Phylogeny of coleoid based on complete mitochondrial genomes and cryptic species identification Fisheries College Ocean University of , Zheng Xiaodong, Xu Ran, Tang Yan, Zhang Xiaoying, Yu Ruihai, Li Qi Shandong, China 266003 [email protected] INTRODUCTIONS OBJECTICESOBJECTIVES

Octopodidae is the largest family in Cephalopoda. Although octopods display a wide Identify the cryptic or undiscovered species with high-throughput methods based on DNA diversity in skin coloration, behavior and life strategies, the hard structures for specimens barcoding are insufficient. This caused difficulties to investigate the phylogenetic relationships only through morphological characteristics. In addition, the higher-level systematic relationships within the octopod group remain unclear and are difficult to establish based Explore optimal DNA barcodes in . on only morphological data. Besides barely octopods fossil record, higher-level systematic relationships within the octopod group species limits and identification are difficult to establish. Effective methods for the explanation of phylogenetic relationships and cryptic species Analyze phylogenetic relationship of coleoid cephalopods based on mitochondrial DNA delimitation are urgently needed. Automatic Barcode Gap Discovery (ABGD) and sequences Bayesian implementation of the PTP model (bPTP) provide a high-throughput solution to identify the cryptic or undiscovered species. Besides, complete mtDNA was utilized to study the phylogenetic position and higher-levels systematic relationships of octopods. Compared to partial mt genes, complete mtDNA sequence can uncover more information Analyze relationships between relative species by comparing morphological and about gene rearrangement and other variation at the genome level for all phyla, and are mitochondrial genome characteristics comprehensively. especially powerful for displaying sufficient interspecies sequence variability and describing species specificity.

RESULTS

1 0.99 Sepia sp. 1 fangsiao II 1 1 with purple rings a 0.8 Sepia sp. 2 Sepia pharaonis 1 1 Sepia kobiensis 1 Sepia recurvirostra 1 0.99 Sepia esculenta Sepiidae 1 Sepia aculeata 1 Sepia aculeata 1 1 0.5 Sepia esculenta 0.88 Sepiella japonica 1 1 Sepia recurvirostra Metasepia tullbergi 0.68 1 Sepiidae 1 Metasepia tullbergi Sepia sp. 1 1 1 - 1 0.58 2- 1 Euprymna morsei Sepia sp. 2 1 Sepiolidae 1 a c Euprymna berryi Sepia sp. 3 b 1 0.98 1 c 1 1 Loliolus beka I 1 Sepia kobiensis 1 1 1 Loliolus beka II Sepiella japonica 1 1 Loliolus japonica 1 Euprymna morsei - 0.95 1 1 Sepiolidae 0.93 Uroteuthis chinensis Euprymna berryi 1 1 0.71 Uroteuthis sp. 1 Loliginidae Illlex argentinus Ommastrephidae 1 1 1 Uroteuthis edulis 1 1 Uroteuthis sibogae Uroteuthis duvaucelii 1 1 1 Uroteuthis duvaucelii Uroteuthis sibogae Naut i l us macromphal us - 1 - Sepioteuthis lessoniana 1 L oliolus beka I 0.67 Naut i l us macromphal us 1 Illlex argentinus Potential cryptic species morphological identification after discrimination by ABGD and bPTP analysis. Ommastrephidae 1 L oliolus beka II Vampyrot eut hi s i nf ernal i s 1 0.84 Loliolus japonica Loliginidae A mphioctopus ovulum Vampyrot eut hi s i nf ernal i s 1 1 0.84 Amphioctopus aegina 0.81 Uroteuthis chinensis Oct opus vul gari s KARNI S1 D T

1 1 L 1 at p8 4 Amphioctopus kagoshimensis Uroteuthis edulis Oct opus vul gari s cox3 nad3 cox2 d nad2 cox1 at p6 nad5 nad4 a cob nad6 nad1 r r nL r r nS 1 1 n 1 Amphioctopus cf. rex Sepioteuthis lessoniana Oct opus bi macul at us F H S2 P L1L2 V MCYWQGE 1 KARNI S1 D T 1 1 L at p8 4 1 nad3 d 0.99 Amphioctopus cf. neglectus Callistoctopus aspilosomatis Oct opus bi macul at us cox3 nad2 cox1 cox2 nad5 n ad4 a cob nad6 nad1 r r nL r r nS 1 at p6 n 1 NaAumtpihli uosc tmoapcusr ofmapnhgasliuaso F H S2 P L1L2 V MCYWQGE 1 Amphioctopus marginatus guangdongensis 1 0.98 KARNI S1 D T 1 L at p8 4 Hapalochlaena sp. 2 1 Amphi oct opus f angsi ao cox3 nad3 cox2 d r r nS Octopus sp. 1 nad2 cox1 at p6 nad5 nad4 a cob nad6 nad1 r r nL n 1 0.81 VampyArmoptheiuotchtiosp uisn faeergni anlai s 0.99 Hapalochlaena sp. 1 1 F H S2 P L1L2 V MCYWQGE Callistoctopus luteus KARNI S1 D T 1 L at p8 4

