Euthyneura
With the current study we present a comprehensive molecular phylogenetic analysis of Euthyneura including representatives of all major subgroups. We used different approaches to infer a phylogenetic hypothesis for this taxon of Gastropoda. These approaches yielded for the most part constant results respective to the deeper nodes in the tree and indicated only minor differences at generic or familial level.
The unification of Opisthobranchia and Pulmonata in the group Euthyneura has been widely accepted since its original definition by Spengel [4]. Consequently, the monophyly of Euthyneura has mostly been accepted in recent systematic and phylogenetic investigations [5, 9, 11–13, 15, 23, 24]. Although the defining character of Euthyneura, euthyneury, is considered to be a result of multiple convergence [16, 17], several autapomorphic characters supporting monophyly have been proposed for Euthyneura [11, 16, 17].
Our results contradict a monophyletic Euthyneura, since Pyramidelloidea are included within euthyneurans as sister group to the Amphiboloidea. This is in congruence with Grande et al. [19] who found Pyramidelloidea deeply nested within Pulmonata in their molecular phylogenetic analysis. Although not having included Pyramidelloidea in his molecular phylogenetic analysis of Euthyneura, Thollesson [18] proposed a possible synapomorphy of Pyramidelloidea and Euthyneura in the presence of a gap in the helix G16 of the 16S rRNA gene, thus supporting inclusion of Pyramidelloidea within Euthyneura as proposed here. Studies considering additional pyramidelloid and other basal heterobranch taxa currently undertaken in the group of the senior author will hopefully shed more light on the position of the enigmatic Pyramidelloidea within Heterobranchia (Euthyneura plus Heterostropha sensu Ponder and Waren [10]). Additionally, new character complexes, such as secondary structures of ribosomal genes, could provide further valuable phylogenetic information for the taxa in question, as has been demonstrated recently by Lydeard et al. [25].
Opisthobranchia
Although the separation between Opisthobranchia and Pulmonata is rather distinct because of e. g. characters of the nervous system, it has been difficult to propose sound autapomorphies for a monophyletic Opisthobranchia [12]. Although the subgroups of Opisthobranchia can be clearly distinguished and appear to be monophyletic [21, 26–29], monophyly of the Opisthobranchia altogether could not be shown by most recent phylogenetic analyses, regardless of whether they used morphological or molecular data [18, 19, 21, 22, 30]. The Opisthobranchia appear to be paraphyletic (cp. e. g. [7, 18]) rather than a natural grouping. Our data confirm this latter assumption by rendering the Opisthobranchia either polyphyletic or paraphyletic (depending on the analysis). Filtering our MCMC trees of the Bayesian analysis of the reduced dataset (including all genes) under the constraint of a monophyletic Opisthobranchia revealed that out of 100.000 trees not one single tree supports a monophyletic Opisthobranchia. Application of the Approximately Unbiased test [31] to both reduced datasets also rejected a monophyletic Opisthobranchia at the 5% significance level. In contrast, we can distinguish several clades within Opisthobranchia clearly distinct from each other gradually leading to the pulmonate level of organisation.
One such clade comprises the Nudipleura and Acteonoidea which in our trees appears at the base of the Euthyneura. Acteonoidea have traditionally been regarded as the most primitive opisthobranchs or have even been excluded from Opisthobranchia [22, 27, 32] whereas Nudipleura are derived [21, 22]. However, this unexpected grouping of both taxa has recently continuously been revealed by molecular systematic studies. [20, 21, 33]. Nevertheless, statistical support in our analyses is rather low and good synapomorphies for this sister group relationship are still warranted. Moreover, we observed deviant base composition and rate heterogeneity in Nudipleura which also could attribute to the basal position of this taxon and possibly artificially groups them with Acteonoidea.
