Ancient Endemic or Recent Invader? Phylogenetic Position and the Probable Origin of the Ccladoceran Diaphanosoma macedonicum (Diplostraca, Sididae) from the Ancient Lakes in the Balkans

© 2024 Academia Sinica, Taiwan Open Access Ancient Endemic or Recent Invader? Phylogenetic Position and the Probable Origin of the Ccladoceran Diaphanosoma macedonicum (Diplostraca, Sididae) from the Ancient Lakes in the Balkans Charo López-Blanco1,2,* , Ohrideja Tasevska3, Goce Kostoski3, Eduardo Vicente4, Laura S. Epp5, and Antonio García-Alix2 1Justus Liebig University Giessen, Department of Animal Ecology and Systematics, Heinrich-Buff-Ring 26 (IFZ). D-35392 Giessen, Germany. *Correspondence: E-mail: [email protected] (López-Blanco) 2Department of Stratigraphy and Paleontology, University of Granada, Avda, Fuentenueva s/n, 18071, Granada, Spain. E-mail: [email protected] (García-Alix) 3Department of Zooplankton, Hydrobiological Institute, Naum Ohridski 50, 6000 Ohrid, Northern Macedonia. E-mail: [email protected] (Tasevska); [email protected] (Kostoski) 4Department of Microbiology and Ecology, University of Valencia, Dr. Moliner 50, 46100 Burjassot, Spain. E-mail: [email protected] (Vicente) 5Limnological Institute, University of Konstanz, Mainaustraße 252 78464 Konstanz / Egg, Germany. E-mail: [email protected] (Epp) Received 24 July 2023 / Accepted 13 January 2024 / Published 3 May 2024 Communicated by Ka Hou Chu Ancient lakes contain unique and very vulnerable fauna. Determining and understanding the origin of such biodiversity is a key factor in promoting conservation and management actions in some of the most singular ecosystems on the planet. Lake Ohrid in the Balkans is known as a natural laboratory for speciation, containing a high number of endemic species. However, the identity and origin of the planktonic cladoceran Diaphanosoma is uncertain. Representatives of the genus were long considered to have invaded the lake, but recent morphological studies have suggested that they belonged to the endemic taxon in the Balkans, D. macedonicum. Here, phylogenetic methods based on two mitochondrial gene fragments (COI and 16S) were used to identify Diaphanosoma specimens from the ancient Lake Ohrid and Lake Prespa in the Balkans and compare them with other species in Europe, including those living in nearby water bodies. Molecular evidence showed that D. macedonicum was constrained to the ancient lakes Ohrid, Prespa, and Mikri Prespa, which suggests reproductive isolation within the lakes. Phylogenetic analyses supported previous morphological assessments and situated D. macedonicum within the D. mongolianum species group, which contains three sibling species (D. mongolianum, D. lacustris, and D. macedonicum). Nuclear markers are needed to study intraspecific gene flow in these organisms and discard a potential formation of hybrids. Key words: Ancient lakes, DNA barcoding, Lake Ohrid, Lake Prespa, Endemicity Citation: López-Blanco C, Tasevska O, Kostoski G, Vicente E, Epp LS, García-Alix A. 2024. Ancient endemic or recent invader? Phylogenetic position and the probable origin of the cladoceran Diaphanosoma macedonicum (Diplostraca, Sididae) from the ancient lakes in the Balkans. Zool Stud 63:09. doi:10.6620/ZS.2024.63-09. BACKGROUND Ancient lakes are some of the most singular ecosystems of the planet, as they comprise extant environments that have been carrying water since before the last glacial period or even before the Pleistocene (Gorthner 1994; Martens 1997). As they have persisted during multiple climatic cycles and environmental Zoological Studies 63: 9 (2024) doi:10.6620/ZS.2024.63-09 1 © 2024 Academia Sinica, Taiwan conditions, they contain continuous archives of the most recent history of the planet, and thus they have been the focus of many palaeoecological investigations (e.g., Cohen 2012; Fritz et al. 2012; Karabanov et al. 2004; Stone et al. 2011). They have also fascinated taxonomists and evolutionary biologists, since as a result their internal speciation processes, they contain high-endemic species and some of the rarest taxa on Earth (Cristescu et al. 2010). By way of illustration, the ancient Lake Ohrid in the Balkans is considered the most biodiverse ancient lake in the world in terms of the number of species per surface area (Albrecht and Wilke 2008). The rate of endemicity was estimated at 36% for all taxa and 34% for Animalia (Albrecht and Wilke 2008), but it has probably increased in recent years with the discovery of new endemic taxa, such as the cladoceran Coronatella begoniae (Sinev and LópezBlanco 2018). Regarding the Cladocera, 26 species have been cited in this lake (Stankovic 1960; Kostoski et al. 2004), two of them — Coronatella begoniae (Sinev and