Freshwater invertebrates from Livingston and Horseshoe Islands, Maritime Antarctica

Various processes trigger the formation, disappearance or expansion of lakes and ponds in Antarctica. Such dynamic ecosystems are a challenging environment for their inhabitants. We studied aquatic invertebrates in lakes and ponds on Livingston and Horseshoe Islands, Antarctica. Invertebrate fauna was either poor, in terms of diversity, or completely lacking. The taxa we found were of the polyphyletic group Protozoa; phylla Nematoda, Tardigrada, Annelida (subclass Oligochaeta) and Arthropoda (subclass Collembola, classes Insecta, Branchiopoda and Copepoda). Whenever a species dominated the communities, it was the copepod Boeckella poppei. When other taxa were recorded, their density was relatively low with few exceptions (Macrothrix oviformis, Branchinecta gaini). High density of B. poppei was recorded in circa 65% of all samples and the majority of the lakes/ponds with fauna. Most of the studied water bodies were oligotrophic, likely among the reasons for the poor faunal diversity, together with the harsh environmental conditions in Antarctica, e.g. low temperatures and long periods during which the lakes are frozen or completely dried out. The formation of new lakes and ponds poses the question on the pathways of distribution of aquatic organisms and why some ponds and lakes are densely populated, while others are deprived of aquatic invertebrates.


Introduction
The first freshwater invertebrates from Subantartic islands were collected 150 years ago from Kerguelen Island by A. E. Eaton [1].As part of numerous Antarctic expeditions various freshwater samples are being collected ever since (see review [2]), with efforts increasing over time.The area of Maritime Antarctica (e.g. the South Shetland Islands and the Antarctic Peninsula) makes no exception.The focus of this paper are freshwater lakes and ponds on Livingston Island (South Shetlands) and Horseshoe Island (Antarctic Peninsula).
One of the first known studies on aquatic fauna from lakes on Livingston Island took place during the Chilean Antarctic expedition in 1971 in the western part of the island, on Byers Peninsula [3].The first study on freshwater lakes in the eastern part of the Livingston Island was conducted in the area of the Bulgarian Antarctic Base (BAB) St. Kliment Ohridski during the 90 s of the ХХ th century by Pandourski and Chipev [4].At the beginning of the ХХI st century, Toro et al [5] studied 15 lakes on Byers Peninsula.Hydrobiological data are scares for the Horseshoe Island: samples from four freshwater lakes on the island have been collected as part of the III rd Turkish Antarctic Expedition IOP Publishing doi:10.1088/1755-1315/1305/1/012004 2 (TAE) in 2018-2019 [6].They aimed to study the community composition and the food web structure therein.Published data on aquatic fauna for water bodies on Horseshoe Island are still lagging with respect to geological, geomorphological, chemical and bacteriological data, as well as data on diatom communities [6].
Dartnall [2] collated available data on freshwater fauna in Antarctica and the adjacent territories.According to their review, the most species-rich in freshwater bodies on the South Shetlands Islands (sensu Dartnall [2]) and the Antarctic Peninsula are the phylla Rotifera, Nematoda and Arthropoda vs Tardigrada, Nematoda and Rotifera, respectively (Table 1).Over the last decades, West Antarctica is subjected to warming [7], leading to the retreat of ice and snow cover over larger territories, as well as to the concurrent thawing of permafrost.These processes trigger the formation of new lakes and ponds, thus providing potential new habitats [8] that, if suitable, could lead to the expansion of habitats of well-adapted Antarctic invertebrates (e.g.[9,10]).Our aim was to collect new distributional data on invertebrates from freshwater lakes and ponds from Livingston and Horseshoe Islands, Antarctica.Furthermore, we verified some older records of aquatic invertebrates from these remote, vulnerable and still understudied ecosystems.

Study area and period
Invertebrate fauna was collected in lakes and ponds on Livingston and Horseshoe Islands, Maritime Antarctica.The area of Livingston Island is almost 800 km 2 ; it is a part of the South Shetlands Archipelago and is separated from the Antarctic Peninsula by the Bransfield Strait.Horseshoe Island is a small island on the Antarctic Peninsula with an area of about 60 km 2 ; it is 12 km long and 6 km wide.
Different types of water bodies were sampled: 35 lakes and ponds on Livingston Island and five on Horseshoe Island (Figure 1).They varied in surface area, size, depth, origin and age [11].Most of the lakes we sampled on Livingston Island were in the vicinity of the BAB St. Kliment Ohridski and the Spanish Antarctic Base (SAB) Juan Carlos Primero.The Hexagona Lake and few smaller ponds near Hannah Point were also sampled.Among the sampled lakes of glacial origin on Livingston Island were: Tododrina buza Lake, formed after the retreat of the Sea lion Glacier, situated south of the eponymous locality [12]; Hexagona Lake, named after its hexagonal shape, situated at about 130 m away from the ocean and 3 km north-east of Hannah Point [12]; a nameless deep lake at the base of the Balkan Glacier, formed after the retreat and the melting of the glacier and another similar lake

