ID 510 Cryptic, but not that much: Mediterranean brackishwater (Crustacea: ) moderately follow trend unraveled in freshwater congeners

Kamil Hupalo, Marcos A.L. Teixeira, Tomasz Rewicz, Murat Sezgin, Valenna Iannilli, Gordan S. Karaman, Michal Grabowski, Filipe O. Costa Department of Invertebrate Zoology and Hydrobiology, University of Lodz, Poland,BCentre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal, CSinop University Fisheries Faculty, Marine Biology and Ecology Department, Sinop, Turkey, DENEA C.R. Casaccia, Rome, Italy,EMontegrin Academy of Sciences and Arts, Riste Sjovica Podgorica Črna Gora, Montenegro

ABSTRACT

Background: Amphipods of the genus Gammarus are a salient component of communies in European inland and coastal, marine and brackish waters of the North Atlanc, the Mediterranean and the Black Sea. Exceponal levels of crypc diversity have been revealed for several widespread freshwater Gammarus species in Europe. No comprehensive assessment has yet been made for brackishwater counterparts, such as Gammarus aequicauda and G. insensibilis, which are among the most frequently recorded members of the so-called “G. locusta group” in the Mediterranean and in the Black Sea. Here we probe the diversity of these morphospecies examining the paroning of COI-5P DNA barcodes across mulple populaons along their distribuon range. Results: We generated and compiled 510 bp COI-5P barcodes for a collecon of 168 individuals from 48 locaons of G. aequicauda, G. insensibilis and G. locusta sampled along the European Atlanc coast between Germany and Portugal, and in the Mediterranean including the Black Sea. All five MOTU delimitaon methods applied revealed deep divergence between Black Sea, Mediterranean and Atlanc populaons in both G. aequicauda and G. insensibilis. There were 5-7 disnct MOTUs delimited for G. aequicauda (6-18% p-distance) and 3-4 MOTUs for G. insensibilis (4-14% p-distance). No sympatric MOTUs were detected, and both species displayed several MOTUs within the Mediterranean, but single MOTUs in the Atlanc or within the Black Sea. Significance: Our results indicate crypc diversity within Mediterranean brackishwater Gammarus, although much more moderate than observed for freshwater counterparts. Nevertheless, even considering only the most conservave MOTU numbers, confirmaon of these findings would translate into the triple and quadruple of known species, respecvely. Implicaons are major in light of the ecological relevance of these species and their extensive use as bioindicators and in ecotoxicological studies.

BACKGROUND č there is a high level of cryptic diversity in freshwater morphospecies of Gammarus1,2,3,4 č members of the so-called “Gammarus locusta group” are widespread in brackish habitats of the Mediterranean Sea, North Sea and the Black Sea - no detailed molecular studies has yet been done on that group5 č the goal of our study is to investigate the cryptic diversity and the phylogenetic relations of two most frequent members of the G. locusta group, G.aequicauda and G.insensibilis, over their wide distribution area

METHODOLOGY č COI (510 bp) from 166 individuals of G.aequicauda and G.insensibilis from 47 locations, with G.locusta as an outgroup č molecular operational taxonomic units (MOTUs) were delimited using five different methods and compared6,7,8,9 č a few specimens of MOTUs of G. aequicauda and G. insensibilis were sequenced for additional markers: 16S and 28S in order to reconstruct a time-calibrated phylogeny in BEAST software Map of sites with G.aequicauda and G.insensibilis, the colours represent respective MOTUs inferred from ABGD; black circle represent G.locusta

ABGD GMYC GMYCSingle PTPMultiple BIN

ABGD 0,81 GMYC GMYCSingle PTPMultiple BIN sea level shifts connected with Messinian Salinity Crysis 1 MOTU1 G.aequicauda;Korfu 1 MOTU1 G.insensibilis;Spain 1 sea level shifts during Pleistocene glaciations

1 1 the formation of Mid-Aegean Trench MOTU2 G.aequicauda;Black Sea, Crete

1 isolation by Siculo-Tunisian Strait, MOTUs present 1 in western and eastern of Mediterranean MOTU2 G.insensibilis;Portugal,Morocco

1 1 the split of African Plate in southern Europe 1 MOTU3 G.aequicauda;Italy,Mallorca 1 1 1 MOTU3 G.insensibilis;Italy,Bulgaria

0,96 1 MOTU4 G.aequicauda;Tunisia 1 the formation of Protomediterranean Sea 1 MOTU5 G.aequicauda;Greece,Black Sea Oligocene, partial isolation of Mediterranean Oligocene, partial isolation of Mediterranean basin and formation of Parathetys basin and formation of Parathetys 1 Outgroup G.locusta;Germany 1 Outgroup G.locusta;Germany

40 35 30 25 20 15 10 5 0 million years ago 35 30 25 20 15 10 5 0 million years ago

Bayesian chronograms, only posterior values above 0.7 are annotated, the colours represent respecve MOTUs inferred from ABGD. Text boxes present an interpretaon of respecve nodes

RESULTS & CONCLUSIONS č high level of cryptic diversity with 5-7 distinct MOTUs delimited for G.aequicauda (6-18% p-distance) and 3-4 MOTUs for G.insensibilis (4-14% p-distance); no sympatric MOTUs were detected and both species displayed several MOTUs within the Mediterranean, but single MOTUs in the Atlantic or within the Black Sea č divergence of Gammarus aequicauda is strongly connected with the geological history of the Mediterranean Sea, starting already with the formation of the Protomediterranean Sea; some of the dispersal and divergence events yet to be explained, more data needed č diversification of Gammarus insensibilis lineages is connected with the appearance of the Siculo-Tunisian Strait - different lineages present in the western and eastern part of Mediterranean; divergence within the western clade is recent and happened possibly due to Pleistocene sea level shifts REFERENCES 1. Hou Z et al. (2014) Phylogenetic analyses of reveal different diversification patterns among sister lineages in the Tethyan region. Cladistics. 2. Copilaş-Ciocianu D, Petrusek A (2015) The southwestern Carpathians as an ancient centre of diversity of freshwater gammarid amphipods: insights from the Gammarus fossarum species complex. Molecular ecology. 3. Mamos T et. al. (2016) The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression. Molecular ecology. 4. Grabowski M et al. (2017) Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod. PeerJ. 5. Stock JH (1967). A revision of the European species of the Gammarus locusta-group (Crustacea, Amphipoda). Zoologische Verhandelingen 90, 1–56. 6. Pons J et al. (2006) Sequence-based species delimitation for the DNA of undescribed insects. Systematic biology. FUNDING SOURCES 7. Puillandre N et al. (2012) ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular ecology. 8. Ratnasingham S, Hebert PD (2013) A DNA-based registry for all species: the Barcode Index Number (BIN) system. PloS one. 9. Zhang J et al. (2013) A general species delimitation method with applications to phylogenetic placements. Bioinformatics.