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Phylogenetic analysis of problematic Asian species of Artemia Leach, 1819 (Crustacea, Anostraca), with the descriptions of two new species

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We tested hypotheses of whether populations from the Tibetan Plateau belong to A. tibetiana Abatzopoulos, Zhang & Sorgeloos,1998 and whether a population from Kazakhstan is a new species, using other Asian species of Artemia as outgroups. We conducted a multitrait phylogenetic study based on the complete mitogenome, mitochondrial (COI, 12S, 16S) and nuclear (microsatellites, ITS1) markers, and a suit of uni- and multivariate morphological traits. Our results led to the discovery of two new species: 1) Artemia sorgeloosi: from the Tibetan Plateau in China 2) Artemia amati: from Kazakhstan
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[Research article: edited R2 JCB-2613] 1
DOI: 10.1093/jcbiol/ruad002 2
Running head: ASEM ET AL.: ASIAN ARTEMIA PHYLOGENETICS 3
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Version of Record, first published online, with fixed content and layout in compliance with Art. 5
8.1.3.2 ICZN. LSID: urn:lsid:zoobank.org:pub: D5D5D6AE-67CA-4A65-85B7-54F65243270D 6
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Phylogenetic analysis of problematic Asian species of Artemia Leach, 8
1819 (Crustacea, Anostraca), with the descriptions of two new 9
species 10
11
Alireza Asem1, Chaojie Yang1, Amin Eimanifar2, Francisco Hontoria3, Inmaculada 12
Varó3, Farnaz Mahmoudi4, Chun-Zheng Fu5, Chun-Yang Shen6, Nasrullah 13
Rastegar-Pouyani7, Pei-Zheng Wang8, Weidong Li9, Liping Yao1, Xinyu Meng10, 14
Ya-Ting Dan11, D. Christopher Rogers12 and Gonzalo Gajardo13 15
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1Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources, 17
Hainan Tropical Ocean University, Sanya 572000, China 18
2Independent Senior Scientist, Industrial District, Gaithersburg, MD 20878, USA 19
3Instituto de Acuicultura de Torre de la Sal (IATS, CSIC), 12595 Ribera de Cabanes (Castellón), 20
Spain 21
4Key Laboratory of Utilization and Conservation for Tropical Marine Bioresources of Ministry 22
of Education, Hainan Tropical Ocean University, Sanya 572000, China 23
2
5Institute of Sericulture, Chengde Medical University, Chengde 067000, China 1
6Department of Biology, Chengde Medical University, Chengde 067000, China 2
7Department of Biology, Faculty of Science, Razi University, 6714967346 Kermanshah, Iran 3
8Key Laboratory for Coastal Marine Eco-environment Process and Carbon Sink of Hainan 4
Province, Hainan Tropical Ocean University, Yazhou Bay Innovation Institute, Sanya 572000, 5
China 6
9College of Ecology and Environment, Hainan Tropical Ocean University, Haikou 570000, Chin 7
10College of Life Sciences, Yan'an University, Yan'an 716000, China 8
11College of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China 9
12Kansas Biological Survey, and the Biodiversity Institute, The University of Kansas, Lawrence, 10
KS 66047-3759, USA 11
13Departamento de Ciencias Biológicas y Biodiversidad, Universidad de Los Lagos, Osorno 12
5290000, Chile 13
14
Corresponding authors: A. Asem: e-mail: asem.alireza@gmail.com; A. Eimanifar: 15
e-mail: amineimanifar1979@gmail.com; F. Hontoria: e-mail: hontoria@iats.csic.es; D.C. Rogers: 16
e-mail: branchiopod@gmail.com; G. Gajardo: e-mail: ggajardo@ulagos.cl 17
First authors: A. Asem; C. Yang 18
ABSTRACT 19
Species of Artemia are regionally endemic branchiopod crustaceans composed of sexual species 20
and parthenogenetic lineages, and represent an excellent model for studying adaptation and 21
speciation to extreme and heterogeneous hypersaline environments. We tested hypotheses of 22
whether populations from the Tibetan Plateau belong to A. tibetiana Abatzopoulos, Zhang & 23
Sorgeloos,1998 and whether a population from Kazakhstan is a new species, using other Asian 24
species of Artemia as outgroups. We conducted a multitrait phylogenetic study based on the 25
complete mitogenome, mitochondrial (COI, 12S, 16S) and nuclear (microsatellites, ITS1) 26
markers, and a suit of uni- and multivariate morphological traits. Our results led to the discovery 27
3
of two new species, one from the Tibetan Plateau (Haiyan Lake) in China (Artemia sorgeloosi n. 1
sp.) and a second from Kazakhstan (Artemia amati n. sp.). Our analysis demonstrate that A. 2
tibetiana and A. amati n. sp. are monophyletic, whereas A. sorgeloosi n. sp., and A. tibetiana are 3
polyphyletic. Evolutionary relationships based on mitochondrial and nSSR markers suggest that 4
A. tibetiana may have arisen from a past hybridization event of a maternal ancestor of A. 5
tibetiana with A. sorgeloosi n. sp. or its ancestor. We present the complete mitogenome of A. 6
tibetiana, A. amati n. sp., and A. sorgeloosi n. sp. We also provide a novel taxonomic 7
identification key based on morphology, emphasizing the phenotype as a necessary component 8
of the species concept. 9
10
KEY WORDS: brine shrimps, Crustacea, molecular systematics, mitogenome, morphology, 11
morphometry, speciation, taxonomy 12
13
INTRODUCTION 14
Artemia Leach, 1819 is a genus of cosmopolitan, zooplankton branchiopod crustacean 15
distributed worldwide (except Antarctica) in hypersaline habitats in arid and semi-arid areas 16
(Van Stappen, 2002).The species of Artemia are considered a model to study adaptation and 17
speciation to extreme hypersaline conditions. The genus contains regionally endemic sexual 18
species and many obligate parthenogenetic lineages (Gajardo et al., 2002; Asem et al., 2010, 19
2016). Three species inhabit the Americas (North, Central, and South), including Artemia 20
monica Verrill, 1869, restricted to Mono Lake in USA, A. franciscana Kellogg, 1906, the most 21
widely distributed, and A. persimilis Piccinelli & Prosdocimi, 1968, found in Chile and 22
Argentina, separated from A. franciscana by a latitudinal barrier (see Gajardo et al., 2002, 2004). 23
4
The other four species are native to the Old World: A. salina (Linnaeus, 1758) in the 1
Mediterranean region, A. sinica Cai, 1989 in China, A. urmiana Günther, 1899 in Lake Urmia 2
(Iran) and the Crimean Peninsula, and A. tibetiana Abatzopoulos, Zhang & Sorgeloos, 1998, in 3
the Tibetan or Qinghai-Tibet Plateau. Artemia franciscana has been anthropogenically 4
introduced in Eurasia and Australia (Zheng et al., 2004; Amat et al., 2005; Abatzopoulos et al., 5
2009; Scalone & Rabet, 2013; Eimanifar et al., 2014, 2020; Asem et al., 2018, 2021a; Saji et al., 6
2019; Shen et al., 2021). Obligate parthenogenetic linegaes widely distributed in Eurasia, Africa, 7
and Australia consist of different ploidy (di-, tri-, tetra-, penta- and heteroploids) (Sun et al., 8
1999; Asem & Sun 2014a, b; Asem et al., 2016). 9
Sainz-Escudero et al. (2021) arguably suggested that A. franciscana and A. monica could 10
be synonymous, which contradicts the observation that they do not share haplotypes (see Muñoz 11
et al., 2013), a sign of the absence of gene exchange or reproductive isolation, the “acid test” of 12
the biological species concept (Mayr, 1942). The concept states that populations within a species 13
share a gene pool but are reproductively isolated from populations of other species (see Mayr & 14
Ashlock, 1991; Queiroz, 2005). A sign of such differentiation is provided by the different eggs of 15
A. franciscana and A. monica. Artemia franciscana has typical, smooth eggs, whereas the eggs 16
have discoidal projections and a network of ridges in A. monica (see images in Shepard & Hill, 17
2001). Thus, the synonymizing of A. franciscana and A. monica is still an open question in need 18
of further study. 19
The divergence of Artemia is a complex problem due to habitat isolation and the ionic 20
variation of brines that exert selective local pressures; thus, adaptation to these conditions occurs 21
at different functional levels, from the individual (molecular-cellular-physiological, 22
morphological) to the population level (Gajardo & Beardmore 2012). Additional complexity in 23
5
delimiting species is due to the co-occurrence in certain places of sexual species and 1
parthenogenetic lineages and the gross morphological similarity among them (Asem et al., 2010; 2
Rogers & Hann, 2016; Rogers et al., 2019, 2020). Thus, multiple tools, including nuclear and 3
mitochondrial DNA markers, biometric and morphological characters, electrophoretic isozyme 4
patterns, and the morphological patterns of the frontal knob, gonopod, brood pouch, and 5
cercopods have therefore been used for the classification of different populations and species 6
(Hontoria & Amat, 1992; Pilla & Beardmore, 1994; Amat et al., 1995, 2005; Gajardo et al., 1995, 7
2004; Baxevanis et al., 2006; Hou et al., 2006; Zheng & Sun, 2008; Abatzopoulos et al., 2009; 8
Kappas et al., 2009; Eimanifar, et al., 2014; Asem et al., 2016; Asem & Sun, 2016; Rogers & 9
Hann, 2016; Rogers et al., 2019, 2020). Some situations in Asia, however, have remained 10
unresolved, particularly in Mongolia, Kazakhstan, and the Tibetan Plateau, where some isolated 11
populations appear divergent from the reported regional species. An example of this situation is 12
the questionable proposition of two new species in Mongolia, A. murae Naganawa & Mura, 2017 13
and A. frameshifta Naganawa & Mura, 2017, especially as A. murae is a parthenogenetic lineage 14
