Corresponding author: Matthias Glaubrecht (
Academic editor: Andreas Schmidt-Rhaesa
The freshwater thiarid gastropod
Thailand is situated in one of the most biodiverse areas of the world (e.g.
In addition, Thailand can be divided into geographical regions based on distinct drainage basins; with those in the north, for example, forming the Chao Phraya drainage flowing into the Gulf of Thailand, those in the northeast as part of the Mekong river basin which eventually drains into the South China Sea, or the north-western region as part of the Salween river system. In contrast to these and other major river systems, in the south there are shorter rivers that either run east to the Gulf of Thailand or west to the Andaman Sea. These water bodies in Thailand form hotspots of aquatic biodiversity with various local endemism.
Among the aquatic biota, limnic molluscs are diverse, and include about 280 species of fresh and brackish water gastropods (
Accordingly, non-marine molluscs in Thailand should receive more attention and focus on studies looking into species diversity and contributing to solving fundamental questions and the evolution of faunal diversity. However, biological information on gastropods in Thai river systems and lakes is generally scarce and often lacks recently collected material or available former museum collections which hampers more in-depth studies. This is problematic, as several freshwater snails with their main occurrence in Southeast Asia have a considerable importance as first intermediate hosts for infections in humans and animals. Despite their proven medical importance, in particular the faunistic and systematic knowledge on cerithioidean freshwater snails of the various families acting as one of the most important vectors for digenic human pathogens, is precarious. The
Cerithioidean freshwater taxa were long subsumed under the historical concept of “melaniids”, which was later uncritically replaced by the family assignment to the
To complicate matters,
In Thailand, the
This holds true especially for species assigned to
Distribution of the freshwater thiarid snail
In addition, this snail has become widely invasive in the tropics outside its native range, the spreading being attributed to the aquarium trade. As early as the 1950s, though,
That way, this snail exhibits its potency as neozoon, in combination with its role as important vector for several diseases, supporting the life cycles of digenic parasites infecting humans as well as other animals. Throughout Southeast Asia and in particular in Thailand,
Therefore, being able to ecologically adopt apparently to a broad range of different freshwater habitats,
Shells of
In light of these phenotypical variations found in the shell morphology of
Viewed from the background of a molecular backbone phylogeny we are, finally, able to analyse a suite of questions concerning the nature of cladogenesis, phylogeography and reproductive biology in these snails, in context with the infections by various trematodes, eventually hoping to elucidate the interrelationship and co-existence of human-infectious trematode parasites and their first intermediate snail hosts.
The National Committee on Hydrology separates Thailand into 25 distinct hydrological units or river basins, which are used in this study as an established geographical reference. These units comprise the following rivers and drainage systems: Salween, Mekong, Kok, Shi, Moon, Ping, Wang, Yom, Nan, Chao Phraya, Sakaekrang, Pasak, Tha Chin, Mae Klong, Prachinburi, Bang Pakong, Tonle Sap, Peninsular East Coast, Phetchaburi, Peninsular West Coast, Southeast Coast, Tapi, Songkhla Lake, Pattani and Southwest Coast. These catchment and drainage systems are re-grouped here into seven areas, each with specific characteristics; refer to Figs
Specimens of
To reconstruct in detail the distributional range, in addition to own collecting activities in most parts of the region, material was analysed in several major museum collections, as well as literature records which were sufficiently verifiable as to the species identity (in general documented by descriptions and, even better, figures of shells collected).
Geographic coordinates of newly collected material were taken with a GPS device at the sampling site (WGS84 datum). Where GPS data for sampling sites were unavailable, coordinates were determined as accurately as possible from a map. Localities of the samples were mapped on a dot-by-dot basis on a public domain map (NaturalEarth,
For climatic data, we used information from the climate of the world database (
The snails identified as belonging to
The following biometrical parameters of the adult shells were taken with a digital calliper (accuracy: 0.1 mm): height of shell (h), width of shell (w), length of aperture (la), width of aperture (wa), height of body whorl (hbw), height of the last three whorls (l3w) and number of whorls (nw) (Fig.
Biometrical parameters (
For the two different mitochondrial clades, the Shapiro-Wilk-test was performed on all measured variables for each group individually to test for normal distribution. If at least one group was not normally distributed, we conducted a Wilcoxon signed rank test with continuity correction, to test for significant differences between clades. If the data for both groups were normally distributed, a Levene-test based on absolute deviations from the mean was performed to check for homoscedasticity. In case homoscedasticity was detected, we tested different groups using a two-sample t-test. Otherwise, we performed Welch’s heteroscedastic t-test.
