Molecular phylogeny of porcelain crabs (Porcellanidae: Petrolisthes and allies) from the south eastern Pacific: the genera Allopetrolisthes and Liopetrolisthes are not natural entities

Introduction

Among the Decapoda, crabs from the infraorder Anomura MacLeay, 1838 are renowned for their astounding anatomical, ecological, and behavioral diversity (McLaughlin et al., 2010; Osawa & McLaughlin, 2010; Tudge, Asakura & Ahyong, 2012). During the last decade, various phylogenetic studies have supported monophyly of the Anomura, clarified the position of this clade relative to other decapod lineages, and revealed internal relationships (Porter, Perez-Losada & Crandall, 2005; Ahyong, Schnabel & Maas, 2009; Bracken-Grissom et al., 2013). Recent studies also have uncovered an evolutionary history much more complex than originally recognized (Schnabel, Ahyong & Maas, 2011; Bracken-Grissom et al., 2013). Furthermore, some systematic studies, combined with behavioral and ecological observations, have exposed the evolutionary basis for most peculiar behaviors and the conditions favoring them (territoriality and vicious agonistic behaviors in Allopetrolisthes spinifrons, living in symbiosis with sea anemones—Baeza, Thiel & Stotz, 2001; Baeza, Stotz & Thiel, 2002; colonization of hydrothermal vents and unique feeding behavior and associated body parts such as bacterophorian setae in the ‘yeti crab’ Kiwa hirsuta—Macpherson, Jones & Segonzac, 2006; Goffredi et al., 2008; multiple transitions to crab-like forms from hermit crab ancestors—Tsang et al., 2011). Our knowledge of the evolutionary history of anomuran crabs has increased substantially; nevertheless, the internal relationships between many genera and families still remain unknown.

Among anomuran crabs of the superfamily Galatheoidea Samouelle, 1819, one of the most species-rich clades of anomurans, the family Porcellanidae Haworth, 1825, is of particular interest. Crabs from the family demonstrate a considerable diversity of lifestyles, body sizes, habitats, and coloration. More than 280 recognized species (Osawa & McLaughlin, 2010; Osawa & Uyeno, 2013; Werding & Hiller, 2015) inhabit intertidal or shallow subtidal, cold-, warm-temperate, subtropical, and tropical rocky and coral reefs. Some species live in large aggregations, whereas, others remain solitarily within shelters (Antezana, Fagetti & López, 1965; Viviani, 1969; Baeza & Stotz, 1995). Species with cryptic coloration usually dwell under rocks or in crevices, but other, more colorful species inhabit sea anemones in shallow temperate or tropical reefs (Antezana, Fagetti & López, 1965; Baeza, Thiel & Stotz, 2001). Some colorful species are traded in the marine aquarium industry (e.g., Porcellana sayana—Baeza et al., 2013). The ecological disparity of crabs from this family has already attracted the attention of systematists (Werding, Hiller & Misof, 2001; Hiller et al., 2006; Rodríguez, Hernández & Felder, 2006; Miranda, Schubart & Mantelatto, 2014), evolutionary ecologists (Baeza & Thiel, 2003; Baeza & Asorey, 2012), and ecophysiologists (Gebauer, Paschke & Anger, 2010; and references therein). The same diversity suggests that these crabs are ideal model systems to explore the role of environmental conditions in explaining evolutionary innovations in the marine environment. Phylogenetic studies in the family Porcellanidae are warranted because of the implications for evolutionary ecology, conservation biology, and biodiversity.

In the family Porcellanidae, the genus Petrolisthes (Stimpson, 1858), was originally established to contain various species of porcelain crabs characterized by, among other traits, a rounded or subquadrate carapace (usually about as broad as long), a triangular or trilobate front often prominent and produced beyond the eyes, a basal segment of the antenna not produced forward to meet the anterior margin of the carapace, either not produced inward, or with a distinct in-ward projection forming a partial suborbital margin, ambulatory legs (pereopods) of moderate length with the propodus bearing movable spinules on the posterior margin and with the dactylus ending in a simple spine, and a telson almost invariably composed of seven plates (Stimpson, 1858; Haig, 1955). The morphology and taxonomic terminology for the group is shown in Fig. S1.

