Marraffini, M
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.
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.
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)
|
Chordata
|
Colonial
|
5-10
|
Watersipora subtorquata (I)
|
Bryozoa
|
Colonial
|
5-10
|
Mytilus galloprovincialis (I)
|
Mollusca
|
Solitary
|
7-10
|
Bugula neritina (I)
|
Bryozoa
|
Colonial
|
5-8
|
Ciona savignyi (I)
|
Chordata
|
Solitary
|
4-8
|
Diplosoma listerianum (I)
|
Chordata
|
Colonial
|
3-7
|
Botryllus "schlosseri"(I)
|
Chordata
|
Colonial
|
2-6
|
Ascidia ceratodes (N)
|
Chordata
|
Solitary
|
7-10
|
Balanus crenatus (N)
|
Arthropoda
|
Solitary
|
1-2
|
Corynactis californica (N)
|
Cnidaria
|
Solitary
|
1-2
|
Metridium senile (N)
|
Cnidaria
|
Solitary
|
3-6
|
Aplidium californium (N)
|
Chordata
|
Colonial
|
5-10
|
Distaplia occidentalis (N)
|
Chordata
|
Colonial
|
3-7
|
Barentsia ramosa (N)
|
Entoprocta
|
Colonial
|
2-6
|
Eudistylia polymorpha (N)
|
Annelida
|
Solitary
|
10-20
|
Mytilus californianus (N)
|
Mollusca
|
Solitary
|
7-10
|
Organisms_recruited_during_the_experiment.'>Table SM2. Organisms recruited during the experiment. Listed in order of appearance and asterisks indicate species that were not originally used in treatments.
Organism
|
|
|
Date of Appearance
|
Successful Recruit
|
Phyla
|
Lowest Taxonomic Level
|
Native Status
|
DOY
|
Day of Experiment
|
Size (mm)
|
Annelida
|
Spirorbis sp.*
|
Native
|
7/16/12
|
14
|
N/A
|
Chordata
|
Diplosoma listerianum
|
NIS
|
7/16/12
|
14
|
2
|
Chordata
|
Ascidiacea (colonial)*
|
Unknown
|
7/16/12
|
14
|
2
|
Chordata
|
Botrylloides violaceus
|
NIS
|
7/30/12
|
28
|
2
|
Bryozoa
|
Watersipora subtorquata
|
NIS
|
7/30/12
|
28
|
1
|
Chordata
|
Botryllus schlosseri
|
NIS
|
8/13/12
|
42
|
2
|
Chordata
|
Ciona savignyi
|
NIS
|
8/13/12
|
42
|
2
|
Annelida
|
Salmacina tribranchiata*
|
Native
|
8/13/12
|
42
|
2
|
Arthropoda
|
Balanus crenatus
|
Native
|
9/10/12
|
70
|
2
|
Bryozoa
|
Bugula neritina*
|
NIS
|
9/24/12
|
84
|
1
|
Annelida
|
Serpula columbiana*
|
Native
|
9/24/12
|
84
|
2
|
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
|
Overall GLMM
|
4
|
100
|
Recruits
|
R, PC, PNS, R:PC, Time, Block
|
0.5
|
0.85
|
0.46
|
Early Recruitment
|
S2
|
57
|
Recruits
|
R, PC, PNS, R:PC, Time, Block
|
0.5
|
0.66
|
0.32
|
Stachowicz Comparison
|
7
|
71
|
Recruits
|
R, PC, PNS, R:PC, Time, Block
|
0.5
|
0.66
|
0.31
|
Invasions
|
S3
|
30
|
NIS Recruits
|
R, PC, PNS, R:PC, Time, Block
|
0.5
|
0.61
|
0.21
|
Table SM4. Estimated parameters from GLMM for the first 57 days. Model for cumulative recruitment during the first 57 days of the experiment.
|
Parameter
|
Estimate
|
Std. Error
|
P value
|
Variance
|
Fixed
|
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
|
--
|
Random
|
Block
|
--
|
--
|
--
|
6.631e-01
|
|
Time
|
--
|
--
|
--
|
1.797e-05
|
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).
