Morphological Differentiation of Artemia urmiana Günther, 1899 (Crustacea: Anostraca) in Different Geographical Stations from the Urmia Lake, Iran

The genus Artemia (Crustacea: Anostraca) is a complex of bisexual and parthenogenetic species, which have very similar morphological characters. Artemia has widely distribution on the 5 continents in many salt lakes, coastal lagoons and solar salt-works (Van staapen, 2002). The Urmia Lake is the second large lake (average total surface 5000 km2) in Iran, which is located in West Azerbaijan Province. It is the habitat of the endemic Artemia urmiana Günther, 1899. This characteristic was confirmed by Clark and Bowen (1976) who demonstrated the reproductive isolation of the species from other bisexual strains. Van Stappen (2002) prepared last check list of distribution and zoogeography of Artemia, in this manuscript many of specimens introduced as unknown population and labeled by "?" symbol. This shows that taxonomy and systematics of Artemia is still puzzled The most relevant methods are comparison of biometrical and morphological characteristics, electrophoretic patterns of different isozymes, cross-fertility tests and electron microscopic survey of morphology such as frontal knob and penis (Hontoria and Amat, 1992; Abreu-Grobois and Beardmore, 1982; Mura, 1990; Triantaphyllidis et al., 1997a,b; Torrentera and Belk, 2002).

Brine shrimp Artemia is an economical taxon which the main application of its have been shown in aquaculture industry (Sorgeloos, 1980; Sorgeloos et al., 1998, 2001; Bengtson et al., 1991). According to base concepts of biosystematics, species and populations have special characters therefore, biosystematical studies can lead to useful economical approach.

In this study, our objective is to study morphological differentiation of A. urmiana in different geographical locations and to investigate if there are isolated populations of A. urmiana in the Urmai Lake. 

Field study: In this study 4 stations were selected in the middle, northern and southern parts of the Urmia Lake (Fig. 1). Four primary ecological factors were measured for each station: Salinity (0.5 m from surface and 0.5 m from depth), pH (0.5 m from surface and 0.5 m from depth), depth and transparency. Table 1 shows chemical comparison of the Urmia Lake water, Dead Sea, Great Salt Lake and oceans.

Morphological study: Male and female samples were analyzed separately because they are sexually dimorphic. Study of sexual dimorphism is very important in stydy of morphological differentiation (Asem et al., 2005).

Thirty male and female specimens of Artemia urmiana were randomly collected from each of the four different harvesting sites in north, south and in the middle of the Urmia Lake: N2; M3-2; M1-2; S2 (Fig. 1). Twelve characters for males and thirteen for females were measured (total length, abdominal length, morphological head width, distance between compound eyes, diameter of left eye, diameter of right eye, abdominal width, distance between 3rd abdominal segment to 8th abdominal segment, length of telson, length of furca, length of right antenna, length of left antenna, ovisac width) and number of setae per furca were counted. (Hontoria and Amat 1992, Pilla and Beardmore, 1994; Triantaphyllidis et al., 1997b; Cohen et al., 1999; Zhou et al., 2003; Camargo et al., 2003; Amat et al., 2005). Morphometric and meristic characters used in this study are shown in Fig. 2 and Table 2.

Table 1: Chemical comparison of Urmia Lake water with Dead Sea, Great Salt Lake and oceans (Daneshvar and Ashasi sorkhabi, 1997)

Fig. 1: Geographical location of the studied area, Urmia Lake-Iran

Fig. 2: Body measurements in Artemia (Version of Gajardo et al., 1998)

Table 2: The metric and meristic character use in this study

Statistical analysis: Morphological differentiation between samples of A. urmiana from four different geographical locations in the Urmia Lake was investigated by ANOVA (Tukey, p<0.05), Principal Components Analysis (PCA) and Discriminant Function Analysis (DA). In addition, DA used to appoint percent of corrected classification among stations. This was achieved by 2 methods: Calculated percent of overall correct classification as well as percent of correct classification between each pair stations. All statistical analysis was done by SPSS 11.5.

 

Primary physico-chimical parameters for 4 sampling locations are shown in Table 3 and morphometric and meristic characters as well as statistical comparisons of the results are summarized in Table 4.

According to Table 4, morphometric characters show different degrees of variation among stations. TL, AL and LF are three characters that have high variation among male samples so they are significantly different (p<0.05) in all the male samples. Meristic characters don't show significant difference among all the male and female samples (p<0.05).

