Philippine Journal of Science 148 (S1): 1-13, Special Issue on Genomics ISSN 0031 - 7683 Date Received: 31 Jan 2019

Cytochrome C Oxidase Subunit 1 (COI) Profile of the Philippine Helicostylinae (: : )

Gizelle A. Batomalaque1,4,*, Gerard Clinton L. Que1, Tyrill Adolf B. Itong5, Anna Regina L. Masanga1, Emmanuel Ryan C. de Chavez3, and Ian Kendrich C. Fontanilla1,2

1Insitute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101 Philippines 2Natural Sciences Research Institute, University of the Philippines Diliman, Quezon City 1101 Philippines 3Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños 4031 Laguna, Philippines 4Department of Biodiversity, Earth and Environmental Sciences, College of Arts and Sciences, Drexel University, Philadelphia, PA 19104 USA 5College of Science, University of the Philippines Cebu, Cebu City 6000 Philippines

The Philippines is the center of radiation of the subfamily Helicostylinae, with around 253 recognized species. Despite their morphological diversity, research on their biology and is lacking. We present here the first mitochondrial COI profiles of 32 species of Philippine helicostyline land snails. With the addition of sequences downloaded from GenBank, we tested the utility of the COI for species identification. Relative distributions of intraspecific and interspecific distances overlapped; hence, no barcoding gap was observed. However, 90% of uncorrected interspecific comparisons can distinguish species at 14% genetic distance or lower. Furthermore, the COI barcodes could not discriminate several co-distributed species that have similar conchological features, which should be flagged for taxonomic re-evaluation.

Keywords: DNA barcoding, Helicostylinae, mitochondrial COI, Philippine land snails

INTRODUCTION exhibit a range in shell forms from discoidal, depressed and keeled, globose, to elongated conical forms (Parkinson The Helicostylinae, a subfamily under family Camaenidae et al. 1987). Within the Philippines, different helicostyline (sensu Bouchet et al. 2017) and order Stylommatophora, species vary in distributions, with most occurring in are hermaphroditic ground and tree snails whose center single islands (e.g., Anixa siquijorensis in Siquijor Is. of diversity is the Philippine Islands (Parkinson et al. and (Calocochlea) chrysocheila in Luzon 1987, Abbott 1989, de Chavez et al. 2015) and whose Is.) and some occurring in multiple adjacent islands (e.g., distribution extends to Taiwan, the Moluccas, and the Leytia fragilis in Samar and Leyte islands and Trachystyla smaller islands off the coast of Borneo (Schileyko 2004, cryptica in the islands of Samar, Leyte, and Mindanao). Schilthuizen et al. 2013). Members of this subfamily There are about 245 species (Batomalaque n/p, Faustino 1930, Richardson 1983, Abbott 1989) belonging to *Corresponding author: [email protected]

