« Niphargus diversity from South-West France through the lens of the barcoding fragment of cytochrome-oxidase I (Crustacea, Amphipoda) »

Niphargus diversity from South-West France through the lens of the barcoding
fragment of cytochrome-oxidase I (Crustacea, Amphipoda)
REXHEPI B.1,*
, KUŠAR G.1
, LEFEBVRE F.2
, ALEZINE T.3
, CHEVILLOT X.3
, OLIVIER M. J.4
, MALARD F.4
, DOUADY
C. J.4
, STOCH F.5
, CHAUVIN J.6
, ZAGMAJSTER M.1
, FIŠER C.1
1University of Ljubljana, Biotechnical Faculty, Department of Biology, SubBioLab, Ljubljana, Slovenia
2SEPANSO Aquitaine, Brive-la-Gaillarde, France
3SEPANSO Aquitaine, Bordeaux, France
4Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, Villeurbanne, France
5Evolutionary Biology & Ecology, Université libre de Bruxelles (ULB), Brussels, Belgium
6
IKERLAN-Recherche, Ayherre, France
*Corresponding author. E-mail: behare.rexhepi@bf.uni-lj.si
Niphargus is the most speciose amphipod genus inhabiting groundwater of the Western Palearctic,
counting over 450 described species. The genus is known for its problematic taxonomy, including large
within-, yet sometimes negligible between-species variation, often associated with cryptic diversity. With
the onset of the molecular methods, it became clear that morphological variation captures between one
half to one third of genus’s genetic species diversity, raising the need to revise past taxonomic and
faunistic efforts using molecular markers. Research efforts of several laboratories in Europe have been
dedicated to this task during the past two decades. Hence, Niphargus was a subject of intense exploration
in parts of Great Britain and of Pyrenees, Alpine and Carpathian arch, Dinaric region and Caucasus.
Surprisingly, France remained relatively understudied, despite the long tradition in Niphargus research
and the fact that the very first molecular studies on Niphargus originated from this country. Here, we fill
this gap and revise diversity of Niphargus in France using molecular markers. We accumulated a large
number of samples originating from dedicated sampling campaigns of stygofauna in the south-western
parts of the country, samples collected during exploration of isopods and other occasional sampling
events. The samples covered altogether over 260 locations. Of these, we selected 434 individuals, whose
morphology corresponded to 26 nominal species. We isolated DNA and amplified the Folmer’s fragment
of the mitochondrial COI marker. To test for species diversity, we employed unilocus species delimitation
Assemble Species by Automated Partitioning (ASAP) and identified 136 Molecular Operational
Taxonomic Units (MOTUs). Notwithstanding the limits of ASAP and similar statistical species
delimitation techniques, more than 80 MOTUs need to be investigated as putative new species, indicating
that Niphargus diversity in France is probably as underestimated as in the rest of this genus range. We
detected up to 6 MOTUs within the morphological species identified as N. ciliatus. Moreover, we
assessed phylogenetic origin of Niphargus collected in France. We selected one individual per MOTU
and amplified three additional nuclear markers (two 28S fragments and Histone 3, subunit 2). The
phylogenetic hypothesis unveiled that Niphargus from France comprise lineages derived from deep splits
within the genus and recent speciation events. The study closed an important knowledge gap in
molecular-spatial coverage of Niphargus and the available barcodes will contribute to future integration
of Niphargus into biodiversity studies.