# Molecular Phylogeny and Morphological Distinctions of Two Popular Bivalves,Ctenoides scaber andCtenoides mitis

Authors: Lindsey F. Dougherty; Jingchun Li
Journal: Journal of Marine Biology
 (2017)

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## Abstract

One of the most well-known species in the bivalve family Limidae (d’Orbigny, 1846) is the brightly coloredCtenoides scaber (Born, 1778), commonly known as the rough file clam or flame scallop. Distinguishing this bivalve from its close relative,C. mitis(Lamarck, 1807), can be difficult using only morphological features and has led to much taxonomic confusion throughout the literature. In this study, morphological characters were compared to a molecular phylogeny constructed using three genes (COI, 28S, and H3) in order to differentiateC. scaber andC. mitis.The phylogeny recovered two well-supported clades that differ significantly in shell rib numbers, but not tentacle colors. The two species were then placed in a larger phylogenetic context of the Limidae family, which revealed the need for further systematic revision across genera. As these bivalves are popular in aquaria, cannot be tank-raised, and have been overcollected in the past, proper species identification is important for assessing sustainable collection practices.


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## Body

## 1. Introduction

The rapid increase of home aquaria owners (~1.5–2 billion people) and the lucrative global trade, valued at up to $330 million annually [1], have led to the overcollecting of many marine organisms [2, 3]. More than 500 species of marine invertebrates at numbers between 9 and 10 million are traded as ornamental species annually [1]. Although there have been some advances in breeding ornamental marine species [4], the majority are still being collected from the wild. The impacts of ornamental fisheries on invertebrate species with regard to their reproductive age, growth rate, population density/distribution, and population connectivity are largely unknown [5].One of the obvious challenges for evaluating the impact of ornamental fisheries is to correctly document the numbers of species collected from various localities [6], especially given that cryptic species in marine ecosystems are common [7], and taxonomy in many marine invertebrate groups awaits systematic revision [8]. Currently, a large number of marine invertebrates have not been evaluated for IUCN (International Union for Conservation of Nature) status, despite their popularity in the aquarium trade.One popular invertebrate group in the ornamental fisheries trade is the bivalve family Limidae, colloquially known as file shells or file clams. They are well-known to SCUBA divers [9] and shell collectors, and several charismatic species are routinely collected for the aquarium trade [10]. Numerous studies have been conducted on their behavior [11–16], morphology [17–19], habitat [20–22], reproduction [23, 24], physiology [25], and vision [26–31]. However, systematics studies have only been done on a small number of species in this family [10, 19, 32–35]. It is also noted that conchological traits alone are not always sufficient to establish clear boundaries between taxa [36].One of the most well-known species in Limidae isCtenoides scaber (Born, 1778), which in the past has been protected with imposed daily bag limits in the state of Florida due to overcollection [10]. It is known as the “flame scallop” due to its bright red coloration. According to a single retailer (Blue Zoo Aquatics, Hawthorne, CA, USA), around 10C. scaber are sold each month(pers. comm.). Multiplying this average by the numerous retailers and wholesalers worldwide may warrant unsustainable collection levels. Furthermore, collection of these bivalves is difficult, as they inhabit rocky areas or crevices in corals [24] and substantial damage can be done to the local habitat when they are harvested. Aquaculture of the species is not possible because spawning cannot be induced in the laboratory, and larvae from wild-collected specimens did not survive the past 36 hours [24].Documentation and identification ofCtenoides scaber in aquarium trade appear to be highly inconsistent and inaccurate. For example, the Aquariumtradedata.org database [6] uses shipment declarations and commercial invoices from the United States Fish and Wildlife Service to track aquarium trade data. The database listed 4,820 and 9,670C. scaber being imported to the US in 2008 and 2011, respectively. But in 2009, only 22 individuals were documented. In addition, the documented countries of origin are the Philippines and Indonesia, even though the trueC. scaber is a Caribbean species.Compounding the unknown collection rates ofCtenoides scaber is the difficulty in differentiating it from the presumably sister speciesC. mitis. The classical distinction between the two species was thatC. scaberhad red pallial tentacles andC. mitishad white pallial tentacles [10]. However, this was based only on populations from the Florida Keys, and variation in tentacle color outside the Florida Keys required further scrutiny [10]. Mikkelsen and Bieler [10] found that the number of radial ribs (C. scaber28–78, mean 55, andC. mitis59–149, mean 89) differed between the two species, but they were not able to confirm any of the other previously cited differences, including shell size, shape, opacity, or distribution. In addition, several questions remain unanswered, including how much of tentacle color variations are due to phenotypic plasticity, and whether the two species are actually reproductively isolated.Therefore, the goal of this study was to assess the phylogenetic status ofCtenoides scaber andC. mitis and to investigate their diagnostic morphological characteristics. Multigene molecular phylogenies were constructed for the two species as well as the Limidae family. Museum and aquaria specimens were examined to determine tentacle coloration and shell rib counts. Our results will aid proper identification of the two species for future collection management and provide a better understanding of the overall phylogeny of Limidae bivalves.


