a new subspecies of microsnail from masungi georeserve...
TRANSCRIPT
PRIMARY RESEARCH PAPER | Philippine Journal of Systematic Biology
DOI 10.26757/pjsb2020c14003
Volume 14 Issue 3 - 2020 | 1 © Association of Systematic Biologists of the Philippines
A new subspecies of microsnail from Masungi Georeserve,
Rizal, Philippines
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
1Graduate School, University of the Philippines Los Baños, College,
Laguna 4031 2Animal Biology Division, Institute of Biological Sciences, University
of the Philippines Los Banos, College, Laguna 4031 3Institute of Biology, College of Science, University of the Philippines,
Diliman, Quezon City 1101
*Corresponding email: [email protected]
Date Submitted: 24 February 2020
Date Accepted: 06 July 2020
Introduction
The genus Hypselostoma Benson, 1856 is classified under
the family Hypselostomatidae Zilch, 1959 (sensu Schileyko
1998). However, Bouchet et al. (2017) considered the family as
a synonym of Gastrocoptidae Pilsbry, 1918 following
Gittenberger (1973), while several studies recognized
Vertiginidae (Thompson & Lee 1988; Panha 1997; Hwang
2014). Páll-Gergely et al. (2019) recognized the
Hypselostomatidae. It is widely distributed throughout southern
China and the Malayan peninsula, extending towards the
Philippines and the Ryukyu Islands (Thompson & Auffenberg
1984; Páll-Gergely et al. 2015). Hypselostoma species are
described as having depressed conic spires with an upturned or
ascending last whorl and continuous peristome (Pilsbry 1918)
and protrusions or teeth along the aperture (Tongkerd et al.
2004), which is of great taxonomic importance. Pilsbry (1917)
provided the first monograph of this genus followed by Haas
(1937) and Tomlin (1939) with additional species and
subspecies described by Benthem Jutting (1949, 1950, 1962).
The Philippine species were also discussed by von Moellendorff
(1888), Quadras and von Moellendorff (1894, 1896), Pilsbry
(1917), and Haas (1937). Common features among the
Philippine group are the spiral striations on the shell and the
cross orientation of apertural teeth (Pilsbry 1918). Currently, a
total of eight species occur in the Philippines, with H. luzonicum
Moellendorff, 1888 and H. polyodon Moellendorff, 1896 having
five and two subspecies, respectively (Pilsbry 1917; Haas 1937;
Thompson & Auffenberg 1984).
The most recently described Philippine species is H.
latispira Thompson & Auffenberg, 1984 from Baguio City,
Benguet Province (Thompson & Auffenberg 1984). Thus far, it
is known only from the type locality until now. In this paper, a
new subspecies of H. latispira was discovered from Masungi
Georeserve, Rizal Province which is more than 200 km away
Harold B. Lipae1*, Angelique L. Estabillo2, Ian Kendrich C. Fontanilla3,
and Emmanuel Ryan C. de Chavez2
Volume 14 Issue 3 - 2020 | 2 Philippine Journal of Systematic Biology Online ISSN: 2508-0342
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).
For comparison, samples of H. l. latispira from Baguio
City, Benguet Province were also collected on March 23, 2019.
Ninety-eight snails were taken from the limestone boulders in a
residential area in Barangay Dominican Hill, Baguio City
(16.40°N, 120.58°E, 1546 masl). Live samples were placed in
Petri dishes with wet tissues and scraped food materials (lichen,
moss, algae) from the rocks and were reared in the laboratory.
All shells were measured in the laboratory.
Type specimens of H. latispira were also loaned from the
Florida State Museum, Gainesville, Florida, USA for further
evaluation. Four paratypes (dry) were examined
morphologically to compare with the samples collected from
the Masungi Georeserve.
Shell Measurements
Adult individuals were measured for the shell height, shell
width, aperture height, and aperture width using a digital
Vernier caliper following Tanmuangpak et al. (2015). The
number of whorls was also counted. Photomicrographs of the
snails were also taken using a camera (ToupTek XCAM
1080PHB, Hangzhou, China) attached to a stereo microscope
(Olympus SZX7, Tokyo, Japan).
Shell Morphometrics
Linear morphometrics were done using Principal
Component Analysis (PCA). Measurement data were exported
to Paleontological Statistics software (PAST version 3, Hammer
et al. 2016) and were color coded based on location. PCA was
performed under the covariation option to determine the shell
characters that would maximize the separation of the two
groups. Subsequently, the number of components was examined
which was generated using eigenvalues and percent variance.
