1 Research Article
2 Early Development of Monoplex pilearis and Monoplex parthenopeus (Gastropoda: 3 Cymatiidae) - Biology and Morphology
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5 Ashlin H. Turner*, Quentin Kaas, David J. Craik, and Christina I. Schroeder*
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7 Institute for Molecular Bioscience, The University of Queensland, Brisbane, 4072, Qld, Australia
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9 *Corresponding authors:
10 Email: [email protected], phone: +61-7-3346-2023
11 Email: [email protected], phone: +61-7-3346-2021
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12 Abstract
13 Members of family Cymatiidae have an unusually long planktonic larval life stage (veligers) which
14 allows them to be carried within ocean currents and become distributed worldwide. However, little
15 is known about these planktonic veligers and identification of the larval state of many Cymatiidae
16 is challenging at best. Here we describe the first high-quality scanning electron microscopy images
17 of the developing veliger larvae of Monoplex pilearis and Monoplex parthenopeus (Gastropoda:
18 Cymatiidae). The developing shell of Monoplex veligers was captured by SEM, showing plates
19 secreted to form the completed shell. The incubation time of the two species was recorded and
20 found to be different; M. parthenopeus took 24 days to develop fully and hatch out of the egg
21 capsules, whereas M. pilearis took over a month to leave the egg capsule. Using scanning electron
22 microscopy and geometric morphometrics, the morphology of veliger larvae was compared. No
23 significant differences were found between the shapes of the developing shell between the two
24 species; however, it was found that M. pilearis was significantly larger than M. parthenopeus upon
25 hatching. Although statistical analysis did not find morphological differences, this study concludes
26 biological differences do exist between these two closely related species of Monoplex.
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29 Keywords: Cymatiidae, veligers, scanning electron microscopy, geometric morphometrics
30 Abbreviations: Scanning electron microscopy (SEM), Principal Component Analysis (PCA),
31 Monoplex spp. abbreviated as M. where applicable.
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32 Introduction
33 The features of the protoconch are widely used to identify gastropods, especially Caenogastropoda
34 (Solsona 1999). The protoconch is the first stage of shell growth in gastropods, which begins in
35 embryo and can be retained into adulthood (Muthiah 2000a; Muthiah 2000b). The protoconch is
36 generally classified into two phases, protoconch I and protoconch II (Sang et al. 2019). Protoconch
37 I is the first stage of the protoconch formed in embryo and is generally smooth and unornamented.
38 (Robertson 1971; Jablonski and Lutz 1983; Sang et al. 2019) Protoconch II can be ornate and
39 useful for identifying species differences and is formed before metamorphosis to mature shell
40 (teleochonch) (Robertson 1971; Jablonski and Lutz 1983; Sang et al. 2019). The differences in
41 the protoconch are used to distinguish cryptic species in Naticidae (Littorinimorpha) and
42 Neritiliidae (Neritimorpha), among others (Solsona 1999). The protoconch is so distinctive,
43 beyond species identification, its structure allows for differentiation between life cycle types
44 within Gastropoda (Solsona 1999) and this determination of the larval development type of a
45 fossilized or extinct specimen, solely based on protoconch features, can be invaluable for
46 paleontologists and evolutionary biologists (Solsona 1999; Kano 2008).
47 The primary biological function of the protoconch may be to provide protection for planktonic
48 larva (hereafter veligers) (Hickman 1999; Hickman 2001). Although this statement at first appears
49 self-evident, many planktonic veligers are eaten whole by zooplankton predators and the shell is
50 of limited protection (Pennington and Chia 1985; Hickman 1999; Hickman 2001). Nonetheless,
51 studies using scanning electron microscopy (SEM) have identified shell features that may result in
52 a protective advantage for larval gastropods (Hickman 1999; Hickman 2001). These features
53 include spiral ridging to reduce fracturing, ‘beaks’ or protrusions that protect the veliger’s aperture,
54 and strategically reinforced notches around the apertural opening (Hickman 1999; Hickman 2001).
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55 Although these adaptations have been observed in several veliger species, shell structure and a
56 description of protoconch features are lacking in Monoplex veligers.
57 Species within Monoplex have an unusually long planktonic larval state, allowing them to become
58 geographically widespread through major ocean currents (Scheltema 1966; Pechenik 1984;
59 Muthiah 2000a; Muthiah 2000b). However, few reports of the morphological features of the
60 protoconch development during this larval state are available. It is believed Monoplex planktonic
61 veligers develop or calcify their shell very little during this growth stage (Pechenik 1984). Due to
62 the planktonic development taking several years, it is difficult to study the growth of veligers in
63 the laboratory, and their wide distribution with ocean currents makes monitoring development in
64 field studies impractical. Muthiah and Sampath (Muthiah 2000a) offer the only account of larval
65 development of Monoplex pilearis in the laboratory, with images taken using low-resolution
66 brightfield microscopy. They described the reproductive behavior of 22 M. pilearis specimens in
67 laboratory conditions and the early development of eggs over 45 days (Muthiah 2000a; Muthiah
68 2000b).
69 Low-resolution SEM images of veligers believed to be Monoplex parthenopeus revealed that the
70 protoconch is not heavily calcified during the planktonic stage (Scheltema 1966; Pechenik 1984).