## 2. Methods

### 2.1. Sampling

Museum specimens were loaned from the University of Florida Museum of Natural History (see Supplementary Table1 for catalogue numbers). Species identifications were given based on museum labels (Ctenoides scaber: n=3;C. mitis: n=8). Additional “flame scallops” (presumablyC. scaber, n=18) were purchased through Aqua Imports (Boulder, CO, USA), which were collected in Haiti (n=10) and the Florida Keys (n=8). The outgroupsCtenoides ales (n=1) andLimariasp. (n=2) were also purchased through Aqua Imports, which were collected in the Philippines. All store-bought specimens were deposited in the University of Colorado Museum of Natural History (Supplementary Table 1).


### 2.2. Molecular Data

Genomic DNA was extracted from the mantle edge (c.25 mg) of each specimen using the E.N.Z.A. Mollusc DNA Kit (Qiagen) according to the manufacturer’s protocol. The mitochondrial gene COI, the nuclear ribosomal gene 28S, and the nuclear histone protein-encoding gene H3 were amplified for each specimen. Primers used to amplify the genes are listed in Table 1. The GoTaq® Hot Start Green Master Mix was used at concentrations following the manufacturers’ instructions. The Polymerase Chain Reaction (PCR) cycling parameters for COI were as follows: initial denaturation of 94°C for 1 min and then 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension of 72°C for 6 min. For 28S, an initial denaturation of 94°C for 4 min was used, followed by 36 cycles of 94°C for 40 s, 53°C for 40 s, and 72°C for 1 min and a final extension of 72°C for 10 min. For H3, an initial denaturation of 94°C for 1 min was used, followed by 35 cycles of 94°C for 30 s, 49°C for 30 s, and 72°C for 30 s with a final extension of 72°C for 6 min. PCR products were visualized using agarose gel electrophoresis (1% agarose). PCR purification and sequencing were conducted by Quintara Biosciences (Berkeley, CA, USA). All sequences were submitted to Genbank and their accession numbers are listed in Supplementary Table 1.Table 1
PCR primers used for gene amplification onCtenoides scaber and C. mitis.
Gene
Primer name
Sequence (5′-3′)
Source
COI
LCO1490
GGT CAA CAA ATC ATA AAG ATA TTGG
Folmer et al. (1994)
HCO2198
GTA AAT ATA TGR TGD GCTC
Folmer et al. (1994)
CTE 54F
AAG GGG GAT TGC CTT TAG CC
This Study
CTE 601R
GCC GTG TTT ACA TGG CGA TCG GT
This Study
CTE 32F
TGT TGG GGT TTT GGT CGT CT
This Study
CTE 664R
GGT ACA AAA CAG GGT CCC CC
This Study
28S
Limoida_121F
TCA GAC GAG ATT ACC CGC TGA ATT TAA GC
This Study
Sc28S_950R
TCT GGC TTC GTC CTA CTC AAG CAT AG
This Study
H3
H3aF
ATG GCT CGT ACC AAG CAG ACV GC
Colgan et al. (1998)
H3aR
ATA TCC TTR GGC ATR ATR GTG AC
Colgan et al. (1998)


