A New Subspecies of Microsnail from Masungi Georeserve, Rizal, Philippines

Home , Gastrocoptidae, Truncatellinidae, Vallonia gracilicosta

PRIMARY RESEARCH PAPER | Philippine Journal of Systematic Biology DOI 10.26757/pjsb2020c14003

A new subspecies of microsnail from Masungi Georeserve, Rizal, Philippines

Harold B. Lipae1*, Angelique L. Estabillo2, Ian Kendrich C. Fontanilla3, and Emmanuel Ryan C. de Chavez2

Abstract

A new subspecies of microsnail, Hypselostoma latispira masungiensis subsp. nov., is described based on shell morphology and molecular characters. This new subspecies is distinguished from H. l. latispira from Baguio City, Benguet Province by having relatively larger major width size, additional apertural teeth (interpalatal plica), larger body whorl and apertural width, and clustering based on location. The collected samples from Masungi Georeserve, Rizal Province appear to be an ecophenotype as indicated by the novel site congruent to the clade separation of Masungi and Baguio H. latispira. Neighbor-joining and maximum likelihood trees also demonstrated that the two sample groups clustered separately, with bootstrap support of 84% and 78%, respectively. However, pairwise distance comparison revealed that there is only an average of 0.0131 ± 0.0126 genetic distance (99.98%) between the two populations, suggesting that they are most likely similar species; thus, the proposal of making it a subspecies. This is the first report on the new distributional record outside the type locality and a new subspecies of H. latispira.

Keywords: land snail, karst, interpalatal plica, pairwise distance comparison

Introduction 1984; Páll-Gergely et al. 2015). Hypselostoma species are described as having depressed conic spires with an upturned or The genus Hypselostoma Benson, 1856 is classified under ascending last whorl and continuous peristome (Pilsbry 1918) the family Hypselostomatidae Zilch, 1959 (sensu Schileyko and protrusions or teeth along the aperture (Tongkerd et al. 1998). However, Bouchet et al. (2017) considered the family as 2004), which is of great taxonomic importance. Pilsbry (1917) a synonym of Gastrocoptidae Pilsbry, 1918 following provided the first monograph of this genus followed by Haas Gittenberger (1973), while several studies recognized (1937) and Tomlin (1939) with additional species and Vertiginidae (Thompson & Lee 1988; Panha 1997; Hwang subspecies described by Benthem Jutting (1949, 1950, 1962). 2014). Páll-Gergely et al. (2019) recognized the The Philippine species were also discussed by von Moellendorff Hypselostomatidae. It is widely distributed throughout southern (1888), Quadras and von Moellendorff (1894, 1896), Pilsbry China and the Malayan peninsula, extending towards the (1917), and Haas (1937). Common features among the Philippines and the Ryukyu Islands (Thompson & Auffenberg Philippine group are the spiral striations on the shell and the cross orientation of apertural teeth (Pilsbry 1918). Currently, a 1Graduate School, University of the Philippines Los Baños, College, total of eight species occur in the Philippines, with H. luzonicum Laguna 4031 Moellendorff, 1888 and H. polyodon Moellendorff, 1896 having 2 Animal Biology Division, Institute of Biological Sciences, University five and two subspecies, respectively (Pilsbry 1917; Haas 1937; of the Philippines Los Banos, College, Laguna 4031 Thompson & Auffenberg 1984). 3 Institute of Biology, College of Science, University of the Philippines, The most recently described Philippine species is H. Diliman, Quezon City 1101 latispira Thompson & Auffenberg, 1984 from Baguio City,

Benguet Province (Thompson & Auffenberg 1984). Thus far, it *Corresponding email: [email protected] is known only from the type locality until now. In this paper, a

Date Submitted: 24 February 2020 new subspecies of H. latispira was discovered from Masungi Georeserve, Rizal Province which is more than 200 km away Date Accepted: 06 July 2020

Lipae et al.: New microsnail subspecies from Masungi Georeserve from the type locality of its nominotypical subspecies, and herein proposed based on morphological and molecular characters.