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23 genera (Schileyko 2004, Bouchet et al. 2017). The amplification using Taq DNA Polymerase and dNTPack current taxonomy of the helicostylines is based on shell (Roche, USA), and universal (forward LCO 1490 morphology, although the reproductive anatomy for GGTCAACAAATCATAAACATATTGG, reverse HCO some species has been described (Schileyko 2004). No 2198 TAAACTTCAGGGTGACCAAAAAATCA; Folmer molecular work has been done to evaluate their current et al. 1994) and stylommatophoran-specific (forward STY_ classification, and phylogenetic relationships among the LCOii ACGAATCATAAGGATATTGGTAC, reverse species are unknown. STY_HCO GAATTAAAATATATACTTCTGGGTG; Fontanilla et al. 2017) primers for the mitochondrial The mitochondrial cytochrome c oxidase subunit 1 (COI) cytochrome c oxidase subunit I (COI) gene. A 50 μL PCR gene has been the gene of choice for DNA barcoding in mix consisted of the following components: 5 μL of 10X (Hebert et al. 2003, Meyer and Paulay, 2005, PCR buffer; 1 μL of 10 mM dNTP; 2.5 μL each of 10 mM Park et al. 2011, Siddall et al. 2012, Perez et al. 2014). forward and reverse primers; 22.75 μL distilled water; However, its utility in species discrimination in low- 0.25 μL Taq-polymerase (5 units/μL); 10 μL Q-buffer vagility species (Davison et al. 2009, Virgilio et al. 2010) (Qiagen, USA); 2 μL of 15 mM MgCl2; and 4 μL of 10 appears to be fraught with high error rates due to lack of mM DNA. Amplification protocol consisted of 2 min at baseline differences established through morphology and 94°C followed by 38 cycles of 30 sec at 94°C, 30 sec at a DNA sequence database. In land snails, several studies 45°C, 60 sec at 65°C, and a final extension of 5 min at have shown high levels of mtDNA sequence divergence 72°C. PCR products were visualized in a 1% agarose gel in intraspecific populations (Watanabe and Chiba 2001, using EtBr UV illumination. Pfenniger and Posada 2002, Davison et al. 2009). Amplified PCR products were extracted using QIAquick® In this paper, we present the first COI profiles of the Gel Extraction Kit (Qiagen, USA). Purified samples were Philippine helicostyline land snails, and we test the sent to 1stBASE, Malaysia for sequencing. utility of 463-bp COI barcodes in distinguishing among morphological species. Sequence Alignment and DNA Barcoding Analysis Sequences were assembled using the STADEN package v.1.5.3 (Staden et al. 2000), and aligned using BioEdit MATERIALS AND METHODS v.5.0.5 (Hall 1999). Only unambiguously aligned nucleotide sites were included in the analyses. All sequences were deposited in GenBank (Table 1). Taxon Sampling and Identification A total of 134 specimens attributed to 35 camaenid An additional 292 stylommatophoran COI sequences species were collected from 27 localities across the from GenBank were analyzed together with those from Philippines (Table 1), representing approximately 15% this study for a total of 423 sequences to test their utility of the total nominal species of Helicostylinae. The 41 in species discrimination. Sequences of species under camaenid species comprised 35 species under subfamily family Polygyridae were used as outgroups, following the Helicostylinae and three under subfamily . recent molecular helicoid phylogeny of Sei et al. (2017). Cuttings of foot tissue were preserved in 95% (v/v) Sequences were viewed and trimmed to 463 nucleotides ethanol, while the vouchers were preserved in 70% (v/v) common to all taxa using BioEdit v.7.0.9 (Hall 1999) ethanol. Identification of species was based on shell and aligned using the ClustalW (Thompson et al. 1994) morphology, using literature (Springsteen and Leobrera accessory program. Haplotypes were counted using 1986, Abbott 1989), and by examination of reference DnaSP v. 6.12.01 (Rozas et al. 2017). collections in the University of the Philippines Diliman The substitution model was determined using ModelTest- Invertebrate Museum (UPDIM), Quezon City, Philippines. NG v.0.1.5 (Darriba et al. 2015), and the model with the best log-likelihood score was chosen using the Akaike DNA Extraction and Sequencing Information Criterion (Akaike 1973, 1974; Hurvich and DNA was extracted using the relatively rapid and Tsai 1993). The Xia Test (Xia et al. 2003, Xia and Lemey inexpensive modified NaOH-lysis method (Fontanilla et 2009) for substitution saturation was also performed in al. 2017). In this method, tissue slices were ground using DAMBE v. 6.4.81 (Xia 2013, 2017). Pairwise comparisons glass beads with 200 μL of 0.1 N NaOH and centrifuged of likelihood scores were performed on the dataset using with 300 μL chloroform-isoamyl alcohol (24:1). The uncorrected p-distances and GTR+Γ corrected distances upper phase was then collected and centrifuged with generated by PAUP* v4.10b (Swofford 2003), with ~300 μL isopropanol. The pellets were washed with GTR+Γ (Tavare 1986) determined as an optimal model ethanol, air-dried, and finally re-suspended in 150 by ModelTest-NG. Comparisons of pairwise distances μL TE buffer. DNA extracts were subjected to PCR