71 That report does not provide images nor a description of the protoconch features for aid in
72 identifying future specimens (Pechenik 1984). SEM is the gold standard in terms of resolution of
73 protoconch features (Solsona 1999; Kano 2008), although brightfield imaging has also been
74 reported in some instances (Muthiah 2000a). Early work used detailed ink sketches in publications
75 due to the lack of high-resolution microscopy imaging, leaving the scientific community a legacy
76 of protoconch features which were significantly better than the microscopy images of the time
77 (Beu 1987). Magnification of current brightfield microscopy is commonly on the scale of 1,000 –
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78 2,000x, whereas SEM can reach greater than 50,000x magnification. For many species, this type
79 of high-resolution image of the protoconch is simply unavailable, limiting the ability of unknown
80 specimens to be properly identified and classified (Scheltema 1966; Pechenik 1984). In the study
81 of trans-Atlantic transport of veligers (Pechenik 1984), a definite identification would be of great
82 assistance, as well as of use in further studies examining the long-distance transport of various
83 planktonic veligers. The gastropod veligers collected by plankton tows in the mid-Atlantic were
84 tentatively identified as M. (Cymatium) parthenopeus (Scheltema 1966; Pechenik 1984). The proof
85 of live veligers being carried by the Gulf Stream provided an explanation for the world-wide
86 distribution of some members of Cymatiidae, yet a species level identification of these veligers
87 was challenging (Scheltema 1966; Pechenik 1984).
88 Geometric morphology is a suite of statistical methods to quantify the differences in shape between
89 a given set of specimens (Rohlf 1993). These methods quantify differences in morphology by
90 reducing shapes to a set of Cartesian coordinates based on consistent landmarks or by using outline
91 curves (Conde-Padin 2007; Avaca et al. 2013; Marquez 2017; Doyle 2018). This powerful method
92 enables quantitative distinction between shape and morphological landmarks (Avaca et al. 2013).
93 Quantitative comparison of shell shapes has been used to identify subtypes within and between
94 adult gastropod species and subspecies, including Buccinanops deformis, Littorina saxatilis, and
95 Littorina littorea (Conde-Padin 2007; Avaca et al. 2013; Marquez 2017; Doyle 2018). This
96 technique has not yet been applied to protoconch features, and here we investigated if geometric
97 morphometrics can be used to identify veliger gastropods.
98 Herein we report the first high-resolution SEM images of M. pilearis and M. parthenopeus
99 veligers, including images of the developing shell during the earliest life stages. In addition, further
100 information on the egg clutches and early development is described, adding to the works of
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101 Pechenik and Muthiah (Pechenik 1984; Muthiah 2000a; Muthiah 2000b). Statistical analysis was
102 performed in an attempt to quantify morphological differences between the protoconch I of two
103 veliger species. To the best of our knowledge, this paper is the first attempt at using geometric
104 morphology metrics to describe the planktonic life stage of any species of Gastropoda.
105 Methods and Materials
106 Specimen Collection
107 Two specimens of M. pilearis were collected from Amity Point, North Stradbroke Island,
108 Queensland, Australia (Fig 1) and seven specimens of M. parthenopeus were collected from the
109 oyster lease of Greg Knight, North Stradbroke Island, Queensland, Australia. Specimens were kept
110 in aquaria and fed rock oysters at ad libitum. The female M. pilearis laid eggs on March 19, 2018,
111 whereas the female M. parthenopeus laid eggs on April 18, 2018. The water quality of the aquaria
112 over the course of the study is summarized in Table 1. The water flow through the tank was set on
113 a slow drip to avoid washing the veligers into the filter. The ammonia and nitrite levels were kept
114 at undetectable levels for the duration of the study.
115 Scanning Electron Microscopy
116 Veligers were collected approximately14 days after they began to emerge from the egg clutch, the
117 first week of May 2018. Specimens were preserved with 2.5% glutaraldehyde, washed twice in
118 phosphate buffered saline, and then dehydrated in a 50% and 100% ethanol solution before being
119 mounted on a slide prepared with poly-L-lysine. The prepared samples were gold-coated using
120 SPI-Module sputter coater and imaged using a JCM-5000 Neoscope scanning electron microscope.
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121 Morphometric Methods
122 The SEM images of each specimen were selected based on orientation. Shells suitable for analysis
123 were in a lateral orientation and rotated to ensure all shells were in the same orientation relative to
124 each other, aperture toward the bottom of the frame. In contrast to shells oriented laterally, shells
125 falling with the aperture facing upwards were generally damaged, preventing consistent images to
126 be obtained. Attempts at manually reorienting the shells resulted in significant damage. Therefore,
127 for all the images suitable for analysis, a 5x5 grid was placed over each shell using Adobe
128 Illustrator. Landmarks are specific points that are consistently placed between specimens or
129 images, intended to be placed on the same corresponding structures of each specimen, and are used
130 for further morphometric analysis. Due to the lack of prominent features on the veliger shells,
131 consistent placement of landmarks on the images was difficult. The grid remedied this problem,
132 allowing for identical placement of 23 landmarks on each image. After placing the grid but before
133 placing the landmarks, the program tpsUtil was used to convert sample images into a TPS file
134 (Rohlf 2006b). TPS files are the standard for storage of datasets comprising multiple
135 measurements and spatial data.
