Electronic Supplementary Material Note S Figures S-S4 Tables S, S Note S: Description of glaucinid reproductive system Glaucinins have diaulic (i.e., having two pallial gonoducts, 30) reproductive systems [0]. After developing in a proximal hermaphroditic otestis, autosperm and eggs are sent distally to the respective male and female reproductive tracts via the muscular ampulla (Fig. ; 0,, ). The male system consists of a long duct (prostate) connected to a protrusible penis, which is housed within a penial sheath, opening at the gonopore (Fig. ). In Glaucus atlanticus the penis is armed with a cuticular spine, whereas it is unarmed in G. marginatus [0]. The female system consists of a proximal receptaculum seminis, which stores oriented allosperm, and female gland complex consisting of a proximal caule gland, followed by a membrane gland, and finally a distal mucous gland [3]. All of these structures open into the iduct. Fertilization occurs in the iduct, immediately distal to the receptaculum seminis and proximal to the female gland complex. The iduct extends distally to the vagina (here it may contain egg strings and/or unoriented sperm), and the vaginal opening is located at the gonopore [9]. In G. atlanticus and two of the G. marginatus lineages (A and C), the iduct connects to a bursa copulatrix, a thinwalled epithelial sac opening immediately proximal to the vagina (Figs., S, S). In G. marginatus lineages B and D, respectively the sister lineages of A and C, the bursa copulatrix is absent; the thin vaginal duct leads directly to the female gland complex and receptaculum seminis (Figs., S, S). Supplementary references: 30. Ghiselin, M. T. 966 The evolution of hermaphroditism among animals Quart. Rev. Biol. 44, 89-08 3. Klussmann-Kolb, A. 00 The reproductive systems of the Nudibranchia (Gastropoda, Opisthobranchia): Comparative histology and ultrastructure of the nidamental glands with aspects of functional morphology Zool. Anz. 40, 9-36.
Supplementary Figure Legends (Figure S is on p. 4) Figure S. Two reproductive morphologies in Glaucus spp. viewed via dissections. The topology of the Glaucus clade is presented at left with color-coding corresponding to four G. marginatus lineages (A-D) and G. atlanticus (E) (Figs.,, S3). Images on the right are dorsal view photographs of glaucinid reproductive systems with most or all of the otestis remed. Dashed black circles outline the gonopore, black arrows indicate position of the vagina, and the bursa copulatrix (when present) is indicated with red letters. The penis is everted in all G. marginatus specimens (A-D) and inverted in G. atlanticus (E). A. G. marginatus (Indo-Pacific). B. G. marginatus (South Pacific). C. G. marginatus (Eastern North Pacific). D. G. marginatus (Central North Pacific). E. G. atlanticus. Abbreviations: am, ampulla;, bursa copulatrix;, female gland complex; ot, otestis;, penis;, prostate;, vagina (dashed red arrow). Scale bars =.0 mm. (Figure S is on p. 5) Figure S. Two reproductive morphologies in Glaucus spp. viewed via histological sections. The topology of the Glaucus clade is presented at left with color-coding corresponding to four G. marginatus lineages (A-D) and G. atlanticus (E) (Figs.,, S3). Images on the right are photographs of histological sections of distal glaucinid reproductive systems. The bursa copulatrix (when present) is indicated with red letters. A. G. marginatus (Indo-Pacific) dorsal view, penis inverted. The bursa copulatrix is located immediately proximal to the vagina. A. G. marginatus (Indo-Pacific) dorsal view. Detail of bursa copulatrix containing unoriented allosperm. B. G. marginatus (South Pacific) ventral view, outer body wall remed, penis everted. No bursa copulatrix is present; proximal to the vagina, the iduct leads to the female gland complex. B. G. marginatus (South Pacific). Detail of iduct. C. G. marginatus (Eastern North Pacific) dorsal view, penis everted. The bursa copulatrix is located immediately proximal to the vagina. C. G. marginatus (Eastern North Pacific). Detail of bursa copulatrix containing unoriented allosperm. D. G. marginatus (Central North Pacific) dorsal view, penis everted. No bursa copulatrix is present; proximal to the vagina, the iduct leads to the female gland complex. D. G. marginatus (Central North Pacific). Detail of iduct. E. G. atlanticus (ventral view), outer body wall remed, penis inverted. The vagina opens immediately to the bursa copulatrix and the iduct. E. G. atlanticus. Detail of the vagina. E3. G. atlanticus sagittal section, lateral view. Detail of the bursa copulatrix containing unoriented allosperm, which is separate from the iduct containing part of an egg string. Abbreviations:, bursa copulatrix;, female gland complex;, penis;, prostate; ot, otestis;, iduct; rs, receptaculum seminis;, vagina. Scale bars: a-a = 00 µm; b =.0 mm; b = 500 µm; c-c = 00 µm; d-d = 00 µm; e =.0 mm; e =.0 mm; e3 = 500 µm. (Figure S3 is on p. 6) Figure S3. Bayesian consensus phylogram of aeolid nudibranchs based on four loci (mt 6S rdna and COI; nuclear 8S rdna and Histone-H3). Statistical support percentages are shown abe internal branches in the form: Bayesian posterior probability/maximum likelihood bootstrap value. Asterisks indicate values of 00. Clades Aeolidida and Doridida (outgroup) are indicated to the far right of the topology. Superfamilies Aeolidioidea (Aeolidiidae+Glaucidae, blue), Fionoidea (green), and Flabellinoidea (orange) are indicated in color and labeled near
internal branches. Presence (+) or absence (-) of the bursa copulatrix is indicated in red on the topology. Glaucus lineage color coding appears as in Figs.,, S3. Four cryptic lineages of G. marginatus are denoted by bold letters: A, Indo-Pacific; B, South Pacific; C, Eastern North Pacific; D, Central North Pacific. (Figure S4 is on p. 7) Figure S4. Glaucus clade individual topologies for four loci. Bayesian phylograms depict within-glaucus relationshi for A, mt 6S rdna; B, mt COI; C, nuclear 8S rdna and D, Histone-H3. Glaucus atlanticus clades are collaed (see Fig. S3). Four cryptic lineages of G. marginatus were recered in mt 6S rdna, three of which were also recered in mt COI. Cryptic lineages of G. marginatus are denoted by bold letters: A, Indo-Pacific; B, South Pacific; C, Eastern North Pacific; D, Central North Pacific. All nodal support values (Bayesian and Maximum likelihood) = 00. 3