López-Blanco 2018) and Phreatalona smirnovi (Van Damme et al. 2009) — being endemic, which situates the endemicity rate at ca. 7.4%. The low number of endemic cladocerans encountered in this lake might either be ascribed to (i) a strong dispersal ability that could explain more cosmopolitan distributions or (ii) failure to estimate real biodiversity by conventional taxonomy based on morphology. This draws attention to the case of Diaphanosoma Fischer, 1850, in the ancient Lake Ohrid, because data obtained in the last few decades depicts contradictory theories about its identity, origin, and status in this ecosystem. Some information indicates that this species might be a recent invader of the lake, while further morphological analyses have suggested it to be an ancient endemism. Representatives of the genus Diaphanosoma were not known in this lake until the 1990s, when new invaders D. birgei lacustris Kořinek and Leptodora kindtii (Focke, 1844) were recorded (Guseska et al. 2019). Analysis of cladoceran subfossils in a sedimentary record in Lake Ohrid only showed sub-fossils from Diaphanosoma in the uppermost sample (mean age 1975) (López-Blanco et al. 2020). However, the sub-fossil analysis did not allow identification at the species level due to the poor taxonomical information provided by the postabdominal claws. Identification of Diaphanosoma in this lake has been unclear, pointing either to D. birgei lacustris (Gušeska et al. 2005 2014 2019) or to D. brachyurum (Kostoski et al. 2010; Tasevska et al. 2017). Recently, Korovchinsky (2022) presented detailed morphological descriptions indicating that Diaphanosoma in Lake Ohrid belong to the endemic Balkan taxon D. macedonicum. Nevertheless, this morphological analysis was only based on parthenogenetic females, while males and gamogenetic females are needed to confirm the species identity and improve the formal species description. In this study, we provide new genetic data from mitochondrial loci (16S and the COI barcoding regions) originating from specimens collected mainly in the Balkan Peninsula with two main aims: 1) to confirm the identity of the Diaphanosoma species in the ancient Lake Ohrid and Lake Prespa and assess its current status in these ecosystems, and 2) to help determine the phylogenetic position of the D. macedonicum within the phylogeny of the genus. The data provided in our study will contribute to a better understanding of ancient lake biodiversity in a poorly studied area like the Balkans and provide a baseline for management actions for some of the most precious ecosystems of the world. MATERIALS AND METHODS Diaphanosoma Fischer, 1850, currently includes 40 valid euplanktonic species, most of which inhabit relatively warm water bodies all over the world (Korovchinsky 2018). Five of them, D. brachyurum (Liévin, 1848), D. lacustris Korinek, 1981, D. mongolianum Ueno, 1938, D. orghidani Negrea, 1982, and D. macedonicum Korovchinsky et Petrovski 2014, occur in European inland waters (Błędzki and Rybak 2016; Korovchinsky 2018). All of them except D. macedonicum have a relatively large distribution covering several countries and biogeographical areas (Fig. 1). Only D. macedonicum has shown a very limited distribution in the Balkan Peninsula, since it was only cited in the pelagic zones of the lakes Dojran and Prespa (Korovchinsky and Petkovski 2014) and recently was also found in Mikri Prespa (Alexiou et al. 2021). Field collection and morphological analyses The Diaphanosoma specimens studied here were collected from Lake Ohrid, Lake Prespa, and geographically close water bodies in North Macedonia and Greece. In addition, material from outside the Balkan Peninsula belonging to D. mongolianum, D. lacustris and D. brachyurum was used for phylogenetic comparison (Table 1). Samples were collected by pulling a 90 µm plankton net in the pelagic zone of the lakes/reservoirs cited in table 1. The material was preserved in a 95% ethanol solution and stored at 4°C for further analyses. Samples were sorted in the lab and identified at the species level using specific keys for European zooplankton (Alonso 1996; Błędzki and Rybak 2016; Margaritora 1983). page 2 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan Specimens from Lake Ohrid identified as D. macedonicum (see Korovchinsky 2022) were morphologically compared with described conspecifics from lakes Prespa and Dojran (Korovchinsky and Petkovski 2014). DNA Sequencing Total genomic DNA was extracted from single individuals following the DNeasy Blood & Tissue (Qiagen) protocol, but using half of the recommended volume for each step. Once in the lysis buffer, each specimen was energetically crushed with sterile and disposable needles to ensure that the