### 2.3. Phylogenetic Analysis

Sequences ofCtenoides scaber,C. mitis, C. ales,andLimariasp. were assembled using CodonCode Aligner© software 7.0.1 (Centerville, MA, USA) and aligned using MUSCLE [38]. Gene substitution models were selected using Partitionfinder 2 [39] based on Akaike Information Criterion. The GTR+G model was selected for COI, the GTR+G model was selected for 28S, and the GTR model was selected for H3. Maximum-likelihood (ML) and Bayesian phylogenetic reconstructions were conducted for the three genes individually as well as the concatenated dataset. The ML analyses were conducted using the RAxML BlackBox online server with 100 bootstraps [40, 41]. Bayesian analyses were conducted using MrBayes [42]. For each dataset, two runs were conducted simultaneously and trees were sampled every 10 iterations. Bayesian searches were run for 500,000 iterations for the COI, 28S, and H3 datasets. For the concatenated dataset, the analysis was run for 1,000,000 iterations. Convergence was ensured for each Bayesian run. For all datasets, a 50% majority rule consensus tree was generated after a 10% burnin and visualized in FigTree v1. 3.1 [43] (Figure 1).Figure 1
Bayesian topologies ofCtenoides scaber andC. mitis.(a) Concatenated gene tree. (b)–(d) Individual gene tree for COI, 28S, and H3, respectively. Shaded individuals belong to theC. mitisclade and nonshaded ones belong to theC. scaber clade. Posterior probabilities (PP) that are greater than 99% were denoted with black dots on the nodes. Bootstrap values that are greater than 90% (based on maximum likelihood analysis) were labeled above the branches.
(a)
COI + 28S +H3
(b)
COI
(c)
28S
(d)
H3To assess the phylogenetic status ofCtenoides scaber andC. mitis in a larger comparative phylogenetic framework, Genbank sequences of the same three genes from all other available Limidae bivalves were downloaded (Supplementary Table 1). Sequences from two representativeC. scaber andC. mitis individuals (based on theCtenoides phylogeny result), as well asC. ales and twoLimariasp., were aligned with the Genbank sequences to create a concatenated dataset. ML and Bayesian phylogenies were constructed using this dataset based on methods described above (Figure 2).Mytilus edulisandPecten maximuswere used as outgroups.Figure 2
Bayesian phylogeny based on a concatenated genes (COI, 28S, and H3) showing phylogenetic position ofCtenoides scaber andC. mitis(black stars) within the Limidae family. Node labels indicating posterior probabilities and bootstrap values. TheCtenoidesphoto(C. scaber)was taken by J. Li. TheAcesta photo(Acestasp.) is Figure 2C of Gagnon et al. [37] and was reprinted with permission from the authors. TheLimaphoto (L. caribaea) was reprinted with permission from the American Museum of Natural History. TheLimaria photo(Limariasp.) was taken by L. Dougherty.


### 2.4. Morphological Analysis

Specimens were photographed using a Canon EOS Rebel® T5 Digital SLR camera with a Canon EFS F/2.8 60 mm Macro Lens. Variations in tentacle coloration were recorded for live specimens or museum vouchers that contained photographs (Figure3). Shell height and rib number were analyzed using ImageJ version 1.51n [44]. The shell height of each individual was measured as the average value (mm) of the left and right valves from the umbo to the furthest distal point. The shell rib number of each individual was recorded as the average of the left and right valves, including the auricles, and was counted near the growth edge on the main body of the shell. Shell height versus shell rib number was plotted for all individuals based on phylogroups and tentacle colors. The Mann-Whitney U test was used to determine whether the ratios were significantly different between groups.Figure 3
Tentacle color variations inCtenoides scaber andC. mitis.C. scaber colorations included red/orange (a), red/orange with white stripes (b), white with red/orange stripes (c), or white (d).C. mitis colorations were white (e), white/orange (f), or white with red bands (g). Photo credit: L. Dougherty and J. Li.
(a)
(b)
(c)
(d)
(e)
(f)
(g)


## 2.1. Sampling

Museum specimens were loaned from the University of Florida Museum of Natural History (see Supplementary Table1 for catalogue numbers). Species identifications were given based on museum labels (Ctenoides scaber: n=3;C. mitis: n=8). Additional “flame scallops” (presumablyC. scaber, n=18) were purchased through Aqua Imports (Boulder, CO, USA), which were collected in Haiti (n=10) and the Florida Keys (n=8). The outgroupsCtenoides ales (n=1) andLimariasp. (n=2) were also purchased through Aqua Imports, which were collected in the Philippines. All store-bought specimens were deposited in the University of Colorado Museum of Natural History (Supplementary Table 1).


