoe Island, San JuanCounty, Washington, USA, dredged 10–20 mdepth); and M. weeksiae N.L.Gardner (type locality:Pacific Grove, Monterey County, California, USA).They queried the distinctions between M. tenuisand M. weeksiae and among M. multiramosa,M. platyphylla, and M. spinulosa. Recently, on thebasis of their analyses of mitochondrial cytochromeoxidase 1 (COI-5P), nuclear internal transcribedspacers (ITS), large subunit of the ribosomal cis-tron (LSU), and RUBISCO large subunit (rbcL)DNA sequences, Wynne and Saunders (2012) rec-ognized only one species, M. platyphylla, distributedfrom British Columbia, Canada to central Califor-nia. All but two of their samples came from BritishColumbia, one from Monterey County, Californiaand another identified as M. weeksiae from BoilerBay, Oregon, USA (GenBank rbcL sequence-AF257384).

Our preliminary analysis of Membranoptera rbcLsequences from northern Washington (San JuanCounty) to San Diego, California indicated the pres-ence of three species. To determine the correctapplication of names and to compare rbcLsequences with those of the entire plastid genomeas a diagnostic marker for red algal species, wesequenced the plastomes from the holotype ofM. weeksiae, the neotype of M. tenuis collected by N.L. Gardner in the absence of the type collection atLund, and a herbarium specimen from Friday Har-bor, Washington identified by Kylin as M. platyphylla.From the mitogenomes of these specimens we alsoanalyzed the cytochrome oxidase I, II, and III locifor all three species. In addition, a portion of therbcL gene from the holotype of M. dimorpha, an iso-type of M. edentata Kylin (type locality: Carmel CityPoint, Monterey County, California; currentlyconsidered a synonym of M. platyphylla), and twoisotypes of M. multiramosa were also analyzed.

MATERIALS AND METHODS

DNA extractions and PCR. DNA was isolated from herbar-ium specimens at Hartnell College following the precaution-ary contamination guidelines outlined by Hughey andGabrielson (2012) and the protocol in Lindstrom et al.(2011). Silica gel-preserved contemporary specimens wereprocessed at the University of North Carolina, Chapel Hillfollowing the protocol in Hughey et al. (2001). DNA fromherbarium type and nontype specimens was PCR amplifiedusing the primer pair F753 (Freshwater and Rueness 1994)and R900 (50-GCGAGAATAAGTTGAGTTACCTG-30) follow-ing the thermocycling methods in Lindstrom et al. (2011).This primer pair represents nucleotide positions 774–895 ofthe rbcL gene. Silica gel-preserved material was amplified withprimer pairs F57/R753 and F753/RrbcS (Freshwater andRueness 1994) based on the protocols in Hughey et al.(2001).

Phylogenetic analysis. Alignment of the rbcL sequences wasaccomplished using the G-INS-1 Progressive Method inMAFFT (Katoh and Standley 2013). Maximum likelihoodanalysis of the 13 Membranoptera sequences (Table 1) was per-formed using RAxML (Stamatakis 2014) with 1,000 bootstrapreplicates and default parameters in Galaxy (Giardine et al.2005, Blankenberg et al. 2010, Goecks et al. 2010) with Phy-codrys rubens (Linnaeus) Batters as the outgroup. Sequencesof Grinnellia americana (C.Agardh) Harvey, Delesseria sanguinea(Hudson) J.V.Lamouroux, Cumathamnion sympodophyllumM.J.Wynne & K. Daniels, and C. decipiens (J.Agardh)M.J.Wynne & G.W.Saunders were included in the analysis forphylogenetic context. Bayesian analysis was executed withMrBayes 3.2.1 (Huelsenbeck et al. 2001, Ronquist andHuelsenbeck 2003) using the search parameters in Lindstromet al. (2015a). The phylogenetic trees were visualized withTreeDyn 198.3 at Phylogeny.fr (Dereeper et al. 2008).

Genomics. Genomic libraries were constructed followingthe protocol outlined in Hughey et al. (2014) that was devel-oped by the High-Throughput Genomics Center (Seattle,Washington, USA). The genomic analysis was performedusing standard Illumina 36 base pairs (bp) paired-endsequencing methods. Data were assembled with default denovo settings in CLC Cell 4.3.0 (!2015 CLC bio, a QIAGENCompany, Valencia, California, USA) and Velvet 1.2.08 (Zer-bino and Birney 2008) on the Bio-Linux platform (Fieldet al. 2006) following the assembly steps in Hughey et al.(2014). The M. weeksiae plastome was assembled by perform-ing a Standard Nucleotide Blast search of the assembly con-tigs against the complete plastid genomes of Calliarthrontuberculosum (Postels & Ruprecht) E.Y.Dawson (GenBankaccession KC153978), Grateloupia taiwanensis S.M.Lin &H.Y.Liang (KC894740), and Chondrus crispus Stackhouse(HF562234). The 13 resulting contigs were oriented andjoined via targeted PCR and direct Sanger sequencing. Theplastid genomes of M. platyphylla and M. tenuis were assem-bled by aligning the contigs from M. platyphylla andM. tenuis to the M. weeksiae plastome using a StandardNucleotide Blast at NCBI. Following orientation of the con-tigs, the gaps were closed in M. platyphylla and M. tenuis bymapping the reads against M. weeksiae using Geneious R8(Biomatters, Inc., Newark, NJ, USA). The plastid genomefeatures were annotated using NCBI open reading frame(ORF)-finder and alignments obtained via BLASTX. ThetRNAs were identified using the tRNAscan-SE 1.21 web ser-ver (Schattner et al. 2005), and rRNAs using the RNAmmer1.2 server (Lagesen et al. 2007). Locally Collinear Block(LCB) alignments were generated using ProgressiveMauvewith a seed of 21 with the “Use seed families” optionselected (Darling et al. 2010). The three complete plastid

COMPLETE PLASTOMES OF MEMBRANOPTERA 33

TABLE1.

Vouch

erspecim

ens,

collection

inform

ation,Gen

Ban

kaccession

numbers,

and

the

iden

tification

ofspecim

ensofMem

bran

optera

used

inthis

study.

*den

otestypematerial.Unless

otherwisenoted,Gen

Ban

kaccessionnumbersrepresentrbcL

sequen

ceaccessions.

Vouch

ers

Locality

Date,

Collector

Gen

Ban

kaccession

Iden

tification

Source

UC

2010

057/

*M.edentata

Carmel,CityPt.,California

21June19

39,G.M

.Sm

ith

KU82

1124

M.platyphylla

Thisstudy

UC

2840

46/*M

.multiramosa

Moss

Beach

,California

26April19

24,N.L.Gardner

KU82

1125

M.platyphylla

Thisstudy

UC

1883

856/

*M.multiramosa

Moss

Beach

,California

26April19

24,N.L.Gardner

KU82

1126

M.platyphylla

Thisstudy

UC

2840

25/*M

.dimorphum

NeahBay,Washington

May

1917

,N.L.Gardner

KU82

1127

M.platyphylla

Thisstudy

UC

1856

248

Friday

Harbor,Wash.