carapace and tissues broke down. Following DNA extraction, DNA was quantified in a Nanodrop spectrophotometer, and 4–5 µL of DNA was added to a PCR, following the procedure published in López-Blanco et al. (2024) (final volume of 20 µL consisting of 2 µL of 10x Thermopol Buffer, 1.4 µL of MgCl, 1.4 µL of dNTP, 6.6 µL of dH2O, 0.2 µL of TMAC, 1.2 µL of BSA, 1.4 µL of forward and reverse primers, and 0.4 of Taq Polymerase [New England Biolabs, 5000 U/mL]). Fragments of the mitochondrial cytochrome c oxidase subunit I (COI) were amplified and sequenced using either the universal primers for arthropods (Folmer et al. 1994) or specific primers designed for zooplankton, Zplank-F and Zplank-R (Prosser et al. 2013). The ribosomal 16S rRNA (16S) fragment was amplified and sequenced using the primer pair 16Sch-a and 16Sbr (Sacherová and Hebert 2003). The polymerase chain reaction amplification conditions followed the methods of Prosser et al. (2013): five cycles of (94°C for 40s, 45°C for 40s, 72°C for 1 min), then 35 cycles of (94°C for 40s, 51°C for 40s and 72°C for 1 min), and then a final extension of 72°C for 5 min. PCR products were visualized on a 1% agarose gel, and visually positive PCR products were selected for sequencing. The amplified PCR products were sequenced in an ABI 3730 XL sequencer (Life Technologies, Carlsbad, CA, USA) using a Big Dye Terminator kit ver. 3.1 (Life Technologies). The chromatograms for each DNA sequence were checked using the software SEQUENCHER 5.4.6 (Gene Codes, Ann Arbor, MI). The primer sequences were removed before any further analysis. Phylogenetic analyses The authenticity of the sequences was verified Fig. 1. (A) Map showing the distribution of the genus Diaphanosoma in Europe (modified after Błędzki and Rybak 2016) and (B) close-up on the Balkan Peninsula, where D. macedonicum was cited. Note that D. brachyurum is widely distributed in Europe, and it is also present in the distribution area of D. orghidani. page 3 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan using BLASTN 2.10 (Zhang et al. 2000) against the National Center for Biotechnology Information (NCBI) nucleotide database. These sequences, together with more than 200 sequences retrieved from GenBank (Table 2), were employed in our phylogenetic reconstructions using Moina cf. micrura (Accession number: MH708070), Simocephalus cf. serrulatus (Accession number: KC617159), and Daphnia cf. pulicaria (Accession number: EU152322) as suitable outgroup. The outgroup selection was made using the same taxa as those used in a recent COI-based phylogeny of Diaphanosoma s. l., published in Dumont et al. (2021), in order to make the results comparable. Protein-coding COI sequences were aligned in Bioedit (Hall 1999) by leaving default settings, and then the alignments were inspected visually and adjusted if gaps were detected. The software jModelTest 2.1.7 (Darriba et al. 2012) of the Cyber Infrastructure for the Phylogenetic Research project (CIPRES; www.phylo.org) was used to select the best-fit model of sequence substitution (COI: HKY+G) under the corrected Akaike Information Criterion (AICc) (Cavanaugh 1997). Uncorrected and K2P genetic distances (550 replicates) were calculated in MEGA for the major COI clades inferred from the Table 1. List of specimens sequenced in this study, location of the sampling sites, and GenBank accession numbers for COI gene Sample ID Species Site Latitude Longitude Elevation (m asl) GenBank accession number COI 16S DiLa_27079 D. lacustris Reservoir La Serena, Spain 38°54'43.17"N 5°25'55.74"W 319 OR050432 DiLa_27080 D. lacustris Reservoir La Serena, Spain 38°54'43.17"N 5°25'55.74"W 319 OR050433 OR039415 DiLa_27081 D. lacustris Reservoir La Serena, Spain 38°54'43.17"N 5°25'55.74"W 319 OR050434 OR039416 DiLa_27082 D. lacustris Reservoir La Serena, Spain 38°54'43.17"N 5°25'55.74"W 319 OR050435 OR039417 DiMa_25980 D. macedonicum Lake Ohrid, Northern Macedonia 41°05'41.29"N 20°47'14.89"E 690 OR050417 DiMa_25981 D. macedonicum Lake Ohrid, Northern Macedonia 41°05'41.29"N 20°47'14.89"E 690 OR050418 DiMa_25982 D. macedonicum Lake Ohrid, Northern Macedonia 41°05'41.29"N 20°47'14.89"E 690 OR050419 OR039407 Dima_25985 D. macedonicum Lake Ohrid, Northern Macedonia 41°05'41.29"N 20°47'14.89"E 690 OR050422 OR039408 DiMa_25983 D. macedonicum Lake Ohrid, Northern Macedonia 41°05'41.29"N 20°47'14.89"E 690 OR050420 DiMa_25984 D. macedonicum Lake Ohrid, Northern Macedonia 41°05'41.29"N 20°47'14.89"E 690 OR050421 DiMa_26646 D. macedonicum Lake Prespa, Northern Macedonia 40°52'26.91"N 20°58'27.56"E 842 OR050428 OR039412 DiMo_27098 D. mongolianum Lake Vegoritida, Greece 40°44'31.75"N 21°48'22.93"E 510 OR050436 OR039418 DiMo_27099 D. mongolianum Lake Vegoritida, Greece 