Geometric morphometrics were also done to determine
shell shape variations between the two groups. Photos of the
snails (dorsal and lateral views) were taken and converted to a
tps file using tpsUtil (http://life.bio.sunysb.edu/morph/). The file
was opened in tpsDig (http://life.bio.sunysb.edu/morph/) to
assign landmark points on the shell. A combination of Types I
and II landmarks was used for both views (Fig. 2). Type I
landmarks are those which correspond to homologous
structures, juxtaposed with tissues, and have evolutionary
significance, whereas Type II landmarks are points that capture
the general shape of the shell. Eighteen landmark points were
used for the lateral view: landmarks 1, 2, 3, 5, 6, 8, 14, 15, 17
Figure 1. Map of Luzon Island showing Baguio City, Benguet
Province and Masungi Georeserve.
Volume 14 Issue 3 - 2020 | 3 © Association of Systematic Biologists of the Philippines
Lipae et al.: New microsnail subspecies from Masungi Georeserve
and 18 as Type I, and 4, 7, 9, 10, 11, 12, 13 and 16 as Type II
landmark points. For the dorsal view, a total of 13 landmarks
were used: 1-6 and 7-13 as Types I and II, respectively. The
converted images were then analyzed using the MorphoJ
software (Klingenberg 2011). Procrustes superimposition was
used to initially extract the shape information of the data to
eliminate factors (i.e., size, position and rotation of the shells) in
the morphometric data and to detect problems in the data set
such as outliers. Generation of covariance matrices was done to
perform PCA and canonical variate analysis to search for
features which would indicate the separation between the
sample groups.
Phylogenetic Analysis
DNA Extraction and PCR Amplification
The molecular analysis was conducted at the DNA
Barcoding Laboratory, Institute of Biology, University of the
Philippines Diliman. Snail specimens from Masungi Georeserve
(n=4) and Baguio City (n=4) were crushed using two clean
microscope slides, then the shell fragments and soft tissue were
separated using a toothpick. DNA extraction was done by
following the manufacturer’s instruction of PureLink Genomic
DNA Kit. From the extracted DNA, portion of the
mitochondrial (16S rDNA) ribosomal gene was amplified using
Polymerase Chain Reaction (PCR). The PCR cocktail for 16S
fragment used the 16S arm forward primer (5’-
CTTCTCGACTGTTTATCAAAAACA-3’) (Bonnaud et al.
1994) and 16S BR primer (5’-CCGGTCTGAACTCAGATC
ACGT-3’) (Palumbi et al. 1991). With 13 µL per sample, the
PCR mix were as follows: 7.312 µL distilled water, 2.5 µL of
5X BiolineTM PCR buffer with DNTPs and MgCl, 0.375 µL
each of 10 mM 16S forward and reverse primers, 0.5 µL of
100% DMSO, 0.375 µL of 25 mM MgCl2, 0.06 of 5U/µl
BiolineTM MyTaq DNA Polymerase and 2 µL of sample DNA.
The cycling profile was as follows: denaturation of one cycle at
94°C for 2 min, followed by 39 cycles composed of 94°C for 30
s denaturation, 45°C for 30 s annealing and 68°C for 1 min
extension. Final extension step was set at 72°C for 2 min. The
PCR products were visualized in 1% agarose gel with ethidium
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
The Staden Package 4.0 (Staden et al. 2000) was used to
assemble the 16S mitochondrial ribosomal DNA fragments.
Using Basic Local Alignment Search Tool (BLAST, http://
blast.ncbi.nih.gov/Blast.cgi, Altschul et al. 1990), the closest
species from GenBank for each individual was identified.
Sequences from family Vertiginidae in which Hypselostoma
latispira belongs, and families Valloniidae and Strobolopsidae,
from which the closest species matches according to BLAST,
were obtained from GenBank and aligned with the samples
using the ClustalW multiple alignment in BioEdit Sequence
Alignment Editor 7.0.9.0 (Hall 1999).
Molecular Phylogenetic Tree Generation
The aligned sequences were tested for saturation using the
Xia test (Xia 2013) in DAMBE (Data Analysis in Molecular
Biology and Evolution). Since the sequences were found to have
hypervariability, the aligned sequences were uploaded to
GBlocks server (http://molevol.cmima.csic.es/castresana/
Gblocks_server.html) to remove ambiguously aligned internal
positions of the DNA and to conserve the informational regions.
