In spite of contemporary morphological taxonomy appraisals, apparent high morphological similarity raises uncertainty about the species status of certain Pagurus hermit crabs. This is exemplified between two European species, Pagurus excavatus (Herbst, 1791) and Pagurus alatus (Fabricius 1775), whose species status is still difficult to resolve using morphological criteria alone.
To address such ambiguities, we used combinations of Maximum Likelihood (ML) and Bayesian Inference (BI) methods to delineate species boundaries of P. alatus and P. excavatus and formulate an intermediate Pagurus phylogenetic hypothesis, based upon single and concatenated mitochondrial (cytochrome oxidase I [COI]) and nuclear (16S and 28s ribosomal RNA) gene partitions. The molecular data supported the species status of P. excavatus and P. alatus and also clearly resolved two divergent clades within hermit crabs from the Northeast Atlantic Ocean and the Mediterranean Sea.
Despite the abundance and prominent ecological role of hermit crabs, Pagurus, in North East Atlantic Ocean and Mediterranean Sea ecosystems, many important aspects of their taxonomy, biology, systematics and evolution remain poorly explored. The topologies presented here should be regarded as hypotheses that can be incorporated into the robust and integrated understanding of the systematic relationships within and between species of the genus Pagurus inhabiting the Northeast Atlantic Ocean and the Mediterranean Sea.
Citation: Matzen da Silva J, dos Santos A, Cunha MR, Costa FO, Creer S, Carvalho GR (2011) Multigene Molecular Systematics Confirm Species Status of Morphologically Convergent Pagurus Hermit Crabs. PLoS ONE 6(12): e28233. https://doi.org/10.1371/journal.pone.0028233
Editor: Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, Canada
Received: July 3, 2011; Accepted: November 4, 2011; Published: December 9, 2011
Copyright: © 2011 Matzen da Silva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Fundação para a Ciência e Tecnologia (Portugal) provided a doctoral fellowship (SFRH/BD/25568/2006) to Joana Matzen da Silva. This research was partially supported by the European Centre for Arctic Environmental Research ARCFAC-026129-2008-21 and LusoMarBol FCT research grant PTDC/MAR/69892/2006. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Although hermit crabs are one of the most morphologically and ecologically diverse group of decapod crustaceans, their evolutionary history at many taxonomic levels is far from being resolved –. More than 1000 species, 127 genera and 6 families are currently reported for the superfamily Paguroidea , that inhabit diverse biotopes from intertidal to deep seas . However, the true taxonomic and ecological heterogeneity associated with hermit crabs is likely to be underestimated because many species and life-histories appear to be undescribed . One of the most diverse groups belong to the family Paguridae, with species distributed widely through all oceans .
The genus Pagurus Fabricius, 1775 is considered to be ancient, with fossils being assigned to the genus as early as the lower Cretaceous  and from the Cenozoic . Pagurus is the least complex of all paguroids, sharing a reduced or virtually nonexistent rostrum, no sexually modified appendages other than the regular female egg-bearing pleopods, and having no penis (or sexual tubes) . Despite its comparatively morphological conservatism, Pagurus exhibits a high degree of species proliferation. Recently, 172 species (of which five are fossil records) are recognised  , that possess specialized adaptations for housing stability, relying upon gastropod shells for protection. Such commensalism has constrained morphological evolution over 150 million years ,  by requiring a decalcified asymmetrical abdomen capable of looping into gastropod shells. Despite the abundance of hermit crabs in the North East Atlantic and Mediterranean Sea  many important aspects of their taxonomy , biology –, ecology , –, systematics and evolution , ,  are poorly documented.