40°44'31.75"N 21°48'22.93"E 510 OR050455 OR039419 DiSp_26589 Diaphanosoma sp. Kastoria, Greece 40°30'39.52"N 21'18'53.07"E 624 OR050423 DiSp_26590 Diaphanosoma sp. Kastoria, Greece 40°30'39.52"N 21'18'53.07"E 624 OR050424 DiSp_26591 Diaphanosoma sp. Lake Vegoritida, Greece 40°44'31.75"N 21°48'22.93"E 510 OR050425 OR039409 DiSp_26592 Diaphanosoma sp. Lake Dojran, Northern Macedonia 41°12'36.78"N 22°44'40.65"E 140 OR050426 OR039410 DiSp_26648 Diaphanosoma sp. Lake Kastoria, Greece 40°30'39.52"N 21'18'53.07"E 624 OR050429 OR039413 DiSp_26649 Diaphanosoma sp. Lake Kastoria, Greece 40°30'39.52"N 21'18'53.07"E 624 OR050430 OR039414 DiSp_26650 Diaphanosoma sp. Lake Vegoritida, Greece 40°44'31.75"N 21°48'22.93"E 510 OR050431 DiSp_27125 D. mongolianum Albufera de Valencia, Spain 39°20'11.41"N 0°21'03.91"W 0 OR050437 OR039420 DiSp_27126 D. mongolianum Albufera de Valencia, Spain 39°20'11.41"N 0°21'03.91"W 0 OR050438 OR039421 DiSp_27127 D. mongolianum Albufera de Valencia, Spain 39°20'11.41"N 0°21'03.91"W 0 OR050439 OR039422 DiSp_E132 Diaphanosoma sp. Reservoir La Serena, Spain 38°50'30.27"N 5°06'20.40"W 350 OR050454 DiSp_E187 Diaphanosoma sp. Reservoir Sotonera, Spain 42°07'35.62"N 0°38'22.75"W 428 OR050440 DiSp_E188 Diaphanosoma sp. Reservoir Sotonera, Spain 42°07'35.62"N 0°38'22.75"W 428 OR050441 DiMo_E192 D. mongolianum Reservoir Moneva, Spain 41°11'00.81"N 0°49'29.41"W 632 OR050442 DiMo_E193 D. mongolianum Reservoir Moneva, Spain 41°11'00.81"N 0°49'29.41"W 632 OR050443 DiMo_E194 Diaphanosoma sp. Reservoir Moneva, Spain 41°11'00.81"N 0°49'29.41"W 632 OR050444 DiBra_E201 D. brachyurum Reservoir Ebro, Spain 42°58'27.69"N 4°01'11.67"W 835 OR050445 DQ470593 DiBra_E202 D. brachyurum Reservoir Ebro, Spain 42°58'27.69"N 4°01'11.67"W 835 OR050446 DiBra_E203 D. brachyurum Reservoir Ebro, Spain 42°58'27.69"N 4°01'11.67"W 835 OR050447 DiSp_E204 Diaphanosoma sp. Reservoir Vicarias, Spain 41°22'24.25"N 2°11'01.19"W 805 OR050448 DiSp_E206 Diaphanosoma sp. Reservoir Vicarias, Spain 41°22'24.25"N 2°11'01.19"W 805 OR050449 DiSp_E217 Diaphanosoma sp. Reservoir Mequinenza, Spain 41°22'58.55"N 0°07'29.64"E 153 OR050450 DiSp_E218 Diaphanosoma sp. Reservoir Mequinenza, Spain 41°22'58.55"N 0°07'29.64"E 153 OR050451 DiSp_E222 Diaphanosoma sp. Reservoir Ortigosa, Spain 42°11'12.53"N 2°40'35.03"W 1013 OR050452 DiSp_E223 Diaphanosoma sp. Reservoir Ortigosa, Spain 42°11'12.53"N 2°40'35.03"W 1013 OR050453 SiCry_26602 Sida crystallina Lake Ohrid, Northern Macedonia 41°05'41.29"N 20°47'14.89"E 690 OR050427 OR039411 page 4 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan phylogenetic analyses. Phylogenetic analyses were performed on the COI dataset using the Maximum likelihood (ML) and Bayesian Inference (BI) methods. ML analysis was conducted using the web server offering RAxML-NG available at https://raxml-ng.vital-it.ch/#/ by applying the HKY+G model. Bayesian phylogenetic analyses were performed in MRBAYES 3.2.2 (Ronquist et al. 2012) with the substitution models selected by jModelTest. The BI analysis was conducted using Markov chain Monte Carlo (MCMC) sampling in MrBayes (Ronquist et al. 2012) under the HKY+G substitution model. Markov chain Monte Carlo (MCMC) methods used random starting trees and employed four independent runs, each with one cold chain and three incrementally heated chains. Trees were sampled every 100 generations for one million generations, and the first 25% of all the trees sampled before convergence were discarded as burn-in. The 50% majority-rule consensus tree was generated from the remaining trees. Node support of the inferred trees was evaluated by bootstrapping for ML and by Bayesian posterior probabilities (BPPs) for BI. Species clusters and supports were visualized in FIGTREE 1.4.3 (Rambaut 2010). Given the restricted phylogenetic information provided for COI within the D. mongolianum group and in order to clarify the position of the D. macedonicum clade, 16S sequences were added to COI sequences (Table 1) and two datasets were compiled (one per gene partition). The 16S fragments were automatically aligned using the MAFFT algorithm (Katoh 2002) with default options. Concatenated COI plus 16S alignments were performed using CONCATENATOR (Pina-Martins and Paulo 2008). The Sididae species Sida crystallina (Table 1) and Penilia avirostris (Accession numbers COI: KT208814 and 16S: DQ470595) were included as suitable outgroup in phylogenetic tree reconstruction based on concatenated data. jModelTest was run again and the phylogenetic analysis was performed Table 2. List of COI sequences downloaded from GenBank, indicating the species name, the phylogenetic position in our COI tree, the region of collection and the accession number. Sequences in the table were downloaded from