1 d Hapalochlaena lunulata NAamupthiiloucst ompaucsr oamepghianlaus cox3 nad3 nad2 cox1 cox2 nad5 nad4 a cob nad6 nad1 r r nL r r nS at p6 n 1 1 0.98 Octopus minor I Ci st opus t ai wani cus 0.55 Hapalochlaena maculosa 1 Oct opus vul gari s F H S2 P L1L2 V MCYWQGE Octopus minor II KARNI S1 D T 1 l KARNI S1 D l T at p8 4 d Amphioctopus fangsiao I 1 VamCpiysrtootpeuust htiasi wiannfiecrunsal i s l Naut i l us macromphal us a 1 cox3 nad3 nad2 cox1 cox2 nad5 nad4 4 cob nad6 nad1 r r nL r r nS

at p8 n

Amphioctopus ovulum at p6 d 1 OcCtiosptuosp ubsi mcahciunleantsuiss cox3 nad3 nad2 cox1 cox2 at p6 nad5 nad4 a cob nad6 nad1 r r nL r r nS Amphioctopus fangsiao II 1 F H n S2 P L1L2 v MCYWQGE Amphioctopus fangsiao I KARNI S1 F1D F2 H S2T P L1L2 V MCYWQGE L3

1 0.99 l KARNI S1 D at p8 T 4 1 Oct opus vul gari s d Octopus nanhaiensis L VamCpi ysrtotpeuust hcihsi nienfseirsnal i s cox3 nad3 nad2 cox1 cox2 nad5 nad4 a cob nad6 nad1 r r nL r r nS

at p6 4 0.95 0.61 at p8 n Amphioctopus fangsiao II Oct opus cyanea- XX cox3 nad3 cox2 d 1 Amphi oct opus f angsi ao nad2 cox1 at p6 nad5 nad4 a cob nad6 nad1 r r nL r r nS Cistopus taiwanicus F1 F2 H n S2 P L1L2 V MCYWQGE L3 0.67 1 F H S2 P L1L2 V MCYWQGE 0.910.92 1 Hapalochlaena lunulata Oct opus bi macul at us KARNI S1 D T Cistopus chinensis OctOocptuosp ucsy avnuel ag-aXrXi s at p8 1 1 Oct opus cyanea- XD cox3 nad3 nad2 cox1 cox2 nad5 nad4 cob nad6 nad1 r r nL r r nS 1 0.75 Hapalochlaena sp. 1 Amphi oct opus aegi na at p6 Octopus vulgaris Octopodidae 0.69 F H S2 P L1L2 V MCYWQGE Hapalochlaena sp. 2 Octopodidae NAamupthi loucst opmuasc rfoamnpghsail auos KARNI S1 D T 1 Oct opus bi macul at us L 0.54 Oct opus cyanea- XD at p8 4 Oct opus mi nor cox3 nad3 cox2 d Octopus sp. 2 1 nad2 cox1 at p6 nad5 nad4 a cob nad6 nad1 r r nL r r nS 0.58 Ci st opus t ai wani cus n 0.99 Hapalochlaena maculosa F H S2 P L1L2 V MCYWQGE

Thaumoctopus mimicus 1 Amphi oct opus aegi na KARNI S1 D T VamApmyprhoitoecuttohpiuss ifnafnegrsniaaloi s L 1 Amphioctopus aegina Oct opus mi nor at p8 4 1 nad3 d Cal l i st oct opus l ut eus cox3 nad2 cox1 cox2 nad5 nad4 a cob nad6 nad1 r r nL r r nS Ci st opus chi nensi s at p6 n Thaumoctopus mimicus 2 1 F H P MCYWQGE 0.96 S2 L1L2 V 1 Amphioctopus kagoshimensis Ci st opus t ai wani cus KARNI S1 D T

Octopus cyanea AOmcpthoipoucst ovpuulsg areigsi na L Cal l i st oct opus l ut eus KARNI S1 D at p8 4 T d

