Introduction

Material and methods

Data resources

Results and Discussion

A total of 447 individuals, morphologically identified to 129 species, were successfully sequenced for at least one marker, including 47 species of shrimps, 57 brachyuran crabs, one achelate lobster, four axiid mudshrimp, one gebiid mudshrimp and 19 anomuran crabs (Table 1, Figs 1, 2). Shrimps included the infra-orders Caridea, Dendrobranchiata and Stenopodidea. Amongst successfully sequenced individuals, 99 were identified to genus, but could not be confidently assigned to a species based on morphology. The Automatic Barcode Gap Discovery method delimited 141 OTUs with COI and 140 OTUs with 16S; likewise, our COI sequences were assigned to 146 Barcode Index Numbers (BINs) in BOLD. The larger number of OTUs and BINs suggest there are ~10 potentially cryptic species or species with unusually high levels of genetic diversity in this dataset.

Figure 1.  

Pie chart indicating the number of individuals present on this study for each decapod family.

Figure 2.  

Representative decapod crustacean specimens from Bocas del Toro, Panama. A. Sicyonia laevigata, ULLZ13484; B. Alpheus thomasi, ULLZ18292; C. Tozeuma carolinense, ULLZ18291; D. Microprosthema semilaeve, ULLZ10770; E. Pseudobiffarius caesari, ULLZ13480; F. Panulirus argus juvenile, ULLZ13319; G. Pachycheles tuerkayi, ULLZ6098; H. Clibanarius antillensis, ULLZ16971; I. Acanthonyx petiverii, ULLZ12015; J. Achelous sebae, ULLZ17128; K. Pilumnus holosericus, ULLZ17614; L. Aratus pisonii, ULLZ14799.

Eighty seven of our consensus OTUs matched COI sequences already in GenBank with an identity of > 95% (see Table 1). Of these, our identification and the name on the GenBank sequence were concordant for 77 of the OTUs, including seven cases in which our identification provided better taxonomic resolution than the GenBank sequence. In many cases, these represent samples of the same taxa from other Caribbean regions confirming the conspecific status of animals from different parts of the same biogeographic region. In ten cases where our identification was not concordant with the name of a COI GenBank sequence >95% identical, the discrepancy typically occurred at the species level while the higher taxonomic ranks remained concordant. Two OTUs did not have sequences in COI: one was a singleton identified as Leander paulensis and the other included two specimens identified as Pilumnus reticulatus and P. pannosus. The remaining 51 OTUs for which we have COI sequences were < 95% identical to another sequence in GenBank and therefore considered to be new additions.

The results for the 16S analysis were relatively similar: 99 consensus OTUs were > 97% identical to 16S sequences available in GenBank and for 89 of them, the morphological identification coincided with the name of the GenBank sequence, whereas the other ten OTUs showed discrepancies at the species level with the GenBank sequence, but remained concordant at higher taxonomic ranks. Four singleton OTUs, identified as Stenopus scutellatus, Inachoides sp., Austinixa aidae and Speleophorus nodosus did not have sequences in 16S. The remaining 16S sequences (belonging to 37 OTUs) were > 97% similar to other sequences in GenBank; thus these were considered new additions.

Our dataset contributed 56 new BINs to BOLD and provided 38 new species for at least one marker in GenBank (Table 1). One hundred and thirty seven of our 140 OTUs are associated with only one morphospecies name. This coincides with our visual observations of the COI and 16S neighbour-joining trees, which showed that our morphospecies identifications are largely concordant with clusters of very similar sequences. These clusters differed from other such clusters by ~0.10 substitutions per site in COI and ~0.05 in 16S (Fig. 3). Such concordance between morphospecies and OTUs, and the magnitude of the observed interspecific divergence are similar to those reported by Costa et al. (2007) and Matzen da Silva et al. (2011). Nevertheless, there are several cases where animals could not be identified to species or in which a single species/species-complex name appeared in different OTUs. These included the following:

a

b

Figure 3.