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Table 1. Species of Philippine camaenids collected with their corresponding GenBank accession numbers. Subfamily Species name Locality Collector/s GenBank accession no. Helicostylinae amoena (Pfeiffer, 1845) Alaminos, I.K.C. Fontanilla, G.A. KM279469 Pangasinan, Luzon Batomalaque, E. de Vera KM279470 KM279471 KM279472 KM279473 Chloraea hennigiana Moellendorff, 1893 Alaminos, I.K.C. Fontanilla, G.A. KM279464 Pangasinan, Luzon Batomalaque, E. de Vera KM279465 KM279466 KM279467 KM279468 Chloraea fibula Not Uploaded to GenBank chrysalidiformis (Sowerby, 1833) Puerto Galera, A.U. Luczon KM056693 Mindoro KM056694 KM056695 bicolorata (Lea, 1840) Laguna, Luzon C.P. Española KM056744 Cochlostyla daphnis (Broderip, 1841) Borbon, Cebu R.J.C. Canoy KM056706 Santander, Cebu P. Olvis KM056707 Cochlostyla fauna (Broderip, 1841) Bantayan Is., Cebu P. Olvis KM056713 KM056714 KM056715 Cochlostyla imperator (Pfeiffer, 1848) Sibulan Is., Polillo E.R.C. de Chavez KM056725 KM056726 Cochlostyla intermedia (Quadras and Moellendorff, 1896) Benguet, Luzon D. Constantino-Santos, KM056728 I.K.C. Fontanilla, A.U. Luczon KM056729 KM056730 KM056731 Cochlostyla marinduquensis (Hidalgo, 1887) Gasan, Marinduque B.O. Sosa III, R.D.C. KM279486 Pedales KM279487 KM279488 KM279489 KM279490 KM279491 KM279492 KM279493 KM279494 Cochlostyla pithogaster (Férussac, 1821) Sta. Cruz, B.O. Sosa III, R.D.C. KM279495 Marinduque Pedales KM279496 KM279497 KM279498

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Table 1. continuation . . . . KM279499 KM279500 KM279501 KM279502 KM279503 KM279504 Torrijos, Marinduque KM279505 KM279506 Cochlostyla portei (Pfeiffer, 1861) Polillo Is., Polillo E.R.C. de Chavez, KM056727 I.K.C. Fontanilla, G.A. Batomalaque, J.F. KM056739 Halili KM056740 KM056741 KM056742 KM056743 Cochlostyla ticaonica (Broderip, 1841) Balamban, Cebu T.A.B. Itong KM279485 Cochlostyla ventricosa (Bruguière, 1792) Medellin, Cebu P. Olvis KM056751 KM056750 Cochlostyla woodiana (Lea, 1840) Amaga, Polillo E.R.C. de Chavez KM056753 Cochlostyla worcesteri Bartsch, 1909 Bantayan Is., Cebu P. Olvis KM056711 KM056710 Corasia puella (Broderip, 1841) Balamban, Cebu T.A.B. Itong KM279479 KM279480 KM279481 KM279482 KM279483 KM279484 KM279478 KM279477 Corasia reginae (Grateloup, 1840) Kidapawan, D.A.E. Ramos KM056692 Mindanao Dryocochlias metaformis (Férussac, 1821) Rizal, Luzon G.A. Batomalaque KM056756 KM056757 Helicostyla amagaensis de Chavez, 2015 Amaga, Polillo E.R.C. de Chavez KM056719 KM056720 KM056721 Helicostyla butleri (Pfeiffer, 1842) Benguet, Luzon D. Constantino-Santos KM056705 Helicostyla corticolor Kobelt, 1911 Mountain Province, G.A. Batomalaque KM056712 Luzon Helicostyla (Calocochlea) generalis (Pfeiffer, 1854) Anawan, Polillo E.R.C. de Chavez KM056690 KM056691 Helicostyla (Calocochlea) pan (Broderip, 1841) Batoan, Bohol P. Olvis KM056683 KM056684 Helicostyla (Calocochlea) polillensis (Pfeiffer, 1861) Laguna, Luzon E.R.C. de Chavez KM056732 Polillo Is., Polillo E.R.C. de Chavez KM056737 KM056738

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Table 1. continuation . . . .