the following publications: (Alexiou et al. 2021; Elías-Gutiérrez et al. 2008; Lakatos et al. 2015; Liu et al. 2018; Prosser et al. 2013; Richter et al. 2007) Species name in GenBank Phylogenetic group Site GenBank accession number Publication Diaphanosoma heberti Diaphanosoma cf. heberti D. heberti Mexico KC617624 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti Mexico KC617623 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti Mexico KC617622 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti Mexico KC617621 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti Mexico KC617620 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti Mexico KC617619 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti Mexico KC617618 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti Mexico KC617617 Prosser et al. (2013) Diaphanosoma cf. heberti D. heberti USA HM884025 Direct Submission Diaphanosoma cf. heberti D. heberti USA HM884019 Direct Submission Diaphanosoma cf. heberti D. heberti USA HM884018 Direct Submission Diaphanosoma cf. heberti D. heberti Mexico EU702164 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702163 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702162 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702161 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702160 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702159 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702158 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702157 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702156 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702155 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702154 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702153 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702152 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702151 Elías-Gutiérrez et al. (2008) Diaphanosoma cf. heberti D. heberti Mexico EU702150 Elías-Gutiérrez et al. (2008) page 5 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan Species name in GenBank Phylogenetic group Site GenBank accession number Publication Diaphanosoma brevireme Diaphanosoma brevireme D. brevireme Mexico KC617616 Prosser et al. (2013) Diaphanosoma brevireme D. brevireme Mexico KC617615 Prosser et al. (2013) Diaphanosoma brevireme D. brevireme Mexico KC616986 Prosser et al. (2013) Diaphanosoma brevireme D. brevireme Mexico KC616984 Prosser et al. (2013) Diaphanosoma brevireme D. brevireme Mexico KC616983 Prosser et al. (2013) Diaphanosoma brevireme D. brevireme Brazil KY659309 Domingos et al. (2017) Diaphanosoma brevireme D. brevireme Mexico EU702149 Elías-Gutiérrez et al. (2008) Diaphanosoma brevireme D. brevireme Mexico EU702148 Elías-Gutiérrez et al. (2008) Diaphanosoma brevireme D. brevireme Mexico EU702147 Elías-Gutiérrez et al. (2008) Diaphanosoma brevireme D. brevireme Mexico EU702146 Elías-Gutiérrez et al. (2008) Diaphanosoma brevireme D. brevireme Mexico EU702145 Elías-Gutiérrez et al. (2008) Diaphanosoma spinulosum Diaphanosoma spinulosum D. spinulosum Mexico KC617336 Prosser et al. (2013) Diaphanosoma spinulosum D. spinulosum Mexico KC617335 Prosser et al. (2013) Diaphanosoma spinulosum D. spinulosum Mexico KC617334 Prosser et al. (2013) Diaphanosoma spinulosum D. spinulosum Mexico KC617333 Prosser et al. (2013) Diaphanosoma spinulosum D. spinulosum Mexico KC617332 Prosser et al. (2013) Diaphanosoma spinulosum D. spinulosum Mexico KC617331 Prosser et al. (2013) Diaphanosoma spinulosum D. spinulosum Mexico KC617330 Prosser et al. (2013) Diaphanosoma orientalis Diaphanosoma orientalis D. orientalis Japan LC060059 Lakatos et al. (2015) Diaphanosoma orientalis D. orientalis Japan LC060058 Lakatos et al. (2015) Diaphanosoma orientalis D. orientalis Japan LC060057 Lakatos et al. (2015) Diaphanosoma macrophthalma Diaphanosoma cf. macrophthalma D. orientalis Japan LC060056 Lakatos et al. (2015) Diaphanosoma cf. macrophthalma D. macrophthalma Japan LC060055 Lakatos et al. (2015) Diaphanosoma cf. macrophthalma D. macrophthalma Japan LC060054 Lakatos et al. (2015) Diaphanosoma cf. macrophthalma D. macrophthalma Japan LC060053 Lakatos et al. (2015) Diaphanosoma macrophthalma D. macrophthalma China KU720105 Direct Submission Diaphanosoma dubium Diaphanosoma cf. dubium D. dubium Japan LC060052 Lakatos et al. (2015) Diaphanosoma cf. dubium D. dubium Japan LC060051 Lakatos et al. (2015) Diaphanosoma cf. dubium D. dubium Japan LC060050 Lakatos et al. (2015) Diaphanosoma cf. dubium D. dubium Japan LC060049 Lakatos et al. (2015) Diaphanosoma cf. dubium D. dubium Japan LC060048 Lakatos et al. (2015) Diaphanosoma dubium D. dubium China KY788978 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788977 