30June19

24,H.Kylin

KT26

6849

M.platyphylla

Thisstudy

CO1,

2,3-

KU82

1146

rRNA-KU82

1149

UC

1599

881

ShellBeach

,California

July

1971

,J.West

KU82

1128

M.platyphylla

Thisstudy

UC

2840

42Parry

Bay,Can

ada

June19

25,R.Cornell

KU82

1129

M.platyphylla

Thisstudy

UC

1856

249

NarrowsMarina,

Wash.

20July

1976

,R.Se

tzer

KU82

1130

M.platyphylla

Thisstudy

UC

2017

775

YaquinaBay,Orego

n03

April19

66,C.K.Kjeldsen

KU82

1131

M.platyphylla

Thisstudy

NCU

6466

22BotanyBeach

,Can

ada

7Augu

st20

02,P.W

.Gab

rielson

KU82

1132

M.platyphylla

Thisstudy

NCU

6466

25BotanyBeach

,Can

ada

7Augu

st20

02,P.W

.Gab

rielson

KU82

1133

M.platyphylla

Thisstudy

NCU

6466

24BotanyBeach

,Can

ada

20Augu

st20

01,P.W

.Gab

rielson

KU82

1134

M.platyphylla

Thisstudy

GWS0

0841

3BeaverIs.,Can

ada

12June20

07,G.W

.Saunderset

al.

JX11

0926

M.platyphylla

Wyn

nean

dSaunders(201

2)LAF-6-17

-99-1-8

Boiler

Bay,Orego

n17

June19

99,S.

Fred

ericq

AF2

5738

4M.platyphylla

Lin

etal.(200

1)UC

2017

754

Bodeg

aHead,California

10March

1988

,C.K.Kjeldsen

KU82

1135

M.tenuis

Thisstudy

UC

4209

94SanFran

cisco,California

Augu

st19

20,N.L.Gardner

KU82

1136

M.tenuis

Thisstudy

UC

1599

882

Partridge

Ban

k,Wash.

06July

1965

,D.HallWest

KU82

1137

M.tenuis

Thisstudy

UC

2664

39WestofCan

oeIs.,Wash.

July

1910

,N.L.Gardner

KP67

5983

M.tenuis

Thisstudy

CO1,

2,3-

KU82

1148

rRNA-KU82

1151

UC

9584

4Santa

Cruz,

California

Augu

st19

20,C.L.Anderson

KU82

1138

M.tenuis

Thisstudy

UC

1856

265

Cayuco

sPt.,California

06March

1971

,C.Link

KU82

1139

M.tenuis

Thisstudy

NCU

6466

23Eagle

Pt.,SanJuan

Is.,Wash.

09July

2001

,J.Norton,M

Hutchinson

KU82

1140

M.tenuis

Thisstudy

UC

2648

04/*M

.weeksiae

PacificGrove,California

26March

1896

,J.M.Weeks

KJ513

670

M.weeksiae

Thisstudy

CO1,

2,3-

KU82

1147

rRNA-KU82

1150

UC

1856

263

S.en

dCarmel

Bch

.,California

27June19

71,R.Se

tzer

KU82

1141

M.weeksiae

Thisstudy

UC

1856

268

S.en

dCarmel

Bch

.,California

17April19

76,R.Se

tzer

KU82

1142

M.weeksiae

Thisstudy

UC

1856

274

SanNicolasIs.,California

08October

1978

,L.Hart,J.Engle

KU82

1143

M.weeksiae

Thisstudy

UC

6969

61SanDiego

,California

March

1945

,E.Wilson

KU82

1144

M.weeksiae

Thisstudy

UC

1455

006

S.en

dCarmel

Bch

.,California

27April19

71,I.A.Abbott

KU82

1145

M.weeksiae

Thisstudy

GWS0

1391

0New

Brunswick,

Can

ada

16Se

ptember

2009

,G.W

.Saunders

JX11

0924

M.fabriciana

Wyn

nean

dSaunders(201

2)LAF-8-23

-05-1-1

St.Mich.dePlouign.,Fran

ce23

Augu

st20

05,E.Coppejan

sAF2

5418

1M.alata

Lin

etal.(201

2)LAF-4-4-98

-1-1

Auke

Bay,Alaska

04April19

98,S.C.Lindstrom

AF2

5738

3M.spinulosa

Lin

etal.(200

1)LAF-6-12

-98-1-5

SunsetBay,Orego

n12

June19

98,B.Wysor

AF2

5418

1Cumathamnion

decipiens

Lin

etal.(200

1)

GWS0

1243

2Trinidad

,California

17June20

10,G.Saunders

JX11

0917

Cumathamnion

sympodophyllum

Wyn

nean

dSaunders(201

2)GWS0

1400

5Brittan

y,Fran

ce28

March

2010

,L.Leveq

ue

JX11

0918

Delesseriasanguinea

Wyn

nean

dSaunders(201

2)LAF-4-23

-94-1-1

Tad

ioIs.,NorthCarolina

23April19

94,M.Deals

AF2

5418

4Grinnelliaam

erican

aLin

etal.(200

1)LAF-7-22

-97-1-6

Pem

broke

shire,

Wales

22July

1997

,F.

andM.Hommersand

AF2

5742

9Phycodrys

rubens

Lin

etal.(200

1)

34 JEFFERY R. HUGHEY ET AL.

genomes were deposited in GenBank under accessions:KT266849 (M. platyphylla), KP675983 (M. tenuis), andKJ513670 (M. weeksiae). Protein comparisons were performedmanually by extracting protein sequences from GenBankfiles and aligning them via a Standard Protein Blast atNCBI. The mitochondrial genome cytochrome oxidase I, II,and III loci were also partly sequenced using the same pro-cedure. The cox sequences are deposited in GenBank underKU821146 (M. platyphylla), KU821148 (M. tenuis), andKU821147 (M. weeksiae). Assembled contigs containing stan-dard nuclear markers (SSU, ITS, LSU) were identified usingStandard Nucleotide Blast searches. They were also depos-ited in GenBank, and are listed under accessions KU821149(M. platyphylla), KU821151 (M. tenuis), and KU821150(M. weeksiae).

RESULTS

Membranoptera plastomes and partial mitogen-omes. The assembled plastomes of M. platyphylla(176,159 bp), M. tenuis (176,031 bp), and M. week-siae (176,070 bp) were similar in size and identicalin content with 222 genes (Table 2). The plastomeswere AT rich (73.6%–74.0%) and contained the fol-lowing genes: 19 small and 27 large ribosomal pro-teins, 29 photosystem I and II, 28 tRNA, 24hypothetical chloroplast reading frames (ycf), 15ORFs, 9 cytochrome b/f complex proteins, 9 ATPsynthase, 3 ribosomal RNAs, and 59 other genes.