Systematic problems remain among the Paguridae, such as the polyphyletic genus Pagurus Fabricius, 1775 . To date, most systematic studies on hermit crabs have been based on morphology with relatively few studies utilising molecular tools to resolve species status , – or to determine phylogenetic relationships among major taxa , , . Within the region of the Northeast Atlantic Ocean and Mediterranean Sea Pagurus is represented by 23 species  and high morphological similarity among some species has resulted in the recognition of two groups characteristic of the North Atlantic and Mediterranean Sea . In some cases, morphology suggests very close relationships between congeners, raising uncertainty about their independent species status. Such a situation exists between the “alatus” group, Pagurus excavatus (Herbst, 1791) and Pagurus alatus (Fabricius, 1775) that is still difficult to resolve using morphology alone (Table 1) , . In spite of the prevailing taxonomic challenges, Ingle  has recognized both species, based mainly from the differences observed in the dorsal aspects of shield, antennular peduncle, the shape of the right cheliped, the length of larger pereiopod and male pleopods. Furthermore, due to a lack of life history studies there has always been considerable confusion regarding ongoing synonymies that are assigned among P. alatus, P. excavatus and P. variabilis (A. Milne-Edwards & Bouvier, 1892) , . As currently recognised, P. excavatus is distributed southwards from the southern part of the Bay of Biscay and into the Mediterranean Sea. Pagurus alatus extends northwards into Iceland waters, but remains sympatric with P. excavatus in many southern regions of the area . Here, we use molecular phylogenetic analyses of mitochondrial cytochrome oxidase I (COI), mitochondrial 16S ribosomal RNA (16S), and nuclear 28S ribosomal RNA (28S) DNA partitions to reconstruct the systematics of selected Pagurus species, in order to make inferences on taxonomic status of P. alastus and P. excavatus.
The variation in COI diversity was examined among 11 species (Table 2 and 3). For each species, one to six representative individuals were analysed (Table 4), and where possible, from different geographical areas, yielding a total of 46 sequences. No insertions, deletions, stop codons or sequences indicative of pseudogenes ,  were observed, and BLAST searches confirmed that the sequences corresponded to decapod mtDNA COI. There was also evidence for base composition bias in the sequences, notably a pronounced underrepresentation of guanine at the third codon positions (35.3% T; 18.1% C; 28.5% A; 18.1%G) a phenomenon commonly observed in metazoan mitochondrial . The COI alignment contained a total of 513 bp with 177 variable characters, of which 170 were parsimony informative (33.13%). The high observed percentage of parsimony-informative character suggests that COI is sufficiently diverse for intrageneric phylogeny and clearly resolved all eleven Pagurus species examined in the present study (Figure 1) , . The 11 species comprise a monophyletic clade (Figure 1) with an average between genetic species distance of 17.01% (Table 2). Among the Pagurus species, P. acadianus exhibits the least genetic divergence to P. bernhardus (6.80%) and in contrast, P. arcuatus and P. excavatus exhibited the highest genetic distances (23.10%) (Table 3). P. pubescens exhibited the highest average distance values (Table 3) with a range of 11.50–21.70% (see also Figure 1).
The numbers on branches are ML bootsrap values and posterior probabilities of BI<50% (ML/BI percentages values, respectively). Each oceanographic region/specimens are defined: Northeast Atlantic Ocean (NEA), Northwest Atlantic Ocean (NWA), North Atlantic Ocean (NA), Mediterranean Sea (MED) and Bering Sea (BER) (see Table 4 for complement information).Two major clades have been roman number-coded, I and II: represent two groups defined previously by Ingel (1985) based on adult and larvae morphological characters.
Overall, the phylogenetic algorithms (ML, BI) resulted in congruent topologies, delimiting the designated true Pagurus species (Figure 1). The resulting molecular phylogeny agrees in several respects with the current morphologically based classification of all species. All analyses support the basal placement of P. longicarpus and identify two main clades (Figure 1). In contrast, the relationships among inner clades of Pagurus were poorly resolved. Clade I is represented by four the Northeast Atlantic Ocean and Mediteranean Sea species and clade II by six species of the North Atlantic Ocean (East and West coasts) and Bering Sea specimens.
Molecular systematic assignments
The three independent genes revealed concordant phylogenetic differences between P. alatus and P.excavatus. Pagurus alatus is substantially divergent from P. excavatus, with a mean divergence of 14.9% and 5.1% for COI and 16S sequences respectively (Table 5). The 16S alignment was more conserved than the COI partition yielding 84/462 variable characters, of which 75/462 were parsimony informative. The 16S sequences are AT-rich (71.86%), indicating a moderate compositional bias. The pattern of nucleotide substitution was also biased in favour of transitions over transversions, yielding a ts∶tv = 1.1 and for 28S a ts∶tv = 3.2.
