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INVASIBILITY OF COMMUNITIES
Supplementary Materials
Figure SM1. Experimental Setup. A. attachment of PVC frame that held experimental blocks
attached to floating dock. B. View of diver (Heather Hawk) assisting with installation of a
block. C. View of a tile several weeks after installation with solitary tunicates and Sabellidae
tube worms that were attached using marine epoxy. D. Close up of treatment tiles in in block B
showing that attachment of animals using marine epoxy. Photos A, B, D by Scott Gabara; photo
C by Michelle Marraffini.
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Figure SM2. Total number of recruits after 100 days plotted as a function of original species
richness (A); original percent cover (B), and original percent of native species (C). Data are
binned across other factors to examine the main effect in each panel.
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Table SM1 Approximate sizes of adult organisms used in the experiment. Size is taken as
the longest axis of a solitary animal and the diameter of a colonial animal. For anemones, this is
their width in their non-contracted form.
Species
Phyla
Body Plan
Range of sizes (cm)
Botrylloides violaceus (I)
Watersipora subtorquata (I)
Mytilus galloprovincialis (I)
Bugula neritina (I)
Ciona savignyi (I)
Diplosoma listerianum (I)
Botryllus "schlosseri"(I)
Ascidia ceratodes (N)
Balanus crenatus (N)
Corynactis californica (N)
Metridium senile (N)
Aplidium californium (N)
Distaplia occidentalis (N)
Barentsia ramosa (N)
Eudistylia polymorpha (N)
Mytilus californianus (N)
Chordata
Bryozoa
Mollusca
Bryozoa
Chordata
Chordata
Chordata
Chordata
Arthropoda
Cnidaria
Cnidaria
Chordata
Chordata
Entoprocta
Annelida
Mollusca
Colonial
Colonial
Solitary
Colonial
Solitary
Colonial
Colonial
Solitary
Solitary
Solitary
Solitary
Colonial
Colonial
Colonial
Solitary
Solitary
5-10
5-10
7-10
5-8
4-8
3-7
2-6
7-10
1-2
1-2
3-6
5-10
3-7
2-6
10-20
7-10
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Table SM2. Organisms recruited during the experiment. Listed in order of appearance and
asterisks indicate species that were not originally used in treatments.
Organism
Phyla
Annelida
Chordata
Chordata
Chordata
Bryozoa
Chordata
Chordata
Annelida
Arthropoda
Bryozoa
Annelida
Lowest Taxonomic Level
Spirorbis sp.*
Diplosoma listerianum
Ascidiacea (colonial)*
Botrylloides violaceus
Watersipora subtorquata
Botryllus schlosseri
Ciona savignyi
Salmacina tribranchiata*
Balanus crenatus
Bugula neritina*
Serpula columbiana*
Native Status
Native
NIS
Unknown
NIS
NIS
NIS
NIS
Native
Native
NIS
Native
Date of Appearance
Day of
DOY
Experiment
7/16/12
14
7/16/12
14
7/16/12
14
7/30/12
28
7/30/12
28
8/13/12
42
8/13/12
42
8/13/12
42
9/10/12
70
9/24/12
84
9/24/12
84
Successful
Recruit
Size (mm)
N/A
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2
2
1
2
2
2
2
1
2
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Table S3. Estimates of variation explained by the GLMMs used in this experiment. ICC
refers to interclass correlation, explaining the proportion of variance explained by random
intercept over the variance of the residuals, interpreted as the total variance between groups
(Zuur et al., 2009). Time is in days into the experiment.
Model
Table Time Response
Components
ICC
Rc2
Rm2
R, PC, PNS, R:PC,
Overall GLMM
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100
Recruits
0.5
0.85 0.46
Time, Block
Early
R, PC, PNS, R:PC,
S2
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Recruits
0.5
0.66 0.32
Recruitment
Time, Block
Stachowicz
R, PC, PNS, R:PC,
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Recruits
0.5
0.66 0.31
Comparison
Time, Block
NIS
R, PC, PNS, R:PC,
Invasions
S3
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0.5
0.61 0.21
Recruits
Time, Block
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Table SM4. Estimated parameters from GLMM for the first 57 days. Model for cumulative
recruitment during the first 57 days of the experiment.
Fixed
Random
Parameter
Estimate
Std.
Error
P value
Variance
Intercept
1.0382
0.3650
0.00445
--
Richness (PR)
0.0435
0.0066
3.44e-07
--
Percent Cover (PC)
0.0118
0.0006
<2e-16
Percent Native Species (PPNS)
-0.018
0.0002
<2e-16
R:PC
0.0003
0.0003
<2e-16
Time
-0.0029
0.0019
<2e-16
--
Block
--
--
--
6.631e-01
Time
--
--
--
1.797e-05
--
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Table SM5. Estimated parameters from GLMM for the first 71 days. Model for cumulative
recruitment during the first 71 days of the experiment, for comparison to previous experiments
(6, 7).
Fixed
Random
Parameter
Estimate
Std.
Error
P value
Variance
Intercept
1.2907
0.3387
0.00014
---
Richness (PR)
-0.0349
0.0057
1.6e-09
Percent Cover (PC)
-0.0203
0.0006
<2e-16
Percent Native Species (PPNS)
-0.0034
0.0002
<2e-16
R:PC
-0.0057
0.0002
<2e-16
Time
0.0223
0.0012
<2e-16
--
Block
--
--
--
5.713e-01
Time
--
--
--
6.955-06
--
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Table SM6. Estimated parameters from the best-fit GLMM on NIS recruitment. Modeling
cumulative recruitment of NIS during the first month of the experiment.