Helicostyla (Calocochlea) speciosa (Jay, 1839) Laguna, Luzon E.R.C. de Chavez KM056733 Sibulan Is., Polillo KM056734 KM056735 KM056736 E.R.C. de Chavez, KM056689 I.K.C. Fontanilla, G.A. Batomalaque, J.F. KM056716 Halili KM056717 KM056718 Helicostyla (Calocochlea) valenciennesii (Eydoux, 1838) Laguna, Luzon E.R.C. de Chavez KM056688 Helicostyla (Opalliostyla) aegle (Broderip, 1841) Agusan, Mindanao G. Galan KM056749 Helicostyla (Opallioastyla) mearnsi Bartsch, 1905 Kidapawan, D.A.E. Ramos KM056723 Mindanao KM056745 Kidapawan, J.A. Anticamara KM056746 Mindanao KM056747 Agusan, Mindanao G. Galan KM056748 Hypselostyla camelopardalis (Broderip, 1841) Argao, Cebu P. Olvis KM056708 KM056709 Hypselostyla carinata (Lea, 1840) Anawan, Polillo E.R.C. de Chavez KM056722 Hypselostyla subcarinata (Pfeiffer, 1842) Gasan, Marinduque B.O. Sosa III, R.D.C. KM279507 Pedales KM279508 KM279509 KM279510 KM279511 KM279512 Leytia fragilis (Sowerby, 1841) Ormoc, Leyte E.R.C. de Chavez, KM056724 I.K.C. Fontanilla, M. Hayashi KM056696 KM056697 KM056698 KM056699 KM056700 Phoenicobius brachyodon (Sowerby, 1841) Puerto Galera, A.U. Luczon KM056754 Mindoro KM056755 Rhymbocochlias grandis (Pfeiffer, 1845) Cagayan, Luzon C.P. Española KM056752 Trachystyla cryptica (Broderip, 1841) Agusan, Mindanao G. Galan KM056704 Ormoc, Leyte G.A. Batomalaque, KM056701 F.S. Magbanua, D.A.E. Ramos KM056702 KM056703 Bradybaeninae similaris (Rang, 1831) Baguio, Benguet, G.A. Batomalaque KM056685 Luzon KM056686 KM056687

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Table 1. continuation . . . .

Eulota mighelsiana (Pfeiffer, 1846) Batan, Batanes G.A. Batomalaque, JQ582272 I.K.C. Fontanilla, P.R.L. Sales JQ582273 JQ582274 JQ582275 JQ582276 batanica (Adams and Reeve, 1850) Batan, Batanes G.A. Batomalaque, JQ582280 I.K.C. Fontanilla, P.R.L. Sales JQ582281 JQ582282 JQ582283

were done for scores within species and between species the mean interspecific distance (uncorrected p-distance following the methods of Meyer and Paulay (2005) in = 0.212; GTR+Γ-corrected distance = 0.884), although order to detect the presence of a barcoding gap. the relative distributions overlap (Figures 1 and 2) such that no barcoding gap is observed. However, the most A Neighbor Joining (NJ) tree (Saitou and Nei 1987) was frequent values for the uncorrected intraspecific (0.000) constructed in PAUP* following the parameters of the and interspecific (0.216) distances appeared to be distinct, optimal model (GTR+Γ as determined by ModelTest-NG. as seen in Figure 1 as separate modes for the two datasets. A Maximum Likelihood (ML) tree (Felsenstein 1981) was Furthermore, around 90% of all uncorrected intraspecific also generated using the parallel (MPI) version RAxML v. comparisons fall within 14% distance. Taking only the 8.2.11 (Stamatakis 2014) with 1000 bootstraps (Felsenstein Philippine camaenid sequences generated from this study, 1985, Stamatakis et al. 2008) and the GTR+Γ model with substitution rates and other parameters determined by RAxML v. 8.2.11 (Stamatakis 2014). A Bayesian (BI) tree was generated using the parallel version of MrBayes v. 3.2.6 (Altekar et al. 2004, Ronquist et al. 2012), also following the GTR+Γ model of nucleotide substitution, using two runs consisting of four chains each. MrBayes was run for 10 million generations and convergence was evaluated using the standard deviation between each run and the Potential Scale Reduction Factor. The trees were visualized and edited using Dendroscope v. 3.5.9 (Huson and Scornavacca 2012), FigTree v. 1.4.3 (Rambaut 2016), and Tree Explorer v. 2.12 (Tamura 1999). Figure 1. Distribution of intraspecific and interspecific pairwise uncorrected distances.