In the BI analysis, systematic positions of five species were not stable (Figure 2 A), but the trees underpinned by the 16S, 28S and the concatenated data partitions were broadly congruent with the COI hypothesis (Figure 1). The combined molecular analysis, based on strong posterior probabilities values (100%; in Figure 2, CON), clearly reveals the two independent lineages here discussed.
Systematics work to date in Pagurus has primarily been in the area of morphological taxonomy, with few hypotheses presented regarding species relationships. Generating such inferences has been difficult because of generally remarkable similarities in morphology among members of the genus. These similarities are perhaps surprising given the degree of the genetic divergence observed in this study between the most phenotypically closest species, P. alatus and P. excavatus (14.8%). A similar pattern has been observed in penaied shrimps , porcelanids crabs , diogenid hermit crabs  and has been attributed to stabilizing selection on morphological/ecological characters, or that they are on independent evolutionary trajectories. Likewise, strong stabilizing selection acting on morphological differentiation has been associated with life as a commensal of thallassinidean shrimps, accompanied by neutral molecular divergence .
The smallest mean intraspecific divergence values observed (Table 3) are possibly underestimated, because samples were obtained from a single locality. Global-scale phylogeography surveys of COI sequence diversity have estimated average intraspecific diversity values of less than 1% within crustaceans, whereas interspecific values typically are greater than 4% among congeneric species – and especially among decapods that can exhibit congeneric divergence values greater than 15% . Elsewhere, five species of the genus Pagurus from Sea of Japan exhibited lower levels of genetic identity when compared with the genera Metapenaeus and Penaeus . Here, the high genetic diversity observed among the pagurid species is in line with the observed morphological variability in informal morphological groups among adults and larvae described by Ingle (1985) and McLaughlin and Gore (1988), respectively.
Ingle  delineated two main groups of Northeast Atlantic Ocean and Mediterranean Sea Pagurus based on three adults, and additional larval morphological characters  that are fully congruent with the current molecular systematic analysis. Furthermore, P. armatus, P. ochotensis and P. bernhardus that were assumed to be most related, based on morphology , share the most basal position in clade II. In addition, all species from clade I and II agree with two distinct larvae groups described by McLaughlin and Gore  based on the characteristics and species assigned. The inconsistent phylogenetic position of P. bernhardus, observed in Mantelatto et al.  was still unresolved here (Figure 1), represented by the weak support of the two focal nodes (<50% bootstrap support) within clade II. However, the observed COI molecular divergence does support a sister group relationship between P. acadianus and P. bernhardus, a taxonomic relationship derived also from morphological comparisons , . In summary, the phylogenetic patterns observed for Pagurus are consistent with morphological groups established by Ingle  and McLaughlin and Gore  but further analyses would be required to establish the precise cladistic relationships within the genus as a whole.
Molecular systematic assignments
Concordance across molecular and morphological characters provides a reliable indicator of longstanding evolutionary independence and consequently provides an operational criterion for species recognition . In the present study, morphological differences between P. alatus and P. excavatus were evident when two morphological variants were compared directly (Table 1), supporting molecular findings that justify separation at the species level. It is important to mention that a restricted collection from one biogeographical region of a species with a wide distribution cannot represent the complete range of morphological variation that is often found in decapods , , . We also know that phenotypic plasticity in morphological traits (especially organisms with commensal behaviours) might be strongly influenced by environmental factors between different areas –. The precise cladistic relationships among the five species were not stable (Figure 2, A), most likely due to lower taxon sampling and also the inability of COI to accurately resolve deep nodes. However, the combined molecular analysis (Figure 2, CON) clearly reveals the evidence for long-standing evolutionary independence between P. excavatus and P. alatus.
Molecular data have not been used before to investigate systematic relationships among Pagurus of the Northeast Atlantic Ocean and Mediterranean Sea. Despite the perceived limitations regarding the use of morphological characters for inferring evolutionary relationships among commensal species, our molecular data support the morphological taxonomy. Our data may indicate the possible existence of two monophyletic groups (clade I and II), supporting previous assertions based on larval and adult morphological criteria. However, the current data confirm the complexity of the relationships within Pagurus, highlighting the absence of complete and integrated morphological descriptions for the diverse and heterogeneous members of the genus. Since the present taxonomic and geographic coverage is incomplete, the topologies presented here should be regarded as working hypotheses. Pagurus have diversified into a wide variety of marine habitats and exemplify classic commensal, anti-predator evolutionary traits. Thus, the group provides an excellent model for studying the interplay between speciation, neutral molecular divergence and potential stabilising selection on body form.
