Supplemental Data. Wolfenstetter et al. (2012). Plant Cell /tpc

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1 Supplemental Figure 1 INT1 CDS: 0001 ATGACATTGA CGATCCCAAA CGCACCTGGG AGCTCCGGGT ACTTGGATAT 0051 GTTCCCAGAG AGAAGGATGT CGTATTTTGG GAATTCTTAC ATTCTGGGTT 0101 TGACTGTCAC TGCCGGAATC GGTGGCCTTC TGTTCGGTTA TGATACAGGT 0151 GTAATTTCCG GTGCCCTTTT GTACATTAAG GATGATTTCG AAGTTGTTAA 0201 GCAGAGCAGT TTCTTACAGG AAACTATTGT AAGTATGGCT TTAGTTGGTG 0251 CCATGATTGG TGCTGCAGCT GGAGGTTGGA TCAATGACTA CTACGGTCGT 0301 AAAAAGGCGA CTCTTTTTGC AGATGTTGTC TTTGCAGCTG GAGCTATCGT 0351 TATGGCTGCT GCTCCTGATC CGTATGTTTT GATATCGGGT CGTCTCTTGG 0401 TCGGTTTAGG AGTTGGTGTT GCTTCTGTGA CTGCTCCGGT TTATATAGCG 0451 GAAGCATCTC CATCGGAAGT TAGAGGTGGA CTTGTGAGCA CCAACGTATT 0501 GATGATCACT GGTGGACAGT TTCTTTCATA TCTCGTTAAT TCTGCTTTCA 0551 CACAGGTTCC AGGAACATGG AGATGGATGC TTGGTGTTTC AGGCGTTCCC CCTAGG (AvrII) 0601 GCTGTGATTC AGTTCATCCT CATGCTTTTC ATGCCAGAAT CCCCTCGATG 0651 GCTTTTCATG AAAAATCGTA AAGCGGAAGC CATCCAAGTG CTCGCCAGAA 0701 CCTATGACAT CTCGCGGTTA GAAGACGAGA TTGATCATCT TTCAGCGGCT 0751 GAAGAGGAAG AAAAACAAAG GAAGCGCACT GTTGGCTACT TGGATGTTTT GCCGGC (NaeI) 0801 CAGATCCAAA GAATTGAGAC TTGCATTTCT TGCCGGAGCA GGACTTCAGG 0851 CGTTTCAGCA GTTCACTGGT ATCAATACAG TTATGTACTA CAGCCCTACG 0901 ATAGTCCAAA TGGCTGGATT TCACTCAAAC CAGCTCGCTT TGTTCCTGTC 0951 TCTCATTGTT GCTGCCATGA ATGCTGCTGG AACAGTCGTG GGGATTTACT 1001 TCATCGATCA TTGTGGAAGG AAAAAGCTAG CTCTCTCAAG TTTATTTGGC 1051 GTCATTATAT CACTCTTAAT CCTCTCAGTC TCGTTTTTCA AACAATCTGA 1101 GACATCATCT GATGGAGGGC TCTATGGTTG GCTCGCTGTG CTTGGCTTAG 1151 CTCTATACAT TGTTTTCTTT GCACCGGGAA TGGGACCAGT CCCATGGACG 1201 GTGAACTCAG AGATATACCC TCAACAATAC CGAGGCATAT GCGGAGGCAT 1251 GTCAGCTACA GTTAACTGGA TCAGCAATTT GATTGTGGCA CAAACATTCT 1301 TGACAATCGC GGAAGCTGCT GGAACAGGAA TGACTTTCTT GATTTTGGCG 1351 GGAATTGCGG TTCTTGCTGT AATCTTTGTG ATTGTGTTTG TCCCTGAGAC AGGCCT (StuI) 1401 TCAAGGGCTA ACGTTTTCGG AAGTTGAACA GATTTGGAAA GAAAGAGCTT 1451 ATGGAAATAT CAGTGGCTGG GGCAGTAGCA GTGATAGCAA CAACATGGAA 1501 GGGTTACTCG AGCAGGGATC TCAATCTTAA INT1 protein sequence: 001 MTLTIPNAPG SSGYLDMFPE RRMSYFGNSY ILGLTVTAGI GGLLFGYDTG 051 VISGALLYIK DDFEVVKQSS FLQETIVSMA LVGAMIGAAA GGWINDYYGR 101 KKATLFADVV FAAGAIVMAA APDPYVLISG RLLVGLGVGV ASVTAPVYIA 151 EASPSEVRGG LVSTNVLMIT GGQFLSYLVN SAFTQVPGTW RWMLGVSGVP 201 AVIQFILMLF MPESPRWLFM KNRKAEAIQV LARTYDISRL EDEIDHLSAA 251 EEEEKQRKRT VGYLDVFRSK ELRLAFLAGA GLQAFQQFTG INTVMYYSPT 301 IVQMAGFHSN QLALFLSLIV AAMNAAGTVV GIYFIDHCGR KKLALSSLFG 351 VIISLLILSV SFFKQSETSS DGGLYGWLAV LGLALYIVFF APGMGPVPWT 401 VNSEIYPQQY RGICGGMSAT VNWISNLIVA QTFLTIAEAA GTGMTFLILA 451 GIAVLAVIFV IVFVPETQGL TFSEVEQIWK ERAYGNISGW GSSSDSNNME 501 GLLEQGSQS* 1