136 Landmarks were placed in the same locations of the image, based on the grid placed earlier over
137 the image, and in the same order using tpsDig2 software (Rohlf 2006a). Supplementary Figure 1
138 shows examples of a landmarked specimens. Images displaying shells broken over a landmark
139 area were rejected. Two different landmarking techniques were used. The primary dataset used 23
140 landmarks to cover the majority of the veliger outline. The alternative dataset used 9 landmarks to
141 mark the extremities of the protoconch I shell. Both landmarking methods used the same methods
142 with the tps suite as described below (Rohlf 2007).
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143 In the primary dataset the number of landmarked M. parthenopeus specimens was 76 and of M.
144 pilearis ten specimens. For the alternative dataset, reduction in number of landmarks allowed for
145 inclusion of specimens with light damage not in landmarked areas. This second analysis included
146 80 images of M. parthenopeus and eleven images of M. pilearis. The M. pilearis egg clutch did
147 not hatch out fully, so fewer veligers were able to be sampled and fewer specimens able to be
148 successfully imaged without significant damage to the shell.
149 Partial warp scores were calculated using tpsRelw software (Rohlf 2007b). The first step in
150 calculation of a partial warp is the generation of an average or consensus shape. This calculation
151 is done by rotating and superimposing landmarks to minimize the distances between corresponding
152 landmarks for all the specimens of the species. Next the principal warps were calculated and
153 represented by a thin-plate spline function; the hypothetical average specimen of the species of
154 interest. This average was used as the reference specimen for further calculations. How much
155 change was required to fit each individual specimen to the average was recorded as the partial
156 warp scores.
157 The partial warps are inherently based on the deviation from the average for the species. Partial
158 warp scores define total local deformation as compared to the theoretical average (or consensus)
159 coordinates (Conde-Padin 2007; Avaca et al. 2013). However, there are some disadvantages to
160 using partial warps to quantify individual specimens. Namely, the average reference specimen
161 does not take into account individual peculiarities of specimens.
162 To explore further the quantification of shape of M. pilearis and M. parthenopeus veligers, the
163 relative warp scores were calculated using tpsRelw software (Rohlf 2007b). Relative warp scores
164 were calculated starting from the partial warps, but weighted by the uniform shape component of
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165 the deformation. This weighting corrects for the individual peculiarities of a specimen and makes
166 the relative warps mathematically stable. Therefore, relative warps are not subject to the drawbacks
167 of partial warps. The relative warp scores of a specimen represent all shape-based variation in that
168 specimen, independent of size (Avaca et al. 2013). The eigenvalues of the relative warps quantify
169 how much variance each relative warp accounts for. In the case of this study, eight relative warps
170 were sufficient to account for over 90% of the variation in the primary dataset. In the alternate
171 dataset, four relative warps accounted for over 94% of the variation. Regression of these shape
172 variables onto the species variable and generation of weight matrixes was done using tpsRegr
173 software (Rohlf 1990; Rohlf 1993; Rohlf 2007a).
174 To test whether it was possible to identify veligers based on their relative warp values, a randomly
175 selected subset of M. parthenopeus and M. pilearis relative warps from the primary dataset were
176 each used as ‘unknowns’. These subsets were then compared with two separate one-way Kruskal-
177 Wallis tests to find whether the given ‘unknown’ dataset was significantly similar to either the
178 complete M. parthenopeus relative warp dataset or the complete M. pilearis relative warp dataset.
179 A more complex linear model was used to determine if a predictive model for species could be
180 built including the first eight relative warps. M. parthenopeus species was assigned arbitrarily a
181 value of 1 and M. pilearis a value of 0 for the purposes of this model. These calculations were also
182 conducted with R 3.2.0 in RStudio.
183 Centroid size was computed using tpsRelw (Rohlf 2007b). Centroid size represents a measure of
184 the size of each specimen independently of shape variation. It is calculated as the root mean square
185 distance between each landmark (Conde-Padin 2007; Avaca et al. 2013). The centroid was
186 calculated for each individual specimen.
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187 The centroid size measurements for each species in the primary dataset follow a normal
188 distribution as suggested by a Shapiro-Wilk normality test (M. parthenopeus centroid size, W =
189 0.97, p = 0.106; M. pilearis centroid size, W = 0.936, p = 0.5185) and mean centroid size between
190 species were compared using a parametric student’s t-test using R 3.2.0 in RStudio. Nonparametric
191 statistics were used to compare centroid size in the alternate dataset after a Shapiro-Wilk test
192 revealed the centroid size was not normal (W = 0.85657, p-value < 0.00002). Non-parametric
193 statistics (Wilcoxon tests and Kruskal-Wallis tests) were used to compare all relative warp scores
194 because their distribution was not normal.
