Micro molluscs generally refer to the mollusc which requires a microscope for observation (Middelfart et al., 2016). Although this mollusc group may have the potential to significantly impact marine biodiversity, it is one of the most neglected assemblages due to their small body size, making the investigation challenging and time-consuming to collect, sort, preserve, and identify (Middelfart et al., 2016). The underestimation of this large biodiversity might also prohibit the precise understanding of the ecological and biogeographical aspects such as the food web structure and genetic connectivity of numerous described species (Middelfart et al., 2016). Thus, increasing the knowledge of the taxonomic diversity of micro molluscs is essential to comprehensively understand not only the evolution of individual species but also the systematics of the whole community.

Philinoidea is one of the micro mollusc groups, and is a superfamily of marine conchifera Cephalaspidea gastropod (Knutson et al., 2020). With 310 currently known species in 10 families (Knutson et al., 2020), Philinoidea is globally distributed in diverse habitats including temperate, tropical, and subtropical shallow water (Gonzales et al., 2011; Malaquias et al., 2015; Zamora-Silva and Malaquias, 2018). As this superfamily has been considered to host a large proportion of biodiversity in Cephalaspidea, their body plan contains substantial morphological variation including its shell and digestive system (Knutson et al., 2020).

The taxonomic relationship within Philinoidea has been debated as the phylogenetic studies have traditionally been conducted mainly based on the external morphology like shell characteristics even though their internal anatomy appears to vary significantly between some species similar in terms of their external features (Oskars, 2013). More recently, the number of molecular phylogenetic analysis of Philinoidea has been increasing (Oskars, 2013; Oskars et al., 2015; Knutson et al., 2020), but most of these focused on the relationship within the family, and few studies have investigated the relationship between different families within Philinoidea. Due to this unresolved phylogeny, there is still uncertainty on how different morphological characteristics might have evolved within this superfamily. Additionally, as their morphology is highly diverse and unique within Philinoidea, it has been considered to hold the key to understanding the evolution of morphological characteristics of Cephalaspidea (Knutson et al., 2020). Thus, with the context of morphology evolution, the increasing knowledge of the taxonomic relationship within and between families of Philinoidea is important. 

The ancestral conchifera gastropod has been considered to have an external calcareous shell, and the internalization of these shells has evolved independently in numerous families (Osterauer et al., 2010). Recent transcriptomic evidence suggested that this internalization of the calcareous shell is concentrated more within Philinoidea than any other superfamilies of Cephalaspidea (Knutson et al., 2020). However, the understanding of the evolution of the shell internalization within Cephalaspidea and Philinoidea remains sparse due to the debatable taxonomic position of Scaphandridae (Knutson et al., 2020), which is one of the few Philinoidean families that possess robust external shell (Eilertsen & Malaquias, 2013). In addition to the shell internalization, Philinoidea seems to be important to infer the evolution of the gizzard and the aligned plate in Cephalaspidea as it contains the minority of Cephalaspidea that lack these characteristics (Brenzinger et al., 2013). Gizzard and the plate play an important role in the digestive system as it is used for grinding or crushing their food (Rudman, 1974; Shepelenko et al., 2015). Previous studies have identified that most of the extant herbivorous Cephalaspidea is equipped with gizzard plates and the external shell (Göbbeler and Klussmann-Kolb, 2011; Oskars et al., 2015) whereas the carnivorous lifestyle could occur with or without the plate, and tends to have an internal shell (Wägele and Klussmann-Kolb, 2005). Despite the potential significance of the evolution of these two evolutionary traits to their habitat and feeding strategies, the functional and mineral aspects of these morphologies have been intensively studied (Shepelenko et al., 2015), and very little is currently known about the evolutionary implications of these morphology. 