2 INT4 CDS: 0001 ATGGTGGAAG GAGGAATTGC GAAAGCAGAC AAAACAGAGT TCACAGAGTG 0051 TTGGAGAACA ACATGGAAGA CACCTTACAT CATGCGACTT GCTCTCTCCG 0101 CCGGAATCGG AGGTCTTCTC TTCGGTTACG ATACCGGAGT CATTTCCGGA 0151 GCTCTTCTTT TCATCAAAGA AGATTTTGAT GAAGTTGATA AGAAAACATG 0201 GCTTCAGTCA ACTATTGTTA GTATGGCAGT GGCTGGAGCC ATCGTCGGAG 0251 CAGCAGTCGG AGGTTGGATC AATGATAAGT TTGGTAGGAG GATGTCGATT 0301 CTTATCGCCG ATGTTCTTTT CTTGATCGGT GCGATTGTGA TGGCATTTGC 0351 TCCGGCTCCT TGGGTTATCA TCGTTGGAAG AATCTTTGTT GGGTTTGGTG 0401 TTGGTATGGC TTCGATGACG TCTCCGTTGT ATATCTCTGA AGCTTCTCCG 0451 GCGAGGATTA GAGGTGCACT TGTTAGTACC AATGGTCTTC TCATCACTGG 0501 TGGACAATTC TTCTCGTACC TCATCAATCT TGCTTTTGTC CATACTCCGG 0551 GAACATGGAG ATGGATGTTG GGTGTTGCGG GAGTTCCAGC TATTGTTCAG CCTAGG (AvrII) 0601 TTTGTGCTTA TGTTGTCGCT ACCTGAGTCT CCAAGGTGGT TATATAGAAA 0651 GGACAGGATT GCGGAATCCA GAGCGATTCT TGAGAGGATT TACCCGGCTG 0701 ACGAAGTGGA GGCGGAGATG GAAGCTTTGA AACTGTCAGT GGAGGCAGAG 0751 AAAGCAGACG AGGCAATCAT TGGAGATAGC TTCTCTGCAA AGCTGAAAGG GCC GGC (NaeI) 0801 AGCTTTCGGG AATCCCGTTG TTCGACGTGG ACTCGCTGCT GGTATCACGG 0851 TGCAAGTGGC TCAGCAGTTT GTGGGGATCA ACACTGTCAT GTACTACAGT 0901 CCTTCTATTG TGCAATTTGC TGGTTACGCC TCTAACAAGA CAGCTATGGC 0951 CTTGTCTCTT ATTACTTCCG GTTTGAATGC TTTGGGTTCT ATTGTGAGCA 1001 TGATGTTTGT CGATCGGTAC GGGAGAAGGA AGCTCATGAT CATCTCTATG 1051 TTTGGGATCA TAGCATGTCT TATCATCTTA GCCACGGTGT TCTCACAAGC 1101 GGCCATCCAC GCCCCCAAGA TCGATGCGTT TGAGTCAAGG ACGTTTGCAC 1151 CAAATGCTAC TTGTTCAGCA TATGCCCCTC TCGCAGCCGA AAATGCTCCT 1201 CCCTCGAGGT GGAACTGCAT GAAGTGTCTC AGGTCCGAAT GTGGATTCTG 1251 CGCCAGCGGG GTACAGCCGT ACGCACCAGG AGCATGTGTG GTGTTGTCAG 1301 ATGACATGAA AGCAACTTGC AGCTCAAGAG GAAGAACATT CTTCAAAGAT 1351 GGATGTCCAA GCAAGTTTGG GTTCTTAGCT ATAGTGTTTT TGGGGCTTTA 1401 CATCGTAGTC TACGCACCAG GTATGGGCAC TGTCCCGTGG ATCGTCAACT 1451 CTGAGATCTA TCCGCTCAGA TACAGAGGTC TTGGTGGAGG AATAGCCGCC 1501 GTCTCGAATT GGGTCTCCAA CCTGATAGTG AGTGAGAGCT TCCTCTCACT 1551 CACCCATGCT CTCGGCTCCT CTGGTACCTT CCTTCTCTTT GCTGGCTTCT AGGC 1601 CCACGATCGG GCTCTTCTTC ATTTGGTTGC TCGTTCCTGA GACCAAAGGG CT (StuI) 1651 CTTCAGTTTG AGGAGGTCGA GAAGTTGCTC GAGGTCGGCT TCAAGCCCAG 1701 TCTCTTGCGG CGGAGGGAGA AGAAGGGCAA AGAAGTCGAT GCTGCTTAA INT4 protein sequence: 001 MVEGGIAKAD KTEFTECWRT TWKTPYIMRL ALSAGIGGLL FGYDTGVISG 051 ALLFIKEDFD EVDKKTWLQS TIVSMAVAGA IVGAAVGGWI NDKFGRRMSI 101 LIADVLFLIG AIVMAFAPAP WVIIVGRIFV GFGVGMASMT SPLYISEASP 151 ARIRGALVST NGLLITGGQF FSYLINLAFV HTPGTWRWML GVAGVPAIVQ 201 FVLMLSLPES PRWLYRKDRI AESRAILERI YPADEVEAEM EALKLSVEAE 251 KADEAIIGDS FSAKLKGAFG NPVVRRGLAA GITVQVAQQF VGINTVMYYS 301 PSIVQFAGYA SNKTAMALSL ITSGLNALGS IVSMMFVDRY GRRKLMIISM 351 FGIIACLIIL ATVFSQAAIH APKIDAFESR TFAPNATCSA YAPLAAENAP 401 PSRWNCMKCL RSECGFCASG VQPYAPGACV VLSDDMKATC SSRGRTFFKD 451 GCPSKFGFLA IVFLGLYIVV YAPGMGTVPW IVNSEIYPLR YRGLGGGIAA 2