that does not fit the binomial specific nomenclature (see Abatzopoulos et al., 2002a; Baxevanis 15
et al., 2006; Asem et al., 2010; Maniatsi et al., 2011). Similarly, A. frameshifta was described 16
based on a single gene sequence (GenBank accession no. LC195588) of the mitochondrial 17
cytochrome oxidase subunit I (COI) gene without any proper taxonomic discussion. The primary 18
complication of the submitted COI sequence of A. frameshifta (LC195588) is the presence of 19
several stop codons (indicating that the sequence is a NUMT), which in this case, renders the 20
sequence used unreliable. Artemia frameshifta is therefore a nomen dubium (Asem et al., 2018, 21
2020, 2022; Eimanifar et al., 2020). Further studies show that the Mongolian populations 22
resemble A. sinica (unpublished data; S. Sun, personal communication, 2019). 23
6
A batch of sexual Artemia eggs from Kazakhstan was collected in 1988 by the Catvis 1
Company (s-Hertogenbosch, The Netherlands), but the origin(s) of the sample remains unknown 2
(Pilla & Beardmore, 1994; Ben Cattel, personal communication, 2017). A comprehensive 3
genetic (allozymes) and morphometric study of sexual Artemia from the Eastern Old World (A. 4
sinica, A. urmiana) found the Artemia from Kazakhstan to be an independent lineage named 5
Artemia sp.” (Pilla & Beardmore, 1994). Studies on the biological characteristics of this 6
population (Triantaphyllidis et al., 1997a; Maccari et al., 2013a) and the mitochondrial COI-7
based phylogenetic analyses demonstrate that this taxon is a separated clade of the Asian species 8
group (Muñoz et al., 2010; Maccari et al., 2013a). Additional surveys have only found 9
parthenogenetic lineages across Kazakhstan (Eimanifar et al., 2014). Although the original 10
collection site for the Kazakhstan Artemia sp. is unknown, the species can still be described (e.g., 11
Thomas & Bek, 2014; Fujita, 2016; Wickert et al., 2016; Wu & Liu, 2016). 12
Abatzopoulos et al. (1998) described A. tibetiana from Lagkor Co (= Lagkor Lake, Tibet, 13
China) without further locality information for the type material. Syntypes most likely containing 14
eggs (two samples collected at different times), nauplii, and adults were identified. Wang et al. 15
(2008) studied the phylogenetic relationships of four Tibetan populations using mitochondrial 16
COI markers and found two clades, one corresponding to the A. tibetiana type locality (Lagkor 17
Co), the other containing collections from various Tibetan sites. Additional COI-based studies 18
also separated the Tibetan populations into two clades, whereas all populations cluster together 19
using the nuclear marker ITS1 (Wang et al., 2008; Maccari et al., 2013a; Eimanifar et al., 2014; 20
Asem et al., 2020). All A. tibetiana populations show inconsistent topologies depending on the 21
markers used (Maccari et al., 2013a; Eimanifar et al., 2014, Asem et al., 2020). 22
We unravel herein the taxonomy and systematics of the undescribed populations from 23
7
Kazakhstan and Tibet, using multiple mitochondrial markers (COI, 16S, 12S), including the 1
complete mitochondrial genome, the nuclear marker ITS1, and microsatellite (SSR) genomic 2
fingerprint analysis, together with morphological and morphometric characters. 3
4
MATERIALS AND METHODS 5
Sampling 6
All examined material is listed in Table 1 and Figure 1. Samples of Artemia eggs were obtained 7
from the type localities of the different Asian species and/or institutional collections. We 8
examined undescribed bisexual populations from Kazakhstan (unknown locality) and Haiyan 9
Lake, China (see Maccari et al., 2013a). We used the American A. franciscana as an outgroup, 10
our material ultimately originating from the type locality of the species. 11
<Table 1> 12
<Fig. 1> 13
Eggs from each locality were cultured under optimal conditions (see Hontoria & Amat, 14
1992). During the molecular study, we found that the Kazakhstan and Haiyan Lake samples were 15
a mixture of bisexual and parthenogenetic individuals by cloning parthenogenetic specimens for 16
four generations. Females were used in the molecular analysis after confirmation of the sexual-17
reproduction mode. 18
19
Mitogenomic analyses 20
Total genomic DNA was extracted from Artemia females from the two Tibetan populations 21
(Lagkor Co and Haiyan Lake) and a female from the Kazakhstan population. Extractions were 22
conducted using the Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, 23
8
China; no. B518221) and following the method provided in Asem et al. (2019a). The quantity of 1
extracted DNA was checked in a Microvolume Spectrophotometer (MaestroGen Inc., Hsinchu 2
City, Taiwan). For each specimen, paired-end (2×150 bp) genomic libraries were prepared with 3
the NEBNext® UltraTM II DNA library preparation kit (New England Biolabs, Ipswich, MA, 4
USA), using the next-generation sequencing (> 10Gb) approach. The sequencing was performed 5
by a single constructed library, pooled on Illumina HiSeq X-ten sequencing flow cell (Novogene, 6
Tianjin, China). 7
Sequence quality control and assembly followed Asem et al., (2021b). Sequencing 8
information is included in Supplementary material Table S1. The Artemia sinica mitogenome 9
served as a reference sequence (MK069595). Programs Geneious R9.1 (Kearse et al., 2012), 10
with default parameters (www.geneious.com) and bowtie v2.2.9 (Langmead & Salzberg, 2012), 11
with “-L 4 -a -m 20 -v 1 -very-sensitive” setting, were used to assemble and map the sequence 12
reads. To check mitogenome sequences validity, each mitogenome was assembled twice, and 13
referred to two different positions of the reference genome with ~5,000bp difference. In addition, 14
the position of each gene was aligned with the reference genome to compare non-match 15
sequences. Our results confirm that all assembled mitogenomes are complete. 16
Phylogenetic analyses considered concatenated datasets of three mitogenomes from the 17
present study and two others from GenBank (GenBank accession no. MK069595, Asem et al., 18
2019a) and A. urmiana (GenBank accession no. MN240408, Asem et al., 2021b), which consist 19
of two ribosomal RNAs (rRNAs) and 13 protein-coding genes (PCGs). The analysis considered 20
maximum likelihood (ML) in MEGA X (Kumar et al., 2018) and Bayesian inference (BI) as 21
implemented in MrBayes 3.2.2 on XSEDE (Miller et al., 2010). Artemia franciscana (GenBank 22
accession no. NC_001620; Perez et al., 1994) was chosen as an outgroup for Asian Artemia 23
9
(Maccari et al., 2013a). For both ML and BI the best fitting nucleotide substitution model of 1
DNA was chosen based on MrModeltest 2.2 (Nylander, 2004). HKY+G was selected as the best-2
fit model (ML: bootstrap replicates: 1,000, maximum parsimony analyses were run using TNT 3
(nearest-neighbor-interchange); BI: mcmcp ngen = 10,000,000, samplefreq = 100, nchains = 4, 4
sump burnin = 25,000). The trees were visualized using FigTree v 1.4.0 (Rambaut, 2012). For 5
the ML bootstraps, values < 70 were regarded as low, 70‒94 as moderate, and ≥ 95 as high 6
(Hillis & Bull, 1993). For the BI posterior probabilities, the values < 0.94 were considered low 7
and ≥ 0.95 as high (Alfaro et al., 2003). 8
9
Molecular analyses 10
DNA extraction. The extraction of the total genomic female DNA followed the Chelex® 100 11
Resin method (Bio-Rad Laboratories, Hercules, CA, USA). The specimens were crushed with a 12
sterilized pipette tip, incubated for 2.5–3 h at 60 °C (tubes were shaken by vortex every 30 min), 13
and eventually for 10 min at 80 °C, and centrifuged at 10,000 rpm for 1 min; the supernatant 14
phase was directly used in the PCR reaction (Asem et al., 2016). All DNAs were stored at –15
80 °C for further genetic analyses. 16
PCR amplification. Three fragments of mitochondrial cytochrome oxidase subunit 1 (COI), 16S 17
ribosomal RNA (16S), and 12S ribosomal RNA (12S) markers, plus the complete sequences of 18
the nuclear internal transcribed spacer 1 (ITS1) marker were amplified. We performed PCRs out 19
in a total volume of 15 μl containing 6 μl of ddH2O, 7.5 μl Taq polymerase (2×TsingKeTM 20
Master Mix, Cat.TSE004; TsingKe Biotechnology, Beijing, China), 0.3 μl DNA template and 21
0.6 μl of each primer. 22
Amplifying of the partial fragment of the mitochondrial COI gene was performed using the 23
10
invertebrate universal primers LCOI490/HC02198 (Folmer et al., 1994). PCR amplification 1
considering the following cycles: 3 min at 95 °C, 35 cycles of one min at 95 °C, 1 min at 40 °C, 2
and 1.5 min at 72 °C, with a final step of 7 min at 72 °C. 3
Analysis of the mitochondrial 16S Ribosome RNA (16S) and 12S Ribosome RNA (12S) 4
fragments considered primers LR-N, LR-J (Yin et al., 2011) and 12S-F/12S-R (Machida et al., 5
2012), respectively. We performed PCR amplification under the following conditions: a cycle of 6
5 min at 94 °C, 35 cycles of 10 s at 94 °C, 30 s at 55–58°C, and 40 s at 72 °C, with a final step of 7
10 min at 72 °C. 8
The complete sequence of the nuclear ITS1 was amplified using the primers 18d-5ˊ/R58 9
(Baxevanis et al., 2006). The thermal cycler PCR conditions were as follows: 4 min at 93 °C, 32 10
cycles of 40 s at 93 °C, 40 s at 62 °C, 1 min at 72 °C, and a final extension of 5 min at 72 °C. 11
Table 2 provides details of the molecular analysis and sequences analyzed for each marker. 12
<Table 2> 13
14