Oct opus coni spadi ceus cox3 nad3 cox2 L

nad2 cox1 nad5 nad4 a cob nad6 nad1 r r nL r r nS

1 at p6 4 0. 66 at p8 n Octopus minor I Oct opus cyanea- XX cox3 nad3 cox2 d Amphioctopus cf. neglectus nad2 cox1 at p6 F nad5 H nad4 S2a cob naPd6 nadL11L2 r r nL V r r MnCSYWQGE 0.98 1 n Ci st opus chi nensi s F H S2 P L1L2 V MCYWQGE Octopus minor-YL 1 Ci st opus t ai wani cus 0.96 0.73 Amphioctopus cf. rex OcOtcotpoupsu sc obniimsapcaudliacteuuss KARNI S1 D T Octopus minor-YL2 0.70 Amphioctopus cf. rex KARNI S1 D at p8 T Oct opus cyanea- XD cox3 nad3 nad2 cox1 cox2 nad5 nad4 L cob nad6 nad1 r r nL r r nS 1 atapt8p6 4 0.51 Amphioctopus marginatus Oct opus cyanea- XX cox3 nad3 cox2 d nad2 cox1 at p6 nad5 n a d4 a cob nad6 nad1 r r nL r r nS F H n S2 P L1L2 V MCYWQGE Octopus minor II Ci st opus chi nensi s Amphi oct opus f angsi ao KARNI S1 D F H S2T P L1L2 V MCYWQGE

1 KARNI S1 D TL at p8 4 Octopus sp. 3 1 AmphioctopusOct opus mi n ocfr . ovulum cox3 nad3 cox2 d 0.85 Octopus vulgaris nad2 cox1 atapt 8p6 nad5 nad4 a cob nad6 nad1 r r nL r r nS Oct opus cyanea- XD KARNInad3S1 D Tn 1 cox3 nad2 cox1 cox2 nad5 nad4 L cob nad6 nad1 r r nL r r nS att p86 F H 4 S2 P L1L2 V MCYWQGE Octopus guangdongensis Oct opus cyanea- XX cox3 nad3 cox2 d 0.66 0.85 1 nad2 cox1 at p6 F nad5 H nad4 a S2 cob nad6 P nad1 L1L2 r r nL V r r nS MCYWQGE Amphi oct opus aegi na n P 1 KARNI S1 D F H TS2 P L1L2 V MCYWQGE

0.58 Octopus cf. sasakii L Cal l i st oct opus l ut eus at p8 4 T 1 Oct opus mi nor cox3 KARNInad3 S1 cox2 D d 1 KARNI S1 nad2 cox1 D at p6 nad5 nad4 a T cob nad6 nad1 r r nL r r nS n 0.55 at p8 l Octopus sp. 1 0.9 Octopus sp. 2 l Oct opus cyanea- XD cox3 nad3 nad2 cox1 cox2 at p8 F nad5 H nad4 S24 cob nad6P nad1 L1L2 r r nL V r r nS MCYWQGE AmphioctopusCi st opus t ai wa marginatusni cus d 1 cox3 nad3 nad2 cox1 cox2 at p6 nad5 nad4 a cob nad6 nad1 r r nL r r nS 1 1 at p6 n Callistoctopus aspilosomatis Octopus sp. 4 F H S2 P L1L2 V MCYWQGE OCcatl ol pi usst ocot noipsups aldui tceus KARNI S1 D F H T S2 P L1L2 v MCYWQGPE

1 KARNI S1 D L T 4

1 at p8 l d 0.99 Oct opus mi nor cox3 nad3 cox2 4 Cistopus chinensis nad2 cox1 at p8 nad5 nad4 a cob nad6 nad1 r r nL r r nS Callistoctopus luteus at p6 d n Ci stCistopusopus chi nchinensisensi s cox3 nad3 nad2 cox1 cox2 at p6 nad5 nad4 a cob nad6 nad1 r r nL r r nS 1 F H S2 n P L1L2 V MCYWQGE Cistopus taiwanicus Oct opus coni spadi ceus F1 F2 H S2 P L1L2 V MCYWQGE L3 1 pompilius 1 Nautilidae Nautilus pompilius Cal l i st oct opus l ut eus Nautilidae Oct opus cyanea- XX

0.2 0.1 Oct opus coni spadi ceus OctopodsOct opus cyanea- XD shared highly consistent gene arrangement and re-rearrangement. Small ABGD and bPTP methods based on COI (a) and 16S (b) reveal cryptic species and changesOct opus mi nor only presented in tRNAs. The trnP in Amphioctopus cf. rex was absent, which undiscovered species in coleoid cephalopods. Two methods detected the clear moved backward in A. ovulum and A. marginatus. In addition, Cistopus chinensis was discrimination of at least 9 cryptic species or new species. Cathel l i st oct oponlyus l ut eus one showing trnL replication.