195 Outline-based morphometric techniques were used to describe the dataset further. Due to the low
196 number of features for landmarks, an outline based morphometric software (DiaOutline) was used
197 to quantify shape (Wisherkerman and Hamilton 2018). Fourier ellipse outline-based morphometric
198 techniques offer several advantages for our dataset (Wisherkerman and Hamilton 2018). Fourier
199 ellipse-based techniques do not require homologous landmarks and are independent of the position
200 of the shape being described (Wisherkerman and Hamilton 2018; Koca et al. 2018). In brief, this
201 technique uses a set of sine and cosine curves (harmonics) to describe the outline of the shape
202 selected (Koca et al. 2018; Wisherkerman and Hamilton 2018). Each harmonic is described by
203 four Fourier coefficients representing the size and shape in two-dimensional space (Haines and
204 Crampton 2000; Koca et al. 2018; Wisherkerman and Hamilton 2018). These Fourier coefficients
205 provide a powerful tool for further analysis. For this analysis, 38 images of M. parthenopeus and
206 12 images of M. pilearis were able to be read and outlined by DiaOutline. See Supplementary
207 Figure 2 for the Fourier ellipse outlines of M. parthenopeus and M. pilearis as read by DiaOutline.
208 Principal component analysis (PCA) of the Fourier coefficients provided by DiaOutline was
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209 performed in R 3.2.0 in RStudio. Further, a MANOVA test was performed to test the significance
210 of any differences in the PCA, also in R 3.2.0 in RStudio.
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211 Results
212 Procreation and Early Development of Monoplex pilearis
213 The egg clutch and number of eggs of M. pilearis were consistent with the earlier observation
214 made by Muthiah (Muthiah 2000a). The egg basket was 5 cm in diameter, eggs were spherical,
215 white, opaque (Fig 2), and the egg clutch itself was clear and similar to thin plastic in texture. One
216 week after the M. pilearis eggs were laid, veligers were found to have cilia and were capable of
217 moving within the egg clutch (Fig 3). After ten days, veligers developed distinct eyespots. At this
218 point, a brown discoloration in the egg clutch became visible owing to the accumulation of
219 veligers.
220 Here and throughout, post-laying refers to the number of days after the egg clutch was deposited
221 by the female. Post-hatching refers to the approximate time all the veligers hatched out of the egg
222 clutch and became free-living in the tank. The shell of M. pilearis at ten days post-laying was not
223 completed, which was not immediately apparent under brightfield microscopy. However, with
224 SEM imaging revealed that veligers had not yet completed the first whorl of their shell (Fig 4A
225 and B). After ten days, the veligers were capable of moving within the egg clutch, although only
226 half the shell was completed. The operculum, in comparison, appeared to be complete and
227 proportional to the size of the veliger.
228 After one month, most of the M. pilearis veligers hatched out of the egg clutch and became free-
229 swimming in the tank (Fig 4C and D). At this stage, shells became darker and notably thicker upon
230 inspection under 4x magnification. The M. pilearis egg clutch was found to have a nematode
231 infestation, with the nematodes presumably feeding on the larval gastropods and eggs.
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232 Procreation and Early Development of Monoplex parthenopeus
233 The egg clutch of M. parthenopeus was similar to the egg clutch of M. pilearis with a plastic
234 texture and a diameter of 6.5 cm. The female M. parthenopeus displayed more protective behavior
235 than the female M. pilearis. The female M. parthenopeus settled on top of the egg clutch and
236 resisted being moved while incubating the eggs (Fig 2). The egg clutch itself comprised
237 approximately 200 individual egg capsules.
238 A total of 20 days after the egg clutch was deposited, all M. parthenopeus veligers hatched out
239 from the egg clutch (Fig 4F-H). M. parthenopeus had a faster development time and hatched with
240 a completed whorl and operculum. The M. parthenopeus egg clutch was not found to have
241 nematodes present until the majority of the veligers had hatched and become free swimming.
242 However, under bright-field microscopy, no obvious morphological differences were visible
243 between M. parthenopeus and M. pilearis veligers.
244 Morphometrics – Statistical Analysis
245 After calculating the centroid data using the landmarked SEM images, it was found the M. pilearis
246 veligers were significantly larger than the M. parthenopeus veligers (Fig 5). This was the case
247 regardless of the number of landmarks used to define the shape. Since there was a significant
248 difference in size between the two species, a correlation test was used to determine if there was a
249 relationship between shape and size that could be confounding statistical analysis. Using the
250 primary dataset, it was found that the size and shape of each species were not correlated
251 (rs = -0.19, p = n.s.) (Fig 6).
252 There were no statistically significant differences in shape between species based on comparing
253 the relative warp scores of the primary dataset (Table 2). A comparison of the partial warp scores
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254 from the primary dataset also could not be used to distinguish the shell morphology of the two
255 species. A limited number of M. pilearis veligers were able to be sampled and landmarked;
256 therefore, their warp scores had smaller variability compared to the larger M. parthenopeus dataset
257 (Supplementary Material Figure 3). The M. pilearis partial warp scores overlapped the M.
258 parthenopeus partial warp scores. A two-dimensional regression analysis of the partial warp scores
259 onto the independent variable (in this case, species) allows for visualization of geometric
260 differences between species (Fig 7). For a full statistical analysis of partial warp scores regressed
261 onto the independent (species) variable, please see Supplementary Tables 1–9 (Rohlf 2007a).