RESULTS Mitochondrial COI was sequenced from 119 specimens of Philippine helicostyline land snails, as well as 12 specimens of other Philippine Camaenidae species (Table 1). These and the additional 292 sequences from GenBank comprised the dataset of 423 sequences (463 nucleotides in length) containing 323 haplotypes for the entire dataset, while 63 unique haplotypes are seen for Philippine helicostyline sequences. The Xia test showed little saturation (lss. = 0.281 is significantly lower than both lss.cSym = 0.698 for a completely symmetrical tree and lss.cAsym = 0.372 for a completely asymmetrical tree). Figure 2. Distribution of intraspecific and interspecific pairwise The mean intraspecific distance (uncorrected p-distance corrected distances based on the GTR+Γ model of DNA = 0.056; GTR+Γ-corrected distance = 0.109) is less than substitution.

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the mean intraspecific distance (uncorrected p-distance = terminal nodes for conspecific groupings, the deeper nodes 0.014; GTR+Γ-corrected distance = 0.026) was less than are not well-supported. the mean interspecific (uncorrected p-distance = 0.181; GTR+Γ-corrected distance = 0.698) but a similar overlap A majority of the helicostyline samples grouped with their in relative distributions of the distances was observed conspecifics with a few exceptions, which constituted (Figures 3 and 4). For the Philippines samples, 98% of three groups (Figure 5). Group 1 is composed of Chloraea all uncorrected intraspecific comparisons fall within 12% amoena and C. hennigiana (NJ/ML/BI: 100/99/0.89), genetic distance. which interfinger with very high sequence similarity (0–0.65% for both corrected and uncorrected interspecific distances). C. amoena has one unique COI haplotype while C. hennigiana has two unique haplotypes; another haplotype is shared between both species. Group 2 is composed of Helicostyla (Calocochlea) speciosa, H. (C.) generalis, H. (C.) polillensis, and H. (C.) valenciennesii and forms a well-supported clade (NJ/ ML/BI:100/100/1.00) with very little interspecific genetic distance (0–7.2% for both corrected and uncorrected distances). H. (C.) speciosa and H. (C.) polillensis have three and two haplotypes, respectively. Two of H. (C.) speciosa’s haplotypes and one of H. (C.) polillensis’ haplotypes are shared with other members of Group 2. H. Figure 3. Distribution of intraspecific and interspecific pairwise (C.) generalis and H. (C.) valenciennesii do not have any uncorrected distances for sequences original to this unique haplotypes and share their COI sequence with other study (Table 1). members of Group 2. Finally, Cochlostyla ventricosa and C. worcesteri comprise Group 3 (NJ/ML/BI: 99/100/1.00), with little interspecific sequence divergence (0.2–1.78% for both corrected and uncorrected distances). One unique COI haplotype is present for each species in Group 3; a third haplotype is shared between both species. Aside from these species, well-supported conspecific groupings were obtained.