Locally collinear blocks were analyzed for 15florideophyte plastid genomes and the outgrouptaxon Porphyra purpurea (Fig. 1). These observationsare described in the discussion section. Some largercontigs were assembled from the mitogenomes ofthe three Membranoptera species, but complete circu-lar chromosomes were not obtained and could

not be assembled due to low copy number andthe presence of inverted repeats that can beproblematic (Phillippy et al. 2008). Nucleotide poly-morphisms, amino acid substitutions, and rbcL,COX1-5P, and COXI, II, III polymorphisms for thethree species of Membranoptera investigated are sum-marized in Table 3.Partial rbcL sequences of type and contemporary speci-

mens. Analysis of a 122-bp-length sequence of therbcL gene from archival herbarium specimens andcontemporary specimens identified three lineages(Fig. 2). The sequence generated from the holotypeof M. weeksiae from Pacific Grove was identical tothree specimens of M. weeksiae collected from CarmelBeach. The holotype of M. weeksiae differed by 1 bpfrom sequences of two specimens from San Diegoand San Nicolas Island in southern California. Theholotype of M. weeksiae differed by 2 bp from sevenidentical sequences of M. tenuis distributed fromWashington to central California, and by 7 bp from11 identical sequences from the type specimens ofM. dimorpha, M. edentata, M. multiramosa, and 7recent collections of M. platyphylla. Membranopteratenuis differed from M. platyphylla by 5 bp. The holo-type ofM. platyphylla (Pteridium serratum f. platyphyllumSetchell & N.L.Gardner, UC 95853) failed to amplify.Phylogenetic analysis results. The rbcL analysis

resulted in a tree with three strongly supportedclades (Fig. 3). The basal clade contained a branchwith two specimens of M. tenuis from Washington,including a sequence obtained from a topotypespecimen of M. tenuis collected by N. L. Gardner.Sister to this species were two specimens of M. week-siae from central (Monterey County) and southern

TABLE 2. Plastid genome content of Membranoptera platyphylla, M. tenuis, and M. weeksiae

Gene groups Genes

Photosystem I psaA, psaB, psaC, psaD, psaE, psaF, psaI, psaJ, psaK, psaL, psaMPhotosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbN, psbT, psbV, psbW,

psbX, psbY, psbZProtochlorophyllide reductase chlIPhycobiliproteins apcA, apcB, apcD, apcE, apcF, cpeA, cpeB, cpcA, cpcB, cpcGCytochrome b/f complex petA, petB, petD, petF, petG, petJ, petM, petNATP synthase atpA, atpB, atpD, atpE, atpF, atpG, atpH, atpIRNA polymerase rpoA, rpoB, rpoC1, rpoC2Ribosomal proteins (SSU) rps1, rps2, rps3, rps4, rps5, rps6, rps7, rps8, rps9, rps10, rps11, rps12, rps13, rps14, rps16,

rps17, rps18, rps19, rps20Ribosomal proteins (LSU) rpl1, rpl2, rpl3, rpl4, rpl5, rpl6, rpl9, rpl11, rpl12, rpl13, rpl14, rpl16, rpl18, rpl19, rpl20,

rpl21, rpl22, rpl23, rpl24, rpl27, rpl28, rpl31, rpl32, rpl33, rpl34, rpl35, rpl36Hypothetical chloroplast orfs ycf3, ycf4, ycf16, ycf19, ycf20, ycf21, ycf22, ycf24, ycf29, ycf33, ycf34, ycf35, ycf36, ycf37, ycf38,

ycf39, ycf40, ycf45, ycf46, ycf54, ycf59, ycf60, ycf61, ycf65Transfer RNAs trnA-ACG, trnA-UGC, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC, trnG-

UUG, trnH-GUG, trnI-GAU, trnK-UUU, trnL-CAA, trnL-UAA, trnL-UAG, trnM-CAU(x2),trnN-GUU, trnP-UGG, trnR-CCG, trnR-UCU, trnS-GCU, trnS-UGA, trnT-UGU, trnV-GAC,trnV-UAC, trnW-CCA, trnY-GUA

Ribosomal RNAs rrsA, rrlA, rrfBOpen reading frames orf7, orf58, orf63, orf68, orf121, orf174, orf198, orf199, orf238, orf263, orf320, orf327, orf382,

orf450, orf621Other genes accA, accB, accD, acpP, argB, carA, cbbx, ccs1, ccsA, cemA, clpC, dnaB, dnaK, dsbD, fabH,

ftrC, ftsH, gltB, groEL, ilvB, ilvH, infB, infC, nblA, ntcA, odpA, odpB, ompR, pbsA, pgmA,preA, rbcL, rbcR, rbcS, rne, secA, secY, syfB, syh, tatC, thiG, trpA, trpG, trxA, tsf, tufA

COMPLETE PLASTOMES OF MEMBRANOPTERA 35

California (San Diego County), including asequence from the lower specimen on the holotypesheet of M. weeksiae. The second clade contained sixsequences assignable to M. platyphylla, including aspecimen collected by H. Kylin from Friday Harbor,Washington. The final clade contained specimensidentified as M. alata (Hudson) Stackhouse,M. fabriciana (Lyngbye) M.J.Wynne & G.W.Saunders,and M. spinulosa from Alaska (AF257383 in Gen-Bank as M. tenuis), type specimens of which havenot been sequenced.

Morphological characterization of M. platyphylla,M. tenuis, and M. weeksiaeBased on examination of type material and speci-

mens in UC, the three species differed in bladewidth (Fig. 4). While some variation in widthdepended on the age of the thallus, M. platyphylla(Fig. 4c) consistently produced blades that mea-sured 2–10-mm wide, whereas the blades of M. week-siae (Fig. 4a) and M. tenuis (Fig. 4b) were less than1.5-mm broad. Membranoptera platyphylla possessed aconspicuous midrib and regular, lateral veins and

FIG. 1. Locally collinear blocks (LCBs) analysis for 16 plastid genomes. The figure depicts linearized alignments identifying conservedgene regions for 15 Florideophyceae and the outgroup taxon Porphyra purpurea. Each plastome is oriented horizontally and homologousblocks are shown as identically colored regions linked across genomes. Regions inverted relative to M. weeksiae are shifted below the gen-ome’s center axis. Sequence similarities within an LCB are proportional to the heights of interior colored bars. Large sections of whitewithin blocks, and gaps between blocks, indicate lineage-specific sequences. The figure was drawn using Mauve 2.3.1. [Color figure can beviewed at wileyonlinelibrary.com]

36 JEFFERY R. HUGHEY ET AL.

their associated reproductive structures (Fig. 4c).The midrib of M. weeksiae was inconspicuous, butextended to branch apices and was flanked by nar-row “wings” lacking lateral veins (Fig. 4a). InM. tenuis, the tips of branches were narrow with themidrib evident only in lower portions of thebranches and with lateral veins also absent(Fig. 4b).