Materials and Methods
Twenty nine hermit crabs of six species, Pagurus alatus (Fabricius, 1775), P. bernhardus (Linnaeus, 1758), P. cuanensis (Bell, 1845), P. excavatus (Herbest, 1791), P. prideaux (Leach, 1815), and P. pubescens (Krøyer, 1838), were collected from the Portugal (Costa Algarvia, Costa de Prata, and Azores), United Kingdom (North Wales), Norway (Bear Island Slide and Svalbard) and Italy (Sicily) between 2006 and 2008. Specimens were harvested as by-catch from rough ground bottom trawls from INRB-IPIMAR, Sicilian fisheries survey and collected by physical searches. Species were identified (Ingle, 1985) prior to the excision and preservation of muscle tissue in 95% ethanol (stored at −20°C) and whole body storage at −20°C. In order to accurately assign specimens to currently accepted (female) species of P. alatus and P. excavatus we used morphological criteria based upon four main groups of characters: the dorsal aspects of shield (Table 1), antennular peduncle, the shape of the right cheliped, length of larger pereiopod and male pleopods (Ingle 1985).
DNA isolation, amplification and sequencing
Total genomic DNA was extracted from approximately 1 mm3 of muscle tissue or whole legs for small specimens using the Chelex dry release method . The COI gene was amplified with alternative sets of primers depending on PCR reaction success following by the protocol develop by Costa et al. . All PCRs were performed in a 25 µl final volume containing 1× PCR buffer, 3–4 mM MgCl2, 0.1–0.3 mM dNTP, 1 U Taq polymerase, 5–10 pmol of each primer, and 2–10 ng of DNA template (Table 6). The thermal cycling conditions are listed in Table 6 for each set of primers. Following amplification, PCR reactions were cleaned by incubation with 10 U Exonuclease I (New England Biolabs ®) and 1 U Shrimp Alkaline Phosphate (Promega ®) at 37°C for 1 hour, followed by heating at 80°C for 5 min. Samples were sequenced by Macrogen Inc. (South Korea) using an Applied Biosystems® 3730 sequencer.
All empirically derived sequences were manually checked for ambiguities in CodonCode Aligner version 1.3.0, aligned using the ClustalX plugin embedded within Mega 4 , prior to manual quality control and megablast annotation.
The most popular barcode marker COI is generally used to study close to moderately deep interspecific taxon relationships of crustaceans –. To provide a comprehensive sister-species coverage and assessment of interspecific variation, Pagurus COI sequences from GenBank were merged with our data (Table 4). In that propose we use using Kimura 2-parameters (K2P) genetic distances within and among species implemented in Mega 4.1, and compared to literature data. Amino acid translations of the target genes were examined to ensure that no gaps or stop codons were present in the alignment.
To identify phylogenetic groups among the resulting eleven putative Pagurus species, the 46 COI sequences comprising 513 bp was analyzed using Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic reconstruction methods. The crab Macropodia longipes (Milne-Edwards & Bouvier, 1899) (Brachyura:Inachidae) and the most phylogenetically closest related species, Dardanus arrosor (Herbst, 1796) (Anomura: Diogenidae) were used as outgroups.
Since Bayesian posterior probability support values (bpp) can often be inflated for certain clades, relative to ML bootstrap values , we constructed trees using both Bayesian approaches and ML. For these searches, we set the substitution model parameters calculated by jModeltest in RAxML 7.0.4  and in MrBayes 3.1 , respectively. Ten independent ML analyses were conducted using GTR+I+G with invariant sites (I) and gamma distributed rates (G)  (see below) as the model (using the GTR+CAT setting) with 4 categories of rate variation (500 bootstrap replicates were undertaken for estimation of node support) for each partition on combined data. In order to find the ML tree, 10 independent runs of RAxML 7.0.4 were conducted. ModelTest  identified the HKY+I+G model  as best indicated by Akaike Information Criterion (AIC) , however, since this model is not implemented in the current version of RAxML, the GTR+I+G model was selected as the closest matching alternative. In MrBayes, two independent Markov chain Monte Carlo (MCMC) analyses were run using four chains for 5×106 generations with the initial 1 million generations (20%) cycles discarded as burn-in. To check that stationarity had been reached, we monitored the fluctuating value of the likelihood graphically with Tracer v1.4 . Once the parameters reach stationarity, a 50% majority rule consensus tree was obtained from the remaining saved trees. The consensus tree was selected from the posterior distribution and visualized using FigTree V.1.0 (http://tree.bio.ed.ac.uk/software/figtree/). Since substitution rates among the four nucleotides and among different nucleotide sites in mitochondrial protein-coding genes as been reported –, codon models of substitution TrNef+G , F81  and HKY+G  were implemented in our BI analyses for first, second and third codon positions respectively.