Fig. S A am - B ot C ot G. marginatus am - D ot am E ot G. atlanticus rs 4
Fig. S A ot - B rs - D C G. marginatus ot 3 G. atlanticus E 5
Fig. S3 Aeolidioidea 93/77 96/7 63/ 9/ 9/ */78 99/87 9/57 */97 */94 89/55 */83 56/ A8 A7 */93 A5 A3 A A */83 A4 A6 A9 A0 B3 B8 - B0 B7 B B B B4 B5 B6 B9 */98 C C C3 C5 C6 C7 C8 C9 C4 C0 - D6 D D D3 D4 D5 4 0 8 5 3 5 6 0 4 6 7 9 Glaucus atlanticus 7 8 4 6 9 30 3 9 3 5 7 8 Favorinus elenalexarium Learchis poica Hermosita hakunamatata Aeolidiella alba Phidiana pugnax Aeolidiella chromosoma Facelina stearnsi Pteraeolidia ianthina Hermissenda crassicornis Nanuca sebastiani Dondice occidentalis Phyllodesmium parangatum Phyllodesmium longicirrum Aeolidia papillosa Spurilla neapolitana Spurilla sargassicola Caloria indica Protaeolidia juliae A C D Glaucus marginatus Aeolidida Fionoidea 98/66 99/63 Flabellinoidea */98 Calma glaucoides Cuthona abronia */7 Cuthona divae */90 Cuthona lagunae Cuthona fulgens Flabellina exoptata Flabellina fusca Flabellina trilineata.4 Cryptobranchia kuragiensis Doridida 0. 6
Fig. S4 A mt 6S rdna A B C D G. marginatus B mt COI A C D G. marginatus G. atlanticus G. atlanticus C D 8S rdna G. marginatus Histone H-3 G. marginatus A B C D A B C D G. atlanticus G. atlanticus 7
Table S. Locality data for Glaucus spp. used in the molecular and morphological analyses. Color coding corresponds to the four glaucinin molecular lineages (Figs., S3). Species Genus Gyre System Latitude ( Decimal) Longitude ( Decimal) atlanticus Glaucus North Atlantic 7.4965-84.9965 atlanticus Glaucus North Atlantic 7.4965-84.9965 atlanticus 3 Glaucus North Atlantic 7.4965-84.9965 atlanticus 4 Glaucus North Atlantic 7.4965-84.9965 atlanticus 5 Glaucus North Atlantic 7.4965-84.9965 atlanticus 6 Glaucus North Atlantic 8.667-85.488 atlanticus 7 Glaucus North Atlantic 7.4997-86.0057 atlanticus 8 Glaucus North Atlantic 8.0004-87.4846 atlanticus 9 Glaucus North Atlantic 4.493 83.5035 atlanticus 0 Glaucus North Atlantic 6.0047 86.0083 atlanticus Glaucus South Pacific -4.65-4.75 atlanticus Glaucus North Pacific 9.355-56.77 atlanticus 3 Glaucus North Pacific 9.5-56. atlanticus 4 Glaucus South Pacific -33.738 5.3 atlanticus 5 Glaucus North Pacific 9.355-56.77 atlanticus 6 Glaucus North Pacific 9.355-56.77 atlanticus 7 Glaucus South Pacific -33.738 5.3 atlanticus 8 Glaucus North Pacific 9.355-56.77 atlanticus 9 Glaucus South Pacific -33.738 5.3 atlanticus 0 Glaucus South Pacific -33.738 5.3 atlanticus Glaucus Indian -3.05 5.7333 atlanticus Glaucus Indian -3.05 5.7333 atlanticus 3 Glaucus Indian -3.05 5.7333 atlanticus 4 Glaucus Indian -3.05 5.7333 atlanticus 5 Glaucus Indian -3.05 5.7333 atlanticus 6 Glaucus Indian -3.05 5.7333 atlanticus 7 Glaucus Indian -30.095 30.86 atlanticus 8 Glaucus Indian -30.095 30.86 atlanticus 9 Glaucus Indian -30.095 30.86 atlanticus 30 Glaucus Indian -30.095 30.86 marginatus A Glaucus Indian -.8 35.59 marginatus A Glaucus Indian -.8 35.59 marginatus A3 Glaucus Indian -.8 35.59 marginatus A4 Glaucus Indian -.8 35.59 marginatus A5 Glaucus North Pacific 9.5-56. marginatus A6 Glaucus North Pacific 9.355-56.77 marginatus A7 Glaucus North Pacific 9.355-56.77 marginatus A8 Glaucus