The final sequence alignment was edited in BioEdit Sequence
Alignment Editor 7.0.9.0 accordingly and was run in DAMBE
to verify its level of saturation. The optimal model for 16S gene,
which was determined using the jModeltest 0.1 (Darriba et al.
2012), was used for the neighbor-joining (NJ) tree (Saitou & Nei
1987) in PAUP version 4.0b10 (Swofford 2002) and Maximum-
Likelihood (ML) (Felsenstein 1981) tree in PhyML version 3.0
(Guindon & Gascuel 2003). Bootstrap resampling for both NJ
and ML trees computed for 1000 replicates and all bootstrap
values were indicated.
Interspecific and Intraspecific Sequence Variations
The interspecific and intraspecific sequence divergence
was also determined by computing the pairwise distance
comparisons in PAUP to further verify if there were distinct
differences within the samples and Hypselostoma genus. The
four 16S rRNA sequences from both Masungi and Baguio were
compared to determine intraspecific sequence divergence while
one 16S rRNA sequence per Hypselostoma species obtained in
the GenBank was compared with the Masungi and Baguio
sequences to determine interspecific sequence divergence.
Figure 2. Dorsal (A) and lateral (B) views with corresponding landmark
points. Red dots are type I landmarks, and yellow dots are type II.
Volume 14 Issue 3 - 2020 | 4 Philippine Journal of Systematic Biology Online ISSN: 2508-0342
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,
respectively, both of which are significant (p < 0.0001).
Molecular Analysis
From the five DNA samples extracted from Masungi and
Baguio, only four from each site were successfully utilized for
the amplification and sequencing of a 317-base pair (bp)
fragment. A total of 31 sequences were aligned: eight (8) from
this study, nineteen (19) from family Vertiginidae found in
GenBank for the in-group taxon, three (3) from family
Valloniidae, and one (1) from family Strobilopsidae for the
outgroup taxon.
The closest 16S sequences match of the samples using
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
Baguio, and Philippine Hypselostoma species and other
Hypselostoma species were obtained using pairwise distance
comparison (Table 2). Only an average of 0.0131 ± 0.0126
corrected pairwise distance was found between the samples
from the two sites, implying that the Masungi and Baguio
samples have an average of 99.9869% 16S rRNA sequence
similarity. Comparison of the sequences from the collected
Hypselostoma samples and Hypselostoma species from the
GenBank showed an average of 0.7778 ± 0.1852 corrected
pairwise distance. The percentage of pairwise distance
Figure 3. PC1 (A) and PC2 (B) loadings of the conchometric
characters. SH – shell height; LW – lesser width; MW – major width;
AH - aperture height; AW – aperture width.
Figure 4. Principal Component Analysis of the linear shell dimensions
of H. latispira from Baguio and Masungi Georeserve.
Volume 14 Issue 3 - 2020 | 5 © Association of Systematic Biologists of the Philippines
Lipae et al.: New microsnail subspecies from Masungi Georeserve
comparison within samples and Hypselostoma species for the
corrected (Fig. 6) were illustrated. The highest sequence
divergence for both corrected and uncorrected distances was
found within the family level (corrected=0.67-0.847 & >1.0;
uncorrected=0.201-0.268) and least within the species level
(corrected and uncorrected=0-0.067). The percentage of
pairwise distance comparison within genus and species showing
the barcoding gap was 0.067-0.335%. However, a single sample
(Baguio 4) was distinct from the rest of the samples based on its
average pairwise differences: with other Baguio samples
(corrected=2.683%; uncorrected=2.528), with Masungi samples
(corrected=3.403%; uncorrected=3.157%), and for both sites
(corrected=3.093%; uncorrected=2.887%).
The NJ tree generated based on 16S rRNA gene (317 bp)
was rooted only to the Valloniidae, excluding the
Strobilopsidae, as an outgroup using the TPM1uf+G model as
determined by jModelTest as the optimum model (Fig. 7). The
H. latispira samples collected from Masungi distinctly separated
from the samples collected from Baguio with 87% bootstrap
support; however, H. latispira Baguio 4 did not cluster with the
other Baguio samples, having 84% bootstrap support, making
the Baguio samples a polyphyletic group. The Baguio cluster
was poorly supported (>50% bootstrap support). The
representative for the outgroup family Strobilopsidae was
observed to be grouped within the in-group taxon with poor
bootstrap support.