## 2.2. Molecular Data

Genomic DNA was extracted from the mantle edge (c.25 mg) of each specimen using the E.N.Z.A. Mollusc DNA Kit (Qiagen) according to the manufacturer’s protocol. The mitochondrial gene COI, the nuclear ribosomal gene 28S, and the nuclear histone protein-encoding gene H3 were amplified for each specimen. Primers used to amplify the genes are listed in Table 1. The GoTaq® Hot Start Green Master Mix was used at concentrations following the manufacturers’ instructions. The Polymerase Chain Reaction (PCR) cycling parameters for COI were as follows: initial denaturation of 94°C for 1 min and then 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension of 72°C for 6 min. For 28S, an initial denaturation of 94°C for 4 min was used, followed by 36 cycles of 94°C for 40 s, 53°C for 40 s, and 72°C for 1 min and a final extension of 72°C for 10 min. For H3, an initial denaturation of 94°C for 1 min was used, followed by 35 cycles of 94°C for 30 s, 49°C for 30 s, and 72°C for 30 s with a final extension of 72°C for 6 min. PCR products were visualized using agarose gel electrophoresis (1% agarose). PCR purification and sequencing were conducted by Quintara Biosciences (Berkeley, CA, USA). All sequences were submitted to Genbank and their accession numbers are listed in Supplementary Table 1.Table 1
PCR primers used for gene amplification onCtenoides scaber and C. mitis.
Gene
Primer name
Sequence (5′-3′)
Source
COI
LCO1490
GGT CAA CAA ATC ATA AAG ATA TTGG
Folmer et al. (1994)
HCO2198
GTA AAT ATA TGR TGD GCTC
Folmer et al. (1994)
CTE 54F
AAG GGG GAT TGC CTT TAG CC
This Study
CTE 601R
GCC GTG TTT ACA TGG CGA TCG GT
This Study
CTE 32F
TGT TGG GGT TTT GGT CGT CT
This Study
CTE 664R
GGT ACA AAA CAG GGT CCC CC
This Study
28S
Limoida_121F
TCA GAC GAG ATT ACC CGC TGA ATT TAA GC
This Study
Sc28S_950R
TCT GGC TTC GTC CTA CTC AAG CAT AG
This Study
H3
H3aF
ATG GCT CGT ACC AAG CAG ACV GC
Colgan et al. (1998)
H3aR
ATA TCC TTR GGC ATR ATR GTG AC
Colgan et al. (1998)


## 2.3. Phylogenetic Analysis

Sequences ofCtenoides scaber,C. mitis, C. ales,andLimariasp. were assembled using CodonCode Aligner© software 7.0.1 (Centerville, MA, USA) and aligned using MUSCLE [38]. Gene substitution models were selected using Partitionfinder 2 [39] based on Akaike Information Criterion. The GTR+G model was selected for COI, the GTR+G model was selected for 28S, and the GTR model was selected for H3. Maximum-likelihood (ML) and Bayesian phylogenetic reconstructions were conducted for the three genes individually as well as the concatenated dataset. The ML analyses were conducted using the RAxML BlackBox online server with 100 bootstraps [40, 41]. Bayesian analyses were conducted using MrBayes [42]. For each dataset, two runs were conducted simultaneously and trees were sampled every 10 iterations. Bayesian searches were run for 500,000 iterations for the COI, 28S, and H3 datasets. For the concatenated dataset, the analysis was run for 1,000,000 iterations. Convergence was ensured for each Bayesian run. For all datasets, a 50% majority rule consensus tree was generated after a 10% burnin and visualized in FigTree v1. 3.1 [43] (Figure 1).Figure 1
Bayesian topologies ofCtenoides scaber andC. mitis.(a) Concatenated gene tree. (b)–(d) Individual gene tree for COI, 28S, and H3, respectively. Shaded individuals belong to theC. mitisclade and nonshaded ones belong to theC. scaber clade. Posterior probabilities (PP) that are greater than 99% were denoted with black dots on the nodes. Bootstrap values that are greater than 90% (based on maximum likelihood analysis) were labeled above the branches.
(a)
COI + 28S +H3
(b)
COI
(c)
28S
(d)
H3To assess the phylogenetic status ofCtenoides scaber andC. mitis in a larger comparative phylogenetic framework, Genbank sequences of the same three genes from all other available Limidae bivalves were downloaded (Supplementary Table 1). Sequences from two representativeC. scaber andC. mitis individuals (based on theCtenoides phylogeny result), as well asC. ales and twoLimariasp., were aligned with the Genbank sequences to create a concatenated dataset. ML and Bayesian phylogenies were constructed using this dataset based on methods described above (Figure 2).Mytilus edulisandPecten maximuswere used as outgroups.Figure 2
Bayesian phylogeny based on a concatenated genes (COI, 28S, and H3) showing phylogenetic position ofCtenoides scaber andC. mitis(black stars) within the Limidae family. Node labels indicating posterior probabilities and bootstrap values. TheCtenoidesphoto(C. scaber)was taken by J. Li. TheAcesta photo(Acestasp.) is Figure 2C of Gagnon et al. [37] and was reprinted with permission from the authors. TheLimaphoto (L. caribaea) was reprinted with permission from the American Museum of Natural History. TheLimaria photo(Limariasp.) was taken by L. Dougherty.