Later, Haig (1960) established three new genera, Allopetrolisthes, Liopetrolisthes, and Clastotoechus for a few of the ‘most aberrant’ species within the genus Petrolisthes. The combination of characters setting the genus Liopetrolisthes, including the type species L. mitra, apart from the closely related Petrolisthes, Allopetrolisthes, and Clastotoechus, includes a carapace subovate and slightly longer than broad, a front trilobated and strongly produced beyond the eyes, a basal antennal segment lacking a strong anterior projection in contact with the carapace margin, chelipeds small and flattened in relation to the carapace and with the carpus armed with prominent teeth on the anterior margin, and a telson composed of five plates (also see Weber, 1991). In turn, Allopetrolisthes differs from species belonging to the remaining closely related genera in exhibiting a combination of the following traits: a carapace rounded and approximately as broad as long, a trilobate front sometimes with two supplementary smaller lobes, a weak anterior projection of the basal antennal segment, which slightly excludes the movable segments from the orbit, a dactylus of the ambulatory legs very short and with posterior movable spinules absent or greatly reduced in size, and a telson composed of five plates (Haig, 1960).

Haig’s (1960) suggestion was followed by scientists throughout the 20th century, and her view has been supported by recent taxonomical studies and the list of porcellanid species from the world (cf. Osawa & McLaughlin, 2010). On the other hand, based on molecular characters (i.e., a fragment of the 16S mitochondrial rRNA gene), both Stillman & Reeb (2001) and Rodríguez, Hernández & Felder (2006) have shown that the genus Petrolisthes, as currently recognized, is paraphyletic on the basis of the nested positions of members from the genera Allopetrolisthes and Liopetrolisthes, among a few others (i.e., Clastotoechus, Megalobrachium, and Parapetrolisthes). Similarly, the studies of larval characters also suggest that the genus Petrolisthes is paraphyletic and can be subdivided in various natural entities (Osawa, 1995; Wehrtmann et al., 1996; Hernández, 1999; Fujita, Shokita & Osawa, 2002; Hernández & Magán, 2012). Certainly, additional taxonomic studies are needed to resolve outstanding systematic problems within the family Porcellanidae (Hiller et al., 2006).

This study represents a contribution to the phylogeny of crabs from the genus Petrolisthes and two of its closely allied genera (i.e., Allopetrolisthes, Liopetrolisthes) restricted to the temperate south eastern Pacific (Fig. 1). I have focused specifically on addressing the hypothesis of monophyly of the three genera above. It was predicted that a molecular phylogeny of the species included within the three genera should segregate the species into three well-supported monophyletic clades. Based upon the large-subunit, 16S mitochondrial rRNA and the Histone 3 [H3] nuclear DNA, a molecular phylogeny of the species native to the temperate south eastern Pacific is presented in order to examine the hypothesis above.

Material and Methods

Hypotheses testing of monophyletic clades

I tested if the different species of the genera Petrolisthes, Liopetrolisthes and Allopetrolisthes segregated and formed different genus-specific monophyletic clades. For this purpose, a constrained tree (in which the monophyly of all three genera was enforced) was obtained in MrBayes with the command constraint. MCMC searches were run and the harmonic mean of the tree-likelihood value was obtained by sampling the post burn-in, posterior distribution as above. Next, Bayes factors were used to evaluate whether or not there was evidence against monophyly (constrained versus unconstrained trees) according to the criteria of Kass & Raftery (1995). Bayes factors compare the total harmonic mean of the marginal likelihood of unconstrained vs. monophyly-constrained models. A higher value of the Bayes factor statistic implies stronger support against the monophyly of a particular group (Kass & Raftery, 1995). Specifically, a value for the test statistic 2 log_e(B₁₀) between 0 and 2 indicates no evidence against H₀; values from 2 to 6 indicate positive evidence against H₀; values from 6 to 10 indicate strong evidence against H₀; and values >10 indicate very strong evidence against H₀ (Kass & Raftery, 1995; Nylander et al., 2004).

Results

The final molecular data matrix was comprised of a total of 690 characters, of which 133 of them were parsimony informative, for a total of 11 ingroup species from the south eastern Pacific pertaining to the genera Petrolisthes, Allopetrolisthes, and Liopetrolisthes and 4 outgroup terminals. Both ‘total evidence’ molecular phylogenetic trees obtained with different inference methods (ML and BI) resulted in the same general topology (Fig. 2).

In the two ‘total evidence’ phylogenetic analyses, with the exception of P. desmarestii, species from the genera Petrolisthes, Allopetrolisthes, and Liopetrolisthes clustered together into a single monophyletic clade strongly supported by a high posterior probability obtained from the BI analysis and was well supported by the bootstrap support values from the ML analysis. Within this clade, P. granulosus was revealed as sister to all other species of Petrolisthes (excluding P. desmarestii), Allopetrolisthes and Liopetrolisthes from the south eastern Pacific. The status of A. punctatus and A. angulosus as a pair of sister species is well supported by the BI and ML analyses. The tree topology recovered P. laevigatus as sister to A. punctatus and A. angulosus. Nonetheless, the sister relationship above was poorly supported by a low posterior probability obtained from the BI analysis and bootstrap support values from the ML analysis, respectively. Interestingly, Allopetrolisthes spinifrons did not cluster together with the two other congeneric species and its position was not well resolved in the two phylogenetic trees.