The ML tree for the phylogeny of 16S rRNA gene (317 bp)
of the samples from Masungi and Baguio, representatives of
Figure 5. Canonical Variate Analysis of the shell shape using the
lateral view (A) and dorsal view (B) of H. latispira from Baguio and
Masungi Georeserve. Mahalanobis distance = lateral view - 5.8160 (p <
0.0001); dorsal view - 3.8037 (p < 0.0001).
Figure 6. Intraspecific Sequence Divergence between Masungi and
Baguio samples and Interspecific Sequence Divergence of
Hypselostoma species obtained in the GenBank and Masungi and
Baguio sequences.
Hypselostoma
Location + Code
GenBank
Species
Accession
Number % Match Family
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
Table 1. BLAST results for the 16S sequences of Hypselostoma samples.
Volume 14 Issue 3 - 2020 | 6 Philippine Journal of Systematic Biology Online ISSN: 2508-0342
Lipae et al.: New microsnail subspecies from Masungi Georeserve
families Vertiginidae, Valloniidae, and Strobilopsidae was
generated using the TPM1uf+G model (Fig. 8). The clusters
reflected were relatively similar with the NJ tree; however, the
polyphyletic group for the Baguio samples had a higher
bootstrap support (78%).
Discussion
Size variations and shape differences in terms of body
whorl and aperture width were observed between the two
populations of H. latispira. The snails from Baguio were
smaller in size and have narrow body whorl and apertural width
while those of the Masungi population were bigger and have
larger body whorl and apertural width. All Masungi samples
appear to be larger as reflected by the individual measurements,
both in linear and geometric morphometrics. At present, any
ecomorphological explanation cannot be provided for such
differences since factors affecting size and shape were not
included in this study. While size variations are generally driven
by genes, several environmental factors may greatly affect the
phenotypes of land snails. Higher plasticity in physiology and
morphology are more observed with increasing latitude and
altitude augmented by greater climatic variability (Naya et al.
2011). For certain species of snails, adult shell sizes decrease
with increasing elevation and decreasing temperature (Burla &
Stahel 1983; Baur & Raboud 1988). Interestingly, the trend
should be opposite since there is higher feeding activity with
high humidity and therefore an increase in body size. However,
smaller shell sizes are also associated with areas having higher
annual precipitation (Goodfriend, 1986; Pfenninger and
Magnin, 2001; Naya et al. 2011). These factors are present in
Baguio (high precipitation, high altitude, low temperature),
which could have potentially affected the size of the H. latispira
in the area. However, this remains to be tested and is an avenue
for further exploration.
The molecular analyses conducted to assess the
relationship between the H. latispira species collected from
Masungi Georeserve, Rizal and Baguio, Benguet showed mixed
results. The distinct separation of Masungi and Baguio samples
in both NJ and ML tree based on their 16S rRNA suggests that
the two samples are phylogenetically different. This implies that
evolutionary changes occurred on the 16S rRNA gene of the two
populations that led to the divergence into two clades. However,
it is also important to note that one specimen from Baguio falls
outside of the two groups. This sample has approximately 3%
genetic difference from the two populations, which could be
explained by incomplete lineage sorting, a common
phenomenon to recent and rapidly radiating species (Funk &
Omland 2003; Tang et al. 2012). Additionally, the specimen
also has strong morphological similarities with the rest of the
Baguio samples. Further taxonomic evaluation is needed for this
specimen to determine its identity.
Interestingly, the genus Hypelostoma did not form a
monophyletic group based on the 16S marker which is a
possible indication of convergent evolution of morphological
features as adaptation to its habitat (Goodacre & Wade 2001). It
is also possible that these are distinct genera based on
biogeography and that the taxon should be flagged for re-
evaluation. However, this must be further investigated and is
beyond the scope of this paper.
Generally, genetic differentiation is more likely to occur
where physical barriers prevent gene flow between populations
(Crispo et al. 2006); in this case the mountain on which Baguio
is situated and the 200-km distance between the two sites may
have acted as physical barriers that can explain the slight genetic
difference within H. latispira. The difference in environmental
conditions between the two sites (e.g. 1000-m elevation
difference) may have played a role in the adaptive divergence of
H. latispira that led to genetic variation. It is also important to
consider how the interaction of dispersal and gene flow would
affect adaptive divergence of the species (Räsänen & Hendry
2008). Moreover, Clarke & Murray (1969) also observed that
due to their low mobility, snails would have some genetic
differences even in short distances (>20 m) that may drive
ecological speciation of the snails. The observed nesting of the
outgroup representative of family Strobilopsidae, Strobilops
Hypselostoma Comparisons
Number of comparisons
Average uncorrected distances ± SD
Range of uncorrected distances (p)
Average corrected distances ± SD
Range of corrected distances
Between genera 38 0.2701 ± 0.0288 0.1829 to 0.3006 0.7778 ± 0.1852 0.3540 to 1.002
Between sampling sites
16 0.0126 ± 0.0112 0.0063 to 0.0315 0.0131 ± 0.0126 0.0061 to
0.0340
Table 2. The range and average uncorrected and corrected distances among the sequences of genus Hypselostoma.