North Pacific 5.08-4.48 marginatus A9 Glaucus North Pacific 5.08-4.48 marginatus A0 Glaucus South Pacific -4.65-4.75 marginatus B Glaucus South Pacific -33.738 5.3 marginatus B Glaucus South Pacific -33.738 5.3 marginatus B3 Glaucus South Pacific -33.738 5.3 marginatus B4 Glaucus South Pacific -33.738 5.3 marginatus B5 Glaucus South Pacific -33.738 5.3 marginatus B6 Glaucus South Pacific -33.738 5.3 marginatus B7 Glaucus South Pacific -33.738 5.3 marginatus B8 Glaucus South Pacific -33.738 5.3 marginatus B9 Glaucus South Pacific -33.738 5.3 8
marginatus B0 Glaucus South Pacific -33.738 5.3 marginatus C Glaucus North Pacific 5.08-4.48 marginatus C Glaucus North Pacific 5.08-4.48 marginatus C3 Glaucus North Pacific 5.08-4.48 marginatus C4 Glaucus North Pacific 5.08-4.48 marginatus C5 Glaucus North Pacific 5.08-4.48 marginatus C6 Glaucus North Pacific 5.08-4.48 marginatus C7 Glaucus North Pacific 5.08-4.48 marginatus C8 Glaucus North Pacific 5.08-4.48 marginatus C9 Glaucus North Pacific 5.08-4.48 marginatus C0 Glaucus North Pacific 5.08-4.48 marginatus D Glaucus North Pacific 9.48-56.883 marginatus D Glaucus North Pacific 9.48-56.883 marginatus D3 Glaucus North Pacific 9.48-56.883 marginatus D4 Glaucus North Pacific 9.48-56.883 marginatus D5 Glaucus North Pacific 9.355-56.7 marginatus D6 Glaucus North Pacific 9.48-56.883 9
Table S. Taxonomy, museum catalog number of voucher specimen, and GenBank accession numbers for sequences for four molecular markers (nuclear 8S rrna, Histone-H3, mitochondrial 6S rrna, COI) from the nudibranch species included in the molecular phylogeny (Figs. b, S3). Museums and universities holding voucher specimens are identified by prefixes before catalog numbers: AMS, Australian Museum, Sydney, Australia; CASIZ, California Academy of Sciences; CPIC, California Polytechnic State University, Pomona, USA; FLMNH, Florida Museum of Natural History, University of Florida, Gainesville, USA; LACM, Los Angeles County Museum of Natural History, Los Angeles, USA; NM, Natal Museum, Pietermaritzburg, South Africa; NUGB, Nagoya University, Nagoya, Japan (outgroup previously in GenBank); UMMZ, University of Michigan Museum of Zoology, Ann Arbor, USA; WAM, Western Australian Museum, Perth, Australia. For Glaucus spp. with no voucher, the specimen size was so small that the whole animal was used for DNA extraction. Whole genomic DNA template for these samples is available at UMMZ. All accession numbers not beginning with JQ come from other studies and were previously published in GenBank. Asterisks indicate no sequence data. Species Genus Family Voucher 8S H3 6S COI papillosa Aeolidia Aeolidiidae CPIC 0077 JQ69993 JQ699385 JQ699475 JQ699565 alba Aeolidiella Aeolidiidae LACM 750 JQ69994 JQ699386 * * chromosoma Aeolidiella Aeolidiidae LACM 7507 JQ69995 JQ699387 JQ699476 JQ699566 glaucoides Calma Calmidae CPIC 0056 JQ69996 JQ699388 JQ699477 JQ699567 indica Caloria Glaucidae FLMNH 49050 JQ69997 JQ699389 DQ4773 DQ4735 kuragiensis Cryptobranchia Dorididae NUGB-L505 AB8779 AB543984 AB38346 AB38457 abronia Cuthona Tergipedidae CASIZ 74485 JQ69998 JQ699390 JQ699478 JQ699568 divae Cuthona Tergipedidae CASIZ 74495 JQ69999 JQ69939 JQ699479 