DISCUSSION

Red algal intergeneric plastome comparisons. Ourgenomic approach is in agreement with themethodology of Janou!skovec et al. (2013), who pro-posed that evolutionary rates among the more than200 genes that comprise the plastome of red algaeare a rich source of information for evaluatingred algal relationships at both deep and species/subspecies levels. To date, plastome sequenceshave been published for 10 florideophycean gen-era: Calliarthron Manza (Corallinaceae, Corallinales,Janou!skovec et al. 2013), Coeloseira Hollenberg(Champiaceae, Rhodymeniales, Kilpatrick andHughey 2016), Chondrus Stackhouse (Gigartinaceae,Gigartinales, Janou!skovec et al. 2013), GelidiumJ.V.Lamouroux (Gelidiaceae, Gelidiales, Lee et al.2016), Gracilaria Greville and Gracilariopsis (Bory deSaint-Vincent) E.Y.Dawson (Gracilariaceae, Gracilari-ales, Hagopian et al. 2004, Campbell et al. 2014, Duet al. 2016, Zhang et al. 2016), Grateloupia C.Agardh(Halymeniales, DePriest et al. 2013, Janou!skovecet al. 2013, Lee et al. 2016), Laurencia J.V.Lamour-oux (Rhodomelaceae, Ceramiales, Verbruggen andCosta 2015), Sporolithon Heydrich (Sporolithaceae,Sporolithales, Lee et al. 2016), and Vertebrata

S.F.Gray (Rhodomelaceae, Ceramiales, Salomakiet al. 2015). For only three of these, Gelidium,Gracilaria, Grateloupia, has more than one speciesplastome been sequenced, and to these we addedthree species of Membranoptera.Plastomes of all three Membranoptera species were

shorter in length (~176 kb) and contained fewergenes (222) than most published Florideophyceae,including C. tuberculosum (178,981 bp/238 genes),C. crispus (180,086/240), Coeloseira compressa (176,291/233), Gelidium elegans (174,749/235), Gel. vagum(179,852/234), Gracilaria salicornia (179,757/234),Grac. chilensis (185,637/236), Grac. tenuistipitata var.liui (183,883/237), Gracilariopsis lemaneiformis (182,505/230), Grateloupia lanceola (188,384/244), Grat.taiwanensis (191,270/266), and Sporolithon durum(191,465/239). However, compared to the twoCeramiales (Rhodomelaceae) that have been ana-lyzed, Vertebrata lanosa (Linnaeus) T.A.Christensen(167,158/223) and Laurencia sp. (174,935/232), theMembranoptera plastomes were longer and the num-ber of genes more varied.Genome alignment showed that gene synteny for

the species of Membranoptera was similar to thatfound in other florideophytes, with the exception ofa single ~24,000 bp inversion in Chondrus, Gelidium,Gracilaria, Gracilariopsis, and Laurencia (Fig. 1).Where it occurs, the inversion begins with the 30Slarge ribosomal protein S6 (rps6) and terminateswith the ATPase, AAA domain-containing protein(ycf46). It includes 37 genes, of which rpl, ycf,tRNA, and orfs predominate, accounting for abouthalf of the genes in the inversion. This inversion isnot present in Membranoptera, Calliarthron, Coeloseira,Grateloupia, Sporolithon, or Vertebrata. In contrast to

TABLE 3. Comparison of the plastomes and genetic markers for M. platyphylla, M. tenuis, and M. weeksiae

M. weeksiae versus M. tenuis M. weeksiae versus M. platyphylla M. tenuis versus M. platyphylla

Nucleotide polymorphisms 1,896 and 158 gaps 10,078 and 2,741 gaps 10,031 and 2,606 gapsNucleotide polymorphismsin coding regions

1,306 (68.9% of totaldifferences)

7,414 (73.6% oftotal differences)

7,469 (74.5% of total differences)

Sequence variation incoding regions

0.89% 5.14% 5.18%

Sequence variation innon-coding regions

2.05% 9.24% 8.88%

Amino acid substitutionsacross all genes

527 (311 conservative,216 radical)

2,391 (1,228 conservative,1,163 radical)

2,298 (1,099 conservative,1,199 radical)

rbcL polymorphisms 17 SNPs (1.2%) 71 SNPs (4.8%) 65 SNPs (4.4%)rbcL amino acid substitutions 11 (9 conservative, 2 radical) 19 (12 conservative, 7 radical) 12 (5 conservative, 7 radical)rbcL SNPs 1st, 2nd,3rd positions

47.0%, 11.8%, 41.2% 31.0%, 8.5%, 60.5% 23.1%, 9.2%, 67.7%

COXI-5P polymorphisms 8 SNPs (1.2%) 50 SNPs (7.5%) 45 SNPs (6.8%)COXI-5P amino acidsubstitutions

0 6 (4 conservative, 2 radical) 6 (4 conservative, 2 radical)

COXI-5P SNPs 1st, 2nd,3rd positions

25%, 12.5%, 62.5% 14%, 2%, 84% 11.1%, 2.2%, 86.7%

COXI, II, III polymorphisms 41 SNPs and 2 gaps (1.3%) 276 SNPs and 8 gaps (8.4%) 268 SNPs and 6 gaps (8.1%)COXI, II, III amino acidsubstitutions

4 (3 conservative, 1 radical) 42 (15 conservative, 27 radical) 43 (16 conservative, 27 radical)

COXI, II, III SNPs 1st,2nd, 3rd positions

0%, 50%, 50% 40%, 37.5%, 22.5% 43.5%, 38.5%, 18%

COMPLETE PLASTOMES OF MEMBRANOPTERA 37

platyphyllaUC284042VancIs TTAGGCACAATCATTATTATGGTTGACCTTGTAATTGGATACACTGCTAT platyphyllaUC1856248FriHar .................................................. platyphyllaUC159981ShelBch .................................................. platyphyllaUC1856249Narrow .................................................. platyphyllaUC2017775YaqBay .................................................. platyphyllaGWS008413BeavIs .................................................. platyphyllaPWG925BotanyBay .................................................. platyphyllaPWG981BotanyBay .................................................. platyphyllaPWG992BotanyBay .................................................. multiramosaUC1883856holoty .................................................. multiramosaUC284046isotype .................................................. dimorphaUC284025holotype .................................................. edentataUC2010057isotype .................................................. ‘weeksiae’BoilerBayOregon .................................................. weeksiaeUC1856274SanNicIs .....T...G...........A................T..T.G...... weeksiaeUC696961SanDiego .....T...G...........A................T..T.G...... weeksiaeUC1455006ScarmlBch .....T...G...........A................T..T.G...... weeksiaeUC1856268ScarmlBch .....T...G...........A................T..T.G...... weeksiaeUC264804Holotype .....T...G...........A................T..T.G...... weeksiaeUC1856263ScarmlBch .....T...G...........A................T..T.G...... tenuisUC2017754BodegaHead .....T...............A................T..T.G...... tenuisUC420994SanFrancisco .....T...............A................T..T.G...... tenuisUC1856265Cayucos .....T...............A................T..T.G...... tenuisUC1599882PatrdgeBank .....T...............A................T..T.G...... tenuisUC266439CanoeIsland .....T...............A................T..T.G...... tenuisUC95844SantaCruz .....T...............A................T..T.G...... tenuisPWG834SanJuanIsland .....T...............A................T..T.G......