Molecular systematic analyses
Use of nuclear genes in addition to mitochondrial genes adds to the number of independent markers in a dataset, thus increasing the chances to understand the systematic relationships between and within P. alatus and P. excavatus (Table 7). Here we analysed partial sequences of nuclear 28S (385 bp), mitochondrial 16S (462 bp), and the barcode region of COI (540 bp). The three gene regions were partitioned separately according to the previously determined model parameters (Table 8) in subsequent BI analyses. Gaps in 16S and 28S sequences were treated as a fifth character-state. BI analysis was conducted for each gene data set and the concatenated partition (CON) with the three gene regions partitioned separately according to the previously determined model parameters (Table 8) as described before. To evaluate the range of intrageneric sequence identity found among Pagurus species, we compared pairwise distances of COI and 16S (Table 5).
Thanks are due to many colleagues and their respective institutions (Dr. Markos Aleksandro, Axel Barlow, Dr Nia Whiteley, Dr. Simon Webster, and Wendy Grail from Bangor University; Dr. Ana Cristina Costa and João Brum from Azores University; Dr. Debbie Bailie from Queens University; Dr. Marco Arculeo from University of Palermo; and Dr. Maria Włodarska-Kowalczuk from Institute of Oceanology PAS from Sopot) that collaborated through this study by making available some essential specimens, literature and sharing interesting scientific discussions. We thank Dr. Judite Alves and Alexandra Cartaxana for archiving and being responsible for the Crustacean collection in the Natural and History Museum of Portugal.
Conceived and designed the experiments: JMdS AdS. Performed the experiments: JMdS. Analyzed the data: JMdS AdS. Contributed reagents/materials/analysis tools: JMdS AdS MRC FOC SC GRC. Wrote the paper: JMdS SC GRC.
- 1. McLaughlin PA (2003) Illustrated keys to families and genera of the superfamily Paguroidea (Crustacea: Decapoda: Anomura), with diagnoses of genera of Paguridae. Memoirs of Museum Victoria 60: 111–144.
- 2. McLaughlin PA, Lemaitre R, Sorhannus U (2007) Hermit crab phylogeny: a reappraisal and its “fall-out”. Journal of Crustacean Biology 27: 97–115.
- 3. McLaughlin PA, Lemaitre R (1997) Carcinization in the Anomura - fact or fiction? I. Evidence from adult morphology. Contributions to Zoology 67: 79–123.
- 4. Mantelatto FL, Pardo LM, Pileggi LG, Felder DL (2009) Taxonomic re-examination of the hermit crab species Pagurus forceps and Pagurus comptus (Decapoda: Paguridae) by molecular analysis. Zootaxa 2133: 20–32.
- 5. De Grave S, Pentcheff ND, Ahyong ST, Chan T-Y, Crandall KA, et al. (2009) A classification of living and fossil genera of decapod Crustaceans. Raffles Bulletin of Zoology 1: 1–109.
- 6. d'Udekem d'Acoz C (1999) Inventaire et distribution des crustacés décapodes de l'Atlantique nord - oriental, de la Méditerranée et des eaux continentales adjacentes au nord de 25°N. Paris: Collection Patrimoines Naturels. 383 p.
- 7. Cunningham CW, Blackstone NW, Buss LW (1992) Evolution of king crabs from hermit crab ancestors. Nature 355: 539–542.
- 8. Jagt JWM, van Bakel BWM, Fraaije RHB, Neumann C (2003) In situ fossil hermit crabs (Paguroidea) from northwest Europe and Russia. Preliminary data on new records. Revista Mexicana de Ciencias Geológicas 23: 364–369.
