The impact of the colonization of the invasive American Artemia franciscana (Crustacea: Anostraca) on genetic differentiation in the United Arab Emirates (Asia)

Artemia franciscana, native to America, has recently colonized non-indigenous populations in Eurasia, Mediterranean regions and Australia. In present we sought to evaluate the potential effects of colonization of A . franciscana on genetic differentiation in the new environments in UAE. We used the COI marker to determine population genetic structure and identify the origins of exotic populations in UAE. Our findings have confirmed the colonization of both localities by A . franciscana . Genetic variation of invasive A . franciscana were exclusively lower than native population in Great Salt Lake and San Francisco Bay. Results have showed the studied population could not possibly have colonized directly from natural American localities, perhaps resulting from secondary introduction events from other non-indigenous populations. Genetic analysis have yielded different demographic patterns for invasive studied populations. Al Wathba Wetland Reserve (AWWR) population have represented demographic expansion. In contrast, Godolphin Lakes (GL) population was at demographic equilibrium. Neutrality tests have documented the excess of both recent and historical mutations in the COI gene pool of invasive AWWR Artemia throughout establishment in the new environment. KF662976, KF662977), respectively 29 . To detect the genealogical relationships and origin of UAE samples with American A . franciscana (more information in Results), a median network was performed using the median-joining algorithm in the Network program ver.


Introduction
The introduction of an exotic species to the natural environments can diminish biodiversity and generate a prominent change on biological community structure 1,2 . Although only nearby 1% of introduced non-natives become invasive 3 , but establishment of invasive species in new habitats have caused widespread ecological effects and economic damage 4 .
Genetic structure of exotic species is one of the most important factors in their successful colonization and dispersion 5 . Regarding to the genetic contexts, the capacity of a non-native species to adapt to a new environmental conditions depends on the potential of genetic diversity of the species 6 . It is expected exotic species are experienced loss of genetic variation during colonization in non-native habitats due to the founder effect which resulted by genetic drift following small size of population 7 .
The brine shrimp genus Artemia is widely used in larviculture 8  Since 1950, American A. franciscana from two major natural sources in the USA, the Great Salt Lake (GSL) and San Francisco Bay (SFB), were exported overseas for larviculture and fishery hatcheries [11][12][13] . Because of Artemia cysts have been harvested in a limited number of natural hypersaline lakes, growing importance of Artemia in aquaculture needed to culture Artemia in other saltwater sources. Due to higher reproductive rate and adaptation ability of A.
franciscana 14,15 , this species has been selected to culture in non-indigenous natural and artificial environments for industrial aquaculture and fishery activates, so currently it has a wide geographical distribution in the world containing inland salt lakes, coastal saltworks, salt ponds and lagoons. Now, A. franciscana has been colonized in numerous regions across Eurasia, especially in the Mediterranean 9, 11-13,16-20 and Australia 21 .
Two Artemia sites have been reported from the United Arab Emirates 22,23 . Saji et al. 20 have documented invasive A. franciscana in Al Wathba Wetland Reserve. There is no evidence that Artemia had been introduced intentionally into these localities for commercial activity, but it has prepared a suitable habitat for the greater flamingos and other native shore birds 20 . In 1998, before introduction of the greater flamingos in Godolphin Lakes, cysts of Artemia were distributed in the water body 23 . Although previous studies have referred existence of Artemia in Godolphin Lakes to A. franciscana¸ there is a lack of phylogenetic proof to support this claim.
The aim of the present study was to perform a phylogenetic analysis of populations from Godolphin Lakes to confirm the taxonomical status of Artemia in this locality. Here we sequenced the mitochondrial COI gene and calculated the genetic diversity and population genetic structure of Artemia populations in UAE to compare evolutionary progresses and genetic differentiation of invasive species in new environments.

Study area and sampling
In total, 82 cysts of Artemia were collected from two geographical localities in United Arab Emirates including Godolphin Lakes (GL) and Al Wathba Wetland Reserve (AWWR). (Fig. 1).
The sampling sites with their abbreviations, geographical coordinates and number of specimens analyzed were summarized in Table 1.
Total DNA was extracted from each decapsulated Artemia cyst following the Chelex® 100 Resin method (Bio-Rad Laboratories, USA). The samples were crushed, incubated for 3 h at 56°C and finally 10 min at 80°C (tubes were vortexed every 30 min). The tubes were centrifuged at 10,000 rpm for 2 min. and the supernatant phase was used as a template in the PCR reaction 13,24 . The extracted DNAs were stored at -80°C for further studies.
A fragment of mitochondrial marker cytochrome c oxidase subunit I (COI) was amplified using the universal primers LCO1490/HC02198 25

Sequence alignment and phylogenetic analyses
Sequences were aligned using MEGA X with default setting 26 . To identify taxonomical status of the collected samples, the COI reference sequences from bisexual species and parthenogenetic populations were downloaded from GenBank ( Table 2) and utilized to draw phylogenetic trees. The phylogenetic tree was generated based on Bayesian inference (BI) as performed in MrBayes 3.2.2 on XSEDE 27 . The best fitting nucleotide substitution model was estimated using MrModeltest 2.2 28 and HKY+G was selected as the best-fit model.