Neighbour-Joining trees for cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) from specimens identified in this study and GenBank as Tozeuma carolinense. The accession number is provided for the GenBank sequences. Likewise, reference numbers are provided for specimens deposited in the University of Louisiana at Lafayette (ULLZ#) and the Oxford University Museum of Natural History (OUMNH). The Jukes-Cantor distance between specimens is proportional to the length of the branches separating them, as indicated in the scale bars at the bottom-left.

a: Neighbour-Joining tree for cytochrome c oxidase subunit I  
b: Neighbour-Joining tree for 16S ribosomal RNA  

For Synalpheus spp., 42 individuals fell into eight OTUs. Seven OTUs matched GenBank sequences from in-depth studies of these taxa (Duffy et al. 2000, Morrison et al. 2004, Hultgren et al. 2014, Chak et al. 2017). These were S. hoetjesi, S. paraneptunus, S. yano, S. aff. longicarpus, S. elizabethae and S. cf. rathbunae. The final OTU did not match anything in GenBank (identity < 90% in both markers). Our failure to detect more than one additional species in this group suggests that it has been well-sampled in Bocas del Toro.

Specimens of Alpheus spp. were split into 18 OTUs. Thirteen OTUs were identified to species, including two OTUs assigned to the same species name (Alpheus paracrinitus); one of these OTUs had four individuals, whereas the other was a singleton. A. paracrinitus has long been considered an unresolved species complex, including at least four species (Knowlton et al. 1993, Anker 2001). The other five OTUs were identified to genus (Alpheus sp.) or as members of a species complex (e.g. A. packardii complex). Eleven Alpheus OTUs were new for BOLD, adding 11 new BINs to the database. Likewise, eleven Alpheus OTUs were < 95% identical to COI or < 97% to 16S sequence in GenBank and, thus, constitute new additions to the database; moreover, our sequences added five new Alpheus species names for 16S in GenBank. Only one OTU, identified as A. paraformosus, matched a different species name, A. formosus, in GenBank (sequence from Leray and Knowlton 2015). Most of the OTUs identified as members of the A. packardii complex are < 95% and < 97% identical to COI and 16S sequence in GenBank, respectively. One other OTU, identified as Alpheus sp., matched GenBank sequences that were also identified only to genus. Clearly, we are far from having a complete barcode database for this speciose taxon.

The shrimps Tozeuma carolinense (Fig. 2C) fell into two OTUs, one with eight and the other with one specimen. Both of these OTUs were new for BOLD, adding two new BINs to the database. The 16S sequences for one of these OTUs matched a T. carolinense sequence in GenBank with > 99% identity. However, both of our T. carolinense OTUs were distinct from the available COI sequences in GenBank with > 95% identity (Fig. 3, Table 1), suggesting that this morphologically distinctive species may include several cryptic species. A similar situation occurred for Sicyonia laevigata (Fig. 2A), Pagurus criniticornis and Alpheus paracrinitus; all these names being assigned to specimens that grouped in two OTUs: one with multiple species and the other being a singleton.

OTUs with multiple species names: One OTU included sequences from two specimens, morphologically identified as different species (Pilumnus dasypodus and P. caribaeus). This OTU matched P. dasypodus sequences in GenBank with > 99% identity for both markers. Another OTU identified as P. caribaeus in our dataset matched GenBank sequences of that species. One other OTU comprised specimens morphologically identified as 2 species (P. pannosus and P. reticulatus).

Pseudogenes in Decapod Barcoding

We found no indels in our COI sequences and no stop codons in the corresponding amino acid sequence. Both the COI and 16S sequences showed a range of GC content (GC%) from 23.97-46.48% (Fig. 4), which is similar to the findings of other studies (e.g. Costa et al. 2007, Matzen da Silva et al. 2011). As mitochondrial genes are expected to show a different AT bias than pseudogenes (Bensasson 2001, Song et al. 2008, Matzen da Silva et al. 2011, Liu et al. 2016), those sequences with significantly deviant GC% are potentially more likely to be pseudogenes; nevertheless, a careful examination of all our sequences failed to detect any strong evidence of pseudogenes. The overall concordance between our morphological identifications and the molecular identification of OTUs, based on 16S and COI, further supports the conclusion that pseudogenes were rare or absent in this dataset.

Figure 4.  

Histogram of the GC content for COI and 16S in three major groups of decapods evaluated in this study. Shrimps included the infra-orders Caridea, Dendrobranchiata and Stenopodidea.