All available type specimens and the other examined material was photographed by remote shooting with EOS Utility 2.12.2.1 for Windows (Canon Inc., Tokyo, Japan) and Digital Photo Professional 3.12.51.2 for Windows (Canon Inc.) using a digital camera (EOS 5D MKII with Canon macro photograph lens
A total of 1,169 standardized images of adult, unbroken shells could be included in our geometric morphometrics data set. Using tpsUtil version 1.74 (
The content of the brood pouch was counted as best proxy for differences in the thiarid reproductive strategy following the method described in
Sequences from 131 specimens of
Collection voucher numbers, geographic coordinates of sampling sites and GenBank accession numbers for specimens of
Voucher Number | Latitude | Longitude | GenBank accession number | |
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15°47’29,7”N, 101°13’30,7”E | – |
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15°47’29,7”N, 101°13’30,7”E | – |
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Outgroup:
Forward and reverse strands were assembled using the program Geneious (Biomatters Limited, Auckland, New Zealand) and corrected by eye. The protein coding
For information on vouchers and GenBank accession numbers, see Table
Bayesian Inference (
The
Heuristic
Heuristic
Bayesian posterior probabilities (
We used the General Mixed Yule-coalescent (
We dated the divergence times for the main clades of
4 syntypes (MHNG 1093/72/1-4).
Originally given as “Timor” by
In addition, in the past some authors employed “
The distributional range of
In Thailand, this species occurs in most lentic and lotic water bodies ranging throughout the various regions, provinces and river systems. There,
In the following we document here in detail the geographical origin of material studied from Thailand, in comparison with the syntypes as well as topotypical material from Timor as reference (see above). Data on other localities indicated in Fig.
The final alignment of the
For 16S sequences, p-distances among specimens from Thailand ranged from 0% to 10.4% and for Timor Leste, pairwise p-distance between specimens were very low, ranging from 0% to 0.1%.
All three phylogenetic analyses recovered two deeply divergent clades of specimens assigned to
Bayesiam 50% majority-rule consensus tree showing two major mitochondrial clades in
All specimens from Timor Leste were included in clade A together with specimens mostly from the southern to southern-central parts of Thailand (Fig.
In contrast, specimens of
When analysed by drainage systems, we found that all specimens from the north-western part of Thailand, which is drained through the Salween river system into the Andaman Sea, were included in clade B. Likewise, specimens from the headwaters of the Ping, Wang, Yom and Nan rivers belonging to the Chao Phraya system, with few exceptions, were assigned to clade B in the phylogenetic analyses. In the lower courses of northern to northern-central Thai drainages, such as e.g. the Chao Phraya and Mae Klong drainages that run into the Gulf of Thailand, specimens assigned to both clades are present.
Similarly, specimens belonging to both mitochondrial clades are present in the Mekong drainage, whereas specimens assigned to clade A predominate in the smaller rivers in the Thai parts of the Malay Peninsula to the north and south of the Isthmus of Kra that either drain into the Gulf of Thailand or the Andaman Sea (Fig.
In contrast to this geographical pattern in
Evolutionary relationships among haplotypes were inferred applying a median-joining network approach that showed the two mitochondrial clades A and B to be separated by > 60 steps (
Molecular analysis of
The
The results of the BEAST analysis assuming a strict molecular clock and a divergence rate of 1% per million years (Fig.
The shells of
As shown in Fig.