3 501 VSNWVSNLIV SESFLSLTHA LGSSGTFLLF AGFSTIGLFF IWLLVPETKG 551 LQFEEVEKLL EVGFKPSLLR RREKKGKEVD AA* Supplemental Figure 1. Arabidopsis INT1 and INT4 coding sequences and modifications introduced to allow domain swapping. In the CDS, N-terminal sequences that were mutually replaced via long PCR primers are highlighted in yellow. Newly introduced, unique restriction sites (AvrII, NaeI and StuI) are indicated in bold face above the original (WT) sequence. Nucleotides altered by PCR are shown in red. Fragments mutually replaced via these restriction sites are highlighted in green (central loop) or blue (C-terminus). Start ATGs and stop codons are underlined. The resulting protein sequences are shown using the same color code; acidic di-leucine regions are shown in pink. The newly inserted restriction sites did not affect the INT1 or INT4 amino acid sequences. 3

4 Supplemental Figure 2 Supplemental Figure 2. Immunoblot analyses to reveal intactness of GFP-fusion proteins in transformed Arabidopsis protoplasts. (A) Analysis of soluble (S) and membrane (M) protein fractions prepared from GFP and GFP-INT1 expressing wildtype (WT) protoplasts. GFP can only be detected in the soluble fraction whereas the membrane-localized GFP-INT1 fusion is exclusively found in the membrane fraction. (B) Analysis of membrane fractions prepared from WT protoplasts expressing INT1 constructs. A soluble fraction with GFP alone (lane 1) was used as a control. (C) Analysis of membrane fractions prepared from WT protoplasts expressing INT4 constructs. (D) Analysis of membrane fractions prepared from WT protoplasts expressing SUC2 constructs. 4

5 (E) Analysis of membrane fractions prepared from WT protoplasts expressing SWEET1 constructs. (F) Analysis of membrane fractions prepared from WT and ap-3 ß protoplasts expressing INT1, SUC4 and ESL1 constructs. The positions of molecular mass markers (in kilodaltons, kd) are indicated on the left. Fusion proteins were detected using an anti-gfp antibody. 5

6 Supplemental Figure 3 Supplemental Figure 3. Subcellular localization of INT1 C(C4)-GFP and INT4 C(C1)-GFP. (A) The fusion of GFP to the C-terminus of INT1 C(C4) does not alter the subcellular localization of this chimera and yields the same localization as GFP-INT1 C(C4) (see Figure 1H in the printed version of the manuscript) in intact protoplasts (left) and isolated vacuoles (right). (B) The fusion of GFP to the C-terminus of INT4 C(C1) does not alter the subcellular localization of this chimera and yields the same localization as GFP-INT4 C(C1) (see Figure 1I in the printed version of the manuscript) in intact protoplasts (left) and isolated vacuoles (right). Red color shows autofluorescence of chlorophyll. Bars are 10 µm. 6

7 Supplemental Figure 4 Supplemental Figure 4. Subcellular localization of GFP-INT4(C1) and GFP- INT1(C4). (A) Addition of the INT1 C-terminus (C1) to the intact INT4 protein had almost no effect on the sorting of the resulting GFP-INT4(C1) protein to the plasma membrane as indicated by the maximum projection (left) and the optical section (middle) of intact protoplasts. Only in lysed protoplasts (right) faint fluorescence of the tonoplast (yellow arrows) is detected indicating that a small amount of GFP-INT4(C1) is rerouted to the vacuole. (B) Addition of the INT4 C-terminus (C4) to the intact INT1 protein traps the resulting GFP-INT1(C4) protein in the ER network as indicated by the maximum projection (left) and the optical section (middle) of intact protoplasts. No fluorescence is observed in the tonoplast (right). Red color shows autofluorescence of chlorophyll. Bars are 10 µm. 7

8 Supplemental Figure 5 Supplemental Figure 5. Subcellular localization of GFP-INT1 (LLE SSS). (A) Mutation of the LLE sequence in the C-terminus of INT1 into SSS causes labeling of the plasma membrane in intact Arabidopsis protoplasts. (B) No GFP fluorescence is seen in the tonoplast after osmotic lysis of the plasma membrane. Red color shows autofluorescence of chlorophyll. Bars are 10 µm. 8

9 Supplemental Figure 6 Supplemental Figure 6. Subcellular localization of SWEET1-GFP and GFP- SWEET1 analyzed 72 h after transformation. Optical sections of protoplasts expressing SWEET1-GFP (A) or GFP-SWEET1 (B). No GFP fluorescence is detected in the plasma membrane even three days after transformation. Red color shows autofluorescence of chlorophyll. Bars are 10 µm. 9