## 2.4. Morphological Analysis

Specimens were photographed using a Canon EOS Rebel® T5 Digital SLR camera with a Canon EFS F/2.8 60 mm Macro Lens. Variations in tentacle coloration were recorded for live specimens or museum vouchers that contained photographs (Figure3). Shell height and rib number were analyzed using ImageJ version 1.51n [44]. The shell height of each individual was measured as the average value (mm) of the left and right valves from the umbo to the furthest distal point. The shell rib number of each individual was recorded as the average of the left and right valves, including the auricles, and was counted near the growth edge on the main body of the shell. Shell height versus shell rib number was plotted for all individuals based on phylogroups and tentacle colors. The Mann-Whitney U test was used to determine whether the ratios were significantly different between groups.Figure 3
Tentacle color variations inCtenoides scaber andC. mitis.C. scaber colorations included red/orange (a), red/orange with white stripes (b), white with red/orange stripes (c), or white (d).C. mitis colorations were white (e), white/orange (f), or white with red bands (g). Photo credit: L. Dougherty and J. Li.
(a)
(b)
(c)
(d)
(e)
(f)
(g)


## 3. Results

### 3.1. Phylogenetic Analysis

For theCtenoides only analyses, both the concatenated tree (Figure 1(a)) and the COI gene tree (Figure 1(b)) recovered two strongly supported clades. One clade contained individuals with the classicCtenoides mitiscoloration (pure white tentacles) and the other contained specimens with the classicC. scaber coloration (pure red tentacles). Therefore, the two groups were tentatively defined as theC. mitis andC. scaber clades, respectively (Figure 1). However, it is worth noting that both clades also contained individuals without the typical coloration of their corresponding species (Figure 2, discussed in the next section). For the museum specimens, one preidentifiedC. scaber was placed in theC. mitis clade. For the aquarium store“C. scaber” specimens, 14 were placed in theC. scaber clade while the other 4 were placed in theC. mitis clade.Both 28S and H3 gene trees supported the monophyly of theC. mitis clade but could not resolve the relationships between theC. mitis clade and theC. scaberindividuals (Figures 1(c) and 1(d)), possibly due to the slower rate of evolution of the two genes.The family level phylogeny for Limidae (Figure2) showed strong support for the generaLimaria,Lima,Acesta, andCtenoides. However, internal relationships within these genera were occasionally poorly resolved, especially inAcesta. TheC. mitis andC. scaber clades were recovered as sister groups to one another.