- 9. McLaughlin PA, Komai T, Lemaitre R, Rahayu DL (2010) Annotated checklist of anomuran decapod crustaceans of the world (exclusive of the Kiwaoidea and families Hirostylidae and Galatheidae of the Galathedoidea) Part I - Lithoidea, Lomisoidea and Paguroidea. The Raffles Bulletin of Zoology 23: 5–107.
- 10. Ingle RW (1985) Northeastern Atlantic and Mediterranean hermit crabs (Crustacea: Anomura: Paguroidea: Paguridae). The genus Pagurus Fabricius, 1775. Journal of Natural History 19: 745–769.
- 11. Neil SJ, Elwood RW (1985) Behavioural modifications during egg-brooding in the hermit crab, Pagurus bernhardus L. Journal of Experimental Marine Biology and Ecology 94: 99–114.
- 12. Appel M, Elwood RW (2009) Gender differences, responsiveness and memory of a potentially painful event in hermit crabs. Animal Behaviour 78: 1373–1379.
- 13. Jackson NW, Elwood RW (1989) How animals make assessments: information gathering by the hermit crab Pagurus bernhardus. Animal Behaviour 38: 951–957.
- 14. Jackson NW, Elwood RW (1989) Memory of information gained during shell investigation by the hermit crab, Pagurus bernhardus. Animal Behaviour 37: 529–534.
- 15. Elwood RW, Glass CW (1981) Negotiation or aggression during shell fights of the hermit crab Pagurus bernhardus? Animal Behaviour 29: 1239–1244.
- 16. Elwood RW, Stewart A (1985) The timing of decisions during shell investigation by the hermit crab, Pagurus bernhardus. Animal Behaviour 33: 620–627.
- 17. Lancaster I (1988) Pagurus bernhardus (L.) - an introduction to the natural history of hermit crabs. Field Studies 7: 189–238.
- 18. Rivera JM (2006) Morphology of the first zoea of the hermit crab Pagurus edwardsi (Dana, 1852), (Decapoda: Anomura: Paguridae) obtained in the laboratory. Revista de Biolgía Marina y Oceanografia 41: 239–244.
- 19. Kellogg CW (1976) Gastropod shells: A potentially limiting resource for hermit crabs. Journal of Experimental Marine Biology and Ecology 22: 101–111.
- 20. Taylor PR (1982) Environmental resistance and the ecology of coexisting hermit crabs: Thermal tolerance. Journal of Experimental Marine Biology and Ecology 57: 229–236.
- 21. Taylor PR (1981) Hermit crab fitness: The effect of shell condition and behavioral adaptations on environmental resistance. Journal of Experimental Marine Biology and Ecology 52: 205–218.
- 22. Ellis JR, Rogers SI, Freeman SM (2000) Demersal assemblages in the Irish Sea, St George's channel and Bristol channel. Estuarine, Coastal and Shelf Science 51: 299–315.
- 23. Young AM, Torres C, Mack JE, Cunningham CW (2002) Morphological and genetic evidence for vicariance and refugium in Atlantic and Gulf of Mexico populations of the hermit crab Pagurus longicarpus. Marine Biology 140: 1059–1066.
- 24. Mantelatto FL, Robles R, Biagi R, Felder DL (2006) Molecular analysis of the taxonomic and distributional status for the hermit crab genera Loxopagurus Forest, 1964 and Isocheles Stimpson, 1858 (Decapoda, Anomura, Diogenidae). Zoosytema 28: 495–506.
- 25. Zaslavskaya NI, Kornienko ES, Korn OM (2009) Genetic similarity between Pagurus gracilipes (Stimpson, 1858) (Decapoda: Paguridae) and another pagurid and diogenid hermit crabs from Russion waters of the sea of Japan. Invertebrate Zoology 6: 147–155.
- 26. Scelzo MA, Fantucci MZ, Mantelatto FL (2010) Spermatophore and Gonopore Morphology of the Southwestern-Atlantic Hermit Crab Pagurus exilis (Benedict, 1982) (Anomura, Paguridae). Zoological Studies 49: 421–433.