10 Supplemental Figure 7 Supplemental Figure 7. Unrooted phylogenetic tree of AP complex subunits and related proteins from man, yeast and Arabidopsis. The phylogenetic tree was calculated with confirmed or predicted, and subunits from different adaptor protein (AP) complexes. The subunit of the Arabidopsis AP-3 complex clearly clusters together with the subunits of the human and yeast AP-3 complexes. Bootstrap values of 1,000 samplings are shown at each branch. The bar indicates the evolutionary distance. Sequences were aligned using the ClustalW2 software package ( (Larkin et al., 2007) using the default settings and stored in the PHYLIP output format. An unrooted phylogenetic tree was calculated by maximum likelihood using PHYML 3.0 at the bioinformatics platform of the ( Los Alamos National laboratory (Guindon and Gascuel, 2003) using the JTT model for amino acid substitutions (1000 bootstrap samplings). A tree was built using the SeaView 4 software package (Gouy et al., 2010). The full alignment used for tree calculation is shown in Supplemental Data Set 1 online. 10

11 Supplemental Figure 8 Supplemental Figure 8. Co-localization of GFP-SUC4 and the cis-golgi marker Wave127R in ap-3 protoplasts. (A) ap-3 protoplast co-expressing a Wave127R construct (left) and a construct for GFP-SUC4 (middle). The right image shows a merge of these two images. (B) Higher magnification of the areas boxed in (A) show the donut shape of two Golgis viewed from the top and the flat shape of a Golgi viewed from the side. Bars are 10 µm in (A) and 1 µm in (B). 11

12 Supplemental Figure 9 Supplemental Figure 9. Co-localization of GFP-SUC4 and the Golgi/TGN marker Wave25R in ap-3 protoplasts. All images show GFP-SUC4 (green) and Wave25R (red) fluorescence in ap-3 protoplast. The Wave25R-labeled structures can be distinguished from the GFP- SUC4-labeled structures. Blue color shows chlorophyll autofluorescence. Bars are 2 µm. 12

13 Supplemental Figure 10 Supplemental Figure 10. Co-localization of GFP-SUC4 and the cis-golgi marker CD3-967-mCherry in a WT protoplast and effect of BFA on a GFP-SUC4 expressing protoplast. (A) Optical section of a protoplast co-expressing GFP-SUC4 and CD3-967-mCherry. The markers localize to the tonoplast or the Golgi, respectively. Chloroplast autofluorescence is shown in blue. (B) Optical section of a protoplast expressing GFP-SUC4 in the presence of BFA (25 µg ml -1 ). GFP-SUC4 localizes no longer to the tonoplast but rather accumulates in a vesicular ER. Chloroplast autofluorescence is shown in red. Bars are 10 µm. 13

14 Supplemental Figure 11 Supplemental Figure 11. Re-establishment of the original subcellular localization for INT4, INT1 and SUC4 in BFA treated protoplasts after removal of BFA. In transformed protoplasts treated with BFA the localization of the fusion proteins in the plasma membrane (A) or in the tonoplast (B) and (C) is lost. 6 h after removal of the inhibitor the original subcellular localization is re-established. (A) Protoplasts expressing GFP-INT4 in presence of 0.25% DMSO (left), 25 µg ml -1 BFA (middle) and after removal of BFA (right). (B) Protoplasts expressing GFP-INT1 in presence of 0.25% DMSO (left), 25 µg ml -1 BFA (middle) and after removal of BFA (right). (C) Protoplasts expressing GFP-SUC4 in presence of 0.25% DMSO (left), 25 µg ml -1 BFA (middle) and after removal of BFA (right). All images show single optical sections of lysed [left and right image in (B) and left image in (C)] or intact protoplasts [all images in (A), middle image in (B) and middle and right image in (C)]. Red color shows autofluorescence of chlorophyll. Bars are 10 µm. 14