Philine rubrata is a species of Philine, belonging to the family Philinidae, described first by Gosliner (Gosliner, 1988). This species is characterized by their internalized shell, pigmented body, absence of the gizzard plate, and the elongate posterior skirt, which are uncommon morphological characteristics in Philine; the majority of Philine species has been shown to have gizzard plate and white body (Gonzales et al., 2011). The investigation of this species might help to understand the evolution and the association of shell internalization and gizzard plate loss with lifestyle in Philinoidea. With a focus on P. rubrata, this project attempts to clarify the taxonomic relationship between families of Philinoidea and the evolution of shell internalization and gizzard plate through the integration of morphological description on the reconstructed molecular phylogenetic tree based on publicly available 16S rRNA gene of 65 species from 7 families (Aglajidae, Alacuppidae, Gastropteridae, Lanoidea, Philinidae, Philinoglossidae, and Scaphandridae). This is the first study to integrate morphological and molecular information to understand the evolutionary scenario at the scale of Superfamily, and covering diverse taxon from a large geographical range.

Morphological method

Autonomous Reef Monitoring Structures (ARMS), the settlement plates developed by US National Oceanic And Atmospheric Administration (NOAA), were deployed at One Mile Jetty, Dunwich, North Stradbroke Island (Figure 1). ARMS consists of ten 25cm x 25cm plates, and it has been internationally used to survey marine biodiversity. To ensure the settlement and growth of the benthic community, ARMS plates were hung at the jetty for approximately 8 weeks during summer conditions from 27th January 2021 to 24th March 2021. As the jetty is a floating pontoon, the plates were maintained at a constant depth below the sea surface. The jetty has been used as the main dock for the Stradbroke Flyer water taxis, which regularly operate between Dunwich and Cleveland, and other irregular boats. After the retrieval and transport of these plates to the lab at the University of Queensland St.Lucia campus, both upper and lower slides of each plate were photographed by students, tutors, and lecturers from the University of Queensland. These images of the plates have been uploaded to the website “Invertebrates of the Great Barrier Reef (GBRI)” (Degnan, n.d.). P. rubrata was found at the bottom side of the plate, and identified based on previously published paper (Gosliner, 1988). Before the preservation, the specimen was placed in filtered seawater with drops of 1M MgCl2  to relax its body for clear morphological observations. Once the tissue seemed to be relaxed, the specimen was transferred and preserved in 95% ethanol for later dissection. The specimen was observed and dissected carefully using Olympus SZX9 Stereomicroscope and Nikon SMZ-18 Motorised Stereo Microscope. The dissection of the internal shell and gizzard was attempted, and the radula extraction was performed using a bleach solution. 

Molecular phylogeny method

Sequence retrieval and alignment 

The accession number of the 16S rRNA sequence of different Philinoidea species was retrieved from Genbank, and added to the excel spreadsheet. It was aimed to cover all the families of Philinoidea, but could not include Antarctophilinidae, Colpodaspididae, and Philinorbidae due to the scarce availability of the sequence data of these families. 65 species from 7 families (Aglajidae, Alacuppidae, Gastropteridae, Lanoidea, Philinidae, Philinoglossidae, and Scaphandridae) were included in this phylogenetic analysis, and the information of each species are summarized in Table 1. The excel spreadsheet containing the information of species and accession number was read and imported into a python program, which was originally provided by Associate Professor Mikael Boden during the Bioinformatics course (SCIE2100) at the University of Queensland and has been modified for this phylogenetic analysis project. The sequence for each species was retrieved based on the list of accession numbers, and the multiple sequence alignment was run using the ClustalW2 method (Larkin, 2007).

Phylogenetic analysis with the Model and Method selection

The FASTA file containing multiple sequence alignment was imported into R studio for further phylogenetic analysis. This R script was coded to estimate phylogenetic trees based on the Neighbor-Joining (NJ) and Unweighted Pair Group Method with Arithmetic mean (UPGMA), and Maximum likelihood approach (ML). The distance method (NJ or UPGMA) with a higher parsimony score was used to analyze the phylogenetic data using the ML method and the R package ape and phangorn. Based on the evolutional model testing, Generalised Time Reversible (GTR) was used to compute the likelihood of the tree. Once the ML tree was built, a bootstrap analysis was performed, and FigTree v1.4.4. was used to annotate the produced tree. Then, along this ML tree, morphological characteristics of interest including shell, gizzard plate, and body-color were mapped. The excel spreadsheet, python project, retrieved sequence, and R scripts used in this phylogenetic analysis are available at https://github.com/marinehisatake/BIOL3211_project.git. 