Volume 14 Issue 3 - 2020 | 7 © Association of Systematic Biologists of the Philippines
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.
Volume 14 Issue 3 - 2020 | 8 Philippine Journal of Systematic Biology Online ISSN: 2508-0342
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
samples are 99.98% similar in terms of their 16S rRNA
sequences, a significant evidence that the samples from the
different sites are the same species. Some Southeast Asian land
snails also exhibit low genetic divergence. Gyliotrachela
hungerfordiana (Möllendorff, 1981), which is a widespread
species, revealed high genetic similarity between individuals in
relatively close areas with an intraspecific divergence of less
than 0.10 (Hoekstra & Schilthuizen 2011) similar to Bornean
Georissa spp. (0.03-0.11) (Khalik et al. 2019). Abu-Bakar et al.
(2014) also showed low species and intraspecific genetic
diversities (4.3–10.1%) in some snails using 16S rRNA from an
island off Peninsular Malaysia. As in the case of the H. latispira
populations considered in this study, connectivity between the
sites would greatly contribute to increased gene flow and high
similarity. However, there are no contiguous forests over
limestone that exist which would directly connect the two
locations except for a few fragments of karst forests along the
eastern side of the central part of Luzon Island. The closest karst
ecosystem to Masungi is in Rodriguez, Rizal and Biak-na-bato,
Bulacan which are >20 km and around 90 km away,
respectively. Baguio, on the other hand, is more than 150 km
away from these two karst forests and the Southern Sierra
Madre mountains in eastern Luzon would be the possible
stepping stones for this species which could probably explain to
some degree the low genetic distance between the populations.
Figure 9. Paratype specimen of Hypselostoma latispira latisipira.
Shell Height (mm)
Major width (mm)
Lesser width (mm)
Aperture width (mm)
Aperture width (mm)
SH/MW (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
Table 3. Shell measurements of the type specimens of H. l. latispira and H. l. masungiensis.
Volume 14 Issue 3 - 2020 | 9 © Association of Systematic Biologists of the Philippines
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
have also reported that using this method is not useful in
identifying taxa with wide range distributions (Bergsten et al.
2012). Although there are studies that would still argue for the
reliability of intra- and interspecific genetic distance for
delineation purposes (Barrett and Hebert 2005), it should be
reckoned that for this method of species identification to be
accurate and conclusive an extensive genetic sampling is
required which is beyond the scope of this study. There are only
five 16S rRNA Hypselostoma species sequences available in the
GenBank that render the sample size comparably insufficient.
This would mean that the lack of molecular researches on these
micro land snails would still restrict phylogenetic elucidation of
the species.
Taxonomy
Hypselostoma latispira masungiensis Lipae & de Chavez,
subsp. nov.
(Fig. 10)
Type material. Holotype (SH: 2.6 mm, LW: 3.2 mm, MW: 5
mm, AH: 1.8 mm, AW: 2 mm) UPLBMNHLS150, Philippines,
Luzon Island, Rizal Province, Municipality of Baras, Masungi
Georeserve, limestone outcrops of 600 steps, 631 masl, 14° 35'
38.724” N, 121° 19' 2.424” E, coll. H.B. Lipae, 6 March 2019.
Paratypes: UPLBMNHLS151 (paratype1), UPLBMNHLS152
(paratype2), UPLBMNHLS153 (paratype3), UPLBMNHLS154
(paratype4), coll. H.B. Lipae, same data as preceding.
Diagnosis. This subspecies is distinguished from H. l. latispira
(Fig. 9) by having a more prominent infraparietal plica and an
additional apertural tooth (interpalatal plica) (Fig. 11). H. l.
masungiensis also has larger major width and taller apertural
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.
Figure 11. Apertural teeth comparison between nominotypical (left)
and novel subspecies (right). PL - parietal lamella, IF - infraparietal
lamella, CL - columellar lamella, LPP - lower palatal plica, ITP - in-
terpalatal plica, UPP - upper palatal plica.