### 3.2. Morphological Analysis

In theCtenoides scaberclade, four variations in tentacle coloration were observed: solid red tentacles (n=5, Figure 3(a)), red tentacles with white stripes (n=2, Figure 3(b)), white tentacles with red stripes (n=4, Figure 3(c)), and solid white tentacles (n=3, Figure 3(d)). The red coloration inC. scaber varied from dark orange to red but is recorded throughout as “red” for simplicity. In theCtenoides mitisclade,three variations in tentacle coloration were observed: solid white tentacles (n=4, Figure 3(e)), light orange tentacles (n=1, Figure 3(f)), and white tentacles with red bands (banded), which may be a coloration observed only in juveniles (n=2, Figure 3(g)).Tentacle coloration patterns of museum and aquarium store specimens based on locality and phylogenetic position are summarized in Table2. Based on the two classical morphologies (red tentacles forC. scaber and white tentacles forC. mitis), of the 10 Florida Keys specimens, 8 fell into their expected clade. Exceptions were twoC. mitis, a light orange individual and a banded individual (likely a juvenile phenotype). Of the 12 Caribbean specimens, only two fell into the expected clades (oneC. scaber and oneC. mitis). The other specimens had tentacle color variations that were not typical of either group (Table 2). The atypical colorations did not show any obvious phylogenetic signals.Table 2
Tentacle color patterns based on locality and phylogenetic position.
Florida Keys
Caribbean
Ctenoides scaberclade
Solid red (n=4)
Solid red (n=1)
Red/white stripes (n=2)
White/red stripes (n=5)
Solid white (n=2)
Ctenoides mitis clade
Solid white (n=4)
Solid white (n=1) White/red bands (n=1)
Light orange (n=1)
White/red bands (n=1)Shell rib analysis revealed that the distribution of shell rib numbers in theC. scaber clade (n=17) was significantly lower than that of theC. mitis clade (n=12, Figure 4(a), Mann-Whitney U Test, P<0.00001). Rib numbers of individuals from theC. scaber clade ranged from 37 to 58 (mean 48.3) and rib numbers in theC. mitis clade ranged from 73 to 103 (mean 85.8). Red and striped tentacles corresponded to the low shell rib number group, while light orange and banded tentacles corresponded to the high shell rib group (Figure 4(b)). White tentacles corresponded to both the low and high shell rib groups. Figure 5 shows a representative shell fromC. scaber (a) andC. mitis (b).Figure 4
(a) Relationships between shell rib numbers and shell height. Black dots represent individuals belonging to theCtenoides scaber clade and open circles represent those from theC. mitis clade. The distributions are significantly different (P<0.00001). (b) Number of shell ribs versus shell height for individuals with tentacle color information available. Red, white, striped, light orange, and banded tentacles are represented by black dots, open circles, grey dots, grey open circles, and triangles, respectively.
(a)
(b)Figure 5
(a) Representative valve ofC. scaber; individual UCM 48137 (University of Colorado Museum of Natural History). Shell height 35.4 mm; shell ribs n=50. (b) Representative valve ofC. mitis; individual UCM 48138. Shell height 34.7 mm; shell ribs n=80. Scale bar = 10 mm.
(a)
(b)In summary, our phylogenetic analysis recovered two well-supported clades corresponding toCtenoides scaber andC. mitis. Genetic variations within each clade are relatively low, but that could be the result of limited geographical sampling. Morphological analyses showed that tentacle color alone cannot be used to determine species, as colors vary inter- and intraspecifically (Figure 3). Shell rib counts gave statistically different distributions for the two species (Figure 4), but the two distributions have some degree of overlap.


## 3.1. Phylogenetic Analysis

For theCtenoides only analyses, both the concatenated tree (Figure 1(a)) and the COI gene tree (Figure 1(b)) recovered two strongly supported clades. One clade contained individuals with the classicCtenoides mitiscoloration (pure white tentacles) and the other contained specimens with the classicC. scaber coloration (pure red tentacles). Therefore, the two groups were tentatively defined as theC. mitis andC. scaber clades, respectively (Figure 1). However, it is worth noting that both clades also contained individuals without the typical coloration of their corresponding species (Figure 2, discussed in the next section). For the museum specimens, one preidentifiedC. scaber was placed in theC. mitis clade. For the aquarium store“C. scaber” specimens, 14 were placed in theC. scaber clade while the other 4 were placed in theC. mitis clade.Both 28S and H3 gene trees supported the monophyly of theC. mitis clade but could not resolve the relationships between theC. mitis clade and theC. scaberindividuals (Figures 1(c) and 1(d)), possibly due to the slower rate of evolution of the two genes.The family level phylogeny for Limidae (Figure2) showed strong support for the generaLimaria,Lima,Acesta, andCtenoides. However, internal relationships within these genera were occasionally poorly resolved, especially inAcesta. TheC. mitis andC. scaber clades were recovered as sister groups to one another.