15 Supplemental Table 1. Quantification of the subcellular localizations seen with the different constructs analyzed. Figure Construct Plasmid name Number of cells analyzed Subcellular localization PM ER Golgi Tono 1B INT1-GFP psw C/8B GFP-INT1 psw H GFP-INT1 C(C4) psw S2A INT1 C(C4)-GFP psw F GFP-INT1 N(N4) psw G GFP-INT1 L(L4) psw S3B GFP-INT1(C4) psw C GFP-INT1 9 psw B GFP-INT1 30 psw D GFP-INT1(ER AA) psw E GFP-INT1(LLE AAA) psw S4 GFP-INT1(LLE SSS) psw PM ER Golgi Tono 1D INT4-GFP psw E GFP-INT4 psw I GFP-INT4 C(C1) psw SB INT4 C(C1)-GFP psw S3A GFP-INT4(C1) psw x 4F GFP-INT4(RRREKK AAAAAA) psw G GFP-INT4(LLE AAA) psw I GFP-INT4(FK AA) psw J GFP-INT4 23 psw PM ER Golgi Tono 5B SUC2-GFP psw A GFP-SUC2 psw F SUC2 14-GFP psw E GFP-SUC2 14 psw D SUC2(C1)-GFP psw xx 5C GFP-SUC2(C1) psw xx 5H SUC2 14(C1)-GFP psw xxx 5G GFP-SUC2 14(C1) psw I GFP-SUC2 14(C1)(LLE AAA) psw PM ER Golgi Tono 6C SWEET1-GFP psw x 6A GFP-SWEET1 psw B GFP-SWEET1 33 psw D GFP-SWEET1(C1) psw E GFP-SWEET1 33(C1) psw PM ER Golgi Tono 7E GFP-SUC4 in WT psw C ESL1-GFP in WT ppw I GFP-SUC4(C1) in WT psw B GFP-INT1 in ap-3 psw F GFP-SUC4 in ap-3 psw D ESL1-GFP in ap-3 ppw J GFP-SUC4(C1) in ap-3 psw

16 Each of our fusion proteins showed a clear localization to one specific compartment (PM, plasma membrane; ER, endoplasmic reticulum; Tono, tonoplast), which is indicated in blue ( ). Additional comparably weak fluorescence in other membranes is indicated by (x) negligible, (xx) very weak and (xxx) weak. For fusion proteins destined to PM or Tono a faint staining was often observed in the ER/Golgi as well, due to newly synthesized proteins that have not been completely targeted yet. This staining is indicated by (+). 16

