Original PaperDiscrepancy between Species Borders at Morphological and Molecular Levels in the Genus Cochliopodium (Amoebozoa, Himatismenida), with the Description of Cochliopodium plurinucleolum n. sp.
Introduction
The genus Cochliopodium Hertwig et Lesser, 1874, comprises lens-shaped amoebae covered by a flexible layer of carbohydrate scales (tectum) located only on the dorsal surface of the cell (Bark, 1973, Kudryavtsev et al., 2005). When the cell is viewed from the top during locomotion, it shows a thick central granuloplasmic mass surrounded by a broad peripheral sheet of hyaloplasm. It is widely accepted that the size and shape of scales comprising the tectum of Cochliopodium are species-specific, so that details of scale structure allow the unambiguous identification of morphospecies (Bark, 1973, Kudryavtsev, 1999, Kudryavtsev, 2005). However, it was demonstrated previously that there is a group of morphospecies in Cochliopodium that can be distinguished based on size, formation of cysts and several characteristics of the locomotive form such as uroidal filaments and shape of the hyaloplasm (Kudryavtsev, 2006, Kudryavtsev et al., 2004), while sharing nearly identical scales. Namely, these are C. barki, C. minutoidum and several strains morphologically resembling C. minus. C. megatetrastylus and C. pentatrifurcatum were also recently added to this group of morphospecies (Anderson and Tekle 2013, Tekle et al. 2013), although scales of the latter species are surprisingly different from the rest of the group. The distinction of these morphospecies may be facilitated by using gene sequence data (Anderson and Tekle 2013; Kudryavtsev et al. 2005, 2011). Therefore it seems unavoidable to decipher how morphological and ultrastructural differences are related to the genetic divergence between strains. Yet, the number of sequenced species is relatively small compared to the total number of around 20 morphologically defined Cochliopodium spp., and this is the main obstacle for understanding the evolutionary relationships within the genus and borders between morphospecies.
In the present paper we revise the phylogenetic relationships in a clade of Cochliopodium comprising the closely related species C. megatetrastylus, C. minus, C. minutoidum and C. pentatrifurcatum (hereinafter referred to as C. minus-clade) and describe a new soil species C. plurinucleolum whose scale structure is very similar to the members of this clade. However, molecular trees show only distant relationships between this species and members of the C. minus-clade.
Section snippets
Microscopical Observations of Cochliopodium Strains
Cochliopodium plurinucleolum n. sp. Trophic amoebae were highly variable, showing most frequently oval, fan-shaped, or crescent-shaped locomotive forms (Fig. 1A-B). Many amoebae temporarily adopted a triangular, drop-shaped locomotive form with length greater than breadth caused by adhesion of the posterior end to the substratum (Fig. 1C). The granuloplasm in above view was entirely surrounded by a thin hyaloplasmic sheet (Fig. 1A-C), which never exceeded 1/5th of the total body length and was
Morphological Identification of the Studied Strains
Cochliopodium plurinucleolum n. sp. Light and electron microscopic analyses unambiguously identify the studied strains 8 and 86 as a species of the genus Cochliopodium due to the presence of a tectum and a characteristic locomotive form. Several morphological characteristics specific for this species distinguish it from all other currently described Cochliopodium spp., including C. barki that is morphologically most similar to C. plurinucleolum, and is one of the few isolates reported so far to
Methods
Establishing cultures: Soil from the upper 20 cm of an intensively managed grassland plot at the Berchidda-Monti long term observatory, managed by the University of Sassari, Italy, 40°46′N, 9°10′E (Bagella et al. 2013) was collected after 2 mm sieving and transferred in thermo isolated containers to the lab. For inoculation, 50 g of soil mixed with 50 ml of sterile distilled water were shaken for 20 min and allowed to settle for 15 min. Ten enrichment cultures were established by transferring 5 μl of
Acknowledgements
SG was supported by research grants involved in the EU-project ‘EcoFINDERS’ No. 264465. This study was partially supported by the Russian Foundation for Basic Research grant 12-04-01835-a to AK, as well as a grant of St-Petersburg State University, and utilized equipment of core facility centers “Development of Molecular and Cell Technologies” and “Culturing of Microorganisms” of SPSU. We are grateful to Dr. Pier Paulo Roggero for providing the soils used to isolate C. plurinucleolum, Dr. Rolf
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- 1
Correspnding author; e-mail [email protected] (S. Geisen).
- 2
These authors contributed equally to the study.
- 3
Present address: Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 50, 6700 AB, Wageningen, The Netherlands.