Sequence alignment and population genetic analyses. We aligned sequences using MEGA X 15
with default parameters (Kumar et al., 2018). The genealogical relationships among haplotypes 16
considered a median network analysis using the median-joining algorithm (Bandelt et al., 1999) 17
available in PopART (http://popart.otago.ac.nz). 18
The overall distance of each marker for populations and between groups mean distances 19
used p-distance available in MEGA X (Kumar et al., 2018). The number of polymorphic sites (S), 20
the total number of mutations (Eta), number of haplotypes (h), haplotype diversity (Hd), 21
haplotype ratio (Hr), nucleotide diversity (Pi), the average number of nucleotide differences (k), 22
and neutrality test (Tajima D) were considered using DnaSP v.5.10 (Librado & Rozas, 2009). 23
11
1
SSR analyses. Nine microsatellite loci were selected for genotyping of the studied Artemia 2
populations (Nougué et al., 2015). A list of labeled primers and further information is provided 3
in Supplementary material Table S2. PCRs were performed out in a total volume of 20 μl 4
containing 17 μl Taq polymerase (2×T5 Super PCR Mix, cat.TSE006; TsingKe Biotechnology, 1 5
μl template DNA, and 1 μl of each primer. The thermal cycler PCR conditions were as follows: 6
98 °C for 2 min, 35 cycles of 10 s at 98 °C, 10 s at TM °C, 10 s at 72 °C, and a final elongation 7
phase at 72 °C for 50 min. Supplementary material Table S2 provides details of TM listed for 8
each pair-primer. 9
Genetic variation was evaluated by the number of different alleles (Na), the number of 10
effective alleles (Ne), Shannon's information index (I), observed heterozygosity (Ho), expected 11
heterozygosity (He), unbiased expected heterozygosity (uHe), fixation index (F), polymorphic 12
loci (PL), and pairwise population matrix of Nei’s genetic distance using GenAlEx 6.5 (Peakall 13
& Smouse, 2012). Additionally, GenAlEx was considered for the spatial distribution of 14
populations in the principal coordinate analysis (PCoA). To avoid the misleading effect of 15
convergence of uninformative loci (Nougué et al., 2015; Arbeiter et al., 2017; Hilmarsson et al., 16
2017; Grijalba et al., 2020; Frisch et al., 2021), we considered Aupm21, Appm4 and Appm5 as 17
uninformative loci (see Tab. 6) for Tibetiana populations. Thus, interspecific differentiation of 18
Tibetan populations was separately reanalyzed using PCoA based on five loci. 19
20
Morphological analyses 21
We observed and measured morphometrical differences between the Asian Artemia (see 22
Fig. 1). using specimens cultured under laboratory conditions (Amat & Hontoria, 1992). Nauplii 23
12
were cultured at a salinity of 70 ppt, 24 ± 1 °C, 12 h light/12 h dark photoperiod, and fed with a 1
mixture (1:1) of the microalgae Dunaliella salina and Tetraselmis suecica. Specific 2
measurements (defined in Hontoria & Amat, 1992) of randomly selected adults (30 females and 3
30 males from each population) were made under a dissecting microscope. In males, the width of 4
the genital segment at the widest part from ventral view was measured instead of the brood 5
pouch width. Discriminant-function analysis was used to check the morphometrical 6
differentiation among females and males. The computer program SPSS 28 7
(https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-28010) was used to 8
perform the statistical analysis. 9
In order to characterize some key features, we examined and photographed three or four 10
specimens focusing on the detailed morphologies of the frontal knob, gonopod, and brood pouch 11
using a scanning electronic microscope (SEM). After washing the samples with phosphate buffer 12
and redistilled water, they were dehydrated in 30, 50, 80, 90, and 100% ethanol at 30 min 13
intervals. Afterwards, samples were transferred into isoamyl acetate:ethanol (1:3, 1:2, and 1:0 for 14
30 min, respectively). After being critical point dried in CO2 and covered by gold, specimens 15
were observed and photographed with a KYKY-2800B SEM (KYKY Technology, Beijing, 16
China) (see Asem & Sun, 2016). Forty individuals (males and females separately) of each 17
population were randomly examined for inter- and intra-specific phenotypic variations of 18
head/frontal knob (in males), brood pouch (in females) and cercopods (in males and females) 19
using a ZEISS Stemi 508 stereomicroscope (Carl Zeiss, Oberkochen, Germany). 20
21
TAXONOMY 22
Phylum Arthropoda von Siebold, 1848 23
13
Subphylum Crustacea Brünnich, 1772 1
Class Branchiopoda Latreille, 1817 2
Order Anostraca Sars, 1867 3
Family Artemiidae Grochowski, 1896 4
Genus Artemia Leach, 1819 5
6
Artemia sorgeloosi Asem, Eimanifar, Hontoria, Rogers & Gajardo n. sp. 7
(Fig. 2) 8
<Fig. 2> 9
Etymology: In honor of Prof. Patrick Sorgeloos (Ghent University, Ghent, Belgium), for his 10
contribution to promote Artemia research, and for training generations of young scientists from 11
around the world. 12
Type material: Holotype: male (NACRCCAS ANO_260), paratypes: five males 13
(NACRCCAS ANO_261-NACRCCAS ANO_265) and five females (NACRCCAS ANO_266-14
NACRCCAS ANO_270). Type specimens were deposited at the National Zoological Museum of 15
China, Institute of Zoology, Chinese Academy of Science (IZCAS). 16
Type locality: Haiyan Lake, Qinghai, China (36°48'N, 100°41'E) 17
Diagnosis: Species separated from other Asian species by triangular brood pouch with lateral 18
lobes in female (see Fig. 15a). Frontal knob with dense spines distributed on lateral and ventral 19
axes, medial axis glabrous (Fig. 15c). 20
Description: Female. Mean body length (head anterior margin to telson posterior margin) 12.16 21
± 0.84 mm. Brood pouch triangular with lateral lobes (see Fig. 15a), average width 2.21 ± 0.19 22
mm. Cercopod lobed/polysetate (see Fig. 16), average number of setae of each lobe ~10. Male. 23
14
Mean body length 10.43 ± 0.44 mm. Gonopod basal spine present (Fig. 15b), genital segment 1
width 0.85 ± 0.05 mm. Frontal knob subspherical, with dense spines distributed across ventral 2
and lateral axes, spines arranged as single, twins, and triplets with homogeneous distribution 3
along lateral axis, single spines predominating and rare twin and triplet spines on distal axis, with 4
medial axis glabrous (Fig. 15c). Cercopod lobed/polysetae (Fig. 16), average number of setae per 5
lobe ~12 (measurements in Supplementary material Tables S10, S11). 6
DNA sequences: COI: sequences (GenBank accession nos. MZ189919–953), 16S: sequences 7
(GenBank accession nos. MZ168838–872), 12S: sequences (GenBank accession nos. 8
MZ169003–037), ITS1: sequences (GenBank accession nos. MW995301–330), mitogenome: 9
sequence (GenBank accession no. OP156999). 10
11
Artemia amati Asem, Eimanifar, Hontoria, Rogers & Gajardo n. sp. 12
(Fig. 3) 13
<Fig. 3> 14
Etymology: In honor of Prof. Francisco Amat (Torre de la Sal Aquaculture Institute, Ribera de 15
Cabanes, Spain) for his valuable contributions in the field of Artemia biology. 16
Type material: holotype: male (NACRCCAS ANO_271), paratypes: five males 17
(NACRCCAS ANO_272-NACRCCAS ANO_276) and five females (NACRCCAS ANO_277-18
NACRCCAS ANO_282). Type specimens are deposited at National Zoological Museum of 19
China, Institute of Zoology, Chinese Academy of Science (IZCAS). 20
Type locality: Kazakhstan (48°N, 68°E), exact locality unknown. 21
Diagnosis: Species separated from Asian congeners by rounded-ellipsoidal brood pouch with 22
lateral lobes in female (Fig. 15a). Frontal knob with extremely sparse spine distribution along 23
15
lateral and ventral axes, with medial axis glabrous (Fig. 15c). 1
Description: Female. Mean body length 9.91 ± 0.52 mm. Brood pouch rounded-ellipsoidal with 2
lateral lobes (Fig. 15a) and mean width 1.77 ± 0.12 mm. Cercopod lobed/polysetae (Fig. 16), 3
mean number of setae of each lobe ~14. Male. Mean body length 9.98 ± 1.06 mm. Gonopod 4
basal spine present (Fig. 15b), genital segment width 0.92 ± 0.11 mm. Frontal knob subspherical 5
shape with sparse spine distribution: single and twin spines toward lateral axis, triplet and twin 6
spines on ventral axis, with medial axis glabrous (Fig. 15c). Cercopod lobed/polysetate (Fig. 16), 7
mean number of setae per lobe ~17 (measurements in Supplementary material Tables S10, S11). 8
DNA sequences: COI: sequences (GenBank accession nos. MZ189884–918), 16S: sequences 9
(GenBank accession nos. MZ1688038–837), 12S: sequences (GenBank accession nos. 10
MZ168968–002), ITS1: sequences (GenBank accession nos. MW995271–300), mitogenome: 11
sequence (GenBank accession no. OP142420). 12
13
Mitogenomic analyses 14
The NGS-assembled fragments of the complete mitochondrial genome (mitogenome) of samples 15
from A. tibetiana, A. amati n. sp., and A. sorgeloosi n. sp. have a typical circular DNA with 13 16
PCGs, 2 rRNAs, and 22 tRNAs and a control region (CR), with a total length of 15,626 bp 17
(GenBank accession no. OP168928), 15,679 bp (GenBank accession no. OP142420), and 15,803 18
bp (GenBank accession no. OP156999), respectively (Supplementary material Tables S3–S7). 19
The nucleotide compositions of concatenated sequences of PCGs and rRNAs 20
(PCGs+rRNAs) are shown in Table 3. Although A. sinica shows lower G+C content (36.09%) 21
than other Asian species (including A. urmiana 38.07, A. tibetiana 38.25, A. amati n. sp. 38.09, 22
A. sorgeloosi n. sp. 37.98), GC-skew has similar values among Asian Artemia. The distributional 23
16