DISCUSSION

Figure 4. Distribution of intraspecific and interspecific pairwise DNA barcoding has become a routine method for corrected distances based on the GTR+Γ model of DNA identifying species and detecting cryptic diversity (Hebert substitution for sequences original to this study (Table 1). et al. 2003, 2004; Scheffer et al. 2006). Threshold values for discriminating species are approximated from the barcoding gap and may vary for different taxa. Hebert et All tree-construction methods yielded similar tree al. (2003) set the threshold value to 3% for characterizing topologies (Figure 5; Appendix Figure I). Helicostylinae different species, and then set a threshold value of 2.7% for was rendered non-monophyletic, forming a polytomy birds (Hebert et al. 2004). For fish, Ward et al. (2008) set it with other camaenid species. Two clades fall outside the at 3–3.5%, while Meyer and Paulay (2005) set a threshold majority of helicostylines: the Chloraea clade and the value of 1.99–2.85% for cowries (marine gastropods). No genera Phoenicobius and Chrysallis – here on referred to evidence of a barcoding gap was observed in this study as the Palawan-Mindoro (PM) clade since these species due to the overlap in relative distributions of intraspecific are restricted to the islands of Palawan and Mindoro. and interspecific distances. Such overlaps have also been The Chloraea clade is sister to species of subfamily observed in several mollusk groups such as the marine Aegistinae (sensu Schileyko, 2004; see Appendix Figure gastropod family Cypraeidae (Meyer and Paulay 2005) II), while the PM clade is sister to Nesiohelix swinhoe and stylommatophoran land snails (Davison et al. 2009). and Satsuma batanica, although these relationships are In their analysis of different stylommatophoran families, not well-supported. The clade containing a majority of Davison et al. (2009) observed that intraspecific variations the Helicostylinae is here on referred to as the crown ranged from 10% to 30% depending on the species and the Helicostylinae. Although there is high support at the

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Figure 5. Maximum likelihood tree of the Philippine Camenidae based on 463 bp of the mitochondrial cytochrome oxidase subunit I (COI) gene under the GTR+Γ model. The tree is rooted on polygrid Vespericola columbiana depressa. The trifurcating node is indicated by the red star. Helicostylinae is shown to be non-monophyletic, having three separate clades – the Chloraea clade that is sister to Aegistinae species, the crown Helicostylinae that contains most of the helicostyline species, and the PM clade that consists of Chrysallis and Phoenicobius. The highlighted groups consist of sequences from more than one morphospecies but are very similar or almost identical. Photos of representative specimens are not to scale: a – Chloraea hennigiana KM279468 (shell height = 10.3 mm); b – C. amoena KM279471 (shell height = 8.8 mm); c – Helicostyla (Calocochlea) speciosa juvenile KM056735 (shell height = 26.4 mm); d – H. (C.) polillensis KM056738 (standard shell height = 42.7 mm); e – H. (C.) valenciennesii juvenile KM056688 (shell height = 22.2 mm); f – Cochlostyla ventricosa KM056751 (shell height = 42.4 mm); g – C. worcesteri KM056711 (shell height = 34.5 mm). Tips marked with an asterisk (*) represent sequences that were downloaded from GenBank. Values on nodes represent NJ and ML bootstraps, respectively, based on 1000 bootstrap samples; values less than 50 % are not shown. The scale bar represents five substitutions for every ten nucleotides.