Much has been made of the problem with pseudogenes in decapods (Schneider-Broussard and Neigel 1997, Williams and Knowlton 2001, Song et al. 2008, Schubart 2010, Matzen da Silva et al. 2011, Raupach and Radulovici 2015, but see Schizas 2012). They are undoubtedly more common in decapods than in some other groups of marine invertebrates, where they have seldom been reported. It is also clear that pseudogenes can cause significant problems in phylogenetic reconstructions (Schubart 2010). However, we argue that the problems they pose for DNA barcoding are limited and that the difficulty in determining if a sequence is a pseudogene or not without resorting to cloning, means that barcode datasets for decapods may never be entirely free of pseudogene sequences unless mitochondrial DNA is directly targeted during DNA extraction with a mitochondrial DNA isolation kit.

Song et al. (2008) described a 3-step method for eliminating obvious pseudogenes in protein coding sequences: eliminate sequences with indels and stop codons, eliminate sequences with unusual or highly divergent amino acid sequences and eliminate sequences with unusual GC bias. The first two steps are only applicable to protein coding sequences like COI and cannot be applied to 16S, another commonly used barcoding gene for many groups [e.g. amphibians (Vences et al. 2005), hydrozoans (Zheng et al. 2014) and other marine invertebrates (Brasier et al. 2016)]. The third step is hindered by the difficulty in determining what GC% value is sufficiently different to be considered suspect. The GC% values of our sequences ranged from 24.0-46.5% and their histograms, in general, did not show any distinct gap or dip that could be considered as a clear threshold between coding sequences and pseudogenes. The small gaps and asymmetric distributions observed in some histograms were always associated with interspecific differences in GC%; for example, the large GC% of Dardanus fucosus, Calcinus tibicens and Paguristes tortugae explains the asymmetry and gaps in the anomuran histograms. A review of crustacean barcodes found that the GC% varies significantly amongst families (Matzen da Silva et al. 2011), ranging from ~30-50% with family-specific averages ranging from 33-42% (Matzen da Silva et al. 2011). If AT/GC bias is similar across the mitochondrion, one might expect that the GC% of COI and 16S are correlated and deviations from the trend-line could be another way to identify potential pseudogenes. Our data show an overall positive correlation between the two, considerable scatter around the trend-line and a small cluster of carideans that fall somewhat below the other carideans, but still within the overall variation for the decapods (Fig. 5).

Figure 5.  

Scatterplot of the GC content (GC%) in COI versus 16S for every individual of this study, successfully sequenced for both markers. In general, the GC% of the two markers appears to be as correlated as expected for two mitochondrial genes and none of the specimens seems to deviate enough to be considered a suspect pseudogene. Major groups are indicated with colours: Brachyura: red, Anomura: blue, Shrimps (Caridea, Dendrobranchiata, and Stenopodidea): green, Axiidea: light-grey, Gebiidae: black, Achelata: yellow.

Our limited ability to identify pseudogene sequences without cloning indicates that pseudogenes are likely to infiltrate metabarcoding datasets generated by high-throughput sequencing, as well as datasets generated by sanger sequencing. One concern about the inclusion of pseudogenes in these kinds of biodiversity studies is that they may over-estimate the number of OTUs reported (Schneider-Broussard and Neigel 1997, Williams and Knowlton 2001, Song et al. 2008, Schubart 2010, Matzen da Silva et al. 2011, Raupach and Radulovici 2015). This could certainly happen, but a more common occurrence is that either co-amplification of the gene and its pseudogenes reduces the quality of the reads, resulting in an unusable sequence or a single sequence significantly out-amplifies the other, resulting in a single sequence from each species. If this is the pseudogene, the results could complicate phylogenetic analyses, but are unlikely to impact the results of DNA barcoding studies. One situation where pseudogenes certainly will not impact the efficacy of a DNA barcoding approach is in the identification of unknown samples through comparisons with sequences from carefully identified material. If a sequence is known to come from a specific species, whether or not it is a pseudogene, that sequence can be used to generate a positive identification of unknown material. Therefore, rather than discard potential pseudogene sequences, they should be included in barcode databases as a potentially informative resource (see also the arguments by Schizas 2012).