Volume 14 Issue 3 - 2020 | 10 Philippine Journal of Systematic Biology Online ISSN: 2508-0342
Lipae et al.: New microsnail subspecies from Masungi Georeserve
Description. Shell relatively medium- to large-size for the
genus, 4.4-5.0 mm wide. Brown with white peristome.
Depressed-conical in shape; about 0.51-0.55 times. Minor
diameter of shell 0.61-0.64 times major diameter. Sculpture
consisting of low irregularly spaced growth striations and
wrinkles giving the surface a fine, undulating appearance.
Peristome slightly expanded and nearly rounded in shape.
Apertural dentition consisting of 6 teeth deeply inserted within.
Parietal lamella largest, about as high as wide, weakly sinuous.
Infraparietal lamella protruding. Columellar lamella half as long
as parietal lamella. Upper palatal plica tubercular and slightly
smaller than lower palatal plica. Interpalatal plica small and
tubular.
Measurements (in mm): SH = 2.2-2.6, LW = 2.9-3.2, MW =
4.4-5.0, AH = 1.5-1.8, AW = 1.7-2.0 (n = 82).
Etymology. The subspecies name is derived from the type
locality – Masungi.
Type Locality. Philippines, Luzon Island, Rizal Province,
Municipality of Baras, Masungi Georeserve, 14° 35' 24” N,
121° 18' 36” E.
Conclusions and Recommendations
The H. latispira collected from Masungi Georserve is
hereby proposed as a new subspecies based on shell size, shell
shape variations, presence of additional apertural tooth
(interpalatal plica), and novel site congruent clade separation of
Masungi and Baguio H. latispira. A new distribution of H.
latispira outside its type locality is also reported here.
Moreover, a total of eight novel sequences of H. latispira were
produced in this study. It is recommended, however, to include
other genes to further determine their molecular differences and
further confirm the topology of the two populations. Also,
relevant environmental data must be gathered to reflect the
ecomorphological variations of the snail in the two areas.
Further surveys must also be conducted in the karst systems
between the two sites to reveal the real distributional range of
H. latispira.
This paper hopes to provide additional knowledge on the
existing but outdated literature on Philippine microsnails.
Moreover, the discovery of a new subspecies further highlights
the importance of karst landscapes as areas of high conservation
value.
Acknowledgements
We would like to thank the Masungi Georeserve
Foundation for allowing us to conduct a study in the Masungi
Georeserve and the Department of Environment and Natural
Resources for granting us a permit for collection (R4A-WGP-
2018-R17-034). We would also like to thank the University of
the Philippines System Enhanced Creative Work and Research
Grant (ECWRG 2018-02-013) for funding this research. Special
thanks to the Florida State Museum, Gainesville, Florida for
loaning the paratype specimens of H. l. latispira. Within the
University of the Philippines, we would like to thank Dr.
Ayolani V. de Lara for the use of the Malacology Laboratory,
Dr. Zenaida Baoanan for lending their H. l. latispira collections,
Dr. Ireneo L. Lit, Jr. and Prof. Annalee Soligam-Hadsall for the
invaluable input on the taxonomy part.
Literature Cited
Abu-Bakar, S., N.M. Razali, F. Naggs, C. Wade, S. Mohd-Nor
& S.A.Tan, 2014. The mitochondrial 16s rRNA reveals
high anthropogenic influence on land snail diversity in a
preliminary island survey. Molecular Biology Reports, 41
(3): 1799-1805.
Altschul, S.F., W. Gish, W. Miller., E.W. Myers & D.J. Lipman,
1990. Basic local alignment search tool. Journal of
Molecular Biology, 215(3): 403-410.
Barrett, R.D.H. & P.D.N. Hebert, 2005. Identifying spiders
through DNA barcodes. Canadian Journal of Zoology-
Revue Canadienne De Zoologie, 83: 481–491.
Baur, B. & C. Raboud, 1988. Life history of the land snail
Arianta arbustorum along an altitudinal gradient. Journal of
Animal Ecology, 57: 71-87.
Bonnaud, L., R. Boucher-Rodoni & M. Monnerot, 1994.
Phylogeny of decapod cephalopods based on partial 16S
rDNA nucleotide sequences. Comptes rendus de l’Académie
de sciences, Serie III, Sciences de la Vie, 317(6): 581-588.
Burla, H. & W. Stahel, 1983. Altitudinal variation in Arianta
arbustorum (Mollusca, Pulmonata) in the Swiss Alps.
