Two new endemic species of freshwater shrimps from the ancient Skadar Lake system revealed with DNA barcoding

Aleksandra JabłońskaA, Tomasz MamosA, Andrzej ZawalB, Vladimir PešićC, Michał GrabowskiA ADepartment of Invertebrate Zoology & Hydrobiology, University of Lodz, Poland BDepartment of Invertebrate Zoology & Limnology, University of Szczecin, Poland CDepartment of Biology, University of Montenegro, Montenegro

Skadar Lake system consists of the largest lake in Balkan Peninsula, as well as of marshlands and numerous karst springs. It is a well known biodiversity and endemism hotspot. The area of the lake varies seasonally from 370 to 600 km2, while its average depth is only 5 meters. The Skadar Lake basin is fed by few rivers and sublacustrine springs. Previously known as ancient, the lake appeared to be young as it originated only ca. 1200 ya. In contrast, its spring system is much older, and may date back even to Miocene. The local endemics are associated mainly with those springs. Among other aquatic invertebrates we investigated shrimps inhabiting the Skadar Lake system so far classified with the use of morphological features as Atyaephyra desmarestii (Atyidae) and Palaemon antennarius (Palaemonidae).

Skadar Lake Balkan Peninsula

Apennine Peninsula Adriatic Sea

Skadar Lake SL1 Zeta Morača SL2 We surveyed the area in spring and autumn 2014, as well as in summer 2015. The samples were taken Sitnica from 65 sites located in the Skadar Lake system. At 21 sites we found shrimps including 101 individuals of Atyidae and 55 of Palaemonidae The collected shrimps were identified based on morphology and

Cem Černojevica SL3 then genotyped for a ca. 650 bp (Atyaephyra) and ca. 600 bp (Palaemon) long fragmentsof SL4 Montenegro Albania SL5 SL13 SL7 SL12 mtDNA COI marker. The amplified fragments were sequenced and then used for a set of analyses, SL6 SL8 SL9 SL10 SL11 including reconstruction of time-calibrated phylogeny and demographic analyses (mismatch distribution). SL14 SL21

SL15 SL16

SL17 SL18

SL19 A B

SL20 Drin

Sampling sites Bojana 1 Sites with Atyaephyra Palaemon antennarius Sites with Palaemon from Apennine Peninsula Sites with both genera

Map of Skadar Lake with sampling sites

1 Atyaephyra from 1 demographic model spatial model

observed observed A expected expected the Skadar Lake system 99% Cl 99% Cl 95% Cl 95% Cl 90% Cl 90% Cl 300 300 SSD = 0.0006, p = 0.89 SSD = 0.0006, p = 0.72 HRI = 0.0351, p = 0.57 HRI = 0.0351, p = 0.57

200 200 1 Palaemon

0,92 Number of pairs of Number Number of pairs of Number from

100 100 1 the Skadar Lake system

0 0 1

0 2 4 6 8 10 0 2 4 6 8 10 Pairwise differences Pairwise differences KJ174018 A. thyamisensis demographic model spatial model KJ174008 A. strymonensis

B observed observed 0,99 expected expected KJ174004 A. stankoi 99% Cl 99% Cl 95% Cl 95% Cl 1 90% Cl 800 800 90% Cl 1 Kj174000 A. stankoi KT157027.1P. zariquieyi SSD = 0.0002, p = 0.59 SSD = 0.0002, p = 0.59 HRI = 0.3093, p = 0.62 HRI = 0.3093, p = 0.62 KJ174012 A. orientalis 1 600 600 1 KJ173992 A. thyamisensis KT157028.1P. zariquieyi Kj173997 A. desmarestii 400 400 1

Number of pairs

Number of pairs Jx289920 A. desmarestii 1 Fj594341 A. desmarestii 200 200 Fj594344 A. desmarestii Jx289934 A. desmarestii 0 0 14 12 10 8 6 4 2 0 [Ma] 0 1 2 3 4 0 1 2 3 4 Pairwise differences Pairwise differences 25 20 15 10 5 0 [Ma] Mismatch distribution analysis, under demographic and spacial expansion models Time-calibrated reconstruction of phylogeny of Atyaephyra (A) and Palaemon (B) species, of the Skadar Lake Atyaephyra (A) (Tajima’s D = -2.11, p = 0.001; Fu’s Fs = -25.18, p = 0) based on the COI sequences and Palaemon (B) (Tajima’s D = -2.18, p = 0; Fu’s Fs = -6.92, p = 0)

DNA barcoding of Skadar Lake shrimps suggested the investigated Atyaephyra and Palaemon representatives may be two species new to science. All the Atyaephyra individuals could not be ascribed to any of the formely existing species, deviating from each of them in at least some morphological characters. Phylogenetic analysis revealed that the new species is most closely related to the south-Balkan A. stankoi and A. thyamisensis, from which it diverged probably in early Pliocene. In contrast, the present diversity of this probably new species dates back only to Middle Pleistocene, when its population has expanded both in demographic and spatial terms. Our data suggest that it is the Skadar Lake system endemic and that it occurs mainly in the areas under influence of spring and riverine waters. The species may be a relic of the old spring system and marshlands occupying the basin before the present lake come into existence.The Skadar Lake population of Palaemon occured to be a sister cryptic species to P. antennarius from the Apennine Peninsula as they diverged probably in early Pleistocene while its expansion started ca. 3 Mya.