JQ699569 fulgens Cuthona Tergipedidae CASIZ 74484 JQ699300 JQ69939 JQ699480 * lagunae Cuthona Tergipedidae no voucher JQ69930 JQ699393 JQ69948 * occidentalis Dondice Glaucidae LACM 003-4.5 JQ69930 JQ699394 JQ69948 JQ699570 stearnsi Facelina Glaucidae no voucher JQ699303 JQ699395 JQ699483 JQ69957 elenalexiarum Favorinus Glaucidae LACM 5338 JQ699304 JQ699396 JQ699484 HM6755 exoptata Flabellina Flabellinidae LACM 53895 JQ699305 JQ699397 JQ699485 JQ69957 fusca Flabellina Flabellinidae LACM 7497 JQ699306 JQ699398 JQ699486 JQ699573 trilineata Flabellina Flabellinidae CPIC 00058 JQ699307 JQ699399 JQ699487 GQ904 atlanticus Glaucus Glaucidae UMMZ 30975 JQ699308 JQ699400 JQ699488 JQ699574 atlanticus Glaucus Glaucidae UMMZ 30975 JQ699309 JQ69940 JQ699489 JQ699575 atlanticus 3 Glaucus Glaucidae no voucher JQ69930 JQ69940 JQ699490 JQ699576 atlanticus 4 Glaucus Glaucidae UMMZ 30975 JQ6993 JQ699403 JQ69949 JQ699577 atlanticus 5 Glaucus Glaucidae UMMZ 30975 JQ6993 JQ699404 JQ69949 JQ699578 atlanticus 6 Glaucus Glaucidae no voucher JQ69933 JQ699405 JQ699493 JQ699579
atlanticus 7 Glaucus Glaucidae UMMZ 30976 JQ69934 JQ699406 JQ699494 JQ699580 atlanticus 8 Glaucus Glaucidae UMMZ 30977 JQ69935 JQ699407 JQ699495 JQ69958 atlanticus 9 Glaucus Glaucidae UMMZ 30978 JQ69936 JQ699408 JQ699496 JQ69958 atlanticus 0 Glaucus Glaucidae UMMZ 30979 JQ69937 JQ699409 JQ699497 JQ699583 atlanticus Glaucus Glaucidae UMMZ 30980 JQ69938 JQ69940 JQ699498 JQ699584 atlanticus Glaucus Glaucidae UMMZ 3098 JQ69939 JQ6994 JQ699499 JQ699585 atlanticus 3 Glaucus Glaucidae UMMZ 3098 JQ69930 JQ6994 JQ699500 JQ699586 atlanticus 4 Glaucus Glaucidae AMS C.46956 JQ6993 JQ69943 JQ69950 JQ699587 atlanticus 5 Glaucus Glaucidae UMMZ 30983 JQ6993 JQ69944 JQ69950 JQ699588 atlanticus 6 Glaucus Glaucidae UMMZ 30983 JQ69933 JQ69945 JQ699503 JQ699589 atlanticus 7 Glaucus Glaucidae AMS C.46956 JQ69934 JQ69946 JQ699504 JQ699590 atlanticus 8 Glaucus Glaucidae UMMZ 30983 JQ69935 JQ69947 JQ699505 JQ69959 atlanticus 9 Glaucus Glaucidae AMS C.46956 JQ69936 JQ69948 JQ699506 JQ69959 atlanticus 0 Glaucus Glaucidae AMS C.46956 JQ69937 JQ69949 JQ699507 JQ699593 atlanticus Glaucus Glaucidae WAM S59354 JQ69938 JQ69940 JQ699508 JQ699594 atlanticus Glaucus Glaucidae WAM S59354 JQ69939 JQ6994 JQ699509 JQ699595 atlanticus 3 Glaucus Glaucidae WAM S59354 JQ699330 JQ6994 JQ69950 JQ699596 atlanticus 4 Glaucus Glaucidae WAM S59354 JQ69933 JQ69943 JQ6995 JQ699597 atlanticus 5 Glaucus Glaucidae WAM S59354 JQ69933 JQ69944 JQ6995 JQ699598 atlanticus 6 Glaucus Glaucidae WAM S59354 JQ699333 JQ69945 JQ69953 JQ699599 atlanticus 7 Glaucus Glaucidae NM W7469 JQ699334 JQ69946 JQ69954 JQ699600 atlanticus 8 Glaucus Glaucidae NM W7469 JQ699335 JQ69947 JQ69955 JQ69960 atlanticus 9 Glaucus Glaucidae NM W7469 JQ699336 JQ69948 JQ69956 JQ69960 atlanticus 30 Glaucus Glaucidae NM W7469 JQ699337 JQ69949 JQ69957 JQ699603 marginatus A Glaucus Glaucidae CASIZ 76985 JQ699338 JQ699430 JQ69958 JQ699604 marginatus A Glaucus Glaucidae CASIZ 76985 JQ699339 JQ69943 JQ69959 JQ699605 marginatus A3 Glaucus Glaucidae CASIZ 76985 JQ699340 JQ69943 JQ69950 JQ699606 marginatus A4 Glaucus Glaucidae CASIZ 76985 JQ69934 JQ699433 JQ6995 JQ699607 marginatus A5 Glaucus