Materials and Methods

Study Site Specimens were collected from the limestone boulders of Masungi Georeserve in Baras, Rizal Province last March 6, 2019. The georeserve is situated 47 km east of Manila and is part of the Southern Sierra Madre mountain range (Fig. 1). It covers over 1,500 ha of karst forest with limestone formations of varying shapes and sizes. Three different sites were identified and searched for the microsnail species namely: “600 steps” (14.59°N, 121.31°E, 614 masl); “Nanay” (14.59°N, 121.31°E, 631 masl); and “Tatay” (14.59°N, 121.32°E, 573 masl). The three sites are characterized by having moderate amounts of grasses, herbs and shrubs, and some vines. Snails were carefully removed from the limestone boulders and were stored in conical tubes provided with damp tissue paper. In total, 82 snails were sampled and brought to the laboratory for sorting and further processing. All examined specimens from the Masungi Georeserve were kept in the UPLB Malacology Laboratory and type specimens (1 holotype and 4 paratypes) were deposited at the UPLB Museum of Natural History (UPLBMNHLS150-154). Figure 1. Map of Luzon Island showing Baguio City, Benguet For comparison, samples of H. l. latispira from Baguio Province and Masungi Georeserve. City, Benguet Province were also collected on March 23, 2019. Shell Morphometrics Ninety-eight snails were taken from the limestone boulders in a Linear morphometrics were done using Principal residential area in Barangay Dominican Hill, Baguio City Component Analysis (PCA). Measurement data were exported (16.40°N, 120.58°E, 1546 masl). Live samples were placed in to Paleontological Statistics software (PAST version 3, Hammer Petri dishes with wet tissues and scraped food materials (lichen, et al. 2016) and were color coded based on location. PCA was moss, algae) from the rocks and were reared in the laboratory. performed under the covariation option to determine the shell All shells were measured in the laboratory. characters that would maximize the separation of the two Type specimens of H. latispira were also loaned from the groups. Subsequently, the number of components was examined Florida State Museum, Gainesville, Florida, USA for further which was generated using eigenvalues and percent variance. evaluation. Four paratypes (dry) were examined Geometric morphometrics were also done to determine morphologically to compare with the samples collected from shell shape variations between the two groups. Photos of the the Masungi Georeserve. snails (dorsal and lateral views) were taken and converted to a

tps file using tpsUtil (http://life.bio.sunysb.edu/morph/). The file Shell Measurements was opened in tpsDig (http://life.bio.sunysb.edu/morph/) to Adult individuals were measured for the shell height, shell assign landmark points on the shell. A combination of Types I width, aperture height, and aperture width using a digital and II landmarks was used for both views (Fig. 2). Type I Vernier caliper following Tanmuangpak et al. (2015). The landmarks are those which correspond to homologous number of whorls was also counted. Photomicrographs of the structures, juxtaposed with tissues, and have evolutionary snails were also taken using a camera (ToupTek XCAM significance, whereas Type II landmarks are points that capture 1080PHB, Hangzhou, China) attached to a stereo microscope the general shape of the shell. Eighteen landmark points were (Olympus SZX7, Tokyo, Japan). used for the lateral view: landmarks 1, 2, 3, 5, 6, 8, 14, 15, 17

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Lipae et al.: New microsnail subspecies from Masungi Georeserve

bromide under UV illumination. The gel extraction of the purified DNA samples was done using Bioline Isolate II PCR and Gel Kit. The purified DNA samples were sent to Macrogen Korea, South Korea for the standard sequencing (Sanger method).