Oct opus coni spadi ceus

- 1 Dosidicus gigas 1 D osidicus gigas 1 Sthenoteuthis oualaniensis Sthenoteuthis oualaniensis 1 ML 1 Ommastrephes bartramii BI 1 Ommastrephes bartramii 1 Todarodes pacificus 1 Todarodes pacificus 0.98 Illex argentinus 0.98. Illex argentinus -1. 1 Architeuthis dux Architeuthi-s dux 1 Watasenia scintillans 1 Watasenia scintillans a b Bat hyteuthis abyssicola Bathyteuathis abyssicola Utoteuthis duvaucelii Utoteuthis duvaucelii 1 1 1 Uroteuthis edulis Uroteuthis edulis 1 1 1 Uroteuthis chinensis 1 1 Uroteuthis chinensis 0.53 Lo liolus beka 0.53 Loliolus beka 1 1 Doryteuthis opalescens D oryteuthis opalescens 1 1 1 1 Heterololigo bleekeri Heterololigo bleekeri 1 1 Se pioteuthis lessoniana S epioteuthis lessoniana Semirossia patagonica Idiosepius sp. Idiosepius sp. Semirossia patagonica 1 1 ND1 and ND5 with relatively higher viabilities may be more 1 1 Sepiella inermis 1 S epiella japonica 1 suitable molecular genetic markers for identification of octopods. Sepiella japonica 1 Sepia officinalis 1 Sepia officinalis 1 Sepia esculenta Sepia esculenta 1 1 1 0.99 Sepia lycidas 1 Sepia lycidas 1 1 0.99 .1 Sepia pharaonis 1 Sepia pharaonis Sepia aculeata Sepia aculeata 1 Sepia apama 1 Sepia apama Sepia latimanus Sepia latimanus Vamp yroteuthis infernalis Vampyroteuthis infernalis REFERENCES 1 Amphioctopus cf. ovulum 1 Amphioctopus cf. ovulum 11 Amphioctopus cf. rex A mphioctopus cf. rex • Tang Y, Zheng X, Ma Y, et al. The complete mitochondrial genome of Amphioctopus ma rg in a tu s (Cephalopoda: Octopodidae) and the exploration for the 1 1 1 Amphioctopus marginatus 11 Amphioctopus marginatus optimal DNA barcoding in Octopodidae [J]. Conservation Genetics Resources, 2017:1-4. doi:10.1007/s12686-017-0777-2 1 1 Amphioctopus aegina 1 Amphioctopus aegina • Zhang X, Zheng X, Ma Y, et al. Complete mitochondrial genome and phylogenetic relationship analyses of Amphioctopus aegina (Gray, 1849) (Cephalopoda: Amphioctopus fangsiao I Amphioctopus fangsiao I 1 1 Octopodidae) [J]. Mitochondrial DNA, 2017, 28(1):17.doi:10.3109/19401736.2015.1106522 1 1 Amphioctopus fangsiao II 1 Amphioctopus fangsiao II 1 1 O ctopus bimaculatus • Cheng R, Zheng X, Lin X, et al. Determination of th e complete mitochondrial DNA sequence of Octopus minor [J]. Molecular biology reports, 2012, 39(4): Octopus bimaculatus 1 1 O ctopus vulgaris 3461-3470. 1 Octopus vulgaris 1 Cistopus chinensis • Cheng R, Zheng X, Ma Y, et al. The Complete Mitochondrial Genomes of Two Octopods Cistopus chinensis and Cistopus ta iwa n icu s: Revealing the Cistopus chinensis 1 1 1 Cistopus taiwanicus Phylogenetic Position of the Genus Cistopus within the OrderOctopoda [J]. 2013, 8(12): e84216. doi:10.1371/jurnal.pone.0084216 Cistopus taiwanicus Octopus minor O ctopus minor 1 1 • Ma Y, Zheng X, Ch e ng R, et al. The complete mitochondrial genome of Octopus conispadiceus (Sasaki, 1917)(Cephalopoda: Octopodidae) [J]. Mitochondrial 1 1 Callistoctopus luteus Callistoctopus luteus DNA, 2016, 2(24): 2058-2059. doi: 10.3109/19401736.2014.928866 1 Octopus conispadiceus 1 Octopus conispadiceus Allonautilus scrobiculatus A llonautilus scrobiculatus 1 1 Nautilus macromphalus N autilus macromphalus Photographs

0.2 0.2 Phylogenetic analysis within Cephalopoda based on amino-acids of 13 protein-coding genes. Amphioctopus genus and Cistopus genus were two monophyletic groups. Octopus genus was divided into two clusters, which was further demonstrated its polyphyletism. O.minor showed a very closer relationship to C. luteus, but exhibited a distant relationship to the other species in Octopus sensu stricto genus.