pattern of the studied Artemia based on G+C/A+T and GC/AT-skew values of PCG+rRNA 1
sequences are displayed on scatter plots in Figure 4. The result of G+C/A+T shows the evident 2
dissimilarity of SIN and other taxa. By contrast, GC/AT-skew showed a homogeneous 3
distribution between Asian Artemia. 4
<Table 3> 5
<Fig. 4> 6
Both ML and BI methods yielded concordant topologies. According to the phylogenetic 7
analysis, each Asian Artemia species represents a distinctive clade, A. sinica being a basal clade 8
(Fig. 5a). The haplotype distribution pattern of mitogenome is shown in Figure 5b. The 9
phylogenetic tree and haplotype distribution network demonstrate that Tibetan Artemia (A. 10
tibetiana and A. sorgeloosi n. sp.) are placed into two distinct maternal lineages. 11
<Fig. 5> 12
The genetic distances among Artemia mitogenomes (PCG+rRNA genes) are reported in 13
Figure 5c, in which the lowest and highest distances, respectively, correspond to A. tibetiana-A. 14
amati n. sp. (0.0068) and A. tibetiana-A. sinica (0.1788). Additionally, the genetic distance 15
values between Tibetan Artemia (A. tibetiana-A. sorgeloosi n. sp.) was 0.0897. 16
17
Molecular analyses 18
Population genetic structure. The haplotype network distribution analyses based on the three 19
mitochondrial markers (COI, 16S, and 12S), demonstrate no shared haplotype among species. 20
Thus, every species is recognized by a unique haplotype for each marker (Figs. 6–8). The ITS1 21
nuclear exhibits shared haplotypes between A. urmiana-A. amati n. sp. (2/30–12/30 sequences; 22
23%) and A. tibetiana-A. sorgeloosi n. sp. (21/30–11/30 sequences; 53%). Instead, A. sinica has 23
17
private ITS1 haplotypes (Fig. 9). 1
<Figs. 6, 7, 8, 9> 2
Results of the heat-map values for overall genetic distance based on mitochondrial and 3
nuclear markers and between species for each marker are presented in Figures 10 and 11. 4
Artemia urmiana and A. sorgeloosi n. sp. show the highest overall distances in all three 5
examined mitochondrial markers (COI 0.007; 16S 0.008; 12S 0.010) and the nuclear marker 6
(ITS1 0.007), respectively. For each marker, the lowest 12S distance is shared between Artemia 7
sinica, A. sorgeloosi n. sp., A. amati n. sp., and A. tibetiana (0.004). Regarding COI, 16S, and 8
ITS1, the lowest distances correspond to A. tibetiana (0.001), A. amati n. sp. (0.001), and A. 9
tibetiana (0.001), respectively. The highest genetic distances, considering all markers, are 10
observed among A. sinica and other species, whereas the lowest distances for each marker was 11
between A. amati n. sp. and A. tibetiana (COI 0.0070; 16S 0.0038; 12S 0.0091; ITS1 0.0029). 12
<Figs. 10, 11> 13
The computed genetic indices based on four molecular markers are shown in Table 4. The 14
highest and lowest genetic variations were observed in A. urmiana and A. tibetiana/A. amati n. 15
sp., respectively. Our results reveal negative and significant Tajima's D values for all markers in 16
A. urmiana, whereas it is negative and non-significant for A. sinica. Values are negative and 17
significant for both A. amati n. sp. and A. sorgeloosi n. sp., except for the nuclear ITS1. Artemia 18
tibetiana only exhibits significant D values, except for the mitochondrial 12S. 19
<Table 4> 20
21
SSR genomic fingerprint analysis. The principal coordinate analysis (PCoA) given in Figure 12 22
shows the two first coordinates that explain 33.95% and 20.07% of the overall variance on the 23
18
species level, respectively. The analysis shows four clusters corresponding to A. urmiana, A. 1
sinica, A. amati n. sp., and the two Tibetan species (A. tibetiana and A. sorgeloosi n. sp.). The 2
highest differentiation is between A. urmiana-A. sinica (first coordinate) and the lowest is 3
between A. tibetiana and A. sorgeloosi n. sp. Artemia tibetiana and A. sorgeloosi n. sp., however, 4
exhibit high divergence as expressed by the non-overlapping area (A. tibetiana 72% and A. 5
sorgeloosi n. sp. 76%) regarding the second coordinate (Fig. 12b). The Nei’s genetic distance 6
also confirms the results of PCoA (Fig. 13). The Tibetan species (A. tibetiana and A. sorgeloosi 7
n. sp.) were separately reanalyzed regarding five informative polymorphic loci, and results show 8
they belong to independent clusters (Figure 12C). 9
<Figs. 12, 13> 10
The calculated genetic indices based on nine microsatellite loci are summarized in Table 5. 11
The highest and lowest total genetic variations were observed in A. urmiana and A. tibetiana, 12
respectively. Significant differences were detected in number of alleles and their size ranges in 13
Locus Aupm16 between the Tibetan species, A. tibetiana (NA 2; 122–130) and A. sorgeloosi n. 14
sp. (NA 12; 118–158). Additionally, Locus Aupm21 is absent in the East Asian Artemia (A. 15
sinica, A. tibetiana, and A. sorgeloosi n. sp.) (Table 6). 16
<Tables 5, 6> 17
18
Morphological analyses 19
The centroids and observations of the different species in females and males are plotted in Figure 20
14 for the two first discriminant functions calculated by the analyses. Five discriminant functions 21
are derived in each analysis (females and males separately). When included in the model, all 22
significantly (P < 0.05) account for the variance (Supplementary material Tables S8, S9, 23
19
respectively). The three first discriminant functions, however, account for the largest part of the 1
variation (93.9% in the male analysis and 89.1% in females). The morphologies of the females of 2
A. tibetiana, A. amati n. sp. and A. sorgeloosi n. sp. cluster together in a very close group (Fig. 3
14a). When males are considered (Fig. 14b), A. sinica individuals add to the mentioned cluster, 4
and they are also close to this group. The morphology of males and females belonging to A. 5
franciscana and A. urmiana are also differentiated and discriminated by the analyses. 6
<Fig. 14> 7
The morphological discrimination among the three Asiatic species (A. urmiana, A. sinica, 8
and A. tibetiana) is apparent along the first discriminant function in females, and in a lesser 9
extent in males. This function is highly influenced by the length of the first antenna and the 10
characteristics of the cercopods (length and number of inserted setae). The mean measurements 11
of the morphological variables for each species are shown in Supplementary material Tables S10, 12
S11 for females and males, respectively. The mean length of the first antenna is over 1 mm in A. 13
tibetiana, A. amati n. sp., and A. sorgeloosi n. sp. females, and over 1.5 mm in males. They are 14
nevertheless ⁓0.9 mm in A. urmiana and A. sinica females, and ⁓1 mm in A. urmiana males. The 15
cercopod is very short in A. urmiana individuals, sometimes rudimentary (mean 0.17 mm in 16
females, 0.12 mm in males), and with 1.5 as the mean number of setae inserted in each cercopod 17
in females, and males presenting a mean number of setae in each cercopod of two. Artemia 18
tibetiana, A. amati n. sp., and A. sorgeloosi n. sp. males and females show a mean cercopod 19
length between 0.37 and 0.57 mm, and > 10 mean number of setae on each cercopod. The 20
general shape, given by the relationship of the abdominal length vs. the total length, and the 21
abdominal length, mostly influence the second discriminant function in both analyses. The 22
second discriminant function clearly discriminates the A. franciscana population. Artemia 23
20
franciscana has a shorter abdomen than the rest of the studied species, which makes a smaller 1
part of the total length, both in males and females. The grouped classification of females and 2
males are summarized in Table 7. Regardless, A. sorgeloosi n. sp. (93.3%), females of Asian 3
Artemia are correctly classified in their groups. Only males of A. urmiana and A. sinica are 4
correctly classified in their groups. Meanwhile, considerable high percentages of correct 5
classification (90%–96.7%) are in non-complete grouped species. 6
<Table 7> 7
The morphological characteristics of the brood pouch, gonopod, and frontal knobs are 8
represented and described in Figures 15, 16 and Table 8. 9
<Figs. 15, 16> 10
<Table 8> 11
Supplementary material table S12 summarizes general information and results of 12
molecular markers and morphological characteristics of each species. 13
14
DISCUSSION 15
We used a reliable multitrait molecular and morphological approach to compare conflicting 16
Artemia populations from the Tibetan Plateau and Kazakhstan with identified Asian Artemia 17
species: A sinica, A. tibetiana, and A. urmiana. The morphological, mitochondrial, and nuclear 18
marker datasets consistently identified two independent clades differentiated from the established 19
Asian species: A. sorgeloosi n. sp. and A. amati n. sp. These new species clarify a long history of 20
confusion mediated by the type of molecular marker considered, the location sampled, the 21
presence of parthenogenetic lineages, and the ecological conditions of these peculiar sites. 22
Although there is no considerable difference among the length of concatenated sequences 23
21
of protein-coding genes and rRNAs genes of Asian Artemia, Tibetan species show slight 1
mitogenome differences (15803 bp vs. 15626 bp) compared to the differences in the control 2
region (1790 bp vs. 1616 bp). Differences in control region demonstrate that it is not conserved 3
in Asian Artemia species, and such differences may be relevant considering that this region 4