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788974 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788973 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788947 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788946 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788945 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788944 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788941 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788940 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788939 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788938 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788933 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788932 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788931 Liu et al. (2017) Diaphanosoma dubium D. dubium China KY788930 Liu et al. (2017) Diaphanosoma dubium D. dubium China KU720106 Direct Submission Diaphanosoma dubium D. dubium Taiwan AB549201 Direct Submission Table 2. (Continued) page 6 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan Species name in GenBank Phylogenetic group Site GenBank accession number Publication Diaphanosoma amurensis Diaphanosoma cf. amurensis D. amurensis Japan LC060047 Lakatos et al. (2015) Diaphanosoma cf. amurensis D. amurensis Japan LC060046 Lakatos et al. (2015) Diaphanosoma cf. amurensis D. amurensis Japan LC060045 Lakatos et al. (2015) Diaphanosoma cf. amurensis D. amurensis Japan LC060044 Lakatos et al. (2015) Diaphanosoma cf. amurensis D. amurensis Japan LC060043 Lakatos et al. (2015) Diaphanosoma birgei Diaphanosoma cf. birgei D. heberti Brazil KU315486 Direct Submission Diaphanosoma birgei D. birgei Mexico EU702144 Elías-Gutiérrez et al. (2008) Diaphanosoma birgei D. birgei Mexico EU702143 Elías-Gutiérrez et al. (2008) Diaphanosoma birgei D. birgei Mexico EU702142 Elías-Gutiérrez et al. (2008) Diaphanosoma birgei D. birgei Canada EU702141 Elías-Gutiérrez et al. (2008) Diaphanosoma excisum Diaphanosoma excisum D. excisum China KY788972 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788971 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788970 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788969 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788968 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788967 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788966 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788965 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788964 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788963 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788962 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788961 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788960 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788959 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788958 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788957 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788956 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788955 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788954 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788953 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788952 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788951 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788950 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788949 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788948 Liu et al. (2017) Diaphanosoma excisum D. excisum China KY788923 Liu et al. (2017) Diaphanosoma orghidani Diaphanosoma cf. orghidani D. macrophthalma China KY788942 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788883 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788870 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788869 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788868 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788867 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788866 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788865 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788852 Liu et al. (2017) Diaphanosoma cf. orghidani D. orghidani China KY788851 Liu et al. (2017) Diaphanosoma cf.. orghidani D. macrophthalma China KY788841 Liu et al. (2017) Diaphanosoma cf. orghidani D. macrophthalma China KY788840 Liu et al. (2017) Diaphanosoma cf. orghidani D. macrophthalma China KY788839 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788829 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788828 Liu et al. (2017) Table 2. (Continued) page 7 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan Species name in GenBank Phylogenetic group Site GenBank accession number Publication Diaphanosoma orghidani D. orghidani China KY788827 