Genetica, 62: 95-108.
Benthem Jutting, W.S.S., 1949. On a collection of non-marine
Mollusca from Malaya in the Raffles Museum, Singapore,
with an appendix on cave snails. Bulletin of the Raffles
Museum, Singapore, 19: 50-77.
Benthem Jutting, W.S.S., 1950. The Malayan species of
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.
Volume 14 Issue 3 - 2020 | 11 © Association of Systematic Biologists of the Philippines
Lipae et al.: New microsnail subspecies from Masungi Georeserve
Benthem Jutting, W.S.S., 1962. Coquilles terrestres nouvelles
de quelques collines calcaires du Cambodge et du Sud
Vietnam. Journal de Conchyliologie, 102: 3-15.
Bergsten, J., D.T. Bilton, T. Fujisawa, M. Elliott, M.T.
Monaghan, M. Balke & A.P. Vogler, 2012. The effect of
geographical scale of sampling on DNA barcoding.
Systematic Biology, 61(5): 851-869.
Bouchet P., J.P. Rocroi, B. Hausdorf, A. Kaim, Y. Kano, A.
Nützel, P. Parkhaev, M. Schrödl & E.E. Strong, 2017.
Revised classification, nomenclature and typification of
gastropod and monoplacophoran families. Malacologia, 61
(1-2): 1-526.
Clarke, B. & J. Murray, 1969. Ecological genetics and
speciation in land snails of the genus Partula. Biological
Journal of the Linnean Society, 1(1-2): 31–42.
Climo, F.M., 1979. The systematic status of some land snails
mistakenly assigned to the New Zealand fauna. New
Zealand Journal of Zoology, 6(3): 407-410.
Crispo, E., P. Bentzen, D.N. Reznick, M.T. Kinnison & A.P.
Hendry, 2006. The relative influence of natural selection
and geography on gene flow in guppies. Molecular
Ecology, 15: 49–62.
Darriba, D., G. Taboada, R. Doallo & D. Posada, 2012.
jModelTest 2: more models, new heuristics and parallel
computing, Nature Methods, 9(8): 772
Felsenstein, J., 1981. Evolutionary trees from DNA sequences:
a maximum likelihood approach. Journal of Molecular
Evolution, 17: 368-376.
Funk, D.J. & K.E. Omland, 2003. Species-level paraphyly and
polyphyly: frequency, causes, and consequences, with
insights from animal mitochondrial DNA. Annual Review of
Ecology Evolution and Systematics, 34: 397–423.
Goodacre, S.L. & C.M. Wade, 2001. Molecular evolutionary
relationships between partulid land snails of the Pacific.
Proceedings of the Royal Society B: Biological Sciences,
268(1462): 1-7.
Goodfriend, G.A., 1986. Variation in land snail shell form and
size and its causes: a review. Systematic Biology, 32(2): 204
-223.
Guindon, S. & O. Gascuel, 2003. A simple, fast, and accurate
algorithm to estimate large phylogenies by maximum
likelihood. Systematic Biology, 52(5): 696-704.
Haas, F., 1937. Neue und Kritische Pupilliden.-Archiv für
Molluskenkunde, 69: 2-12.
Hall, T.A., 1999. BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows 95/98/
NT. Nucleic Acids Symposium Series, 41: 95-98.
Hammer, O., D.A.T. Harper & P.D. Ryan, 2016. PAST:
Paleontological Statistics software package for education
and data analysis. Paleontologica Electronica, 4(1):1-9.
Hoekstra, P. & M. Schilthuizen, 2011. Phylogenetic
relationships between isolated populations of the limestone-
dwelling microsnail Gyliotrachela hungerfordiana
(Gastropoda: Vertiginidae). Journal of Zoological
Systematics and Evolutionary Research, 49(4): 266–272.
Hwang, C.-C., 2014. Hypselostoma kentingensis (Gastropoda:
Vertiginidae) sp. nov., a new species of land snail from
southern Taiwan. Bulletin of Malacology, 37: 27-36.
Khalik, M. Z., K.P. Hendriks, J.J. Vermeulen & M.
Schilthuizen, 2019. Conchological and molecular analysis
of the “non-scaly” Bornean Georissa with descriptions of
three new species (Gastropoda, Neritimorpha,
Hydrocenidae). ZooKeys, 840: 35-86.