Acknowledgements

References

Anker A (2001)
Two new species of snapping shrimps from the Indo-Pacific, with remarks on colour patterns and sibling species in Alpheidae (Crustacea: Caridea)
.
The Raffles Bulletin of Zoology
49
(
1
):
57
‑
72
.
Anker A, Hurt C, Knowlton N (2007)
Three transisthmian snapping shrimps (Crustacea: Decapoda: Alpheidae: Alpheus) associated with innkeeper worms (Echiura: Thalassematidae) in Panama
.
Zootaxa
1626
(
1
):
1
‑
23
. https://doi.org/10.11646/zootaxa.1626.1.1
Anker A, Hurt C, Knowlton N (2008)
Revision of the Alpheus formosus Gibbes, 1850 complex, with redescription of A. formosus and description of a new species from the tropical western Atlantic (Crustacea: Decapoda: Alpheidae)
.
Zootaxa
1707
:
1
‑
22
. https://doi.org/10.11646/zootaxa.1707.1.1
Anker A (2010)
A new genus and three new species of alpheid shrimps (Crustacea, Decapoda, Caridea) from the tropical American coasts
.
Zootaxa
2652
(
1
):
47
‑
63
. https://doi.org/10.11646/zootaxa.2652.1.4
Anker A, Pachelle PPG, De Grave S, Hultgren KM (2012)
Taxonomic and biological notes on some Atlantic species of the snapping shrimp genus Synalpheus Spence Bate, 1888 (Decapoda, Alpheidae)
.
Zootaxa
3598
:
1
‑
96
.
Appeltans W, Ahyong S, Anderson G, Angel M, Artois T, Bailly N, Bamber R, Barber A, Bartsch I, Berta A, Błażewicz-Paszkowycz M, Bock P, Boxshall G, Boyko C, Brandão S, Bray R, Bruce N, Cairns S, Chan T, Cheng L, Collins A, Cribb T, Curini-Galletti M, Dahdouh-Guebas F, Davie PF, Dawson M, De Clerck O, Decock W, De Grave S, de Voogd N, Domning D, Emig C, Erséus C, Eschmeyer W, Fauchald K, Fautin D, Feist S, Fransen CJ, Furuya H, Garcia-Alvarez O, Gerken S, Gibson D, Gittenberger A, Gofas S, Gómez-Daglio L, Gordon D, Guiry M, Hernandez F, Hoeksema B, Hopcroft R, Jaume D, Kirk P, Koedam N, Koenemann S, Kolb J, Kristensen R, Kroh A, Lambert G, Lazarus D, Lemaitre R, Longshaw M, Lowry J, Macpherson E, Madin L, Mah C, Mapstone G, McLaughlin P, Mees J, Meland K, Messing C, Mills C, Molodtsova T, Mooi R, Neuhaus B, Ng PL, Nielsen C, Norenburg J, Opresko D, Osawa M, Paulay G, Perrin W, Pilger J, Poore GB, Pugh P, Read G, Reimer J, Rius M, Rocha R, Saiz-Salinas J, Scarabino V, Schierwater B, Schmidt-Rhaesa A, Schnabel K, Schotte M, Schuchert P, Schwabe E, Segers H, Self-Sullivan C, Shenkar N, Siegel V, Sterrer W, Stöhr S, Swalla B, Tasker M, Thuesen E, Timm T, Todaro MA, Turon X, Tyler S, Uetz P, van der Land J, Vanhoorne B, van Ofwegen L, van Soest RM, Vanaverbeke J, Walker-Smith G, Walter TC, Warren A, Williams G, Wilson S, Costello M (2012)
The magnitude of global marine species diversity
.
Current Biology
22
(
23
):
2189
‑
2202
. https://doi.org/10.1016/j.cub.2012.09.036
Bensasson D (2001)
Mitochondrial pseudogenes: evolution's misplaced witnesses
.
Trends in Ecology & Evolution
16
(
6
):
314
‑
321
. https://doi.org/10.1016/s0169-5347(01)02151-6
Bonnet NK, Rocha R (2011)
The family Ascidiidae Herdman (Tunicata: Ascidiacea) in Bocas del Toro, Panama. Description of six new species
.
Zootaxa
2864
(
1
):
1
‑
33
. https://doi.org/10.11646/zootaxa.2864.1.1
Brasier M, Wiklund H, Neal L, Jeffreys R, Linse K, Ruhl H, Glover A (2016)
DNA barcoding uncovers cryptic diversity in 50% of deep-sea Antarctic polychaetes
.
Royal Society Open Science
3
(
11
). https://doi.org/10.1098/rsos.160432