17 Supplemental Table 2. List of primers used in this work. Name of the primer C INT1 NcoI 5 ERD6B5 ERD6B3 INT1 3 PciI INT1 3 stop XbaI INT1 3'StuI R INT1 5 PciI INT1 AvrII F INT1 AvrII R INT1BglN INT1BlgC INT1 del9r INT1 del30r INT1 ER/AAr INT1 LLE/AAAr INT1 LLE/SSSr INT1 NaeI F INT1 NaeI R INT1 nco INT1 N Term PciI INT1 p5 INT1 pci INT1 StuI F(long) INT1 StuI R INT4 3 NcoI INT4 3 NcoI wo C Term INT4 3'StuI R INT4 5 NcoI INT4 5 SbfI INT4 AvrII F INT4 AvrII R INT4 del23r INT4 FK/AA R INT4 LLE/AAA F INT4 LLE/AAA R INT4 NaeI F INT4 NaeI R INT4 nco INT4 N Term PciI INT4 pci INT4 RRREKK/AAAAAAr INT4 StuI F(long) INT4 StuI R pint1 3 SbfI SUC2 3 PciI SUC2 3 PciI wo C Term SUC2 5 PciI SUC4 PciI F SUC4 StuI PciI R SWEET1 3 NcoI SWEET1 5 NcoI SWEET1 wo C Term 3 NcoI 5` 3` Sequence GCCGCCATGGTAACGTTTTCGGAAGTTGAACAG CACCATGACGATGTCGGAGAACTCAAG GGATCMTTGTAGAAAATCTGTAAGAGAAGC ACATGTTGGCAGATTGAGATCCCTGCTCGAG ATTCTAGATTAAGATTGAGATCCCTGCTCGA TAGGCCTGCAGATTGAGATCCCTGCTCGAGTAACC ACATGTCATTGACGATCCCAAACG CTTTTCATGCCAGAATCCCCTAGGTGGCTTTTCATGAAAAATCGTAAAGC CCACCTAGGGGATTCTGGCATGAAAAG CCATGGAAGTAGATCTCTGAGTTCACCGTCCACG GAGATCTACCCTCAACAATACCGAG ACATGTTTATTATTCCATGTTGTTGCTATCACTGC ACATGTTTATTACCAAATCTGTTCAACTTCCGAAAACG ACATGTTGGCAGATTGAGATCCCTGCTCGAGTAACCCTTCCATGTTGTTGCTATCACTGCTA CTGCCCCAGCCACTGATATTTCCATAAGCTGCTGCTTTCCAAATCTGTTCAACTTCCG ACATGTTGGCAGATTGAGATCCCTGCGCGGCTGCCCCTTCCATGTTGTTGCTATCACTGC ACATGTTGGCAGATTGAGATCCCTGCGAGGATGACCCTTCCATGTTGTTGCTATCACTGC GCCGGCGCAGGACTTCAGGCGTTTCAG CTGAAACGCCTGAAGTCCTGCGCCGGCAAGAAATGCAAGTCTCAATTC CCATGGATTGAGATCCCTGCTCGAGTAACC ACATGTCATTGACGATCCCAAACGCACCTGGGAGCTCCGGGTACTTGGATATGTTCCCAGAG AGAAGGATGTCGACATGGAAGACACCTTACATCATGCGACTTG