Sequence Analysis Figure 2. Dorsal (A) and lateral (B) views with corresponding landmark The Staden Package 4.0 (Staden et al. 2000) was used to points. Red dots are type I landmarks, and yellow dots are type II. assemble the 16S mitochondrial ribosomal DNA fragments. Using Basic Local Alignment Search Tool (BLAST, http:// and 18 as Type I, and 4, 7, 9, 10, 11, 12, 13 and 16 as Type II blast.ncbi.nih.gov/Blast.cgi, Altschul et al. 1990), the closest landmark points. For the dorsal view, a total of 13 landmarks species from GenBank for each individual was identified. were used: 1-6 and 7-13 as Types I and II, respectively. The Sequences from family Vertiginidae in which Hypselostoma converted images were then analyzed using the MorphoJ latispira belongs, and families Valloniidae and Strobolopsidae, software (Klingenberg 2011). Procrustes superimposition was from which the closest species matches according to BLAST, used to initially extract the shape information of the data to were obtained from GenBank and aligned with the samples eliminate factors (i.e., size, position and rotation of the shells) in using the ClustalW multiple alignment in BioEdit Sequence the morphometric data and to detect problems in the data set Alignment Editor 7.0.9.0 (Hall 1999). such as outliers. Generation of covariance matrices was done to perform PCA and canonical variate analysis to search for Molecular Phylogenetic Tree Generation features which would indicate the separation between the The aligned sequences were tested for saturation using the sample groups. Xia test (Xia 2013) in DAMBE (Data Analysis in Molecular Biology and Evolution). Since the sequences were found to have Phylogenetic Analysis hypervariability, the aligned sequences were uploaded to DNA Extraction and PCR Amplification GBlocks server (http://molevol.cmima.csic.es/castresana/ The molecular analysis was conducted at the DNA Gblocks_server.html) to remove ambiguously aligned internal Barcoding Laboratory, Institute of Biology, University of the positions of the DNA and to conserve the informational regions. Philippines Diliman. Snail specimens from Masungi Georeserve The final sequence alignment was edited in BioEdit Sequence (n=4) and Baguio City (n=4) were crushed using two clean Alignment Editor 7.0.9.0 accordingly and was run in DAMBE microscope slides, then the shell fragments and soft tissue were to verify its level of saturation. The optimal model for 16S gene, separated using a toothpick. DNA extraction was done by which was determined using the jModeltest 0.1 (Darriba et al. following the manufacturer’s instruction of PureLink Genomic 2012), was used for the neighbor-joining (NJ) tree (Saitou & Nei DNA Kit. From the extracted DNA, portion of the 1987) in PAUP version 4.0b10 (Swofford 2002) and Maximum- mitochondrial (16S rDNA) ribosomal gene was amplified using Likelihood (ML) (Felsenstein 1981) tree in PhyML version 3.0 Polymerase Chain Reaction (PCR). The PCR cocktail for 16S (Guindon & Gascuel 2003). Bootstrap resampling for both NJ fragment used the 16S arm forward primer (5’- and ML trees computed for 1000 replicates and all bootstrap CTTCTCGACTGTTTATCAAAAACA-3’) (Bonnaud et al. values were indicated. 1994) and 16S BR primer (5’-CCGGTCTGAACTCAGATC ACGT-3’) (Palumbi et al. 1991). With 13 µL per sample, the Interspecific and Intraspecific Sequence Variations PCR mix were as follows: 7.312 µL distilled water, 2.5 µL of The interspecific and intraspecific sequence divergence 5X BiolineTM PCR buffer with DNTPs and MgCl, 0.375 µL was also determined by computing the pairwise distance each of 10 mM 16S forward and reverse primers, 0.5 µL of comparisons in PAUP to further verify if there were distinct

100% DMSO, 0.375 µL of 25 mM MgCl2, 0.06 of 5U/µl differences within the samples and Hypselostoma genus. The BiolineTM MyTaq DNA Polymerase and 2 µL of sample DNA. four 16S rRNA sequences from both Masungi and Baguio were The cycling profile was as follows: denaturation of one cycle at compared to determine intraspecific sequence divergence while 94°C for 2 min, followed by 39 cycles composed of 94°C for 30 one 16S rRNA sequence per Hypselostoma species obtained in s denaturation, 45°C for 30 s annealing and 68°C for 1 min the GenBank was compared with the Masungi and Baguio extension. Final extension step was set at 72°C for 2 min. The sequences to determine interspecific sequence divergence. PCR products were visualized in 1% agarose gel with ethidium