Glaucidae UMMZ 30984 JQ69934 JQ699434 JQ6995 JQ699608 marginatus A6 Glaucus Glaucidae UMMZ 30985 JQ699343 JQ699435 JQ69953 JQ699609 marginatus A7 Glaucus Glaucidae UMMZ 30985 JQ699344 JQ699436 JQ69954 JQ69960 marginatus A8 Glaucus Glaucidae UMMZ 30986 JQ699345 JQ699437 JQ69955 JQ6996 marginatus A9 Glaucus Glaucidae UMMZ 30986 JQ699346 JQ699438 JQ69956 JQ6996 marginatus A0 Glaucus Glaucidae UMMZ 30987 JQ699347 JQ699439 JQ69957 JQ69963 marginatus B Glaucus Glaucidae AMS C.46957 JQ699348 JQ699440 JQ69958 * marginatus B Glaucus Glaucidae AMS C.46957 JQ699349 JQ69944 JQ69959 *
marginatus B3 Glaucus Glaucidae AMS C.46957 JQ699350 JQ69944 JQ699530 * marginatus B4 Glaucus Glaucidae AMS C.46957 JQ69935 JQ699443 JQ69953 * marginatus B5 Glaucus Glaucidae AMS C.46957 JQ69935 JQ699444 JQ69953 * marginatus B6 Glaucus Glaucidae AMS C.46957 JQ699353 JQ699445 JQ699533 * marginatus B7 Glaucus Glaucidae AMS C.46957 JQ699354 JQ699446 JQ699534 * marginatus B8 Glaucus Glaucidae AMS C.46957 JQ699355 JQ699447 JQ699535 * marginatus B9 Glaucus Glaucidae AMS C.46957 JQ699356 JQ699448 JQ699536 * marginatus B0 Glaucus Glaucidae AMS C.46957 JQ699357 JQ699449 JQ699537 * marginatus C Glaucus Glaucidae UMMZ 30988 JQ699358 JQ699450 JQ699538 JQ69964 marginatus C Glaucus Glaucidae UMMZ 30988 JQ699359 JQ69945 JQ699539 JQ69965 marginatus C3 Glaucus Glaucidae UMMZ 30988 JQ699360 JQ69945 JQ699540 JQ69966 marginatus C4 Glaucus Glaucidae UMMZ 30988 JQ69936 JQ699453 JQ69954 JQ69967 marginatus C5 Glaucus Glaucidae UMMZ 30988 JQ69936 JQ699454 JQ69954 JQ69968 marginatus C6 Glaucus Glaucidae UMMZ 30988 JQ699363 JQ699455 JQ699543 JQ69969 marginatus C7 Glaucus Glaucidae UMMZ 30988 JQ699364 JQ699456 JQ699544 JQ69960 marginatus C8 Glaucus Glaucidae UMMZ 30988 JQ699365 JQ699457 JQ699545 JQ6996 marginatus C9 Glaucus Glaucidae UMMZ 30988 JQ699366 JQ699458 JQ699546 JQ6996 marginatus C0 Glaucus Glaucidae UMMZ 30988 JQ699367 JQ699459 JQ699547 JQ69963 marginatus D Glaucus Glaucidae UMMZ 30989 JQ699368 JQ699460 JQ699548 JQ69964 marginatus D Glaucus Glaucidae UMMZ 30989 JQ699369 JQ69946 JQ699549 JQ69965 marginatus D3 Glaucus Glaucidae UMMZ 30989 JQ699370 JQ69946 JQ699550 JQ69966 marginatus D4 Glaucus Glaucidae UMMZ 30989 JQ69937 JQ699463 JQ69955 JQ69967 marginatus D5 Glaucus Glaucidae UMMZ 30990 JQ69937 JQ699464 JQ69955 JQ69968 marginatus D6 Glaucus Glaucidae UMMZ 30989 JQ699373 JQ699465 JQ699553 JQ69969 crassicornis Hermissenda Glaucidae LACM 007-. JQ699374 JQ699466 JQ699554 JQ699630 hakunamatata Hermosita Glaucidae LACM 7503 JQ699375 JQ699467 JQ699555 JQ69963 poica Learchis Glaucidae CPIC 0037 JQ699376 JQ699468 JQ699556 JQ69963 sebastiani Nanuca Glaucidae CPIC 005 JQ699377 JQ699469 JQ699557 JQ699633 pugnax Phidiana Glaucidae LACM 004-. JQ699378 JQ699470 JQ699558 * longicirrum Phyllodesmium Glaucidae LACM 5383 JQ699379 JQ69947 JQ699559 JQ699634 parangatum Phyllodesmium Glaucidae CASIZ 74440 JQ699380 JQ69947 JQ699560 JQ699635 juliae Protaeolidia Aeolidiidae LACM 5385 JQ69938 * JQ69956 * ianthina Pteraeolidia Glaucidae LACM 53995 JQ69938 JQ699473 JQ69956 * neapolitana Spurilla Aeolidiidae LACM 004-94.3 JQ699383 JQ699474 JQ699563 JQ699636 sargassicola Spurilla Aeolidiidae CPIC 0078 JQ699384 * JQ699564 *