Table 1. List of Philinoidea species included in the molecular phylogenetic study.

The diversity and phylogenetic relationship of Philinoidea remain unclear, mainly due to the difficulties of studying a microscopic body, making the collection, sorting, and identification time-consuming (Middelfart et al., 2016). As this study covered a broader species and geographical coverage of Philinoidea by assembling the publicly available sequence data of the 16S rRNA gene, the suggested phylogenetic relationship could serve as a guideline for further investigations. 

Regarding the produced phylogenetic trees (Figures 9, 10, and 11), both NJ and ML trees have shown that there is a sister group relationship between Philinidae and Aglajidae (BS = 28%) with Scaphandridae and Gastropteridae as the outgroup; this result seems to be consistent with previous studies (Grande et al., 2004; Eilertsen & Malaquias, 2013; Oskars et al., 2015). Besides, both trees have shown weak evidence of the sister group relationship of P. rubrata and P. orca with Aglajidae clades (BS = 23% and BS = 3% respectively); the rest of the Philinidae species were identified as the outgroup to these two species. The species in Philinidae Clade 1 such as P. aperta and P. orientalis have been commonly called “true” Philinidae (Gonzales et al., 2011), and it is characterized by the presence of internal shell, white body, and well-developed gizzard and gizzard plates (Eilertsen and Malaquias, 2013). In contrast, P. rubrata and P. orca are known for their colorful pigmented body (as shown in Figures 2, 3, 4, 5, and 6) and the lack of gizzard plate (Gonzales et al., 2011; Eilertsen and Malaquias, 2013). Since these body plans are dominantly found within Aglajidae (Zamora-Silva and Malaquias, 2018), it can be suggested that P. rubrata and P. orca might be more closely related to Aglajidae rather than Philinidae. This has been hypothesized by a previous study, which had very weak evidence due to the inclusion of few Aglajidae species (Gonzales et al., 2011). Nevertheless, as the bootstrap value of positioning these colored Philinidae near Aglajidae was not significantly high (BS < 50%), further studies with more sample numbers might be required.

Figure 12 shows the distribution and evolution of 3 morphological characteristics in the ML tree of Philinoidea. The clade of Scaphandridae, which has been considered to be the key Philinoidean family to understand the shell evolution due to the possession of the robust external shell, was nested within the larger clade of Gastropteridae, which is known to have an internalized shell, with weak evidence (BS = 22%). Similarly, Lanoidea (containing only one Retusophiline sp.), which is also known for the possession of an external shell (Valdés et al., 2016), was nested within the clade of Philinidae, the family characterized by an internal shell (BS = 4 %). Hence, based on the parsimonious interpretation, the last common ancestor of Philinoidea might have had an internalized shell, and the shell externalization evolved independently twice in different lineages. This could be considered as the product of Devolution as the ancestral body plan of Cephalaspidea is typically characterized by the presence of an external calcareous shell (Osterauer et al., 2010). Due to the small bootstrap value, a validation experiment of this evolutionary interpretation would be required. Since it has been suggested that the blocking of the TGF-β pathway could induce a shell internalization in external shell-bearing species (Link et al., 2019), the validation could be made by investigating whether the gain or blocking of the TGF-β pathway might occur more likely.