Table 3: The primary ecological factors for each station

Fig. 3: Principal Component Analysis for female samples

Differentiation among female samples: PCA shows that according to factor 1, station M1-2 comparatively was separated from other stations. Station S2 and N2 created one mixed group that with regard to factor 2, this collection fairly was separated from station M3-2 (Fig. 3).

In component 1, ED_RI, LA_RI, LA_LE and ED_LE characters and in component 2, TL and AL characters are the most important characters for morphological differentiation among female samples (Table 5) The first 2 factors show 66.88 and 12.07% of variety and altogether 2 components show 78.95% of variation (Table 5) DA comparatively confirmed PCA finding. According to DA, any of station didn't completely divide together (Fig. 4). But stations M1-2 and M3-2 show most separation in comparison with other stations. Stations S2 and N2 mixed in one group that with regard to function 2 this collection absolutely was separated from station M3-2. In function 1, TL, AL, LF and LTE characters and in function 2, ED_RI and LA_RI characters are the most important characters for morphological differentiation among female samples (Table 5). The first second function show 85.9 and 14.10% of variation and on the whole the 2 functions show 100% of diversification (Table 5) Correctly classified between each pair stations were tested by DA:

• Correctly classified between M1-2 and N2 equal 91.7%.
• Correctly classified between M1-2 and S2 equal 86.7%.
• Correctly classified between M1-2 and M3-2 equal 98.3%.
• Correctly classified between M3-2 and N2 equal 78%.
• Correctly classified between M3-2 and S2 equal 73.3%.
• Correctly classified between N2 and S2 equal 66.7%.

In sum, correctly classified for four female samples equal 60%; so station M3-2, 66.7%, station S2, 33.3%, station N2, 46.7 and station M1-2, 93.3% pleased in your group (Table 6).

Differentiation among male samples: In PCA, according to factor 1, station M1-2 was divided from other stations. Station S2 and M3-2 made one mixed group that with regard to factor 2 this collection positively was divided from station N2 (Fig. 5). In component 1, TL, AL, LTE and LF characters and in component 2, ED_LE, LA_LE, ED_R and AL_R characters are the most important traits for morphological differentiation in male samples (Table 5) The first 2 factors show 52.65 and 27.98% of variation and in total, 2 components show 80.64% of variety (Table 5).

According to DA, station M1-2 completely divided to other three stations. Stations S2 and M3-2 mixed in one group that didn't show most separation from station M3-2 (Fig. 6). In function 1, LTE, LF, AL and TL characters and in function 2, ED_RI, AL_RI, AL_LE and ED_LE characters are the most important characters for morphological differentiation among male samples (Table 5) The first and second functions show 83.1 and 13.5% of the total variation and in sum the 2 functions show 96.7% of variation (Table 5) Correctly classified between each pair stations were tested by DA:

• Correctly classified between M1-2 and N2 equal 100%.
• Correctly classified between M1-2 and S2 equal 100%.
• Correctly classified between M1-2 and M3-2 equal 100%.
• Correctly classified between M3-2 and N2 equal 90%.
• Correctly classified between M3-2 and S2 equal 73.3%.
• Correctly classified between N2 and S2 equal 88.3%.

In all correctly classified for four males sample equal 78.3% in the other side station M3-2, 53.3%; station, S2 70%; N2, 90 and station M1-2 100% pleased in your group (Table 6).

Table 4: Mean (mm) ±S.D of morphometric and meristic characters for male and female samples from four stations (same letters shows non-significant differences between means in each row of main column, p>0.05)

Table 5: Portion of each trait in two first components and functions for morphological differentiation

Table 6: Percentage of complete original female and male grouped classification by discriminant function Analysis (a) classification results

Fig. 4: Discriminat function analysis for female samples

Fig.5: Principal component analysis for male samples

Fig. 6: Discriminat function analysis for male samples

 

Study of morphological differentiation is one of the most useful methods in Artemia taxonomy and biosystematics.