Bucklin A, Steinke D, Blanco-Bercial L (2011)
DNA barcoding of marine metazoa
.
Annual Review of Marine Science
3
(
1
):
471
‑
508
. https://doi.org/10.1146/annurev-marine-120308-080950
Bucklin A, Lindeque P, Rodriguez-Ezpeleta N, Albaina A, Lehtiniemi M (2016)
Metabarcoding of marine zooplankton: prospects, progress and pitfalls
.
Journal of Plankton Research
38
(
3
):
393
‑
400
. https://doi.org/10.1093/plankt/fbw023
Buhay J (2009)
“COI-like” sequences are becoming problematic in molecular systematic and DNA barcoding studies
.
Journal of Crustacean Biology
29
(
1
):
96
‑
110
. https://doi.org/10.1651/08-3020.1
Cancian de Araujo B, Schmidt S, Schmidt O, von Rintelen T, Kilmaskossu A, Panjaitan R, Balke M (2018)
From field courses to DNA barcoding data release for West Papua - making specimens and identifications from university courses more sustainable
.
Biodiversity Data Journal
6
:
e25237
. https://doi.org/10.3897/bdj.6.e25237
Cardoso P, Erwin TL, Borges PAV, New TR (2011)
The seven impediments in invertebrate conservation and how to overcome them
.
Biological Conservation
144
(
11
):
2647
‑
2655
. https://doi.org/10.1016/j.biocon.2011.07.024
Chak STC, Duffy JE, Hultgren K, Rubenstein D (2017)
Evolutionary transitions towards eusociality in snapping shrimps
.
Nature Ecology & Evolution
1
(
4
):
96
. https://doi.org/10.1038/s41559-017-0096
Coleman CO (2015)
Taxonomy in times of the taxonomic impediment – examples from the community of experts on amphipod crustaceans
.
Journal of Crustacean Biology
35
(
6
):
729
‑
740
. https://doi.org/10.1163/1937240x-00002381
Collin R, Díaz MC, Norenburg J, Rocha RM, Sánchez JA, Schulze A, Schwartz M, Valdés A (2005)
Photographic identification guide to some common marine invertebrates of Bocas Del Toro, Panama
.
Caribbean Journal of Science
41
(
3
):
638
‑
707
.
Collin R, Venera-Pontón D, Driskell A, Macdonald K, Boyle M (2018)
Planktotrophic brachiopod larvae from the Pacific and Caribbean of Panama
.
Diversity
11
:
2
. https://doi.org/10.3390/d11010002
Collin R, Venera‐Pontón D, Driskell A, Macdonald K, Chan KK, Boyle M (2019)
Documenting neotropical diversity of phoronids with DNAbarcoding of planktonic larvae
.
Invertebrate Biology
138
(
2
). https://doi.org/10.1111/ivb.12242
Costa FO, deWaard JR, Boutillier J, Ratnasingham S, Dooh RT, Hajibabaei M, Hebert PDN (2007)
Biological identifications through DNA barcodes: the case of the Crustacea
.
Canadian Journal of Fisheries and Aquatic Sciences
64
(
2
):
272
‑
295
. https://doi.org/10.1139/f07-008
Costello M, Bouchet P, Boxshall G, Fauchald K, Gordon D, Hoeksema B, Poore GB, van Soest RM, Stöhr S, Walter TC, Vanhoorne B, Decock W, Appeltans W (2013)
Global coordination and standardisation in marine biodiversity through the World Register of Marine Species (WoRMS) and related databases
.
PLOS One
8
(
1
):
e51629
. https://doi.org/10.1371/journal.pone.0051629
Cowen R, Gawarkiewicz G, Pineda J, Thorrold S, Werner F (2007)
Population connectivity in marine systems: an overview
.
Oceanography
20
(
3
):
14
‑
21
. https://doi.org/10.5670/oceanog.2007.26
Cowen R, Sponaugle S (2009)
Larval dispersal and marine population connectivity
.
Annual Review of Marine Science
1
(
1
):
443
‑
466
. https://doi.org/10.1146/annurev.marine.010908.163757
De Grave S, Anker A (2017)