262 Figure 7 indicates minor differences in shell shape, especially around the aperture, yet these
263 differences were insufficient to cause statistical significance between the morphology of M.
264 parthenopeus and M. pilearis according to the relative warp scores (Table 2).
265 Further insight was obtained by examining the bending energies of an individual specimen
266 compared to the reference shape and determining which landmarks contributed most to specimen
267 variation from the reference. The first three landmarks on both species in the primary dataset had
268 partial warp values outside of the other landmark partial warp values from 0.5 to -0.5. The other
269 landmarked areas with the most variability in both species were landmarks 7, 8, 10, 11, 17, and
270 20–23. Since the partial warp values quantify differences from the average, the more extreme
271 values in the first three landmarks demonstrate that those three areas are the most variable.
272 To test whether it was possible to identify veligers based on their relative warp values, a randomly
273 selected subset of M. parthenopeus and M. pilearis relative warps from the primary dataset were
274 each used as ‘unknowns’. These subsets were then compared with two separate one-way Kruskal-
275 Wallis tests to find whether the given ‘unknown’ dataset was significantly similar to either the
276 complete M. parthenopeus relative warp dataset or the complete M. pilearis relative warp dataset.
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277 The Kruskal-Wallis tests demonstrated that the ‘unknown’ relative warps were not statistically
278 different from either the M. pilearis or M. parthenopeus relative warps. A more complex linear
279 model was used to determine if a predictive model for species could be built including the first
280 eight relative warps. M. parthenopeus species was assigned arbitrarily a value of 1 and M. pilearis
281 a value of 0 for the purposes of this model. The linear model was not able to predict species based
282 on the first eight relative warps. Higher order non-linear models were not attempted due to earlier
283 results demonstrating no statistically significant differences between any of the relative warps of
284 M. pilearis and M. parthenopeus. The results of the linear model and all statistical analysis are
285 given in Supplementary Material Tables 10 and 11 with Supplementary Equation 1.
286 Using the alternate dataset, similar results were obtained as with the extensively landmarked
287 dataset. See Supplementary Tables 1-9 for full data from alternate dataset. Significant difference
288 was only seen in the first relative warp score (W = 138, p = 0.03) (Table 2). The centroid size
289 calculations were similar to the previous results, showing a significant size difference between the
290 two species (W = 0, p-value < 0.0001).
291 All statistical analyses for elliptical Fourier analysis were successfully performed using the
292 DiaOutline software with PCA. There was no clear distinction between species shape as defined
293 by PCA, see Fig. 8. When Fourier ellipses were used to define outline shape, a MANOVA test
294 found no statistical differences between M. parthenopeus and M. pilearis shape (approx. F = 1.263,
295 p = 0.2778).
296 Discussion
297 Incubation Behavior and Early Development
298 This study elaborates on the early development and protoconch structure of M. parthenopeus and
299 M. pilearis. An interesting contrast between species was the incubation behavior by the female M.
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300 parthenopeus, which was not observed in the female M. pilearis. The female M. pilearis moved
301 away from the egg clutch almost as soon as it was laid, whereas the female M. parthenopeus
302 displayed more aggressive protective behavior, incubating the egg clutch for several weeks. In
303 addition, the M. parthenopeus egg clutch did not suffer from the same infestation of nematodes as
304 the M. pilearis egg clutch. Indeed, heavy nematode predation reduced the number of M. pilearis
305 veligers available for sampling and subsequent imaging. This observation suggests the incubation
306 behavior of M. parthenopeus had an initial protective effect on the eggs.
307 Previous work established the protective effect of brooding behavior on developing egg capsules
308 in Gastropoda (Chaparro et al. 2008; Andrade-Villagran et al. 2018; Chaparro et al. 2019).
309 Buccinum scalariforme has been observed to guard egg masses for approximately three days after
310 laying, while various Crepipatella spp. incubated eggs from two days to two weeks (Montory et
311 al. 2014; Andrade-Villagrán et al. 2018; Chaparro et al. 2019). One purpose of maternal brooding
312 behavior in Crepipatella dilatata is to protect the eggs from salinity changes in an estuarine
313 environment; the mother being able to completely seal off the eggs from outside water flow
314 (Chaparro et al. 2008). However, in Cymatiidae, complete isolation of the eggs from the outside
315 is not anatomically possible because brooding mothers deposit the eggs on the substrate and cover
316 the eggs with their foot and shell. This type of maternal brooding behavior, while different from
317 Crepipatella spp., clearly still has benefits for the developing larvae and is found in multiple
318 species of Cymatiidae (Laxton 1969). Laxton observed brooding behavior of several months in
319 Charonia lampas (previously referred to as C. capax and C. rubicunda) as well as Cabestana
320 spengleri (Laxton 1969; Board 2018). He also noted the brooding behavior in M. parthenopeus
321 (synonymized with Monoplex australasiae), recording the incubation time as one month (Laxton
322 1969; Board 2018). Given that this incubation behavior was recorded for multiple other
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323 Cymatiidae and previous work did not explicitly note any brooding behavior of M. pilearis
324 (Muthiah 2000b), the absence of brooding behavior in female M. pilearis is worth considering
325 (Laxton 1969). Despite absence of brooding behavior noted in the past and present studies done in
326 aquaria, it is premature to assume that M. pilearis never incubates egg masses because laboratory
327 conditions possibly influenced natural behavior (Muthiah 2000b).