platyphyllaUC284042VancIs TCAATCAATGGCAATTTGGTCTCGTAAAAATGATATGATTTTACATTTAC platyphyllaUC1856248FriHar .................................................. platyphyllaUC159981ShelBch .................................................. platyphyllaUC1856249Narrow .................................................. platyphyllaUC2017775YaqBay .................................................. platyphyllaGWS008413BeavIs .................................................. platyphyllaPWG925BotanyBay .................................................. platyphyllaPWG981BotanyBay .................................................. platyphyllaPWG992BotanyBay .................................................. multiramosaUC1883856holoty .................................................. multiramosaUC284046isotype .................................................. dimorphaUC284025holotype .................................................. edentataUC2010057isotype .................................................. ‘weeksiae’BoilerBayOregon .................................................. weeksiaeUC1856274SanNicIs .................................................. weeksiaeUC696961SanDiego .................................................. weeksiaeUC1455006SCarmlBch .............................................C.... weeksiaeUC1856268SCarmlBch .............................................C.... weeksiaeUC264804Holotype .............................................C.... weeksiaeUC1856263SCarmlBch .............................................C.... tenuisUC2017754BodegaHead .................................................. tenuisUC420994SanFrancisco .................................................. tenuisUC1856265Cayucos .................................................. tenuisUC1599882PatrdgeBank .................................................. tenuisUC266439CanoeIsland .................................................. tenuisUC95844SantaCruz .................................................. tenuisPWG834SanJuanIsland ..................................................

FIG. 2. Alignment of rbcL gene sequences for Membranoptera spp. nucleotide positions 790–889 bp. Abbreviations correspond to namesand localities in Table 1. Dots represent nucleotides that are identical to those in the uppermost line.

38 JEFFERY R. HUGHEY ET AL.

Membranoptera and other Florideophyceae, theremainder of the Coeloseira and Laurencia plastomesappears to be reversed (Fig. 1). As is the case forother Florideophyceae, except Calliarthron, Membra-noptera does not code for chlL, chlN, and chlB. TheMembranoptera plastome contains the group II intronreverse transcriptase maturase protein, a featurethat is uniquely shared among florideophytes(Janou!skovec et al. 2013).Intrageneric plastome comparisons in Membra-

noptera. Detailed analysis at the nucleotide andamino acid levels demonstrate that M. weeksiae is sis-ter to M. tenuis and both are similarly distant fromM. platyphylla (Tables S1–S3 in the Supporting Infor-mation). These relationships are also supported by

the standard DNA markers (Table 4). The rbcLsequence variation exhibited between M. tenuis andM. weeksiae (1.2%) is within the range of interspecificvariations for rbcL (0%–7.2%) but varies by authoraccording to other evidence (Freshwater and Rue-ness 1994, Hughey and Hommersand 2008). Twospecies of Chondracanthus (Gigartinaceae, Gigarti-nales) differed by 0–1 bp for 1,398 bp of the rbcLgene, but differed in morphology, showed significantITS sequence variation above typical species levels(4.3%) and passed the test of sympatry (Hughey andHommersand 2008). Cumathamnion sympodophyllum(Delesseriaceae, Ceramiales), originally monotypic, ismorphologically distinct, but very closely related toC. decipiens based on DNA sequence divergence data

FIG. 3. Maximum likelihood phylogram based on rbcL gene sequences of species of Membranoptera. Bootstrap support (nreps=1,000)/Bayesian posterior probabilities are cited at the nodes. The legend shows the scale for nucleotide substitutions.

FIG. 4. Habits of Membranoptera species. (a) M. weeksiae; upper specimen on holotype sheet (UC264804). Scale bar = 2.0 cm. (b)M. tenuis (UC266439). Scale bar = 1.0 cm. (c) M. platyphylla (UC1856248); genomic analysis was performed on the specimen on the toprow, second from right, scale bar = 2.0 cm.

COMPLETE PLASTOMES OF MEMBRANOPTERA 39

(Wynne and Saunders 2012). The generitype differsby only 5 of 1,358 bp (0.37%) for the rbcL gene com-pared to C. decipiens. The 1.2% variation in rbcLexhibited between M. weeksiae and M. tenuis, alsomembers of Delesseriaceae, is thus sufficient to rec-ognize these two morphologically cryptic taxa as dis-tinct.

The Membranoptera plastome data are comparableto interspecific divergences between Gracilaria sal-icornia and G. tenuistipitata var. liui (divergencieswere 10.2% for rbcL and 21.9% for the plastomes),G. salicornia and G. chilensis (11.5% rbcL and 22.3%plastomes), G. tenuistipitata var. liui and G. chilensis(7.8% rbcL and 10.7% plastomes) (Hagopian et al.2004, Campbell et al. 2014, Lee et al. 2016); thosefor Grateloupia lanceola and Grat. taiwanensis were5.9% and 11.6%, respectively (DePriest et al. 2013,Janou!skovec et al. 2013); and Gelidium elegans andGel. vagum were 8.4% and 15.6%, respectively (Leeet al. 2016). In most of these examples, plastomesequences showed approximately twice the variationcompared to rbcL sequences. The interspecificgenetic variation between rbcL and plastomesequences of Membranoptera species indicate thatrbcL has about the same level of variation as theplastome for the closely related M. weeksiae andM. tenuis, but slightly underestimates divergence forthe two compared to M. platyphylla (Table 3). TheRuBisCO Spacer, and the CO1 gene exhibited varia-tion similar to that of the rbcL gene (Table 4). Com-parison of the top 20 most variable genes from theplastome showed seven genes in common amongthe three species of Membranoptera that are usefulfor species-level comparisons: rbcS, orf621, petF,rpl9, ycf35, ycf37, and infB (Table S4 in the Sup-porting Information).

Membranoptera species in the NE Pacific. On thebasis of our DNA sequencing results, three speciesof Membranoptera are present in the NE Pacific southof Alaska, namely M. platyphylla, M. tenuis, andM. weeksiae. Wynne and Saunders (2012) recognizedonly one species over the same geographic range.They correctly placed M. dimorpha and M. multi-ramosa in synonymy under M. platyphylla. We con-firm this placement with DNA sequences from typespecimens, as well as from M. edentata Kylin (typelocality: Carmel City Point, Monterey County, Cali-fornia, USA), a synonym of M. multiramosa. Onceagain, the sequencing of type material has facili-tated the correct application of names (Hughey andGabrielson 2012).The main reason for the differing taxonomic con-

clusions between Wynne and Saunders (2012) andherein was the narrow geographic range that theysampled. All, but two, of the Wynne and Saundersspecimens were collected from southern BritishColumbia, with one from Oregon and the otherfrom central California. The range of M. platyphyllaextends over 2,000 km, from northern VancouverIsland to Shell Beach, San Luis Obispo County, Cali-fornia, and that of M. tenuis is 1,750 km, fromCanoe Island, San Juan County, Washington toCayucos, San Luis Obispo County, California.M. weeksiae is a California endemic that extends only700 km from Pacific Grove, Monterey County toSan Diego, San Diego County, but whose northernrange overlaps that of M. tenuis from Pacific Groveto its southern limit at Shell Beach.Membranoptera platyphylla is the most morphologi-

cally variable of the three species, with thalli rangingfrom a minimum width of 1.5 mm up to 10 mm.Setchell and Gardner (1903) described specimens

TABLE 4. Genetic marker differences in base pairs and percentages, comparing genome data among Membranoptera platy-phylla, M. tenuis, and M. weeksiae herbarium specimens. Markers below the line are the most variable genes identified acrossthe plastid genomes of the three species.