An annotated checklist of marine caridean and stenopodidean shrimps (Malacostraca: Decapoda) of the Caribbean coast of Panama
.
Nauplius
25
https://doi.org/10.1590/2358-2936e2017015
Drew L (2011)
Are we losing the science of taxonomy?
BioScience
61
(
12
):
942
‑
946
. https://doi.org/10.1525/bio.2011.61.12.4
Duffy JE, Morrison C, Ríos R (2000)
Multiple origins of eusociality among sponge-dwelling shrimps (Synalpheus)
.
Evolution
54
(
2
):
503
‑
516
. https://doi.org/10.1111/j.0014-3820.2000.tb00053.x
Ebach MC, Valdecasas AG, Wheeler QD (2011)
Impediments to taxonomy and users of taxonomy: accessibility and impact evaluation
.
Cladistics
27
(
5
):
550
‑
557
. https://doi.org/10.1111/j.1096-0031.2011.00348.x
Efron B, Halloran E, Holmes S (1996)
Bootstrap confidence levels for phylogenetic trees
.
Proceedings of the National Academy of Sciences
93
(
23
):
13429
‑
13429
. https://doi.org/10.1073/pnas.93.23.13429
Felsenstein J (1985)
Confidence limits on phylogenies: an approach using the bootstrap
.
Evolution
39
(
4
). https://doi.org/10.2307/2408678
Gascuel O (1997)
BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data
.
Molecular Biology and Evolution
14
(
7
):
685
‑
695
. https://doi.org/10.1093/oxfordjournals.molbev.a025808
Geiger M, Moriniere J, Hausmann A, Haszprunar G, Wägele W, Hebert P, Rulik B (2016)
Testing the Global Malaise Trap Program – How well does the current barcode reference library identify flying insects in Germany?
Biodiversity Data Journal
4
:
e10671
. https://doi.org/10.3897/bdj.4.e10671
Geller J, Meyer C, Parker M, 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
. https://doi.org/10.1111/1755-0998.12138
Giangrande A (2003)
Biodiversity, conservation, and the 'Taxonomic impediment'
.
Aquatic Conservation: Marine and Freshwater Ecosystems
13
(
5
):
451
‑
459
. https://doi.org/10.1002/aqc.584
Goodheart J, Ellingson R, Vital X, Galvão Filho H, McCarthy J, Medrano S, Bhave V, García-Méndez K, Jiménez L, López G, Hoover C, Awbrey J, De Jesus J, Gowacki W, Krug P, Valdés Á (2016)
Identification guide to the heterobranch sea slugs (Mollusca: Gastropoda) from Bocas del Toro, Panama
.
Marine Biodiversity Records
9
(
1
):
56
. https://doi.org/10.1186/s41200-016-0048-z
Hajibabaei M, Singer GC, Hebert PN, Hickey D (2007)
DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics
.
Trends in Genetics
23
(
4
):
167
‑
172
. https://doi.org/10.1016/j.tig.2007.02.001
Harris SE, Bellino M (2013)
DNA Barcoding from NYC to Belize
.
Science
342
(
6165
):
1462
‑
1463
. https://doi.org/10.1126/science.1230006
Hebert PN, Cywinska A, Ball S, deWaard J (2003)
Biological identifications through DNA barcodes
.
Proceedings of the Royal Society of London. Series B: Biological Sciences
270
(
1512
):
313
‑
321
. https://doi.org/10.1098/rspb.2002.2218
Hebert PN, Gregory TR (2005)
The promise of DNA barcoding for taxonomy
.
Systematic Biology
54
(
5
):
852
‑
859
. https://doi.org/10.1080/10635150500354886
Hultgren K, Hurt C, Anker A (2014)
Phylogenetic relationships within the snapping shrimp genus Synalpheus (Decapoda: Alpheidae)
.
Molecular Phylogenetics and Evolution
77
:
116
‑
125
. https://doi.org/10.1016/j.ympev.2014.03.008
Knowlton N, Weigt LA, Solórzano LA, Mills DK, Bermingham E (1993)
Divergence in proteins, mitochondrial DNA, and reproductive compatibility across the isthmus of Panama.
Science
260
(
5114
):
1629
‑
1632