- 27. Spivak ED, Schubart CD (2003) Species status in question: a morphometric and molecular comparison of Cyrtograpsus affinis and C. altimanus (Decapoda, Brachyura, Varunidae). Journal of Crustacean Biology 23: 212–222.
- 28. Schubart CD (2009) Mitochondrial DNA and decapod phylogenies; the importance of pseudogenes and primer optimization. In: Martin JW, Crandall KA, Felder DL, editors. Decapod Crustacean Phylogenetics. New York: Taylor and Francis Group. pp. 47–65.
- 29. 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. PNAS 105: 13486–13491.
- 30. Wolstenholme DR (1992) Animal mitochondrial DNA: structure and evolution. International Review of Cytology 141: 173–216.
- 31. Cruickshank RH (2002) Molecular markers for the phylogenetics of mites and ticks. Systematic and Applied Acarology 7: 3–14.
- 32. Schwentner M, Timms BV, Bastrop R, Richter S (2009) Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers – An Australian origin for Limnadopsis. Molecular Biology and Evolution 53: 716–725.
- 33. Palumbi SR, Benzie J (1991) Large mitochondrial differences between morphologically similar penaeid shrimp. Molecular Marine Biology and Biotechnology 1: 27–34.
- 34. Hiller A, Kraus H, Almon M, Werding B (2006) The Petrolisthes galathinus complex: Species boundaries based on color pattern, morphology and molecules, and evolutionary interrelationships between this complex and other Porcellanidae (Crustacea: Decapoda: Anomura). Molecular Phylogenetics and Evolution 40: 547–569.
- 35. Malay MCMD, Paulay G (2009) Peripatric speciation drives diversification and distributional pattern of reef hermit crabs (Decapoda: Diogenidae: Calcinus). Evolution 64: 634–662.
- 36. Harrison JS (2004) Evolution, biogeography, and the utility of mitochondrial 16S and COI genes in phylogenetic analysis of the crab genus Austinixa (Decapoda: Pinnotheridae). Molecular Phylogenetics and Evolution 30: 743–754.
- 37. Costa FO, deWaard JR, Boutillier J, Ratnasingham S, Dooh RT, et al. (2007) Biological identifications through DNA barcodes: the case of the Crustacea. Canadian Journal of Fisheries and Aquatic Sciences 64: 272–295.
- 38. Radulovici AE, Sainte-Marie B, Dufresne F (2009) DNA barcoding of marine crustaceans from the Estuary and Gulf of St Lawrence: a regional-scale approach. Molecular Ecology Resources 9: 181–187.
- 39. Lefébure T, Douady CJ, Gouy M, Gibert J (2006) Relationship between morphological taxonomy and molecular divergence within Crustacea: Proposal of a molecular threshold to help species delimitation. Molecular Phylogenetics and Evolution 40: 435–447.
- 40. Matzen da Silva J, Creer S, Dos Santos A, Costa AC, Cunha MR, et al. (2011) Systematic and evolutionary insights derived from mtDNA COI barcode diversity in the Decapoda (Malacostraca). PLoS ONE 6: e19449.
- 41. McLaughlin PA, Gore RH (1988) Studies on the Provenzanoi and other pagurid groups: I. The larval stages of Pagurus maclaughlinae García-Gómez, 1982 (Decapoda: Anomura: Paguridae), reared under laboratory conditions. Journal of Crustacean Biology 8: 262–282.
- 42. d'Udekem d'Acoz C, Pinho MR, Melo O, Gonçalves J, Martins H (2001) New records and notes on little know shrimps (Crustacea, decapoda) from Azorean Waters. Arquipélago 18A: 61–64.
- 43. Avise JC, Wollenberg K (1997) Phylogenetics and the origin of species. PNAS 94: 7748–7755.
- 44. Cuesta JA, Schubart CD (1998) Morphological and molecular differentiation between three allopatric populations of the littoral crab Pachygrapsus transversus (Gibbes, 1850) (Brachyura: Grapsidae). Journal of Natural History 32: 1499–1508.
- 45. Agrawal AA (2001) Phenotypic plasticity in the interactions and evolution of species. Science 294: 321–326.
- 46. Price TD, Qvarnstrom A, Irwin DE (2003) The role of phenotypic plasticity in driving genetic evolution. Proc R Soc Lond B 270: 1433–1440.