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gene used. No barcoding gap was observed in species of be necessary to resolve the polytomy. Polygyridae, and the mean genetic distance within species ranged from 0.9% to 19.1% (Perez et al. 2014). The Three groups contained species interfingering with very camaenid Camaena cicatricosa had 0–6.725 nucleotide high sequence similarity. The first group, Chloraea differences between populations, but no correlation was amoena and C. hennigiana, might also represent a found between genetic distance and geographical distance single species. Both species share the same shell shape, (Zhou et al. 2017). Although no barcoding gap was size, and thickness, but C. amoena has both banded and observed in the Philippine camaenids, it should be noted unbanded forms while C. hennigiana does not have any that a majority (90%) of the uncorrected intraspecific banding pattern. The second is composed of Helicostyla comparisons in this study yielded distances of 14% (Calocochlea) speciosa, H. generalis, H. polillensis, and lower. On the other hand, 99% of the uncorrected and H. valenciennesii. These species are co-distributed interspecific comparisons yielded distances above 14%. in Luzon and Polillo Is. and may represent at least three Most species within the Camaenidae could therefore be closely related species or possible cases of hybridization. distinguished within 14% sequence divergence using the Although they share the same semi-globular shell shape COI gene as a marker. and ground color (yellow to tan/ light brown), their banding patterns, shell thickness, and microsculpture The tree topology obtained in this study, which is based on differ. The third group is composed of Cochlostyla the mitochondrial COI, can be interpreted as a preliminary ventricosa and C. worcesteri – both occurring in Cebu Is. phylogenetic framework for the Helicostylinae, although and having turriform shell shape but differing in banding most of the deeper nodes are not well-supported. Our result pattern (C. ventricosa has thin brown bands that are showed that the Helicostylinae is not monophyletic, but visible even on eroded shells). This group could possibly this topology might change with the addition of more loci. represent a single polymorphic species. Furthermore, The Chloraea and PM clades represent entirely different the specimens were collected from the same locality and evolutionary histories from the rest of the Helicostylinae. therefore could exhibit polymorphisms of a single species. Among the genera whose reproductive anatomies have We have not observed cases of possible cryptic diversity, been described and illustrated by Schileyko (2004), where individuals of a single species occurred in different Chloraea, Chrysallis, and Phoenicobius are the only parts of the tree, thus representing two morphologically helicostylines whose accessory glands (mucus glands in similar but genetically different species. Schileyko 2004) are not globular or sub-globular. They are instead elongated (as in Chloraea and Phoenicobius) A wide range of intraspecific polymorphisms is not like those of Guamampa and Tricheulota (of subfamily uncommon in land snails (Goodfriend 1986; Chiba Aegistinae sensu Schileyko, 2004) or divided into several 1993, 1996; Davison and Chiba 2006; Perez et al. 2014). tubules (as in Chrysallis) like those of Nesiohelix and For example, Cepaea nemoralis exhibits different color Plectotropis (of subfamily Aegistinae sensu Schileyko, morphs (yellow, pink, or brown) and banding patterns 2004). It has been shown that the and dart-related (unbanded, midbanded, many-banded), which vary with organs (accessory glands included) were lost independently habitat (Cameron and Cook 2012). The occurrence of in some camaenid species (Hirano et al. 2014). However, highly-supported clusters and low sequence divergence whether these accessory gland morphologies are of different species could mean either one or all of three synapomorphic characters or not is yet to be tested. things. First, these species could be results of natural Aside from reproductive anatomy, the separation of the hybridization between sympatric species, as exhibited by PM clade may be reflective of the Philippines’ geologic their similarity in general shell color and form, and the history. The islands of Palawan and Mindoro were part occurrence of seemingly intermediate forms. Woodruff of the continental block, while the rest of the Philippine and Gould (1987) documented a controlled interspecific islands emerged through tectonic-volcanic activity (Hall hybridization of two species of the land snail Cerion in 2002). This is not to say that a biogeographic boundary the Florida Keys. They observed the intermediacy of exists between these islands (Palawan and Mindoro) forms and enhanced variation in the hybrids, noting that and the rest of the Philippines since some helicostylines such phenomena could occur naturally between sympatric occur in the oceanic islands as well as Mindoro (e.g., species. Second, the morphologically different species Cochlostyla pithogaster and Helicostyla speciosa, which that clustered together may be a single species – or a are both under the crown Helicostylinae). It must be collection of closely related species – with very plastic noted that the phylogeny presented here is only based on shell morphology. Such was observed by Chiba (1999) the mitochondrial COI. Gene trees generated from other on the land snail , which is endemic to the mitochondrial loci or nuclear loci would have a different oceanic Bonin Islands off the coast of Japan. These topology due to different evolutionary rates and histories. snails were found in the same site but had morphological A wider taxonomic sampling and more genetic loci would features that appeared to be more correlated with the type