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788786 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788785 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788784 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788783 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788782 Liu et al. (2017) Diaphanosoma orghidani D. orghidani China KY788781 Liu et al. (2017) Diaphanosoma orghidani D. orghidani Greece MW259043 Alexiou et al. (2021) Diaphanosoma orghidani D. orghidani Greece MW259042 Alexiou et al. (2021) Diaphanosoma orghidani D. orghidani Greece MW259038 Alexiou et al. (2021) Diaphanosoma orghidani D. orghidani Greece MW259011 Alexiou et al. (2021) Diaphanosoma mongolianum Diaphanosoma mongolianum D. mongolianum China KY788836 Liu et al. (2017) Diaphanosoma mongolianum D. mongolianum China KY788835 Liu et al. (2017) Diaphanosoma mongolianum D. mongolianum China KY788834 Liu et al. (2017) Diaphanosoma mongolianum D. mongolianum China KY788837 Liu et al. (2017) Diaphanosoma mongolianum D. mongolianum Greece MW259036 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259035 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259034 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259033 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259032 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259031 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259030 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259029 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259027 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259026 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259025 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259023 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259022 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259021 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259020 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259019 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259018 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259017 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259016 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259015 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259014 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259013 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259012 Alexiou et al. (2021) Diaphanosoma mongolianum D. mongolianum Greece MW259010 Alexiou et al. (2021) Diaphanosoma brachyurum Diaphanosoma brachyurum D. brachyurum China KY788792 Liu et al. (2017) Diaphanosoma brachyurum D. brachyurum China KY788791 Liu et al. (2017) Diaphanosoma brachyurum D. brachyurum Italy MH321371 Unpublished Diaphanosoma brachyurum D. brachyurum Italy MH321370 Direct Submission Diaphanosoma brachyurum D. brachyurum Italy MH321369 Direct Submission Diaphanosoma brachyurum D. brachyurum Italy MH321368 Direct Submission Diaphanosoma brachyurum D. brachyurum Italy MH321367 Direct Submission Diaphanosoma brachyurum D. brachyurum Italy MH321366 Direct Submission Diaphanosoma brachyurum D. brachyurum Italy MH321365 Direct Submission Diaphanosoma brachyurum D. brachyurum Italy MH321364 Direct Submission Diaphanosoma brachyurum D. brachyurum Germany EF189666 Richter et al. (2007) Diaphanosoma macedonicum Diaphanosoma macedonicum D. macedonicum Greece MW259047 Alexiou et al. (2021) Diaphanosoma macedonicum D. macedonicum Greece MW259046 Alexiou et al. (2021) Diaphanosoma macedonicum D. macedonicum Greece MW259045 Alexiou et al. (2021) Table 2. (Continued) page 8 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan as indicated before, but now on the combined dataset. The best-fit model of sequence substitution (TIM1 + G) under the AICc was selected for the rest of the phylogenetic analysis performed using the concatenated matrix, repeating the same procedure as indicated before for the COI dataset. Alignments for the COI and concatenated datasets are provided in supplementary data 1 and 2. RESULTS Morphological comparison Morphologically, the specimens of D . macedonicum found in Lake Ohrid were quite similar to those from the two previously investigated lakes, Prespa and Dojran (see Korovchinsky and Petkovski 2014), differing only in minor features (Fig. 2). The former were smaller, and their integument lacked reinforced chitinization; denticles of posteroventral margin were on average more abundant. Morphological characteristics (body length, head length, diameter of the eye, swimming antennae length, and upper antennal length) of the specimens from Lake Ohrid were closer to those of Lake Prespa than to the specimens of Lake Dojran (Fig. 2). Phylogenetic and coalescence analysis Specimens retrieved from the Iberian and Balkan