Hontoria and Amat (1992) studied adult female Artemia belonging to 25 populations from all over the America. They were able to clearly separate the populations of 2 different species, A. franciscana and A. persimilis. Discrimination based on morphometric characters within the Eastern Old World group clearly separates A. urmiana from the other populations (Pilla and Beardmore, 1994). Gajardo et al., (1998) studied 6 populations from Chile, the analysis showed that A. franciscana and A. persimilis are morphologically divergent. They analysed the data combining both males and females for the individuals grouped by the type of population to which they belong. Cohen et al., (1999) showed that adult females studied by multivariate discriminant analysis, provided evidence that populations from La Pampa and Buenos Aires provinces (Argentina) belonged to the species A. persimilis. Triantaphyllidis et al. (1997a) studied eleven bisexual populations according to morphological characters. These populations divided into four different groups: the A. franciscana group, the A. tunisiana group, the A. urmiana group and a broader group which includes Eastern Old World populations. They carried out same study about Morphological differentiation of 15 parthenogenetic Artemia populations. Discriminant analysis revealed five main groups of morphological patterns for 15 populations (Triantaphyllidis et al., 1997b). Morphometric characterization of A. franciscana populations from the Colomsbian Caribbean shown that Male and female Colombian Caribbean populations were separated from the North American populations Camargo et al. (2003).

With regard to biosystematics and taxonomic concepts, if species show sexual dimorphism, separate analyses for male and female data must be performed (Fowler et al., 1998; Manley, 1996). Male and female samples were analyzed separately because the Artemia urmiana populations from these four stations were shown sexually dimorphic (Asem et al., 2005).

In the PCA, the female samples were almost clustered in one group in all sites and it was not possible to make any separation among them. But in the DA, the station M1-2 was relatively separated from the other groups. In this analysis: 98.3% of original groups were correctly classified between M1-2/M3-2, 91.7% of original groups were correctly classified between M1-2/N2, 86.7% of original groups were correctly classified between M1-2/S2.

On the other hand, the PCA shows that male samples in stations N2, S2 and M2-3 were almost clustered in one group and this mixed group was separated from station M1-2 with regard to the factor1. This finding was also confirmed by DA. In the latter analysis 100% of original groups were correctly classified between M1-2/M3-2, M1-2/N2 and M1-2/S2 stations. Male morphometric characters separated population groups more clearly than the female characters. This result was also confirmed by result of (Camargo et al. (2003) according to which classification based on male characters provides better group membership than females.

Therefore, based on the results, there are minimum 2 ecological populations of Artemia urmiana in the Urmia Lake. With regard to these results it isn't possible to make taxonomic decisions on these 2 populations and further investigation is needed to make the final decision.

With regard to the PCA and DA Scatterplots, there is no geographical arrangement between stations. Among the female samples, stations N2 and S2 assembled as a mixed group but these stations are geographically distant and among the male samples M3-2 and S2 mixed in one group and stations M3-2 and M1-2 were separated but these 2 stations are geographically very close. If we accept that Ionic composition of the habitat can produce ecological isolation and can result in morphological and biometrical differences (Bowen et al., 1985, 1988; Hantoria and Amat, 1992) therefore we can suggest, speciation of A. urmiana in the Urmia Lake goes by ecological speciation.

According to previous studies (Hontoria and Amat 1992, Pilla and Beardmore 1994, Triantaphyllidis et al., 1997b, Gajardo et al., 1998, Cohen et al., 1999, Camargo et al., 2003, Amat et al., 2005) Artemia materials have been harvested from the samples that grow in the same laboratory conditions. If we accept that salty composition of the habitat can produce ecological isolation and can result in morphological and biometrical differences (Bowen et al., 1985, 1988; Hontoria and Amat, 1992), accordingly we can suggest that the main mechanism of evolution in the genus Artemia is a kind of ecological speciation. Therefore study of effects of ecological processes in morphological differentiation and evolution of Artemia is of paramount importance. According to this scenario, collecting Artemia samples from their natural habitat has priority to the materials growing in the same conditions in laboratory. The use of the horizontal conditions for all species and populations of Artemia deviate us from correct knowledge of the ecological speciation process. In addition, maybe 2 populations have significant differences in morphology, but same laboratory conditions make limitation and disadvantageous conditions with ionic concentration and composition. Therefore, in these conditions, 2 populations couldn't disclose their differentiation. This hypothesis is completely acceptable with regard to ecological concepts. But provide of Artemia field sample is difficult because Artemia has short life cycle in natural habitat, also most habitats are temporary ponds and lagoons and if collecting of samples are difficult, therefore each population must grow in general ionic conditions according to natural habitat with different salinity treatments. These 2 methods associate main evolution and speciation processes in nature. 

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