Starting off from the type series of
We were not able to find any correlation of shell morphology with molecular genetic clusters as described above, or any other geographical or ecological factor matching these distinct phenotypes in
For ranges and mean values of measured shell parameters for the different predefined groups, i.e. shell morphs/geographic groups or genetic clades, see Table
Biometric data for different shell morphs/geographic groups (see also Figs
Min | Max | Mean | Median | Standard deviation | |
---|---|---|---|---|---|
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Morph A | 9.29 | 29.83 | 18.93 | 18.90 | 3.69 |
Morph B | 8.56 | 32.38 | 19.73 | 20.06 | 3.87 |
Morph C | 10.53 | 26.88 | 19.03 | 18.94 | 3.02 |
Morph B+C | 8.56 | 32.38 | 19.62 | 19.81 | 3.76 |
Timor | 11.67 | 28.53 | 19.68 | 19.69 | 3.75 |
Clade A | 8.56 | 32.38 | 19.24 | 19.52 | 3.86 |
Clade B | 9.45 | 30.67 | 19.66 | 19.68 | 3.64 |
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Morph A | 3.73 | 13.28 | 8.26 | 8.44 | 1.71 |
Morph B | 3.49 | 14.46 | 8.35 | 8.55 | 1.61 |
Morph C | 4.39 | 11.58 | 7.94 | 7.98 | 1.32 |
Morph B+C | 3.49 | 14.46 | 8.28 | 8.39 | 1.58 |
Timor | 5.04 | 12.18 | 8.15 | 8.20 | 1.42 |
Clade A | 3.73 | 13.28 | 8.05 | 8.13 | 1.51 |
Clade B | 3.49 | 14.46 | 8.46 | 8.61 | 1.64 |
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Morph A | 4.38 | 14.39 | 9.23 | 9.31 | 1.84 |
Morph B | 4.23 | 15.35 | 9.31 | 9.46 | 1.75 |
Morph C | 4.94 | 18.96 | 9.06 | 8.98 | 1.72 |
Morph B+C | 4.23 | 18.96 | 9.27 | 9.38 | 1.74 |
Timor | 5.16 | 13.6 | 9.13 | 9.12 | 1.62 |
Clade A | 4.38 | 14.39 | 9.06 | 9.10 | 1.72 |
Clade B | 4.23 | 18.96 | 9.41 | 9.50 | 1.77 |
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Morph A | 1.63 | 8.92 | 4.30 | 4.29 | 0.87 |
Morph B | 1.68 | 8.91 | 4.25 | 4.25 | 0.90 |
Morph C | 2.42 | 8.41 | 4.31 | 4.23 | 1.01 |
Morph B+C | 1.68 | 8.91 | 4.26 | 4.25 | 0.92 |
Timor | 2.40 | 6.07 | 4.09 | 4.14 | 0.71 |
Clade A | 1.63 | 8.92 | 4.18 | 4.21 | 0.87 |
Clade B | 1.68 | 8.91 | 4.32 | 4.29 | 0.91 |
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Morph A | 5.91 | 19.55 | 12.48 | 12.47 | 2.44 |
Morph B | 5.77 | 20.37 | 12.60 | 12.78 | 2.35 |
Morph C | 6.81 | 15.83 | 11.99 | 11.92 | 1.87 |
Morph B+C | 5.77 | 20.37 | 12.50 | 12.65 | 2.29 |
Timor | 6.53 | 17.78 | 12.35 | 12.48 | 2.22 |
Clade A | 5.91 | 17.81 | 12.25 | 12.38 | 2.29 |
Clade B | 5.77 | 20.37 | 12.69 | 12.81 | 2.33 |
|
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Morph A | 7.93 | 26.43 | 16.84 | 17.04 | 3.29 |
Morph B | 7.73 | 28.74 | 16.93 | 17.13 | 3.28 |
Morph C | 9.20 | 21.34 | 15.97 | 15.95 | 2.49 |
Morph B+C | 7.73 | 28.74 | 16.77 | 16.85 | 3.19 |
Timor | 9.46 | 23.89 | 16.56 | 16.40 | 3.12 |
Clade A | 7.93 | 26.22 | 16.49 | 16.54 | 3.22 |
Clade B | 7.73 | 28.74 | 17.01 | 17.13 | 3.17 |
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Morph A | 1.77 | 2.95 | 2.31 | 2.29 | 0.25 |
Morph B | 1.22 | 3.13 | 2.37 | 2.38 | 0.22 |
Morph C | 1.50 | 3.05 | 2.41 | 2.41 | 0.24 |
Morph B+C | 1.22 | 3.13 | 2.38 | 2.39 | 0.22 |
Timor | 1.92 | 2.87 | 2.41 | 2.41 | 0.18 |
Clade A | 1.50 | 3.13 | 2.39 | 2.41 | 0.22 |
Clade B | 1.22 | 2.93 | 2.34 | 2.35 | 0.23 |
|
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Morph A | 1.27 | 2.54 | 2.05 | 2.04 | 0.13 |
Morph B | 1.22 | 2.53 | 2.03 | 2.03 | 0.15 |
Morph C | 1.39 | 2.65 | 2.02 | 2.03 | 0.16 |
Morph B+C | 1.22 | 2.65 | 2.03 | 2.03 | 0.15 |
Timor | 1.66 | 2.28 | 2.03 | 2.04 | 0.13 |
Clade A | 1.27 | 2.65 | 2.05 | 2.06 | 0.16 |
Clade B | 1.22 | 2.38 | 2.02 | 2.01 | 0.13 |
Results of biometric (
Between genetic clades at least one of the groups was found to be not normally distributed (Shapiro-Wilk-test, p < 0.05) for shell width and l3w/w. By contrast normal distribution was found for lw3 and shell height. Subsequent Levene-testing identified the height and l3w data sets as homoscedastic (p > 0.05), hence a two-sample t-test was performed, identifying significant differences (p < 0.025) between the means for the two clades for lw3 and no significant differences for shell height. For shell width and l3w/w a Wilcoxon signed rank test was performed, revealing significant differences (p < 0.025) for the mean of both shell parameters. However, similar to the situation when comparing the different shell morphs/geographical groups, it has to be noted that the ranges of all measured shell parameters widely overlap and, therefore, do not allow to derive diagnostic characteristics for the two main clades found in the phylogenetic analyses (see boxplots in Fig.
Results of biometric (
A principal component analysis (
Principal components (PC) 1–6 had all at least one group that proved to be not normally distributed (Shapiro-Wilk-test, p < 0.05). Subsequent Kruskal-Wallis-testing was significant (p < 0.05) in PC1–5 and not significant in PC6. Hence, no further testing was done for PC6. The Bonferroni-corrected Dunn-test identified the mean value for specimens from Timor to be significantly different (p > 0.025) from all other morphs on PC1. By contrast, examining PC2 and PC4 with the same test, proved morph A and B to be the only groups not significantly different (with regard to mean values) from one another. Finally, on PC3 and PC5 the Bonferroni-corrected Dunn-Test revealed the mean value of morph C not to be significantly different from all other groups, but the means of morph A and B to be significantly different to that of the specimens from Timor.
Finally, when morph C was integrated into morph B (since these were only differentiated on the basis of slight differences in banding pattern), PC1–5 supported only the group consisting of specimens from Timor to have significantly different means from all other specimens (data not shown). The scatter plot in Fig.
For PC1 and PC3–6 at least one of the groups (clade A/clade B) was not normally distributed (Shapiro-Wilk-test, p > 0.05). Hence, we conducted Wilcoxon singed rank tests for all these PC, with none showing significant differences between groups (p > 0.05). By contrast, in PC2 both groups showed normally distributed data. Therefore, Levene-testing based on deviations from the mean followed and was found significant (p < 0.05). Accordingly, we conducted Welch’s two sample t-test, revealing significant differences between the means of the two clades on PC2. The scatter plot in Fig.
Females of
Frequency of ontogenetic stages in the subhemocoelic brood pouches of female
The frequency of these different size classes in the subhemocoelic brood pouch of the total of n = 1,007 dissected females of
Frequency of ontogenetic stages in the subhemocoelic brood pouches of female
In all examined populations, the number of early and late embryonic stages was above 50%, in most cases even above 75%; see Fig.
Composition of contents of the subhemocoelic brood pouches of female
When considering the overall distribution of different size classes in the different morphs/geographic clusters or mitochondrial clades, the resulting histograms (Fig.
We also compared the size class composition of offspring in the subhemocoelic brood pouches of
Composition of contents of the subhemocoelic brood pouches of female
The distribution of gravid vs. non-gravid specimens according to the 17 rivers systems exhibits some variation (Fig.
Whether reproduction is seasonal, or whether there is any influence of the month of collecting on our data, can currently not be answered with certainty. In an attempt to correlate reproduction (i.e. the frequency of gravid vs. non-gravid females) with climatic effects such as, for example, rainy season resulting in high water levels in rivers and streams, we have used published meteorological data (e.g. minimum/maximum temperature and precipitation) for stations representing the different climatic regions of Thailand, viz. Chiang Mai for northern inland region, Ko Samui for the Gulf of Thailand and Phuket for the Andaman Sea localities (see map in Fig.
Proportions of gravid vs. non-gravid specimens of
As evolutionary biologists working with molluscs, we should aim at testing the universality of known and disputed speciation mechanisms, and it is with a clear focus on these mechanisms we should choose our molluscan models to increase their frequency as a source of data in order to decipher the underlying mechanisms of biodiversity.
The combination of molecular genetics and phenotypic analyses in concert with information on the geographical occurrence and additional data, e.g. on biological properties such as reproductive strategies, provides a powerful tool for the study of species differentiation, or diversification indicating speciation. It allows truly biological species to be distinguished, not only as perceivable taxonomic or even genetic units, but also as natural entities of evolutionary significance; if we want to make here the careful distinction between a species taxon (with identifying characteristics) and species entity (as a group of coevolving populations); see for the theoretical background of applications of species concepts in freshwater molluscs
In freshwater gastropods high levels of morphological disparity and taxonomic diversity are frequently correlated, but often only because traditionally disparity was equated with diversity, as has been exemplified for limnic
As has been discussed by the latter author with focus on freshwater gastropods, the widely adopted typological practice during the 19th and way into the 20th century of naming allopatric populations, in isolated fashion and often based on single specimens only, as if representing putatively distinct (morpho-) species, has led to a plethora of species and subspecies names. Freshwater gastropods were found to exhibit a pronounced individual conchological variability, which has been attributed to the environmental conditions of their habitats that widely fluctuate on a temporal and spatial scale (e.g.
In the course of the systematic revision of these thiarids, based on an evolutionary systematic approach (see
However, an assessment of the significance of distinct phenotypic traits is in general lacking, as is an understanding of the genetic basis of phenotypical variation in particular for gastropods. For the limnic pomatiopsid
Owing to the earlier typological approach that resulted in the traditional overestimation of taxonomical diversity due to conchological disparity, but also in context of the genetically apparently closely related but morphologically highly distinct thiarids found across the distributional ranges throughout Southeast Asia and Australasia, we have to ask whether we are indeed dealing with actually many diverse species as separate evolutionary entities rather than only few, though highly polymorphic species with maybe several sympatric morphs exhibiting different ecophenotypical adaptations in shell response to the many variable environments where thiarids are usually to be found.
In the present study, we examined phenotypically distinguishable shell morphs of yet another thiarid from Thailand, traditionally assigned to
We found
Applying a drainage-based phylogeographical as well as a biometrical approach, we were unable to find for the populations in Thailand any correlation of the morphs distinguished in this study based on discernable shell features as well as overall “Gestaltwahrnehmung” with any criteria deducible from our observations given above, neither with geographical occurrence or preferred habitat and substrate nor with the molecular genetic substructuring detected (see below). So, all available evidence points at the coexistence of different morphologies or disparate phenotypes in
Biometric analyses are found useful tools for the study of characteristics that shape morphologically distinct entities, thus allowing to look into evolutionary pattern (e.g.
Although there are some differences in the biometric parameters and in the geometric morphometrics of Thai
The measurement of shell height of
The results of geometric morphometrics revealed the overall shell shape of
In contrast to shell morphology (morphs A–C, or
Therefore, our analyses would potentially allow for a more narrow species delimitiation within what has been to date traditionally treated in Thailand as
However, the p-distance of 13.8 % for
While we found representatives of clade A in the northern tributaries of rivers such as the Chao Phraya and Mae Klong that run into the Gulf of Thailand, with only few others occurring at some localities in the south of Thailand (Figs
However, although being more frequent in the northern provinces, some representatives of clade B also occur in more southern locations, such as in the provinces Surat Thani (
As we found in our molecular analyses this major split of clade A and B in Thai
For the distributional pattern found in
While the separation of
The fact that today the distributional boundaries of the two
In the Thai populations of
As in this later case, it could be hypothesized that any environmental factor might affect the reproductive strategy also in
As in most (if not all) thiarids,
Also in malacology there are some classical case studies, such as the New Zealand freshwater hydrobiid
It would be tempting to anticipate a similar phenomenon of
Given the prediction supported here that thiarid gastropods reproduce largely (if not completely) via parthenogenesis, the application in particular of the biological species concept is not made easy in case of thiarids.
In case of the thiarids it remains to be seen in how far they are actually prone exclusively to parthenogenesis. For example, for populations of
In view of the pronounced phenotypic plasticity reported herein for the Thai
The development of an accurate and rapid method for the detection of males in aphallic thiarids, in order to evaluate the frequency of parthenogenesis in individual populations and species or higher-level taxa, respectively, remain an essential desideratum in biosystematics research on these snails. In addition, it remains to be analysed thoroughly whether and in how far there is a correlation of partially or completely parthenogenetic populations with parasite infections by digenic trematodes, for example, in the thiarids
Our preliminary analyses of the brood pouch content in the latter species under study here revealed that infected females tend to have fewer embryos than non-infected specimens, which might be a hint to the influence of parasite load on the reproductive mode of this major intermediate host. Therefore, given the human infection aspects of these trematode-carrying gastropods, our study not only has implication for human health in Thailand. We also hope that with studying trematode infections in the various conchologically disparate and molecular genetically distinct lineages of
This research was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph. D. Program (Grant No. PHD/0093/2556) to Nuanpan Veeravechsukij and Duangduen Krailas. Both and Matthias Glaubrecht also thank the Deutsche Akademische Austauschdienst (DAAD) and the Deutsche Forschungsgemeinschaft (DFG; grant GL 297/29-1) for financial support of this study. We are grateful to the Department of Biology, Faculty of Science, Silpakorn University for support. We also thank Vince Kessner (Adelaide River, Australia) very much for collecting thiarids in Timor Leste and for providing material of