## 3.2. Morphological Analysis

In theCtenoides scaberclade, four variations in tentacle coloration were observed: solid red tentacles (n=5, Figure 3(a)), red tentacles with white stripes (n=2, Figure 3(b)), white tentacles with red stripes (n=4, Figure 3(c)), and solid white tentacles (n=3, Figure 3(d)). The red coloration inC. scaber varied from dark orange to red but is recorded throughout as “red” for simplicity. In theCtenoides mitisclade,three variations in tentacle coloration were observed: solid white tentacles (n=4, Figure 3(e)), light orange tentacles (n=1, Figure 3(f)), and white tentacles with red bands (banded), which may be a coloration observed only in juveniles (n=2, Figure 3(g)).Tentacle coloration patterns of museum and aquarium store specimens based on locality and phylogenetic position are summarized in Table2. Based on the two classical morphologies (red tentacles forC. scaber and white tentacles forC. mitis), of the 10 Florida Keys specimens, 8 fell into their expected clade. Exceptions were twoC. mitis, a light orange individual and a banded individual (likely a juvenile phenotype). Of the 12 Caribbean specimens, only two fell into the expected clades (oneC. scaber and oneC. mitis). The other specimens had tentacle color variations that were not typical of either group (Table 2). The atypical colorations did not show any obvious phylogenetic signals.Table 2
Tentacle color patterns based on locality and phylogenetic position.
Florida Keys
Caribbean
Ctenoides scaberclade
Solid red (n=4)
Solid red (n=1)
Red/white stripes (n=2)
White/red stripes (n=5)
Solid white (n=2)
Ctenoides mitis clade
Solid white (n=4)
Solid white (n=1) White/red bands (n=1)
Light orange (n=1)
White/red bands (n=1)Shell rib analysis revealed that the distribution of shell rib numbers in theC. scaber clade (n=17) was significantly lower than that of theC. mitis clade (n=12, Figure 4(a), Mann-Whitney U Test, P<0.00001). Rib numbers of individuals from theC. scaber clade ranged from 37 to 58 (mean 48.3) and rib numbers in theC. mitis clade ranged from 73 to 103 (mean 85.8). Red and striped tentacles corresponded to the low shell rib number group, while light orange and banded tentacles corresponded to the high shell rib group (Figure 4(b)). White tentacles corresponded to both the low and high shell rib groups. Figure 5 shows a representative shell fromC. scaber (a) andC. mitis (b).Figure 4
(a) Relationships between shell rib numbers and shell height. Black dots represent individuals belonging to theCtenoides scaber clade and open circles represent those from theC. mitis clade. The distributions are significantly different (P<0.00001). (b) Number of shell ribs versus shell height for individuals with tentacle color information available. Red, white, striped, light orange, and banded tentacles are represented by black dots, open circles, grey dots, grey open circles, and triangles, respectively.
(a)
(b)Figure 5
(a) Representative valve ofC. scaber; individual UCM 48137 (University of Colorado Museum of Natural History). Shell height 35.4 mm; shell ribs n=50. (b) Representative valve ofC. mitis; individual UCM 48138. Shell height 34.7 mm; shell ribs n=80. Scale bar = 10 mm.
(a)
(b)In summary, our phylogenetic analysis recovered two well-supported clades corresponding toCtenoides scaber andC. mitis. Genetic variations within each clade are relatively low, but that could be the result of limited geographical sampling. Morphological analyses showed that tentacle color alone cannot be used to determine species, as colors vary inter- and intraspecifically (Figure 3). Shell rib counts gave statistically different distributions for the two species (Figure 4), but the two distributions have some degree of overlap.