Markers

Membranoptera species

M. weeksiae versus M. tenuis M. weeksiae versus M. platyphylla M. tenuis versus M. platyphylla

rbcL (1,467 bp) 17 bp/1.2% 71 bp/4.8% 65 bp/4.4%psbA (1,083 bp) 1 bp/0.0% 29 bp/2.7% 30 bp/2.8%UPA (2,879 bp) 7 bp/0.2% 28 bp/1.0% 25 bp/0.9%psaA (2,259 bp) 14 bp/0.6% 86 bp/3.8% 88 bp/3.9%psaB (2,205 bp) 17 bp/0.8% 92 bp/4.2% 89 bp/4.0%Rubisco Spacer (84 bp) 1 bp/1.2% 5 bp + 1 gap/7.1% 4 bp + 1 gap/6.0%LSU (2,739 bp) 3 bp/0.1% 53 bp + 8 gaps/2.2% 56 bp + 6 gaps/2.3%SSU (1,776 bp) 0 bp/0.0% 21 bp + 2 gaps/1.3% 21 bp + 2 gaps/1.3%ITS1&2 (1,011 bp) 15 bp + 4 gaps/1.9% 142 bp + 37 gaps/17.7% 141 bp + 25 gaps/16.4%CO1 (664 bp) 8 bp/1.2% 50 bp/7.5% 45 bp/6.8%COB (1,139 bp) 12 bp/1.1% 97 bp/8.5% 94 bp/8.3%cox 2-3 (144 bp) 4 bp/2.8% 17 bp/11.8% 17 bp/11.8%

rbcS (417 bp) 11 bp/2.6% 35 bp/8.4% 33 bp/7.9%petF (312 bp) 11 bp/3.5% 40 bp/12.8% 37 bp/11.9%orf621 (1,971 bp) 33 bp/1.7% 251 bp/12.7% 257 bp/13.0%rpl9 (468 bp) 10 bp/2.1% 45 bp/9.6% 45 bp/9.6%ycf35 (402 bp) 7 bp/1.7% 41 bp/10.2% 38 bp/9.5%ycf37 (474 bp) 10 bp/2.1% 42 bp/8.7% 38 bp/8.0%infB (2,178 bp) 36 bp/1.7% 161 bp/7.4% 170 bp/7.8%

40 JEFFERY R. HUGHEY ET AL.

of Pteridium? serratum f. platyphyllum (basionym ofM. platyphylla), as having a minimum width of 2 mmjust below the apices to 5 mm in more proximalparts. Kylin (1924), working with specimens col-lected at Friday Harbor, Washington, describedmale plants as 2–3-mm wide, tetrasporangial plants4–8-mm wide, and female plants 5–10-mm wide. Allof these specimens were characterized by the pres-ence of conspicuous lateral veins arising from themidrib. Gardner (1926) described M. multiramosaand M. dimorpha, noting microscopic lateral veins inboth species. He distinguished the former by its rel-atively wide thin fronds, its profuse branchingthroughout (for which it was named), and its den-ticulate margins and fimbriate outgrowths from thecystocarps (the last two characters shared with M. di-morpha). Membranoptera dimorpha was distinguishedby the production of blades from both the margins(a generic character of Membranoptera) and from themidrib. Membranoptera tenuis and M. weeksiae, charac-terized by narrow fronds less than 1.5-mm wide andlacking lateral veins, are often difficult to distin-guish morphologically but are distinct, requiringDNA sequence comparisons for positive identifica-tion. In fact, M. tenuis was not included in the Mar-ine Algae of California (Abbott and Hollenberg 1976).M. weeksiae differs from M. tenuis near its branchapices where the branches remain winged, whereasthose of M. tenuis lack “wings.” Both are ofteneroded to the midrib in basal portions. Membranop-tera weeksiae and M. tenuis are essentially subtidalspecies, whereas M. platyphylla can occur in theintertidal region. We suggest that the ancestors ofM. tenuis and M. weeksiae were separated during oneor more of the ice ages, with M. tenuis persisting inthe region of Puget Sound and with M. weeksiae per-sisting south of Point Conception. The distributionof the two species may have overlapped onlyrecently in Monterey, California.

Below we summarize our taxonomic findings.Membranoptera platyphylla (Setchell & N.L.Gard-

ner) Kylin 1924: 15–16, figs. 7, 8Basionym: Pteridium? serratum f. platyphyllum Setch-

ell & N.L.Gardner 1903: 325Holotype: UC 95853, Washington, Kitsap County,

Pleasant Beach, on wooden float, leg. N. L. Gardner344.

Heterotypic synonyms:Membranoptera multiramosa N.L.Gardner 1926: 209,

Pl. 19, fig. 1Holotype: UC 284045, California, San Mateo

County, Moss Beach, April, epilithic in shelteredhabitats in the lowest intertidal, leg. N. L. Gardner4911; Isotype: UC 284046.

Membranoptera dimorpha N.L. Gardner 1926: 211–212, Pl. 17, fig. 1, Pls. 20, 21

Holotype: UC 284025, Washington, Clallam County,Neah Bay, May, epilithic in the lowest intertidal, leg.N. L. Gardner 3884.

Membranoptera edentata Kylin 1941: 30–31, Pl. 10,fig. 27Holotype: LD s. n., California, Monterey County,

Carmel City Point, 21.vi.1939, on deeply shadedoverhanging rock face, leg. G. M. Smith; Isotype: UC2010057.Membranoptera weeksiae Setchell & N.L.Gardner,

in Gardner 1926: 209–210, Pl. 19, fig. 2Holotype: UC 264804, California, Monterey County,

Pacific Grove, 26.iii.1896, epilithic in low intertidaland shallow subtidal, leg. J. M. Weeks s. n.; Isotype:UC 284046.Membranoptera tenuis Kylin 1924: 16–17, fig. 9, a

and bNeotype: UC 266439, Washington, San Juan

County, west of Canoe Island, vii.1910, leg. N. L.Gardner 2294.Comment: According to Patrik Fr€oden, Assistant

Curator at LD, no specimen from Canoe Island col-lected by H. Kylin could be found (24.iii.2015 e-mailto MHH). Therefore, in accordance with Article9.16 (McNeill et al. 2012) we have designated theN.L. Gardner specimen cited above from the typelocality as neotype.

We thank Dr. S. Schafer at NCBI for assistance with the anno-tations. We also greatly appreciate the laboratory help pro-vided by J. Black at Hartnell College. This work was fundedby US NSF grant DEB0937978 to J.M. Lopez Bautista, M.H.Hommersand, and S. Fredericq, and generous support froma private family trust from P.W.G.

Abbott, I. A. & Hollenberg, G. J. 1976. Marine Algae of Califor-nia. Stanford University Press, Stanford, California,827 pp.

Adey, W. H., Hernandez-Kantun, J. J., Johnson, G. & Gabrielson,P. W. 2015. DNA sequencing, anatomy and calcification pat-terns support a monophyletic, subarctic, carbonate reef-form-ing Clathromorphum (Hapalidiaceae, Corallinales,Rhodophyta). J. Phycol. 51:189–203.