B C
between the Spanish Refugio and the SAB Juan Carlos Primero (see Table 2 in the Results section), as well as four of the studied lakes on Horseshoe Island.The ponds we sampled between the Sinemorets (64 m a.s.l.) and Belozem Hills (41 m a.s.l.: [12]) had been formed on top of thawing permafrost and were all small and shallow.Smaller and shallower ponds were sampled also in the area of Hannah Point, the SAB Juan Carlos Primero, Punta Ereby, Caleta Argentina, Sally Rocks (all on Livingston Island) and one pond on Horseshoe Island.Most of the samples from Livingston Island were collected within the XXX th and the XXXI st Bulgarian Antarctic Expeditions (BAEs) in January-February 2022 and 2023, respectively.We also included previous data from the region, collected in February (V th BAE) and December 1997, February 1998 (VI th BAE) and January 2020 (XXVIII th BAE).All samples from Horseshoe Island were collected during the VI th TAE in February 2020.

Invertebrate sampling
Aquatic invertebrates were collected using a hand-held net or Apstein plankton net, depending on the depth, substratum stability and accessibility of the studied lakes or shallow ponds.Both nets were with mesh size of 40 μm.Samples for two of the deeper lakes in the vicinity of the BAB St. Kliment Ohridski on Livingston Island were collected from a boat, while in the remaining shallow ponds or B C farther lakes invertebrates were collected through directly entering the lakes.For all bigger lakes additional samples were also collected from the lake shore.A total of 65 samples were collected: 58 from Livingston Island and seven from Horseshoe Island.No protected species have been collected.

Water parameters
When feasible, physical and chemical water parameters were measured following [14].Those included temperature, pH, dissolved oxygen and oxygen saturation, salinity and conductivity, measured in situ with portable WTW series 330i.Water samples were collected for laboratory determination of nutrients using WTW PhotoFlex series.Most of these measurements were done during the XXX th and the XXXI st BAEs.

Results
We sampled a total of 40 ponds and lakes.Only lakes with salinity 0.00‰ were included in the current study.The water temperature varied from 0.1 to 11.6°C; with dissolved oxygen ranging from 8.4 to 15.58 [mg.dm -3 ]; pH -from 6.99 to 9.79; conductivity varied greatly: from 22.50 to 309 μS.cm -1 .The concentrations of various forms of nitrogen (NO 2 -N; NO 2 ; NO 3 -N; NO 3 ; NH 4 -N and NH 4 ), as well as PO 4 -P and PO 4 were all very low (below 0.8 [mg.dm -3 ]) or outside of the lower limit of the used method.No data on the water parameters were collected from the sites on Horseshoe Island.No invertebrates were found in 13 of the 65 samples (Table 2).In the other lakes were recorded from one to four taxa per site.
Table 2. Lakes/ponds with month & year of sampling, coordinates and (min) number of taxa in each lake.*denotes approximate coordinates taken from maps; **denote approximate coordinates owing to sampling more than one site or on more than one occasions.In bold are highlighted water bodies for which these are the first data on their invertebrates.LI -Livingston Island; HI -Horseshoe Island.Specimens of five phylla were recorded.Most of the recorded taxa were present with low densities except for the three crustacean species: Boeckella poppei (Mrázek, 1901) that was recorded from both islands (Table 3.a), Macrothrix oviformis Ekman, 1900 (Table 3.b) and Branchinecta gaini Daday, 1910 (Table 3.c), both found only in the lakes on Horseshoe Island.Forty-two of the samples were notably dominated by one species, the calanoid B. poppei (Table 3).The cladoceran M. oviformis dominated in one of the samples in Pond 1 and was recorded in four samples (two from Lake 1 and two from Pond 1) on Horseshoe Island.3.d) and representatives of class Clitellata and phylum Rotifera.The record of the rotifer from the Hexagona Lake needs to be confirmed as it was based only on a single specimen.