Lipae et al.: New microsnail subspecies from Masungi Georeserve

Results

Conchometric Analysis A total of 82 and 98 samples were collected from Masungi and Baguio, respectively. All characters in PC1 and PC2 showed positive loadings except for major width (MW), which is negative on the latter (Fig. 3). Among all the characters included, MW also has the highest loading value (PC1). PC1 also accounted for most of the variance (80.45%), which is followed by PC2 (10.14%). The two populations of H. latispira have slightly overlapped with each other based on the linear morphometrics with the red plane (Baguio) on the left and the blue plane (Masungi) showing that some individuals have similar measurements in between the two populations (Fig. 4). Nonetheless, the scatter plot diagram demonstrated that the two populations have aggregated separately from each other as influenced by MW being the character with the greatest contribution in separating the two populations. Geometric morphometric analysis was also done in the study to determine the shell shape variations between the two populations. Results revealed that the two populations were consistently separated based on the canonical variant analysis of the two views (Fig. 5). The lateral and dorsal views have Mahalonobis distance values of 5.8160 and 3.8037, Figure 3. PC1 (A) and PC2 (B) loadings of the conchometric respectively, both of which are significant (p < 0.0001). characters. SH – shell height; LW – lesser width; MW – major width; AH - aperture height; AW – aperture width. Molecular Analysis From the five DNA samples extracted from Masungi and Hypselostoma species were obtained using pairwise distance Baguio, only four from each site were successfully utilized for comparison (Table 2). Only an average of 0.0131 ± 0.0126 the amplification and sequencing of a 317-base pair (bp) corrected pairwise distance was found between the samples fragment. A total of 31 sequences were aligned: eight (8) from from the two sites, implying that the Masungi and Baguio this study, nineteen (19) from family Vertiginidae found in samples have an average of 99.9869% 16S rRNA sequence GenBank for the in-group taxon, three (3) from family similarity. Comparison of the sequences from the collected Valloniidae, and one (1) from family Strobilopsidae for the Hypselostoma samples and Hypselostoma species from the outgroup taxon. GenBank showed an average of 0.7778 ± 0.1852 corrected The closest 16S sequences match of the samples using pairwise distance. The percentage of pairwise distance BLAST was obtained (Table 1). There were no conspecific matches from the species from GenBank (percent similarities ranged from 83- 84%); therefore, the H. latispira samples have novel 16S rRNA sequences. The Xia test was implemented in DAMBE to test for substitution saturation after removing the hypervariable regions of the 16s rRNA using the GBlocks server (Table 2). The index of substitution (0.3299) was less than the index of substitution for symmetrical (0.6798) and asymmetrical tree (0.3596), rendering the data set useful for molecular phylogenetic analysis. Sequence divergences between samples from Masungi and Figure 4. Principal Component Analysis of the linear shell dimensions Baguio, and Philippine Hypselostoma species and other of H. latispira from Baguio and Masungi Georeserve.

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Lipae et al.: New microsnail subspecies from Masungi Georeserve

Figure 6. Intraspecific Sequence Divergence between Masungi and Baguio samples and Interspecific Sequence Divergence of Hypselostoma species obtained in the GenBank and Masungi and Figure 5. Canonical Variate Analysis of the shell shape using the Baguio sequences. lateral view (A) and dorsal view (B) of H. latispira from Baguio and Masungi Georeserve. Mahalanobis distance = lateral view - 5.8160 (p < The NJ tree generated based on 16S rRNA gene (317 bp) 0.0001); dorsal view - 3.8037 (p < 0.0001). was rooted only to the Valloniidae, excluding the comparison within samples and Hypselostoma species for the Strobilopsidae, as an outgroup using the TPM1uf+G model as corrected (Fig. 6) were illustrated. The highest sequence determined by jModelTest as the optimum model (Fig. 7). The divergence for both corrected and uncorrected distances was H. latispira samples collected from Masungi distinctly separated found within the family level (corrected=0.67-0.847 & >1.0; from the samples collected from Baguio with 87% bootstrap uncorrected=0.201-0.268) and least within the species level support; however, H. latispira Baguio 4 did not cluster with the (corrected and uncorrected=0-0.067). The percentage of other Baguio samples, having 84% bootstrap support, making pairwise distance comparison within genus and species showing the Baguio samples a polyphyletic group. The Baguio cluster the barcoding gap was 0.067-0.335%. However, a single sample was poorly supported (>50% bootstrap support). The (Baguio 4) was distinct from the rest of the samples based on its representative for the outgroup family Strobilopsidae was average pairwise differences: with other Baguio samples observed to be grouped within the in-group taxon with poor (corrected=2.683%; uncorrected=2.528), with Masungi samples bootstrap support. (corrected=3.403%; uncorrected=3.157%), and for both sites The ML tree for the phylogeny of 16S rRNA gene (317 bp) (corrected=3.093%; uncorrected=2.887%). of the samples from Masungi and Baguio, representatives of

Table 1. BLAST results for the 16S sequences of Hypselostoma samples.