Blankenberg, D., Von Kuster, G., Coraor, N., Ananda, G., Lazarus,R., Mangan, M., Nekrutenko, A. & Taylor, J. 2010. Galaxy: aweb-based genome analysis tool for experimentalists. Curr.Protoc. Mol. Biol. 89:19.10.1–10.21.

Campbell, M. A., Presting, G., Bennett, M. S. & Sherwood, A. R.2014. Highly conserved organellar genomes in the Gracilari-ales as inferred using new data from the Hawaiian inva-sive alga Gracilaria salicornia (Rhodophyta). Phycologia 53:109–16.

Darling, A. E., Mau, B. & Perna, N. T. 2010. ProgressiveMauve:multiple genomic alignment with gene gain, loss, and rear-rangement. PLoS ONE 5:e11147.

DePriest, M. S., Bhattacharya, D. & L#opez-Bautista, J. M. 2013.The plastid genome of the red macroalga Grateloupia taiwa-nensis (Halymeniaceae). PLoS ONE 8:e68246.

Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Cheve-net, F., Dufayard, J. F. et al. 2008. Phylogeny.fr: robust phylo-genetic analysis for the non-specialist. Nucleic Acids Res. 36:W465–9.

Du, Q., Bi, G., Mao, Y. & Sui, Z. 2016. The complete chloroplastgenome of Gracilariopsis lemaneiformis (Rhodophyta) givesnew insight into the evolution of family Gracilariaceae. J. Phy-col. 52:441–50. doi:10.1111/jpy.12406.

Field, D., Tiwari, B., Booth, T., Houten, S., Swan, D., Bertrand, N.& Thurston, M. 2006. Open software for biologists: fromfamine to feast. Nat. Biotechnol. 24:801–3.

COMPLETE PLASTOMES OF MEMBRANOPTERA 41

Freshwater, D. W. & Rueness, J. 1994. Phylogenetic relationshipsof some European Gelidium (Gelidiales, Rhodophyta) speciesbased on rbcL nucleotide sequence analysis. Phycologia33:187–94.

Gabrielson, P. W. 2008a. Molecular sequencing of Northeast Paci-fic type material reveals two earlier names for Prionitis lyallii,Prionitis jubata and Prionitis sternbergii, with brief commentson Grateloupia versicolor (Halymeniaceae, Rhodophyta). Phy-cologia 47:89–97.

Gabrielson, P. W. 2008b. On the absence of previously reportedJapanese and Peruvian species of Prionitis (Halymeniaceae,Rhodophyta) in the Northeast Pacific. Phycol. Res. 56:105–14.

Gabrielson, P. W., Lindstrom, S. C. & O’Kelly, C. J. 2012. Keys tothe seaweeds and seagrasses of southeast Alaska, British Columbia,Washington and Oregon. Phycological Contribution #8, Phy-coID, Hillsborough, North Carolina, iv + 192 pp.

Gabrielson, P. W., Miller, K. A. & Martone, P. T. 2011. Morpho-metric and molecular analyses confirm two distinct species ofCalliarthron (Corallinales, Rhodophyta), a genus endemic tothe Northeast Pacific. Phycologia 50:298–316.

Gardner, N. L. 1926. New Rhodophyceae from the Pacific coastof North America. I. Univ. Cal. Publ. Bot. 13:205–27.

Giardine, B., Riemer, C., Hardison, R. C., Burhans, R., Elnitski,L., Shah, P., Zhang, Y. et al. 2005. Galaxy: a platform forinteractive large-scale genome analysis. Genome Res. 15:1451–5.

Goecks, J., Nekrutenko, A., Taylor, J. & The Galaxy Team. 2010.Galaxy: a comprehensive approach for supporting accessible,reproducible, and transparent computational research in thelife sciences. Genome Biol. 11:R86.

Hagopian, J., Reis, M., Kitakima, J., Bhattacharya, D. & de Oli-veira, M. C. 2004. Comparative analysis of the completechloroplast genome of the red alga Gracilaria tenuistipitatavar. liui provides insights into the evolution of rhodoplastsand their relationship to other chloroplasts. J. Mol. Evol.59:464–77.

Hernandez-Kantun, J. J., Rindi, F., Adey, W. H., Heesch, S., Pe~na,V., Le Gall, L. & Gabrielson, P. W. 2015. Sequencing typematerial resolves the identity and distribution of the generit-rype Lithophyllum incrustans, and related European species L.hibernicum and L. bathyporum (Corallinales, Rhodophyta). J.Phycol. 51:791–807.

Hind, K. R., Gabrielson, P. W., Lindstrom, S. C. & Martone, P. T.2014a. Misleading morphologies and the importance ofsequencing type specimens for resolving coralline taxonomy(Corallinales, Rhodophyta): Pachyarthron cretaceum is Corallinaofficinalis. J. Phycol. 50:760–4.

Hind, K. R., Gabrielson, P. W. & Saunders, G. W. 2014b. Molecu-lar-assisted alpha taxonomy reveals pseudocryptic diversityamong species of Bossiella (Corallinales, Rhodophyta) in theeastern Pacific Ocean. Phycologia 53:443–56.

Hind, K. R., Miller, K. A., Young, M., Jensen, C., Gabrielson, P.W. & Martone, P. T. 2015. Resolving cryptic species of Bos-siella (Corallinales, Rhodophyta) using contemporary andhistorical DNA. Am. J. Bot. 102:1–19.

Huelsenbeck, J. P., Ronquist, F., Nielsen, R. & Bollback, J. P.2001. Bayesian inference of phylogeny and its impact on evo-lutionary biology. Science 294:2310–4.

Hughey, J. R. & Gabrielson, P. W. 2012. Comment on “AcquiringDNA from dried archival red algae (Florideophyceae) forthe purpose of applying available names to contemporarygenetic species: a critical assessment”. Botany 90:1191–4.

Hughey, J. R., Gabrielson, P. W., Rohmer, L., Tortolani, J., Silva,M., Miller, K. A., Young, J. D., Martell, C. & Ruediger, E.2014. Minimally destructive sampling of type specimens ofPyropia (Bangiales, Rhodophyta) recovers complete plastidand mitochondrial genomes. Sci. Rep. 4:5113.

Hughey, J. R. & Hommersand, M. H. 2008. Morphological andmolecular systematic study of Chondracanthus (Gigartinaceae,Rhodophyta) from Pacific North America. Phycologia 47:124–55.

Hughey, J. R., Silva, P. C. & Hommersand, M. H. 2001. Solvingtaxonomic and nomenclatural problems in Pacific

Gigartinaceae (Rhodophyta) using DNA from type material.J. Phycol. 37:1091–109.

Hughey, J. R., Silva, P. C. & Hommersand, M. H. 2002. ITS1sequences of type specimens of Gigartina and Sarcothalia andtheir significance for the classification of South AfricanGigartinaceae (Gigartinales, Rhodophyta). Eur. J. Phycol.37:209–16.