For the evolution of the gizzard plate, Figure 12 has presented that both Philinidae Clade 1 and Scaphandridae, which possess gizzard plate (Gonzales et al., 2011; Price et al., 2011), were nested within the large clade where most species are characterized by the absence of the gizzard plate. Based on the parsimonious evolutionary interpretation, the absence of gizzard plate could be identified as the morphological characteristic of the last common ancestor of Philinoidea, and the development of gizzard plate might have occurred independently in different lineages including Philinidae Clade 2, Philinoglossidae (Plusclua cuica), and Scaphandridae; this evolutionary scenario is illustrated in Figure 13 (a). Previous studies have suggested that the herbivory with external calcareous shell and gizzard plate are plesiomorphic characteristics of Cephalaspidea (Zamora-Silva et al., 2016). Hence, this might suggest an evolutionary history of Philinoidea where the common ancestor of Philinoidea have lost their plesiomorphic condition of Cephalaspidea, and regained these conditions independently in different lineages during the diversification (Figure 13 (a)). Considering the physiological aspects, however, the convergent gain of this plate might occur less likely than the independent loss between different lineages (Figure 13 (b)) as the plate is typically composed of chitin or mineralized calcium (Shepelenko et al., 2015), indicating its energetically expensive development. As the nesting of Philinidae Clade 1 and Scaphandaridae had significantly weak evidence (BS = 28% and BS = 22% respectively), the phylogeny might need to be interpreted with caution. Thus, a comparative mineral analysis of the gizzard plate with species of other superfamilies might be beneficial.

The present study was conducted to comprehensively understand the evolution of shell internalization and lack of gizzard plate of Philinoidea with a focus on P. rubrata. Previous studies have hypothesized that carnivory has arisen independently from ancestral herbivory Cephalaspidea (Malaquias et al., 2009), and dietary specialization, by the development of new morphological characteristics, played a significant role in adaptive radiation, creating rich species diversity of Cephalaspidea. For Aglajidae, it has been shown that the development of morphological features such as shell internalization, developed chemical detection with cephalization, and unique digestive features including eversion of the buccal bulb have potentially allowed them to occupy new ecological niches and to become carnivorous; thus, resulting in adaptive radiation as they become capable of exploiting new habitat (Malaquias et al., 2009). Given P. rubrata might be closely associated with Aglajidae, the shell internalization and lack of gizzard plate might have led them to have a more active carnivorous mode of life with a streamlined flexible body and weight reduction. Besides, P. rubrata has an autapomorphic condition of radula with fewer denticles than other Philine species (Price et al, 2011), which could be interpreted as a common reduction of teeth observed during the evolution of the carnivorous feeding lifestyle of Mollusc (Eisapour, 2015). In addition, it also exhibits cephalization, indicated by the well-developed eyes (Figure 2 and 5), stout-like anterior shields, parapodium lobes, and posterior mantle cavity, which have been suggested to improve the burrowing and crawling ability of some carnivorous Philinidae species to look for diverse prey including Annelid and Bivalves at various habitats where might have been inaccessible for the shelled animals (Morton and Chiu, 1990). Hence, the evolution of these morphological characteristics including internal shell and gizzard plate of the common ancestor of Philinoidea (before the diversification) might have facilitated ancestral P. rubrata to occupy a unique niche as a carnivorous predator, triggering adaptive radiation through the exploitation of new habitats; this might potentially explain the diverse habitats where different Philinoidea species have been found.

The phylogenetic tree was created purely based on the sequence data of the 16S rRNA gene available on the NCBI database as shown in Table 1. However, this dependence on the database might have potentially caused the low value of bootstrap at several branches. Since this study involved multiple studies conducted by different research groups, different sequencing techniques were used, which might indicate the different sequence data quality. Although the resolution of the phylogenetic tree provided by the comparison of the 16S rRNA is suggested to be sufficiently high (Zhi et al., 2012), the computed phylogenetic tree needs to be interpreted with caution as it might have been over or underestimated by overlooking the whole genomic diversity. Thus, further research with a phylogenetic tree analysis using multiple genetic markers or whole-genome sequencing might provide a better resolution of the relationship and evolution of these morphological characteristics between different species.

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