Hypselostoma GenBank Accession % Match Family Location + Code Species Number Baguio 1 Vallonia gracilicosta GQ921547 83.72% Valloniidae Baguio 2 Vallonia gracilicosta GQ921547 83.46% Valloniidae Baguio 3 Vallonia gracilicosta GQ921547 83.72% Valloniidae Baguio 4 Vallonia gracilicosta GQ921547 83.80% Valloniidae Masungi 1 Vallonia gracilicosta GQ921547 84.48% Valloniidae Masungi 2 Vallonia gracilicosta GQ921547 84.48% Valloniidae Masungi 3 Vallonia gracilicosta GQ921547 84.48% Valloniidae Masungi 5 Vallonia gracilicosta GQ921547 84.48% Valloniidae

Lipae et al.: New microsnail subspecies from Masungi Georeserve

Table 2. The range and average uncorrected and corrected distances among the sequences of genus Hypselostoma. Range of Range of Hypselostoma Number of Average uncorrected Average corrected uncorrected corrected Comparisons comparisons distances ± SD distances ± SD distances (p) distances

Between genera 38 0.2701 ± 0.0288 0.1829 to 0.3006 0.7778 ± 0.1852 0.3540 to 1.002

Between sampling 0.0061 to 16 0.0126 ± 0.0112 0.0063 to 0.0315 0.0131 ± 0.0126 sites 0.0340 families Vertiginidae, Valloniidae, and Strobilopsidae was results. The distinct separation of Masungi and Baguio samples generated using the TPM1uf+G model (Fig. 8). The clusters in both NJ and ML tree based on their 16S rRNA suggests that reflected were relatively similar with the NJ tree; however, the the two samples are phylogenetically different. This implies that polyphyletic group for the Baguio samples had a higher evolutionary changes occurred on the 16S rRNA gene of the two bootstrap support (78%). populations that led to the divergence into two clades. However, it is also important to note that one specimen from Baguio falls Discussion outside of the two groups. This sample has approximately 3% genetic difference from the two populations, which could be Size variations and shape differences in terms of body explained by incomplete lineage sorting, a common whorl and aperture width were observed between the two phenomenon to recent and rapidly radiating species (Funk & populations of H. latispira. The snails from Baguio were Omland 2003; Tang et al. 2012). Additionally, the specimen smaller in size and have narrow body whorl and apertural width also has strong morphological similarities with the rest of the while those of the Masungi population were bigger and have Baguio samples. Further taxonomic evaluation is needed for this larger body whorl and apertural width. All Masungi samples specimen to determine its identity. appear to be larger as reflected by the individual measurements, Interestingly, the genus Hypelostoma did not form a both in linear and geometric morphometrics. At present, any monophyletic group based on the 16S marker which is a ecomorphological explanation cannot be provided for such possible indication of convergent evolution of morphological differences since factors affecting size and shape were not features as adaptation to its habitat (Goodacre & Wade 2001). It included in this study. While size variations are generally driven is also possible that these are distinct genera based on by genes, several environmental factors may greatly affect the biogeography and that the taxon should be flagged for re- phenotypes of land snails. Higher plasticity in physiology and evaluation. However, this must be further investigated and is morphology are more observed with increasing latitude and beyond the scope of this paper. altitude augmented by greater climatic variability (Naya et al. Generally, genetic differentiation is more likely to occur 2011). For certain species of snails, adult shell sizes decrease where physical barriers prevent gene ﬂow between populations with increasing elevation and decreasing temperature (Burla & (Crispo et al. 2006); in this case the mountain on which Baguio Stahel 1983; Baur & Raboud 1988). Interestingly, the trend is situated and the 200-km distance between the two sites may should be opposite since there is higher feeding activity with have acted as physical barriers that can explain the slight genetic high humidity and therefore an increase in body size. However, difference within H. latispira. The difference in environmental smaller shell sizes are also associated with areas having higher conditions between the two sites (e.g. 1000-m elevation annual precipitation (Goodfriend, 1986; Pfenninger and difference) may have played a role in the adaptive divergence of Magnin, 2001; Naya et al. 2011). These factors are present in H. latispira that led to genetic variation. It is also important to Baguio (high precipitation, high altitude, low temperature), consider how the interaction of dispersal and gene flow would which could have potentially affected the size of the H. latispira affect adaptive divergence of the species (Räsänen & Hendry in the area. However, this remains to be tested and is an avenue 2008). Moreover, Clarke & Murray (1969) also observed that for further exploration. due to their low mobility, snails would have some genetic The molecular analyses conducted to assess the differences even in short distances (>20 m) that may drive relationship between the H. latispira species collected from ecological speciation of the snails. The observed nesting of the Masungi Georeserve, Rizal and Baguio, Benguet showed mixed outgroup representative of family Strobilopsidae, Strobilops