- 47. Brian JV, Fernandes T, Ladle RJ, Todd PA (2006) Patterns of morphological and genetic variability in UK populations of the shore crab, Carcinus maenas Linnaeus, 1758 (Crustacea: Decapoda: Brachyura). Journal of Experimental Marine Biology and Ecology 329: 47–54.
- 48. Hajibabaei M, DeWaard JR, Ivanova NV, Ratnasingham S, Dooh RT, et al. (2005) Critical factors for assembling a high volume of DNA barcodes. Philosophical Transactions of the Royal Society B-Biological Sciences 360: 1959–1967.
- 49. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA 4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596–1599.
- 50. Mathews LM, 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: 268–281.
- 51. Shih H-T, Ng PKL, Schubart CD, Chang H-W (2007) Phylogeny and phylogeography of the genus Geothelphusa (Crustacea: Decapoda, Brachyura, Potamidae) in southwestern Taiwan based on two mitochondrial genes. Zoological Science 24: 57–66.
- 52. Schubart CD, Cuesta JA, Felder DL (2005) Phylogeography of Pachygrapsus transversus (Gibbes, 1850): The effect of the American continent and the Atlantic Ocean as gene flow barriers and recognition of Pachygrpsus socius Stimpson 1871 as a valid species. Nauplius 12: 99–113.
- 53. von Rintelen K, von Rintelen T, Glaubrecht M (2007) Molecular phylogeny and diversification of freshwater shrimps (Decapoda, Atyidae, Caridina) from ancient Lake Poso (Sulawesi, Indonesia) - The importance of being colourful. Molecular Phylogenetics and Evolution 45: 1033–1041.
- 54. Groeneveld JC, Gopal K, George RW, Matthee CA (2007) Molecular phylogeny of the spiny lobster genus Palinurus (Decapoda: Palinuridae) with hypotheses on speciation in the NE Atlantic/Mediterranean and SW Indian Ocean. Molecular Phylogenetics and Evolution 45: 102–110.
- 55. Huelsenbeck JP, Rannala B (2004) Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Systematic Biology 53: 905–913.
- 56. Stamatakis A (2008) The RAxML. 7.0.4 ed. Munchen: The Exelixis Lab, Department of Computer Science.
- 57. Huelsenbech JP, Ronquist F (2001) Mr Bayes:Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755.
- 58. Yang ZH (1994) Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: Approximate methods. Journal of Molecular Evolution 39: 306–314.
- 59. Posada D (2008) jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25: 1253–1256.
- 60. Hasegawa M, Kishino K, Yano T (1985) Dating the human-ape splitting by a molecular clock of mitochondrial DNA. Journal Molecular Evolution 22: 160–174.
- 61. Akaike H (1973) Information theory and an extension of maximum likelihood principle. In: Petrov BNaC F, editor. Second Internationall Symposium on Information Theory. Budapest Akademiai Kiado. pp. 267–281.
- 62. Drummond AJ, Rambaut A (2007) A beast: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 1–8.
- 63. Kumar S (1996) Patterns of nucleotide substitution in mitochondrial protein coding genes of vertebrates. Genetics 143: 537–548.
- 64. Yang ZH, Yoder AD (1999) Estimation of the transition/transversion rate bias and species sampling. Journal of Molecular Evolution 48: 274–283.
- 65. Yang Z, RN , Nielsen R, Goldman N, Pedersen AMK (2000) Codon-Substitution Models for Heterogeneous Selection Pressure at Amino Acid Sites. Genetics 155: 431–449.
- 66. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10: 512–526.
- 67. Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal Molecular Evolution 17: 368–376.
- 68. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytrocrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.
- 69. Mathews LM, Schubart CD, Neigel JE, Felder DL (2002) Genetic, ecological, and behavioural divergence between two sibling snapping shrimp species (Crustacea: Decapoda:Alpheus). Molecular Ecology 11: 1427–1437.
- 70. Schubart CD, Neigel JE, Felder DL (2000) Use of the mitochondrial 16S rRNA gene for phylogenetic and population studies of Crustacea. Crustacean Issues 12: 817–830.
- 71. Whiting MF (2002) Mecoptera is paraphyletic: multiple genes and phylogeny of Mecoptera and Siphonaptera. Zoologica Scripta 31: 93–104.