Klingenberg, C.P., 2011. MorphoJ: an integrated software
package for geometric morphometrics. Molecular Ecology
Resources, 11: 353-357.
Meyer, C.P. & G. Paulay, 2005. DNA barcoding: Error rates
based on comprehensive sampling. PLoS Biology, 3(12):
e422.
Moritz, C. & C. Cicero, 2004. DNA barcoding: Promise and
pitfalls. PLoS Biology, 2(10): e354.
Naya, D.E., T. Catalan, P. Artacho, J.D. Gaitan-Espitia & R.F.
Nespolo, 2011. Exploring the functional association
between physiological plasticity, climatic variability, and
geographical latitude: lessons from land snails. Evolutionary
Ecology Research, 13(6): 647-659.
Páll-Gergely, B., A. Hunyadi, A. Jochum & T. Asami, 2015.
Seven new hypselostomatid species from China, including
some of the world's smallest land snails (Gastropoda,
Pulmonata, Orthurethra). ZooKeys, 523: 31–64.
Páll-Gergely, B., M. Schilthuizen, A. Örstan & K. Auffenberg,
2019. A review of Aulacospira Möllendorff, 1890 and
Pseudostreptaxis Möllendorff, 1890 in the Philippines
(Gastropoda, Pupilloidea, Hypselostomatidae). ZooKeys,
842: 67–83.
Palumbi, S.R., A. Martin, S. Romano, W.O. Mcmillan, L. Stice
& G. Grabowski, 1991. The simple fool’s guide to PCR.
Version 2. University of Hawaii Press, Honolulu. 45 pp.
Panha, S., 1997. Three new species of Hypselostoma from
Thailand (Pulmonata: Vertiginidae). Malacological Review,
30: 61-69.
Pfenninger, M. & F. Magnin, 2001. Phenotypic evolution and
hidden speciation in Candidula unifasciata ssp.
(Helicellinae, Gastropoda) inferred by 16S variation and
quantitative shell traits. Molecular Ecology, 10: 2541-2554.
Pilsbry, H., 1916-1918. Pulmonata, Pupillidae (Gastrocoptinae).
Manual of Conchology, second series: Pulmonata, vol. 24, i
-xii, 1-380, pls. 1-24. The Conchological Department,.
Volume 14 Issue 3 - 2020 | 12 Philippine Journal of Systematic Biology Online ISSN: 2508-0342
Lipae et al.: New microsnail subspecies from Masungi Georeserve
Academy of Natural Sciences, Philadelphia.
Räsänen, K. & A.P. Hendry, 2008. Disentangling interactions
between adaptive divergence and gene flow when ecology
drives diversification. Ecology Letters, 11(6): 624–636.
Rohlf, J.A., (2016, August 16). Morphometrics at SUNY Stony
Brook. Retrieved from http://life.bio.sunysb.edu/morph
Saitou, N. & M. Nei, 1987. The neighbor-joining method: A
new method for reconstructing evolutionary trees.
Molecular Biology and Evolution, 4: 406-425.
Schileyko, A.A., 1998. Treatise on recent terrestrial pulmonate
molluscs. Part 2. Gastrocoptidae, Hypselostomatidae,
Vertiginidae, Truncatellinidae, Pachnodidae, Enidae,
Sagdidae. Ruthenica Supplement, 2: 129–262.
Staden, R., K. Beal & J. Bonfield, 2000. The Staden package,
1998. Methods in Molecular Biology, 132: 115-130.
Swofford, D.L., 2002. PAUP* (Phylogenetic Analysis Using
Parsimony *and other methods) 4.0b10, Sinauer
Associates, Sunderland, MA.
Tang, Q.‐Y., S.‐Q. Liu, D. Yu, H.‐Z. Liu & P.D. Danley, 2012.
Mitochondrial capture and incomplete lineage sorting in the
diversification of balitorine loaches (Cypriniformes,
Balitoridae) revealed by mitochondrial and nuclear genes.
Zoologica Scripta, 41: 233-247.
Thompson, F.G. & K. Auffenberg, 1984. Hypselostoma
latispira, a new pupillid land snail from the Philippine
islands. Proceedings of the Biological Society of
Washington, 97(1): 86-89.
Thompson, F.G. & H.G. Lee, 1988. Hypselostoma holimanae
new species, a pupillid land snail from Thailand. The
Nautilus, 102: 78-81.
Xia, X., 2013. DAMBE5: A comprehensive software package
for data analysis in molecular biology and evolution.
Molecular Biology and Evolution, 30: 1720-1728.