controls the regulation and initiation of DNA replication and transcription (Geng et al., 2016). Its 5
mutations can thus alter the expression and energy of mitogenomic activity (Coskun et al., 2003; 6
Asem et al., 2021b), which may be effective in adaptation to particular sites. 7
The mitogenomic phylogenetic tree topology demonstrates that A. tibetiana and A. amati n. 8
sp. (Kazakhstan) are monophyletic taxa with a common ancestor with A. urmiana, whereas A. 9
tibetiana and A. sorgeloosi n. sp., are polyphyletic. Previous phylogenetic analyses based on the 10
mitochondrial COI and the nuclear ITS1 marker considered the American A. franciscana and 11
Asian Artemia as sister clades, with A. franciscana as a basal for this group. Additionally, A. 12
sinica was reported as the basal clade for Asian Artemia (Baxevanis et al., 2006; Kappas et al., 13
2009; Maniatsi et al., 2011; Eimanifar et al., 2014, 2020; Saji et al., 2019; Asem et al., 2018, 14
2021a, b). Our findings, using the complete mitochondrial sequences, also suggest that A. sinica 15
and the American A. franciscana would be divided with a basal node (“ancestral node,” 16
according to Omland et al., 2008), in which A. sinica is basal to other Asian species of Artemia. 17
In addition, the similarity of GC content between A. sinica and A. franciscana is remarkable. 18
Based on this evolutionary relationship, A. sinica (or its ancestor) could be considered an 19
intermediate evolutionary step for the dispersal of Artemia from the New World to Asia, in line 20
with the anostracan biogeography model (Rogers, 2015). 21
The low genetic distance and intra and interspecific variation we observed among Asian 22
species of Artemia (for the markers considered) is in line with the reported variation for other 23
22
anostracans (e.g., Aguilar et al., 2017). Artemia is, however, a special case. Artemia is a recently 1
evolved and established group (see Bellec et al., 2019; Xu et al., 2021; Sun & Cheng, 2022), and 2
the Asian sexual species are recent within the genus (Eimanifar et al., 2015). Artemia 3
populations and species are regionally adapted to the stringent local conditions of hypersaline 4
habitats (except A. franciscana). Regional or local conditions reduce the gene flow and genetic 5
variability underlying adaptive physiological or morphological traits if eggs are not dispersed 6
between adjacent lakes by wind or birds (Rogers, 2015). Highly adaptive traits in Artemia are 7
controlled by multiple genes, such as salinity tolerance (De Vos et al., 2021). Closely related 8
sibling Artemia species, however, show extensive functional-genetic differences (e.g., A. 9
francisana, A. monica, and A. persimilis) (Rogers, 2015). In this context, the morphological 10
differentiation discussed below is relevant and in line with the idea that biodiversity is a 11
phenotypic concept and so a necessary component of the species concept (Freudenstein et al., 12
2017). The haplotype network distribution generated in this work by the three mitochondrial 13
marker fragments (COI, 16S, 12S) demonstrates that each Asian Artemia species exhibits private 14
haplotypes, a sign of reproductive isolation. 15
Baxevanis et al. (2006) showed that Asian species (A. urmiana, A. sinica, and A. tibetiana) 16
and parthenogenetic lineages belong to different clades based on the nuclear ITS1 marker. The 17
marker, however, does not separate A. urmiana and parthenogenetic lineages due to shared 18
haplotypes (Asem et al., 2016). Likewise, an ITS1-based phylogenetic tree could not separate A. 19
amati n. sp.-A. urmiana, and the Tibetan species (A. sorgeloosi n. sp.-A. tibetiana) (Maccari et 20
al., 2013a; Eimanifar et al., 2014). Our study demonstrates the existence of shared ITS1 21
haplotypes between A. amati n. sp.-A. urmiana and A. sorgeloosi n. sp.-A. tibetiana. Shared 22
haplotypes in ITS gene(s) sequences, however, were observed among species in other taxa, e.g., 23
23
Sphaerium Scopoli, 1777 (Bivalvia: Veneroida) (Lee & Foighil, 2003), Sinularia May, 1898 1
(Anthozoa: Octocorallia) (McFadden et al., 2009), Dendrolimus Germar, 1812 (Insecta: 2
Lepidoptera) (Dai et al., 2012), and Cordulegaster Leach, 1815 (Insecta: Odonata) (Froufe et al., 3
2013). Therefore, these findings demonstrate that, except in A. sinica, the ITS1 gene tends to be 4
somewhat conserved among Asian taxa, and is not so informative to identify the taxonomic 5
status of Asian Artemia. 6
Nine microsatellites (nSSR) loci also differentiated A. urmiana, A. amati n. sp., and A. 7
sinica, and five informative microsatellite loci separated A. sorgeloosi n. sp. from A. tibetiana. 8
Regarding genetic diversity in COI, A. urmiana exhibited high variation in agreement with other 9
studies (Eimanifar et al., 2014, 2020), and the highest interspecific variation among all the 10
markers considered in this study, whereas A. tibetiana and A. amati n. sp. had the lowest 11
variation. Negative and significant values of the D Tajima test in A. urmiana and A. tibetiana 12
considering mitochondrial markers (except 12S) suggest the existence of rare alleles at high 13
frequencies, coincidently with a population expansion after a probable bottleneck or the effect of 14
selection (Asem et al., 2019b). 15
Asian species were suggested to be sibling species or subspecies (Sorgeloos, 1991) given 16
the reported full interfertility among A. urmiana, A. amati n. sp., and A. sinica (Pilla, 1990; Pilla 17
& Beardmore, 1994). Our morphological studies, however, do not support these species as 18
sibling or cryptic forms (sensu Mayr & Ashlock, 1991; Bickford et al., 2006; Beheregaray & 19
Caccone, 2007), considering the significant morphological differentiation of the cercopod, 20
frontal knob, and brood pouch. All studied Asian populations can be distinguished using 21
morphological characters. Morphological differentiation correlates well with the independent 22
gene pools of A. urmiana, A. sinica, A. tibetiana, A. amati n. sp., and A. sorgeloosi n. sp., and is 23
24
further corroborated by the mitochondrial and microsatellite markers. Despite close genetic 1
distance, lack of recognized hybrid zone(s) and isolated gene pools (including both 2
mitochondrial and nuclear SSR gene pools) demonstrate the lack of gene exchange (reproductive 3
isolation) among Asian taxa in nature, and as such they cannot be described as subspecies/semi-4
species or lineages of a species (sensu Mayr & Ashlock, 1991; Helbig et al. 2002; Freudenstein 5
et al., 2017). 6
Reproductive characterization and crossbreeding tests in Artemia have been helpful in 7
assessing reproductive isolation (offspring quantity or infertility), despite the difficulty of 8
comparing results due to the lack of standardized protocols. Pilla & Beardmore (1994) 9
documented complete interfertility among A. urmiana, A. amati n. sp., and A. sinica (see also 10
Pilla 1990), while Zhou et al. (2003) showed interfertility between A. sinica and A. tibetiana, 11
which is in line with the finding of isolating barriers between A. urmiana and A. sinica (Zheng & 12
Sun, 2008), and between A. sinica and A. tibetiana (Abatzopoulos et al., 2002b; Zheng & Sun, 13
2008). Assuming the relevance of verifying reproductive isolation, it is necessary to point out 14
that multiple parameters can influence Artemia reproductive potential, including temperature, 15
food nutritive quality/quantity, salinity, and ionic composition of the medium (Abatzopoulos et 16
al., 2003; Velasco et al., 2016, 2018), and there could be other unknown parameters as well. As a 17
result, different studies have obtained dissimilar results for same species/population(s), due to 18
lack of a standardized culture medium desirable for all species/populations that can support the 19
conditions of natural habitats. Baxevanis & Abatzopoulos (2004) demonstrated that two different 20
laboratory clones of a tetraploid parthenogenetic Artemia (M. Embolon, Greece) exhibited the 21
best reproductive performance in different salinities (40 g l–1 vs. 80 g l–1). More recently, Sellami 22
et al. (2021) documented significant alterations in reproductive characteristics in successive 23
25
generations of A. salina (Sebkha, Tunisia). It is important to consider the reproductive variation 1
observed in Artemia cultures, even between conspecific populations. Additionally, cross-2
breeding studies consider individual pairs in vials that do not represent the natural conditions 3
where individuals can select their mates (Zapata et al., 1990; Rode et al., 2011; Tapia et al., 2015; 4
Rogers, 2019). Nevertheless, these studies have been a preliminary and valuable approach to 5
assessing reproductive capacity and reproductive isolation across generations (Gajardo et al., 6
2001). While reproductive isolation (especially infertility) is one of the taxonomic principles 7
used to determine species status, it seems that laboratory cross-breeding tests cannot necessarily 8
demonstrate infertility and/or cross-breeding in nature due to variations in laboratory culture 9
conditions. Helbig et al. (2002) suggested that if there is no hybrid zone where reinforcement of 10
reproductive isolation occurs, laboratory interfertility results, even for subsequent generation(s), 11
cannot demonstrate reproductive isolation in nature. Because the distribution of anostracans is 12