AAAAATCGAAGCTTCGGTTCAGACC ACATGTCATTGACGATCCCAAACGCACCTG GTTTGTCCCTGAGACTCAAGGCCTAACGTTTTCGGAAGTTG CGTTAGGCCTTGAGTCTCAGGGACAAACACAATC CCATGGCAGCAGCATCGACTTCTTTGC CCATGGCTTTGGTCTCAGGAACGAGCAACC TAGGCCTGCAGCAGCATCGACTTCTTTGCCCTTCTTC CCATGGTGGAAGGAGGAATTG TACCTGCAGGATGGTGGAAGGAGGAATTGC GTTGTCGCTACCTGAGTCTCCTAGGTGGTTATATAGAAAGGACAGGATTG CCACCTAGGAGACTCAGGTAGCGACAAC CCATGGTTATTACAACTTCTCGACCTCCTCAAACTGAAGC CCATGGCAGCAGCATCGACTTCTTTGCCCTTCTTCTCCCTCCGCCGCAAGAGACTGGGCGCGG CGCCGACCTCGAGCAACTTCTC CGAGAAGGCGGCCGCGGTCGGCTTCAAGCCCAGTCTCTTGCG GCCGACCGCGGCCGCCTTCTCGACCTCCTCAAACTGAAGCCCTTTG GCCGGCATCACGGTGCAAGTGGCTCAG CTGAGCCACTTGCACCGTGATGCCGGCAGCGAGTCCACGTCGAACAAC CCATGGCAGCATCGACTTCTTTGCCCTTCT ACATGTTGGAAGGAGGAATTGCGAAAGCAGACAAAACAGAGTTCACAGAGTGTTGGAGAAC ATATTTTGGGAATTCTTACATTCTGGGTTTG ACATGTTGGAAGGAGGAATTGCGAAAGCAG CCATGGCAGCAGCATCGACTTCTTTGCCCGCCGCCGCCGCCGCCGCCAAGAGACTGGGCTTGA AGCCG CGTTCCTGAGACCAAAGGCCTTCAGTTTGAGGAGGTCGAG CTGAAGGCCTTTGGTCTCAGGAACGAGCAACC TACCTGCAGGCTTTCTTATTAAGCTCTTC ATACATGTTGTGAAATCCCATAGTAGCTTTG ATACATGTCCGGAGAAGGCAAAACCGTC ATACATGTTGAGCCATCCAATGGAG ACATGTCTACTTCCGATCAAGATCGCCGTC ACATGTTTAAAGGCCTGGGAGAGGGATGGGCTTCTGAATCCTTG CCATGGCAACTTGAAGGTCTTGCTTTCC CCATGGCAATCGCTCACACTATCTTCG CCATGGCTCCTTTGTTTCCACAGTAG 17

18 SUPPLEMENTAL REFERENCES Gouy, M., Guindon, S., and Gascuel, O. (2010). SeaView version 4: A multiplatform graphical user interface for the sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27: Guindon, S., and Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52: Larkin, M.A., Blackshields, G., Brow n, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., and Higgins, D.G. (2007). ClustalW and ClustalX version 2. Bioinformatics 23: