Phylogenetic relationships of the operculate land snail genus Cyclophorus Montfort, 1810 in Thailand

Highlights

• This work is the first molecular phylogenetic study of Cyclophorus in Thailand.

• The phylogenetic trees obtained in general confirmed the species level classification.

• We found cryptic species of Cyclophorus in Thailand.

Abstract

Operculate land snails of the genus Cyclophorus are distributed widely in sub-tropical and tropical Asia. Shell morphology is traditionally used for species identification in Cyclophorus but their shells exhibit considerable variation both within and between populations; species limits have been extremely difficult to determine and are poorly understood. Many currently recognized species have discontinuous distributions over large ranges but geographical barriers and low mobility of snails are likely to have led to long periods of isolation resulting in cryptic speciation of allopatric populations. As a contribution towards solving these problems, we reconstructed the molecular phylogeny of 87 Cyclophorus specimens, representing 29 nominal species (of which one was represented by four subspecies), plus three related out-group species. Molecular phylogenetic analyses were used to investigate geographic limits and speciation scenarios. The analyses of COI, 16S rRNA and 28S rRNA gene fragments were performed using neighbour-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. All the obtained phylogenetic trees were congruent with each other and in most cases confirmed the species level classification. However, at least three nominate species were polyphyletic. Both C. fulguratus and C. volvulus appear to be species complexes, suggesting that populations of these species from different geographical areas of Thailand are cryptic species. C. aurantiacus pernobilis is distinct and likely to be a different species from the other members of the C. aurantiacus species complex.

Introduction

The Cyclophoridae are dioecious terrestrial caeonogastropod snails with a long fossil record extending from the Mesozoic era (Gordon and Olson, 1995, Kongim et al., 2006) and with a wide current geographical distribution: Southern Europe, Central America, Asia, Africa, various Pacific islands and Australia (Kobelt, 1902, Solem, 1959). The most broadly used classification for the Cyclophoridae is that of Kobelt (1902). He classified the cyclophorids based on shell, opercular and radular characters, but whilst this undoubtedly reflects some of the broad relationships, it is of little value below the subfamily level (Solem, 1956). Currently, the Cyclophoridae is comprised of about 870 species arranged in three subfamilies and 35 genera (Kobelt, 1902, Kobelt, 1908, Wenz, 1938, Vaught, 1989, Bouchet and Rocroi, 2005, Lee et al., 2008b). The genus Cyclophorus Montfort, 1810 is the most species rich genus in the family Cyclophoridae with over 100 described species distributed from South Asia to Southeast Asia and including the south of China, Korea and Japan (Reeve, 1862, Kobelt, 1902, Kobelt, 1908, Gude, 1921, Pilsbry, 1916, Pilsbry, 1926, van Benthem Jutting, 1948, van Benthem Jutting, 1949, Solem, 1959, Solem, 1966, Minato and Habe, 1982). Members of Cyclophorus have a distinctive large solid, low conical shell form with a thin and multispiral operculum. They are “ground-dwelling” in leaf litter, under logs, etc. and occur in a wide range of forest habitats from evergreen rainforest to monsoon deciduous forest. In Thailand, the highest densities occur in limestone forest (Kobelt, 1902, Kobelt, 1908, Gude, 1921, Solem, 1959).

The validity of the nominal Cyclophorus species level classification is not clear because of the degree of shell variation within and between nominated species and the limited number of characters available in Cyclophorus shells. Kobelt (1902) classified Cyclophorus into eight subgenera while Vaught (1989) rearranged Cyclophorus into five subgenera. Few descriptions of the internal anatomy, including reproductive organs have been published for Cyclophorus (Tielecke, 1940, Kasinathan, 1975). Available information demonstrates a high degree of similarity for internal anatomy within Cyclophorus that does not provide robust characters for recognizing species level categories (Welber, 1925, Kongim et al., 2006). Thus there has been little advance on the use of characters based on shell morphology, including shell size, shape, colour pattern and peristome morphology (e.g. Reeve, 1862, Kobelt, 1902, Kobelt, 1908, Gude, 1921). Environmental factors can greatly affect shell morphology (Uit de Weerd et al., 2004, Lee et al., 2008a, Lee et al., 2008b, Elejalde et al., 2009) and homoplasy confounds the recognition of biological species of Cyclophorus. Species limits in Cyclophorus are notoriously difficult to establish with numerous geographically isolated populations exhibiting seemingly minor differences in their morphology.

There is evidence to suggest that Cyclophorus was important in the diet of Stone Age cave dwellers in Oriental regions (Rabett et al., 2011). In the present day, these edible snails have been utilized for food in many parts of Thailand, Laos and Vietnam (Oakley, 1964, Paz and Solheim, 2004). However, the number of Cyclophorus snails seems to have dropped (Hildyard, 2001) most likely due to a range of contributing factors such as changes in the environmental conditions around snails’ habitats especially in limestone areas and improper snail harvesting (Clements et al., 2006). Recent work has focused on systematic research and development of conservation strategies (Clements et al., 2008). In addition to its intrinsic scientific interest, a reliable taxonomy is important for avoiding the mistaken treatment of multi-species genera as a single taxon that may fail to effectively regulate their conservation. There is therefore practical conservation value in the recognition of Cyclophorus cryptic species (Kongim et al., 2006, Prasankok et al., 2009).

Over the past decade, sequence data has been used to help clarify problems in systematics and evolution where morphological and physiological characters have proven to be ambiguous (Vogler and Monaghan, 2007). Studies on land snail phylogeny using molecular DNA sequences have suggested that such approaches are potentially useful (e.g. Harasewych et al., 1998, Wade et al., 2006, Colgan et al., 2007, Lee et al., 2008a, Lee et al., 2008b). The genes most commonly used for land snail systematics, at various taxonomic levels are the mitochondrial cytochrome oxidase subunit I (COI) and16S rRNA genes and the nuclear ribosomal RNA genes (e.g. Harasewych et al., 1998, Wade et al., 2006, Colgan et al., 2007, Liew et al., 2009). They provide a highly effective tool to resolve taxonomic problems and identify cryptic species (Paquin and Hedin, 2004). These genes have been found to be suitable for use at the generic level in cyclophorids, revealing that the evolution of morphological and ecological traits occurs at extremely high rates during adaptive radiation, especially in fragmented environments (Sanders et al., 2006, Lee et al., 2008a, Lee et al., 2008b). The work reported here is the first molecular phylogenetic study of the genus Cyclophorus in Thailand.