very much a case akin to island biogeography (Rogers, 2015); however, such a hybrid zone is 13
unlikely. 14
Morphometry of adults using discriminant analysis presented species-specific 15
morphological characters. We show that A. urmiana has a distinctive morphology compared to 16
the other Asian species, as originally depicted by Pilla & Beardmore (1994). Pilla & Beardmore 17
(1994), however, did not find A. amati n. sp. morphologically distinct from A. sinica. This was 18
also demonstrated with A. tibetiana and the different ploidies of the parthenogenetic Artemia 19
lineages (Triantaphyllidis et al., 1997a, b; Baxevanis et al., 2005; Abatzopoulos et al., 2009; 20
Maccari et al., 2013b). These studies showed that the morphometry of the species and 21
parthenogenetic lineages are very similar, except for A. urmiana. Discriminant analysis (DA) 22
calculates multidimensional functions to separate as much as possible the a priori defined groups 23
26
of observations. The DA output, when provided adequate characters, is not comparable to most 1
of our molecular results, which employ analyses that join the different observations as much as 2
possible. Consequently, the closely related observations in the discriminant analysis score plot 3
must have a similar morphometric characteristic, but the output does not necessarily have to 4
match molecular characters that may evolve at a different pace. The incongruences between 5
molecular and morphometric data may also be due to the different pace and regulations driving 6
the molecular and phenotypic processes. Using different approaches to assessing phylogenetic 7
relationship are therefore helpful in obtaining a closer image of the real situation as Wiens (2004) 8
claims. Our morphometric results confirm that the specimens of each Asian species (especially 9
females) are predominantly classified in their own group. 10
According to Rogers (2015), anostracan biogeography is an example of island 11
biogeography and anostracans live in “islands of water in a sea of land.” As these habitats may 12
dry up, however, the resting egg banks developed from multiple generations over multiple years 13
will cause the mixing of multiple generations, counter-balancing the lack of genetic variability at 14
any time. Rogers’ (2015) model, based on the monopolization hypothesis of De Meester et al. 15
(2002) argues that anostracan species must have evolved allopatrically (geographically isolated) 16
in unoccupied, insular habitats (see references cited in both articles). The monopolisation 17
hypothesis argues that the founder population can grow rapidly and locally adapts hampering 18
gene flow, allowing it to monopolize resources (De Meester et al., 2002). Evolution may thus 19
occur cladogenically via small, genetically isolated founder populations, which will monopolize 20
a given basin due to strong priority effects. Simultaneously, evolution may occur anagenically 21
through local adaptation to changing environmental conditions. The monopolization hypothesis 22
drives speciation and by reversing competitive dominance patterns, leads to selection mediated 23
27
priority events since competitive equilibrium dynamics cannot readily develop in astatic habitats. 1
The likelihood of a hybrid contact zone is therefore very low. 2
Artemia tolerates wide periodic seasonal salinity changes in natural habitats (Litvinenko & 3
Boyko 2008; Ben Naceur et al., 2009). Many studies have demonstrated that different salinity 4
levels and temperature regimes can affect morphological characters of Artemia (especially size 5
of cercopod and its number of setae) in both natural habitats and laboratory conditions (Amat, 6
1980; El-Bermawi et al., 2004; Litvinenko & Boyko, 2008; Ben Naceur et al., 2011a, b, 2012; 7
Vetriselvan & Munuswamy, 2011; Krishnakumar & Munuswamy, 2014), although alteration 8
patterns of brood pouch and frontal knob have not been well studied. The variability of 9
morphology and morphometry make classic taxonomic identification keys difficult. Therefore, 10
we present a regional identification key for Asian Artemia based on laboratory cultured 11
specimens, following Hontoria & Amat (1992). We show that all Asian species can be identified 12
by the morphology of the cercopod, brood pouch, and the frontal knob. The morphology of the 13
frontal knob is species specific, but SEM is required to observe the necessary characters. 14
Additionally, the cercopod morphology is species specific in A. urmiana because salinity 15
changes do not affect its pattern in natural environmental conditions (Günther, 1899; Asem & 16
Rastegar-Pouyani, 2007, 2008) or laboratory conditions (Beardmore and Abreu-Grobois, 1983; 17
Triantaphyllidis et al., 1997b, Baxevanis et al., 2005). 18
19
Key to the Asian species of Artemia 20
1a Cercopod not reduced, bearing 9–20 polysetae; frontal knob subspherical in
dorsal view; brood pouch with lateral lobes in ventral view…………………... 2
1b Cercopod reduced, rudimentary, bearing 1–3 oligosetae; frontal knob
subconical in dorsal view; brood pouch without lateral lobes in ventral view …….
28
A. urmiana
2a Frontal knobs with spines mostly separated by > 3× their length, and medial
surface glabrous …..………………………………………………………….….. 3
2b Frontal knobs with spines mostly separated by < 3× their length, and medial
surface glabrous or spinose …..………………………………………………….. 4
3a Frontal knob with single spines, in pairs, or in triplets, homogeneously
dispersed on lateral and distal surfaces; brood pouch subtriangular .….. A. sinica
3b Frontal knob laterally with single or paired spines, distal surface with spines
paired or in triplets; brood pouch subellipsoidal ……...……….... A. amati n. sp.
4a Frontal knob with medial surface not glaborous…………………………… 5
4b Frontal knob medially glabrous, lateral surface with spines scattered
homogeneously as single, paired, or triplets, distal surface predominantly with
spines single, rarely in pairs or triplets; brood pouch triangula. A. sorgeloosi n. sp.
5a Frontal knob laterally with spines in triplets, distomedially with spines in pairs,
medially with single spines; brood pouch subtriangular ………….…. A. tibetiana
5b Frontal knob predominately with spines in triplets; brood pouch triangular,
with lateral lobes in ventral view ……………………..……….….. A. franciscana
1
Two new species, one from the Tibetan Plateau (Haiyan Lake) in China (Artemia 2
sorgeloosi n. sp.) and the other from Kazakhstan (Artemia amati n. sp.), are described, whereas 3
the Tibetan species (A. sorgeloosi n. sp. and A. tibetiana) represent distinctive mitogenomic 4
polyphyletic clades that lack shared mitochondrial haplotypes. Nuclear markers (nSSR) reinforce 5
their independent status, despite their close evolutionary relationship, which may be explained by 6
an historical hybridization event(s). The ancestral hybrid population(s) differentiated in a 7
different environment from their parental species evolving to become independent taxa (Abbott 8
et al., 2013). The mitochondrial phylogenetic trees topology suggests that the maternal 9
divergence time of the A. tibetiana clade occurred after A. sorgeloosi n. sp. Therefore, we 10
conservatively hypothesize that A. tibetiana may have resulted from a past ancestral 11
29
hybridization of a maternal ancestor of A. tibetiana with A. sorgeloosi n. sp. or its ancestor. A 1
similar process led to the speciation of the Indo-West Pacific soft-coral genus Sinularia 2
(McFadden et al., 2009). 3
Regarding the lack of new records of A. amati n. sp. from Kazakhstan (or elsewhere) in the 4
last three decades, and due to the effect of recent anthropogenic habitat conversion, we 5
hypothesize that the species has become extinct in the wild. The live resting eggs of A. amati n. 6
sp. are available at the Artemia Reference Center (ARC 1039) (Ghent University, Belgium), the 7
Institute of Aquaculture Torre de la Sal (IATS 693) (Spain), and the Hainan Tropical Ocean 8
University (HTOU 0009) (China). We thus suggest that A. amati n. sp. should be listed 9
following the IUCN Red-List guidelines (International Union for Conservation of Nature, 2001) 10
as Extinct in the Wild (EW). Numerous records from the Tibetan Plateau are reported as A. 11
tibetiana or “unidentified species” (Zheng & Sun, 2013). We recommend a comprehensive study 12
on the phylogeography of Artemia to determine the diversity and distribution of A. tibetiana and 13
A. sorgeloosi n. sp. on the Tibetan Plateau. 14
15
SUPPLEMENTARY MATERIAL 16
Supplementary materials are available at Journal of Crustacean Biology online. 17
S1 Table. Summary of data on output quality for the studied Artemia. 18
S2 Table. Information of the multiplex groups of microsatellite loci. 19
S3 Table. Gene characteristics in the mitochondrial genome assembly of A. tibetiana. 20
S4 Table. Gene characteristics in the mitochondrial genome assembly of A. amati n. sp.. 21
S5 Table. Gene characteristics in the mitochondrial genome assembly of A. sorgeloosi n. sp.. 22
S6 Table. Gene characteristics in the mitochondrial genome assembly of A. sinica. 23
30
S7 Table. Gene characteristics in the mitochondrial genome assembly of A. urmiana. 1
S8 Table. Canonical function results for the discriminant analysis of the female morphometric 2
characters. 3
S9 Table. Canonical function results for the discriminant analysis of the male morphometric 4
characters. 5
S10 Table. Mean measurements of the female morphometric variables of different Asian species 6