328 The different development times of the two species was also noted. M. parthenopeus emerged
329 from the eggs with a nearly complete whorl about twelve days after the egg clutch was first
330 deposited and after a total of 20 days, there were no M. parthenopeus veligers left in the egg clutch.
331 M. pilearis took ten days to finish partially developing the shell and a total of 30 days to begin
332 emerging from the egg clutch. It took approximately 40 days for all the M. pilearis veligers to
333 hatch out of the egg clutch. M. parthenopeus could in general emerge earlier as a smaller veliger,
334 whereas M. pilearis spends a longer time incubating and emerges as a larger veliger. This finding
335 may provide a selective advantage for M. pilearis veligers; a larger size could assist in deterring
336 zooplankton predators. However, selection pressure may also account for shorter incubation times,
337 such as evident in M. parthenopeus. Veligers may benefit by spending less time immobile and
338 relatively vulnerable in the egg clutch.
339 Previous literature has established that the larval shell may indeed protect from predation by
340 zooplankton (Hickman 1999; Hickman 2001). In fact, morphological features of the shell such as
341 spiral sculpting on the shell may reduce breakage of the shell when subjected to stress (Hickman
342 1999; Hickman 2001). However, the Monoplex veligers studied herein were not of a sufficient age
343 to have developed some of the shell details present in older veligers, such as ridges, sculpting, or
344 an aperture beak (Hickman 1999; Hickman 2001). The veligers in this study were recently hatched
345 from embryo stage and only exhibited protoconch I (Robertson 1971; Jablonski and Lutz 1983;
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346 Sang et al. 2019). Protoconch II generally has more ornamentation and might provide more
347 features for morphometric landmarking (Robertson 1971; Jablonski and Lutz 1983; Sang et al.
348 2019). Previous literature remarked on ‘granulation’ upon the surface of the shell of some larval
349 gastropods, presumably mineralized microprotruberances (Hickman 1999). The above images of
350 Monoplex veligers revealed that these two species did not exhibit these same microprotruberances;
351 rather the texture of the shell appeared cratered and wrinkled. The texture observed in Monoplex
352 veliger shells may be a result of the shell formation process (Fig 4). Images capturing the leading
353 edge of the calcifying whorl of M. pilearis was of particular interest. Plates, presumably of calcium
354 carbonate, seemed to be secreted at the leading edge and then fused to form the complete shell.
355 These plates resulted in a pitted appearance of the larval shell (Fig 4E).
356 Earlier SEM images of developing volute veliger shells of Odontocymbiola pescalia
357 (Penchaszadeh et al. 2017) were different from the Monoplex veligers described herein. Neither
358 the volute veligers nor the Monoplex veligers exhibited the microprotruberances described by
359 Hickman (Hickman 1999), yet the volute veliger shell did not demonstrate the ‘plate’ or ‘pitted’
360 appearance of Monoplex veligers (Penchaszadeh et al. 2017). It is worth noting that volute larvae
361 exhibit direct development as opposed to the long planktonic development of Monoplex (Pechenik
362 1984; Hickman 1999; Penchaszadeh et al. 2017). The differences in shell secretion and formation
363 may be related to the differences in life-cycle of these species (Pechenik 1984; Hickman 1999;
364 Penchaszadeh et al. 2017).
365 Statistical Analysis and Limitation of Morphometrics
366 No significant difference in shape was found between the two species using geometric
367 morphometrics. However, a few interesting results came from the examination of the bending
368 energies of the partial warp scores. Since the partial warp values quantify differences from the
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369 average, the more extreme values in the first three landmarks demonstrate that those three areas
370 are the most variable. Landmarks 1–3 and 20–23 are around the aperture area (see Supplementary
371 Figure 1), again supporting the conclusion that the aperture area has a good deal of variation within
372 species and between species. However, these differences might be due to some shell damage and
373 therefore these statistics may be of limited use for specimen identification.
374 Using the alternate dataset with less landmarks, there was significant difference found in only the
375 first relative warp. The other relative warps showed very high p-values. The first relative warp was
376 not highly significant with a p-value only slightly under 0.03. This might be a result of the lower
377 number of landmarks, which only take into account the extremities of the shell. Given that other
378 morphometric techniques found no significant differences in shape, this casts doubt on the one
379 significant p-value in this dataset. This result is likely an artifact of analysis and not a useful tool
380 for future identification.
381 Fourier ellipse description with PCA did not identify statistically significant differences between
382 the protoconch I of the two veliger species. There are some limitations identified with Fourier
383 ellipse morphometrics; one being that higher-order harmonics are given less importance in the
384 analysis (Wisherkerman and Hamilton 2018). This may reduce the effects that outline details have
385 on downstream statistical analysis (Wisherkerman and Hamilton 2018). Despite this limitation, the
386 main problem in this case study is likely the limited number of satisfactory images obtained for
387 both species. Not only that, the featureless nature of the shells and their similar appearance make
388 even advanced morphometric techniques of little value here.