Fixed
Random
Parameter
Estimate
Std.
Error
P value
Variance
Intercept
-0.0585
0.4528
0.897
--
Richness (PR)
-0.013
0.0218
0.551
--
Percent Cover (PC)
-0.0256
0.0023
<2e-16
Percent Native Species (PNS)
-0.004
0.0009
8.39e-06
R:PC
-0.0051
0.0009
4.95e-08
Time
0.0676
0.0095
1.01e-12
--
Block
--
--
--
0.6764
Time
--
--
--
0.0003
--
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Genetic Methods
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When morphological identification was not feasible, genetic methods were used to
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confirm the identity of recruited individuals (genus or species) and organisms in community
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manipulations. A sample of DNA was extracted from fresh tissue using Promega© Wizard 96-
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well DNA extraction kits following manufacturer’s protocol, with the additional step of tissue
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maceration by bead beating before overnight incubation. Polymerase chain reaction (PCR)
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amplified the COI region with primers jgLCO1490 and jgHCO2198 [1x Green Gotaq Master
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Mix, Promega (Cat #PRM7123), 0.2 M BSA, 1.5 mM MgCl2, 0.2 μM of each primer] using a
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three-step thermocycler program (three minute at 94C followed by 30 cycles of 94C for one
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minute as a denaturation step, 47C for one minute as the annealing temperature, and an
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extension step at 72C for one minute) using universal COI primers (Geller et al. 2013). PCR
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products deemed successful as bright single bands visualized by agarose electrophoresis were
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sequenced by the Sanger method (Elim Biopharmaceuticals, Hayward, CA). Geneious© software
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(by Biomatters Ltd.) was used to assemble forward and reverse reads, and edited sequences were
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compared to known sequences in Genbank (National Center for Biotechnology Information) and
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a reference library developed in the San Francisco invasive species monitoring program (Geller
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et al. 2013). Similarity of sequences from unknown specimens to a reference sequence of 95%
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or greater was considered a match for identification.
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Results
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Ninety-three samples were successfully amplified by PCR and sent for Sanger
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sequencing. Of these samples 61 sequences, showed strong quality sequences that could be
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used for analysis (Table S4). This analysis helped resolve the identification of two species
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(an anemone and Balanus sp.) too small for morphological identification. These specimens
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had results from Genbank but the identifications were to species never recorded in this
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area and the matches were suspect. Comparisons to the Geller database revealed better
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matches and identifications of species known to inhabit these areas. Several specimens
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remained unresolved as the samples failed to amplify or to give a clean, high quality
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sequence. Further work is needed to resolve the identity of these samples. The unresolved
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species were not assigned native or NIS status and were removed from analyses on
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invasion success.
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References
Geller, J. , Meyer, C., Parker, M. and Hawk, H. 2013. Redesign of PCR primers for
mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in
all-taxa biotic surveys. Molecular Ecology Resources 13(5) 851-861.
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Table S4. Field and molecular identifications of organisms used in treatments. Shows comparisons with Genbank
database based on the best sequence.
Number of
Molecular
Accession
Percent
Field Identification
Final Identification
Samples
Identification
Number
Identical
Anemone (small
4
Aiptasia pulchella
HG423158.1
95
Unknown
brown)
Amphibalabus
Balanus sp.
5
JQ035518.1
89
Unknown
reticulatus
Lamillaria sp.
4
Assiminea pecos
DQ533844.1
83
Lamillaria sp.
Botrylloides violaceus
7
Botrylloides violaceus
GQ365691
100
Botrylloides violaceus
Colonial Tunicate
3
Botryllus schlosseri
JN083284
100
Botryllus schlosseri
Botryllus schlosseri
3
Botryllus schlosseri
JN083238
99
Botryllus schlosseri
Bugula sp. (brown)
1
Bugula neritina
KC129832.1
99
Bugula neritina
Bugula sp. (white)
1
Bugula stolonifera
KC129849.1
100
Bugula stolonifera
Encrusting Bryozoa
2
Celleporaria oculata
AY168607.1
94
Unknown
Corynactis californica
1
Corynactis californica
AB441257.1
100
Corynactis californica
HM473366.
Sabellid sp.
3
Eudistylia vancouveri
90
Eudistylia polymorpha
1
Sabellid sp. (smaller
2
Megalomma splendida
HM473463
98
Megalomma splendida
tube)
Mytilus galloprovincalis
6
Mytilus galloprovincalis GQ480284.1
100
Mytilus galloprovincalis
Sponge (yellow)
3
Haliclona xena
JN242209.1
99
Haliclona sp.
Anemone (small)
5
Metridium senile
HG423143
99
Metridium senile
Mytilus californianus
4
Mytilus californianus
GQ902188.1
99
Mytilus californianus
Watersipora sp.
2
Watersipora sutorquata DQ417456.1
99
Watersipora sutorquata
Tube (Amphipod)
1
Ericthonius brasiliensis
JX545463
89
Amphipoda
Diplosoma listerianum
4
Diplosoma listerianum KF791868.1
99
Diplosoma listerianum
Total
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Table S5. Field and molecular identifications of organisms unresolved by Genbank. Shows comparisons with Genbank
database and Geller lab database based on the best sequence.
Field
Genbank
Accession
Percent
Geller Lab
Percent
Finial
Identification Identification
Number
Identical Identification
Identification Identification
Anemone
Aiptasia pulchella HG423158.1 95
Diadumene lineata
100
Diadumene lineata
(small brown)
Balanus sp.
Amphibalabus
JQ035518.1 89
Balanus crenatus
98
Balanus crenatus
reticulatus
(haplotype A)
94
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