Identification and phylogenetic reconstruction of Neritina snails
Material and Methods
Sequences were newly obtained from 46 specimens of Neritina snails representing 10
species and also from a specimen of Septaria porcellana for outgroup comparison
(online supplementary table 1). Vanuatu material was collected during the SANTO
2006 Expedition organized by Museum National d'Histoire Naturelle, Paris (MNHN),
Pro Natura International, and Institut de Recherche pour le Développement.
The
expedition operated under a permit granted to Dr P. Bouchet by the Environment Unit of
the Government of Vanuatu.
The National Geographic Society Committee for
Research and Exploration made possible my participation in the expedition, and the
Total Foundation covered most of the field logistics.
Live snails for the DNA analysis were boiled in 80ºC–95ºC water for 0.5–1 min, and
the animals were extracted from the shells and preserved in pure ethanol.
Identification of specimens was based on type specimens and original descriptions
whenever available. However, rigorous assignment to a species was difficult or
impossible in several cases and some seemed to be undescribed. Voucher material has
been deposited at the Department of Biological Production and Environmental Science,
University of Miyazaki, Japan, or MNHN. All shell, operculum, radula and cephalic
part of the animal were kept undamaged in all specimens for future taxonomic studies.
Photographs of shells are available from the author upon request.
Total genomic DNA was obtained from the preserved foot tissue using a Qiagen
DNeasy kit. Fragments of the COI gene were amplified using a polymerase chain
reaction (PCR) and the ‘universal’ primers LCO1490
(5’–GGTCAACAAATCATAAAGATATTGG–3’) and HCO2198
(5’–TAAACTTCAGGGTGACCAAAAAATCA–3’) (Folmer et al. 1994).
PCR
reactions were carried out in a final volume of 25 µl [2.5 µl genomic DNA template (c.
100ng), 17.5 µl ddH2O, 2.5 µl Takara ExTaq buffer, 2 µl dNTPs, 0.2 µl of each primer
(20 µM stock), and 0.1 µl Takara ExTaq enzyme]. After an initial denaturation for 2.5
min at 94ºC, the reaction solution were run for 35 cycles with the following parameters:
denaturation for 30 s at 94ºC, annealing for 40 s at 42ºC, followed by extension for 60 s
at 72ºC. PCR products were visualised by electrophoresis on 1.5% TBE agarose gel,
which was stained with ethidium bromide, and photodocumented. Successful PCR
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products were cleaned using ExoSAP-IT (USB) following the described protocol.
Both strands were directly cycle-sequenced using the amplification primers with a
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI310
automated sequencer at University of Miyazaki.
Alignment of COI sequences across
OTUs was unambiguously made by eye in MacClade 4.08 (Maddison & Maddison
2005), as no insertion or deletion was observed.
All sequences have been deposited in
the DDBJ/EMBL/Genbank with accession numbers in online supplementary table 1
(AB477472–AB477514).
Phylogenetic relationships among Neritina species were investigated using the
Bayesian and maximum likelihood (ML) criteria.
In the Bayesian inference performed
with MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003), the general time reversible model
was used and invariant site frequency and gamma-shape parameter were estimated from
the data (GTR + I + G).
The shape, proportion of invariant sites, state frequency and
substitution rate parameters were estimated separately for each codon positions. Two
parallel runs were made for 5,000,000 generations (with a sample frequency of 100)
using default value of four Markov chains.
The first 20,000 trees for each run were
discarded to ensure the four chains reached stationarity. The consensus tree and
posterior probabilities were computed from the remaining 60,000 trees (30,000 trees x
two runs). The ML analyses were performed using heuristic searches in PAUP 4.0b10
(Swofford 2002). Parameter settings for the ML search were identified by the Akaike
Information Criterion (AIC, implemented in Modeltest 3.7; Posada & Crandall, 1998).
The reliability of inferred topologies was assessed by bootstrap resamplings of the
sequences with 500 replicates; ML bootstrap analysis was performed using GARLI
v0.951 (Zwickl 2006) with the same parameter settings identified by AIC.
Competing
alternative phylogenies were obtained by searching under topological constrains and
subsequently compared with the SH test (Shimodaira & Hasegawa 1999) for ML as
implemented in PAUP* under RELL option.
Results
Bayesian and likelihood phylogenies based on COI sequences from 46 specimens of
Neritina unanimously identified 11 evolutionarily significant units (ESUs) in the genus
(figure 2f). The 11 ESUs can also be distinguished from one another by conchological
features, except the morphologically identical sister clades of N. asperulata with
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parapatric distributions, one in the Southeast Asia and Micronesia and the other in
Melanesia. Of the six attaching juveniles from Guadalcanal and Santo included in the
analysis, five with the black spiral lines appeared in the Melanesian subclade of N.
asperulata and one with the scale-like pattern was identified as N. petitii. Two other
juveniles from Guam and Japan having elongate shells with a continuous peristome
were also found attached on N. pulligera and were included in the molecular analysis.
They clustered either with the adult snails of the Asian-Micronesian subclade of N.
asperulata or an unidentified species (Neritina sp. 3) sister to N. petitii. Each of the
four ESUs has a widespread geographic distribution with the maximum distance of
1000–6300 km between study specimens, while their COI sequences are hardly
differentiated despite those remote occurrences (0–0.74% in corrected ML distance).
The unequivocal clade of N. petitii, N. sp. 3 and four other species with free-living
juveniles (Bayesian posterior probability of 100%) rendered the attaching species
non-monophyletic, while the Shimodaira-Hasegawa test did not reject the monophyly of
attaching species (P = 0.105).