of Artemia included in the discriminant analysis. 7
Table S11. Mean measurements of the male morphometric variables of different Asian species of 8
Artemia included in the discriminant analysis. 9
Table S12. Summary of the basic characters of each species studied. 10
11
ACKNOWLEDGEMENTS 12
The authors thank Prof. Shi-Chun Sun (Ocean University of China, China) for preparing the eggs 13
samples of Artemia from Yuncheng Lake and Lagkor Co. We thank the anonymous reviewers 14
for their comments and suggestions. The help of Dr. Wei Liu (Institute of Oceanology, Chinese 15
Academy of Sciences, China) and Mr. Wang Zhixu (Hainan Tropical Ocean University, China) 16
for their assistance in SEM work is highly appreciated. This study has been supported by the 17
Major Science and Technology Projects in Hainan Province in 2019 (ZDKJ2019011-03-02). 18
19
20
REFERENCES 21
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1890 (Branchiopoda: Anostraca) in Europe: An integrated and interdisciplinary approach. 24
31
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Abatzopoulos, T.J., Beardmore, J.A., Clegg, J.S. & Sorgeloos, P. 2002a. Artemia: basic and 2
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Abatzopoulos, T.J., El-Bermawi, N., Vasdekis, C., Baxevanis, A.D. & Sorgeloos, P. 2003. 4
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18
19
20
21
22
48
1
Figure 1. Localities of the populations of the studied Artemia populations. Map © 2022 2
Microsoft Corporation. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, A. amati n. 3
sp.; SOR, A. sorgeloosi n. sp.. 4
5
6
7
8
9
10
11
12
49
1
Figure 2. Artemia sorgeloosi n. sp.: female in dorsal view (A), male in dorsal view (B1, B2). 2
Scale bars = 1 mm. 3
4
5
6
7
8
9
50
1
2
Figure 3. Artemia amati n. sp.: female in dorsal view (A), male in dorsal view (B). Scale = 1 3
mm. 4
5
6
7
51
1
2
Figure 4. Distribution of species on scatter plots (up) and heat-maps structure of Euclidean 3
distance between species (down) based on AT- and GC-skew values of PCGs+rRNAs (FRA has 4
been used as outgroup to exhibit clear position of species). URM, Artemia urmiana; SIN, A. 5
sinica; TIB, A. tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.; FRA, A. franciscana. 6
7
8
9
52
1
Figure 5. Phylogenetic tree of Asian species of Artemia based on mitochondrial genome 2
(PCGs+rRNAs) sequences (scale bar is referred to BI analysis). The maximum-likelihood 3
bootstrap (left) and Bayesian support values (right) are shown for each major node (A). 4
Haplotype network distribution (B) and heat-map displaying pairwise genetic distance 5
comparisons between-species (C) following PCGs+rRNAs sequences. URM, A. urmiana; SIN, A. 6
sinica; TIB, A. tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.; FRA, A. franciscana. 7
8
9
10
11
12
53
1
Figure 6. The relationship of the distribution of COI haplotypes among Asian species of Artemia. 2
The size of each circle is proportional to the frequency of specimens. Hatched circles indicate 3
intermediate or unsampled haplotypes. Black dots and numbers between haplotypes represent the 4
number of nucleotide substitutions. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, 5
A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 6
7
8
54
1
Figure 7. The relationship of the distribution of 16S haplotypes among Asian species of Artemia. 2
The size of each circle is proportional to the frequency of specimens. Hatched circles indicate 3
intermediate or unsampled haplotypes. Black dots and numbers between haplotypes represent the 4
number of nucleotide substitutions. URM, Artemia urmiana; SIN, A. sinica; TIB, A. tibetiana; 5
AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 6
55
1
Figure 8. The relationship of the distribution of 12S haplotypes among Asian species of Artemia. 2
The size of each circle is proportional to the frequency of specimens. Hatched circles indicate 3
intermediate or unsampled haplotypes. Black dots and numbers between haplotypes represent the 4
number of nucleotide substitutions. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, 5
A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 6
56
1
Figure 9. The relationship of the distribution of ITS1 haplotypes among Asian species of 2
Artemia. The size of each circle is proportional to the frequency of specimens. Hatched circles 3
indicate intermediate or unsampled haplotypes. Black dots and numbers between haplotypes 4
represent the number of nucleotide substitutions. URM, A. urmiana; SIN, A. sinica; TIB, A. 5
tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 6
7
8
9
57
1
Figure 10. Heat-map values for overall genetic distance among Asian species of Artemia based 2
on mitochondrial and nuclear markers. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; 3
AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 4
5
6
58
1
Figure 11. Heat-map pairwise comparison of between-species genetic distance among Asian 2
species of Artemia based on mitochondrial and nuclear markers. URM, A. urmiana; SIN, A. 3
sinica; TIB, A. tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 4
59
1
Figure 12. Principal coordinates analysis (PCoA) among Asian species of Artemia based on SSR 2
markers. using nine (A) and five (B) loci. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; 3
AMA; A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 4
5
6
60
1
Figure 13. Heat-map pairwise population matrix of Nei’s genetic distance among Asian species 2
of Artemia based on SSR. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, A. amati n. 3
sp.; SOR, A. sorgeloosi n. sp.. 4
5
61
1
Figure 14. Female (A) and male (B) morphometric discrimination plotted for the two first 2
canonical functions (larger symbols show centroids of each species). URM, A. urmiana; SIN, A. 3
sinica; TIB, A. tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.; FRA, A. franciscana. 4
5
62
1
2
Figure 15. SEM photographs of the brood pouch in ventral view (A), gonopod in ventral view 3
(B) and frontal knob in frontal view (C) of Asian species of Artemia. URM, A. urmiana; SIN, A. 4
sinica; TIB, A. tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.. 5
6
7
63
1
2
Figure 16. Schematic illustration of male head (dorsal view) and female-male cercopods (dorsal 3
view) of Asian species of Artemia. Scale bars = 1 mm. URM, A. urmiana; SIN, A. sinica; TIB, A. 4
tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp..5
64
Table 1. Studied populations of Artemia. 1 Hainan Tropical Ocean University, 2 Ocean University of China, 3 Lagkor Co = Lagkor
Lake; in Tibetan language Co means Lake (Zheng & Sun 2013), 4 Geographic data only refer to Kazakhstan, 5 Institute of Aquaculture
Torre de la Sal, 6 The geographical coordinate (36°03'N, 100°11'E) has been cited by Van Stappen et al., (2007) for Haiyan Lake is
unavailable on map, correct location followed Dong et al., 2010 and L. Sui (personal communication, 2022).
Species Type locality Geographic
coordinates Abbreviation Sampling
year Accession
Artemia urmiana
Urmia Lake, Iran
37°42'N,
URM
2004
HTOU
1
, China
Artemia sini
ca
Yuncheng Lake, China
34°59'N, 111°00'E
SIN
2003
OUC
2
, China
Artemia tibetiana
Lagkor
Co
3
, China
3
03'N, 84°13'E
TIB
2004
OUC, China
Artemia amati
n. sp.
?, Kazakhstan
48°0'N,
68°0'E
4
AMA
1988
IATS
5
, Spain
Artemia sorgeloosi
n. sp.
Haiyan Lake, China
36°48'N, 100°41'E
6
SOR
2001
IATS, Spain
Artemia franciscana
Great Salt Lake, USA
41°10
'N, 112°35'W
FR
A
1992
IATS, Spain
Table 2. GenBank accession numbers for type localities and molecular markers (* 577 bp length belongs to A. sinica). URM, A.
urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp..
G
ene
Size (bp)
URM
SIN
TIB
AMA
SOR
COI 577*–629 N = 35
MZ189779
813
N =35
MZ1898149
848
N =35
MZ189849
883
N =35
MZ189884
918
N =35
MZ189919
953
16S 436 N =35
MZ168698
732
N =35
MZ168733
767
N =35
MZ168768
802
N =35
MZ1688038
837
N =35
MZ168838
872
12S 424 N =35
MZ168873
907
N =25
MZ168908
932
N =35
MZ168933
967
N =35
MZ168968
002
N =35
MZ169003
037
ITS1
1086 N =30
MW995181
210
N =30
MW995211
240
N =30
MW995241
270
N =30
MW995271
300
N =30
MW995301
330
65
Table 3. Base nucleotide compositions of Asian species of Artemia based on concatenated
sequences of PCGs and rRNAs. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, A.
amati n. sp.; SOR, A. sorgeloosi n. sp..
Species GenBank
accession no.
Length
(bp)
G+C A+T GC–skew AT–skew Reference
URM
MN240408
12,440
38.07
61.93
0.0549
0.1649
Asem
et al
., 2021b
SIN
MK069595
12,412
36.09
63.91
0.0490
0.1691
Asem
et al
., 2019a
TIB
OP168928
12,399
38.25
61.75
0.0559
0.1649
this study
AMA
OP142420
12,399
38.09
61.91
0.0522
0.1662
this study
SOR
OP156999
12,399
37.98
62.02
0.0490
0.1645
this study
66
Table 4. Population genetic diversity indices and Tajima D values of Asian species of Artemia
for mitochondrial and nuclear markers. S, number of polymorphic (segregating) sites; Eta, total
number of mutations; H, No. of Haplotypes; Hr, Haplotype ratio; Hd, Haplotype (gene) diversity;
Pi, Nucleotide diversity; K, Average number of nucleotide differences; D, Tajima D. URM, A.
urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp..
Gene Index URM SIN TIB AMA SOR
COI
Nsa
35
35
35
35
35
Nsi
576
576
576
575
576
S
42
14
7
8
15
Eta
42
14
7
8
15
H
26
13
7
8
10
Hr
0.74
0.37
0.20
0.22
0.28
Hd
0.983
±
0.010
0.891
±
0.029
0.363
±
0.104
0.627
±
0.088
0.855
±
0.029
Pi
0.00722
±
0.