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Lipae et al.: New microsnail subspecies from Masungi Georeserve

Figure 7. Neighbor-joining (NJ) gene tree based on the 317 nucleotides of the 16S rRNA gene of Hypselostoma species from Masungi Georeserve and Baguio, representative species from family Vertginidae as in-group taxon, family Valloniidae (Vallonia costata, Vallonia gracilicosta and Zoogenetes harpa) and family Strobilopsidae (Strobilops labyrinthica) as outgroup taxon. Values on nodes represent percent NJ bootstraps based on 1000 replicates; values less than 50% are not shown. Scale bar represents 1 nucleotide substitution for every 10 nucleotides.

Figure 8. Maximum Likelihood (ML) gene tree based on the 317 nucleotides of the 16S rRNA gene of Hypselostoma species from Masungi Georeserve and Baguio including outgroup and ingroup taxa. Values on nodes represent percent NJ bootstraps based on 1000 replicates; values less than 50% are not shown. Scale bar represents 5 nucleotide substitutions for every 100 nucleotides.

Lipae et al.: New microsnail subspecies from Masungi Georeserve labyrinthicus (Say, 1817) with the in-group family Vertiginidae in both NJ and ML trees should be further clarified using another marker to resolve the placement of the Strobilopsidae. Climo (1979) also noted the uncertain taxonomic status of the group relative to the other families. Meanwhile, the pairwise divergence computed between the Masungi and Baguio samples suggested a completely opposite result from the generated phylogenetic trees. Since a barcoding gap was observed between intraspecific and interspecific variations of H. latispira, a threshold value (0.067) for delineating species was obtained. Threshold values are genetic distance values that can be computed from the pairwise distance comparison when a barcoding gap between the intraspecific and interspecific genetic variations is observed (Meyer & Paulay 2005). The threshold value would allow delineation and identification of a species, and it would vary across taxa. The average genetic difference of the Masungi and Baguio samples (0.0131 ± 0.0126) implies that the H. latispira Figure 9. Paratype specimen of Hypselostoma latispira latisipira. samples are 99.98% similar in terms of their 16S rRNA sequences, a significant evidence that the samples from the sites would greatly contribute to increased gene flow and high different sites are the same species. Some Southeast Asian land similarity. However, there are no contiguous forests over snails also exhibit low genetic divergence. Gyliotrachela limestone that exist which would directly connect the two hungerfordiana (Möllendorff, 1981), which is a widespread locations except for a few fragments of karst forests along the species, revealed high genetic similarity between individuals in eastern side of the central part of Luzon Island. The closest karst relatively close areas with an intraspecific divergence of less ecosystem to Masungi is in Rodriguez, Rizal and Biak-na-bato, than 0.10 (Hoekstra & Schilthuizen 2011) similar to Bornean Bulacan which are >20 km and around 90 km away, Georissa spp. (0.03-0.11) (Khalik et al. 2019). Abu-Bakar et al. respectively. Baguio, on the other hand, is more than 150 km (2014) also showed low species and intraspecific genetic away from these two karst forests and the Southern Sierra diversities (4.3–10.1%) in some snails using 16S rRNA from an Madre mountains in eastern Luzon would be the possible island off Peninsular Malaysia. As in the case of the H. latispira stepping stones for this species which could probably explain to populations considered in this study, connectivity between the some degree the low genetic distance between the populations.