Janou!skovec, J., Liu, S. L., Martone, P. T., Carr#e, W., Leblanc, C.,Coll#en, J. & Keeling, P. J. 2013. Evolution of red algal plastidgenomes: ancient architectures, introns, horizontal genetransfer, and taxonomic utility of plastid markers. PLoS ONE8:e59001.

Katoh, K. & Standley, D. M. 2013. MAFFT Multiple SequenceAlignment Software Version 7: improvements in perfor-mance and usability. Mol. Biol. Evol. 30:772–80.

Kilpatrick, Z. M. & Hughey, J. R. 2016. Mitochondrial and plastidgenome analysis of the marine red alga Coeloseira compressa(Champiaceae, Rhodophyta). Mitochondrial DNA Part B:Resour. 1:456–8. doi:10.1080/23802359.2016.1182452.

Kylin, H. 1924. Studien €uber die Delesseriaceen. Lunds Univ.!Arsskr. N. F. Avd. 20:1–111.

Kylin, H. 1941. Californische Rhodophyceen. Lunds Univ. !Arsskr.N. F. Avd. 37:1–71.

Lagesen, K., Hallin, P., Rødland, E. A., Staerfeldt, H. H., Rognes,T. & Ussery, D. W. 2007. RNAmmer: consistent and rapidannotation of ribosomal RNA genes. Nucl. Acids Res. 35:3100–8.

Lee, J., Kim, K. M., Yang, E. C., Miller, K. A., Boo, S. M., Bhat-tacharya, D. & Yoon, H. S. 2016. Reconstructing the complexevolutionary history of mobile plasmids in red algal gen-omes. Sci. Rep. 6:23744.

Lin, S. M., Fredericq, S. & Hommersand, M. H. 2001. Systematicsof the Delesseriaceae (Ceramiales, Rhodophyta) based onLSU rDNA and rbcL sequences, including the Phycodryoi-deae subfam. nov. J. Phycol. 37:881–9.

Lin, S. M., Fredericq, S. & Hommersand, M. H. 2012. Molecularphylogeny and developmental studies of Apoglossum andParaglossum (Delesseriaceae, Rhodophyta) with a descriptionof Apoglosseae trib. nov. Eur. J. Phycol. 47:366–83.

Lindstrom, S. C., Gabrielson, P. W., Hughey, J. R., Macaya, E. C.& Nelson, W. A. 2015a. Sequencing of historic and modernspecimens reveals crypticdiversity in Nothogenia (Scinaiaceae,Rhodophyta). Phycologia 54:97–108.

Lindstrom, S. C., Hughey, J. R. & Aguilar-Rosas, L. E. 2015b. Fournew species of Pyropia (Bangiales, Rhodophyta) from thewest coast of NorthAmerica: the Pyropia lanceolata speciescomplex updated. Phytokeys 52:1–22. doi:10.3897/phy-tokeys.52.5009.

Lindstrom, S. C., Hughey, J. R. & Martone, P. T. 2011. New, res-urrected and redefined species of Mastocarpus (Phyl-lophoraceae, Rhodophyta) from the northeast Pacific.Phycologia 50:661–83.

Martone, P. T., Lindstrom, S. C., Miller, K. A. & Gabrielson, P.W. 2012. Chiharaea and Yamadaia (Corallinales, Rhodophyta)represent reduced and recently derived articulated corallinemorphologies. J. Phycol. 48:859–68.

McNeill, J., Barrie, F. R., Buck, W. R., Demoulin, V., Greuter, W.,Hawksworth, D. L., Herendeen, P. S. et al. 2012. Interna-tional code of nomenclature for algae, fungi, and plants(Melbourne Code). Reg. Veg. 154:1–240.

van der Merwe, E., Miklasz, K., Channing, A., Maneveldt, G. W. &Gabrielson, P. W. 2015. DNA sequencing resolves species ofSpongites (Corallinales, Rhodophyta) in the Northeast Pacificand South Africa, including S. agulhensis sp. nov. Phycologia54:471–90.

Phillippy, A. M., Schatz, M. C. & Pop, M. 2008. Genome assemblyforensics: finding the elusive mis-assembly. Genome Biol. 9:R55.

Ronquist, F. & Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian phy-logenetic inference under mixed models. Bioinformatics19:1572–4.

Salomaki, E. D., Nickles, K. R. & Lane, C. E. 2015. The ghost plas-tid of Choreocolax polysiphoniae. J. Phycol. 51:217–21.

42 JEFFERY R. HUGHEY ET AL.

Schattner, P., Brooks, A. N. & Lowe, T. M. 2005. The tRNAscan-SE, snoscan and snoGPS web servers for the detection oftRNAs and snoRNAs. Nucleic Acids Res. 33:686–9.

Setchell, W. A. & Gardner, N. L. 1903. Algae of NorthwesternAmerica. Univ. Cal. Pub. Bot. 1:1–418.

Sissini, M. N., de Oliveira, M. C., Gabrielson, P. W., Robinson, N.M., Okolodkov, Y. B., Riosmena-Rodriguez, R. & Horta, P. A.2014. Mesophyllum erubescens (Corallinales, Rhodophyta)–somany species in one epithet. Phytotaxa 190:299–319.

Stamatakis, A. 2014. RAxML Version 8: a tool for phylogeneticanalysis and post-Analysis of large phylogenies. Bioinformatics30:1312–3.

Verbruggen, H. & Costa, J. F. 2015. The plastid genome of thered alga Laurencia. J. Phycol. 51:586–9.

Wynne, M. J. & Saunders, G. W. 2012. Taxonomic assessment ofNorth American species of the genera Cumathamnion, Delesse-ria, Membranoptera and Pantoneura (Delesseriaceae, Rhodo-phyta). Algae 27:155–73.

Zerbino, D. R. & Birney, E. 2008. Velvet: algorithms for de novoshort read assembly using de brujin graphs. Genome Res.18:821–9.

Zhang, Y., Guo, Y. M., Li, T. J., Chen, C. H., Shen, K. N. & Hsiao,C. D. 2016. The complete chloroplast genome of Gracilariop-sis lemaneiformis, an important economic red alga of the fam-ily Gracilariaceae. Mitochondrial DNA Part B: Resour. 1:2–3.

Supporting Information

Additional Supporting Information may befound in the online version of this article at thepublisher’s web site:

Table S1. Number of SNPs observed and per-cent variation calculated between M. weeksiae ver-sus M. tenuis, M. weeksiae versus M. platyphylla,M. tenuis versus M. platyphylla for plastid codinggenes.

Table S2. rbcL SNPs, codon position, aminoacid substitution, and substitution type observedfor M. weeksiae versus M. tenuis, M. weeksiae versusM. platyphylla, M. tenuis versus M. platyphylla.

Table S3. COXI-5P SNPs, codon position,amino acid substitutions, and substitution typesobserved for M. weeksiae versus M. tenuis andM. weeksiae versus M. platyphylla, M. tenuis versusM. platyphylla.

Table S4. Top 20 genes with highest percentgenetic variation between species of Membra-noptera. Genes formatted in bold occur in all com-parisons.

COMPLETE PLASTOMES OF MEMBRANOPTERA 43