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of microhabitat they were in. Thus, the diversification identification. IEEE Trans Automat Contr 19(6): in shell color and shell shape was possibly caused by 716–723. microhabitat differentiation (Chiba 1999, 2009). Third, ALTEKAR G, DWARKADAS S, HUELSENBECK JP, these species may simply have clustered together because RONQUIST F. 2004. Parallel Metropolis-coupled there is not enough species or individuals that would cause Markov chain Monte Carlo for Bayesian phylogenetic them to separate in the tree. In these cases, the utility of inference. Bioinformatics 20: 407–415. the mitochondrial COI as a species discrimination tool is limited. The species that interfingered in the tree must BOUCHET P, ROCROI J, HAUSDORF B, KAIM A, be re-examined in other aspects, such as targeting more KANO Y, NÜTZEL A, PARKHAEV P, SCHRÖDL genes (both mitochondrial and nuclear) and examining M, STRONG EE. 2017. Revised classification, their reproductive anatomy and ecology. nomenclator and typification of gastropod and monoplacophoran families. Malacologia 61(1–2): Including more loci will be worth pursuing to come up 1–526. with a phylogenetic framework for the Helicostylinae. Furthermore, anatomical and ecological characteristics CAMERON RAD, COOK LM. 2012. Habitat and the could also provide valuable information in elucidating shell polymorphism of Ceparea nemoralis (L.): relationships in this less understood group. We therefore Interrogating the Evolution Megalab database. J Moll recommend exploring these avenues in greater detail for a Stud 78: 179–184. comprehensive phylogenetic framework of the subfamily. CHIBA S. 1993. Modern and historical evidence of natural hybridization between sympatric species in Mandarina (: Camaenidae). Evolution 47(5): 1539–1556. ACKNOWLEDGMENTS CHIBA S. 1996. Ecological and morphological This research was funded by the Natural Sciences diversification within single species and character Research Institute (NSRI) of the University of the displacement in Mandarina, endemic land snails of Philippines (UP) Diliman (Grant numbers: BIO-11-2- 02 and BIO-13-1-05). We thank the Institute of Biology, the Bonin Islands. J Evol Bio 9: 277–291. UP Diliman for the logistical support. We thank the local CHIBA S. 1999. Character displacement, frequency- government units and local offices of the Department dependent selection, and divergence of shell colour in of Environment and Natural Resources for granting land snails Mandarina (Pulmonata). Biol J Linn Soc us permits, and private landowners for allowing us to 66: 465–479. collect samples. CHIBA S, 2009. Morphological divergence as a result of common adaptation to a shared environment in land snails of the Hirasea. J Molluscan Stud NOTES ON APPENDICES 75: 253–259. The complete appendices section of the study is DARRIBA D, POSADA D, STAMATAKIS A. 2015. accessible at http://philjournsci.dost.gov.ph ModelTest-NG. Retrieved from https://github.com/ ddarriba/modeltest DAVISON A, CHIBA S. 2006. Labile ecotypes accompany REFERENCES rapid cladogenesis in an adaptive radiation of Mandarina (Bradybaenidae) land snails. Biol J Linn ABBOTT RT. 1989. Compendium of landshells: A full- Soc 88: 269–282. color guide to more than 2,000 of the world’s terrestrial shells. Melbourne, FL: American Malacologist, Inc. DAVISON A, BLACKIE RLE, SCOTHERN GP. 2009. 240p. DNA barcoding of stylommatophoran land snails: A test of existing sequences. Mol Ecol Resour 9(4): AKAIKE H. 1973. Information theory and an extension 1092–1101. of the maximum likelihood principle. In: Petrov BN, Csaki F eds. 2nd International Symposium on DE CHAVEZ ERC, FONTANILLA IKC, Information Theory. Budapest, Hungary: Akademia BATOMALAQUE GA, CHIBA S. 2015. A new Kiado. Helicostyla species (Bradybaenidae: Helicostylinae) from Patnanungan Island, Philippines. Asia Life Sci AKAIKE H. 1974. A new look at the statistical model 24(1): 37–49. FAUSTINO LA. 1930. Summary of Philippine land shells.

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APPENDIX

Figure I. Maximum likelihood tree with GenBank numbers of all Figure II. Maximum likelihood tree showing the species of novel sequences in this study. Values on nodes represent Aegistinae sister to the Chloraea clade. Values on ML bootstraps based on 1000 bootstrap samples; values nodes represent ML bootstraps based on 1000 bootstrap less than 50 % are not shown. The scale bar represents samples; values less than 50% are not shown. The scale five substitutions for every ten nucleotides. bar represents five substitutions for every 10 nucleotides.

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