389 There may be further reasons that morphometrics was insufficient in this case, despite being an
390 effective tool to determine shape difference within and between other gastropod species (Avaca et
391 al. 2013; Marquez 2017; Doyle 2018). A study published in 2007 used similar morphometric
19
392 techniques from this study to differentiate shells from Littorina saxatilis, and the authors claimed
393 that they were able to discriminate between two distinct shell morphologies within the same
394 species (Conde-Padin 2007). The study concluded that the two differing shell morphologies of
395 Littorina saxatilis were based on habitat and exposure to wave action (Conde-Padin 2007). The
396 successful ability of the software to distinguish between subtypes of other gastropod species, even
397 down to environment subtypes within the same species, begs the question of why this study found
398 no statistical difference between M. parthenopeus and M. pilearis veligers.
399 The work described here was the first to use morphometrics on SEM images of veliger gastropods.
400 The use of SEM provided the high-resolution images that made morphometric analysis possible,
401 yet came with several drawbacks. One is that it was impossible to arrange all specimens in the
402 same orientation and impossible to ensure all specimens were free of damage. Many sample
403 images were excluded from the landmark-based morphometrics because of slightly different
404 orientation or damage to the fragile shells, limiting the sample size and reducing the statistical
405 power of our observations. The aperture of the specimen was generally the most prone to damage
406 yet appeared to be the most distinctive feature when intact. In previous studies using adult
407 gastropods, imaging was far more straightforward and shells were arranged in identical
408 orientations with no damage (Conde-Padin 2007; Avaca et al. 2013; Marquez 2017; Doyle 2018).
409 In addition, sample preparation and mounting when using SEM is far more labor-intensive,
410 creating a logistical barrier to making an extensive sample set. These factors limit the statistical
411 power of this study and possibly of future studies using SEM images coupled with morphometric
412 analysis. Despite these difficulties, the other limiting factor in this study was the feature-less nature
413 of weeks-old veligers. At this stage in development, there are no features that would provide
414 distinctive morphology. Previous landmark-based studies were able to place multiple landmarks
20
415 with consistency on the shell itself, negating the need to use a grid as a reference (Conde-Padin
416 2007; Avaca et al. 2013; Marquez 2017; Doyle 2018). The veligers in this study of both species
417 had few, if any, distinctive features. Morphological differences become more distinctive at later
418 stages of growth, though these species develop slowly (Beesley 1998).
419 Future work could repeat a similar workflow with veligers older than one month. One caveat to
420 such an experiment is that rearing veligers past a month in a laboratory have had limited success
421 for most Cymatiidae (Muthiah 2000a; Strathmann and Strathmann 2007). It should not be
422 overlooked that morphological identification is challenging or close to impossible with some
423 veligers, as has been noted in several ecologically important bivalves (Claxton and Boulding 1998;
424 Hendriks et al. 2005). These morphologically cryptic species are typically identified using
425 molecular biology techniques (Claxton and Boulding 1998; Sparagano et al. 2002; Abalde et al.
426 2003; Wang et al. 2006). In summary, we propose that future work aiming at species level
427 identification of Cymatiidae veligers should focus on a molecular biology approach.
428 Conclusion
429 This study reports the biology and morphology of veligers of the Cymatiidae species M. pilearis
430 and M. parthenopeus, including the first high-resolution SEM images of the veligers. Differences
431 were observed in guarding behavior and incubation time of the two species and it was found M.
432 pilearis larvae emerge slower and larger than M. parthenopeus. Calculations from the SEM images
433 demonstrated significant centroid size differences between the two species upon emerging from
434 the egg capsule. SEM images also show the secretion of ‘plates’ which fuse to form the completed
435 shell, though no other distinctive shell features were observed herein.
21
436 Morphometric analysis in this case was unable to differentiate between SEM images of the two
437 species, likely due to the highly conserved morphology and limited number of intact samples. This
438 study has demonstrated that despite the differences in size, differences in shape of protoconch I
439 appear to be insufficient to make a species level identification when the veligers are two weeks
440 old. In the case where a specimen of unknown origin or age is obtained, a reliable identification to
441 the species level might not be possible even with high-resolution SEM.
442 Future studies examining the morphology and growth of Cymatiidae veligers should note that the
443 veligers must have developed at least past two weeks for identification. Longer development times
444 are likely required for the formation of protoconch II, making identification of field-collected
445 specimens of unknown age more difficult. It is challenging at best to rear sufficient numbers of
446 veligers to an age where protoconch features become distinctive enough to allow for statistically
447 rigorous morphometric differentiation.