References
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. 1994 DNA primers for
amplification of mitochondrial cytochrome c oxidase subunit I from diverse
metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299.
Maddison, D. R. & Maddison, W. P. 2005 MacClade 4: Analysis of phylogeny and
character evolution. Version 4.08. Sinauer Associates, Sunderland, Massachusetts
Posada, D. & Crandall, K. A. 1998 MODELTEST: testing the model of DNA
substitution. Bioinformatics 14, 817–818. (doi: 10.1093/bioinformatics/14.9.817)
Ronquist, F & Huelsenbeck, J. P. 2003 MRBAYES 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19, 1572–1574. (doi:
10.1093/bioinformatics/btg180)
Shimodaira, H. & Hasegawa, M. 1999 Multiple comparisons of log-likelihood with
applications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116.
Swofford, D. L. 2002 PAUP*. Phylogenetic Analysis Using Parsimony (*and other
methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Zwickl, D. J. 2006 Genetic algorithm approaches for the phylogenetic analysis of large
biological sequence datasets under the maximum likelihood criterion. PhD Thesis,
3
University of Texas, Austin.
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Online Supplementary Table 1.
Specimens used in phylogenetic reconstruction of
Neritina snails, arranged systematically, with collection sites and voucher numbers of
specimens and DDBJ/EMBL/Genbank accession numbers.
Asterisks indicate
hitchhiking individuals/species.
Species
Locality
Voucher Accession no.
Neritina asperulata (Récluz, 1843)*
Hoshino, Ishigaki Is., Okinawa, Japan
Takana, Iriomote Is., Okinawa, Japan
Ooura, Nago, Okinawa Is., Japan
Kawasan R., Matutinao, Cebu, Philippines
Asmafines R., Agat, Guam, Micronesia
Takana, Iriomote Is., Okinawa, Japan
Lungga R., Guadalcanal, Solomon Islands
Mbonehe R., Guadalcanal, Solomon Islands
Wounaouss R., Santo Is., Vanuatu (VM5)
Baldwin Bay, Santo Is., Vanuatu (VM29)
Yamada R., Kominato, Amami Is., Japan
Hoshino, Ishigaki Is., Okinawa, Japan
Malinao, Santa Lucia, Palawan, Philippines
Asmafines R., Agat, Guam, Micronesia
Lauli'i, Upolu, Samoa
Takana, Iriomote Is., Okinawa, Japan
Tabaru R., Yonaguni Is., Okinawa, Japan
Asmafines R., Agat, Guam, Micronesia
Patunmali R., Santo Is., Vanuatu (VM50)
Hoshino, Ishigaki Is., Okinawa, Japan
Takana, Iriomote Is., Okinawa, Japan
Asmafines R., Agat, Guam, Micronesia
Senipehn R., Pohnpei, Micronesia
Nankep R., Pohnpei, Micronesia
YK#148
YK#149
YK#520
YK#518
YK#510
YK#776*
YK#781*
YK#782
YK#783*
YK#777*
YK#778*
YK#664
YK#779*
YK#667
YK#662
YK#187
YK#505
YK#146
YK#507
YK#504
YK#511
YK#150
YK#506
YK#512
YK#513
YK#147
YK#508
YK#577
YK#780*
YK#784
YK#665
YK#666
YK#502
YK#145
YK#503
YK#519
YK#261
YK#151
YK#514
YK#515
YK#663
YK#152*
YK#153
YK#516
YK#509
YK#517
AB477472
AB477473
AB477474
AB477475
AB477476
AB477477
AB477478
AB477479
AB477480
AB477478
AB477481
AB477482
AB477478
AB477483
AB477484
AB477485
AB477486
AB477487
AB477488
AB477489
AB477490
AB477491
AB477492
AB477493
AB477494
AB477495
AB477496
AB477497
AB477498
AB477499
AB477495
AB477500
AB477501
AB477502
AB477502
AB477503
AB477504
AB477505
AB477506
AB477507
AB477508
AB477509
AB477510
AB477511
AB477512
AB477513
Yarabu, Ishigaki Is., Okinawa, Japan
YK#164
AB477514
Lungga R., Guadalcanal, Solomon Islands
Mataniko R., Guadalcanal, Solomon Islands
Sarakata R., Santo Is., Vanuatu (VM27)
Wounaouss R., Santo Is., Vanuatu (VM5)
Puelapa R., Santo Is., Vanuatu (VM49)
Neritina canalis Sowerby, 1825
Neritina delestennei Récluz, 1853
Neritina iris Mousson, 1849
Neritina petitii (Récluz, 1841)*
Neritina powisiana (Récluz, 1843)
Neritina pulligera (Linnaeus, 1767)
Neritina sp. 1
Neritina sp. 2
Neritina sp. 3*
(outgroup)
Septaria porcellana (Linnaeus, 1758)
Patunmali R., Santo Is., Vanuatu (VM50)
Fuluasou R., Apia, Upolu, Samoa
Chinase, Naze, Amami Is., Japan
Hoshino, Ishigaki Is., Okinawa, Japan
Asmafines R., Agat, Guam, Micronesia
Creoles R., Grand Port, Mauritius
Yuwangama, Yamatoson, Amami Is., Japan
Takana, Iriomote Is., Okinawa, Japan
Malinao, Santa Lucia, Palawan, Philippines
Inabucayon R., Palawan Is., Philippines
Nankep R., Pohnpei, Micronesia
Hoshino, Ishigaki Is., Okinawa, Japan
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