Peninsulas cover all the Diaphanosoma spp. cited in Europe. D. lacustris, D. mongolianum, D. brachyurum and D. orghidani were detected in the Iberian Peninsula, while D. orghidani, D. macedonicum and D. mongolianum were found in the Balkan Peninsula. Sequences of Diaphanosoma specimens recovered from the ancient lakes Ohrid and Prespa were grouped together with the only available sequences of D. macedonicum from Mikri Prespa (Alexiou et al. 2021). Molecular data from these three locations together formed the D. macedonicum clade in our phylogenetic tree (Fig. 3). In Lake Dojran, although cited in the scientific literature (Alexiou et al. 2021; Korovchinsky and Petkovski 2014), we have not found specimens of D. macedonicum but instead of D. mongolianum. Mean ingroup K2P genetic distances for D. macedonicum are 2.36 ± 0.31%, while the K2P distances between the closest groups, i.e., D. lacustris and D. mongolianum, reached 6.9 ± 0.11% and 8.4 ± 0.15%, respectively (Table 3). The phylogenetic analysis presented in figure 3 showed that D. macedonicum, together with D. lacustris and D. mongolianum, is part of the mongolianum group. The term mongolianum group is used hereinafter to describe the assemblage formed by three species: D. lacustris, D. mongolianum, and D. macedonicum. However, the phylogenetic position of D. macedonicum within this group was not well resolved and/or denotes discrepancies between the Bayesian and maximum likelihood methods when using only COI markers (Fig. 3). These differences vanished when using the concatenated dataset, as shown in figure 4. Here, there are no discrepancies between the two methods, and the level of support is higher in both the Bayesian (MrBayes posterior probability; pp = 0.99 for D. macedonicum; pp = 1.00 for the mongolianum group) and maximum likelihood methods (ML bootstrap values, bs = 86% for D. macedonicum and bs = 99% for the mongolianum group). The distinction between the species of interest and others within the genus is further corroborated by the genetic distances presented in table 3. DISCUSSION Identification and phylogenetic position The molecular results presented here indicate that the Diaphanosoma collected in the ancient lakes Ohrid and Prespa belong to the endemic species of the Balkan peninsula, D. macedonicum. This is confirmed by short genetic distances (COI: K2P < 3%) in comparison to the only available sequences of this taxon (Alexiou et al. 2021) and by the larger genetic differentiation (COI: K2P > 6%) with sister groups, such as D. lacustris and D. mongolianum. These small K2P distances are expected for the genus since Dumont et al. (2021) also found relatively small intraspecific genetic distances, mostly less than 2%, within temperate climate species of Diaphanosoma. Besides the identity of Diaphanosoma in Ohrid and Prespa, our phylogenetic analysis (Figs. 3, 4) supported previous morphological evidence (Korovchinsky and Petkovski 2014) that considered D. macedonicum as a member of the D. mongolianum species group, comprising three sibling species (D. mongolianum, D. lacustris, and D. macedonicum). Molecular results indicate that D. macedonicum is restricted to the pelagic zones of lakes Ohrid, Prespa, and Mikri Prespa (Fig. 4). The sampled populations in lower-altitude lakes in the Balkans, including Lake Dojran, belong to D. mongolianum (Fig. 3). However, D. macedonicum was reported from Lake Dojran (Alexiou et al. 2021; Korovchinsky and Petkovski 2014) based on morphological identification. Figure 2 shows morphological differences (body length, head length, diameter of the eye, swimming antennae length and upper antennal length) between the Ohrid, page 9 of 16Zoological Studies 63: 9 (2024) © 2024 Academia Sinica, Taiwan et Petkovski, 2014 (Crustacea: Cladocera: Sididae) in Lake Ohrid. Arthropoda Sel 31:179–182. doi:10.15298/arthsel.31.2.06. Korovchinsky NM. 2018. Cladocera: Ctenopoda, Families Sididae, Holopediidae & Pseudopenilidae (Branchiopoda: Cladocera). In: Identification guides to the plankton and benthos of inland waters. Backhuys Publ. & Margraf Publ., The Netherlands. Korovchinsky NM, Petkovski KT. 2014. 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Adaptations to the hyporheic in Aloninae (Crustacea: Cladocera): Allocation of Alona protzi Hartwig, 1900 and related species to Phreatalona gen. nov. Hydrobiologia 618:1–34. doi:10.1007/s10750-0089607-6. Zhang Z, Schwartz S, Wagner L, Webb M. 2000. A greedy algorithm for aligning DNA sequences. J Comput Biol 7:1–2. doi: 10.1089/10665270050081478. Supplementary materials Supplementary data 1. Alignment COI dataset. (download) Supplementary data 2. Alignment concatenated dataset. (download) page 16 of 16Zoological Studies 63: 9 (2024)