22
448 Acknowledgements We thank Kathryn Green and Nicolas Condon from the Center for
449 Microscopy and Microanalysis at the University of Queensland, St. Lucia; the UQ Biological
450 Resources Department personnel for care of the tritons in aquaria, Mr. Greg Knight and the
451 employees of his oyster lease, the Clout family of Kooringal Oysters, and everyone at Moreton
452 Bay Rock Oyster Company for donating specimens of M. parthenopeus. Thanks also to Dr. David
453 Anning for assisting in collecting M. pilearis and Dr. John Healy, Curator of Molluscs at the
454 Queensland Museum, for his expertise and assistance in specimen identification. This research
455 was funded by Australian Research Council (ARC) Discovery Project DP150103990 awarded to
456 Q.K. and D.J.C. A.H.T. was supported by a University of Queensland Research Training Tuition
457 Offset Scholarship and a University of Queensland Training Program Living Allowance
458 Scholarship, C.I.S. was supported by an ARC Future Fellowship (FT160100055) and an Institute
459 for Molecular Bioscience (IMB) Industry Fellowship and D.J.C. was supported by an ARC
460 Australian Laureate Fellowship (FL150100146).
23
461 Data Availability:
462 All data generated or analyzed during this study are included in the published article and
463 supplementary material. All images generated during this study are available from the authors
464 upon reasonable request.
465
466 Conflict of Interest Statement:
467 The authors declare they have no conflict of interest.
468
469 Ethical Approval:
470 This article does not contain any studies with human participants or animals performed by any of
471 the authors. See University of Queensland Animal Ethics Committee definition of animal research.
24
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571 Figure Captions:
572 Figure 1 Site map of collection site of M. parthenopeus and M. pilearis. Collection was carried
573 out at Amity Point, North Stradbroke Island and oyster leases on Moreton Island, Queensland,
574 Australia. Map adapted from Google Maps
575 Figure 2 Egg clutches of M. pilearis and M. parthenopeus. A) View from above and, B) view
576 from side of female M. pilearis with freshly laid egg clutch. C) Female M. parthenopeus incubating
577 egg clutch and D) M. parthenopeus egg clutch after larvae have emerged from eggs and begun to
578 form shells, approximately 12 days post laying
579 Figure 3 Brightfield microscopy image of M. pilearis veligers ten days post-laying. A) View
580 of bilobate veliger from the side and, B) view with cilia and eyespots immediately facing the lens
581 Figure 4 SEM comparison of M. pilearis and M. parthenopeus. A) SEM images (200x
582 magnification) of multiple M. pilearis veliger at ten days post laying. B) SEM image of M. pilearis
583 at 270x magnification, ten days post laying. C) M. pilearis veliger seven days after emergence
584 from egg clutch. Shown at 440x magnification. D) M. pilearis veliger seven days after emergence
585 from the egg clutch, apertural view, shown at 400x magnification. E) Image (1000x magnification)
586 of the growing edge of the M. pilearis larval shell, demonstrating secreted ‘plates’ fusing to form
587 a completed shell. F) M. parthenopeus veligers seen under brightfield microscopy 12 days post
588 laying; G) M. parthenopeus veligers 12 days after eggs were laid and immediately after emerging
589 from egg clutch and, H) M. parthenopeus, 12 days after eggs deposited and immediately upon
590 emerging from the egg clutch, operculum view
591
30
592 Figure 5 Comparison of the centroid sizes of M. parthenopeus and M. pilearis veliger lateral
593 view images. The data shown is from the primary dataset. The centroid sizes of M. parthenopeus
594 and M. pilearis were statistically significant according to two-sample normal Student’s t-test with
595 a p-value of < 0.00001.
596 Figure 6 Correlation of centroid size and relative warp of M. parthenopeus veliger side view
597 images. Values of Relative Warp 1 plotted against centroid size for all M. parthenopeus veliger
598 specimens. Data show no correlation between centroid size and shape variables (Spearman
599 correlation test, rs = - 0.057, p = 0.599)
600 Figure 7 Partial warp score regression of M. pilearis and M. parthenopeus veliger side view
601 images. Graphs of shape data (as partial warp scores) regressed onto individual species a) M.
602 pilearis and b) M. parthenopeus
603 Figure 8 PCA analysis of M. pilearis and M. parthenopeus outlines. As given by DiaOutline,
604 PC 1 and PC 2 shown. M. pilearis data space shown in green, M. parthenopeus data space in red.
605
31
606 Figure 1
607 608
32
609 Figure 2
610 611
33
612 Figure 3
613 614
615
616
34
617 Figure 4
618
35
619 Figure 5
620
621 622
623
36
624 Figure 6
625 626
627
628
37
629 Figure 7
630 631
38
632 Figure 8.
633
39
634 635 Table 1 Water quality specifications during the time both species laid eggs and when the 636 veligers hatched (150 days)
637
a pH Temperature (˚C) Dissolved O2 (%) Salinity (PSU ) NO3 (ppm) Average 8.2 ± 0.7 26 ± 2 79 ± 26 35.3 ± 3 5.1 ± 5 638 aPractical Salinity Units
639
640
641
642
40
643 Table 2 Results of statistical analysis comparing relative warp scores. All data non-normal, 644 Wilcoxon rank sum tests performed.
645 Dataset 1 n = 86 Alternate Dataset n = 93 Sample Wa P value Sample Wa P value RW1b 423 0.3749 RW1b 138 0.026 RW2 341 0.7916 RW2 258 0.7075 RW3 386 0.7174 RW3 241 0.7174 RW4 363 0.9716 RW4 231 0.8555 RW5 360 >0.9999 RW6 380 0.7804 RW7 339 0.7694 RW8 366 0.937 RW9 321 0.578 646 aWilcoxon Score 647 bRW = relative warp 648 649
41