In the two phylogenetic analyses, two species from the genus Liopetrolisthes, L. mitra and L. patagonicus, were recovered as well supported sister species. Petrolisthes violaceus was recovered as sister to the genus Liopetrolisthes with moderate to high support. Lastly, P. tuberculatus and P. tuberculosus were recovered as sister species with strong support from both ML and BI analyses.

Unexpectedly, P. desmarestii did not cluster together with other congeneric species. Indeed, P. desmarestii was recovered as sister to a clade including all the remaining species of Petrolisthes, Allopetrolisthes, and Liopetrolisthes, and also containing Polyonyx gibbesi and Megalobrachium soriatum.

Overall, the ‘total evidence’ phylogenetic analyses demonstrated that species from the genera Petrolisthes, Allopetrolisthes, and Liopetrolisthes altogether did not segregate according to genera and did not form well-supported, monophyletic clades, as should be expected according to adult morphology. Similarly, the Bayes factor analysis revealed no support for the separation of the studied species into three different genera (Petrolisthes, Allopetrolisthes, and Liopetrolisthes). Comparisons of the unconstrained tree (harmonic mean = − 3496.7) versus the tree wherein Petrolisthes, Allopetrolisthes, and Liopetrolisthes were imposed as monophyletic clades (harmonic mean = − 3402.17), indicated strong support for the unconstrained tree (2ln(B₁₀) = 9.09).

Phylogenetic trees obtained with ML and BI using only a single, either mitochondrial (16S) or nuclear (H3), marker resulted in similar general topologies (Figs. 3 and 4). As expected, these single-marker phylogenetic trees were less resolved than those produced by the ‘total evidence’ ML and BI phylogenetic analyses. Nonetheless, the single-gene analyses retrieved various monophyletic clades observed in the ‘total evidence’ analyses described above. For instance, in both the ML and BI trees based on the 16S and H3 gene fragments, both L. mitra and L. patagonicus, and A. angulosus and A. punctatus, were well supported as sister species. Petrolisthes desmarestii was recovered as sister to a clade including all the remaining species of Petrolisthes, Allopetrolisthes, and Liopetrolisthes, and additionally containing Polyonyx gibbesi and Megalobrachium soriatum (Figs. 3 and 4).

Discussion

This study presents a two-locus molecular phylogeny of crabs from the genera Petrolisthes, Liopetrolisthes, and Allopetrolisthes. This pool of species represents the totality of the members from the genus Petrolisthes and allies (Allopetrolisthes and Liopetrolisthes) native to the south eastern Pacific (Haig, 1960; Viviani, 1969; Weber, 1991). The analyses with two different phylogenetic reconstruction methods recognized only one monophyletic group consisting of two species of Liopetrolisthes (L. mitra and L. patagonicus) and also supported two members from the genus Allopetrolisthes as sister species (A. angulosus and A. punctatus). The position of A. spinifrons, the remaining congeneric species, was not well resolved. In disagreement with hypotheses based solely upon adult morphology (e.g., Haig, 1960), a well-resolved grouping of all of the species belonging to a particular genus and segregation of species from different genera was not revealed by these analyses. Also, Bayesian factors analyses strongly supported unconstrained trees over trees in which monophyly of Petrolisthes, Liopetrolisthes, and Allopetrolisthes was imposed. Overall, the present results do not support the separation of these species into three different genera as proposed by Haig (1960) that was based upon adult morphology alone. Instead, the present study agrees with previous larval and molecular phylogenetic studies indicating that the division of Petrolisthes, Liopetrolisthes, and Allopetrolisthes within the Porcellanidae is not natural (Osawa, 1995; Wehrtmann et al., 1996; Hernández, 1999; Fujita, Shokita & Osawa, 2002; Stillman & Reeb, 2001; Rodríguez, Hernández & Felder, 2006; Hernández & Magán, 2012). This study argues in favor of future phylogenetic studies using various types of evidence (molecular, adult morphology, larval anatomy) to improve our knowledge of the natural relationships within these species/genera complexes and their position in the Porcellanidae.