00584
0.00377
±
0.00230
0.00088
±
0.0013
8
0.00239
±
0.00152
0.00610
±
0.00243
K
4.158
2.171
0.508
1.375
3.516
D
2.132*
1.169
ns
1.999*
0.857
ns
0.113
ns
16S
Nsa
35
35
35
35
35
Nsi
433
434
434
434
433
S
37
12
14
2
6
Eta
37
12
14
2
6
H
28
10
1
3
3
8
Hr
0.80
0.28
0.37
0.08
0.22
Hd
0.978
±
0.016
0.696
±
0.064
0.575
±
0.101
0.375
±
0.086
0.701
±
0.062
Pi
0.00810
±
0.00693
0.00373
±
0.00271
0.00184
±
0.00306
0.00089
±
0.00082
0.00332
±
0.00165
K
3.509
1.617
0.800
0.387
1.439
D
2.176*
1.4
07
ns
2.474**
0.399
ns
0.034
ns
12S
Nsa
35
25
35
35
35
Nsi
418
419
419
419
419
S
38
11
14
8
14
Eta
38
12
14
8
14
H
39
10
13
8
12
Hr
0.82
0.40
0.37
0.22
0.34
Hd
0.980
±
0.016
0.780
±
0.073
0.775
±
0.060
0.813
±
0.035
0.748
±
0.072
Pi
0.00981
±
0.00736
0.00402
±
0.00297
0.00418
±
0.
00317
0.00376
±
0.00208
0.00369
±
0.00317
K
4.101
1.683
1.751
1.576
1.546
D
1.987*
1.598
ns
1.568
ns
0.553
ns
1.764
ns
ITS1
Nsa
30
30
30
30
30
Nsi
1048
1062
1059
1052
1028
S
56
24
7
30
51
Eta
57
24
7
35
5
3
H
23
14
7
17
19
Hr
0.76
0.46
0.23
0.56
0.63
Hd
0.979
±
0.014
0.860
±
0.057
0.508
±
0.108
0.844
±
0.066
0.871
±
0.060
Pi
0.00507
±
0.00446
0.00375
±
0.00206
0.00061
±
0.00079
0.00335
±
0.00253
0.00561
±
0.00417
K
5.313
3.979
0.648
3.520
5.763
D
2.365**
1.217
ns
1.870*
2.197**
2.
127*
67
Table 5. Population genetic indices of Asian species of Artemia based on nine microsatellite loci.
Na, number of alleles; Ne, number of effective alleles; I, Shannon's Information Index; Ho,
observed heterozygosity; He, expected heterozygosity; uHe, unbiased expected heterozygosity; F,
fixation index; PL, polymorphic loci. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA,
A. amati n. sp.; SOR, A. sorgeloosi n. sp..
Species
Na Ne I Ho He uHe F %PL
URM 6.556
(1.396)
3.340
(0.992)
1.174
(90.245)
0.449
(0.076)
0.532
(0.095)
0.543
(0.097)
0.110
(0.049)
100.00
SIN 3.111
(0.964)
1.598
(0.387)
0.520
(0.199)
0.200
(0.082)
0.268
(0.097)
0.274
(0.099)
0.203
(0.128)
77.78
TIB 2.222
(0.465)
1.376
(0.254)
0.426
(0.143)
0.296
(0.105)
0.243
(0.083)
0.248
(0.085)
–0.176
(0.054)
66.67
AMA 3.111
(0.611)
2.055
(0.380)
0.691
(0.180)
0.341
(0.108)
0.386
(0.096)
0.394
(0.098)
0.174
(0.147)
77.78
SOR 3.444
(1.226)
2.068
(0.701)
0.630
(0.251)
0.330
(0.120)
0.312
(0.112)
0.319
(0.114)
–0.091
(0.115)
55.56
Table 6. Characteristics of the alleles (number and size) among Asian species of Artemia in
different loci (NA, number of allele). URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA,
A. amati n. sp.; SOR, A. sorgeloosi n. sp..
Locus
URM
SIN
TIB
AMA
S
OR
NA
Size range
(bp)
NA
Size range
(bp)
NA
Size range
(bp)
NA
Size range
(bp)
NA
Size range
(bp)
Apcpm1
3
105
116
4
107
118
4
107
116
3
107
114
5
107
142
Appm4
2
82
87
1
82
1
82
1
82
1
82
Aupm5
6
163
180
2
154
160
1
160
1
160
1
160
Aupm7
8
116
141
2
119
122
2
128
131
3
105
128
3
128
137
Aupm15
8
83
97
2
87
89
3
87
98
3
91
95
1
87
Aupm16
16
114
176
10
74
126
2
122
130
4
106
139
12
118
158
Appm20
6
98
109
4
98
105
4
98
113
7
93
114
3
98
111
Aupm21
7
97
118
2
106
112
Appm26
3
182
192
3
182
239
3
181
192
4
181
223
5
179
215
68
Table 7. Percentage of complete original female and male of species of Asian Artemia grouped
classification by discriminant analysis. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana;
AMA, A. amati n. sp.; SOR, A. sorgeloosi n. sp.; FRA, A. franciscana (A. franciscana used as
an outgroup).
Female classification results (98.9%
of original grouped cases correctly
classified
).
Origin
Predicted group membership
SIN
URM
FRA
TIB
SOR
AMA
Total
Original Count
SIN
30
0
0
0
0
0
30
URM
0
30
0
0
0
0
30
FRA
0
0
30
0
0
0
30
TIB
0
0
0
30
0
0
30
SOR
0
0
0
1
28
1
30
AMA
0
0
0
0
0
30
30
%
SIN
100.0
0.0
0.0
0.0
0.0
0.0
100.0
URM
0.0
100.0
0.0
0.0
0.0
0.0
100.0
FRA
0.0
0.0
100.0
0.0
0.0
0.0
100.0
TIB
0.0
0.0
0.0
100.0
0.0
0.0
100.0
SOR
0.
0
0.0
0.0
3.3
93.3
3.3
100.0
AMA
0.0
0.0
0.0
0.0
0.0
100.0
100.0
Male classification results
(97.2% of original grouped cases correctly
classified
).
Origin
Predicted group membership
SIN
URM
FRA
TIB
SOR
AMA
Total
Original Count
SIN
30
0
0
0
0
0
30
URM
0
30
0
0
0
0
30
FRA
0
0
30
0
0
0
30
TIB
0
0
0
29
1
0
30
SOR
0
0
0
0
29
1
30
AMA
1
0
0
0
2
27
30
%
SIN
100.0
0.0
0.0
0.0
0.0
0.0
100.0
URM
0.0
100.0
0.0
0.0
0.0
0.0
100.0
FRA
0.0
0.0
100.0
0.0
0.0
0.0
100.0
TIB
0.0
0.0
0.0
96.7
3.3
0.0
100.0
SOR
0.0
0.0
0.0
0.0
96.7
3.3
100.0
AMA
3.3
0.0
0.0
0.0
6.7
90.0
100.0
69
Table 8. Description of brood pouch (up), gonopod (middle), and frontal knob (down) in Asian
species of Artemia. URM, A. urmiana; SIN, A. sinica; TIB, A. tibetiana; AMA, A. amati n. sp.;
SOR, A. sorgeloosi n. sp.; FRA, A. franciscana (* because of A. franciscana is widely
introduced in Asia and spreading, it was considered as a species in Asian).
Organ
Species
Description
Reference
Brood pouch
(female)
URM
triangular without lateral lobes in ventral view
this study
SIN
rounded
-
triangular with lateral lo
bes in ventral view
this study
TIB
rounded
-
triangular with lateral lobes in ventral view
this study
AMA
rounded
-
ellipsoidal with lateral lobes in ventral view
this study
SOR
triangular with lateral lobes in ventral view
this study
FRA*
triangular or rounded with lateral lobes in ventral view Torrentera
& Dodson
1995
Gonopod (male)
URM
basal spine present
this study
SIN
basal spine present
this study
TIB
basal spine present
this study
AMA
basal spine present
this study
SOR
basal spine presen
t
this study
FRA basal spine present Torrentera
& Dodson
1995,
Beardmore
& Abreu-
Grobois
1983
Frontal knob
(male)
URM subconical in dorsal view, with dense spine distribution:
triplet spines toward external axis, twin spines toward
medial/internal axis
, single spines are rare
this study
SIN subspherical in dorsal view, with sparse spine distribution:
single, twin and triplet spines with homogeneous
dispersion in external/medial axes, with glabrous in
internal
axis
this study
TIB subspherical in dorsal view, with dense spine distribution:
triplet spines toward external axis, twin spines toward
internal/medial axes, single spines in internal axis
this study
AMA subspherical in dorsal view, with extremely sparse spine
distribution: single and twin spines toward external axis,
triplet and twin spines in medial axis, with glabrous in
internal
axis
this study
SOR subspherical in dorsal view, with dense spine distribution:
single, twin and triplet spines with homogeneous
dispersion in external axis, predominance with single
spines and rare twin and triplet spines in medial axis,
with
this study
70
glabrous in internal
axis
FRA Subconical or subspherical in dorsal view/ with dense
spine distribution and homogeneous dispersion with
single,
twin and triplet
spines
Torrentera
& Dodson
1995
Cercopod
(female/male)
URM
rudimentary/oligosetae
this study
SIN
lobed/
polysetae
this study
TIB
lobed/
polysetae
this study
AMA
lobed/
polysetae
this study
SOR
lobed
/polysetae
this study
FRA lobed/polysetae Beardmore
& Abreu-
Grobois
1983
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