Table 3. Shell measurements of the type specimens of H. l. latispira and H. l. masungiensis.

Shell Height Major width Lesser width Aperture width Aperture width SH/MW (mm) (mm) (mm) (mm) (mm) (mm) H. l. latispira Holotype 2.30 4.50 2.90 1.70 0.51 0.65 Paratype 2.30 4.20 2.80 - 0.55 0.67 Paratype 2.40 4.20 3.00 1.80 0.56 0.71 Paratype 2.20 4.40 3.00 1.70 0.51 0.68 Paratype 2.40 4.40 3.00 1.90 0.54 0.68 H. l. masungiensis Holotype 2.60 5.00 3.20 2.00 0.52 0.64 Paratype 2.50 4.70 3.00 1.80 0.53 0.64 Paratype 2.60 4.70 2.90 1.90 0.55 0.62 Paratype 2.60 4.90 3.00 1.80 0.53 0.61 Paratype 2.50 4.90 3.00 1.80 0.51 0.61

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Lipae et al.: New microsnail subspecies from Masungi Georeserve

Nevertheless, it must be put into consideration that there are limitations in the effectivity of using the intra- and interspecific genetic distance for species identification. Several studies have criticized the effectivity of threshold value for delineating species for it is only effective in “globally comprehensive and well-circumscribed datasets” (Meyer & Paulay 2005), and that the barcoding gap is usually absent and instead an overlap is observed when large proportion of closely related taxa is analyzed (Moritz & Cicero 2004). Other studies Figure 11. Apertural teeth comparison between nominotypical (left) have also reported that using this method is not useful in and novel subspecies (right). PL - parietal lamella, IF - infraparietal identifying taxa with wide range distributions (Bergsten et al. lamella, CL - columellar lamella, LPP - lower palatal plica, ITP - in- 2012). Although there are studies that would still argue for the terpalatal plica, UPP - upper palatal plica. reliability of intra- and interspecific genetic distance for delineation purposes (Barrett and Hebert 2005), it should be Type material. Holotype (SH: 2.6 mm, LW: 3.2 mm, MW: 5 reckoned that for this method of species identification to be mm, AH: 1.8 mm, AW: 2 mm) UPLBMNHLS150, Philippines, accurate and conclusive an extensive genetic sampling is Luzon Island, Rizal Province, Municipality of Baras, Masungi required which is beyond the scope of this study. There are only Georeserve, limestone outcrops of 600 steps, 631 masl, 14° 35' five 16S rRNA Hypselostoma species sequences available in the 38.724” N, 121° 19' 2.424” E, coll. H.B. Lipae, 6 March 2019. GenBank that render the sample size comparably insufficient. This would mean that the lack of molecular researches on these Paratypes: UPLBMNHLS151 (paratype1), UPLBMNHLS152 micro land snails would still restrict phylogenetic elucidation of (paratype2), UPLBMNHLS153 (paratype3), UPLBMNHLS154 the species. (paratype4), coll. H.B. Lipae, same data as preceding.

Taxonomy Diagnosis. This subspecies is distinguished from H. l. latispira (Fig. 9) by having a more prominent infraparietal plica and an Hypselostoma latispira masungiensis Lipae & de Chavez, additional apertural tooth (interpalatal plica) (Fig. 11). H. l. subsp. nov. masungiensis also has larger major width and taller apertural (Fig. 10) height. It also has a wider body whorl and narrow apertural width than H. l. latispira from Baguio.

Figure 10. Hypselostoma latispira masungiensis subspecies nov.