A second well supported opisthobranch clade in the current analysis of the large taxon set comprises the Cephalaspidea, Umbraculoidea and Aplysiomorpha plus Thecosomata and Gymnosomata (Pteropoda). This close grouping of Cephalaspidea, Aplysiomorpha and Pteropoda has been suggested lately by several studies [17, 33]. The position of Umbraculoidea within this clade, although well supported in our current analyses, should be considered with caution since only one umbraculoid taxon has been included. Moreover, no morphological synapomorphies are known to support a close relationship of Umbraculoidea and Aplysiomorpha/Pteropoda. More umbraculoid taxa must be considered in future studies to clarify the phylogenetic position of this taxon.
The phylogenetic position of Sacoglossa within Opisthobranchia/Euthyneura has been a matter of debate [20–22]. Although morphologically well defined as a monophylum [26, 27], different analyses assign Sacoglossa equivocally to different clades within the Euthyneura. Our data suggest a close affinity to primitive Pulmonata. However, Sacoglossa are definitely a taxon that needs to be paid more attention to in future phylogenetic studies.
The same holds true for the Acochlidiacea, an enigmatic taxon inhabiting interstitial marine habitats and even freshwater systems, a feature unique within Opisthobranchia. Due to their very small size (often less than 1mm in length) hardly anything is known about their morphology and life history. Recent morphological investigations utilising modern computer generated 3-D reconstructions [34, 35] have shed some light on the anatomy of these enigmatic gastropods, however, more studies are warranted. The phylogenetic position of the Acochlidiacea is still unclarified. Vonnemann et al. [21] found them as a basal taxon within Opisthobranchia, while our results imply a position within Pulmonata.
Pulmonata
The monophyly of Pulmonata is widely accepted on the basis of morphological characters [6, 7, 11, 13, 16]. In contrast, our data suggest paraphyly of Pulmonata due to the inclusion of Sacoglossa, Pyramidelloidea and possibly Acochlidiacea. If our phylogenetic hypothesis is correct, one must postulate that pulmonate autapomorphies (such as acquisition of a pneumostome and pulmonary vessels as well as the presence of a procerebrum and dorsal bodies) have secondarily been reduced in Sacoglossa, Acochlidiacea and Pyramidelloidea.
Paraphyly or even polyphyly of Pulmonata was recovered by molecular systematic studies [14, 19, 36].
Within Pulmonata we can distinguish two monophyletic clades: Eupulmonata and Hygrophila. The unification of Siphonarioidea and Amphiboloidea in the Thalassophila based on morphological data [13] could not be confirmed by our data.
The Eupulmonata comprise a group of marine, semi-terrestrial and truly terrestrial gastropods showing a high diversity with regards to species number and life history characters. Monophyly of this taxon is strongly supported by Wade and Mordan [15] and the present results, however no morphological apomorphy is known to date [9, 10]. Relationships within Eupulmonata have been disputed in the past. Monophyly of Stylommatophora is undoubted [11, 13–15, 17, 37] and can be supported by several autapomorphies [11, 13]. Our results also show a monophyletic Stylommatophora. Haszprunar and Huber [16] proposed a close relationship of Ellobiidae, Trimusculidae and Stylommatophora on the basis of apomorphic characters of the nervous system, whereas Dayrat and Tillier [11, 12] consider Stylommatophora and Systellommatophora (Onchidioidea and Veronicelloidea) to be closely related. Neither of these two hypotheses are confirmed by our findings since we find a close relationship between ellobioid taxa, Otinoidea, Onchidioidea and Trimusculoidea. The phylogenetic status of the Ellobioidea cannot be conclusively clarified from our data since they appear to be paraphyletic in the two analyses utilising all sequence data. Exclusion of 16S-sequences and 3rd codon positions of COI-sequences, however, recovers them monophyletic.