. https://doi.org/10.1126/science.8503007
Kough A, Paris C, Butler M (2013)
Larval connectivity and the international management of fisheries
.
PLOS One
8
(
6
):
e64970
. https://doi.org/10.1371/journal.pone.0064970
Lassmann T, Sonnhammer E (2005)
Kalign--an accurate and fast multiple sequence alignment algorithm
.
BMC Bioinformatics
6
:
298
. https://doi.org/10.1186/1471-2105-6-298
Leray M, Knowlton N (2015)
DNA barcoding and metabarcoding of standardized samples reveal patterns of marine benthic diversity
.
Proceedings of the National Academy of Sciences
112
(
7
):
2076
‑
2081
. https://doi.org/10.1073/pnas.1424997112
Liu G, Cui X, Li H, Cai L (2016)
Evolutionary direction of processed pseudogenes
.
Science China Life Sciences
59
(
8
):
839
‑
849
. https://doi.org/10.1007/s11427-016-5074-x
Mantelatto F, Terossi M, Negri M, Buranelli R, Robles R, Magalhães T, Tamburus AF, Rossi N, Miyazaki M (2017)
DNA sequence database as a tool to identify decapod crustaceans on the São Paulo coastline
.
Mitochondrial DNA Part A
29
(
5
):
805
‑
815
. https://doi.org/10.1080/24701394.2017.1365848
Mathews L, Anker A (2009)
Molecular phylogeny reveals extensive ancient and ongoing radiations in a snapping shrimp species complex (Crustacea, Alpheidae, Alpheus armillatus)
.
Molecular Phylogenetics and Evolution
50
(
2
):
268
‑
281
. https://doi.org/10.1016/j.ympev.2008.10.026
Matzen da Silva J, Creer S, dos Santos A, Costa A, Cunha M, Costa F, Carvalho G (2011)
Systematic and evolutionary insights derived from mtDNA COI barcode diversity in the Decapoda (Crustacea: Malacostraca)
.
PLOS One
6
(
5
):
e19449
. https://doi.org/10.1371/journal.pone.0019449
Meyer C, Geller J, Paulay G (2005)
Fine scale endemism on coral reefs: archipelagic differentiation in turbinid gastropods
.
Evolution
59
(
1
):
113
‑
125
. https://doi.org/10.1111/j.0014-3820.2005.tb00899.x
Mora C, Tittensor D, Adl S, Simpson AB, Worm B (2011)
How many species are there on earth and in the ocean?
PLOS Biology
9
(
8
):
e1001127
. https://doi.org/10.1371/journal.pbio.1001127
Morín J, Venera-Pontón D, Driskell A, Sánchez J, Lasker H, Collin R (2019)
Reference DNA barcodes and other mitochondrial markers for identifying Caribbean octocorals
.
Biodiversity Data Journal
7
https://doi.org/10.3897/bdj.7.e30970
Morrison CL, Rios R, Emmett Duffy J (2004)
Phylogenetic evidence for an ancient rapid radiation of Caribbean sponge-dwelling snapping shrimps (Synalpheus)
.
Molecular Phylogenetics and Evolution
30
(
3
):
563
‑
581
. https://doi.org/10.1016/s1055-7903(03)00252-5
Naro-Maciel E, Reid B, Holmes K, Brumbaugh D, Martin M, DeSalle R (2011)
Mitochondrial DNA sequence variation in spiny lobsters: population expansion, panmixia, and divergence
.
Marine Biology
158
(
9
):
2027
‑
2041
. https://doi.org/10.1007/s00227-011-1710-y
Puillandre N, Lambert A, Brouillet S, Achaz G (2011)
ABGD, Automatic Barcode Gap Discovery for primary species delimitation
.
Molecular Ecology
21
(
8
):
1864
‑
1877
. https://doi.org/10.1111/j.1365-294x.2011.05239.x
Ratnasingham S, Hebert PDN (2007)
BOLD: the barcode of life data system (http://www.barcodinglife.org)
.
Molecular Ecology Notes
7
(
3
):
355
‑
364
. https://doi.org/10.1111/j.1471-8286.2007.01678.x
Ratnasingham S, Hebert PDN (2013)
A DNA-based registry for all animal species: the barcode index number (BIN) system.