Lipae et al.: New microsnail subspecies from Masungi Georeserve

Description. Shell relatively medium- to large-size for the Acknowledgements genus, 4.4-5.0 mm wide. Brown with white peristome. Depressed-conical in shape; about 0.51-0.55 times. Minor We would like to thank the Masungi Georeserve diameter of shell 0.61-0.64 times major diameter. Sculpture Foundation for allowing us to conduct a study in the Masungi consisting of low irregularly spaced growth striations and Georeserve and the Department of Environment and Natural wrinkles giving the surface a fine, undulating appearance. Resources for granting us a permit for collection (R4A-WGP- Peristome slightly expanded and nearly rounded in shape. 2018-R17-034). We would also like to thank the University of Apertural dentition consisting of 6 teeth deeply inserted within. the Philippines System Enhanced Creative Work and Research Parietal lamella largest, about as high as wide, weakly sinuous. Grant (ECWRG 2018-02-013) for funding this research. Special Infraparietal lamella protruding. Columellar lamella half as long thanks to the Florida State Museum, Gainesville, Florida for as parietal lamella. Upper palatal plica tubercular and slightly loaning the paratype specimens of H. l. latispira. Within the smaller than lower palatal plica. Interpalatal plica small and University of the Philippines, we would like to thank Dr. tubular. Ayolani V. de Lara for the use of the Malacology Laboratory, Dr. Zenaida Baoanan for lending their H. l. latispira collections, Measurements (in mm): SH = 2.2-2.6, LW = 2.9-3.2, MW = Dr. Ireneo L. Lit, Jr. and Prof. Annalee Soligam-Hadsall for the 4.4-5.0, AH = 1.5-1.8, AW = 1.7-2.0 (n = 82). invaluable input on the taxonomy part.

Etymology. The subspecies name is derived from the type Literature Cited locality – Masungi. Abu-Bakar, S., N.M. Razali, F. Naggs, C. Wade, S. Mohd-Nor Type Locality. Philippines, Luzon Island, Rizal Province, & S.A.Tan, 2014. The mitochondrial 16s rRNA reveals Municipality of Baras, Masungi Georeserve, 14° 35' 24” N, high anthropogenic influence on land snail diversity in a 121° 18' 36” E. preliminary island survey. Molecular Biology Reports, 41 (3): 1799-1805. Conclusions and Recommendations Altschul, S.F., W. Gish, W. Miller., E.W. Myers & D.J. Lipman, 1990. Basic local alignment search tool. Journal of The H. latispira collected from Masungi Georserve is Molecular Biology, 215(3): 403-410. hereby proposed as a new subspecies based on shell size, shell Barrett, R.D.H. & P.D.N. Hebert, 2005. Identifying spiders shape variations, presence of additional apertural tooth through DNA barcodes. Canadian Journal of Zoology- (interpalatal plica), and novel site congruent clade separation of Revue Canadienne De Zoologie, 83: 481–491. Masungi and Baguio H. latispira. A new distribution of H. Baur, B. & C. Raboud, 1988. Life history of the land snail latispira outside its type locality is also reported here. Arianta arbustorum along an altitudinal gradient. Journal of Moreover, a total of eight novel sequences of H. latispira were Animal Ecology, 57: 71-87. produced in this study. It is recommended, however, to include Bonnaud, L., R. Boucher-Rodoni & M. Monnerot, 1994. other genes to further determine their molecular differences and Phylogeny of decapod cephalopods based on partial 16S further confirm the topology of the two populations. Also, rDNA nucleotide sequences. Comptes rendus de l’Académie relevant environmental data must be gathered to reflect the de sciences, Serie III, Sciences de la Vie, 317(6): 581-588. ecomorphological variations of the snail in the two areas. Burla, H. & W. Stahel, 1983. Altitudinal variation in Arianta Further surveys must also be conducted in the karst systems arbustorum (Mollusca, Pulmonata) in the Swiss Alps. between the two sites to reveal the real distributional range of Genetica, 62: 95-108. H. latispira. Benthem Jutting, W.S.S., 1949. On a collection of non-marine This paper hopes to provide additional knowledge on the Mollusca from Malaya in the Raffles Museum, Singapore, existing but outdated literature on Philippine microsnails. with an appendix on cave snails. Bulletin of the Raffles Moreover, the discovery of a new subspecies further highlights Museum, Singapore, 19: 50-77. the importance of karst landscapes as areas of high conservation Benthem Jutting, W.S.S., 1950. The Malayan species of value. Boysidia, Paroboysidia, Hypselostoma and Gyliotrachela (Gastropoda. Pulmonata, Vertiginidae) with a catalogue of all the species hitherto described. Bulletin of the Raffles Museum, Singapore, 21: 5-47.

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Lipae et al.: New microsnail subspecies from Masungi Georeserve

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