The Basommatophora sensu Nordsieck [13] and Bouchet et al. [38] are paraphyletic in our analyses. No topology in our set of 100.000 MCMC trees of the Bayesian analysis of the reduced dataset supports a monophyletic Basommatophora. Moreover, testing an alternative tree topology with a constrained monophyletic Basommatophora with the Approximately Unbiased test [31] rejected monophyly of Basommatophora at the significance level of 5%. This is not surprising since the monophyly of Basommatophora has already been disputed by Hubendick [39]. Within Basommatophora the Hygrophila are a well supported and defined group of pulmonates. Our results support earlier hypotheses of a common origin of these freshwater taxa [12, 13, 17, 40]. For the first time, however, crucial taxa like Latia are included in a molecular study. Within Hygrophila two clades can be distinguished; on the one hand the Chilinoidea (sensu Boss [3], including Chilinidae and Latiidae), and on the other the higher limnic Basommatophora. Chilina and Latia are sister-species, a relationship that has already been mentioned earlier [13, 39, 41]. Higher limnic Basommatophora can be divided into four well distinguishable families [39] while interfamilial relationships of these taxa vary in the current analyses thus rendering them beyond the scope of this study. These relationships are discussed in other publications [42, 43].
Former taxonomic classifications of Pulmonata united a group of primitive taxa (Ellobiidae, Otinidae, Amphibolidae, Siphonariidae, Chilinidae and Latiidae) in the Archaeopulmonata [44]. Other authors followed this classification [6, 3, 45]. However, our results clearly indicate a paraphyletic nature of these taxa and therefore we propose the disuse of the name Archaeopulmonata.
Colonisation of freshwater and terrestrial habitats
One key step in the evolution of Euthyneura was the invasion of freshwater and terrestrial habitats by Pulmonata resulting in a multitude of taxa worldwide adapted to these habitats. It is undisputed that the first pulmonate gastropods were marine organisms [12, 46]. Marine pulmonates can be found in truly marine submersed environments (such as Williamia) and, more frequently, in upper littoral zones (Siphonaria, Myosotella, Trimusculus). Certain taxa even occur in estuaries or mangroves (Onchidiidae, certain Ellobiidae, Amphibolidae). Terrestrial pulmonates inhabit a wide spectrum of habitats, ranging from caves to lowlands to mountains and from boreal to tropical ecological zones. The same holds true for freshwater taxa, which occur in different types of limnic environments worldwide.
Our reconstruction of character evolution relating to habitat type (Fig. 4) shows that within Pulmonata the freshwater habitat has only been conquered once by the Hygrophila. Calculation of posterior probabilities for the different habitat types at specific nodes indicates that the ancestor of Hygrophila probably already lived in a freshwater habitat. Acknowledging that there are extremely few onchidioidean species reported to live in brackish or freshwater it would not change the picture that the major radiation into freshwater happened only once leading to the Hygrophila. The same is true for the few acochlidiaceans that are known from freshwater habitats. Freshwater members of both taxa were not included in our data set, for the time being, we would also hypothesize a similar evolutionary pathway excluding a terrestrial step.
In non-pulmonates, the situation is different. 'Prosobranchs', such as the superfamily Cerithoidea, comprise predominantly marine taxa. Nevertheless, freshwater was invaded independently in several cerithoid lineages [47]. Even the evolution of life history traits such as viviparity in the freshwater invaders was correlated with this evolutionary step [48].
The ancestor of Eupulmonata and Hygrophila, in contrast, appears to have lived in a marginal zone like supralittoral zones, estuaries or mangroves. Therefore we conclude, that colonisation of freshwater in Pulmonata occurred via an aquatic pathway directly from the marine realm and not via a terrestrial step.
The terrestrial habitats have most probably also been invaded via marginal zones, as can be seen from the Bayesian reconstruction of ancestral character states in Eupulmonata. Involvement of freshwater systems in colonisation of terrestrial habitats is less likely, since the freshwater taxa (Hygrophila) are clearly separated from Eupulmonata and the posterior probability for a freshwater habitat is basically non-existant at the base of the Eupulmonata.
Terrestriality on the contrary is thought to have evolved much more than 10 times independently (discussed in Barker [49]). Barker [49] already emphasized that recent ellobiid terrestriality was clearly derived from marine littoral ancestors and not secondarily from terrestrial ancestors.
Invasion of marine habitats by Otina ovata within Eupulmonata and Turbonilla sp. obviously is the result of a secondary colonisation originating in marginal zones.