An integrative description of Macrobiotus paulinae sp. nov. (Tardigrada: Eutardigrada: Macrobiotidae: hufelandi group) from Kenya

Abstract

In this paper we describe Macrobiotus paulinae, a new species of the hufelandi group from the Kenyan highlands. In addition to the traditional taxonomic description, aided with morphometrics as well as light and scanning microscopy imaging, we also provide nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) DNA fragment of the new species. The sequences allowed not only a more accurate description but also provided an independent verification of the taxonomic status of Ma. paulinae sp. nov. Such integrative approach requires a considerable number of individuals and eggs, which we have partially subsidised by employing an in vitro culture of the new species. Our analyses revealed that Ma. paulinae sp. nov. is most similar to Macrobiotus madegassus Maucci, 1993 and Macrobiotus modestus Pilato & Lisi, 2009, however it differs from these species, as well as from all other known species of the hufelandi group, by the presence of seven paired dorso-lateral patches of cuticular granulation and the presence of chorionic filaments growing out of terminal discs of egg processes. Macrobiotus paulinae sp. nov. is an example of a species with a miniaturised buccal apparatus (i.e. with reduced peribuccal lamellae and oral cavity armature, and stylet supports inserted on the buccal tube more anteriorly than in typical Ma. hufelandi group species), and it therefore resembles two recently described two-macroplacoided Minibiotus species: Mi. acadianus Meyer & Domingue, 2011 and Mi. julianae Meyer, 2012. The re-examination of the type material for these two species confirmed that they are equipped with peribuccal lamellae and therefore we transfer them to the genus Macrobiotus, specifically to the hufelandi group.

The first record of Macrobiotus vladimiri Bertolani, Biserov, Rebecchi & Cesari, 2011 (Tardigrada: Eutardigrada: Macrobiotidae: hufelandi group) from Poland

“…For many years, specimens exhibiting these morphological traits were commonly and erroneously identified as the nominal species. Following the revision of this group by Bertolani and Rebecchi (1993), researchers began to recognize differences in morphological details, and as a result, numerous new species within the complex were identified (e.g., Bertolani and Rebecchi, 1993;Guidetti et al, 2013;Stec et al, 2015;Bąkowski et al, 2016).…”

Integrative redescription of a common Arctic water bear Pilatobius recamieri (Richters, 1911)

“…1a, b). The sample was processed following a protocol described in detail in Stec et al (2015). Isolated specimens were divided into four groups, destined for different analyses: (i) imaging of entire individuals by light microscopy (external and internal morphology and morphometry; 35 specimens), (ii) imaging of entire individuals by scanning electron microscopy (SEM, fine external morphology; 5 specimens), (iii) buccopharyngeal apparatus extraction and imaging by SEM (fine morphology of the apparatus; 4 specimens), and (iv) DNA extraction (including new molecular data for Pilatobius; 4 specimens).…”

“…Specimens for SEM imaging were prepared according to Stec et al (2015) and examined under high vacuum in a Versa 3D DualBeam SEM at the ATOMIN facility of Jagiellonian University. Buccopharyngeal apparatuses were extracted following a protocol by Eibye-Jacobsen (2001) with modifications described thoroughly in Gąsiorek et al (2016), and examined under high vacuum in a Versa 3D DualBeam SEM at the ATOMIN facility of Jagiellonian University.…”

“…DNA was extracted from individual animals following a Chelex Ò 100 resin (Bio-Rad) extraction method by Casquet et al (2012) with modifications described in detail in Stec et al (2015). We attempted to sequence four DNA fragments differing in effective mutation rate: the small and large ribosome subunit (18S rRNA and 28S rRNA, respectively), nuclear markers typically used for phylogenetic inference at high taxonomic level (e.g., Bertolani et al 2014); the internal transcribed spacer (ITS-2), a noncoding nuclear fragment with high evolution rate, suitable for intraspecific comparisons as well as comparisons between closely related species (e.g., Gąsiorek et al 2016); and finally, the cytochrome oxidase subunit I (COI), a protein-coding mitochondrial marker, widely used as a standard barcode gene with intermediate effective mutation rate (e.g., Bertolani et al 2011).…”

“…We attempted to sequence four DNA fragments differing in effective mutation rate: the small and large ribosome subunit (18S rRNA and 28S rRNA, respectively), nuclear markers typically used for phylogenetic inference at high taxonomic level (e.g., Bertolani et al 2014); the internal transcribed spacer (ITS-2), a noncoding nuclear fragment with high evolution rate, suitable for intraspecific comparisons as well as comparisons between closely related species (e.g., Gąsiorek et al 2016); and finally, the cytochrome oxidase subunit I (COI), a protein-coding mitochondrial marker, widely used as a standard barcode gene with intermediate effective mutation rate (e.g., Bertolani et al 2011). All fragments were amplified and sequenced according to the protocols described in Stec et al (2015); primers and original references for specific polymerase chain reaction (PCR) programs are listed in Table 1. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of Jagiellonian University, Kraków, Poland.…”