Discussion
The studied Antarctic lakes and ponds varied greatly in age, basin volume permanence, size and depth, physical and chemical parameters.To the best of our knowledge, here we present the first data on invertebrate fauna of 34 of the water bodies (in 11 of these water bodies we found no invertebrates) in this remote and still understudied region.The recorded fauna belonged to the polyphyletic group Protozoa, as well as to the phyla Nematoda, Tardigrada, Annelida (subclass Oligochaeta) and Arthropoda (subclass Collembola, family Chironomidae of class Insecta, classes Branchiopoda and Copepoda).
We found a relatively low lake/pond invertebrate diversity with up to four taxa per water body.The majority of the studied water bodies were oligotrophic, likely among the reasons for the poor faunal diversity, together with the harsh environmental conditions in Antarctica, i.e. low temperatures and IOP Publishing doi:10.1088/1755-1315/1305/1/0120047 long periods during which the lakes are frozen or completely dried out.Such dynamic ecosystems could be a challenging environment for their inhabitants.Overall, Antarctic lakes are known to be species-poor ecosystems and one of the McMurdo Dry Valley lakes is considered of "high zooplankton diversity" with a total of ten taxa [15].
Regardless of the differences in the characteristics and in the environmental conditions of the studied lakes and ponds, in most of them (with two exceptions, one of the two sites in Lake 4 and Pond 1 on HI) dominated a crustacean of order Calanoida, class Copepoda.Despite the low concentrations of available nutrients in the water column, for 23 of the ponds and lakes were recorded very dense populations of the calanoid B. poppei.This is the only species of the genus that is common in both Maritime and Continental Antarctica.Paggi [16] found that B. poppei dominated in 86% of the 30 studied ponds (all with invertebrates) on King George Island.Our results are very similar when we consider only the samples with fauna (B.poppei dominated in 79.31%).This species is known to be a successful coloniser of newly-formed lakes and ponds [10] and we recorded it in almost 70% of the collected samples.Various adaptations make B. poppei the ultimate coloniser in Antarctic lakes, e.g.protective colouration [17,18]; resting eggs that can survive >100 years [19]; well-functioning pathways of distribution [9,20].We provide new distributional records for this calanoid species for 19 of the studied lakes and ponds.
Branchinecta gianni was recorded in all seven samples from Horseshoe Island, it dominated in one of them.It is the only Antarctic species of the genus, which is presently represented by 48 species and two subspecies [21].Branchinecta gianni is found from Southern Patagonia to the Antarctic Peninsula [22] and it is thought to distribute in Antarctica zoophoretically [23].The same species was recorded in more than half (54%) of the studied ponds on King George Island [16] and on Livingston Island in the area of the Byers Peninsula [5].We did not record the species from the water bodies in the eastern part of Livingston Island.However, the ecological conditions in our study area are notably distinct from the ones on the Byers Peninsula, owing to the fact that this peninsula is the largest ice and snow free area in the Shetlands Archipelago as opposed to the studied by us water bodies, formed following the quite recent retreat (over the last decades) of ice and snow cover.Further, the Byers Peninsula has an active lithosol soil layer of shattered rocks overlaying the permafrost [5].
We found the cladoceran Macrothrix oviformis only in two of the lakes on Horseshoe Island and it dominated in one of the samples.Currently, the genus Macrothrix is represented by 47 species, four of which are found in Antarctica [24].Macrothrix oviformis is distributed in the southernmost parts of continental South America, Tierra del Fuego, Falkland Islands, South Georgia, South Orkney Islands and the Antarctic Peninsula [24,25].According to Kotov [25], the specimens of the genus recorded from Byers Peninsula belong to this species.We did not find the species in our samples from Livingston Island.Macrothrix oviformis, alike other parthenogenetic crustaceans, is often found in marginal habitats associated with higher latitudes and altitudes [26].Parthenogenesis is considered by some authors an advantage in polar regions and low temperatures [27].
Parochlus steinenii is one of the two native Antarctic dipterans; it occurs throughout the South Shetland Islands, sub-Antarctic South Georgia and the sub-Antarctic Magellanic Ecoregion of southern South America but has not been found on the Antarctic Peninsula [28,29].It has been previously recorded from Byers Peninsula [5], in the western "green" part of the Livingston Island but this is its first record from the eastern "white" part of the island.Parochlus steinenii lays almost 200 eggs per batch on average, sometimes over multiple batches; its aquatic larvae require two years for their development and hatch immediately after spring thaws [30], all adaptations that assist its survival in harsh polar conditions.
Unlike the relatively rich rotifer communities recorded by Paggi ([16]: 12 taxa) on King George, and for the South Shetland Islands given by Dartnall [2: 21 taxa], rotifers on the Antarctic Peninsula (six taxa in the review by Dartnall [2]) and Livingston Island are relatively rare and their taxonomical identity is still understudied [5].We found only one specimen of Rotifera but this record requires further verification.Protozoans and nematods in our samples likely originated from the bottom sediments of the studied water bodies.
Larger organisms, such as fish and other aquatic vertebrates, are not found in these lakes and ponds.Such simple food webs could likely make these water bodies more sensitive to changing environmental conditions.Further warming might change the distributional range of Antarctic organisms and could lead to invasions of non-native species.The present and any future formation of new lakes and ponds, especially in West Antarctica, poses the question on the pathways of distribution of aquatic organisms and why some ponds and lakes are densely populated, while others are deprived of aquatic invertebrates.

Figure 1 .
Figure 1.Map of Antarctica (upper right corner), the South Shetlands Islands and the Antarctic Peninsula (A), Livingston Island (B) and the Horseshoe Island (C) with the sampled lakes and ponds marked with pins.Modified after Davies et al [13].
Three taxa were found only in one sample: the chironomid Parochlus steinenii (Gerke, 1889) (Table

Table 3 .
Aquatic invertebrate taxa recorded from lakes/ponds on Livingston and Horseshoe Islands.For each taxon is given the number of samples, in which it was recorded.Photos of taxa 3.a÷3.dare presented below the table.l denotes larva, a denotes adult.