The set of species considered in the present study allows a few relevant systematic questions for the group to be addressed. For instance, the two species from the genus Liopetrolisthes, L. mitra and L. patagonicus, clustered together and formed a well supported monophyletic group. Liopetrolisthes mitra inhabits the body surface of the black sea urchin Tetrapygus niger from Ancon, Peru (∼11.8°S latitude) to Bahia San Vicente, Chile (∼36°S latitude) while L. patagonicus dwells among the spines of the red sea urchin Loxechinus albus from Ancon, Peru (∼11.8°S latitude) to the strait of Magellan, Chile (∼54°S latitude) (Weber, 1991). This suggests that the genus Liopetrolisthes likely diversified in the south eastern Pacific in sympatry although it remains to be addressed whether or not speciation in this genus was host-driven. Importantly, although the genus Liopetrolisthes represents a natural clade in the present phylogenetic analyses, its generic status, different from Petrolisthes, is not supported as the two species in the genus clustered within a clade that included other members from the genus Petrolisthes (also, see below).

The present study also retrieved P. tuberculosus and P. tuberculatus as a single well supported monophyletic clade. The close relationship between the two species was recognized early on by Ortmann (1897) who named them as belonging to the ‘Gruppe des Petrolisthes tuberculatus,’ a view supported by Haig (1960). The two species are characterized by a strongly trilobate front, two narrow lobes that project strongly from the anterior margin of the basal segment of the antennule, and a row of uneven, serrate teeth on the anterior margin of the cheliped carpus (Haig, 1960; Viviani, 1969). Given the particular distinctiveness of the two species, Haig (1960) suggested that they should form a separate genus or subgenus. However, at present, it seems inadvisable to split P. tuberculosus and P. tuberculatus from the remaining species in the genus until Petrolisthes is analyzed from locations worldwide.

Lastly and unexpectedly, P. desmarestii, the largest known species of porcelain crab (Haig, 1960; Antezana, Fagetti & López, 1965), did not cluster together with the other congeneric representatives included in this study. Petrolisthes desmarestii was recovered as sister to a clade that included Petrolisthes, Allopetrolisthes, Liopetrolisthes as well as Polyonyx gibbesi and Megalobrachium soriatum. The clustering of P. desmarestii with members from the genera Polyonyx and Megalobrachium likely resulted from incomplete taxon sampling in Porcellanidae. Nonetheless, Petrolisthes desmarestii is unique among other congeneric representatives from the south eastern Pacific because of the carapace, covered with fine plications, the presence of a single epibranchial spine on the carapace, a triangular front, a carpus of the cheliped with four or five broad, serrate-edged teeth on the anterior margin, and a manus covered with small flattened tubercles (Haig, 1960). The traits above, in particular, the presence of an epibranchial spine on the carapace, teeth on the anterior margin of the cheliped carpus, and the postero-distal spines on the merus of the first ambulatory pereopod, suggest that P. desmarestii belongs to either the ‘Gruppe des Petrolisthes galathinus’ or ‘Gruppe des Petrolisthes lamarcki’ recognized by Ortmann (1897), both containing more than twenty species in the eastern Pacific and western Atlantic (Haig, 1960; Hiller et al., 2006). A preliminary molecular phylogenetic analysis based on the 16S mtDNA ribosomal gene (Bayesian inference, 95 terminals, GTR+G model, not shown here) provides support for the close relationship between P. desmarestii and members from the P. galathinus species complex. These results suggest more than a single colonization event in the south eastern Pacific during the evolutionary history of porcelain crabs.

In general, this study has shown that the separation of Petrolisthes + Allopetrolisthes + Liopetrolisthes into three taxonomic entities is not natural based on molecular characters of the studied species set. Crabs from these three genera demonstrate a considerable diversity of lifestyles, body sizes, microhabitats, and coloration (Haig, 1960; Antezana, Fagetti & López, 1965; Baeza & Thiel, 2003; Baeza & Thiel, 2007). Studies describing the life history and ecology of Petrolisthes, Allopetrolisthes, and Liopetrolisthes within a phylogenetic framework are underway (e.g., Baeza & Thiel, 2003; Baeza & Asorey, 2012; Gebauer, Paschke & Anger, 2010). This approach is expected to prove most useful in understanding the role of environmental conditions in driving the evolution of morphological, ecological, and behavioral traits in the marine environment (e.g., Baeza & Thiel, 2003; Baeza & Asorey, 2012). The present study included only porcellanid species from the south eastern Pacific; nevertheless, the amphi-American nature of Petrolisthes and allies (see Haig, 1960) suggests that this group might also be a model to study speciation mechanisms, as in other transisthmian clades of fish (Bermingham, McCafferty & Martin, 1997), sea urchins (Lessios, 2008), caridean shrimps (Williams et al., 2001), and brachyuran crabs (Windsor & Felder, 2014).

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