Results
The Bayesian inference (BI) phylogenetic tree have utilized all studied Artemia samples from GL and AWWR were clustered in the clade of A. franciscana (Fig. 2 The differentiation of haplotype frequencies was exclusively non-significant between both exotic populations in UAE (Table 3). Figure 3 showed the haplotype distribution network of the A. franciscana among native and invaded habitats. The 170 COI sequences of A. franciscana have represented 14 distinct haplotypes, that H1, H10 and H12 were the major types, grouped with 57.06% (97 ind.), 18.24% (31 ind.) and 12.94% (22 ind.), respectively (Table S1). In addition, the majority of sequences belonged to H1, composed of 47.42% AWWR, 46.39% GL, 4.12% SFB and 2.07% GSL sequences, respectively ( Fig. 3 and Table 4).

7
The haplotypes distribution and frequency in each locality were determined in Table 5. The majority of haplotype frequency of the native American A. franciscana from SFB and GSL were located in H10 (83.87%) and H12 (95.45%), respectively. Two localities from UAE have possessed the greatest H1 haplotype frequency consisting of AWWR (88.46%: 46 individuals out of 52) and GL (86.54%: 45 individuals out of 52).
The estimated genetic indices for the studied localities were displayed in Table 6. The highest-ranking amounts of Hr (0.206), Pi (0.0038 ± 0.002) and K (2.334 ± 1.013) were documented in GSL, whereas, the SFB locality had the highest quantities of Hd (0.480 ± 0.087) and Hexp (0.277 ± 0.136)., The lowest genetic variations has observed in the AWWR location, excepting Hr which the lowest value was noticed in GL (0.096). Neutrality tests have yielded negative values with significant and non-significant levels.

The mismatch distributions for invasive UAE and American populations of A. franciscana
have showed that GSL, SFB and GL localities had a multimodal pattern, while AWWR location revealed a pattern likely to be unimodal (Fig. 4).
The lowest and non-significant values of the pairwise genetic differentiation index (FST) were observed between UAE populations (2.68%). The significant population differentiations were represented between UAE and American populations (P < 0.01). Additionally, the lowest and significant value of FST was determined between native American populations (SFB-GSL: 58.39%) ( Table 7).
The permutation test has indicated that the difference of GST and NST was exclusively significant between UAE populations where NST (0.027) was higher than GST (0.0001) ( Table 8).
AMOVA analysis has documented that more than a third of genetic variation (37.20%) is attributed within populations (Table 9).

Discussion
The occurrence of Artemia in UAE had been recorded in two geographical sites including in Al Wathba Wetland Reserve 22 and Godolphin Lakes 23 . A molecular phylogenetic study has confirmed that population in Al Wathba Wetland Reserve belonged to an exotic American A. Additionally, there is no meaningful relationship between American and invasive populations sequences in other haplotypes ( Fig. 3 and Table 4). On the other hand the highest and significant FST values between American and exotic populations have strongly supported that GSL and SFB could not be an introduced source for UAE localities ( Table 7) Regarding to non-significant value of neutrality tests and multimodal of mismatch distribution, Godolphin Lakes have shown a demographic equilibrium. American Artemia (GSL and SFB) has presented negative and non-significant value for neutrality tests and also mismatch distribution have revealed multimodal. These findings have evidenced that these populations were at demographic equilibrium. In contrast, Asem et al. 21 found that GSL represented a unimodal mismatch distribution and have recorded this population was under demographic expansion. This difference can be attributed using sequences in different period from GSL. Asem et al. 56 have proved ecological variation could alter genetic structure of Artemia from Urmia Lake. It seems a comprehensive study needs to be performed on population genetic of both native American Artemia in GSL and SFB to estimate ability of genetic variation in two major sources in long-term.
The NST showed high and significant value (0.027) than GST (0.0001) between invasive populations in UAE. A significantly higher NST over GST generally point to the existence of phylogeographic structure (Pons and Petit, 1996). The results have indicated that there was a differentiation of geographical structure between exotic populations of Godolphin lakes and Al Wathba Wetland Reserve, while there was no phylogeographic differentiation between GSL and SFB populations and also among UAE and American populations.     Legends for figures