|
Parameter
|
Estimate
|
Std. Error
|
P value
|
Variance
|
Fixed
|
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
|
--
|
Random
|
Block
|
--
|
--
|
--
|
5.713e-01
|
|
Time
|
--
|
--
|
--
|
6.955-06
|
Table SM6. Estimated parameters from the best-fit GLMM on NIS recruitment. Modeling cumulative recruitment of NIS during the first month of the experiment.
|
Parameter
|
Estimate
|
Std. Error
|
P value
|
Variance
|
Fixed
|
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
|
--
|
Random
|
Block
|
--
|
--
|
--
|
0.6764
|
|
Time
|
--
|
--
|
--
|
0.0003
|
Genetic Methods
When morphological identification was not feasible, genetic methods were used to confirm the identity of recruited individuals (genus or species) and organisms in community manipulations. A sample of DNA was extracted from fresh tissue using Promega© Wizard 96-well DNA extraction kits following manufacturer’s protocol, with the additional step of tissue maceration by bead beating before overnight incubation. Polymerase chain reaction (PCR) amplified the COI region with primers jgLCO1490 and jgHCO2198 [1x Green Gotaq Master Mix, Promega (Cat #PRM7123), 0.2 M BSA, 1.5 mM MgCl2, 0.2 μM of each primer] using a three-step thermocycler program (three minute at 94C followed by 30 cycles of 94C for one minute as a denaturation step, 47C for one minute as the annealing temperature, and an extension step at 72C for one minute) using universal COI primers (Geller et al. 2013). PCR products deemed successful as bright single bands visualized by agarose electrophoresis were sequenced by the Sanger method (Elim Biopharmaceuticals, Hayward, CA). Geneious© software (by Biomatters Ltd.) was used to assemble forward and reverse reads, and edited sequences were compared to known sequences in Genbank (National Center for Biotechnology Information) and a reference library developed in the San Francisco invasive species monitoring program (Geller et al. 2013). Similarity of sequences from unknown specimens to a reference sequence of 95% or greater was considered a match for identification.
Results
Ninety-three samples were successfully amplified by PCR and sent for Sanger sequencing. Of these samples 61 sequences, showed strong quality sequences that could be used for analysis (Table S4). This analysis helped resolve the identification of two species (an anemone and Balanus sp.) too small for morphological identification. These specimens had results from Genbank but the identifications were to species never recorded in this area and the matches were suspect. Comparisons to the Geller database revealed better matches and identifications of species known to inhabit these areas. Several specimens remained unresolved as the samples failed to amplify or to give a clean, high quality sequence. Further work is needed to resolve the identity of these samples. The unresolved species were not assigned native or NIS status and were removed from analyses on invasion success.
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.
Table S4. Field and molecular identifications of organisms used in treatments. Shows comparisons with Genbank database based on the best sequence.
Field Identification
|
Number of Samples
|
Molecular Identification
|
Accession Number
|
Percent Identical
|
Final Identification
|
Anemone (small brown)
|
4
|
Aiptasia pulchella
|
HG423158.1
|
95
|
Unknown
|
Balanus sp.
|
5
|
Amphibalabus reticulatus
|
JQ035518.1
|
89
|
Unknown
|
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
|
Sabellid sp.
|
3
|
Eudistylia vancouveri
|
HM473366.1
|
90
|
Eudistylia polymorpha
|
Sabellid sp. (smaller tube)
|
2
|
Megalomma splendida
|
HM473463
|
98
|
Megalomma splendida
|
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
|
61
|
|
|
|
|
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 Identification
|
Genbank
Identification
|
Accession Number
|
Percent Identical
|
Geller Lab Identification
|
Percent Identification
|
Finial Identification
|
Anemone (small brown)
|
Aiptasia pulchella
|
HG423158.1
|
95
|
Diadumene lineata
|
100
|
Diadumene lineata
|
Balanus sp.
|
Amphibalabus reticulatus
|
JQ035518.1
|
89
|
Balanus crenatus (haplotype A)
|
98
|
Balanus crenatus
|
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