PLOS One
8
(
7
):
e66213
. https://doi.org/10.1371/journal.pone.0066213
Raupach M, Radulovici A (2015)
Looking back on a decade of barcoding crustaceans
.
ZooKeys
539
:
53
‑
81
. https://doi.org/10.3897/zookeys.539.6530
Raupach M, Barco A, Steinke D, Beermann J, Laakmann S, Mohrbeck I, Neumann H, Kihara T, Pointner K, Radulovici A, Segelken-Voigt A, Wesse C, Knebelsberger T (2015)
The application of DNA barcodes for the identification of marine crustaceans from the North Sea and adjacent regions
.
PLOS One
10
(
9
):
e139421
. https://doi.org/10.1371/journal.pone.0139421
Rocha RM, Faria SB, Moreno TR (2005)
Ascidians from Bocas del Toro, Panama. I. Biodiversity
.
Caribbean Journal of Science
41
(
3
):
600
‑
612
.
Schill S, Raber G, Roberts J, Treml E, Brenner J, Halpin P (2015)
No reef is an island: integrating coral reef connectivity data into the design of regional-scale marine protected area networks
.
PLOS One
10
(
12
):
e144199
. https://doi.org/10.1371/journal.pone.0144199
Schizas NV (2012)
Misconceptions regarding nuclear mitochondrial pseudogenes (Numts) may obscure detection of mitochondrial evolutionary novelties
.
Aquatic Biology
17
(
1
):
91
‑
96
. https://doi.org/10.3354/ab00478
Schneider-Broussard R, Neigel JE (1997)
A large-subunit mitochondrial ribosomal DNA sequence translocated to the nuclear genome of two stone crabs (Menippe)
.
Molecular Biology and Evolution
14
(
2
):
156
‑
165
. https://doi.org/10.1093/oxfordjournals.molbev.a025748
Schubart CD (2010)
Mitochondrial DNA and decapod phylogenies: the importance of pseudogenes and primer optimization
.
Decapod Crustacean Phylogenetics. Crustacean Issues
18
:
47
‑
65
. https://doi.org/10.1201/9781420092592-c4
Sluys R (2013)
The unappreciated, fundamentally analytical nature of taxonomy and the implications for the inventory of biodiversity
.
Biodiversity and Conservation
22
(
4
):
1095
‑
1105
. https://doi.org/10.1007/s10531-013-0472-x
Song H, Buhay JE, Whiting MF, Crandall KA (2008)
Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified
.
Proceedings of the National Academy of Sciences
105
(
36
):
13486
‑
13491
. https://doi.org/10.1073/pnas.0803076105
Vences M, Thomas M, van der Meijden A, Chiari Y, Vieites DR (2005)
Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians.
Frontiers in zoology
2
(
1
):
5
. https://doi.org/10.1186/1742-9994-2-5
Vernooy R, Haribabu E, Muller MR, Vogel JH, Hebert PDN, Schindel DE, Shimura J, Singer GAC (2010)
Barcoding life to conserve biological diversity: beyond the taxonomic imperative
.
PLOS Biology
8
(
7
):
e1000417
. https://doi.org/10.1371/journal.pbio.1000417
Wägele H, Klussmann-Kolb A, Kuhlmann M, Haszprunar G, Lindberg D, Koch A, Wägele JW (2011)
The taxonomist - an endangered race. A practical proposal for its survival
.
Frontiers in Zoology
8
(
1
). https://doi.org/10.1186/1742-9994-8-25
Williams ST, Knowlton N (2001)
Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus
.
Molecular Biology and Evolution
18
(
8
):
1484
‑
1493
. https://doi.org/10.1093/oxfordjournals.molbev.a003934
Zheng L, He J, Lin Y, Cao W, Zhang W (2014)
16S rRNA is a better choice than COI for DNA barcoding hydrozoans in the coastal waters of China
.
Acta Oceanologica Sinica
33
(
4
):
55
‑
76
. https://doi.org/10.1007/s13131-014-0415-8

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