## 4. Discussion

DistinguishingCtenoides scaber fromC. mitis is challenging, as is evidenced by observed misidentification in museum specimens and by the hobbyist community. The two species have many characteristics in common; they share ecological similarities with similar maximum depths and overlapping geographical distributions, and they are often found in the same location, suggesting overlapping ecological niches [10]. Internal anatomy, including mantle and eye structures, is also similar between the two species [17, 29, 30, 45].BothC.scaber andC. mitisexhibit tentacle color variations. Mikkelsen and Bieler [10] placed high confidence in species identification using tentacle coloration in the Florida Keys. Our results confirmed the white and very light orange tentacle colorations inC. mitis, and the red coloration inC. scaber from the Florida Keys. However, outside of the Florida Keys,C. scaberexhibits many tentacle color variations without showing obvious genetic distinction from the red individuals (Figure 3).As individuals of both species can possess solid white tentacles, tentacle color alone is not sufficient to determine species. This study confirmed that theC. scaber group has significantly fewer shell ribs than theC. mitisgroup. However, the overlap between the two species (between 59 and 78 ribs) means that no universal rule can be established to determine species based on shell ribs alone. Shell roughness is another characteristic which may be used to distinguish the two species, as the radial ribs ofC. mitis are finer thanC. scaber(Figure 5). However, shell roughness, size, and shape do vary to some extent across individuals, and, without instrumentation, species differentiation based on roughness could be challenging. Overall, the morphological diversity of shell ribs combined with the variety of tentacle coloration schemes stresses the importance of using molecular phylogenetics to verify species identification in future studies.It is unclear whether the differential rib numbers between the two species has ecological significance. One possible hypothesis is that the high and low rib numbers are related to different predator defense mechanisms. The bright coloration (the result of carotenoids) in the tentacles ofC. scaber [46] is hypothesized to be warning coloration for distasteful tissue. AsC. mitis generally lacks brightly colored tentacles, they might have developed higher number of shell ribs to increase shell strength [47, 48]. However, this hypothesis does not explain the presence of white-coloredC. scaber, so the effects of rib number and rib “roughness” on shell strength need to be examined further.Based on the phylogenetic results, it is likely thatC. scaber andC. mitis are recently diverged sister species. However, the speciation mechanism between the two has not been well studied. Ecological speciation has been demonstrated in numerous shallow-water marine invertebrates, with a divergence in life history being the most conspicuous difference between sibling species [7]. In bivalves, these differences include bathymetry limits, unique dietary adaptations, or host/symbiont associations [49].Ctenoides scaber andC. mitis have overlapping geographical ranges and are often collected from the same substrate and at the same depth [10]. This strong similarity in microhabitat makes it difficult to hypothesize what ecological factor(s) could result in divergent selection between the two species. One potential difference is the prevalence of whiter tentacles inC. mitis, which may be the result of fewer carotenoids in the diet. Future research is needed to determine if the two species have different dietary adaptations and how they utilize carotenoids.Alternatively, the speciation could have been allopatric rather than ecological, with geographical isolation occurring following secondary contact. Although absolute geographical barriers are fewer in the sea, abiotic factors such as ocean currents or temperature can result in population isolation [50]. More information regarding each species’ historical geographical range would be necessary to examine this hypothesis. It should be noted that the sampling in this study did not cover the entire geographical range of both species, so it is possible that additional genetically distinct clades or morphotypes may exist.Although our family-level phylogeny based on limited taxon sampling was able to resolve phylogenetic relationships betweenCtenoides scaber andC. mitis, it demonstrates the need for a more comprehensive phylogenetic reconstruction. To our knowledge, phylogenetic relationships among/within the nine extent Limidae genera are not well established. This is partially because taxon coverage in past studies are poor and partially because taxonomical classification of Limidae is largely based on shell morphologies, some of which may not reflect true phylogenetic signals. For example, a recent study shows that the generaLimatulaandAntarctolimaare actually nested withinCtenoides [35]. We have also found that taxonomical inconsistencies are common in Genbank entries of Limidae species.Limidae species exhibit many morphological and behavioral traits that are quite unique for bivalves. For example, a diverse spectrum of visual abilities can be found in the family, ranging from eyeless(Limaria) to pit eyes(Lima) to closed-lens eyes(Ctenoides) [29–31]. Members of the family also show distinct defense mechanisms, including nest-building behavior [20], swimming, tentacle autotomy [14, 18, 20, 21], and, potentially, defensive chemicals [30]. The evolution of these traits cannot be addressed without a comprehensive phylogeny.Finally, conservation concerns must be addressed.Ctenoides scaber is highly collected for the aquarium trade, but estimates of their abundance are lacking. How many of the collected file clams are actuallyC. scaber and notC. mitisis unknown, as is the abundance of both of the species throughout the western Atlantic. Considering the popularity of these organisms with shell collectors, SCUBA divers, and within the aquarium trade, a closer look at their conservation status is warranted.



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*Source: 1624014-2017-12-06.xml*